Universidade de Lisboa

Faculdade de Medicina

Ana Rita Cascão Rodrigues

Doutoramento em Ciências Biomédicas

Especialidade em Imunologia

2012

Universidade de Lisboa

Faculdade de Medicina

Ana Rita Cascão Rodrigues

Tese orientada por:

Professor Doutor João Eurico Fonseca

Professor Doutor Luís Ferreira Moita

Doutoramento em Ciências Biomédicas

Especialidade em Imunologia

2012

Todas as afirmações efectuadas no

presente documento são da

exclusiva responsabilidade do seu

autor, não cabendo qualquer

responsabilidade à Faculdade de

Medicina da Universidade de

Lisboa pelos conteúdos nele

apresentados.

A impressão desta dissertação

foi aprovada pela Comissão

Coordenadora do Conselho

Científico da Faculdade de

Medicina da Universidade de

Lisboa em reunião de 15 de

Maio de 2012.

Dissertação apresentada à

Faculdade de Medicina da

Universidade de Lisboa, para

obtenção do grau de Doutor em

Ciências Biomédicas.

To Science

“The important thing in science is not so much to obtain new facts

as to discover new ways of thinking about them.”

Sir William Bragg (1862 - 1942)

Nobel Prize in Physics

XIV

Table of Contents

Index of Figures XVIII

Index of Tables XX

Acknowledgments XXII

List of Abbreviations XXIV

Sumário XXVI

Summary XXX

Chapter I Introduction -1-

1. Rheumatoid arthritis - 3 -

1.1 Definition - 3 -

1.2 Pathogenesis - 5 -

1.3 Immune cells - 8 -

1.3.1 Macrophages - 8 -

1.3.2 Neutrophils - 8 -

1.3.3 T cells - 9 -

1.3.4 B cells - 10 -

1.4 Cytokines - 10 -

1.4.1 IL-1β - 10 -

1.4.2 TNF - 12 -

XV

1.4.3 IL-6 - 13 -

1.4.4 IL-17 - 14 -

1.5 Treatment options - 14 -

1.5.1 Glucocorticoids - 15 -

1.5.2 Conventional DMARDs - 15 -

1.5.3 Biologic DMARDs - 15 -

2. IL-1β Immunological and inflammatory functions - 18 -

2.1 IL-1 family - 18 -

2.2 IL-1β pathway - 18 -

2.2.1 Priming - 18 -

2.2.2 Processing - 19 -

2.2.3 Secretion - 21 -

2.2.4 Signaling - 21 -

2.3 IL-1β and autoinflammatory diseases - 22 -

2.4 IL-1β and rheumatoid arthritis - 24 -

2.5 Targeting IL-1β in rheumatoid arthritis: the case of anakinra - 27 -

Chapter II Aims -31-

Chapter III Results -35-

Cytokine pattern in very early rheumatoid arthritis favours B cell activation and survival

- 39 -

XVI

Identification of a cytokine network sustaining neutrophil and Th17 activation in untreated

early rheumatoid arthritis - 41 -

Caspase-1 is active since the early phase of rheumatoid arthritis - 43 -

Effective treatment of rat adjuvant-induced arthritis by celastrol - 45 -

Gambogic acid is a potent anti-inflammatory and anti-proliferative drug in a rat model of

adjuvant-induced arthritis - 47 -

Chapter IV Discussion -49-

Chapter V Conclusions -67-

References -71-

XVII

XVIII

Index of Figures

Figure 1 – Representative scheme illustrating immune cells and cytokine networks in

rheumatoid joints. - 7 -

Figure 2 – An overview on the multiple roles of IL-1β in rheumatoid arthritis physiopathology.

- 12 -

Figure 3 – A simplified scheme showing main pathophysiologic pathways in rheumatoid arthritis

and the effects of some currently available biologic agents. - 17 -

Figure 4 – IL-1β processing pathways. - 19 -

Figure 5 – Proposed model for cytokine networks in the very early phase of rheumatoid arthritis.

- 54 -

Figure 6 – Proposed mechanism for excessive IL-1β production since early rheumatoid arthritis.

- 57 -

Figure 7 – Proposed mechanism for celastrol and gambogic acid anti-inflammatory effects.

- 62 -

XIX

XX

Index of Tables

Table 1 – The 2010 American College of Rheumatology / European League Against

Rheumatism classification criteria for rheumatoid arthritis. - 4 -

Table 2 – The 1987 American College of Rheumatology criteria for the classification of

rheumatoid arthritis. - 5 -

XXI

XXII

Acknowledgments

First and foremost, I would like to acknowledge both my supervisors, Professor

Doutor João Eurico Fonseca and Professor Doutor Luís Ferreira Moita, for having

accepted me as a PhD student, for all the scientific meetings and discussions, for all

the guidance and opportunities. Thank you both for all the support.

Financial support was generously granted by Sociedade Portuguesa de

Reumatologia, Pfizer and Fundação para a Ciência e a Tecnologia through the PhD

fellowship (SFRH/BD/40513/2007).

My thanks also go to my PhD thesis committee, Professor Doutor Luís Graça,

Professor Doutor Carlos São José and Doutora Margarida Carneiro, for all the

meetings, discussions and suggestions that improved the work. But most of all, thank

you for your unconditional support, motivation and trust.

I would like also to thank all the members from Rheumatology Department,

Hospital de Santa Maria, all the medical doctors and nurses, for the effort to collaborate

in the development of my work, especially in sample collection and in the organization

of clinical data. In particular, a thanking note to Enfª. Lurdes Narciso, Doutora Helena

Canhão, Drª. Ana Maria Rodrigues, Dr. Joaquim Polido Pereira, Drª. Elsa Sousa, Drª.

Cristina Ponte, Drª. Raquel Marques, Drª. Ana Filipa Mourão (Rheumatology

Department, Hospital Egas Moniz) and Dr. Márcio Navalho (Radiology Department,

Hospital da Luz) with whom I have worked closer.

I am also very grateful to all the patients and healthy volunteers for their

willingness in providing us with samples making this project possible.

I wish to acknowledge Doctor Vineet Gupta from the Department of Medicine,

University of Miami for giving us the library of drugs used in this work.

Many thanks to all my colleagues from Instituto de Medicina Molecular that have

directly worked with me, especially Rita Moura, Bruno Vidal, Ana Lopes, Helena

Raquel, Ana Neves Costa, Ana Luísa Caetano and Saba Abdulghani. This work greatly

benefited from the close collaboration and invaluable friendship over the years.

Many thanks also to all my friends for everyday support and friendship.

Finally, I would like to deeply thank my parents and Zé for their unconditional love

and guidance in life.

Thank you all for turning this challenging journey into an enjoyable one!

Obrigada!

XXIII

XXIV

List of Abbreviations

ACPA Anti-citrullinated protein antibodies

ACR American College of Rheumatology

AIA Adjuvant-induced arthritis

APRIL A proliferation-inducing ligand

ASC Adaptor molecule apoptosis associated speck-like protein containing a

caspase recruitment domain

BLyS B-lymphocyte stimulator

CAPS Cryopyrin-associated periodic fever syndromes

CARD Caspase recruitment domain

CINCA/NOMID Chronic infantile neurological cutaneous and articular syndrome/neonatal

onset multisystem inflammatory disease

DAMP Danger associated molecular pattern

DMARD Disease-modifying anti-rheumatic drug

EA Early arthritis

EAE Experimental autoimmune encephalomyelitis

ERA Early rheumatoid arthritis

EULAR European League Against Rheumatism

FCAS Familial cold-induced autoinflammatory syndrome

FcγR Fc gamma-receptor

HLA Human leukocyte antigen

IFNγ Interferon gamma

IL Interleukin

IL-1R IL-1 receptor

IL-1Ra IL-1 receptor antagonist

IL-1RAcP IL-1 receptor accessory protein

IL-1RI IL-1 receptor type I

IL-1RII IL-1 receptor type II

LRR Leucine rich repeat

MAP Mitogen activated protein

MCP-1 Monocyte chemotactic protein-1

MHC Major histocompatibility complex

XXV

MMP Metalloproteinase

MTX Methotrexate

MWS Muckel-Wells syndrome

MyD88 Myeloid differentiation primary response gene 88

NACHT Nucleotide-binding domain or NAIP, CIITA, HET-E and TP1

NF-kB Nuclear factor kappa-light-chain-enhancer of activated B cells

NLR NOD-like receptor

NOD Nucleotide-binding oligomerization domain

NSAID Non-steroid anti-inflammatory drug

PAMP Pathogen associated molecular pattern

PRR Pattern recognition receptor

PYD Pyrin domain

RANK Receptor activator of nuclear factor

RANKL RANK ligand

RF Rheumatoid factor

RORγ Retinoic acid receptor-related orphan receptor gamma

ROS Reactive oxygen species

VEA Very early arthritis

VEGF Vascular endothelial growth factor

VERA Very early rheumatoid arthritis

TCR T cell receptor

TfR1 Transferrin receptor 1

Th T helper

TIR Toll-IL-1 receptor domain

TLR Toll-like receptor

TNF Tumor necrosis factor

XXVI

Sumário

A artrite reumatóide é uma doença inflamatória crónica imunomediada, com

envolvimento predominante da membrana sinovial das pequenas articulações e

destruição progressiva de cartilagem e osso, que induz incapacidade funcional e

aumento da morbilidade e mortalidade dos doentes. O diagnóstico precoce da artrite

reumatóide e a administração imediata de uma terapêutica adequada são cruciais para

a prevenção da progressão da doença e da destruição articular, a qual pode ocorrer

numa fase muito inicial. A característica mais debilitante da artrite reumatóide é a

hiperplasia sinovial. Esta inflamação sinovial é mediada pela interação entre diversas

células do sistema imunitário (macrófagos, neutrófilos, células T e B) e complexas

redes de citocinas (particularmente interleucina (IL)-1β, fator de necrose tumoral (TNF)

e IL-17).

A IL-1β é considerada um dos elementos centrais na patologia da artrite

reumatóide, por induzir um aumento na secreção de citocinas e quimiocinas e

produção de metaloproteinases (MMPs) bem como expansão e sobrevivência de

células T, que por sua vez conduzem a um aumento na produção de autoanticorpos

pelas células B, à diferenciação de células T auxiliares (Th)17 e à osteoclastogénese,

com consequente erosão óssea. É importante salientar que a produção de IL-1β é

fortemente regulada quer a nível da transcrição, quer a nível da pós-tradução,

envolvendo o factor nuclear kappa B (NF-kB), o inflamassoma e a caspase-1. É

interessante notar que estudos anteriores documentaram que a desregulação destes

mecanismos resulta numa resposta inflamatória exacerbada e desnecessária,

característica de doenças autoinflamatórias e autoimunes, como é o caso da artrite

reumatóide. Estes mecanismos poderão ser particularmente relevantes na fase muito

inicial da doença. Contudo, a maioria dos estudos relativos à fisiopatologia da artrite

reumatóide têm sido focados na fase estabelecida da doença, sendo o conhecimento

acerca das interações celulares e dos mecanismos regulatórios imunológicos que

participam na fase inicial da artrite reumatóide ainda muito limitado.

O principal objectivo deste trabalho foi o estudo dos elementos iniciadores do

processo inflamatório na artrite reumatóide, incluindo aqueles que estão relacionados

com a atividade do inflamassoma. Assim, a primeira tarefa realizada neste estudo foi a

análise da libertação de citocinas e quimiocinas na fase muito inicial da artrite

reumatóide. Para tal, um grupo de doentes com poliartrite com menos de seis

semanas de evolução, não tratada, foi prospectivamente seguido. Estes doentes foram

XXVII

avaliados antes do tratamento, após terapêutica de curta duração com dose baixa de

corticosteróides e após metotrexato (MTX). O acompanhamento clínico destes

doentes permitiu a identificação de dois subgrupos: um que evoluiu para artrite

reumatóide e um outro que evoluiu para outras doenças crónicas inflamatórias

articulares ou que incluiu formas autolimitadas de artrite. Adicionalmente, foi analisado

um segundo grupo de doentes com artrite reumatóide na fase estabelecida da doença,

sob terapêutica com MTX. É interessante notar que, na fase muito inicial da artrite

reumatóide, foram observados no soro dos doentes níveis aumentados de citocinas

relacionadas com o recrutamento (IL-6), maturação e sobrevivência das células B (a

proliferation-inducing ligand (APRIL) e B-lymphocyte stimulator (BLyS)), bem como um

perfil de citocinas relacionadas com o recrutamento de neutrófilos (IL-8) e com a

polarização (IL-1β e IL-6) e ativação (IL-17A e IL-22) de células Th17,

comparativamente com doentes que não evoluem para artrite reumatóide e com

doentes com artrite reumatóide estabelecida. Além disso, a análise de amostras de

líquido sinovial de doentes tratados com MTX na fase estabelecida da artrite

reumatóide revelou também um perfil de citocinas semelhante ao da fase muito inicial

desta doença. De salientar que o tratamento com corticosteróides e MTX, embora

clinicamente eficaz na redução das manifestações inflamatórias, não pareceu afetar o

padrão de citocinas em circulação.

A elevada concentração sérica e sinovial de IL-1β observada desde a fase muito

inicial da artrite reumatóide pode ser explicada pelo aumento nos níveis de caspase-1

ativada que foi também observado, quer em doentes com artrite reumatóide com

menos de 1 ano de evolução e não tratados, quer nos doentes com artrite reumatóide

estabelecida, tratados com MTX. O papel central da IL-1β faria supor que esta citocina

seria um alvo ideal na terapêutica da artrite reumatóide. Contudo, os resultados dos

ensaios clínicos em doentes com artrite reumatóide estabelecida com um bloqueador

do recetor da IL-1β (anakinra) e com um anticorpo monoclonal (canakinumab) não

demonstraram o mesmo nível de eficácia dos antagonistas do TNF. Colocámos a

hipótese de que a IL-1β desempenhe um papel complementar ao do TNF e mais

relevante na fase inicial da doença. Por este motivo, a interferência na fase inicial da

artrite com a via reguladora da libertação de IL-1β e de TNF poderia constituir uma

opção terapêutica promissora. Para testar esta hipótese, usou-se uma biblioteca de

drogas na linha celular macrofágica THP1 no sentido de identificar as drogas que

simultaneamente diminuam a produção de ambas as citocinas. As drogas

selecionadas foram posteriormente estudadas in vivo num modelo de rato Wistar de

artrite induzida por adjuvante para analisar as suas propriedades anti-inflamatórias.

XXVIII

Nestas experiências in vivo foi observado que duas das drogas selecionadas, o

celastrol e o ácido gambógico, têm propriedades anti-inflamatórias e anti-proliferativas,

tratando eficazmente os ratos artríticos devido ao seu efeito inibitório sobre a IL-1β e o

TNF, induzido pela diminuição da ativação quer da caspase-1, quer do NF-kB. Por fim,

com o intuito de distinguir entre um papel direto da IL-1β como elemento efetor ou

como iniciador do processo inflamatório, pela indução da diferenciação das células

Th17, foi também testada in vivo uma droga inibitória da atividade transcricional do

retinoic acid receptor-related orphan receptor gamma (RORγ)T (digoxina) no mesmo

modelo animal, permitindo a análise do seu efeito anti-inflamatório. Após o tratamento

com a digoxina foi observada uma melhoria nos sinais inflamatórios dos ratos artríticos

quando a droga foi administrada na fase inicial do desenvolvimento da artrite, tendo-se

verificado um efeito menos rápido e menos eficiente na progressão da doença

comparativamente com as drogas acima mencionadas. Contrariamente à digoxina, o

celastrol e o ácido gambógico são também eficientes quando administrados em fases

tardias do desenvolvimento da artrite, sendo este resultado muito importante no

contexto da prática clínica. Estes resultados sugerem que a inibição da polarização

das células Th17 per se parece ser uma estratégia com uma eficácia limitada, pelo

menos no que diz respeito à fase estabelecida da doença.

No seu conjunto, os resultados da presente tese suportam a hipótese de que a IL-

1β desempenha um papel mais relevante na fase inicial do que na fase tardia da artrite

reumatóide. Estes novos dados têm assim uma implicação clínica importante, ao

sugerirem que a introdução precoce de terapêuticas direcionadas às vias reguladoras

de IL-1β poderão ser particularmente eficazes na indução da remissão na fase inicial

da doença, devendo esta estratégia ser avaliada em doentes com artrite reumatóide.

Palavras-chave: Artrite reumatóide, Inflamassoma, Caspase-1, NF-kB, IL-1β, TNF, IL-

17A, Modelo de rato AIA, Drogas

XXIX

XXX

Summary

Rheumatoid arthritis is a chronic inflammatory and immuno-mediated disease with

a significant involvement of the synovial membrane of multiple small joints and

progressive destruction of cartilage and bone, which leads to functional impairment and

a significant increase of both morbidity and mortality of patients. Early diagnosis and an

accurate prompt therapeutic strategy are crucial to prevent rheumatoid arthritis

progression and joint destruction that can occur immediately after its onset. The most

debilitating characteristic of rheumatoid arthritis is the synovial hyperplasia of joints,

which is mediated by the interplay of several immune cells (macrophages, neutrophils,

T and B cells) and complex cytokine networks (particularly interleukin (IL)-1β, tumor

necrosis factor (TNF) and IL-17).

IL-1β is considered to be a crucial element in rheumatoid arthritis pathology due to

its ability to enhance cytokine and chemokine secretion, metalloproteinases (MMPs)

production, T cell expansion and survival which further increases antibody production

by B cells, T helper (Th)17 cells differentiation, and osteoclastogenesis with

consequent bone loss. Importantly, the production of IL-1β is tightly regulated both at

the transcriptional and post-translational levels, involving nuclear factor kappa-light-

chain-enhancer of activated B cells (NF-kB), inflammasomes and caspase-1.

Interestingly, it has been shown that perturbation of these regulatory mechanisms

results in an exaggerated inflammatory response that underlies autoinflammatory and

autoimmune diseases, such as rheumatoid arthritis. These mechanisms might be

particularly relevant in the early phase of the disease. Nevertheless, the majority of the

studies concerning rheumatoid arthritis physiopathology have focused on the

established phase of this disease, while the knowledge regarding the cellular

interactions and the immunologic regulatory cascades involved in its onset are still

scarce.

The main goal of this work was to study the early triggers of the inflammatory

process in rheumatoid arthritis, including those involved in inflammasome activation.

Accordingly, the first task in this study was the analysis of the early cytokine and

chemokine environment in the early phase of rheumatoid arthritis. To that end, a cohort

of untreated polyarthritis patients with less than six weeks of disease duration was

prospectively followed up. These patients were evaluated at baseline (without

treatment) and after short-term therapy with low dose of corticosteroids and

methotrexate (MTX). The clinical follow up of these patients allowed the identification of

two subgroups of patients: one that evolved into rheumatoid arthritis and another which

XXXI

evolved into other chronic inflammatory joint diseases or had self-limited forms of

arthritis. Additionally, a second cohort of rheumatoid arthritis patients with established

disease and under MTX therapy was also analyzed. Interestingly, we found that in the

very early phase of rheumatoid arthritis there are increased levels of circulating

cytokines related with the recruitment (IL-6), maturation and survival (a proliferation-

inducing ligand (APRIL) and B-lymphocyte stimulator (BLyS)) of B cells, as well as a

cytokine profile that supports neutrophil recruitment (IL-8) and Th17 cells polarization

(IL-1β and IL-6) and activation (IL-17A and IL-22) when compared with other very early

chronic inflammatory joint diseases and with the established phase of the disease.

Furthermore, the analysis of synovial fluid samples from established rheumatoid

arthritis patients treated with MTX also revealed a pattern of cytokines similar to that of

the very early phase of the disease. Of note, treatment with corticosteroids and MTX,

although clinically effective in reducing inflammatory manifestations, did not seem to

affect the cytokine content in circulation.

The increased concentration of IL-1β observed since the early phase of the

disease both in peripheral blood and in the synovial fluid may be explained by the

increased levels of active caspase-1 that were also observed both in early untreated

rheumatoid arthritis patients with less than 1 year of disease evolution and in

established rheumatoid arthritis patients treated with MTX. The crucial role of IL-1β

suggests that this cytokine could be a promising target for rheumatoid arthritis

treatment. However, clinical trials with IL-1 receptor targeting (anakinra) and with IL-1

monoclonal antibody (canakinumab) have not revealed encouraging results in the

established phase of rheumatoid arthritis compared with TNF antagonists. Therefore,

we raised the hypothesis that IL-1β plays a complementary role to that of TNF and

more relevant in the early stage of the disease rather than in the established phase.

Consequently, interference, during the early phase of arthritis, with pathways regulating

both IL-1β and TNF could constitute a promising therapeutic option. To test this

hypothesis, a drug screen was performed in a THP1 macrophage-like cell line in order

to identify those that simultaneously down-regulate the production of both cytokines.

The drugs that were selected were further studied in vivo in a wistar rat model of

adjuvant-induced arthritis to analyze their potential anti-inflammatory properties. The in

vivo experiments revealed that two of the selected drugs, celastrol and gambogic acid,

have anti-inflammatory and anti-proliferative properties and are able to effectively treat

arthritic rats, by an inhibitory effect of IL-1β and TNF, induced by a down-regulation of

caspase-1 and NF-kB activation. Finally, to distinguish between the direct role for IL-1β

as an effector or as an upstream initiator, by driving the differentiation of Th17 cells in

XXXII

the inflammatory process, a drug which inhibits retinoic acid receptor-related orphan

receptor gamma (RORγ)T transcriptional activity (digoxin) was also tested in vivo in the

same animal model in order to observe its anti-inflammatory effects. We found that

digoxin was able to ameliorate the inflammatory signs of the arthritic rats only if

administered in the early phase of arthritis development, albeit with a slower and less

efficient effect on disease progression compared with the drugs above mentioned. In

contrast to digoxin, celastrol and gambogic acid are also effective if administered in the

later phase of arthritis development, which is crucial for rheumatoid arthritis

management. These results suggest that the isolated inhibition of Th17 cells

polarization might be a strategy with limited efficacy, at least in the established phase

of the disease.

Overall, the results of the present thesis support the hypothesis that IL-1β plays a

more relevant role in the early rather than late phase of rheumatoid arthritis. Thus,

these original results have important clinical implications, by suggesting that an earlier

intervention of therapies targeting IL-1β pathways might be of beneficial clinical use to

induce early remission and this strategy should be evaluated in rheumatoid arthritis

patients.

Key-words: Rheumatoid arthritis, Inflammasome, Caspase-1, NF-kB, IL-1β, TNF, IL-

17A, AIA rat model, Drugs

XXXIII

Chapter I

Introduction

- 2 -

Introduction

- 3 -

1. Rheumatoid arthritis

1.1 Definition

Rheumatoid arthritis is a chronic, systemic, autoimmune inflammatory disease that

mainly affects the synovium of multiple joints, leading to the progressive destruction of

cartilage and bone. This disease is often associated with other co-morbidities, such as

cardiovascular diseases, infections and cancer 1, 2. Rheumatoid arthritis causes

considerable personal and economic costs as it is associated with serious disability,

reduced quality of life, loss of work capacity 3 and diminished life expectancy 4.

Epidemiological studies have shown that rheumatoid arthritis affects about 1% of the

worldwide population and 75% of those afflicted are women 5, suggesting an influence

of hormonal factors in the occurrence of the disease. Moreover, its incidence peaks

among those aged between 30 and 50 years old and because it is a chronic condition,

its prevalence increases with age.

Importantly, the clinical presentation of rheumatoid arthritis can be elusive and

insidious and therefore several months can pass before the clinician makes a definitive

diagnosis. The predominant symptoms are symmetrical pain, stiffness and swelling of

peripheral joints. The clinical course of the disease is also variable, ranging from mild

to severe and highly destructive arthritis. The analysis of the clinical course and of

laboratorial and radiological parameters have defined prognostic factors related to

progressive joint destruction, such as the presence of rheumatoid factor (RF) and anti-

citrullinated protein antibodies (ACPA) as well as the early development of joint

erosions. Such prognostic factors can influence treatment decisions, particularly in the

early phase of the disease. However, they do not drive treatment decisions by

themselves. In fact, tight disease control and treatment driven by a predefined disease

activity target are crucial for an effective long-term management of rheumatoid arthritis.

A prompt diagnosis and an accurate early therapeutic strategy are essential to prevent

rheumatoid arthritis progression and joint erosions, which can occur as early as 4

months after the onset of the disease 6, 7, highlighting the importance of diagnosing

rheumatoid arthritis in the earliest possible phase of its course. To contribute to that

important goal, the American College of Rheumatology and the European League

Against Rheumatism (ACR/EULAR) published the reviewed Rheumatoid Arthritis

Classification Criteria in 2010, which refocuses on the number of involved joints,

serologic abnormalities, elevated acute-phase proteins and symptoms duration (Table

1) 8.

Introduction

- 4 -

Table 1 – The 2010 American College of Rheumatology / European League Against Rheumatism classification criteria for rheumatoid arthritis. * The criteria are aimed at classification of newly

presenting patients; ** Differential diagnoses vary among patients with different presentations. If it is unclear about the relevant differential diagnoses to consider, an expert rheumatologist should be consulted; ‡ Although patients with a score of < 6/10 are not classifiable as having rheumatoid arthritis, their status can be reassessed and the criteria might be fulfilled cumulatively over time; § Joint involvement refers to any swollen or tender joint on examination, which may be confirmed by imaging evidence of synovitis; # “Large joints” refers to shoulders, elbows, hips, knees, and ankles; ## “Small joints” refers to the metacarpophalangeal joints, proximal interphalangeal joints, second through fifth metatarsophalangeal joints, thumb interphalangeal joints and wrists; †† Negative refers to international unit (IU) values that are less than or equal to the upper limit of normal (ULN) for the laboratory and assay; low-positive refers to IU values that are higher than the ULN but ≤ 3 times the ULN for the laboratory and assay; high-positive refers to IU values that are > 3 times the ULN for the laboratory and assay. Where rheumatoid factor (RF) information is only available as positive or negative, a positive result should be scored as low-positive for RF. ACPA – anti-citrullinated protein antibody; ‡‡ Normal/abnormal is determined by local laboratory standards. CRP – C-reactive protein, ESR – erythrocyte sedimentation rate; §§ Duration of symptoms refers to patient self-report of the duration of signs or symptoms of synovitis (e.g. pain, swelling, tenderness) of joints that are clinically involved at the time of assessment, regardless of treatment status.

However, since this new revision occurred after the completion of the experimental

procedures of this thesis, the criteria used for rheumatoid arthritis classification were

the 1987 ACR criteria (Table 2) 9.

Introduction

- 5 -

Table 2 – The 1987 American College of Rheumatology criteria for the classification of rheumatoid arthritis. A patient is said to have rheumatoid arthritis if he or she meets at least four criteria. Patients with

two clinical diagnoses are not excluded. Designation as classic, definite, or probable rheumatoid arthritis is not to be made.

