Laboratorio de Imunogenetica

(IMUNOGEN)

Alessandra Pontillo

DEPARTAMENTO

Imunologia

Inflamassoma e Sistema imune

Inflamação

Adapted from creative-diagnostics.com

Infeccioso

Estéril

Células residentes

no tecido

Células e moléculas

circulantes no sangue

Vaso

Endotelio

IL-1ß

Processo fisiológico patológico

Resposta imune

Eliminaçao do dano/infeçao

Restauraçao da homeostasia

Dano > beneficio

Cronica

Inflamassoma

Adapted from tocris.com

“Cytoplasmic complex triggering IL-1ß secretion

and inflammation” Martinon F, Burns K, Tschopp J. 2002

sensor/receptor

adaptador

efetor

ATIVAÇAO DA

CASPASE-1

Pro-IL-1ß IL-1ß

Inflamassoma(s)

NLRP6 (microbiota

LMW metabolites)

NLRP12 (YoP)

NLRP7 (microbial

acylated ipopeptides)

IFI16 (DNA)

RIG-I (RNA)

NAIP/

??

Adapted from Sharma and Kanneganti, 2017

NLRP3 inflamassoma

Adapted from Marthur 2017

Ativaçao canônica Ativaçao non canônica

Ativação do inflamassoma

Clivagem da caspase-1

Clivagem das proformas das citocinas (pro-IL-1ß, pro-IL-18)

Adapted from Broz 2016

Ativação do inflamassoma

PYD-containing receptors (NLRP3, AIM2, pyrin) precisam de ASC para montar

o inflamassoma

NLRP1 e NLRC4 nao precisam para montar o inflamassoma (e para piroptose)

mas com ASC o processamento das citocinas è mais eficiente

ASC recruta a caspase-1 de uma forma «prionic-like» levando a formaçao de

oligomeros (specks ou puncta)

Funçoes do inflamassoma

IL-18

Citocina inflamatoria

Promove Th17

+IL-12Th1; -IL-

12Th2

IL-1ß

Citocina inflamatoria,

pirogenica

Promove Th1, Th17

Ativa condrocitos e

osteoclastos

GSDM-D

Pore-formation protein

Piroptose

Regulaçao transcricional: NLRC4 e iNOS (PPARg)

Homeostasia (intestino: AIM2, NLRP3, NLRP6, NLRC4)

Resposta imune inflamassoma independente:

- NLRP6, NLRP12 inibição NF-kB

- NLRP3 cofator de transcrição para IL4 (Th2)

Cardiolip

ina,

mtDNA

Citrato

Succinato

ER

stress

Acido

urico

PAMPs

Metabolic stress

UV stress

Hipoxia

Necrose

Uric acid, cristals

Sterile damage

Virus

Bacteria

Fungus

Protozoa

Microbes

Inflammation

Tolerance/

Suppression

Immune

activation

patogenomicrobiota

piroptoseEliminaçao nicho

de replicaçao

Adapted from Tahseen H, 2012

DEFESA CONTRA PATOGENOS

HOMEOSTASIA

RESPOSTA A STRESS

ADJUVANTES/VACINAS

Fisiologia do inflamassoma

Adapted from LatzH, 2013

POPs/COPs

CARD8

Regulação do inflamassoma

Metabolic stress

UV stress

Hipoxia

Necrose

Uric acid, cristals

STERILE DAMAGE

piroptose

DEFEITO GENETICO

DISREGULAÇAO IMUNE

IL-1ß, IL-18 TH17

DOENÇAS AUTO-INFLAMATORIAS

«RARAS»

DOENÇAS MULTIFATORIAIS

”COMUNS”

Disregulação do inflamassoma

Adapted from 2013

Type 1 Diabetes

Celiac disease

IBD

Colon carcinoma

SLE

Ankylosing spondylitis

Vitiligo

Melanoma

Infectious diseases

Gout

Autoimmunity

Obesity and metabolic syndrome

Cardiovascular diseases

Neurodegenerative diseases

Cancer

Transplant

Constitutive activation

of inflammasome

??

Multifactorial diseases

Dysregulation

of IL-1ß (IL-18?)

Risk factors

- Genetics

- Enviroment

- Pathogen

Inflamassoma & Doenças MF

Adapted from LatzH, 2013

Inflamassoma & Doenças “raras”

Rare variants (<1%) Constitutive activation of inflammasome Distinct

Phenotypes & Response to drugs

Adapted from MAsters, 2017

AIFEC/SCAN4 (NLRC4)CAPS (NLRP3)

(Romberg et al., 2014; Canna et al.,

2014).

