Autoimmunity Reviews 14 (2015) 971–983

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Autoimmunity Reviews

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Review

Gene/environment interactions in the pathogenesis of autoimmunity:New insights on the role of Toll-like receptors

Elena Gianchecchi a,b, Alessandra Fierabracci a,⁎a Immunology and Pharmacotherapy Area, Bambino Gesù Children's Hospital, IRCCS, Rome, Italyb Vismederi Srl, Siena, Italy

⁎ Corresponding author at: Immunology and Pharmaco6859 2904.

E-mail address: alessandra.fierabracci@opbg.net (A. Fi

http://dx.doi.org/10.1016/j.autrev.2015.07.0061568-9972/© 2015 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 1 July 2015Accepted 8 July 2015Available online 13 July 2015

Keywords:AutoimmunityEtiopathogenesisToll-like receptorsTLR signaling pathwayCandidate autoimmune genesPrevention–treatment

Autoimmune disorders are increasing worldwide. Although their pathogenesis has not been elucidated yet, acomplex interaction of genetic and environmental factors is involved in their onset.Toll-like receptors (TLRs) represent a family of pattern recognition receptors involved in the recognition and inthe defense of the host from invadingmicroorganisms. They sense awide range of pathogen associatedmolecularpatterns (PAMPs) deriving from metabolic pathways selective of bacterial, viral, fungal and protozoan microor-ganisms. TLR activation plays a critical role in the activation of the downstream signaling pathway by interactingand recruiting several adaptormolecules. Although TLRs are involved in the protection of the host, several studiessuggest that, in certain conditions, they play a critical role in the pathogenesis of autoimmune diseases. Wereview the most recent advances showing a correlation between some single nucleotide polymorphisms orcopy number variations in TLR genes or in adaptor molecules involved in TLR signaling and the onset of severalautoimmune conditions, such as Type I diabetes, autoimmune polyendocrinopathy candidiasis-ectodermaldystrophy, rheumatoid arthritis, systemic lupus erythematosus and systemic sclerosis. In light of the foregoingwe finally propose that molecules involved in TLR pathway may represent the targets for novel therapeutictreatments in order to stop autoimmune processes.

© 2015 Elsevier B.V. All rights reserved.

Contents

1. Toll-like receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9722. TLR signaling mediated through the MyD88-dependent pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9723. MyD88-independent signaling pathway induced by TLR3 and TLR4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9724. The TLR-independent cytosolic pattern-recognition receptors (PRRs) for nucleic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9735. TLR ligands: bacterial, viral, fungal and protozoan PAMPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9736. The negative regulation of TLR signaling pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9747. The possible involvement of nucleic acid PAMPs and endogenous ligands in autoimmunity . . . . . . . . . . . . . . . . . . . . . . . . . . 9748. The involvement of TLRs in autoimmune disease onset . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 975

8.1. Type I diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9758.2. Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9768.3. Rheumatoid arthritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9768.4. Systemic lupus erythematosus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9778.5. Systemic sclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9788.6. Behcet's disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9788.7. Crohn's disease and ulcerative colitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9798.8. Multiple sclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9798.9. Vitiligo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9798.10. Myasthenia gravis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 979

therapyArea, Bambino Gesù Children'sHospital, IRCCS, Viale S. Paolo 15, 00146 Rome, Italy. Tel.:+39 06 6859 2656; fax+39 06

erabracci).

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9. Conclusive remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 980Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 980References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 980

1. Toll-like receptors

TLRs represent a family of pattern recognition receptors [1] whichare type I integral trans-membrane glycoproteins [2,3]. They show atrimodular structure [3], with an extracellular N-terminal domain andan intracellular C-terminal region. The first is constituted by about16–28 leucine rich repeats (LRRs) and has the function to recognizePAMPs [3], whereas the second, also called Toll/IL-1 receptor (TIR)domain, is similar to the cytoplasmic region of the interleukin-1 receptor(IL-1R) [4–7]. TIR domain has a critical role for TLR function (vide infra)[3].