1.2 Pathogenesis

The etiopathogenesis of rheumatoid arthritis is not yet well understood. The

current lack of knowledge is partially due to the fact that rheumatoid arthritis is a

complex multifactorial process, which involves an interplay among genotype,

environmental triggers and chance. Among the genetic factors, the strongest

association with the occurrence of the disease has been established with the major

histocompatibility complex (MHC) class II human leukocyte antigen (HLA)-DRB1*0404

and DRB1*0401 alleles 10. Specifically, in the Portuguese population and in some other

Mediterranean populations, an association with the DRB1*1001 allele was also found

11. HLA-DRB1 alleles are involved in MHC molecule-based antigen presentation and

are responsible for self-peptide selection and T cell repertoire. As T cell specificity is

mediated by an interaction between the T cell receptor (TCR) and the peptide

presented by MHC molecules, it has been suggested that rheumatoid arthritis is

caused by the presentation of an unidentified arthritogenic antigen 12, either exogenous

or endogenous 13. Smoking is the most established environmental risk factor for

rheumatoid arthritis 14. Several studies have shown that smoking is a risk factor for the

presence of autoantibodies, RFs or ACPAs 15, which are predictor factors for a severe

form of rheumatoid arthritis. Interestingly, the observation that autoantibodies may be

Introduction

- 6 -

present for several years before the clinical onset of rheumatoid arthritis suggests that

these risk factors are acting several years before the disease development. Findings

from studies of gene-environmental factors interaction showed that smoking increases

the risk of rheumatoid arthritis in individuals with susceptible HLA-DRB1 alleles 16.

Moreover, smoking and HLA-DRB1 alleles synergistically increase the risk of having

ACPA 17. Therefore, the prevailing view is that gene-environment interactions may lead

to altered post-translational modifications, such as citrullination, which consequently

promote a loss of tolerance to these altered self-proteins. However, there has been no

consistent evidence that a single environmental factor could explain per se the onset of

this disease.

The most debilitating feature of rheumatoid arthritis is joint destruction, which is

derived from an uncontrolled inflammatory process. Inflammation is usually tightly

regulated, involving both the mediators which initiate and stop it. In the case of chronic

inflammation, such as rheumatoid arthritis, there is an unbalance between the two

opposing groups of mediators resulting in the destruction of cartilage and bone.

The articular cartilage is composed of specialized cells, the chondrocytes,

embedded in a structured extracellular matrix of proteoglycans and collagen type II.

The destruction of cartilage is a consequence of two major events: altered synthesis of

matrix components by chondrocytes and increased enzymatic degradation of the

matrix, with both mechanisms being influenced by the ongoing inflammatory process.

In addition, the formation of a highly invasive synovial tissue, the pannus, leads to

further cartilage destruction and bone erosion. The pannus evolves throughout the

course of the disease. Initially, it is characterized by the presence of activated

mononuclear cells and fibroblasts 18 and increased production of metalloproteinases

(MMPs) by synovial-lining cells 19, 20. Subsequently, the cellular pannus can be

replaced by fibrous pannus composed of a residual vascularized layer of pannus cells

and collagen overlying cartilage 21. Synovial fibroblasts, together with macrophages,

also promote bone erosion by the induction of osteoclastogenesis 22. Another

characteristic feature of rheumatoid arthritis is the formation of an inflamed synovium,

which is characterized by cellular hyperplasia, influx of inflammatory leukocytes,

increased angiogenesis and alterations in the expression of adhesion molecules,

proteinases, proteinase inhibitors and cytokines. Furthermore, the characteristics of the

inflamed synovium also vary with disease progression. During the first weeks of the

disease onset, there is a neutrophil influx, tissue oedema and fibrin deposition 23. Next,

the synovial lining becomes hyperplastic as it consists of macrophage-like and

fibroblast-like synoviocytes, and finally in the sublining layer a pronounced infiltration of

Introduction

- 7 -

mononuclear cells occurs, namely macrophages, neutrophils, dendritic cells, T cells, B

cells and plasma cells. Importantly, synovial-vessel endothelial cells are transformed

into high endothelial venules early in the course of the disease, thus promoting the

infiltration of inflammatory leukocytes 24 and facilitating the oxygenation of this hypoxic

environment 25. Interestingly, several innate immune sensing pathways are found in

rheumatoid arthritis joints, namely Toll-like receptors (TLRs), nucleotide-binding

oligomerization domain (NOD)-like receptors (NLRs) and molecular components of the

inflammasome promoting the detection of tissue damage signals 26, 27. In conclusion,

the synovial inflammation characteristic of rheumatoid arthritis is mediated by the

interplay of several immune cells and cytokine networks (Figure 1).

Figure 1 – Representative scheme illustrating immune cells and cytokine networks in rheumatoid joints. In rheumatoid arthritis, the inflammatory process leads to a cellular infiltration of the synovial

membrane and synovial fluid, with consequent pannus formation, cartilage and bone destruction. Immune cells are responsible for the secretion of high amounts of several cytokines that amplify inflammation by the recruitment and activation of immune cells. ACPA – Anti-citrullinated protein antibodies, APRIL – A proliferation-inducing ligand, BLyS – B-lymphocyte stimulator, IFNγ – Interferon gamma, IL – Interleukin, IL-6R – IL-6 receptor, MCP-1 – Monocyte chemotactic protein-1, MHC – Major histocompatibility complex, MMP – Metalloproteinase, RANKL – Receptor activator of nuclear factor ligand, RF – Rheumatoid factor, TCR – T cell receptor, TGFβ – Transforming growth factor beta, Th – T helper, TNF – Tumor necrosis factor. (see text for details)

Introduction

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1.3 Immune cells

Rheumatoid arthritis pathophysiology is characterized by the presence of several

different interacting immune cells both from the innate immune system, such as

macrophages and neutrophils, as well as from the adaptive immune system, namely T

and B cells.

1.3.1 Macrophages

Macrophages are pivotal cells in the pathophysiology of rheumatoid arthritis and

one of the most abundant cell types in the inflamed synovium. These cells migrate as

monocytes from peripheral blood, infiltrate and accumulate in the synovium.

Macrophages promote the inflammatory process and joint destruction through the

production of several proinflammatory cytokines, such as interleukin (IL)-1β and tumor

necrosis factor (TNF), and through osteoclastogenesis 28. Crucial to the mechanism of

osteoclastogenesis is the differentiation of monocytes into bone-resorbing osteoclasts,

a process mediated by the binding of their receptor activator of nuclear factor (RANK)

with its ligand (RANKL) expressed by osteoblasts and some immune cells.

In the synovium, the activation of macrophages can occur by T and B cells,

through direct cell-cell contact or indirectly by the production of cytokines 28-30.

Moreover, macrophages can also be activated by the immune complexes that deposit

in the joints through the low-affinity IgG receptor Fc gamma receptor (FcγR) IIIa 31.

Activation of macrophages is also driven by TLRs and NLRs that recognize a wide

range of pathogen associated molecular pattern (PAMP) and danger associated

molecular pattern (DAMP) 32.

1.3.2 Neutrophils

Neutrophils are the first to infiltrate the synovium 33. In fact, the synovial tissue of

very early rheumatoid arthritis patients can be heavily infiltrated by neutrophils 23.

Moreover, circulating neutrophils from these patients have decreased levels of

spontaneous apoptosis 34. Synovial neutrophils internalize immune complexes that are

present in the joints and become activated. The activated neutrophils release

damaging proteases and MMPs, in addition to producing high amounts of reactive

oxygen species (ROS), thus contributing to chronic inflammation and host tissue

damage 33. Furthermore, neutrophils secrete high amounts of cytokines and

Introduction

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chemokines, such as IL-1β, IL-8, TNF and B-lymphocyte stimulator (BLyS), which are

crucial to amplify inflammation by recruiting and activating more neutrophils and other

immune cells such as macrophages, T and B cells 35-37. The delay observed in the

apoptosis of neutrophils after rheumatoid arthritis onset can be the result of the action

of proinflammatory cytokines, such as TNF, and may create the appropriate conditions

for an inflammatory vicious cycle that might contribute to the self-perpetuation of an

initial acute arthritis episode in these patients 34.

1.3.3 T cells

The participation of T cells in the etiopathogenesis of rheumatoid arthritis has been

supported by several studies 38-43. In fact, the genetic evidence that HLA-DRB1 alleles

contribute to rheumatoid arthritis susceptibility suggests a role for T cells in the

systemic initiation of the inflammatory process. Moreover, T cells can infiltrate the

inflamed synovium and organize themselves into aggregates similar to germinal centre-

like structures 38. However, there is still some controversy regarding their precise role.

Some studies have shown that T cells do not directly cause synovitis in the joint

microenvironment by demonstrating that synovial infiltrating T cells do not show

significant proliferation 44, that the phenotype of these cells suggests a recruitment of

previously stimulated and mature T cells 45, 46, as opposed to an in situ maturation as

previously thought, and also that the synovial cytokine environment has a small

contribution of T cell-derived cytokines 47. Therefore, although T cells probably play a

part in the systemic initiation of rheumatoid arthritis, their direct role in synovitis and

joint destruction is still unclear.

Classically, rheumatoid arthritis has long been classified as a T helper (Th)1 driven

disease 41. However, recent data have pointed out that the Th1 contribution was

relatively small in the global context of rheumatoid arthritis 48. More recently, this Th1

paradigm was redefined by the increasingly substantiated relevance of the IL-17-

secreting Th17 cells subpopulation in the context of this disease. A recent report has

demonstrated that self-reactive T cells stimulate antigen-presenting cells to secrete IL-

6, which in turn is able to induce the differentiation of naïve self-reactive T cells into

Th17 cells 49, via nuclear transcription factor retinoic acid receptor-related orphan

receptor gamma (RORγ)T. Other cytokines also participate in the differentiation of

Th17 cells such as IL-1β and IL-21. In fact, macrophage-derived and dendritic cell-

derived cytokines provide a milieu that supports Th17 differentiation and suppress

regulatory T cells polarization, thus shifting T cell homeostasis towards inflammation.

Introduction

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Furthermore, Th17 cells produce IL-17, a cytokine involved in increased production of

other cytokines, cartilage-destructive enzymes and also expression of bone

destruction-related mediators such as RANKL 50, 51.

1.3.4 B cells

Several reports have also highlighted the participation of B cells in the

etiopathogenesis of rheumatoid arthritis 52-57. These cells can contribute to disease

onset and perpetuation through their ability to function as antigen-presenting cells to

secrete cytokines and to produce autoantibodies, such as RF and ACPA, leading to

immune complex formation, which can occur even before the onset of disease

symptoms 52. These phenomena might be explained by a deficient removal of

autoreactive B cells and an abnormal B cell tolerance checkpoint in the bone marrow

that occur in rheumatoid arthritis patients 53. Interestingly, data have shown that

peripheral B cells from rheumatoid arthritis patients have altered expression of key

molecules, such as high co-stimulatory molecule CD86 and low FcγRIIb (inhibitory

receptor for IgG immune complexes required for feedback inhibition), potentially

contributing to tolerance breakdown and development of humoral autoimmunity 58.

1.4 Cytokines

Several cytokines have also been implicated in the pathophysiology of rheumatoid

arthritis. However, it is still difficult to clearly establish their functional hierarchy and

regulatory networks. Specifically, IL-1β, TNF, IL-6 and more recently IL-17 have been

identified as key players in established rheumatoid arthritis.

1.4.1 IL-1β

IL-1β is a pleiotropic proinflammatory cytokine produced mainly by macrophages

that is crucial for the joint pathology and destruction seen in rheumatoid arthritis 51, 59.

Binding of IL-1β to its receptor leads to activation of the mitogen activated protein

(MAP) kinase pathway and the nuclear factor kappa-light-chain-enhancer of activated

B cells (NF-kB) pathway. Both pathways lead to the downstream secretion of several

cytokines, chemokines and MMPs, which are responsible for the inflammatory process

and cartilage destruction 59-61. In addition, IL-1β is able to activate several cell types,

Introduction

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such as macrophages, T and B cells, fibroblasts, osteoblasts, endothelial and epithelial

cells (Figure 2). IL-1β has been known for a long time to promote T cell responses. For

example, IL-1β derived from antigen-presenting cells acts directly on both naïve and

memory T cells to enhance their expansion and survival 62. Moreover, increased T cell

numbers due to IL-1β also result in an enhanced help for antibody production by B cells

62, thus contributing to rheumatoid arthritis perpetuation. Additionally, recent findings

have also identified a key role for IL-1β in the differentiation of Th17 cells 63, 64, which is

an important T cell subset in rheumatoid arthritis physiopathology. Furthermore, IL-1β

together with TNF have been implicated in bone loss by the induction of RANKL

expression contributing to osteoclast differentiation via RANK/RANKL interaction 65 66.

Therefore, IL-1β can be viewed not only as a regulator of innate immune cells but also

as a major amplifier of adaptive immune cells.

Importantly, IL-1β is present in high levels in the synovial fluid and tissue of

rheumatoid arthritis patients. In fact, some studies suggest that the synovial membrane

mRNA levels of IL-1β, TNF and IL-17 are predictors of damage progression 67. In

addition, animal models have demonstrated that targeting of IL-1β and components of

its receptor is effective in reducing inflammation, particularly in articular cartilage and

bone damage 68, 69. Of note, when transgenic mice expressing human TNF were

crossed with IL-1β deficient mice, inflammation still occurred but bone erosions were

dramatically reduced 70. Additional studies performed in TNF transgenic mice have also

shown that treatment with IL-1 receptor (IL-1R) antagonist fully abolished arthritis even

with confirmed high TNF levels, arguing for a possible dominant role for IL-1β 71, 72.

Introduction

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Figure 2 – An overview on the multiple roles of IL-1β in rheumatoid arthritis physiopathology. IL-1β

is a pleiotropic proinflammatory cytokine that modulates the function of several types of cells leading to the secretion of other proinflammatory cytokines and chemokines contributing to cell activation and recruitment to sites of inflammation. Moreover, IL-1β also participates in cartilage and bone destruction promoting the functional impairment characteristic of rheumatoid arthritis patients. IL – Interleukin, MHC – Major histocompatibility complex, MMP – Metalloproteinase, Th – T helper. (see text for details)

1.4.2 TNF

TNF is implicated in the pathogenesis of rheumatoid arthritis, having diverse

systemic effects due to its ability to stimulate several cells 51. This proinflammatory

cytokine has two receptors; TNF-RI (p55) expressed in most tissues and TNF-RII (p75)

expressed in immune cells. Both receptors can act as TNF inhibitors when they are

enzymatically cleaved from the cell membrane. TNF is secreted by activated

macrophages, neutrophils, T and B cells 73. TNF can contribute to rheumatoid arthritis

as a potent inducer of inflammation acting as an autocrine stimulator, as well as a

paracrine inducer of other proinflammatory cytokines, such as IL-1β, IL-6 and IL-8 74-76.

TNF is also able to induce the expression of adhesion molecules by fibroblasts,

Introduction

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facilitating the infiltration of leukocytes into the joints of rheumatoid arthritis patients 77.

Furthermore, in vitro studies have already shown that TNF is able to degrade cartilage

78 and bone 79, thus contributing to the debilitating loss of articular joint function.

The serum and synovial fluid concentration of TNF is increased in rheumatoid

arthritis patients with active disease 80. It was demonstrated through in vitro studies that

cultures of rheumatoid arthritis synovial mononuclear cells are able to spontaneously

and chronically produce TNF and other proinflammatory cytokines such as IL-1β, IL-6

and IL-8 81, 82. Transgenic mice expressing a modified human TNFA gene

spontaneously develop peripheral arthritis and show a significant improvement after

administration of antibodies specific for human TNF 71. Of note, animal studies have

also revealed that blocking TNF is more effective in early stages of arthritis, in contrast

to IL-1β, for which the treatment outcome was equally effective in early and established

arthritis 69, 83.

1.4.3 IL-6

IL-6 is a multifactorial cytokine that also plays a relevant role in the pathogenesis

of rheumatoid arthritis. This cytokine is produced by monocytes, macrophages, T cells

and synovial fibroblasts 84. IL-6 possesses diverse biological activities, which include

the regulation of immune responses, inflammation, hematopoiesis, hypothalamus-

pituitary-adrenal gland axis activity, and the increase of the adrenocorticotropic

hormone and cortisol production. The proinflammatory properties of IL-6 comprise the

induction of acute-phase proteins in liver cells 85, the increase of neutrophil blood

counts 86, the participation in Th17 polarization 49 and plasma B cells differentiation 87,

88, the increase in the production of chemokines, adhesion molecules 89, and vascular

endothelial growth factor (VEGF), which is thought to be the most important angiogenic

factor in rheumatoid arthritis 90, as well as the induction of RANKL expression 91 and

the production of MMPs and their tissue inhibitors 92-94. These evidences support the

concept that IL-6 aggravates the local inflammatory reaction by amplifying inflammatory

cell infiltration and by inducing osteoclastogenesis and extracellular matrix turnover.

The serum and synovial fluid concentration of IL-6 is increased in rheumatoid

arthritis patients and correlate to disease activity 95, 96. Synovial fibroblastic cells

produce augmented levels of IL-6 upon stimulation with inflammatory cytokines, such

as IL-1β, TNF and IL-17. Subsequently IL-6 is able by itself to increase the proliferation

of these synovial fibroblastic cells 91, 97. The recently shown effective treatment of

rheumatoid arthritis patients with the humanized anti-IL-6 receptor antibody

Introduction

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(tocilizumab) 98, 99, which binds to both the soluble and the membrane-bound IL-6

receptors 100, reinforces the important role of IL-6 in the context of this disease. In fact,

it has already been observed that tocilizumab treatment not only significantly improves

inflammation but also halts radiographic progression 101 in rheumatoid arthritis

patients 102.

1.4.4 IL-17

The IL-17 family is composed of 6 members (IL-17A to F) with distinct biological

roles. IL-17A (IL-17) is however the most studied one and has long been implicated in

several autoimmune diseases. IL-17, produced by Th17 cells, is an important cytokine

in the physiopathology of rheumatoid arthritis, mainly due to its participation in joint

destruction 103, 104. This cytokine promotes the activation of diverse cells, such as

macrophages, synovial fibroblasts, chondrocytes and osteoblasts, and also induces the

secretion of proinflammatory cytokines such as IL-1β, TNF, IL-6 and IL-23, which

amplify positive feedback loops leading to Th17 cells differentiation 105-107. IL-17 also

stimulates the production of chemokines responsible for the recruitment of

macrophages, neutrophils, T and B cells to the arthritic joints 108. Additionally, it has

been shown that IL-17 can synergize with IL-1β and TNF promoting cartilage and bone

damage 109. In fact, this cytokine induces MMPs production by synoviocytes, which are

crucial for cartilage destruction 110. Furthermore, in addition to the stimulation of

osteoclast-mediated bone resorption, it was recently shown that IL-17 is able to inhibit

collagen type I synthesis, further increasing bone destruction 111.

Importantly, IL-17-positive cells were observed in synovial biopsies from

rheumatoid arthritis patients 112 and increased levels of IL-17, which correlated with

disease activity, were also observed in the serum and synovial fluid samples from

these patients 104. Furthermore, in vivo results have also highlighted the relevance of

IL-17 in rheumatoid arthritis by showing that the neutralization of IL-17 in mice

decreased the severity of antigen-induced and collagen-induced arthritis and that IL-

17-deficient mice presented reduced disease severity 113, 114.

1.5 Treatment options

The presentation and the clinical course of rheumatoid arthritis patients as well as

the response to the available treatment options are highly variable. Despite the

Introduction

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existence of several treatment options for the management of rheumatoid arthritis, this

is still an incurable disease with enormous personal and economic costs. The strategy

of disease treatment relies on an early diagnosis and a prompt adequate treatment with

the goal of inducing remission in order to preserve joint function and maintain quality of

life 115-117. Drug-free remission is at this moment impossible for most patients.

Rheumatoid arthritis treatments can be divided into the following categories:

glucocorticoids, conventional disease-modifying anti-rheumatic drugs (DMARDs) and

biologic DMARDs.

1.5.1 Glucocorticoids

Glucocorticoids (namely prednisone) act rapidly to suppress inflammation and,

consequently, pain and swelling 118. Glucocorticoids are useful to control symptoms in

the first weeks of disease diagnosis, until slower-action DMARD can start to have an

effect. Glucocorticoids can have an effect in limiting joint damage, but long term

adverse effects, particularly at higher doses, limits their usefulness 119.

1.5.2 Conventional DMARDs

Conventional DMARDs, which include for instance methotrexate (MTX),

hydroxychloroquine, sulfasalazine and leflunomide, have long been used as the main

therapeutic option for newly diagnosed rheumatoid arthritis 118, 120. Among the different

DMARDs, MTX is the most frequently used and the most likely to induce a long-term

response 121-123. DMARDs have a relatively slow onset of action (1-6 months) but they

are effective in the long term and are considered to have an acceptable safety 118, 124

either as monotherapy or in combination therapy. However, around 30% of patients are

either non-responsive to DMARDs or lose their response secondarily or even develop

adverse effects that are incompatible with their use 125, 126 127. Therefore, the recent

development of biologic DMARDs is a relevant therapeutic alternative for these

DMARD refractory patients.

1.5.3 Biologic DMARDs

Nine different biologic DMARDs are currently approved by the European

Medicines Agency and many others are being tested in clinical trials for the treatment

of rheumatoid arthritis patients with inadequate response to one or more non-biologic

Introduction

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DMARDs (Figure 3). In rheumatoid arthritis, biologic DMARDs have shown efficacy in

improving clinical, functional and radiographic outcomes 117, 128, especially if

administrated early in the disease course 116.

The first line of biologic DMARDs is the TNF antagonist class. TNF antagonists

approved for the treatment of rheumatoid arthritis include monoclonal antibodies

against TNF (infliximab, adalimumab, certolizumab pegol and golimumab) and a

soluble TNF receptor (etanercept). All TNF antagonists have similar efficacy and the

combination therapy with a TNF antagonist plus MTX has been proven to be superior

to the TNF antagonist alone 116, 129, 130. When patients fail to respond to a TNF

antagonist they have approximately a 50% chance of responding to a second one, but

the probability of efficacy of a third switch is very low 119. There are also approved

biologic DMARDs that target specific immune cells such as abatacept (T-cell co-

stimulation blocker) and rituximab (anti-CD20 monoclonal antibody for B-cell depletion).

Both these drugs, given in combination with MTX, may be useful alternatives for

patients who are non-responders to conventional DMARDs (rituximab is not formally

approved in that indication) or TNF antagonists 131, 132 and have already shown the

ability to inhibit progression of structural damage 133-135. Treatment of rheumatoid

arthritis patients with tocilizumab (humanized anti-IL-6 receptor monoclonal antibody) is

highly effective 98, 99, with reduced systemic inflammation, synovitis and radiographic

progression. This biologic DMARD can be used in the setting of DMARD or TNF

antagonist failure 101. There are also biologic agents targeting the IL-1β receptor

(anakinra, an IL-1β receptor antagonist), blocking the circulating IL-1β (rilonacept, a

fusion protein composed of the IL-1β receptor and IgG) and binding to IL-1β directly

(canakinumab, a monoclonal antibody against IL-1β) 136. Specifically, anakinra is

approved for rheumatoid arthritis treatment and it was shown to be effective albeit at a

lower efficacy than a TNF antagonist 137, 138. Rilonacept and canakinumab are still

undergoing clinical trials but they seem to have also lower efficacy as compared to TNF

antagonists 139. Interestingly, denosumab (antibody against RANKL) was also recently

shown to significantly reduce structural damage, but had no effect on the clinical signs

and symptoms of rheumatoid arthritis 140.

Switching among biologic DMARDs is often considered in patients with inadequate

response to initial treatment and the selection of the second biologic DMARD depends

in most cases on the reason of the first failure (e.g. a second TNF antagonist is less

effective when the first TNF antagonist failed due to lack of efficacy instead of adverse

events), but this rationale is somehow empirical and needs further studies 141, 142.

Overall, biologic DMARDs have been well tolerated by patients, despite the occurrence

Introduction

- 17 -

of infections and injection-site reactions, which are among the most common adverse

effects. Curiously, these types of problems occur with a similar frequency among the

different biologics 143. Moreover, the formation of antibodies to biologic agents is a

great concern since it can decrease the efficacy and increase the adverse effects of the

treatment. Of note, biologic therapies do not achieve optimal response in all patients

and even those who initially respond to treatment may experience diminished efficacy

with time. In addition, remission is only possible in 30% of the patients. Therefore,

searching for new, more effective and safer treatments for rheumatoid arthritis is still a

current medical priority.

Figure 3 – A simplified scheme showing main pathophysiologic pathways in rheumatoid arthritis and the effects of some currently available biologic agents. Treatment options for rheumatoid arthritis

have been developed to stop or at least attenuate disease progression. Recently, several biologic therapeutics, targeting immune cells or cytokines, have been introduced for the treatment of rheumatoid arthritis. ACPA – Anti-citrullinated protein antibodies, APRIL – A proliferation-inducing ligand, BLyS – B-lymphocyte stimulator, IFNγ – Interferon γ, IL – Interleukin, IL-1R – IL-1 receptor, IL-6R – IL-6 receptor, MCP-1 – Monocyte chemotactic protein-1, MHC – Major histocompatibility complex, MMP – Metalloproteinase, RANKL – Receptor activator of nuclear factor ligand, RF – Rheumatoid factor, TCR – T cell receptor, TGFβ – Transforming growth factor β, Th – T helper cell, TNF – Tumor necrosis factor. (see text for details)

Introduction

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2. IL-1β Immunological and inflammatory functions

2.1 IL-1 family

IL-1β is the most well characterized cytokine among the 11 members of the IL-1

family. The IL-1 family also comprises IL-1α, IL-1 receptor antagonist (IL-1Ra), IL-18,

IL-33 and IL-1F5 to IL-1F10 144. IL-1β, as discussed above, is a pleiotropic

proinflammatory cytokine with immuno- and inflammatory-modulating activities that are

crucial for host responses to infection and also to injuries and immunological

challenges 145. Several cell types produce this cytokine, although its main source are

cells from the innate immune system, such as monocytes and macrophages 145, 146.

Under normal conditions IL-1β is expressed at low levels, however, in the presence of

an inflammatory stimulus, its secretion increases. The production of IL-1β requires

induction in both transcriptional and post-translational steps 145, 146. This dual

mechanism responsible for the production of IL-1β is tightly regulated and ensures that

its secretion only occurs when it is required, thus preventing an unnecessary

exacerbated inflammatory response. The loss of these regulatory steps results in fever,

rash and arthritis, characteristic of autoinflammatory syndromes, such as

cryopyrinopathies 148. Gout is also associated with altered IL-1β regulation, induced by

the stimulus of uric acid crystals 147. Some autoimmune diseases are also likely to be at

least partially dependent on IL-1β 149.

2.2 IL-1β pathway

2.2.1 Priming

The production of IL-1β requires two signals, one for IL-1β gene expression and

the second for the completion of protein synthesis 150 (Figure 4). IL-1β is expressed as

an inactive 31 kDa precursor, designated as pro-IL-1β, in response to PAMPs or

cytokines, such as TNF and IL-1β itself, which signal through pattern recognition

receptors (PRRs) present in macrophages 151. These signaling pathways lead to the

activation of NF-kB, which induces the transcription of IL-1β promoter 152. This

induction of pro-IL-1β gene expression is commonly referred to as the priming step.

However, it does not act as a secretion stimulus. Instead, the primed cell must

encounter a new PAMP or DAMP to induce the processing and secretion of the active

IL-1β.