NAIAD

(NLRP1)

(Grandemange et al., 2017)

FMF (MEFV)

(Simon et al 2005)

Inflamassoma & Doenças “raras”

Expressao tecido-especifica papel diferente em

celulas diferentes?

Common variants, SNPs (>1-5%) contribution for

complex, multifactorial diseases? Common

pathogenic pathways? therapeutic approaches?

↓ or ↑ inflammasome activation threshold?

↑ or ↓ IL-1ß, IL-18?

↓ or ↑ caspase-1 independent function?

Innate immune sensing, Inflammation, homeostasis

Genes do Inflamassoma & MFD

Adapted from MAsters, 2017

SNP assay

Reaction plate

Genotyping data +

clinical/demographic

data

SNPassoc (R-program)

Haploview

SNPs selection

MFD

ex-vivo, human cells modelAssociation

results

A nossa pesquisa

from Karki R, 2017

A nossa pesquisa

- Conhecimento “básico”

- Ferramentas diagnosticas

- Alvos terapêuticos

INFECTIOUS

DISEASES

Gain-of-function

SNPs

Loss-of-function

SNPs

STERILE

DISEASES

??

2007 2009 2010 20122011 2013 2015 2016 2017 2018

Association studies: an overview

Cell type specific response

R LPS R LPS R LPS0

100

200

300

IL-1ß

(p

g/m

L)

w/o ATP

1mM ATP

T CD8+ B CD19+T CD4+

* *

monocyte Mø DC

Lymphocytes

Unpublished results

Imunogen projects

MS L

B

HD L

B

NLRP1

NLRP3

NLRC4

CASP1

CASP4

IL1B

IL18

200

400

600

Flagelin as adjuvant

in HIV+ patients

NLRP3 in lymphocytes

in HIV+ patients

NLRP3 inflammasome

in the response to

M.tuberculosis

Inflammasome

genetics in Multiple

Sclerosis

Inflammasome

activation in HPV-

associated cervical

cancer

Imunogen projects

Imunogen partners’ projects

Inflammasome in gut

from HIV+ patients

LIM-56

Inflammasome in

gestacional & cerebral

malaria

Claudio Marinho

Inflammasome

genetics in Colon

Cancer

(Modified from Moir S, 2011)

HIV-1 induces IL-1ß release

NLRP3

IL-1ß

IL-1ß

DC responsiveness

Defeito

NLRP3

HIV-1

0

100

200

300

400

500

IL-1

b p

g/m

L

LPS LPS+ATP

CD19+ HD HIV

*

IL-1ß

Lymphocytes inflammation?

NLRP3 3’UTR SNP confers

protection against HIV-1

Outros rec?

Outras funções?

NLRP3 3’UTR rs10754558

protection vs pulmonar TB

0

50

100

150

200200

400

600

IL-1ß

(p

g/m

L)

- - - - - - +++++

LPSBCGR

*

##

##

##

- : w/o ATP+: with 1mM ATP

Rv Bj MP

+

Virulent Hipervirulentnon-vir

-ATP

+ATP

TB

conta

tos

HC

0

20

40

60

seru

m

IL-1ß

(p

g/m

L)

Tuberculosis (TB)

UN +IL-1β0

50

100

150

Bacte

rial g

row

th (

CF

U) *

Edione C Reis, PhD student

Dhemerson Souza Lima, PhD student

Vinicius Nunes Cordeiro Leal, PhD student

Jaine Soares, Ms studen

Fernanda Pereira Fernandes, , Ms studen

Partners Financial support & Fellowships

Autoimmune Diseases

T1D, MS, SLEEx-vivo and in vitro PBMC analysis

Inflammasome activation (IL-1ß, IL-18,

caspase-1 cleavage, «specks» formation

SNPs assyas

Genotyping

Analysis of SNPs

distribution

Blood samples

Basal inflammasome activation

Inflammasome defect against common

stimulus (i.e. LPS+ATP)

NLRP1 rs12150220 protection vs MS

NLRP3 Q705K risk for bad prognosis (>EDSS)

Multiple Sclerosis (MS)

R R+ATP LPS LPS+ATP 0

100

200

300

400

500

IL-1ß

(p

g/m

L)

HCMS

p=0.002

n=16n=7

SLE n=11

p=5,6 exp-4

nsns

MS patients’ monocytes

1 µg/mL LPS ± 1mM ATP

Gain-of-function NLRP3 ↑ L-1ß ↑ ↑ neuronal inflammation

Gain-of-function NLRP1 ??