Toll was the first receptor identified in Drosophila, where it was in-volved in the dorsal ventral patterning in developing embryos [8].Since the observation performed by the group of Hoffmann [9] thatflies mutant for Toll were characterized by an increased susceptibilityto fungal infections, several homologues of Toll receptor were identifiedin mammals and called TLRs [10].

In total 13 and 11 TLRs were identified in mice and in humans, re-spectively [2]. They are evolutionary conserved [1], playing an essentialrole in the recognition of PAMPs [2] from viruses, fungi, protozoanparasites and bacteria (vide infra) [2]. Different PAMPs are recognizedby specific TLRs [5].

Several immune cells, including B lymphocytes, selective popula-tions of T cells, dendritic cells (DCs) and macrophages [5,11] as well asnon-immune cells such as epithelial cells and fibroblasts express TLRs[5]. TLR expression can quickly change in the presence of cytokines,pathogens and environmental factors [5].

It is possible to distinguish TLRs on the basis of their intracellularlocalization. TLR1, TLR2, TLR4, TLR5 and TLR6 can be observed at the cellmembrane, while TLR3, TLR7, TLR8 and TLR9 into cell compartments,like endosomes. TLRs characterized by an intracellular localization recog-nize principally bacterial and viral nucleic acids, which are released andenter in contact with TLRs after being endocytosed and degraded in lateendosomes or lysosomes [5]. It has beenhypothesized that TLR intracellu-lar presence plays an essential role for the discrimination betweenself-DNA and viral DNA, thus avoiding the development of autoimmuneconditions (vide infra) [12].

After the recognition of PAMPs by TLRs, type I interferon (type I IFN),chemokines, inflammatory cytokines and co-stimulatory molecules arereleased by the immune system of the host [4–7].More in detail, TIR do-main plays a critical role in the activation of the downstream signalingpathway [3]. The function of TIR domain was identified in C3H/HeJmouse strain, characterized by a point mutation which caused anamino acid change to histidine at position 712 of the cytoplasmic pro-line residue [13,14]. This amino acid substitution induced a dominantnegative effect on the signaling mediated by TLRs [14,15]. TIR domainactivates the downstream signaling pathway through the interactionand recruitment of several adaptor molecules [3] such as myeloiddifferentiation primary-response protein 88 (MyD88), TIR domain-containing adapter protein (TIRAP) (also defined as MyD88 adaptor-like (MAL)), TIR domain-containing adapter protein inducing IFN-β(TRIF) (also known as TICAM1) and TRIF-related adapter molecule(TRAM) (also defined as TICAM2). This recruitment occurs throughTIR–TIR interactions [16]. Depending on which molecular adaptor isrecruited, a different signaling pathway is activated; in fact whereassome pathways are similar among TLRs, others are specifically activatedby only one TLR (vide infra) [17]. More in detail the recruitment ofMyD88 occurs for all TLRs, except for TLR3. Both TLR3 and TLR4 promotethe recruitment of TRIF adaptor; however while the first can initiateonly the TRIF-dependent pathway, TLR4 can activate also a MyD88-

dependent signaling [3]. The adaptor TIRAP is recruited in the signalingpromoted by TLR1, TLR2, TLR4 and TLR6 allowing, through its TIRdomain, the recruitment of MyD88 [3]. The fundamental role playedby TIRAP in the interaction with MyD88 has been demonstrated inTIRAP-deficientmicewhich showed TLR2- and TLR4-defective signalingpathways similar to those observed in mice deficient for MyD88.Accordingly MyD88/TIRAP-deficient mice did not show any additionaldefect as compared with mice showing a single deficiency affectingthese adaptors [17]. The TLR7/9 pathway is MyD88-dependent anddoes not require TIRAP [17].