Introduction

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Figure 4 – IL-1β processing pathways. The secretion of mature IL-1β involves a dual mechanism at the

level of IL-1β priming and at the level of its processing/activation, which is tightly regulated in order to prevent uncontrolled and unnecessary exacerbated inflammatory responses. IL-1β priming is mainly regulated by NF-kB and IL-1β processing is strongly regulated by caspase-1 and inflammasome complexes. ASC – Adaptor molecule apoptosis associated speck-like protein containing a caspase recruitment domain, ATP – Adenosine-5'-triphosphate; CARD – Caspase recruitment domain, DAMP – Danger associated molecular pattern, IL – Interleukin, IL-1R – IL-1 receptor, LRR – Leucine reach repeats, MyD88 – Myeloid differentiation primary response gene 88, NACHT – Nucleotide-binding domain or NAIP, CIITA, HET-E and TP1, NF-kB – Nuclear factor kappa-light-chain-enhancer of activated B cells, PAMP – Pathogen associated molecular pattern, PYR – Pyrin domain, TLR – Toll-like receptor, ROS – Reactive oxygen species. (see text for details)

2.2.2 Processing

The 31 kDa pro-IL-1β must be processed to become a 17 kDa active IL-1β that

can be secreted by the cell. The pro-IL-1β is cleaved by the intracellular

proinflammatory cysteine protease caspase-1 153.

Caspases are also expressed as inactive proenzymes of 30-50 kDa and consist of

an N-terminal prodomain followed by a 20 kDa and a 10 kDa conserved domains. The

proteolytic activation of caspases includes the cleavage between these two domains

and subsequent assembly of two heterodimers each one containing a p20 and p10

subunits and lacking the prodomain 154. This activation of caspase-1 itself occurs via

Introduction

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recruitment to a large multiprotein complex, termed inflammasome, which is present in

the cytosol 155.

The inflammasome is a molecular scaffold composed of a cytosolic NLR, adaptor

molecules and pro-caspase-1 156. The most well characterized inflammasome is the

NLRP3 (also known as cryopyrin or NALP3) inflammasome. The NLRP3 receptor is

comprised of a C-terminal PAMP/DAMP sensing leucine rich repeat (LRR), a central

nucleotide binding NACHT domain and an N-terminal pyrin domain (PYD) 156. The

NACHT (nucleotide-binding domain or NAIP, CIITA, HET-E and TP1) domain is

responsible for the oligomerization and, consequently, activation of the inflammasome.

The PYD domain of the NLRP3 is responsible for the recruitment of the adaptor

molecule, apoptosis associated speck-like protein containing a caspase recruitment

domain (ASC) or CARDINAL, by homotypic interactions between their PYD domains.

The NLRP3 inflammasome cannot recruit pro-caspase-1 without the adaptor molecule

ASC, because it lacks a caspase recruitment domain (CARD). Therefore, pro-caspase-

1 is recruited to ASC, by a homotypic interaction of CARD domains, facilitating

caspase-1 autocatalytic activation. The activation of the inflammasome itself requires

two signals. The first signal is the priming with TLR and NLR ligands or

proinflammatory cytokines that enhance NF-kB-driven transcription of inflammasome

components 157-159. The NLRP3 is expressed in myeloid cells and is upregulated in

response to a diverse panel of agonists. These agonists include exogenous / foreign

stimulus, such as asbestos, silica, alum, pathogens and pore-forming toxins, and also

endogenous / self-stimulus, such as monosodium urate crystals and calcium

pyrophosphate crystals 147, 160-162. The second signal is the activation and assembling of

the inflammasome, where ROS 163, 164, K+ efflux 165, pore formation at the cellular

membrane and lysosomal disruption 166, 167 seem to play a role. Despite the current

limited insight into the precise mechanism of NLRP3 inflammasome activation, it is

becoming increasingly evident that one signal alone is insufficient to induce its

activation. Also crucial for the regulation of inflammasome activation is its auto-

inhibition, which limits the occurrence of an aberrant inflammatory response. To date,

some mechanisms have been pointed out as possible pathways to inhibit

inflammasome activation, such as NLRs alternative splicing, ASC isoforms 168 and

pyrin 169.

Interestingly, there is also evidence that IL-1β activation can be mediated by

pathways independent of NLRP3 and caspase-1. For example, it was already

demonstrated that neutrophil proteases, such as proteinase 3, are able to cleave pro-

IL-1β, in the absence of NLRP3 and caspase-1 activation 170. Data from animal models

Introduction

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have shown that when monosodium urate crystals were injected intraperitoneally in

caspase-1 deficient mice, there was a moderate reduction of cell influx in the peritoneal

cavity. This observation indicates that other mechanisms besides caspase-1 can also

participate in IL-1β processing and mediate the inflammatory response 147, 171. Such

distinct enzymes able to cleave pro-IL-1β may have different kinetics and sites of

cleavage, resulting in the formation of various peptides with variable biological

activities, and with an in vivo role that is yet poorly understood.

Thus, the primary role of caspase-1 and NLRP3 inflammasome is the tight

regulation of IL-1β activation.

2.2.3 Secretion

After caspase-1 processing of pro-IL-1β, mature IL-1β is rapidly secreted from the

cell 172 by a mechanism still incompletely known. There are already some evidences

that the secretion of this cytokine does not follow the conventional route of endoplasmic

reticulum and Golgi apparatus 173. Some authors believe instead that IL-1β can be

exported out of the cell via distinct non-conventional routes, such as lysosomes,

exososomes, microvesicles and membrane pores, depending on the type and duration

of the inflammatory stimulus and the levels of this cytokine required extracellularly to

mount an effective inflammatory response 174.

2.2.4 Signaling

IL-1β signals by binding first to the ligand-binding chain of the IL-1R type I (IL-1RI).

Subsequently, the co-receptor chain termed IL-1R accessory protein (IL-1RAcP) is

recruited, forming a trimeric functional high affinity complex of IL-1β, IL-1RI and IL-

1RAcP. This binding signal leads to the recruitment of the adaptor myeloid

differentiation primary response gene 88 (MyD88) protein to the Toll-IL-1 receptor (TIR)

cytoplasmic domain. Activation of the MAP kinase and of the NF-kB pathways occurs

next, leading to the NF-kB activation and translocation to the nucleus where it initiates

the expression of many inflammatory genes, such as IL-1 gene itself 175. NF-kB is

inactive in the majority of cells, being located in the cytoplasm in complex with

inhibitory Ikβ proteins. When signaling pathways are activated, the Ikβ protein is

phosphorylated by IKK kinases and degraded and, consequently, NF-kB can enter the

nucleus to modulate target gene expression 176.

Introduction

- 22 -

Surprisingly, a crosstalk mechanism between TLRs and IL-1β/IL-1RI seems to

exist, as during this signalling transduction the TIR domain of the TLRs can also recruit

MyD88 and synergistically contribute to the activation of NF-kB.

There is also a tight regulation at the level of IL-1β signalling. IL-1Ra plays a role in

this regulation since it binds to IL-1RI preventing its activation by IL-1β. The expression

of IL-1Ra is promoted by IL-1β-induced NF-kB signaling, thus providing a negative

feedback loop to control inflammation. Interestingly, mice deficient in IL-1Ra

spontaneously develop diseases such as highly destructive forms of arthritis 177. IL-1R

type II (IL-1RII) also regulates IL-1β signalling since it acts as a decoy receptor, being

unable to signal due to the lack of the cytoplasmic TIR domain. IL-1RII has a greater

affinity than IL-1RI in sequestering IL-1β 178 and is expressed mainly in macrophages

and B cells, being present both in cellular membranes and in a soluble form, acting as

an effective anti-inflammatory agent 179. The accessory protein IL-1RAcP is also

sequestered by IL-1RII, since it is recruited by the IL-1β/IL-1RII complex, thus

preventing its participation in IL-1RI signaling. Of note, both the expression of IL-1RII at

the cellular surface and circulating soluble form appears to affect the degree of

inflammation in several diseases. Importantly, the administration of glucocorticoids

induces the expression IL-1RII at the cell surface and this ability might contribute to

their anti-inflammatory effects 180. Thus, both the balance of IL-1β/IL-1Ra and IL-

1RII/IL-1RI may affect disease severity.

2.3 IL-1β and autoinflammatory diseases

An increased production of IL-1β is necessary for the development of several

autoinflammatory diseases. Autoinflammatory disorders include several heterogeneous

pathologies mainly characterized by fever and inflammation, with local as well as

systemic manifestations, that are often periodic, rather than progressive, and occur in

the absence of infections or autoimmune causes 181.

Some classical autoinflammatory disorders are caused by genetic alterations in

inflammatory pathways and, consequently, are usually manifested early in life. One

classical example of inherited autoinflammatory diseases is cryopyrin-associated

periodic fever syndromes (CAPS). Patients with CAPS often display a broad spectrum

of disease severity. The mildest form is familial cold-induced autoinflammatory

syndrome (FCAS) 182, a more severe form is Muckel-Wells syndrome (MWS) 183 and

Introduction

- 23 -

finally, the most severe form is chronic infantile neurological cutaneous and articular

syndrome/neonatal onset multisystem inflammatory disease (CINCA/NOMID) 184.

CAPS syndromes, despite being phenotypically heterogeneous, are all characterized

by periodic fevers and rashes, arthralgia, conjunctivitis, bone and joint symptoms and

elevated inflammatory markers. Interestingly, blood monocytes from patients with

CAPS secrete more IL-1β spontaneously or in response to low concentration of

inflammatory stimuli than cells from healthy controls 184-187. IL-1β itself, as mentioned

above, can increase the synthesis of NLRP3 components and caspase-1 as well as its

own IL-1β precursor, creating a positive feedback loop which amplifies the

inflammatory process underlying these diseases 188. A significant breakthrough in

understanding the pathogenesis of CAPS was the observation that these patients

possess a gain-of-function mutation in the NLRP3 gene, which promotes

inflammasome assembly and, consequently, leads to the continuous increase of

caspase-1 activation and IL-1β production 189. Currently, more than 70 inherited and de

novo disease associated-mutations have been identified, most of them occurring in the

NACHT domain of the NLRP3 inflammasome 190. Intriguingly, about 50% of the

patients with classic symptoms and biochemical markers of CAPS and other

autoinflammatory diseases do not have these mutations and, thus, the mechanism for

increased production of IL-1β is still not completely understood. Interestingly, some

mice models with knock-in mutations associated with FCAS and MWS exhibit severe

phenotypes 191, 192 and have increased concentration of IL-1β, as well as TNF and IL-17

at sites of inflammation. Myeloid cells derived from these mice do not spontaneously

secrete IL-1β but instead have a lower threshold in response to PAMPs, arguing that

the gain-of-function mutations associated with CAPS affect the myeloid-monocytic

lineage by lowering the threshold of IL-1β production. Moreover, mice deficient in

NLRP3, ASC and caspase-1 are often resistant to IL-1β mediated inflammation. In

general, there is a rapid and sustained resolution of disease severity and reduction of

biochemical and haematological disease markers in these autoinflammatory diseases

after treatment with IL-1β blockers 184, 193, 194.

Interestingly, in acute or chronic gout and pseudogout, the IL-1β pathway also

seems to play a relevant role. In gout, the deposition of monosodium urate crystals

within the joints of patients induces a strong inflammatory response with increased

levels of IL-1β, TNF and IL-8 195 and recurrent attacks of acute painful arthritis 196.

Importantly, some reports have already demonstrated that such monosodium urate

crystals together with free fatty acids are able to activate the NLRP3 inflammasome

and, consequently, account for the increased production of IL-1β 197, 198. Monosodium

Introduction

- 24 -

urate crystals can be phagocytosed by resident macrophages and their internalization

activates the NLRP3 inflammasome leading to neutrophil-dependent inflammation. In

fact, animal studies have shown that injection of crystals in the peritoneum of mice

induces a strong inflammatory response with an increased neutrophil influx, which

depends on both NLRP3 and IL-1R 147. There are other studies, however, which have

revealed that NLRP3 deficient mice exhibited a phenotype similar to the wild-type as

opposed to ASC and caspase-1 deficient mice which exhibited significantly reduced

synovial inflammation in response to monosodium urate crystal-free fatty acid

combination 197. This remaining synovial inflammation observed in caspase-1 deficient

mice might be explained by the high infiltration of neutrophils in gouty joints, which

could contribute to IL-1β precursor processing due to extracellular neutrophilic

enzymes, such as proteinase 3 170. Notably, mice deficient in IL-1β were fully protected

against joint inflammation after intraarticular administration of monosodium urate

crystals 197. This observation is in agreement with recent developments in the clinical

practice, in which some patients with recurrent attacks of gout, which are resistant to

colchicine and other steroid therapies, showed rapid and sustained reduction in pain

and signs of inflammation after IL-1 blockade therapy 199-202. Interestingly, it has been

suggested that the reason for the effectiveness of colchicine is possibly related with its

ability to inhibit microtubules and thus prevent the crystals from entering the cell or

interacting with NLRP3 by a yet unidentified mechanism 203, 204. Likewise, in

pseudogout, which is a similar crystalline-joint disease derived from the deposition of

calcium pyrophosphate crystals, there is a triggering of the NLRP3 inflammasome 147.

Several publications have also reported significant improvement of symptoms of

pseudogout patients with anakinra treatment 205, 206.

2.4 IL-1β and rheumatoid arthritis

Almost all autoimmune diseases, such as rheumatoid arthritis, have an

inflammatory component. In contrast to autoinflammatory disorders, autoimmune

diseases are primarily caused by deregulation of the immune system, especially of the

adaptive immune system, which mistakenly recognizes the body’s own molecular

constituents as foreign antigens. Moreover, these diseases are associated with high-

titer of autoantibodies or antigen-specific T cells.

In addition to the proinflammatory role of NLRP3 inflammasome in innate immune

responses, recent studies strongly suggest that NLRP3 inflammasome-mediated

Introduction

- 25 -

cytokines, such as IL-1β, play an essential role in modulating adaptive immune

responses. As mentioned previously, several studies have indicated that T cell

differentiation into IL-17-producing cells plays a major role in some autoimmune

diseases and that IL-1β appears to be crucial for the polarization of T cells towards

Th17 cells subset 63, 207-209. The role of IL-1β in the generation of Th17 cells is also in

agreement with the inflammatory process developed in mice deficient in IL-1Ra. In this

mouse model the activity of IL-1β is left unopposed and is thus able to drive Th17

differentiation, which results in spontaneous rheumatoid arthritis-like disease

development. In contrast, mice, which are also deficient in IL-17 show no signs of

disease 177, 210, 211. IL-1β also seems to be relevant for B cell activation. In rheumatoid

arthritis patients the depletion of CD20-bearing B cells results in decreased IL-17

production, which may be due to a reduction in IL-1β production by B cells 212. IL-1β/IL-

1R signaling may, therefore, affect the adaptive immune responses and promote

autoreactivity through several pathways: via the production of cytokines by dendritic

cells and consequent T cell priming 149, via induction of T and B cell interaction 213 and

by directly affecting autoreactive effector T cells 214, contributing to rheumatoid arthritis

physiopathology.

Another interesting point is that immunocomplexes can stimulate RF producing B

cells by concomitant B cell receptor and TLR9 stimulation 215. It has also been

demonstrated that innate immune responses mediated by TLRs may regulate

inflammation in rheumatoid synovial tissue, as supported by the fact that TLR2

deficient mice have reduced disease severity 216 and that TLRs are abundantly

expressed in the synovial tissue from rheumatoid arthritis patients 217. These data

reinforce the possible role of inflammasome/caspase-1/IL-1β pathway in rheumatoid

arthritis since it is known that there is a significant crosstalk between TLRs and NLRs.

Importantly, some recent studies have highlighted the importance of

inflammasome pathways as prominent participants in rheumatoid arthritis

physiopathology. For instance, Kastbom et al have shown that genetic variants of

components of NLRP3 inflammasome are associated with a higher susceptibility,

disease severity and a higher requirement for anti-TNF therapies 218, 219. Additionally,

Fontalba et al also revealed that polymorphisms in components of the NLRP3

inflammasome, namely in CARD8, influence NF-kB transcriptional activity and are

associated with rheumatoid arthritis severity 220. Interestingly, Rosengren et al have

demonstrated that the levels of NLRP3 mRNA are increased in rheumatoid arthritis

synovium, specifically in synovial macrophages, which further increase the levels of

NLRP3 mRNA after TNF stimulation. These authors concluded that there are increased

Introduction

- 26 -

levels of NLRP3 in rheumatoid synovium in later phases of the disease due to the

higher number of infiltrated macrophages, which are the main source of IL-1β 221.

However, another study from Kolly et al raised some controversy by showing that,

despite the increase in NLRP3 mRNA levels in rheumatoid arthritis synovium

comparing with osteoarthritis, the levels of NLRP3 protein were similar between the two

diseases 222. Additionally, the authors also determined the levels of caspase-1 and

found that they were increased in rheumatoid arthritis 222. Nonetheless, these authors

have not studied the levels of activated caspase-1, which is physiologically more

relevant than the total amount of the protein. Kolly et al also showed that mice deficient

in IL-1β presented reduced severity of antigen-induced arthritis, while NLRP3 deficient

animals had a severe phenotype similar to wild-type mice 222.

As described earlier, the potent proinflammatory effects of IL-1β in rheumatoid

arthritis physiopathology are mediated not only by the modulation of the adaptive

immune cells but also by activation of several types of cells accompanied by the

production of several inflammatory mediators, such as IL-1β itself, TNF, IL-6, IL-8,

cyclooxygenase 2 and prostaglandin E2, which account for the pain, swelling and

tenderness of joints. In addition, IL-1β contributes to the formation of pannus, leading

to cartilage and bone destruction which directly contribute to patients’ disability.

Interestingly, the levels of IL-1β are increased in rheumatoid arthritis synovial fluid and

correlate with disease severity 223, 224. Moreover, in rheumatoid arthritis patients, IL-1β

is overexpressed in inflamed synovial tissue, in particular in the lining and sublining

cells 225. Furthermore, IL-1β is localized in the synovial pannus, in close proximity to

bone and cartilage 226, and this cartilage exhibits up-regulation of IL-1β mRNA when

compared with normal tissue 227.

Therefore, although the regulatory cascades that mediate the initiation and

escalation of rheumatoid arthritis into a self-sustained immune response are not yet

completely understood, recent studies described herein have outlined the role of

inflammasome pathways as prominent participants in joint inflammation. However, their

exact role in rheumatoid arthritis, particularly in the initial phase of the disease, remains

largely unknown.

Introduction

- 27 -

2.5 Targeting IL-1β in rheumatoid arthritis:

the case of anakinra

Anakinra was approved in 2001 for the treatment of rheumatoid arthritis 228. As

mentioned before, anakinra is an injectable recombinant humanized form of the

endogenous IL-1Ra. It acts by blocking IL-1β/IL-1R binding and, thus, inhibits the

proinflammatory effects of this signal transduction 229-232.

Several trials have shown the benefit of anakinra in treating the signs and

symptoms of rheumatoid arthritis, with a significant improvement in various clinical and

biochemical markers of disease activity, as well as in the Larsen radiographic scores,

in comparison with placebo 228. In fact, after one year of treatment with anakinra,

rheumatoid arthritis patients experienced a reduction in disease activity 233 and also a

clear decrease in joint structural damage progression 233-235. Anakinra was also shown

to be generally well tolerated and safe 228. Nonetheless, this therapy has several

disadvantages, such as the need for a subcutaneous daily injection, because it has a

very short half-life of 4-6 hours. There is also a need for a large (10 to 1000 fold)

excess of IL-1Ra to effectively block the effect of IL-1β, which often results in painful

injection-site reactions 236, 237.

Surprisingly, the efficacy of anakinra for rheumatoid arthritis treatment was lower

than the one observed with TNF antagonists, in contrast to what was anticipated given

the critical role of IL-1β in rheumatoid arthritis physiopathology 238, 228, 239. Therefore,

rheumatologists currently rarely use anakinra for treating rheumatoid arthritis.

The disappointing results of anakinra have raised the question of whether anakinra

failed due to drug design problems or whether IL-1β was the wrong cytokine to target in

rheumatoid arthritis. Of note, all studies that investigated the efficacy of anakinra were

conducted in patients with established rheumatoid arthritis that had already failed other

therapies 240. Unlike other biologic treatments, an early intervention trial was not

performed. Furthermore, the modest clinical benefits of anakinra could reflect the

functional redundancy in TLR and IL-1R signalling pathways. Importantly, a subset of

patients did respond very well to anakinra treatment, supporting the idea that IL-1β is a

crucial cytokine that drives inflammation and joint destruction in rheumatoid arthritis, at

least in certain phases of the disease or in the context of specific patient’s

characteristics. The difference of anakinra efficacy in autoinflammatory versus

autoimmune diseases may in part reflect the fact that autoimmune disorders develop

as a result of a more complex combination of hereditary and environmental factors,

which lead to the regulatory failure of adaptive immune responses. It is therefore

Introduction

- 28 -

crucial to elucidate where IL-1β is positioned in the cascade of inflammatory responses

and to understand whether it directly mediates inflammation or acts instead upstream

of T cells as an initiator. The IL-1β activation pathway, which is regulated by many

steps, clearly has the potential to provide several putative targets for therapy

intervention in rheumatoid arthritis.

- 29 -

The pathological activation of the immune system seems to occur years before the

clinical onset of rheumatoid arthritis. However, the majority of the studies have been

focused in the established phase of rheumatoid arthritis, while the knowledge about the

immune mechanisms involved in disease onset is still very scarce. Importantly, it is

during the early phase of this disease that the immune system might still be malleable

enough to allow some immunologic intervention. A “therapeutic window of opportunity”

has been described in early rheumatoid arthritis in which it is still possible to prevent

permanent damage and to achieve remission in a large proportion of the patients. As

previously reviewed, the efficacy of anakinra for rheumatoid arthritis treatment was

lower than it was anticipated given the evident IL-1β critical role in disease

physiopathology. However, this treatment strategy was not fully explored, as it was not

tested in early rheumatoid arthritis patients and the subset of the patients that

responded very well was not properly characterized. Therefore, further studies are

clearly needed to fully understand the contribution of IL-1β for early rheumatoid arthritis

physiopathology and to test a potential dual target of IL-1β and TNF to be introduced in

the early phase of disease development when there is a unique “therapeutic window of

opportunity” and the possibility to change the outcome of the disease.

- 30 -

Chapter II

Aims

- 32 -

Aims

- 33 -

The main goal of the present work was to study the role of inflammasome

activation and IL-1 secretion in rheumatoid arthritis.

The specific aims of this study were:

I. To quantify cytokine and chemokine levels in sera and synovial fluid

samples from patients with early and established rheumatoid arthritis;

II. To study inflammasome activation by the quantification of caspase-1

activation in blood samples from patients with early and established

rheumatoid arthritis;

III. To identify drugs which down-regulate the production of IL-1β and TNF;

IV. To study the in vivo early and late anti-inflammatory effects of drugs that

simultaneously down-regulate IL-1β and TNF levels using a rat model of

arthritis;

V. To study the early and late anti-inflammatory effects of a drug that

specifically inhibits RORγT in a rat model of arthritis.

- 34 -

Chapter III

Results

- 36 -

Results

- 37 -

In agreement with the Decreto-Lei 388/70, art. 8º, parágrafo 2, the results

presented and discussed in this thesis were published or submitted for publication in

the following scientific peer-reviewed journals:

I. Rita A. Moura*, Rita Cascão*, Inês Perpétuo, Helena Canhão, Elsa Vieira-

Sousa, Ana F. Mourão, Ana M. Rodrigues, Joaquim Polido-Pereira, Mário

V. Queiroz, Henrique S. Rosário, Maria M. Souto-Carneiro, Luis Graca,

João E. Fonseca. Cytokine pattern in very early rheumatoid arthritis

favours B cell activation and survival. Rheumatology (Oxford) 2011;

50(2):278-282

* Rita A. Moura and Rita Cascão contributed equally to this work.

II. Rita Cascão*, Rita A. Moura*, Inês Perpétuo, Helena Canhão, Elsa Vieira-

Sousa, Ana F. Mourão, Ana M. Rodrigues, Joaquim Polido-Pereira, Mário

V. Queiroz, Henrique S. Rosário, Maria M. Souto-Carneiro, Luis Graca,

João E. Fonseca. Identification of a cytokine network sustaining neutrophil

and Th17 activation in untreated early rheumatoid arthritis. Arthritis

Research and Therapy 2010; 12(5):R196

* Rita Cascão and Rita A. Moura contributed equally to this work.

III. Rita Cascão, Joaquim Polido-Pereira, Helena Canhão, Ana M. Rodrigues,

Márcio Navalho, Helena Raquel, Ana F. Mourão, Catarina Resende, João

E. Fonseca, Luis F. Moita. Caspase-1 is active since the early phase of

Rheumatoid Arthritis. Clinical and Experimental Rheumatology 2012;

30(1):144

IV. Rita Cascão, Bruno Vidal, Helena Raquel, Ana Neves-Costa, Nuno

Figueiredo, Vineet Gupta, João E. Fonseca, Luis F. Moita. Effective

treatment of rat adjuvant-induced arthritis by celastrol. Autoimmunity

Reviews 2012 Mar 3. [Epub ahead of print]

Results

- 38 -

V. Rita Cascão, Bruno Vidal, Helena Raquel, Ana Neves-Costa, Nuno

Figueiredo, Vineet Gupta, João E. Fonseca, Luis F. Moita. Gambogic acid

is a potent anti-inflammatory and anti-proliferative drug in a rat model of

adjuvant-induced arthritis. Under revision

Results

- 39 -

Cytokine pattern in very early rheumatoid

arthritis favours B cell activation and survival

Results

- 40 -

Concise report

Cytokine pattern in very early rheumatoid arthritisfavours B-cell activation and survival

Rita A. Moura1,*, Rita Cascao1,*, Ines Perpetuo1, Helena Canhao1,2,Elsa Vieira-Sousa1,2, Ana F. Mourao1,3, Ana M. Rodrigues1,2,Joaquim Polido-Pereira1,2, Mario V. Queiroz2, Henrique S. Rosario4,Maria M. Souto-Carneiro5, Luis Graca6,7 and Joao E. Fonseca1,2

Abstract

Objectives. B cells play an important role in the perpetuation of RA, particularly as autoantibody-

producing cells. The ICs that further develop deposit in the joints and aggravate the inflammatory process.

However, B-cell contribution in the very early stage of the disease remains unknown. The main goal of this

work was to determine the concentration of cytokines potentially relevant for B-cell activation in serum

from very early polyarthritis patients, with <6 weeks of disease duration, who latter on evolved into very

early RA (VERA).

Methods. A proliferation-inducing ligand (APRIL), B-cell activating factor (BAFF) and IL-21 levels were

measured by ELISA in the serum of VERA, other very early arthritis (VEA), established RA patients and

controls. SF samples of established RA were also analysed.

Results. VERA patients have higher levels of APRIL and BAFF as compared with VEA, established RA and

controls. Furthermore, APRIL and BAFF levels are also significantly elevated in RA-SF when compared

with serum.

Conclusions. The increased levels of APRIL and BAFF in VERA patients suggests that B-cell activation

and the development of autoreactive B-cell responses might be crucial in early phases of RA. Therefore,

APRIL and BAFF could be promising targets for therapy in the early phase of RA.

Key words: B cells, VERA, Synovial fluid, APRIL, BAFF.