- = Alzheimer Disease; CNS expression

- PAMPs sensing? Molecular mimetism?

Primed cells?

Unresponsiveness to ATP?

DEFECT in NLRP3

NLRP3

(1q44)

NLRP1

(17p13.2)

NLRC4

(2p22.3)

NAIP

(5q13.2)

NLRP12

(2,4,5,7,8,9,11,13)

(19q13.42)

IL1B

(2q13-q21)

IL1R, IL1RN

(2q11-14)

CARD8

(19q13.33)

NLRP6 (10,14)

(11p15.5)

IL18

(11q23.1)

CASP1

(11q22.3)

PYCARD/ASC

(16p11.2)

MEFV

(16p13.3)

PSTPIP1

(15q24.3)

P2X7R

(12q24.31)

miR-223

(Xq12)

miR-155

(21q21.3)

IFI16

(1q23.1)

AIM2

(1q23.1-q23.2)

Locus dos genes do inflamassoma

Teste de ativaçao do inflamassoma

0

100

200

300

IL-1ß

(p

g/m

L) w/o ATP

1mM ATP

3

* *

**

*

*

0 8 18 24

0

100

200

300

IL-1ß

(p

g/m

L) w/o ATP

1mM ATP

3

**

*

**

**

0 8 18 24

0

100

200

300

IL-1ß

(p

g/m

L)

w/o ATP

1mM ATP

3

***

0 8 18 240

2

4

300IL

-1ß

(p

g/m

L) w/o ATP

1mM ATP

30 8 18 24

A B

C D

monocitos Mø (+25 ng/mL M-CSF 5d)

DC (+50 ng/mL GM-CSF e IL-4 5d) linfocitos

1 µg/mL LPS ± 1mM ATP

mono Mø DC T B pDC

0.0

0.5

1.0

1.5

2.0

gen

e e

xp

ressio

n

(2-∆

Ct )

NLRP3

CASP1

IL1B

*

3 8 18 24 3 8 18 24 3 8 18 24

0

10

20

30

40

50

60

LPS stimulation (hours)

gen

e e

xp

ressio

n

(FC

)

NLRP3

CASP1

IL1B

Mø DCmono

A

B

Teste de ativaçao do inflamassoma

Expressao basal

Modulaçao da expressao por LPS 1 µg/mL

LPS 18h LPS 24h0

50

100

150

200

IL-1ß

(p

g/m

L)

A/A+A/TT/T

p<0,05 p<0,05

LPS 24h LPS 24h +ATP0

100

200

300

IL-1ß

(p

g/m

L)

A/A+A/T

T/T

p=0,05

LPS 3h LPS 24h0

50

100

150

200

IL-1ß

(p

g/m

L) A/T+T/T

A/A

p<0,05

A B C

Ensaio genotipo-guiado

rs12150220_A>T (NLRP1 L155H)

monocitos Mø DC

rs11651270_T>C (NLRP1 M1154V)

LPS 3h LPS 24h0

50

100

150

200

IL-1ß

(p

g/m

L)

C/C

C/T+T/T

LPS 24h LPS 24h+ATP0

100

200

300

400

IL-1ß

(p

g/m

L)

C/C

C/T+T/T

LPS 8h LPS 24h0

50

100

150

IL-1ß

(p

g/m

L)

C/CC/T+T/T

A B Cmonocitos Mø DC

LPS 3h LPS 24h0

50

100

150

200

250

IL-1ß

(p

g/m

L)

C/C+C/G

G/G

p<0,05

LPS 24h LPS 24h +ATP0

100

200

300

IL-1ß

(p

g/m

L)

C/C

C/G+G/G

p=0,05

LPS18h LPS 24h0

50

100

150

200

IL-1ß

(p

g/m

L)

C/G+G/GC/Cp<0,05

A B C

Ensaio genotipo-guiado

monocitos Mø DC

rs10754558_C>G (NLRP3 3’UTR)

SNPs ganho-de-funçao

Diferente efeito de acordo com o tipo de celula