Introduction

B cells play critical roles in RA pathogenesis. They are the

source of RFs and anti-CCP autoantibodies, which

contribute to IC formation in the joints. These cells are

also efficient antigen-presenting cells and contribute to

T-cell activation through expression of co-stimulatory

molecules. B cells simultaneously respond and produce

chemokines and cytokines that promote leucocyte infiltra-

tion into the joints, formation of ectopic lymphoid struc-

tures, angiogenesis and synovial hyperplasia. Moreover,

B-cell depletion therapy with rituximab, as well as the

promising results obtained with atacicept in a Phase Ib

trial [1], confirmed the importance of these cells in estab-

lished RA [2, 3]. However, B-cell participation in the early

phase of the disease is not yet completely understood.

Interestingly, our previous studies showed a significant

decrease of pre-switch memory B cells (IgD+CD27+) in

peripheral blood of very early RA (VERA) patients, with

<6 weeks of disease duration, when compared with

controls [4]. In accordance with the results of another

group, it is highly likely that this B-cell subset migrates

towards the SM, contributing to the onset of the synovitis

1Rheumatology Research Unit, Instituto de Medicina Molecular,Faculdade de Medicina da Universidade de Lisboa, 2RheumatologyDepartment, Centro Hospitalar de Lisboa Norte, EPE, Hospital deSanta Maria, 3Rheumatology Department, Centro Hospitalar de LisboaOcidental, EPE, Hospital Egas Moniz, 4Microvascular Biology andInflammation Unit, Instituto de Medicina Molecular, Faculdade deMedicina da Universidade de Lisboa, Lisbon, 5Center forNeurosciences and Cell Biology, Autoimmunity Group, Coimbra,6Cellular Immunology Unit, Instituto de Medicina Molecular,Faculdade de Medicina da Universidade de Lisboa, Lisbon and7Instituto Gulbenkian de Ciencia, Oeiras, Portugal.

Correspondence to: Joao E. Fonseca, Rheumatology Research Unit,Instituto de Medicina Molecular, Edifıcio Egas Moniz, Faculdade deMedicina da Universidade de Lisboa, Av. Professor Egas Moniz,1649-028 Lisbon, Portugal. E-mail: jefonseca@netcabo.pt

*Rita A. Moura and Rita Cascao contributed equally to this study.

Submitted 18 June 2010; revised version accepted 3 September 2010.

! The Author 2010. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

RHEUMATOLOGY

Rheumatology 2011;50:278–282

doi:10.1093/rheumatology/keq338

Advance Access publication 2 November 2010

BA

SIC

SC

IEN

CE

at Harvard U

niversity on January 25, 2012http://rheum

atology.oxfordjournals.org/D

ownloaded from

process [5]. Our hypothesis is that the cytokine environ-

ment in early RA favours the recruitment, activation and

survival of B cells, and herein we tested this concept in a

cohort of very early polyarthritis patients.

Materials and methods

Patients

Blood samples were collected from 19 untreated very

early polyarthritis patients with <6 weeks of disease

duration, who after a minimum follow-up of 3–4 months

fulfilled the 1987 ACR criteria for RA [6]. These patients

were classified as VERA patients. Further samples were

collected 4–6 weeks after starting a low dose of oral

CSs (5–10 mg of prednisone) (Time 1) and 4 months

after reaching the minimum effective dose of MTX (Time

2) up to a maximum of 20 mg/week required to reduce

the 28-joint DAS (DAS-28) to <3.2 [7]. Also, baseline

blood samples from VERA patients were compared

with 19 other very early arthritis (VEA) patients who, after

the same follow-up, did not evolve into RA and with

24 controls. Additionally, 12 blood and 15 SF samples

were obtained from MTX-treated established RA patients.

Of note, SF samples were only collected from established

RA, since most VERA patients did not have joint effusions

in easily accessible joints (Table 1). The HAQ [8] and

the DAS-28 were applied to all patients. The study was

approved by the local ethics committee (Comissao de

Etica do Hospital de Santa Maria, Lisbon, Portugal)

and all patients gave informed consent. Patient care

was conducted in accordance with the standard clinical

practice in the Rheumatology Department, Hospital de

Santa Maria, Lisbon, Portugal and the study was

performed in accordance with the Declaration of Helsinki

as amended in Edinburgh (2000).

Cytokine quantification

A proliferation-inducing ligand (APRIL), B-cell activating

factor (BAFF) and IL-21 levels were determined by

ELISA (Bender MedSystems GmbH, Vienna, Austria)

according to the manufacturer’s instructions. Samples

were analysed using plate reader Infinite M200 (Tecan,

Mannedorf, Switzerland).

Measurement of autoantibodies

RF-immunoglobulin M (RF-IgM) was determined in all pa-

tients by IMTEC Autoimmune Diagnostics ELISA kit

(Human GmbH, Wiesbaden, Germany) according to the

manufacturer’s instructions and samples were processed

using a ChemWell 2910 automated analyser. Serum levels

of anti-CCP were measured by the ELIA CCP test system

(Phadia GmbH, Freiburg, Germany) and samples were

analysed using an ImmunoCAP 100 instrument.

Statistical analysis

Statistical differences were determined with non-

parametric Kruskal–Wallis and Mann–Whitney tests using

GraphPad Prism (GraphPad, San Diego, CA, USA).

Correlation analysis was performed using Spearman’s TA

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test. Differences were considered to be statistically

significant for P< 0.05.

Results

Characterization of patients and disease evaluation

A total of 38 polyarthritis patients with <6 weeks of

disease duration were consecutively included. VERA

patients had a mean (S.D.) age of 59 (17) years; 84.2%

were female, 42% were RF positive and 32% anti-CCP

positive. The baseline DAS-28 and HAQ were 6.1 (1.8) and

1.4 (0.8), respectively. After treatment with CSs and MTX

there was a significant reduction of both DAS-28 and HAQ

values (Table 1). VEA patients were classified as having

SpA (five cases), SLE (four cases), crystal-induced arthritis

(two cases), SS (one case), paraneoplastic polyarthritis

related to multiple myeloma (one case) and arthritis asso-

ciated with HIV infection (one case), and five patients

entered spontaneous remission before 3 months of

follow-up, remaining without a specific diagnosis and

were thus classified as presenting a self-limited polyarthri-

tis. These early polyarthritis patients represent a subset of

a larger cohort previously described by our group [4].

Furthermore, unpaired blood and SF samples were col-

lected from established RA patients who had similar

DAS-28 and HAQ values to those of VERA patients at

baseline (Table 1).

APRIL and BAFF levels are increased in VERApatients at baseline

At baseline, APRIL and BAFF levels were significantly

higher in VERA patients as compared with both VEA and

controls (Fig. 1A). No differences were observed between

treated or untreated VERA patients, or between VEA and

controls (Table 1). Moreover, no significant differences in

IL-21 levels could be observed in VERA when compared

with either VEA patients and controls, or after therapy with

CSs and MTX (data not shown). Furthermore, there was

no correlation between DAS-28, anti-CCP or RF autoanti-

bodies and APRIL and BAFF serum concentrations (data

not shown).

VERA patients have higher APRIL and BAFF levelsin comparison with established RA patients

In order to compare early RA with the chronic phase,

we also analysed established RA serum samples.

Importantly, VERA patients, in baseline and even after

MTX treatment, had higher circulating levels of both

APRIL (P< 0.05) and BAFF (P< 0.001) in comparison

with established RA. However, regarding IL-21, no differ-

ences could be found in VERA when compared with

established RA (data not shown). Furthermore, no signifi-

cant differences could be observed in established RA in

comparison with controls (data not shown).

Established RA-SF has increased levels of APRIL,BAFF and IL-21

To verify whether in established RA patients a B-cell

activation environment could be present in the joint fluid

despite lower APRIL and BAFF serum levels, we tested

the same cytokine panel in established RA-SF. APRIL,

BAFF and IL-21 levels were in fact increased locally in

the joints of established RA patients in comparison with

RA serum (Fig. 1B).

Discussion

In the present work, a cytokine pattern favouring B-cell

activation is observed in RA patients with <6 weeks of

disease duration, when compared with other causes of

VEA and established RA. Our previous results demon-

strated a significant decrease of pre-switch memory

B cells (IgD+CD27+) in peripheral blood of VERA patients

[4]. This is in agreement with the report from another

group referring to a migration of this B-cell subset towards

the SM [5]. In fact, in established RA, ectopic germinal

centre-like structures develop in the inflamed synovial

tissue that support survival of B cells and autoantibody

production [9]. Therefore, we also analysed established

RA-SF samples and depicted the presence of a

cytokine-based B-cell survival environment that could

explain the maintenance of potentially autoreactive B

cells in the synovium. Our results demonstrated that

VERA patients have increased APRIL and BAFF levels

when compared with VEA and controls. Interestingly,

APRIL was also increased in VERA patients’ serum as

compared with established RA and its levels were even

higher in RA-SF suggesting a local up-regulation in the

synovium. APRIL affects not only the class-switch recom-

bination process [10–12], but also plasma cell differenti-

ation and survival [13, 14], which could thus explain the

maintenance of autoreactive B cells in the joints [15].

Actually, a highly positive association between the infiltra-

tion of plasma cells and SF levels of APRIL has been

demonstrated in RA patients [15, 16]. BAFF, similar to

APRIL, is a fundamental B-cell survival factor and we

have also detected increased serum levels in VERA

patients when compared with established RA. Moreover,

BAFF was also significantly elevated in RA-SF in compari-

son with RA serum. Previous studies have demonstrated

that BAFF increases the chemokine (C-X-C motif) ligand

13 (CXCL13)-dependent chemotaxis of memory B cells

through BAFF receptor (BAFF-R) triggering [17].

Therefore, increased BAFF levels in RA could support

the migration of pre-switch memory B cells towards RA

synovium, thus justifying the decrease of this B-cell sub-

population in circulation. Furthermore, increased IL-21

levels in RA-SF support local plasma cell differentiation

and autoantibody production [18]. Additionally, therapy

with neither CSs nor MTX affected cytokine production

in VERA patients. However, the patients with established

RA that we have studied were on chronic treatment with

MTX and low-dose CSs and this might have influenced the

serum levels of APRIL and BAFF. The effect of low-dose

CSs and MTX on cytokine production in RA patients is

still controversial [19, 20]. So, an absence of effect of

short-term therapy with CSs and MTX on the cytokines

analysed was not entirely unexpected in VERA, but an

effect on chronic-treated patients might in fact occur.

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Conclusions

In conclusion, we have shown increased APRIL and BAFF

levels in VERA and in RA-SF, which could hypothetically

support the maintenance, expansion, activation and

survival of autoreactive B cells from the first weeks of

disease onset. Therefore, we suggest that APRIL and

BAFF may be potential promising treatment targets in

the very early phase of RA.

Rheumatology key messages

. APRIL and BAFF levels are increased from the firstweeks of RA onset.

. APRIL and BAFF may be potential promisingtreatment targets in VERA.

FIG. 1 VERA patients have higher APRIL and BAFF levels than controls and those with VEA at baseline (A). Serum

samples from VEA patients with no therapy and healthy controls were analysed by ELISA technique. Differences were

considered to be statistically significant at P< 0.05. Established RA patients have increased levels of APRIL, BAFF and

IL-21 locally in the joints (B). Serum and SF samples from established RA patients were analysed by ELISA technique.

Dotted lines represent the limit of detection for the assay. Differences were considered to be statistically significant at

P< 0.05.

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Acknowledgements

The authors would like to acknowledge Bruno Vidal for

technical assistance.

Funding: This work was supported by a grant from

Sociedade Portuguesa de Reumatologia/Schering-

Plough 2005. R.A.M. and R.C. were funded by

Fundacao para a Ciencia e a Tecnologia (FCT) SFRH/

BD/30247/2006 and SFRH/BD/40513/2007, respectively.

M.M.S.-C. was funded by Marie Curie Intra-European

Fellowship PERG-2008-239422 and an EULAR Young

Investigator Award.

Disclosure statement: The authors have declared no

conflicts of interest.

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Results

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Identification of a cytokine network sustaining

neutrophil and Th17 activation in untreated

early rheumatoid arthritis

Results

- 42 -

RESEARCH ARTICLE Open Access

Identification of a cytokine network sustainingneutrophil and Th17 activation in untreatedearly rheumatoid arthritisRita Cascão1†, Rita A Moura1†, Inês Perpétuo1, Helena Canhão1,2, Elsa Vieira-Sousa1,2, Ana F Mourão1,3,Ana M Rodrigues1,2, Joaquim Polido-Pereira1,2, Mário V Queiroz2, Henrique S Rosário4, Maria M Souto-Carneiro5,Luis Graca6†, João E Fonseca1,2*†

Abstract

Introduction: Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterized by sustainedsynovitis. Recently, several studies have proposed neutrophils and Th17 cells as key players in the onset andperpetuation of this disease. The main goal of this work was to determine whether cytokines driving neutrophiland Th17 activation are dysregulated in very early rheumatoid arthritis patients with less than 6 weeks of diseaseduration and before treatment (VERA).

Methods: Cytokines related to neutrophil and Th17 activation were quantified in the serum of VERA andestablished RA patients and compared with other very early arthritis (VEA) and healthy controls. Synovial fluid (SF)from RA and osteoarthritis (OA) patients was also analyzed.

Results: VERA patients had increased serum levels of cytokines promoting Th17 polarization (IL-1b and IL-6), aswell as IL-8 and Th17-derived cytokines (IL-17A and IL-22) known to induce neutrophil-mediated inflammation. Inestablished RA this pattern is more evident within the SF. Early treatment with methotrexate or corticosteroids ledto clinical improvement but without an impact on the cytokine pattern.

Conclusions: VERA patients already display increased levels of cytokines related with Th17 polarization andneutrophil recruitment and activation, a dysregulation also found in SF of established RA. 0 Thus, our data suggestthat a cytokine-milieu favoring Th17 and neutrophil activity is an early event in RA pathogenesis.

IntroductionRheumatoid arthritis (RA), the most common chronicautoimmune disease, affects approximately 1% of thepopulation worldwide. This disease comprises a syn-drome of pain, stiffness, and symmetrical synovitiswhich leads to joint destruction, functional disability,and substantial comorbidity due to the involvement ofmultiple organs and systems. The migration of leuko-cytes toward the synovium is crucial for the establish-ment of a chronic inflammatory process in RA [1-3].This multi-regulated mechanism involves interactions

with endothelial cells through cell adhesion moleculesand complex cytokine and chemokine pathways.Neutrophils specifically play an important role in the

onset and perpetuation of RA, not only as interleukin(IL)-producing cells but also as cells responsible for therelease of high amounts of reactive oxygen species anddestructive enzymes, such as metalloproteases, contri-buting to joint erosions [4]. Neutrophils are among thefirst leukocytes to arrive at sites of inflammation. Infact, these cells are the most abundant in the synovialfluid (SF) of patients with active RA, and previousresults from our group showed that the synovial tissueis heavily infiltrated by neutrophils in the first weeks ofRA onset [5]. Interestingly, in animal models of arthritis,neutrophil depletion prevented joint inflammation ifneutrophil-depleting antibodies were given before the

* Correspondence: jefonseca@netcabo.pt† Contributed equally1Rheumatology Research Unit, Instituto de Medicina Molecular, Edifício EgasMoniz, Faculdade de Medicina da Universidade de Lisboa, Av. Professor EgasMoniz, Lisboa, 1649-028, PortugalFull list of author information is available at the end of the article

Cascão et al. Arthritis Research & Therapy 2010, 12:R196http://arthritis-research.com/content/12/5/R196

© 2010 Cascão et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.

induction of arthritis. Moreover, when the depletingantibody was given very early after the induction ofarthritis, complete abrogation of the inflammatorysymptoms was achieved [6].T helper 17 (Th17) cells have also been proposed to

have a relevant role in the early phase of RA throughthe production of IL-17 [7,8]. This cytokine promotesthe recruitment and survival of neutrophils, induces thesecretion of proinflammatory cytokines and the upregu-lation of RANKL (receptor activator of nuclear factor-kappa B ligand), and stimulates the activity of matrixmetalloproteases, leading to cartilage catabolism andbone resorption [9,10]. The recruitment, activation, andeffector function of Th17 cells and neutrophils are dri-ven by a network of cytokines and chemokines secretedby multiple cellular sources. In established RA, it hasbeen reported that IL-1b, IL-6, IL-8, IL-17, and tumornecrosis factor are elevated in the serum and this corre-lates with a higher disease activity [11-13]. Nevertheless,our knowledge of the influence of the cytokine networkon RA onset remains limited. The characterization ofthe cytokine profile at this stage, where the transitionfrom an acute to a chronic inflammatory phase occurs,may lead to the identification of early key players, withpotential implications for early treatment strategies.Thus, the main goal of our work was to determine

whether cytokines driving neutrophil and Th17 cell acti-vation and proinflammatory function were already pre-sent in very early RA (with less than 6 weeks of diseaseduration) and how this early cytokine environment dif-fers from established RA. We also evaluated whether theintroduction of low-dose corticosteroids and methotrex-ate (MTX) therapy had any influence on the cytokineprofile observed at that early stage of the disease. Wefound that cytokines related to Th17 polarization andneutrophil recruitment and activation were elevated inearly RA and that the conventional therapeutic options,though able to control clinical manifestations of the dis-ease, were ineffective in reversing this underlying proin-flammatory drive.

Materials and methodsPatientsBlood samples were obtained from 38 consecutiveuntreated polyarthritis patients with less than 6 weeks ofdisease duration. Some of these patients (19), after aminimum follow-up of 3 months, fulfilled the 1987American College of Rheumatology (ACR) criteria forRA [14]. These patients were classified as very early rheu-matoid arthritis (VERA) patients, and further sampleswere collected 4 to 6 weeks after starting a low dose oforal corticosteroids (5 to 10 mg of prednisone) (time 1)and 4 months after reaching the minimum effective doseof MTX (time 2) (up to a maximum of 20 mg/week) that

was required to reduce the disease activity score using 28joint counts (DAS28) to less than 3.2 [15]. The remainingearly arthritis patients (19), who did not develop RA,were classified as very early arthritis (VEA). Baselineblood samples from VERA and VEA patients were com-pared with 27 healthy donors used as controls. Addition-ally, 12 blood and 15 SF samples were obtained frompatients with established RA. SF samples were also col-lected from 10 patients with osteoarthritis (OA) (Rheu-matology Department, Hospital de Santa Maria, Lisbon,Portugal) (Table 1). Owing to the clinical characteristicsof the VEA patients, SF in easily accessible joints was notavailable in VERA and VEA patients and thus SF was notanalyzed in these groups of patients. The health assess-ment questionnaire (HAQ) [16] and DAS28 were appliedto all patients. The study was approved by the local ethicscommittee, and all patients signed an informed consentform. Patient care was conducted in accordance withstandard clinical practice, and the study was performedin accordance with the Declaration of Helsinki asamended in Edinburgh (2000).

Cytokine quantificationIL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12(p70), IL-17A,IL-22, IL-23, and interferon-gamma levels were mea-sured in the serum and SF by FlowCytomix assay kit(Bender MedSystems, Vienna, Austria) in accordancewith the instructions of the manufacturer. Standardcurves for each cytokine were generated by using refer-ence cytokine concentrations supplied by the manufac-turer. Samples were acquired with a FACS Calibur flowcytometer (BD Biosciences, San Jose, CA, USA). Rawdata of the flow cytometry bead assay were analyzed byFlowCytomix Pro 2.2 software (Bender MedSystems).

Measurement of autoantibodiesRheumatoid factor (RF)-IgM was determined in allpatients by means of an IMTEC Autoimmune Diagnos-tics ELISA [enzyme-linked immunosorbent assay] kit(Human GmbH, Wiesbaden, Germany) in accordancewith instructions of the manufacturer, and samples wereprocessed using a ChemWell 2910 automated analyzer(GMI, Ramsey, Minnesota, USA). Serum levels of anti-cyclic citrullinated peptide (anti-CCP) were measured byELIA™ CCP test system (Phadia GmbH, Freiburg, Ger-many), and samples were analyzed with an ImmunoCAP100 instrument (Phadia GmbH).

Statistical analysisStatistical differences were determined with non-parametric Kruskal-Wallis, Mann-Whitney, andWilcoxon signed-rank tests and GraphPad Prism (Graph-Pad Software, Inc., San Diego, CA, USA). Correlationanalysis was performed with the Spearman test.

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Page 2 of 8

Differences were considered statistically significant forP values of less than 0.05.

ResultsCharacterization of patients and disease evaluationA total of 38 polyarthritis patients with less than 6weeks of disease duration were evaluated. Nineteenpatients fulfilled the 1987 ACR criteria for RA after aminimum follow-up of 3 months and were classified asVERA patients. The mean age of the VERA patients was59 ± 17 years, 84% were female, 42% were RF-positiveand 32% anti-CCP-positive, the initial DAS28 was 6.1 ±1.8, and the initial HAQ was 1.4 ± 0.8. After treatmentwith low doses of prednisone and MTX, there was a sig-nificant reduction of both DAS28 and HAQ values(Table 1). The group of VEA patients included 19patients, and 14 of them later had one of the followingdiagnoses: spondylarthritis (5 cases), systemic lupuserythematosus (4 cases), crystal induced arthritis (2cases), Sjögren syndrome (1 case), paraneoplastic polyar-thritis related to multiple myeloma (1 case), and arthritisassociated with HIV infection (1 case). Five patientsentered spontaneously into remission before 3 monthsof follow-up, remaining without a specific diagnosis andwere thus classified as presenting a self-limited form ofpolyarthritis. The mean age of the VEA patients was 40± 13 years, 79% were female, all patients were RF-nega-tive and anti-CCP-negative, the initial DAS28 was 4.5 ±1.6, and the initial HAQ was 0.8 ± 0.6. Both DAS28 andHAQ values were significantly lower than those ofVERA patients at baseline (Table 1). These early polyar-thritis patients represent a subset of a larger cohort pre-viously described by our group [17].Furthermore, blood samples were collected from 12

patients with established RA; mean age was 60 ± 10years, 92% were female, and 67% were RF-positive and45% anti-CCP-positive (Table 1). Additionally, SF sam-ples were collected from 12 patients with establishedRA; mean age was 57 ± 10 years, and 73% were female

(Table 1). The established RA group of patients had aDAS28 and a HAQ mean scores similar to VERA base-line values.

IL-8 is increased in VERA patients and locally in the jointsof patients with established RAGiven the proposed role of neutrophils in the pathogen-esis of RA [18,19], we quantified the major neutrophilchemoattractant, IL-8, in the serum of VERA patients.At baseline, VERA patients had significantly higherlevels of IL-8 when compared with both VEA andhealthy controls (Figure 1a). After 2 to 4 weeks of low-dose corticosteroids and after 4 months of MTX ther-apy, there were no significant changes in the levels ofcirculating IL-8 (data not shown). Interestingly, VERApatients also had significantly higher circulating levels ofIL-8 in comparison with serum from established RA(Figure 1a). Neutrophils accumulate locally in the jointsof patients with RA [20]. Thus, we quantified the con-centration of IL-8 in the SF of patients with RA andcompared the concentration with that of SF frompatients with OA. We found significantly higher levelsof IL-8 in the SF of patients with RA in comparisonwith OA SF (Figure 1b).

IL-17 levels are dysregulated in both VERA patients andpatients with established RAPrevious studies from our group showed that there is adelay in the apoptosis of circulating neutrophils inVERA patients [21]. Therefore, we analyzed IL-17Alevels in these patients since it has already beendescribed that this cytokine is important for the survivalof neutrophils [22]. Moreover, IL-17A is a signaturecytokine of Th17 cells, a subset proposed to have a keyrole in RA pathogenesis [9,23]. We found that VERApatients had significantly higher levels of IL-17A whencompared with healthy controls, but not with VEApatients (Figure 2a). Furthermore, in our previous work,we found no difference in the frequency and absolute

Table 1 Clinical information about healthy controls and patients with VERA, VEA, RA, or OA

Controls(n = 24)

VERA (n = 19) VEA(n = 19)

RA(n = 12)

RA SF(n = 15)

OA SF(n = 10)

Baseline Time 1 Time 2

Age in years, mean ± SD 40 ± 13 50 ± 17 40 ± 13 63 ± 10 57 ± 10 67 ± 13

Sex, female/male 17/7 16/3 15/4 11/1 11/4 5/5

DAS28, mean ± SD NA 6.1 ± 1.8 4.1 ± 1.6a 3.1 ± 1.6a 4.5 ± 1.6a 5.2 ± 1.0 4.6 ± 1.4 NA

HAQ, mean ± SD NA 1.4 ± 0.8 0.8 ± 0.7a 0.8 ± 0.7 0.8 ± 0.6a 1.5 ± 1.0 1.4 ± 0.8 NA

RF-positive, % ND 42 ND ND 0 67 ND ND

Anti-CCP-positive, % ND 32 ND ND 0 45 ND NDaDisease activity score using 28 joint counts (DAS28) and health assessment questionnaire (HAQ) values were compared between very early rheumatoid arthritis(VERA) and very early arthritis (VEA) patients with reference to VERA baseline values. Differences were considered statistically significant for P values of less than0.05. anti-CCP, anti-cyclic citrullinated peptide; IL, interleukin; MTX, methotrexate; NA, not applicable; ND, not determined; OA, osteoarthritis; RA, rheumatoidarthritis; RF, rheumatoid factor; SD, standard deviation; SF, synovial fluid.

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numbers of CD4+ and CD8+ T-cell subpopulations inthe peripheral blood of these patients when analyzed byflow cytometry [17].Regarding the effects of early therapy, we found that

neither corticosteroids nor MTX affected the level ofIL-17A (data not shown). Moreover, IL-17A was signifi-cantly increased locally within the joints of patients withestablished RA in comparison with control SF frompatients with OA (Figure 2b).

RA has a Th17-cytokine pattern since the very first weeksof onsetHaving found that IL-17A was elevated in VERApatients, we decided to quantify a panel of cytokinesknown to be associated with Th17 polarization. At base-line, VERA patients had significantly higher levels of IL-1b and IL-22 in comparison with both VEA and healthycontrols. In addition, we found that VERA patients have

significantly higher IL-6 levels than healthy controls(Figure 3). Furthermore, the significantly higher circulat-ing levels of IL-6 and IL-22 were maintained in estab-lished RA (Figure 3).Locally, within the joints of patients with RA, the SF

displayed elevated levels of IL-1b and IL-6 in compari-son with OA SF (Figure 4 and Table 2). Moreover, nosignificant differences could be observed for IL-23 incirculation or locally in the joints (data not shown). Wehave also studied cytokines associated with the functionof Th2 (IL-4 and IL-10) and Th1 (IL-2, IL-12 (p70), andINFg) cells. However, no statistically significant differ-ences could be observed for any of these cytokines (datanot shown).

DiscussionSeveral studies have previously demonstrated that neu-trophils play an important role in the onset of RA [21].

Figure 1 Interleukin-8 (IL-8) is increased in the serum of very early rheumatoid arthritis (VERA) patients and in synovial fluid (SF) ofestablished rheumatoid arthritis (RA). (a) The serum concentration of IL-8 was measured in VERA and very early arthritis (VEA) patients as wellas healthy controls and patients with established RA. The serum concentration of IL-8 was increased in VERA patients compared with any othergroup. Dotted line represents the limit of detection for the assay. (b) The concentration of IL-8 was measured in the SF collected from patientswith established RA and from a control group with osteoarthritis (OA). We found a significant increase of IL-8 in RA-SF. Differences wereconsidered statistically significant for P values of less than 0.05 according to the Mann-Whitney test.

Figure 2 Very early rheumatoid arthritis (VERA) patients and synovial fluid (SF) of established rheumatoid arthritis (RA) displayincreased levels of interleukin-17A (IL-17A). (a) The serum concentration of IL-17A was measured in VERA and very early arthritis (VEA)patients as well as healthy controls and patients with established RA. The serum concentration of IL-17A was increased in VERA patientscompared with healthy controls. (b) The concentration of IL-17A was measured in the SF collected from patients with established RA and froma control group with osteoarthritis (OA). In the SF of patients with RA, we observed a significant increase of IL-17A. Dotted lines represent thelimit of detection for the assay. Differences were considered statistically significant for P values of less than 0.05 according to the Mann-Whitneytest.

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This hypothesis is supported by data from animal mod-els [24]. In fact, neutrophils are the most abundant leu-kocytes in the SF of patients with active RA, and inearly RA, these cells show significantly lower levels ofapoptosis when compared with patients with other per-sistent forms of arthritis or with arthritis that has a self-limited disease course [25]. Additionally, previous results

from our group demonstrated that there is a delay inthe apoptosis of circulating neutrophils in VERApatients [21] and that these cells heavily infiltrate thesynovial tissue during RA onset [5].In the present study, we demonstrate that a neutro-

phil- and Th17-driving cytokine pattern is present inuntreated VERA patients with less than 6 weeks of

Figure 3 Cytokines related to T helper 17 (Th17) polarization are increased in the serum of very early rheumatoid arthritis (VERA)patients and synovial fluid of established rheumatoid arthritis (RA). The serum concentrations of interleukin (IL)-1b, IL-6, and IL-22 weremeasured in VERA and very early arthritis (VEA) patients as well as healthy controls and patients with established RA. All three cytokines wereincreased in VERA patients compared with healthy controls. IL-6 was equally elevated in all groups of patients with an inflammatory disease,whereas the other two cytokines were increased only in VERA (IL-1b) or in VERA and RA patients (IL-22). Dotted lines represent the limit ofdetection for the assays. Differences were considered statistically significant for P values of less than 0.05 according to the Mann-Whitney test.

Figure 4 Cytokines related to T helper 17 (Th17) polarization are increased in the synovial fluid (SF) of established rheumatoidarthritis (RA). The concentrations of interleukin (IL)-1b and IL-6 were markedly increased in the SF collected from patients with established RAwhen compared with osteoarthritis (OA). Dotted lines represent the limit of detection for the assays. Differences were considered statisticallysignificant for P values of less than 0.05 according to the Mann-Whitney test.

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disease duration. We consider this observation of inter-est because the knowledge concerning the immunemechanisms associated with the onset of RA is still elu-sive. In fact, the majority of early RA studies includepatients with 3 to 12 months of disease duration oreven more. In accordance with an early participation ofneutrophils in RA, our results revealed that VERApatients have increased levels of IL-8 when comparedwith both VEA and healthy controls, and this couldexplain the preactivated state of circulating neutrophils[18] and their recruitment toward the SF from the veryfirst weeks of RA onset.In addition, Th17 cells are known to be important for

the promotion of neutrophil-mediated inflammation byproducing IL-17A, a cytokine known to indirectly acti-vate neutrophil chemotaxis and extend their survival[10,22]. We found a high serum concentration of IL-17Ain VERA patients as well as locally within the joints ofpatients with established RA. This might indicate that anactivation of Th17 cells from a very early phase of thedisease can promote neutrophil participation in RApathogenesis [22]. However, we found no evidence forchanges in the frequency of T-cell subsets in the periph-eral blood of VERA patients [17]. This observation is notunexpected; the relatively small representation of anti-gen-specific T cells in the circulating pool are the acti-vated T cells that drive the pathology more likely foundwithin the tissues [26]. In a study performed by Kokko-nen and colleagues [27], the levels of several cytokinesand chemokines were analyzed in blood samples from agroup of individuals 3.3 years before RA onset (’pre-patients’) and compared with healthy donors and RApatients with 7.7 ± 3.6 months of disease duration. Aninteresting finding was that IL-17 was present at its high-est concentration in pre-patients and the level of thiscytokine was lower in patients with RA. This is in accor-dance with our own results; we observed an increasedlevel of IL-17 in RA patients with less than 6 weeks ofdisease duration, whereas in patients with establishedRA, the levels were not significantly different from those

of healthy controls. Remarkably, the IL-17 median con-centration observed in our established RA cohort (2.6 pg/mL) was even lower than that of RA patients from thework of Kokkonen and colleagues [27] (6.0 pg/mL).Thus, this observation reinforces the role of IL-17 in theinitial phase of RA, and as the pathogenesis progresses toa chronic stage, other factors are subsequently broughtinto action in the peripheral blood. Unlike Kokkonen andcolleagues, we have not detected differences in Th1- andTh2-related cytokines between both VERA and patientswith established RA in comparison with controls. Thesediscrepancies might be related to the different methodol-ogies used.Additionally, the elevated levels of IL-1b observed in

VERA patients can stimulate endothelial cells, T and Bcells, and fibroblasts in the joints to produce IL-6 andIL-8. But importantly, IL-1b and IL-6, both found to beincreased in VERA patients, are known to promote thedifferentiation of Th17 cells, which in turn secreteIL-17A and IL-22 [28,29], two cytokines that were ele-vated in VERA patients and have an essential functionin the pathogenesis of autoimmune diseases [29].Currently, the treatment of choice for RA at the time

of presentation is MTX. Interestingly, in spite of clinicalimprovement (DAS28 reduced from 6.1 ± 1.8 to 3.1 ±1.6), neither therapy with low-dose corticosteroids norcombined therapy with low-dose corticosteroids andMTX corrected the dysregulated cytokine patternobserved in VERA patients. In fact, low-dose corticos-teroids and MTX have unclear effects on the RA cyto-kine network. For instance, corticosteroids fail to reduceserum levels of IL-1b and IL-8 [30] and MTX does notalter serum IL-1b concentration when compared withpre-treatment levels [31,32]. Our results suggest that theconditions contributing to Th17 cells and neutrophil-mediated inflammation, thus driving early pathogenesis,are not modified with early treatment with low-dosecorticosteroids and MTX.The elevated IL-1b, IL-6, IL-8, and IL-17A levels

observed in the SF of patients with RA confirm a local

Table 2 Cytokine levels in healthy controls and patients with VERA, VEA, established RA, or OA

Cytokine, pg/mL Controls VERA VEA RA RA SF OA SF

IL-1b 4.2(4.2-116.8)

17.7(4.2-99.7)

4.2(4.2-272.7)

4.2(4.2-360.3)

6.5(4.2-322.1)

4.2(4.2-15.5)

IL-6 1.2(1.2-7.2)

5.2(1.2-153.2)

1.2(1.2-84.7)

8.2(1.2-19.7)

6361.0(272.7-24,135.0)

177.6(46.6-4,881.0)

IL-8 9.9(0.5-407.7)

57.6(12.5-1,546.0)

8.9(0.5-665.2)

15.2(0.8-67.5)

735.7(28.3-3,717.0)

48.2(22.0-680.0)

IL-17A 2.5(2.5-333.7)

62.0(2.5-1,714.0)

2.5(2.5-1,477.0)

2.6(2.5-375.6)

21.1(2.5-369.1)

2.5(2.5-9.5)

IL-22 43.3(43.3-153.4)

165.3(43.3-380.6)

43.3(43.3-270.5)

131.7(75.8-250.7)

153.4(75.8-336.0)

151.7(92.4-235.3)

Values are presented as median (range). IL, interleukin; OA, osteoarthritis; RA, rheumatoid arthritis; SF, synovial fluid; VEA, very early arthritis; VERA, very earlyrheumatoid arthritis.

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role for these cytokines in the maintenance of synovitis.Moreover, IL-6 can support a continuous recruitment ofautoreactive B cells toward the synovium [33,34], contri-buting to an exacerbation of the inflammatory processbecause of the production of autoantibodies andimmune complexes.

ConclusionsTaken together, our data reinforce the potential rele-vance of therapies targeting IL-1b [35,36] and IL-6[37,38] in early RA. In addition, the data establish IL-8and IL-17A as other potential therapeutic targets at anearly stage of the disease. Finally, we found that MTXand corticosteroids, though effective in reducing diseaseactivity in VERA patients, do not appear to correctunderlying cytokine dysregulation driving the Th17/neu-trophil-mediated inflammation.

AbbreviationsACR: American College of Rheumatology; anti-CCP: anti-cyclic citrullinatedpeptide; DAS28: disease activity score using 28 joint counts; HAQ: healthassessment questionnaire; IL: interleukin; MTX: methotrexate; OA:osteoarthritis; RA: rheumatoid arthritis; RF: rheumatoid factor; SF: synovialfluid; Th17: T helper 17; VEA: very early arthritis; VERA: very early rheumatoidarthritis.

AcknowledgementsThe authors would like to acknowledge the technical assistance of BrunoVidal. This work was supported by a grant from Sociedade Portuguesa deReumatologia/Schering-Plough 2005. RAM and RC were funded byFundação para a Ciência e a Tecnologia (FCT) SFRH/BD/30247/2006 andSFRH/BD/40513/2007, respectively. MMS-C was funded by Marie Curie Intra-European Fellowship PERG-2008-239422 and a EULAR Young InvestigatorAward.

Author details1Rheumatology Research Unit, Instituto de Medicina Molecular, Edifício EgasMoniz, Faculdade de Medicina da Universidade de Lisboa, Av. Professor EgasMoniz, Lisboa, 1649-028, Portugal. 2Rheumatology Department, CentroHospitalar de Lisboa Norte, EPE, Hospital de Santa Maria, Av. Professor EgasMoniz, Lisboa, 1649-028, Portugal. 3Rheumatology Department, CentroHospitalar de Lisboa Ocidental, EPE, Hospital Egas Moniz, Rua da Junqueira,126, Lisboa,1300, Portugal. 4Microvascular Biology and Inflammation Unit,Instituto de Medicina Molecular, Edifício Egas Moniz, Faculdade de Medicinada Universidade de Lisboa, Av. Professor Egas Moniz, Lisboa, 1649-028,Portugal. 5Chronic Inflammation Group, Center for Neurosciences and CellBiology, Rua Larga 6, Coimbra, 3030, Portugal. 6Cellular Immunology Unit,Instituto de Medicina Molecular, Edifício Egas Moniz, Faculdade de Medicinada Universidade de Lisboa, Av. Professor Egas Moniz, Lisboa, 1649-028,Portugal.

Authors’ contributionsRC and RAM equally performed all of the laboratorial work, data collection,and statistical analysis and wrote the paper. IP contributed to some of thelaboratory experiments. HC, ES, AFM, AMR, and JP-P were responsible for theselection, follow-up, and medical care of patients enrolled in this study andhelped review the paper. MVQ participated as the head of theRheumatology Department of Hospital de Santa Maria, which approved thestudy and patients’ management. HSR and MMS-C made a substantialintellectual contribution to the present work and revised it critically. LG andJEF, as senior authors, conceived of the study, participated in its design andcoordination, and contributed important intellectual input to the draft of themanuscript. All authors read and approved the final manuscript.

Competing interestsThe authors declare that they have no competing interests.

Received: 7 June 2010 Revised: 9 September 2010Accepted: 20 October 2010 Published: 20 October 2010

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doi:10.1186/ar3168Cite this article as: Cascão et al.: Identification of a cytokine networksustaining neutrophil and Th17 activation in untreatedearly rheumatoid arthritis. Arthritis Research & Therapy 2010 12:R196.

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Results

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Caspase-1 is active since the early phase of

rheumatoid arthritis

Results

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144

Letters to the EditorsCaspase-1 is active since the early phase of rheumatoid arthritis

Sirs,We have recently reported increased interleu-kin (IL)-1β levels in very recent onset rheu-matoid arthritis (RA) (1). Together with IL-6, which we also found to be increased in these patients, IL-1β can promote the polarization of Th17 cells, which have an essential role in the pathogenesis of autoimmune diseases (2), such as RA. It has also been reported that RA synovial tissue contains higher concen-trations of cytokines including IL-1β and shows activation of the nuclear transcription factor NF-κB (3), suggesting that IL-1β/IL1 receptor signalling may contribute to arthritis onset and progression (4). Caspase-1 is activated by multiprotein com-plexes known as inflammasomes. One of the best characterised examples is the NLRP3 inflammasome (5), which is able to process immature proinflammatory cytokines, such as pro-IL-1β and pro-IL-18 (6). Interestingly, it has been reported that polymorphisms in the nlrp3 gene are associated with increased susceptibility and a worse prognosis in RA (7, 8). Therefore, we hypothesised that the inflammasome could be an active contributor to the inflammatory response in RA and set out to evaluate the activation of caspase-1 in early and established RA patients.To study the early phase of RA, we collected blood samples from 10 untreated early RA (ERA) and 9 untreated early arthritis patients who have not evolved to RA (EA) with less than 12 months of disease duration. ERA patients had a mean age of 48±9 years and DAS28 of 5.0±1.8. Among ERA patients, 90% were female, 60% were rheumatoid fac-tor (RF) positive and 40% were anti-cyclic citrullinated peptide (anti-CCP) positive. EA patients had mean age of 60±17 years and DAS28 of 5.5±2.3. In the EA group, 67% were female, 22% were anti-CCP positive and all patients were RF negative. To study the chronic phase of RA, we collected blood samples from 11 established RA patients. Established RA patients had a mean age of 54±10 years and DAS28 of 4.5±1.3, 70% were RF positive and 36% were anti-CCP positive, and 80% of them were females. This group of patients had a mean disease dura-tion of 13±8 years and all of them were under methotrexate (MTX) treatment with a mean dose of 14±5 mg/week and a low dose of prednisolone (less than 10 mg). We also col-lected blood samples from 10 age- and sex-matched healthy donors for comparison.We measured caspase-1 activity using the Carboxyfluorescein FLICA Detection kit (Immunochemistry Technologies, LLC, USA) following the reagent instructions. Samples were acquired using a FACS Calibur (BD biosciences, USA) and analysed using FlowJo software (Tree Star Inc). Statistical differenc-es were determined with parametric unpaired t-test and Bonferroni’s multiple comparison

test using GraphPad Prism (GraphPad, San Diego, CA), and differences were considered statistically significant for p<0.05.We found that ERA patients have signifi-cantly higher levels of basal active caspase-1 than in healthy controls (Fig. 1, p=0.0222). Also established RA patients have higher basal levels of active caspase-1 as compared to healthy controls (Fig. 1, p=0.0301). In ad-dition, no differences were found when com-paring EA with healthy controls or between ERA and EA patients, neither between ERA and EA with established RA patients. Of note, we previously did not find any significant differences in the percentages of leukocyte populations between patients and healthy controls (9, 10).These results show that the level of active caspase-1 is increased in circulating leuko-cytes of early and established RA patients, supporting our hypothesis that the caspase-1 pathway is already activated since the early phase of RA, plays a role in this disease and may be a promising treatment target in early RA. Given the relative inefficacy of blocking IL-1β in established RA we thus suggest that this treatment strategy should be re-evalu-ated in early RA patients.The authors would like to acknowledge Ana Lopes and Bruno Vidal for their technical as-sistance. This work was supported by a grant (SFRH/BD/40513/2007) from Fundação para a Ciência e a Tecnologia (FCT) and by an un-restricted research grant from Pfizer. Work in Luis Moita’s laboratory is supported by FCT (PIC/IC/82991/2007 and PTDC/SAU-MII/100780/2008) and FLAD.

R. CASCÃO1, MScJ. POLIDO-PEREIRA1,2, MDH. CANHÃO1,2, MD, PhDA.M. RODRIGUES1,2, MDM. NAVALHO1,3, MDH. RAQUEL1, PhDA. NEVES-COSTA1, PhDA.F. MOURÃO1,4, MDC. RESENDE2, MDJ.A.P. DA SILVA2, MDJ.E. FONSECA1,2*, MD, PhDL.F. MOITA1*, MD, PhD 1Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Lisbon;

2Rheumatology Department, Centro Hospitalar de Lisboa Norte, EPE, Hospital de Santa Maria, Lisbon; 3Radiology Department, Hospital da Luz, Lisbon; 4Rheumatology Department, Cen-tro Hospitalar de Lisboa Ocidental, EPE, Hospital Egas Moniz, Lisbon, Portugal.*Joint senior authors.Address correspondence to: Dr Luis Ferreira Moita, Cell Biology of the Immune System Unit, Instituto de Medicina Molecular, Edifício Egas Moniz, Faculdade de Medicina da Universidade de Lisboa, Av. Professor Egas Moniz, 1649-028 Lisbon, Portugal.E-mail: lmoita@fm.ul.ptCompeting interests: none declared.

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5. HOFFMAN HM, MUELLER JL, BROIDE DH, WAN-DERER AA, KOLODNER RD: Mutation of a new gene encoding a putative pyrin-like protein causes familial cold autoinflammatory syndrome and Muck-le-Wells syndrome. Nat Genet 2001; 29: 301-5.

6. DOWDS TA, MASUMOTO J, ZHU L, INOHARA N, NUNEZ G: Cryopyrin-induced interleukin 1beta se-cretion in monocytic cells: enhanced activity of dis-ease-associated mutants and requirement for ASC. J Biol Chem 2004; 279: 21924-8.

7. KASTBOM A, VERMA D, ERIKSSON P et al.: Ge-netic variation in proteins of the cryopyrin inflam-masome influences susceptibility and severity of rheumatoid arthritis (the Swedish TIRA project). Rheumatology (Oxford) 2008; 47: 415-7.

8. KASTBOM A, JOHANSSON M, VERMA D, SODERKVIST P, RANTAPAA-DAHLQVIST S: CARD8 p.C10X polymorphism is associated with inflammatory activity in early rheumatoid arthritis. Ann Rheum Dis 2010; 69: 723-6.

9. WEINMANN P, MOURA RA, CAETANO-LOPES JR et al.: Delayed neutrophil apoptosis in very early rheu-matoid arthritis patients is abrogated by methotrexate therapy. Clin Exp Rheumatol 2007; 25: 885-7.

10. MOURA RA, WEINMANN P, PEREIRA PA et al.: Alterations on peripheral blood B-cell subpopula-tions in very early arthritis patients. Rheumatology (Oxford) 2010; 49: 1082-92.

Fig. 1. Early and established RA patients have a persistent activation of caspase-1. No-tice that, in contrast to EA patients, both in early and established RA patients there are higher active caspase-1 levels. (Unpaired t-test and Bonferroni’s multiple com-parison test).

© Clinical and Experimental Rheumatology 2012

Results

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Effective treatment of rat adjuvant-induced

arthritis by celastrol

Results

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Autoimmunity Reviews xxx (2012) xxx–xxx

AUTREV-01244; No of Pages 7

Contents lists available at SciVerse ScienceDirect

Autoimmunity Reviews

j ourna l homepage: www.e lsev ie r .com/ locate /aut rev

Review

Effective treatment of rat adjuvant-induced arthritis by celastrol

R. Cascão a, B. Vidal a, H. Raquel a, A. Neves-Costa a, N. Figueiredo a,b, V. Gupta c,J.E. Fonseca a,d,1,⁎, L.F. Moita a,1,⁎a Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, Lisbon, Portugalb Gulbenkian Programme for Advanced Medical Education, Lisbon, Portugalc Division of Nephrology and Hypertension, Department of Medicine, University of Miami, Miami, FL 33136, USAd Rheumatology Department, Centro Hospitalar de Lisboa Norte, EPE, Hospital de Santa Maria, Lisbon, Portugal

⁎ Corresponding authors at: Instituto de Medicina MoPortugal. Tel.: +351 217999544; fax: +351 217999459

E-mail addresses: ritacascao@gmail.com (R. Cascão)nunolfigueiredo@hotmail.com (N. Figueiredo), vgupta2@

1 Joint senior authors.

1568-9972/$ – see front matter © 2012 Elsevier B.V. Alldoi:10.1016/j.autrev.2012.02.022

Please cite this article as: Cascão R, et adoi:10.1016/j.autrev.2012.02.022

a b s t r a c t

a r t i c l e i n f o

Article history:Received 7 February 2012Accepted 29 February 2012Available online xxxx

Keywords:CelastrolDigoxinWistar AIA rat modelIL-1βCaspase-1Th17 cells

We have previously reported an increase in interleukin (IL)-1β and IL-17 levels, and a continuous activationof caspase-1 in early rheumatoid arthritis (RA) patients. These results suggest that drugs targeting IL-1βregulatory pathways, in addition to tumor necrosis factor (TNF), may constitute promising therapeutic agentsin early RA. We have recently used a THP-1 macrophage-like cell line to screen 2320 compounds for thosethat down-regulate both IL-1β and TNF secretion. Celastrol was one of the most promising therapeutic can-didates identified in that study. Our main goal in the present work was to investigate whether administrationof celastrol is able to attenuate inflammation in a rat model of adjuvant-induced arthritis (AIA). Moreover,since IL-1β is known to play a role in the polarization of Th17 cells, we also investigate whether administra-tion of digoxin, a specific inhibitor of Th17 cells polarization, is able to attenuate inflammation in the same ratmodel. We found that celastrol administration significantly suppressed joint inflammation. The histologicaland immunohistochemical evaluation revealed that celastrol-treated rats had a normal joint structure withcomplete abrogation of the inflammatory infiltrate and cellular proliferation. In contrast, we observed thatdigoxin administration significantly ameliorated inflammation but only if administrated in the early phaseof disease course (after 4 days of disease induction), and it was not efficient at inhibiting the infiltration ofimmune cells within the joint and in preventing damage. Thus, our results suggest that celastrol has signifi-cant anti-inflammatory and anti-proliferative properties and can constitute a potential anti-inflammatorydrug with therapeutic efficacy in the treatment of immune-mediated inflammatory diseases such as RA.Furthermore, we find that early inhibition of Th17 cells polarization ameliorates arthritis but it is not as effec-tive as celastrol.

© 2012 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02. Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

2.1. Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.2. IL-1β and TNF secretion assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.3. Cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.4. Animal experimental design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.5. Histological and immunohistochemical evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.6. Intracellular IL-17 staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.7. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

3. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03.1. Celastrol decreases IL-1β and TNF secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03.2. Celastrol inhibits the activation of NF-kB and caspase-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

lecular, Edifício Egas Moniz, Faculdade de Medicina da Universidade de Lisboa, Av. Professor Egas Moniz, 1649-028 Lisbon,., bvidal@fm.ul.pt (B. Vidal), mhraquel@gmail.com (H. Raquel), ananevescostaana@gmail.com (A. Neves-Costa),med.miami.edu (V. Gupta), jecfonseca@gmail.com (J.E. Fonseca), lmoita@fm.ul.pt (L.F. Moita).

rights reserved.

l, Effective treatment of rat adjuvant-induced arthritis by celastrol, Autoimmun Rev (2012),

2 R. Cascão et al. / Autoimmunity Reviews xxx (2012) xxx–xxx

3.3. Celastrol is able to suppress inflammation in Wistar rat adjuvant-induced arthritis . . . . . . . . . . . . . . . . . . . . . . . . . . 03.4. Celastrol prevents joint immune cells infiltration and proliferation as well as cartilage and bone erosions . . . . . . . . . . . . . . . . 03.5. Digoxin delayed the course and reduced the severity of arthritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03.6. Digoxin prevents proliferation but not infiltration of immune cells within joints . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 05. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0List of abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Conflict of interests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Take-home messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

1. Introduction

Rheumatoid arthritis (RA) is a chronic systemic immune-mediated inflammatory disease characterized by synovial hyperplasiacaused by a large proliferative cellular infiltrate of immune cells, highexpression of proinflammatory cytokines and consequent erosion andremodelling of joint cartilage and bone. RA management strategy hasbeen revolutionized in the last decade with the discovery of specifictreatments targeted against cytokines, such as tumor necrosis factor(TNF), and immune (B and T) cells. The current treatment goal forRA is to achieve a state of disease remission but, despite all availabletherapeutic approaches, RA remains an incurable, progressive, debili-tating and destructive disease with only 20% of patients reachingremission [1]. Moreover, these novel treatment strategies are onlyeffective in around 70% of the patients, with many of them eventuallyloosing response to the drugs or being forced to interrupt drug ad-ministration due to adverse effects. Anti-TNF treatment has beenshown to be more effective when introduced early in the diseasecourse [2,3]. However, still a relatively large proportion of patientsfail to respond in these optimal conditions.

We have previously reported increased levels of interleukin (IL)-1β in very recent onset arthritis and in the synovial fluid of estab-lished RA patients [4]. This observation may be explained by activa-tion of caspase-1, the pro-inflammatory enzyme which is activatedby the inflammasome and which is responsible for the processing ofpro-IL-1β, which we have also observed to be increased both inearly and established RA patients [4]. Interestingly, the role of theinflammasomes has been recently addressed in the context of RA. Infact, it has been reported that polymorphisms in NLRP3 and CARD8inflammasome genes are associated with anti-citrullinated proteinantibodies (ACPA)-positive RA, an increased susceptibility for the dis-ease and a worse prognosis for these patients [5–7]. Inflammasomesare activated by several different foreign and self-antigens and recentevidences suggest that these multiprotein complexes may participatein the development of the new syndrome termed ASIA, ‘Autoimmune(Auto-inflammatory) Syndrome [8], which is induced by adjuvantsand assembles a spectrum of immune-mediated diseases triggeredby an adjuvant stimulus [9,10].

The importance of IL-1β in the early phase of RA is furtherhighlighted by reports of its ability to promote the differentiation ofTh17 cells [11,12] through the induction of the transcription factorsIFR4 and RORγt expression [11]. These cells are characterized by theproduction of IL-17, a cytokine that is also up-regulated in the earlyphase of RA [13]. Interestingly, IL-17 serum levels and Th17 frequencyare decreased in Cryopyrin-associated periodic syndromes (CAPS)patients following in vivo IL-1β blockage [14]. Therefore, it is possiblethat IL-1β plays an important role in early rather than late stages ofthe disease and that pathways regulating this cytokine and TNF,such as the inflammasome/caspase-1 and NF-kB, can potentiallyconstitute promising combined therapeutic targets. Based on thisbackground and on the results of a recent drug screen performed inour laboratory for compounds that simultaneously inhibit IL-1β and

Please cite this article as: Cascão R, et al, Effective treatment of ratdoi:10.1016/j.autrev.2012.02.022

TNF secretion (Figueiredo et al., unpublished), we have identifiedcelastrol as a promising therapeutic candidate for arthritis. Celastrol,a pentacyclic-triterpene extract from Trypterigium wilfordii Hook, isused in traditional Chinese medicine and was recently shown topossess anti-tumor [15,16] and anti-inflammatory [17] effects. Ouraim in this study was to investigate whether celastrol administrationis able to attenuate inflammation in a rat model of adjuvant-inducedarthritis (AIA) and which mechanisms might be important for itsprotective effect. Moreover, since IL-1β is known to play a role inthe polarization of Th17 cells, we have also analyzed the anti-inflammatory and anti-proliferative properties of digoxin, a specificinhibitor of RORγt transcriptional activity and consequently inhibitorof Th17 cells polarization [18], in the same rat model. We found thatcelastrol, in contrast to digoxin, has significant anti-inflammatory andanti-proliferative properties and can putatively constitute an anti-inflammatory drug with therapeutic efficacy in the treatment ofimmune-mediated inflammatory diseases such as RA.

2. Methods

2.1. Compounds

Celastrol and digoxin were purchased from Sigma (Missouri, USA).

2.2. IL-1β and TNF secretion assay

THP-1 cells were stimulated with 4% PFA-fixed DH5 Escherichia coli(E. coli) at a Multiplicity of Infection (MOI) of 20 bacterial cells perTHP-1 cell, 1 hour after incubation with celastrol. Cell supernatantswere collected and IL-1β and TNF cytokines quantified by enzymelinked immunosorbent assay (ELISA) technique (R&D systems,Minnesota, USA) according to the provider's instructions.

2.3. Cell culture

THP-1 (ATCC TIB-202) macrophage-like cell line and THP-1/NF-kBreporter cell line were cultured in R10 - RPMImedia 1640 supplemen-ted with 10% (v/v) fetal bovine serum, 1% (v/v) penicillin-streptomycin, 1% (v/v) pyruvate, 1% (v/v) L-glutamine, 1% (v/v) non-essential aminoacids, 1% (v/v) hepes buffer and 2-mercaptoethanolto a final concentration of 0.05 M, as recommended by the AmericanTissue Culture Collection (ATCC). Cells were cultured at 250.000cells/mL, incubated with 10 μM of celastrol for 1 h at 37 °C 5% CO2,and then stimulated with PFA-fixed E. coli (20 E. coli per cell) for 8 hand 24 h at 37 °C 5% CO2. Simultaneously, non-stimulated negativecontrol cells were also cultured at the same density as the stimulatedpopulation for comparison. Caspase-1 activity wasmeasured in THP-1macrophage-like cell line using the Carboxyfluorescein FLICA Detec-tion kit for Caspase Assay (Immunochemistry Technologies, LLC,Minnesota, USA) following the reagent instructions. Briefly, cellsfrom the different assays were protected from light exposure whileincubated for 1 hour at 37 °C with 30X FLICA solution at a 1:30 ratio.

adjuvant-induced arthritis by celastrol, Autoimmun Rev (2012),

3R. Cascão et al. / Autoimmunity Reviews xxx (2012) xxx–xxx

NF-kB activity was measured in THP-1/NF-kB reporter cell line. Lenti-viral particles carrying a NF-kB-responsive GFP-expressing reportergene (Cignal Lenti Reporters, SABiosciences, Maryland, USA) wereused to infect THP-1 cells and to establish a stable cell line. All sampleswere analyzed by flow cytometry using a FACS Calibur (BD biosci-ences, New Jersey, USA). The data collected were further analyzedusing FlowJo software (Tree Star Inc, Oregon, USA).

2.4. Animal experimental design

Wistar AIA rats were purchased from Charles River LaboratoriesInternational (Massachusetts, USA). Female Wistar AIA rats weighing125–150 g were maintained under specific pathogen free (SPF) con-ditions and all experiments were approved by the Animal User andEthical Committees at the Instituto de Medicina Molecular, accordingto the Portuguese law and the European recommendations. Celastroland digoxin were administrated at a dose of 1 μg/g and 2 μg/g bodyweight every day, respectively [18,19]. Drugs and vehicle controlwere dissolved in normal saline solution and injected intraperitone-ally to AIA rats (N=5–10 animals per group) after 4 days (earlytreatment group) and after 11 days (late treatment group) of diseaseinduction, when arthritis was already present. The inflammatoryscore, ankle perimeter and body weight were measured during theperiod of treatment. Inflammatory signs were evaluated by countingthe score of each joint in a scale of 0–3 (0 — absence; 1 — erythema;2 — erythema and swelling; 3 — deformities and functional impair-ment) [20]. The total score of each animal was defined as the sumof the partial scores of each affected joint. Rats were sacrificedafter 19 days of disease evolution and paw samples were collectedfor histological and immunohistochemical evaluation.

2.5. Histological and immunohistochemical evaluation

For histopathological observation, paw, lung, liver, kidney and pan-creas samples were collected at the time of sacrifice. Samples werefixed immediately in 10% neutral buffered formalin solution and thendehydrated with increasing ethanol concentrations (70%, 96% and100%). Paw samples, after being fixed, were also decalcified in 10%formic acid. Samples were next embedded in paraffin, sectioned andstained with hematoxylin and eosin for morphological examination.Paws were also used for immunohistological staining with Ki67 anti-body, a cellular proliferation marker. Tissue sections were incubatedwith primary antibody against rat polyclonal Ki67 (Abcam, Cambridge,UK) andwith EnVision+ (Dako, Glostrup, Denmark). Colourwas devel-oped in solution containing diaminobenzadine-tetrahydrochloride(Sigma, Missouri, USA), 0.5% H2O2 in phosphate-buffered saline buffer(pH 7.6). Slides were counterstained with hematoxylin and mounted.All images were acquired using a Leica DM 2500 (Leica microsystems,Wetzlar, Germany) microscope equipped with a colour camera. Dataregarding the degree of proliferation of synovial cells was scored from0–3 (0 — fewer than three layers; 1 — three to four layers; 2 — five tosix layers; 3 — more than six layers). Lymphoid cell infiltration wasscored from 0–3 (0 — none to diffuse infiltration; 1 — lymphoid cellaggregate; 2— lymphoid follicles; 3— lymphoid follicles with germinalcenter formation) [21].

2.6. Intracellular IL-17 staining

Spleen cells were cultured in complete cell culture media at 37 °C5% CO2. Cells were stimulated for 3 h with brefeldin A (0.01 mg/ml)(Epicenter Technologies, Nebraska, USA), phorbol 12-myristate 13-acetate (PMA) (50 ng/ml) (Sigma, Missouri, USA) and ionomycin(500 ng/ml) (Calbiochem, Darmstadt, Germany). Next, cells werepermeabilised using saponin (Sigma, Missouri, USA) and stainedwith anti-IL17 FITC (Biolegend, California, USA) to detect intracellularIL-17. Lastly, cells were acquired with a FACS LSR Fortessa (BD

Please cite this article as: Cascão R, et al, Effective treatment of ratdoi:10.1016/j.autrev.2012.02.022

biosciences, New Jersey, USA) and data collected were further ana-lyzed using FlowJo software (Tree Star Inc, Oregon, USA).

2.7. Statistical analysis

Statistical differences were determined with non-parametricKruskal-Wallis and Mann–Whitney tests using GraphPad Prism(GraphPad, California, USA). Differences were considered statisticallysignificant for pb0.05.

3. Results

3.1. Celastrol decreases IL-1β and TNF secretion

Based on the hypothesis that drugs which block the secretion ofboth IL-1β and TNF might be particularly effective at decreasingearly disease activity in RA, we have recently used the humanTHP-1 macrophage-like cell line to screen for drugs that can simulta-neously down-regulate the secretion of both cytokines. Among the2320 tested drugs included in the Spectrum collection (MicrosourceDiscovery Systems, Connecticut, USA), we found 45 that significantlydecrease the levels of both IL-1β and TNF secretion (Figueiredo et al.,unpublished). We further narrowed the selection by taking intoaccount the possible human tolerability to chronic exposure andother biological properties that could be of interest in the contextof RA. Celastrol was thus selected for testing in a rat model ofadjuvant-induced arthritis (AIA), due to its prior human use in tradi-tional Chinese medicine and due to its concomitant alleged anti-proliferative effects [15,16]. We started by validating the effect ofcelastrol in the inhibition of IL-1β and TNF secretion in a humanTHP-1 macrophage-like cell line, by using increasing concentrationsof celastrol for 1 hour before challenging them with PFA-fixed E. colifor 6 hours. The conditioned media was then probed for the secretionof either IL-1β or TNF using ELISA technique. Celastrol was very effec-tive at inhibiting the secretion of both cytokines over a wide range oftested concentrations (Fig. 1A).

3.2. Celastrol inhibits the activation of NF-kB and caspase-1

The sequence of events culminating in IL-1β secretion is complex,but it can be summarized in two steps: induction of pro-IL-1β and itsprocessing by activated caspase-1 (reviewed in [22]). Both pro-IL-1βand TNF depend on NF-kB activation for the transcription of theirrespective mRNAs. We therefore tested the effect of celastrol onthese key pathways. To investigate its effect in the activation of NF-kB, we used an NF-kB reporter cell line made by stably infectingTHP-1 cells with a commercial lentiviral GFP reporter under the con-trol of a minimal CMV promoter and tandem repeats of the NF-kBtranscriptional response element (TRE). We found that celastrol wasable to suppress NF-kB reporter activation upon E. coli stimulationin comparison with cells that were also stimulated but did not receivetreatment (Fig. 1B). To test the effect of this drug in caspase-1 proces-sing and activation we used a caspase-1 fluorescent substrate andmeasured the relative active caspase-1 levels using FACS. Also inthis case, celastrol administration decreased the activation ofcaspase-1 (Fig. 1B). We can thus conclude that celastrol inhibits NF-kB and caspase-1 activation.

3.3. Celastrol is able to suppress inflammation in Wistar ratadjuvant-induced arthritis

To study the anti-inflammatory properties of celastrol in vivo, AIArats were treated daily with this drug after the disease had alreadybecome symptomatic. We started the treatment after 4 days of dis-ease induction (early treatment group) and after 11 days of diseaseinduction (late treatment group). The inflammatory score and ankle

adjuvant-induced arthritis by celastrol, Autoimmun Rev (2012),

Fig. 1. (A) IL-1β and TNF secretion are inhibited by celastrol treatment. Conditioned media samples from human THP-1 macrophage-like cell line cultured with growing concentra-tions of celastrol were analyzed by ELISA technique. Differences were considered statistically significant for p valuesb0.05. (B) The activation of NF-kB reporter and caspase-1 isdecreased with celastrol treatment. NF-kB expression was measured by flow cytometry in a THP-1/NF-kB reporter cell line incubated with celastrol and then stimulated for 24 hwith E. coli. Each thin line in the histogram corresponds to untreated but E. coli stimulated cells, the shaded area corresponds to drug-treated and E. coli stimulated cells and thethick line corresponds to untreated non-stimulated cells, used as a control. Caspase-1 activation was measured using flow cytometry in a THP-1 cell line incubated with celastroland then stimulated for 8 h with E. coli. Each thin line in the histogram corresponds to untreated but E. coli stimulated cells used as control and the thick line corresponds to drug-treated and E. coli stimulated cells. (C) Celastrol is able to suppress inflammation throughout time. Notice that after 6 days of treatment the vehicle injected group increased theinflammatory manifestations sharply, whereas in celastrol-treated rats there was minimal inflammatory activity or even complete abrogation of arthritis manifestations. Arrowsindicate the beginning of treatment after 4 and 11 days of disease induction. Differences were considered statistically significant for p valuesb0.05. (D) Celastrol possesses anti-inflammatory properties. Inflammation score in celastrol-treated AIA rats is maintained significantly diminished in comparison with vehicle-treated rats after treatment. Differenceswere considered statistically significant for p valuesb0.05. (E) Histological evaluation of joints after celastrol treatment. Notice that celastrol has completely prevented immunecellular infiltration and bone and cartilage invasion, allowing for a normal joint structure comparable to non-arthritic rats in both early and late treatment groups. Magnification50× and 100×. Bars: 100 μm.

4 R. Cascão et al. / Autoimmunity Reviews xxx (2012) xxx–xxx

perimeter were evaluated during the period of treatment. As shownin Fig. 1C, all animals already presented arthritis by the fourth dayof disease induction, which corresponds to the first day of treatment.After 6 days of treatment the vehicle-injected group increased the in-flammatory manifestations sharply, while in early celastrol-treatedrats there was minimal inflammatory activity or even complete abro-gation of arthritis manifestations. In the late treatment group, drugadministration was started after 11 days of disease evolution, whenanimals presented a mean inflammatory score of 5. Also in thisgroup, by the second day of treatment with celastrol the inflammato-ry manifestations started to significantly decrease over time. Thisresult shows that this drug has anti-inflammatory effects evenwhen administrated in a later phase of arthritis. After 15 (early treat-ment group) and 8 (late treatment group) days of treatment, celastrolshowed significant anti-inflammatory effects, as assessed by the eval-uation of the inflammatory score shown in Fig. 1D and also by theevaluation of ankle perimeter (p=0.007 in early and late treatmentgroups vs. untreated animals).

3.4. Celastrol prevents joint immune cells infiltration and proliferation aswell as cartilage and bone erosions

To evaluate the infiltration of immune cells within joints of AIArats, joint tissue sections stained with hematoxylin and eosin wereperformed. The histological evaluation shown in Fig. 1E revealedthat rats treated with celastrol had a normal joint structure withcomplete abrogation of the inflammatory infiltrate (pb0.0001 inearly and p=0.006 in late treatment group vs. untreated animals).We also studied cell proliferation by staining joint tissue sectionswith Ki67. The immunohistochemical results revealed that rats

Please cite this article as: Cascão R, et al, Effective treatment of ratdoi:10.1016/j.autrev.2012.02.022

treated with celastrol presented a reduced level of immune cellsproliferation in the early treatment group (p=0.0009 vs. untreatedanimals). The late treatment group showed a less effective prolifera-tion decrease (p=0.046 vs. untreated animals), even though thedrug successfully diminished the inflammatory score. Early and latetreatment with celastrol prevented cartilage and bone damage(Fig. 1E). We have not observed significant differences in bodyweight or any other side effects in treated rats, as revealed duringautopsy and histological analysis made in lung, liver, kidney andpancreas (data not shown).

3.5. Digoxin delayed the course and reduced the severity of arthritis

To study the in vivo anti-inflammatory properties of digoxin, AIArats were treated daily with this drug using the same experimentalsetup used for celastrol. As shown in Fig. 2A, after 11 days of treat-ment, we observed a delay in the course of arthritis in the earlydigoxin-treated rats, with a reduction in the severity of inflammatorysigns in comparison with the vehicle-injected group. After 19 daysof disease induction, digoxin showed significant anti-inflammatoryeffects, as assessed by the evaluation of the inflammatory score(Fig. 2B) and also by the evaluation of ankle perimeter (p=0.007 inearly vs. untreated animals). In the late treatment group, there wereno statistically significant differences in the inflammatory scorewhen compared with the vehicle-treated rats (Fig. 2B). This resultsuggests that digoxin has anti-inflammatory effects only when ad-ministrated in the early phase of arthritis. Additionally, we observedthat the percentage of IL-17-producing T cells in digoxin-treatedrats was reduced as compared to vehicle-treated rats (p=0.0286 inearly and p=0.0286 in late treatment group vs. untreated animals).

adjuvant-induced arthritis by celastrol, Autoimmun Rev (2012),

Fig. 2. (A) Digoxin is able to reduce the severity of inflammation throughout time. After 11 days of treatment, the digoxin-injected group started to progressively reduce inflammatorymanifestations. Arrow indicates the beginning of treatment after 4 and 11 days of disease induction. (B) Digoxin possesses anti-inflammatory properties. Inflammation score in earlydigoxin-treated AIA rats is significantly diminished in comparison with vehicle-treated rats after treatment. Of note, when treatment begins in the later phase of inflammation it hasno effect in reducing the inflammatory process. Differences were considered statistically significant for p valuesb0.05. (C) Histological and immunohistochemical evaluation of jointsafter 15 days of treatment. Notice that digoxin reduced immune cellular proliferation only if treatment administration started in the early phase of arthritis but had no effect inimmune cell infiltration within joints. Magnification 50× and 100× in histological images and a magnification 200× in immunohistochemical images. Bars: 100 μm.

5R. Cascão et al. / Autoimmunity Reviews xxx (2012) xxx–xxx

3.6. Digoxin prevents proliferation but not infiltration of immune cellswithin joints

The histological evaluation shown in Fig. 2C revealed that digoxinwas not able to suppress the infiltration of immune cells within joints(p=0.1201 in early and p=0.3475 in late treatment group vs.untreated animals). Furthermore, the immunohistochemical resultsrevealed that rats treated with digoxin presented a reduced level ofimmune cell proliferation in the early treatment group (p=0.0042vs. untreated animals), in contrast with the late treatment groupwhich showed no effect in immune cell proliferation (p=0.4100 vs.untreated animals). Minimal cartilage and bone damage was presentboth in early and late digoxin treated animals (Fig. 2C). Also in thecase of digoxin, we have not observed significant differences inbody weight or any other side effects in treated rats, as revealedduring autopsy and histological results (data not shown).

4. Discussion

In the present study, we demonstrated that AIA can be effectivelytreated through a possible inhibitory effect over IL-1β and TNF secre-tion induced by celastrol. The effect of this compound was profoundas it induced a complete abrogation of joint immune cellular infiltra-tion and proliferation, preventing cartilage and bone damage.

Celastrol is a novel compound that has been shown to inhibitcancer progression and NF-kB activity [15,16,23]. Our results revealthat the anti-inflammatory properties of this drug might not only berelated with its ability to inhibit the activation of NF-kB but alsowith its capacity to inhibit caspase-1 activation. Celastrol has also beenreported to abrogate the release of IL-1β in LPS-stimulated humanperipheral mononuclear cells [24] and to exert anti-inflammatory prop-erties in animal models [17,25]. Interestingly, Pinna et al. described thatcelastrol inhibited pro-inflammatory cytokine secretion from mucosal

Please cite this article as: Cascão R, et al, Effective treatment of ratdoi:10.1016/j.autrev.2012.02.022

inflammatory biopsies from Crohn's patients, possibly due to the abro-gation of cytokine gene transcription [26]. Of note, tripterine isolatedfrom Tripterygium wilfordii Hook F has previously been shown to beeffective on adjuvant [27] and collagen-induced arthritis [25] in rats,supporting the 2002 report showing that an ethanol/ethyl acetateextract of Tripterygium wilfordii Hook F shows therapeutic benefit inpatients with refractory RA [28]. Furthermore, in an in vivo model ofmetastatic bone disease associated with breast cancer, celastrol inhib-ited bone resorption, consistent with the inhibitory effect on osteoclastformation and survival observed in in vitro experiments [19]. Of interest,these data support our findings that celastrol suppresses synovialimmune cells infiltration and proliferation, preventing bone erosions.Importantly, celastrol treatment is effective when administrated bothin the early and more established phase of arthritis which is relevantfor the possible clinical implications of our findings. In RA the infiltrationof immune cells and the proliferation of joint lining synovial fibroblastslead to the formation of the tumor-like pannus tissue, which invadesand destroys joint cartilage and bone. The cell proliferation inhibitoryeffect of celastrol may thus prove to be of interest to prevent and treatthis complication of established RA.

Additionally, we also found that digoxin is able to ameliorateinflammatory signs in the same AIA rat model of arthritis. This is inagreement with the recent report from Huh et al. in which it wasshown that digoxin was able to delay the onset and reduce diseaseseverity in an experimental autoimmune encephalomyelitis (EAE)mice model, through the inhibition of RORγt transcriptional activityand, consequently, of Th17 cells differentiation [18]. However, despiteour observation that digoxin was able to suppress the severity ofinflammatory signs, we also found that it was not able to efficientlyreduce the infiltration of immune cells within the joints. Importantly,we observed that digoxin treatment was only effective if the drug wasadministrated in the early phase of arthritis development, in contrastto what we found in the case of celastrol. In fact, blocking IL-1β and

adjuvant-induced arthritis by celastrol, Autoimmun Rev (2012),

6 R. Cascão et al. / Autoimmunity Reviews xxx (2012) xxx–xxx

TNF simultaneously results in a significant inhibitory effect in arthritisprogression and severity, even when administrated in a later phaseof disease course, with a complete abrogation of the inflammatoryscore, infiltration and proliferation of immune cells within jointsand prevention of structural damage. In contrast, we observed thatdigoxin had a slower and less efficient effect on disease progression.These data indicate that IL-1β, in the context of arthritis, might playa role independent from Th17/IL-17. Besides inducing Th17 cell polar-ization, IL-1β also directly stimulates the influx of neutrophils andmacrophages into the damaged site. These cells in turn can destroythe tissue by the release of proteases and reactive oxygen species[29], and also by the formation of osteoclasts [30] leading to tissuedamage and consequent functional disability characteristic of RA pa-tients. IL-1β, together with IL-6 and TNF, also has a potent capacityto induce the receptor activator of nuclear factor kappa-B ligand(RANKL) expression on synovial fibroblasts/osteoblasts and to facili-tate RANK signaling, thus directly contributing to the bone destruc-tion process. In contrast, IL-17 seems to have a more limited effecton inflammatory cell influx and consequent inflammatory symptoms,as we observed in this study. Moreover, in agreement with this fact, arecent phase II study testing an anti-IL-17A drug in RA did not achieveits primary end point [31]. This does not preclude the important in-volvement of Th17 cells in driving the innate immune inflammationtowards the adaptive (auto)immune chronic inflammation in RA,involving several other cytokines apart from IL-17A [32].

Despite this apparent crucial role of IL-1β signaling in RA, clinicalbenefits after IL-1β inhibition have been modest compared to anti-TNF drugs, at least in moderate to severe long established RA. Further,in 2004 a study which tested the efficacy of combination therapy

Fig. 3. Proposed mechanism for celastrol anti-inflammatory effects. Celastrol showed anti-inof arthritis development and abrogation of joint immune cellular infiltration and proliferatiof caspase-1 and NF-kB activation and, consequently, lead to a decrease in IL-1β and TNF scaspase recruitment domain, CARD — caspase recruitment domain, DAMP — danger associkappa-light-chain-enhancer of activated B cells, NLRP — NOD-like receptor protein, PAMP —

TNF — tumor necrosis factor.

Please cite this article as: Cascão R, et al, Effective treatment of ratdoi:10.1016/j.autrev.2012.02.022

using anakinra and etanercept in 244 long-standing and very activeRA patients who have been treated unsuccessfully with MTX showedthat concomitant IL-1β and TNF inhibition provides no added benefitand increased infections as compared to etanercept alone [33]. Possi-bly, in the context of RA inhibiting the IL-1β pathway at the receptorlevel is not an effective strategy but an upstream inhibition mightwork better, at least, in animal models. On top of that, downregulat-ing TNF and IL-1β production might be safer than inhibitingcompletely TNF and IL-1β.

5. Conclusions

In conclusion, celastrol can putatively constitute an anti-inflammatoryand anti-proliferative drug with therapeutic efficacy in the treatmentof immune-mediated inflammatory diseases such as RA, possibly throughthe down-regulation of caspase-1 and inhibition of NF-kB activation(Fig. 3). Its anti-proliferative effects might be of additional value in RAto counteract the formation of the characteristic pannus leading to thedestruction of cartilage and bone. In addition, we have shown thatdespite the ability to decrease the severity of early inflammatory signsin AIA, digoxin is not effective in reducing the infiltration of immunecells within the joints. We thus suggest that the isolated inhibition ofTh17 polarization might be a strategy with limited efficacy, at least inestablished RA. Further animal experimentation is required to deter-mine the real efficacy and safety of celastrol for arthritis treatment butthese results suggest that it might be worth of entering into phase Iclinical trials. Simultaneously, this study highlights the need for moreresearch on the role of the inflammasome/caspase-1 and NF-kB path-ways in the etiopathology of RA.

flammatory and anti-proliferative properties in vivo, promoting a complete suppressionon, preventing cartilage and bone damage. This compound induced a down-regulationecretion. ASC — adaptor molecule apoptosis associated speck-like protein containing aated molecular pattern, IL — interleukin, IL-1R — IL-1 receptor, NF-kB — nuclear factorpathogen associated molecular pattern, PYR — pyrin domain, TLR — toll-like receptor,

adjuvant-induced arthritis by celastrol, Autoimmun Rev (2012),

7R. Cascão et al. / Autoimmunity Reviews xxx (2012) xxx–xxx

List of abbreviations

RA rheumatoid arthritisTNF tumor necrosis factorIL interleukinACPA anti-citrullinated protein antibodiesAIA adjuvant-induced arthritisCAPS cryopyrin-associated periodic syndromesMOI multiplicity of infectionELISA enzyme linked immunosorbent assaySPF specific pathogen freeTRE transcriptional response elementEAE experimental autoimmune encephalomyelitisRANKL receptor activator of nuclear factor kappa-B ligand

Conflict of interests

The authors declare that they have no competing interests.

Funding

This work was supported by a grant (SFRH/BD/40513/2007)from Fundação para a Ciência e a Tecnologia (FCT). Work in LuisMoita's laboratory is supported by FCT (PIC/IC/82991/2007 andPTDC/SAU-MII/100780/2008) and Fundação Luso-Americana para oDesenvolvimento (FLAD). The Gulbenkian Programme for AdvancedMedical Education was sponsored by Fundação Calouste Gulbenkian,Fundação Champalimaud, Ministério da Saúde e FCT.

Take-home messages

• Celastrol has significant anti-inflammatory and anti-proliferativeproperties.

• Digoxin is not as effective as celastrol treatment for AIA.• Blocking IL-1β and TNF (upstream to their receptors) is effective inarthritis.

• Therapies targeting IL-1β pathway should be re-evaluated in RApatients.

Acknowledgements

The authors would like to acknowledge Ana Lopes for technicalassistance and Ana Luisa Caetano for technical support with FACS.

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adjuvant-induced arthritis by celastrol, Autoimmun Rev (2012),

Results

- 47 -

Gambogic acid is a potent anti-inflammatory

and anti-proliferative drug in a rat model of

adjuvant-induced arthritis

Results

- 48 -

1

GAMBOGIC ACID IS A POTENT ANTI-INFLAMMATORY AND 1

ANTI-PROLIFERATIVE DRUG IN A RAT MODEL OF ADJUVANT-2

INDUCED ARTHRITIS 3

4

R. Cascãoa, B. Vidal

a, H. Raquel

a, A. Neves-Costa

a, N. Figueiredo

a,b, V. Gupta

c, J. E. 5

Fonsecaa,d*

, L. F. Moitaa*

6

7

aInstituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, 8

Lisbon, Portugal. 9

bGulbenkian Programme for Advanced Medical Education, Lisbon, Portugal. 10

cDivision of Nephrology and Hypertension, Department of Medicine, University of 11

Miami, Miami, FL 33136, U.S.A. 12

dRheumatology Department, Centro Hospitalar de Lisboa Norte, EPE, Hospital de 13

Santa Maria, Lisbon, Portugal. 14

* Joint senior authors. 15

16

Corresponding author: 17

Luis Ferreira Moita 18

Cell Biology of the Immune System Unit 19

Instituto de Medicina Molecular 20

Edifício Egas Moniz 21

Faculdade de Medicina da Universidade de Lisboa 22

Av. Professor Egas Moniz 23

1649-028 Lisbon 24

Tel: (+351) 217999544 25

2

Fax: (+351) 217999459 26

Email: lmoita@fm.ul.pt 27

28

ABSTRACT 29

Background: In a previous study, we have reported a continuous activation of caspase-30

1 in rheumatoid arthritis (RA) patients in the early phase of the disease, as well as 31

increased levels of interleukin (IL)-1. These observations raised the hypothesis that 32

drugs targeting IL-1 regulatory pathways, as well as tumor necrosis factor (TNF), may 33

be particularly effective for the treatment of early RA. We have recently identified 34

gambogic acid as one of the most promising therapeutic candidates to simultaneously 35

block the secretion of IL-1 and TNF after screening ~2320 compounds, using a THP1 36

macrophage-like cell line challenged with E. coli. Our main goal here is to investigate 37

whether administration of gambogic acid is able to attenuate inflammation in a rat 38

model of adjuvant-induced arthritis (AIA). Methodology/Principle Findings: 39

Gambogic acid was administered to AIA rats in the early phase of arthritis (4 days after 40

disease induction) for a period of 15 days. The inflammatory score, ankle perimeter and 41

body weight were evaluated during the time of treatment. Rats were sacrificed after 19 42

days of disease progression and paw samples were collected for histological and 43

immunohistochemical evaluation. We found that inflammation in joints was 44

significantly suppressed with administration of gambogic acid. Histological and 45

immunohistochemical evaluation of treated rats revealed normal joint structures with 46

complete abrogation of the inflammatory infiltrate and cellular proliferation. 47

Conclusions/Significance: Our results suggest that gambogic acid has significant anti-48

inflammatory properties and can putatively constitute an anti-inflammatory drug with 49

3

therapeutic efficacy in the treatment of inflammatory diseases such as RA, possibly 50

through the down-regulation of IL-1β and TNF secretion. 51

52

INTRODUCTION 53

Rheumatoid arthritis (RA), which afflicts about 1% of the world population, is the most 54

common of the inflammatory joint diseases. This disease can have a very aggressive 55

course and poor outcome as inferred by the analysis of its social impact (after 10 years, 56

more than 50% of the RA patients are too impaired to perform professional activities) 57

[1] and life expectancy diminishes 10 years due to disease activity and associated 58

comorbidities [2]. RA is a chronic systemic inflammatory disease characterized by 59

synovial hyperplasia caused by a large proliferative cellular infiltrate of leukocytes, high 60

expression levels of proinflammatory cytokines and consequent erosion of joint 61

cartilage and bone. The therapeutic approach of RA has been revolutionized in the last 62

decade with the discovery of specific targeted treatments. However, despite all available 63

therapeutic options, RA remains a progressive, destructive and debilitating disease with 64

only 20% of patients reaching remission [3]. Anakinra, an antagonist of interleukin 65

(IL)-1, was approved for RA treatment in the last decade. However, the real impact on 66

disease activity has been shown in practice to be lower than what was anticipated from 67

clinical trial results, casting doubts on the role of IL-1β as a therapeutic target [4]. 68

Nonetheless, we have previously reported increased levels of IL-1β in very recent onset 69

arthritis and in the synovial fluid of established RA patients [5]. This observation could 70

be explained by the activation of caspase-1 that we also have observed both in early and 71

established RA patients [6]. Therefore, it is possible that IL-1β plays an important role 72

in early rather than late stages of the disease and that pathways regulating this cytokine, 73

such as caspase-1 and NF-kB activation, can potentially constitute promising 74

4

therapeutic targets for specific drugs. The effect might be further boosted if an 75

inhibitory effect on tumor necrosis factor (TNF) can also be achieved. Based on the 76

results of a recent drug screen for compounds that simultaneously inhibit IL-1β and 77

TNF secretion (Figueiredo et al., unpublished), we chose gambogic acid as a promising 78

therapeutic candidate for the treatment of arthritis. Gambogic acid is a polyprenylated 79

xanthone abundant in resin derived from Garcinia hanburyi and G. Morella and is used 80

in Southeast Asia complementary and alternative medicine [7]. Recent studies showed 81

that gambogic acid could inhibit the growth of a wide range of tumor cells [8]. Our aim 82

in this study is to investigate whether gambogic acid administration is able to attenuate 83

inflammation in a rat model of adjuvant-induced arthritis (AIA). 84

85

METHODS 86

Ethics statement 87

All experiments were approved by the Animal User and Ethical Committees at the 88

Instituto de Medicina Molecular, according to the Portuguese law and the European 89

recommendations. 90

91

Compound 92

Gambogic acid was purchased from Santa Cruz Biotechnology (Santa Cruz, USA). 93

94

IL-1 and TNF quantification 95

THP-1 cells were stimulated with 4% PFA-fixed DH5 Escherichia coli (E. coli) at a 96

Multiplicity of Infection (MOI) of 20 bacterial cells per THP-1 cell, 1 hour after 97

incubation with gambogic acid. Cell supernatants were collected and IL-1β and TNF 98

5

cytokines quantified by enzyme linked immunosorbent assay (ELISA) (R&D systems, 99

USA) according to the provider's instructions. 100

101

AIA rat model and assessment of arthritis 102

Female Wistar AIA rats were purchased from Charles River Laboratories International 103

(Massachusetts, USA) and maintained under specific pathogen free (SPF) conditions. 104

Gambogic acid was administrated at a dose of 4µg/g body weight every day. Drug and 105

vehicle control were dissolved in normal saline solution and injected intraperitoneally to 106

AIA rats (N=5-10 animals per group) after 4 days of disease induction, when arthritis 107

was already present, and then for a period of 15 days. The inflammatory score, ankle 108

perimeter and body weight were measured during the time of treatment. Inflammatory 109

signs were evaluated trough the counting of the score of each joint in a scale of 0‐3 (0 – 110

absence; 1 – erythema; 2 – erythema and swelling; 3 – deformities and functional 111

impairment). The total score of each animal was defined as the sum of the partial scores 112

of each affected joint [9]. Rats were sacrificed after 19 days of disease evolution and 113

paw samples were collected for histological and immunohistochemical evaluation. 114

115

Histology and Immunohistochemistry 116

For histopathological observation, paws, lungs, livers, kidneys and pancreas samples 117

were collected at the time of sacrifice. Samples were fixed immediately in 10% neutral 118

buffered formalin solution and then dehydrated using increased ethanol concentrations 119

(70%, 96% and 100%). Paw samples, after being fixed, were also decalcified in 10% 120

formic acid. Samples were next embedded in paraffin, sectioned and stained with 121

hematoxylin and eosin for morphological examination. Paws were also used for 122

immunohistochemical staining with Ki67 antibody, a cellular proliferation marker. 123

6

Tissue sections were incubated with primary antibody against rat polyclonal Ki67 124

(Abcam, UK) and with EnVision+ (Dako, Denmark). Colour was developed in solution 125

containing diaminobenzadine-tetrahydrochloride (Sigma, USA), 0.5% H2O2 in 126

phosphate-buffered saline buffer (pH 7.6). Slides were counterstained with hematoxylin 127

and mounted. All images were acquired using a Leica DM 2500 (Leica microsystems, 128

Germany) microscope equipped with a colour camera. Data regarding the degree of 129

proliferation of synovial cells was scored from 0-3 (0 – fewer than three layers; 1 – 130

three to four layers; 2 – five to six layers; 3 – more than six layers). Lymphoid cell 131

infiltration was scored from 0-3 (0 – none to diffuse infiltration; 1 – lymphoid cell 132

aggregate; 2 – lymphoid follicles; 3 – lymphoid follicles with germinal center 133

formation) [10]. 134

135

Caspase-1 and NF-kB assay 136

THP1 (ATCC TIB-202) macrophage-like cell line and THP1/NF-kB reporter cell line 137

were cultured in R10 - RPMI media 1640 supplemented with 10% (v/v) fetal bovine 138

serum, 1% (v/v) penicillin-streptomycin, 1% (v/v) pyruvate, 1% (v/v) L-glutamine, 1% 139

(v/v) non-essential aminoacids, 1% (v/v) hepes buffer and 2-mercaptoethanol to a final 140

concentration of 0.05M, as recommended by the American Tissue Culture Collection 141

(ATCC). Cells were cultured at 250.000 cells/mL, incubated with 10µM of gambogic 142

acid for 1h at 37ºC 5% CO2, and then stimulated with PFA-fixed E. coli (20 E. coli per 143

cell) for 8h and 24h at 37ºC 5% CO2. Simultaneously, non-stimulated negative control 144

cells were also cultured at the same density as the stimulated population for comparison. 145

Caspase-1 activity was measured in THP1 macrophage-like cell line using the 146

Carboxyfluorescein FLICA Detection kit for Caspase Assay (Immunochemistry 147

Technologies, LLC) following the reagent instructions. Briefly, cells from the different 148

7

assays were protected from light exposure while incubated for 1 hour at 37ºC with 30X 149

FLICA solution at a 1:30 ratio. NF-kB activity was measured in THP1/NF-kB reporter 150

cell line. Lentiviral particles carrying a NF-kB-responsive GFP-expressing reporter gene 151

(Cignal Lenti Reporters, SABiosciences, USA) were used to infect THP-1 cells and to 152

establish a stable cell line. All samples were analyzed by flow cytometry using a FACS 153

Calibur (BD biosciences, USA). The data collected were further analyzed using FlowJo 154

software (Tree Star Inc, USA). 155

156

Statistical analysis 157

Statistical differences were determined with non-parametric Kruskal-Wallis and Mann-158

Whitney tests using GraphPad Prism (GraphPad, USA). Differences were considered 159

statistically significant for p < 0.05. 160

161

RESULTS 162

Gambogic acid reduces IL-1β and TNF production 163

To study the effect of this drug in the inhibition of IL-1β and TNF secretion we treated 164

the human THP-1 macrophage-like cell line with growing concentrations of gambogic 165

acid for 1 hour before challenging them with PFA-fixed E. coli for 6 hours. The 166

conditioned media was then probed for the secretion of either IL-1β or TNF using 167

ELISA. Gambogic acid significantly inhibited the secretion of both cytokines over a 168

wide range of concentrations (Fig. 1), confirming the previously reported effect of 169

gambogic acid in blocking the secretion of these cytokines [11] and validating our 170

earlier finding (Figueiredo et al., unpublished). 171

172

8

173

Figure 1. IL-1β and TNF secretion are inhibited by gambogic acid treatment. Media 174

samples from human THP-1 macrophage-like cell line cultured with growing 175

concentrations of gambogic acid were analyzed by ELISA technique. Differences were 176

considered statistically significant for p values < 0.05. 177

178

Gambogic acid inhibits the activation of NF-kB and Caspase-1 179

Pro-IL-1 and TNF both depend on NF-kB activation for the transcription of their 180

respective mRNAs. Pro-IL-1 processing is further dependent on the activation of 181

caspase-1. We therefore tested the effect of gambogic acid on these key pathways. To 182

investigate the effect of this drug in the activation of NF-kB, we used an NF-kB reporter 183

cell line created by stably infecting THP-1 cells with a commercial lentiviral GFP 184

reporter under the control of a minimal CMV promoter and tandem repeats of the NF-185

kB transcriptional response element (TRE). Gambogic acid was able to suppress NF-kB 186

reporter activation upon E. coli stimulation in comparison with cells that were also 187

stimulated but did not receive treatment (Fig. 2A). To test the effect of this drug in 188

caspase-1 processing and activation we used a caspase-1 fluorescent substrate, and 189

measured relative active caspase-1 levels using FACS. Also in this case, gambogic acid 190

significantly decreased the activation of caspase-1 (Fig. 2B). 191

192

9

193

Figure 2. (A) NF-kB reporter activation is suppressed by gambogic acid treatment. NF-194

kB expression was measured by flow cytometry in a THP-1/NF-kB reporter cell line 195

incubated with gambogic acid and then stimulated for 24h with E. coli. Each thin line in 196

the histogram corresponds to untreated but E. coli stimulated cells, the shaded area 197

corresponds to drug-treated and E. coli stimulated cells and the thick line corresponds to 198

untreated non-stimulated cells as a control. Caspase-1 activation is decreased with 199

gambogic acid treatment. (B) Caspase-1 activation was measured using flow cytometry 200

in a THP-1 cell line incubated with gambogic acid and then stimulated for 8h with E. 201

coli. Each thin line in the histogram corresponds to untreated but E. coli stimulated cells 202

used as control and the thick line corresponds to drug-treated and E. coli stimulated 203

cells. 204

205

Gambogic acid is able to suppress inflammation in Wistar rat adjuvant-induced 206

arthritis 207

To study the in vivo anti-inflammatory properties of gambogic acid, AIA rats were 208

treated daily with this drug after disease had already started to be symptomatic and for a 209

period of 15 days. The inflammatory score and ankle perimeter were evaluated during 210

the period of treatment. As shown in Fig. 3, by the 4th

day all induced animals already 211

presented arthritis. All induced animals received either vehicle or gambogic acid at that 212

time point. After 6 days of treatment the vehicle injected group increased sharply the 213

inflammatory manifestations, whereas in gambogic acid-treated rats there was minimal 214

10

inflammatory activity or even complete abrogation of arthritis manifestations. After 15 215

days of treatment, gambogic acid showed an anti-inflammatory effect as assessed by the 216

evaluation of the inflammatory score (Fig. 4) and ankle perimeter (p=0.007 vs untreated 217

animals). 218

219

220

Figure 3. Gambogic acid is able to suppress inflammation throughout time. After 6 221

days of treatment the vehicle injected group increased inflammatory manifestations, 222

whereas in gambogic acid-treated rats there was a significantly reduction in the 223

inflammatory activity. Arrow indicates the beginning of treatment after 4 of disease 224

induction. Differences were considered statistically significant for p values < 0.05. 225

226

227

228

229

11

230

Figure 4. Gambogic acid possess anti-inflammatory properties. Inflammation score in 231

gambogic acid-treated AIA rats is significantly diminished in comparison with vehicle-232

treated rats after treatment. Differences were considered statistically significant for p 233

values < 0.05. 234

235

Gambogic acid prevents joint inflammatory infiltration and proliferation 236

To evaluate the infiltration of immune cells within joints in AIA rats, joint tissue 237

sections were stained with hematoxylin and eosin. The histological evaluation shown in 238

Fig. 5 revealed that rats treated with gambogic acid had a normal joint structure with 239

complete abrogation of the inflammatory infiltrate (p<0.0001 vs untreated animals). In 240

contrast, vehicle-treated rats exhibited infiltration of inflammatory cells, bone invasion 241

and erosions (Fig. 5). We have not observed significant differences in body weight or 242

any other side effects in treated rats, as revealed during autopsy and histological 243

analysis made in lung, liver, kidney and pancreas (data not shown). We also studied the 244

levels of proliferation of immune cells, by staining joint tissue sections with Ki67. The 245

immunohistochemical results revealed that rats treated with gambogic acid presented 246

reduced proliferation of immune cells within joints (p=0.0098 vs untreated animals). 247

248

12

249

Figure 5. Histological and immunohistochemical evaluation of joints after 15 days of 250

treatment. Non-arthritic control (A), vehicle-treated (B) and gambogic acid-treated (C) 251

AIA rats (magnification 50x and 100x in histological images and a magnification 200x 252

in immunohistochemical images). Notice that gambogic acid has completely prevented 253

immune cellular infiltration, proliferation and bone invasion. 254

255

DISCUSSION 256

Our results demonstrated that treatment with gambogic acid protected Wistar AIA rats 257

from arthritis development with a complete abrogation of joint immune cellular 258

infiltration and proliferation, preventing cartilage and bone damage. 259

Previous reports have already demonstrated that gambogic acid can inhibit the growth 260

of a wide variety of tumor cell lines, possibly due to its ability to induce apoptosis [12] 261

via the transferrin receptor (TfR1) [13]. Additionally, recent data have shown that this 262

drug can inhibit NF-kB signalling pathway in human leukemia cancer cells [8] and in a 263

non-cancerigenous macrophagic cell line [14] also via TfR1. Therefore, the anti-264

inflammatory effects of gambogic acid appear to be mediated by the inhibition of NF-265

13

kB activation pathway which in turn leads to the silencing of most of the inflammatory 266

genes. In our study we demonstrated that the anti-inflammatory properties of this drug 267

in AIA rats might not only be related with its ability to suppress the activation of NF-kB 268

but also to its effect on inhibiting caspase-1 activation. Importantly, in RA the inflamed 269

synovium expands into and destroys the underlying cartilage and bone, resulting in 270

irreversible erosion of the bone and in the loss of normal joint architecture leading to 271

disability [15]. The inhibitory effect of gambogic acid in cellular proliferation can thus 272

prove relevant for the management of RA course. 273

In conclusion, gambogic acid constitutes an effective drug in a rat model of RA, 274

possibly by its ability to down-regulate caspase-1 and NF-kB activation and by blocking 275

synovial hyperplasia due to its anti-proliferative properties. Further animal 276

experimentation is required to explore the safety of this compound for the treatment of 277

inflammatory diseases, such as RA, but these results hold the promise that gambogic 278

acid could be worth of entering into phase I clinical trials. 279

280

Acknowledgements 281

The authors would like to acknowledge Ana Lopes for technical assistance and Ana 282

Luisa Caetano for technical support with FACS. 283

284

Financial Disclosure 285

This work was supported by a grant (SFRH/BD/40513/2007) from Fundação para a 286

Ciência e a Tecnologia (FCT) and by an unrestricted research grant from Pfizer. Work 287

in Luis Moita’s laboratory is supported by FCT (PIC/IC/82991/2007 and PTDC/SAU-288

MII/100780/2008) and Fundação Luso-Americana para o Desenvolvimento (FLAD). 289

14

The funders had no role in study design, data collection and analysis, decision to 290

publish, or preparation of the manuscript. 291

292

Authors’ contributions 293

RC performed all the laboratorial work, data collection, statistical analysis and writing 294

of the paper. BV contributed in animal manipulation, histological and 295

immunohistochemical experiments. HR, ANC and NF participated in some laboratory 296

experiments. VG contributed with the drugs used in the primary screening. LFM and 297

JEF as senior authors conceived the study, participated in its design and coordination, 298

and contributed in the draft of the manuscript with important intellectual input. All 299

authors read and approved the final manuscript. 300

301

REFERENCES 302

1. Yelin E, Callahan LF (1995) The economic cost and social and psychological impact 303

of musculoskeletal conditions. National Arthritis Data Work Groups. Arthritis 304

Rheum 38: 1351-1362. 305

2. Radner H, Smolen JS, Aletaha D (2010) Impact of comorbidity on physical function 306

in patients with rheumatoid arthritis. Ann Rheum Dis 69: 536-541. 307

3. Lindqvist E, Saxne T, Geborek P, Eberhardt K (2002) Ten year outcome in a cohort 308

of patients with early rheumatoid arthritis: health status, disease process, and 309

damage. Ann Rheum Dis 61: 1055-1059. 310

4. Goldbach-Mansky R (2009) Blocking interleukin-1 in rheumatic diseases. Ann N Y 311

Acad Sci 1182: 111-123. 312

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5. Cascao R, Moura RA, Perpetuo I, Canhao H, Vieira-Sousa E, et al. (2010) 313

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untreated early rheumatoid arthritis. Arthritis Res Ther 12: R196. 315

6. Rita Cascão, Joaquim Polido-Pereira, Helena Canhão, Ana M. Rodrigues, Márcio 316

Navalho, et al. (2011) Caspase-1 is active since the early phase of Rheumatoid 317

arthritis. Clin Exp Rheumatol in press. 318

7. Han QB, Cheung S, Tai J, Qiao CF, Song JZ, et al. (2005) Stability and cytotoxicity 319

of gambogic acid and its derivative, gambogoic acid. Biol Pharm Bull 28: 2335-320

2337. 321

8. Pandey MK, Sung B, Ahn KS, Kunnumakkara AB, Chaturvedi MM, et al. (2007) 322

Gambogic acid, a novel ligand for transferrin receptor, potentiates TNF-induced 323

apoptosis through modulation of the nuclear factor-kappaB signaling pathway. 324

Blood 110: 3517-3525. 325

9. da Silva JA, Fonseca JE, Graca L, Moita L, Carmo-Fonseca M (1996) Reinnervation 326

of post-arthritic joints in the rat. Clin Exp Rheumatol 14: 43-51. 327

10. Tsubaki T, Arita N, Kawakami T, Shiratsuchi T, Yamamoto H, et al. (2005) 328

Characterization of histopathology and gene-expression profiles of synovitis in 329

early rheumatoid arthritis using targeted biopsy specimens. Arthritis Res Ther 7: 330

R825-836. 331

11. Zhang L, Yi Y, Chen J, Sun Y, Guo Q, et al. Gambogic acid inhibits Hsp90 and 332

deregulates TNF-alpha/NF-kappaB in HeLa cells. Biochem Biophys Res 333

Commun 403: 282-287. 334

12. Zhang HZ, Kasibhatla S, Wang Y, Herich J, Guastella J, et al. (2004) Discovery, 335

characterization and SAR of gambogic acid as a potent apoptosis inducer by a 336

HTS assay. Bioorg Med Chem 12: 309-317. 337

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13. Kasibhatla S, Jessen KA, Maliartchouk S, Wang JY, English NM, et al. (2005) A 338

role for transferrin receptor in triggering apoptosis when targeted with gambogic 339

acid. Proc Natl Acad Sci U S A 102: 12095-12100. 340

14. Palempalli UD, Gandhi U, Kalantari P, Vunta H, Arner RJ, et al. (2009) Gambogic 341

acid covalently modifies IkappaB kinase-beta subunit to mediate suppression of 342

lipopolysaccharide-induced activation of NF-kappaB in macrophages. Biochem 343

J 419: 401-409. 344

15. Tak PP, Bresnihan B (2000) The pathogenesis and prevention of joint damage in 345

rheumatoid arthritis: advances from synovial biopsy and tissue analysis. 346

Arthritis Rheum 43: 2619-2633. 347

348

349

350

Chapter IV

Discussion

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Discussion

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Rheumatoid arthritis is a chronic immune-mediated inflammatory disease that is

mainly characterized by symmetrical synovitis of the small joints of hands and feet. The

migration and infiltration of leukocytes towards the synovium is crucial for the

establishment of the chronic inflammatory process of the disease 241-243. These multi-

regulated mechanisms involve interactions with endothelial cells through cell adhesion

molecules and complex cytokine networks.

Significant efforts have been made during the last 20 years to identify the cytokine

profile of rheumatoid arthritis. In established rheumatoid arthritis, it has already been

reported that IL-1β, IL-6, IL-8, IL-17A and TNF levels are elevated in the serum of

patients and that these levels are correlated with disease activity 244-246. Nevertheless,

the knowledge about the relative influence of different cytokine pathways on disease

onset remains scarce. The characterization of the cytokine profile at the early stage of

rheumatoid arthritis may lead to the identification of early key players, thus extending

our knowledge in the disease physiopathology and highlighting new potential treatment

targets for early disease.

Accordingly, the first goal and one of the major novelties of the present study was

the analysis of the early cytokine and chemokine environment in the first few weeks of

rheumatoid arthritis onset. For this purpose, a cohort of untreated polyarthritis patients

with less than six weeks of disease duration was followed up, allowing the identification

of a subset of patients that later on evolved into rheumatoid arthritis, defined as very

early rheumatoid arthritis (VERA) patients. The remaining patients either have self-

limited forms of arthritis or evolved into other chronic inflammatory joint diseases, being

classified as very early arthritis (VEA) patients. Clinical and laboratorial evaluations of

VERA patients were performed at baseline without therapy, after short-term treatment

with low-dose corticosteroids and after 4 months of MTX. The panel of cytokines

selected to be studied in VERA patients was selected to include all the major known

rheumatoid arthritis pathogenic players, which encompassed complex cytokine-driven

interactions between resident synovial and infiltrating inflammatory cells, as illustrated

in Figure 1.

Importantly, it was found that VERA patients have increased circulating levels of

cytokines related with the recruitment (IL-6), maturation and survival (a proliferation-

inducing ligand, APRIL and BLyS) of B cells, as well as a cytokine profile that supports

neutrophil recruitment (IL-8) and Th17 cells polarization (IL-1β and IL-6) and activation

(IL-17A and IL-22) when compared with VEA and established rheumatoid arthritis

patients under MTX treatment (Figure 5). Furthermore, the analysis of synovial fluid

samples from established rheumatoid arthritis patients, representative of the

Discussion

- 52 -

inflammatory process that occurs locally in the joints, also revealed a similar cytokine

pattern. Considering the cytokine profile observed in the very early phase of the

disease, it is tempting to consider that after an initial unknown triggering event,

macrophages and neutrophils are attracted to the joints and are activated by monocyte

chemotactic protein (MCP)-1 and IL-8 chemotactic gradients, respectively 247, 248. In

fact, previous results demonstrated that there is a delay in the apoptosis of circulating

neutrophils in VERA patients 34 and that these cells heavily infiltrate the synovial tissue

during disease onset 23. Hence, neutrophils together with macrophages are the most

abundant cells infiltrated into the joints of rheumatoid arthritis patients. Notably,

activated macrophages and fibroblasts are able to produce APRIL, which affects the

class-switch recombination process 249, 250 and induces plasma cell differentiation 249-253,

possibly explaining the maintenance of autoreactive B cells in joints 254. Subsequent

steps in rheumatoid arthritis progression could include the production by autoreactive B

cells of autoantibodies and immune complexes that deposit in joints and activate Fcγ-

receptors expressed by macrophages 255-257, further increasing the inflammatory

process through the production of proinflammatory cytokines, such as TNF and IL-1β

by macrophages 255, 258, 259. In addition, IL-1β can stimulate endothelial cells, T and B

cells, and fibroblasts to produce IL-6 and IL-8. Furthermore, IL-1β together with IL-6 is

able to drive Th17 cells polarization 260, 261. Activated Th17 cells could thus produce

high levels of IL-17A which is able to extend neutrophil survival 262, 263 and stimulate

osteoclastogenesis leading to bone destruction 264-266, and IL-22 which helps B cells in

autoantibody production 267-269. On the other hand, once activated, neutrophils can also

release high amounts of BLyS 35 that in turn can stimulate the function and survival of

autoreactive B cells 270-272. Moreover, increased IL-6 levels support a scenario of

continuous recruitment of autoreactive B cells toward the synovium during rheumatoid

arthritis progression 65, 95, thus contributing to the chronic inflammatory process.

Moreover, in the established phase of the disease, the increased levels of IL-21, mainly

produced by CD4+ T cells, including Th17 cells 273-275, can not only induce plasma cell

differentiation and autoantibody production 276, but also significantly enhance Th17

cells proliferation 277. Interestingly, cytokines associated with the function of Th1 and

Th2 cells were not significantly elevated in the early phase of the disease. This result

suggests that Th17-cell activation is the most relevant T-cell related occurrence in very

early rheumatoid arthritis, reinforcing the notion that this might be a Th17-driven

disease.

Currently, MTX is the gold standard treatment for rheumatoid arthritis at the time of

presentation. Of note, neither initial therapy with low dose corticosteroids nor MTX

Discussion

- 53 -

affected cytokine production in VERA patients. Thus, despite being able to control

clinical manifestations of the disease, these conventional therapies were ineffective in

reversing the proinflammatory cytokine production that underlies inflammation in VERA

patients. Such an effect on cytokine production can presumably occur for long-term

treatments, as is the case of established rheumatoid arthritis patients who do not show

increased levels of circulating cytokines.

These results support the pathogenic role of, not only adaptive immune cells such

as B and Th17 cells, but also of innate immune cells, namely macrophages and

neutrophils in rheumatoid arthritis onset and chronicity. Particularly, these observations

also suggest that B cells and Th17-related cytokines targeted therapies could be

beneficial for rheumatoid arthritis patients in the first weeks of disease onset.

The results of this thesis are additionally supported by recently published data. It

has been reported that in pre-arthritis patients (3.3 years before disease onset), there

are increased levels of several circulating cytokines, such as IL-1β, IL-2, IL-4, IL-6, IL-

7, IL-10, IL-12, IL-17, interferon (IFN)γ and TNF along with MCP-1, all of them

associated with the development of inflammation and possibly predictive of the onset of

rheumatoid arthritis. An interesting finding was that IL-17 concentration was present at

its highest level in pre-arthritis patients and decreased afterwards in rheumatoid

arthritis patients after 7.7 months of disease duration 278. This is in accordance with our

observations that the concentration of IL-17A was increased in VERA patients but

similar to healthy controls in the established phase of the disease. Accordingly, it is

conceivable that in individuals who will develop rheumatoid arthritis, there is a trend

towards Th1/Th2/Th17 lineages and macrophage responses 278-280. Other study using

synovial fluid samples also demonstrated that the cytokines which better distinguished

patients with early synovitis (rheumatoid arthritis and crystal synovitis) from controls

with no inflammation (osteoarthritis) were IL-1β, IL-6 and IL-2. In contrast, the cytokines

that better distinguished persistent synovitis of rheumatoid arthritis from other

synovitides were IL-1β, IL-2 and IL-13 281. Interestingly, Meyer et al recently

demonstrated that in DMARD-naïve rheumatoid arthritis patients (with less than 2

years of disease duration) there are increased circulating levels of Th1 and

monocyte/macrophage-derived cytokines, possibly counter-regulated by cytokines

produced by Th2 cells. In contrast, IL-17 was barely detectable in this cohort of

patients, which is in agreement with the notion that the early phase of rheumatoid

arthritis is primarily driven by a Th17 profile, which is gradually replaced by a Th1

pattern during the progression of the disease. Surprisingly, several studies have

Discussion

- 54 -

recently suggested the plasticity of Th17 cells with a phenotype that may be converted

into both Th17/Th1 and Th1 in response to inflammation 282, 283.

In summary, the emerging view is that the cytokine pattern which arises from

several cell types and which is central in the pathogenesis of rheumatoid arthritis may

shift with time. Our results suggest that IL-1β, IL-6 and IL-17A are the cytokines most

likely to be relevant in the first few weeks of rheumatoid arthritis, where the transition

from the acute to the chronic phase of the inflammatory process occurs, and should

thus be considered as potential promising therapeutic targets for an early intervention

in this disease.

Figure 5 – Proposed model for cytokine networks in the very early phase of rheumatoid arthritis. In

the peripheral blood of very early rheumatoid arthritis patients there is a cytokine milieu that favours B-cell activation and survival and that also sustains neutrophil and Th17 cells activation. APRIL – A proliferation-inducing ligand, BLyS – B-lymphocyte stimulator, IL – Interleukin, MCP-1 – Monocyte chemotactic protein-1, Th – T helper. (see text for details)

Discussion

- 55 -

Several studies have documented the relevance of IL-1β in rheumatoid arthritis

physiopathology through diverse mechanisms. IL-1β, which is mainly produced by

macrophages 146, 147, is able to increase the secretion of several cytokines and

chemokines, to elevate the production of MMPs that are responsible for cartilage

damage 59-61, to enhance the expression and survival of T cells and, consequently,

enhance the help for B cell production of autoantibodies 62, to promote the

differentiation of Th17 cells 63, 64, to activate endothelial cells and chondrocytes 45, 267

and also to induce the differentiation of osteoclasts 65, 66, thus triggering the destructive

inflammatory process characteristic of rheumatoid arthritis, as illustrated in Figure 2.

IL-1 family cytokines, such as IL-1β, are abundantly expressed in rheumatoid

arthritis. In fact, in established rheumatoid arthritis there are augmented levels of IL-1β,

which are associated with a higher disease activity 223, 224, 226, 245, and there is also

activation of NF-kB 284 in the synovial fluid and tissue. Additionally, it has been

suggested that synovial membrane mRNA levels of this cytokine are also predictive of

damage progression 67. An interesting finding of the present study was the observation

that IL-1β levels were increased not only in the synovial fluid of established rheumatoid

arthritis patients, but also in the peripheral blood of patients in the early phase of the

disease.

Furthermore, the NLRP3 inflammasome is able to activate caspase-1 285, 286 which,

in turn activates IL-1β enabling the secretion of this cytokine 287-289. Recently, it has

been reported that polymorphisms in NLRP3 and CARD8 genes are associated with

ACPA-positive rheumatoid arthritis, increased susceptibility for the disease and a

worse prognosis for these patients 218-220.

Therefore, these observations together with the above-mentioned results,

prompted us to hypothesize that pathways which regulate IL-1β secretion, such as

caspase-1 and inflammasome activation, could be actively contributing to the

inflammatory response in rheumatoid arthritis. To address this question, the activation

of caspase-1 was evaluated in untreated early rheumatoid arthritis (ERA) patients with

less than one year of disease duration and established rheumatoid arthritis patients

under treatment with MTX. Likewise, other untreated early arthritis (EA) patients who

did not evolve to rheumatoid arthritis and had less than one year of disease duration

were also analyzed.

Strikingly, it was found for the first time that the levels of active caspase-1 are

increased in circulating leukocytes in rheumatoid arthritis patients since the early phase

of the disease, which could explain the increased levels of IL-1β after rheumatoid

Discussion

- 56 -

arthritis onset (Figure 6). In contrast, there were no differences when comparing EA

patients with healthy controls, corroborating the previous IL-1β results in which IL-1β

levels were increased in the early phase of rheumatoid arthritis but not in early non-

rheumatoid arthritis patients in comparison with healthy controls. Of interest, it has

been already reported in Sjögren’s syndrome-like prone mice that inflammatory

caspases are essential in promoting a proinflammatory microenvironment in the target

tissues of Sjögren’s syndrome before the onset of this autoimmune disease 290. In this

study, it was demonstrated that caspase-11, which activates caspase-1 via a non-

canonic pathway 291, plays a critical role in the susceptibility of mice to Sjögren’s

syndrome-like disease, by up-regulating caspase-1 mediated pathways, such as IL-1β

production. The authors suggest the existence of an abnormal regulation of the

inflammasome in these mice in the early pathogenesis of Sjögren’s syndrome 290. It

may be possible that individuals bearing variants in genes coding for inflammasome

components, which confer susceptibility for rheumatoid arthritis, might produce higher

levels of inflammasome components, thus implying that upon an inflammasome

activating event, more inflammasome complexes may be formed, ultimately producing

more inflammatory cytokines that might trigger the activity of the disease, as already

hypothesized based on a very recent study performed by Sui et al 292.

In conclusion, the results of this thesis support our initial hypothesis that the

caspase-1 pathway is already activated since the early phase of rheumatoid arthritis

and that IL-1β, caspase-1 and the proteins that regulate the activation of

inflammasomes may be potentially promising treatment targets in the early phase of

this disease.

Despite this apparent crucial role of IL-1β signaling in rheumatoid arthritis, clinical

benefits after IL-1β inhibition in these patients have been modest. Several trials have

shown the efficacy of anakinra in treating symptoms of a subset of rheumatoid arthritis,

with significant improvement in many different clinical, biochemical and radiographic

parameters 228. Unexpectedly, the efficacy of this IL-1β blocking therapy was lower

than TNF inhibitors in treating rheumatoid arthritis 228, 238, 239.

Notably, even though the disappointing observations of anakinra have raised some

controversy regarding the importance of IL-1β in rheumatoid arthritis, our results do

support the relevant role of IL-1β signaling in the pathogenesis of this disease since its

onset, suggesting that this treatment strategy should be re-evaluated in early

rheumatoid arthritis patients.

Discussion

- 57 -

Figure 6 – Proposed mechanism for excessive IL-1β production since early rheumatoid arthritis.

The levels of active caspase-1 are increased in circulating leukocytes in both early and established rheumatoid arthritis patients, which could explain the augmented concentration of IL-1β after disease onset, contributing for the excessive and destructive inflammatory process characteristic of these patients. ASC – Adaptor molecule apoptosis associated speck-like protein containing a caspase recruitment domain, APRIL – A proliferation-inducing ligand, BLyS – B-lymphocyte stimulator, IL – Interleukin, MCP-1 – Monocyte chemotactic protein-1, NF-kB – Nuclear factor kappa-light-chain-enhancer of activated B cells, NLRP – NOD-like receptor protein, Th – T helper. (see text for details)

Based on the increased levels of IL-1β and the continuous activation of caspase-1

both in early and established rheumatoid arthritis, we hypothesized that IL-1β plays a

relevant role in the early stages of the disease and that pathways regulating this

Discussion

- 58 -

cytokine and TNF, such as caspase-1 and NF-kB activation, could constitute promising

combined therapeutic targets.

Our laboratory used a THP-1 macrophage-like cell line to screen 2320 drugs for

those that simultaneously inhibit IL-1β and TNF secretion. We have selected celastrol

and gambogic acid as the two most effective drugs in inhibiting both IL-1β and TNF

secretion due to the reduction in caspase-1 and NF-kB activation. Celastrol is a novel

pentacyclic-triterpene compound extracted from Trypterigium wilfordii Hook F, which is

used in traditional Chinese medicine and that was recently shown to be able to

suppress cancer progression 293, 294, to inhibit NF-kB activity 295, to abrogate the

secretion of IL-1β in lipopolysaccharide-stimulated human peripheral mononuclear cells

296 and to inhibit proinflammatory cytokine secretion in mucosal inflammatory biopsies

from Crohn’s patients, possibly due to the blockade of cytokine gene transcription 297.

Gambogic acid is a polyprenylated xanthone abundant in resin derived from Garcinia

hanburyi and Garcinia Morella, which is used in Southeast Asia traditional medicine 298,

and that has recently been shown to inhibit the growth of a wide variety of tumor cell

lines 299, possible due to its ability to induce apoptosis 300, 301, to abrogate NF-kB

signalling pathway in human leukemia cancer cells 299 and in a non-cancerigenous

macrophagic cell line 302, both via the transferrin receptor (TfR1). Celastrol and

gambogic acid were further tested in vivo in a rat model of adjuvant-induced arthritis

(AIA) in order to evaluate their anti-inflammatory properties. These drugs were

administrated to AIA rats in the early phase of arthritis development. Administration

was through a therapeutic approach, since it started in the early phase of arthritis, after

4 days of disease induction, with all animals already displaying signs of inflammation.

Additionally, celastrol was also administrated in the established phase of arthritis, after

11 days of disease induction when the animals already displayed a marked

inflammatory score.

Animal models of rheumatoid arthritis have been extensively used for many years

in the evaluation of anti-arthritic agents 303, 304. The most widely used model is the AIA

in rats 305, 306, which shares key features related to human rheumatoid arthritis making

them a critical tool in drug development. There are some similarities in disease

characteristics when comparing AIA rat model and human rheumatoid arthritis, namely

the presence of acute phase proteins, polyarthritis, synovitis and bursitis/tendonitis,

erosion of cartilage and bone, the influx of neutrophils, the homing of macrophages to

soft tissues, the increased levels of synovial cytokines such as IL-1β, IL-6 and TNF,

and the occurrence of edema. The AIA rat model, when compared with other models of

arthritis such as collagen-induced arthritis, clearly exhibits the greatest magnitude of

Discussion

- 59 -

disease, as measured by edema, cellular influx, cytokine levels and markers of

cartilage and bone destruction. The prolonged time frame and the recurrent nature of

disease activity of human rheumatoid arthritis are not replicated in this model, as is

also the case of some other features of this disease, including RF and synovial

lymphoid follicles that are absent in the AIA rat model. This animal model was used to

test arthritis therapies such as steroids and NSAIDs 303, 304, as well as to assess

immunomodulatory drugs such as MTX, cyclosporine A and therapies designed to

block cyclooxygenase 2, TNF or IL-1β 307-315. Overall, the AIA model has been the most

extensively used arthritic rat model by the pharmaceutical industry because it has an

excellent track record for predicting both activity and toxicity 313. Paw swelling in AIA rat

model is robust, with increases up to 3.5-fold times control volume and, in general,

maximal swelling occurring by day 19 and then plateaus. Thus, studies are generally

completed at this time point.

We found, for the first time, that wistar AIA rats can be effectively treated by both

celastrol and gambogic acid possibly through an inhibitory effect over TNF and IL-1β

secretion, induced by the down-regulation of caspase-1 and NF-kB activation. These

drugs showed anti-inflammatory and anti-proliferative properties, promoting a complete

suppression of arthritis development and abrogation of joint immune cellular infiltration

and proliferation, preventing cartilage and bone damage (Figure 7). Interestingly,

celastrol treatment was effective when administrated both in the early and later phase

of arthritis, which is relevant for the possible clinical implications of our findings. Our

results demonstrated that the anti-inflammatory properties of these drugs might not

only be related to their ability to suppress the activation of NF-kB but also to their ability

to inhibit caspase-1 activation, as observed in the in vitro experiments performed in

THP-1 macrophage-like cell line.

The role of NF-kB in the pathogenesis of rheumatoid arthritis has been already

described. In fact, the inhibition of NF-kB in animal models has shown the ability to

inhibit inflammatory arthritis 316-319, demonstrating that NF-kB may be an important

therapeutic target in this disease. Indeed, NF-kB participates in the transcription of the

genes encoding many proinflammatory cytokines and chemokines, in the regulation of

the different immune cells and in the regulation of the expression of adhesion

molecules and matrix MMPs 320-323. As recently reviewed, the Ikβ kinase IKKβ is

essential for the inflammatory cytokine-induced activation of NF-kB 324. Blocking IKKβ

could therefore be part of a therapeutic strategy for the treatment of inflammation. In

this context, it was demonstrated by Wen and co-workers that the inhibition of IKKβ

inhibits NF-kB signaling in human synovial fibroblasts, chondrocytes and mast cells 325,

Discussion

- 60 -

and that it reduces NF-kB activity in rats with induced polyarthritis 326. Importantly, it

has already been reported that both celastrol and gambogic acid are able to inhibit

IKKβ activity 295, 302.

Our results are also supported by a study from van den Berg et al, in which the

authors suggest potential uncoupled activities of TNF and IL-1β in joint swelling and

ongoing cartilage destruction 327. The authors also point out that the claim of TNF being

a driving force for IL-1 production in rheumatoid arthritis should be viewed with care 327.

Their results in animal models of arthritis suggest that anti-TNF treatment is not

sufficient and that a combination therapy with anti-IL-1 provided the best protection

against disease progression, thus suggesting that this could be a promising strategy for

rheumatoid arthritis treatment 327. Synovial biopsy immunostainings of rheumatoid

arthritis patients have shown distinct TNF and IL-1β localisation patterns 328 and no

significant IL-1β reduction in synovial cells after treatment with TNF antagonists 327.

Additionally, systemic treatment with TNF antagonists does not reduce IL-1β mRNA in

circulating leukocytes of most patients 327. In fact, it has already been reported that

there are different time dependent roles for IL-1β and TNF in the various stages of

collagen-induced arthritis, since anti-TNF therapy showed efficacy only shortly after

disease onset while anti-IL-1 therapy ameliorated both early and established arthritis 83.

TNF is in fact sufficient to drive inflammation but IL-1 is a crucial mediator for

inflammatory cartilage and bone degradation 70. Bendele et al have demonstrated that

a combination of treatment with agents blocking both IL-1 and TNF receptors provides

substantially more clinical benefits than either agent alone in animal models of arthritis

313. This result could be explained by a synergistic effect between IL-1 and TNF. This

synergism has been already reported by others 327, 329-333 and may be explained by the

ability of TNF to induce IL-1 and vice-versa 334. Consequently, a double blocking of IL-1

and TNF strategy could be beneficial for the treatment of inflammatory diseases such

as rheumatoid arthritis. Taking into consideration the apparent advantage of a

combination therapy blocking both IL-1 and TNF, in 2004 a trial tested the efficacy of

combination therapy using anakinra and etanercept in 244 long-standing and very

active rheumatoid arthritis patients who have been treated unsuccessfully with MTX 335.

Results from this study revealed that combination therapy with anakinra and etanercept

provided no added benefit for these patients comparing with etanercept alone and

increased the frequency of infection related adverse effects 335. The reasons for the

discrepancies between preclinical and clinical results are unclear but several theories

are discussed by the authors of this study 335 such as negative interaction between

compounds and the development of anti-anakinra antibodies. Moreover, these

Discussion

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discrepancies might be additionally explained by the fact that anakinra has some drug

concept limitations that can limit its effectiveness in blocking IL-1. Finally, the very

advanced disease stage of the patients could have also limited a sufficient evaluation

of this combination treatment. Our results thus suggest that a combination therapy

inhibiting both IL-1β and TNF production is effective not only in the early but also in the

later phases of arthritis and is safe, since these drugs diminish the levels of both

cytokines, instead of silencing their receptors as is the case of etanercept and

anakinra.

Our findings are also in agreement with data already published, showing that a

tripterine isolated from Trypterigium wilfordii Hook F was effective on adjuvant 336 and

collagen-induced arthritis 337 in rats, supporting the 2002 report from Tao et al revealing

that an ethanol/ethyl acetate extract of Trypterigium wilfordii Hook F possess

therapeutic benefits in patients with refractory rheumatoid arthritis 338. Interestingly, our

results are also supported by data from a study performed in an in vivo model of

metastatic bone disease associated with breast cancer, in which celastrol inhibited

bone resorption, consistent with the inhibitory effect on osteoclast formation and

survival that was already observed in in vitro experiments 339. In rheumatoid arthritis the

infiltration of immune cells and the proliferation of joint lining synovial fibroblasts lead to

the formation of the tumor-like pannus tissue, which invades and destroys the

underlying joint cartilage and bone, resulting in irreversible erosion of the bone and in

the loss of normal joint structure, causing functional disability 225. The cell proliferation

inhibitory properties of celastrol and gambogic acid may thus prove to be of interest to

prevent and treat this complication of established rheumatoid arthritis. This is exactly

compatible with our experiments that have shown an inhibition of the inflammatory

activity, cell proliferation and bone erosions. Although further animal experimentation is

required to fully assess the safety of these drugs for human arthritis treatment, these

results point out that both celastrol and gambogic acid might be worth entering into

phase I clinical trials.

To sum up, these data support our hypothesis that IL-1β dependent inflammatory

effects, preceded by caspase-1 and NF-kB activation, have a role in the very early

pathogenesis of rheumatoid arthritis.

Discussion

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Figure 7 – Proposed mechanism for celastrol and gambogic acid anti-inflammatory effects. Both

celastrol and gambogic acid showed anti-inflammatory and anti-proliferative properties in vivo, promoting a complete suppression of arthritis development and abrogation of joint immune cellular infiltration and proliferation, preventing cartilage and bone damage. These drugs induced a downregulation of caspase-1 and NF-kB activation and, consequently, lead to a decrease in TNF and IL-1β secretion. ASC – Adaptor molecule apoptosis associated speck-like protein containing a caspase recruitment domain, CARD – Caspase recruitment domain, DAMP – Danger associated molecular pattern, IL – Interleukin, IL-1R – IL-1 receptor, MyD88 – Myeloid differentiation primary response gene 88, NF-kB – Nuclear factor kappa-light-chain-enhancer of activated B cells, NLRP – NOD-like receptor protein, PAMP – Pathogen associated molecular pattern, PYR – Pyrin domain, TLR – Toll-like receptor, TNF – Tumor necrosis factor. (see text for details)

Of note, we have found that not only the circulating levels of IL-1β but also of IL-

17A were increased in the early phase of rheumatoid arthritis. Previous studies have

already shown that there are increased levels of IL-17 in the sera and synovial fluid of

rheumatoid arthritis patients and that IL-17 is present in the T cell rich areas of the

synovium 112, 281, 340-342. Thus, anti-IL-17 cytokine therapy may be of interest as an

additional new anti-rheumatic drug for this disease 343. Strikingly, Lalor et al have found

that the activation of caspase-1 in innate immune cells is a critical step in the induction

of IL-17 production and in the development of experimental autoimmune

encephalomyelitis (EAE) in mice. In vivo inhibition of caspase-1 suppressed the

Discussion

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development of Th17 cells and the induction of EAE. These new evidences suggest

that caspase-1 might also be an efficient drug target in immune mediated inflammatory

diseases 344. An interesting report dissecting CAPS inflammatory environment have

demonstrated the central role of IL-1β in Th17 cells differentiation, since these patients

showed significantly increased IL-17 serum levels as well as a higher frequency of

Th17 cells, which were decreased following in vivo IL-1β blockade 345. This expansion

of Th17 phenotype in CAPS patients may be a simple epiphenomenon secondary to

the over-secretion of IL-1β due to NLRP3 mutations or it may be contributing to the

maintenance of chronic inflammation.

To help distinguish between a direct role for IL-1β as an effector in the

inflammatory process or as an upstream initiator of inflammation by driving the

differentiation of Th17 cells, we analyzed the anti-inflammatory effects of digoxin in the

same rat model of arthritis, using the same experimental design as described above. A

very recent paper from Huh et al has shown that digoxin is a specific inhibitor of RORγt

transcriptional activity and consequently an inhibitor of Th17 cells polarization 346.

Digoxin is currently recommended for patients with heart failure who are symptomatic

despite standard therapy and for controlling the ventricular rate in atrial fibrillation, with

an important role in optimizing care in certain acute and chronic cardiac conditions 347.

We found for the first time that digoxin was able to ameliorate the inflammatory

signs of the AIA rat model of arthritis, which is in accordance with the reported effect of

digoxin in delaying the onset and reducing disease severity in an EAE mice model, by

the inhibition of Th17 cells differentiation 346. We observed that digoxin treatment was

effective if administrated in the early phase of arthritis development but not if given in

the later phase of the disease course. Although digoxin was able to efficiently suppress

the severity of inflammatory signs, it was not able to significantly reduce the infiltration

of immune cells within the joints. Contrarily, treatment with the previously mentioned

drugs that block IL-1β and TNF simultaneously, without inhibiting IL-17, have a

significant inhibitory effect in arthritis progression and severity, even when

administrated in a later phase of the disease course, with a complete abrogation of the

inflammatory score, infiltration and proliferation of immune cells. Moreover, digoxin had

a slower and less efficient effect on disease progression comparing with these drugs.

In conclusion, these data suggest that despite the role of IL-1β as a driver of Th17

cells polarization, the inhibition of this pathway is not effective in the treatment of

arthritis. Indeed, IL-1β contributes to rheumatoid arthritis physiopathology not only by

the induction of Th17 cells differentiation but also by promoting the migration of

macrophages and neutrophils to the damaged joints, thus driving the release of

Discussion

- 64 -

proteases and ROS 348 and promoting the differentiation of osteoclasts 349. Likewise, IL-

1β, together with TNF and IL-6, has also a significant capacity to induce RANKL

expression on synovial fibroblasts/osteoblasts and to facilitate RANK signaling, thus

directly contributing to the bone resorption and destructive process. In contrast, IL-17

seems to have a more limited effect on inflammatory cell influx and subsequent

inflammatory signs, as suggested by the results of digoxin treatment. This does not

preclude however the important role of Th17 cells in driving the innate immune

inflammation towards the adaptive autoimmune inflammation in rheumatoid arthritis,

involving several other cytokines besides IL-17. Nevertheless, the results regarding

anti-IL-17 therapy in rheumatoid arthritis are still contradictory. Two different clones of

anti-IL-17 antibody are currently undergoing clinical trials in patients with rheumatoid

arthritis. A study performed by Genovese et al has shown that one of these clones, a

humanized anti–IL-17 monoclonal antibody (LY2439821), added to oral DMARDs

improved signs and symptoms of rheumatoid arthritis with no significant adverse safety

signal noted 350. More recently, the same author reported a phase II placebo-controlled

trial with 237 patients testing the second clone, an anti-IL-17A drug (secukinumab, also

called AIN457) in rheumatoid arthritis where the primary end point was not met 351, 352.

This drug was significantly more effective after 12 weeks, but at week 16 - the pre-

specified point for evaluating the primary outcome (percentage of patients achieving an

ACR20 response) was not met - a rise in placebo response did not allow statistically

significant difference between the treatment and placebo groups 351. Interestingly,

combination therapy with soluble IL-1R and IL-17R is more effective for controlling

synovial inflammation and bone resorption than each therapy alone in an ex vivo model

of rheumatoid arthritis synovium and bone explants 353. These results suggest that the

isolated inhibition of Th17 cells polarization might be a strategy with limited efficacy in

rheumatoid arthritis, at least in the established phase of the disease.

In summary, our results, as compared with other published studies, have shown

that caspase-1 inhibitors are efficient in different experimental models of immune

mediated inflammatory diseases 354-358. The study discussed here provides new and

original evidence for the relevance of IL-1β, including caspase-1/NF-kB/inflammasome

activation, as possible drug targets for immune mediated inflammatory diseases,

especially if introduced in the early phase of the disease.

Discussion

- 65 -

Overall, the results of this thesis suggest that an earlier intervention on the IL-1β

pathway might be of beneficial clinical use to induce early remission in rheumatoid

arthritis patients. It cannot be claimed that IL-1β is the only factor, but these results

imply that interfering with IL-1β in the context of a network of cooperating mediators is

sufficient to down-regulate inflammation and progressive articular destruction.

Therefore, it is worthwhile pursuing the possibility that IL-1β is at least an adjuvant

therapeutic target in the early phase of arthritis.

- 66 -

Chapter V

Conclusions

- 68 -

Conclusions

- 69 -

The study of IL-1β since the early phase of rheumatoid arthritis supports an early

influence of this cytokine in disease progression and has revealed that:

1) In very early rheumatoid arthritis and in the synovial fluid of established

rheumatoid arthritis patients there is a cytokine milieu, which supports the activation,

expansion and survival of autoreactive Th17 and B cells as well as macrophages and

neutrophils since the first weeks of disease onset;

2) IL-1β is one of the cytokines with an increased concentration detected since the

early phase of rheumatoid arthritis until the established phase of the disease;

3) Early short-term treatment with corticosteroids or MTX leads to clinical

improvement, but does not seem to have an impact in the cytokine pattern of very early

rheumatoid arthritis patients;

4) The caspase-1 pathway, which is responsible for IL-1β activation, is activated

since the early phase of rheumatoid arthritis, reinforcing the potential relevance of

therapies targeting IL-1β in the early course of this disease;

5) Drugs targeting IL-1β and TNF simultaneously, upstream to their receptors,

namely celastrol and gambogic acid, have anti-inflammatory and anti-proliferative

properties in the treatment of arthritis and thus might constitute therapeutic approaches

with significant efficacy in the treatment of immune-mediated inflammatory diseases

such as rheumatoid arthritis;

6) Digoxin, which inhibits Th17 cells differentiation, despite being able to decrease

the severity of early inflammatory signs in AIA rat model, is not effective in reducing the

infiltration of immune cells within the joints, suggesting that the isolated inhibition of

Th17 cells polarization might be a strategy with limited efficacy, at least in established

rheumatoid arthritis.

The understanding of the pathogenesis of rheumatoid arthritis remains, however,

far from complete. Therapeutic targets and biological windows of opportunity identified

from advances made in studies as the one presented herein have contributed to the

expansion of treatment options for rheumatoid arthritis, in a series of successful

transitions from bench to bedside. This growing knowledge may ultimately make

complete remission a real goal for rheumatoid arthritis patients.

- 70 -

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- 91 -

A presente dissertação foi realizada na Unidade de Investigação em Reumatologia e

na Unidade de Biologia Celular do Sistema Imunitário do Instituto de Medicina

Molecular, Faculdade de Medicina da Universidade de Lisboa, Lisboa, Portugal.

O trabalho aqui apresentado foi realizado no âmbito do projecto “The role of

inflammasome in the initiation and perpetuation of rheumatoid arthritis: contribution for

new targeted therapies.”, financiado pela Sociedade Portuguesa de Reumatologia e

por uma unrestricted research grant da empresa Pfizer.

Bolsa de Doutoramento da Fundação para a Ciência e a Tecnologia (referência:

SFRH/BD/40513/2007).