2. TLR signaling mediated through the MyD88-dependent pathway

TheMyD88-dependent pathway characterizes the signaling inducedby all TLRs, whereas only TLR3 and TLR4 show a pathway independentfrom MyD88 (vide infra) (Fig. 1) [10,12].

MyD88 adaptor shows the presence of a death domain in theN-terminal region, whereas a TIR domain is localized in the C-terminal.MyD88 interacts with TLRs in their TIR domain and plays a critical func-tion for the induction of inflammation by TLR signaling [10,17]. As regardit has been demonstrated that MyD88 knockout mice did not showany response when stimulated with imidazoquinoline (a TLR7 ligand)or cytosine-guanine dinucleotides (CpG) DNA (a TLR9 ligand) [18–20],and interleukin-6 (IL-6) production after stimulationwith bacterialflagel-lin (a TLR5 ligand) [21]. FurthermoreMyD88-deficient mice did not showany inflammatory molecule production, the proliferation of B lympho-cytes or endotoxin shock upon lipopolysaccharide (LPS) stimulation [10].

After TLR activation, MyD88 allows the recruitment of IL-1 receptor-associated kinase (IRAK)molecule to TLRs by interactingwith the deathdomains of the two molecules [10]. Four members belonging to IRAKfamily have been identified (IRAK-1, IRAK-2, IRAK-4 and IRAK-M)[22]. Among these, IRAK-4 plays the most important role in the signal-ing pathway mediated by MyD88 (Fig. 1) [12,17]. The phosphorylationof IRAK causes its dissociation fromMyD88 and the subsequent associ-ationwith TRAF6, which constitutes an E3 ligase and is amember of theTRAF family [12]. TRAF6 is involved in the activation of TGF-β-activatedkinase 1 (TAK1) [23] and the canonical IκB kinases (IKKs) constituted byIKKα and IKKβ. IKKs cause the phosphorylation of IκB protein andits follow-up degradation by a proteasome-dependent pathwaypermitting nuclear factor κB (NF-κB) translocation into the nucleusand activation (reviewed (rev.) in [17]).

MyD88 can also promote the activation of the mitogen-activatedprotein kinases (MAPKs) p38, extracellular signal-regulated kinase 1/2(ERK1/2) and c-Jun N-terminal kinases (JNKs) [12,17].

As results of the activation of these TLR signaling pathways, severalinflammatory cytokine genes are expressed [17]. The critical role playedby MyD88 in the activation of inflammation has been demonstratedboth in mice deficient for MyD88 (vide supra) and in the presence of avariant form of MyD88 known as MyD88s [10]. MyD88s represents aspliced variant which is characterized by the loss of the intermediatedomain and thus could induce a negative regulation of the inflammato-ry process upon LPS stimulation [24,25].

3.MyD88-independent signaling pathway induced by TLR3 and TLR4

The stimulation of TLR3 and TLR4 can induce the activation of aMyD88-independent signaling pathway designed as TRIF-dependentpathway [12], through the association of TLR with TRIF moleculewhich occurs differently depending on which TLR is activated. In factwhereas TLR3 directly associateswith TRIF, TLR4needs the involvement

Fig. 1. TLR signaling cascade. The signaling pathwaydownstreamof TLR1, TLR2, TLR3, TLR4, TLR6, TLR7 andTLR9 are represented as examples. The activation of TLR2/1, TLR2/6 and TLR4onthe cellmembrane inducesMyD88 and TIRAP recruitment through TIR-TIR interactions.MyD88 leads to the recruitment of IRAKmolecules. IRAK-4has themost important role inMyD88-dependent signaling pathway. IRAK-4 is phosphorylated causing its dissociation from MyD88. Then IRAK-4 associates with TRAF6. The latter can activate IRF5 or TAK1. TAK1 can induceMAPK or IKKb activation. IKKb is involved in the nuclear translocation and activation of NF-kB. A MyD88-independent signaling cascade, called TRIF-dependent signaling pathway, isactivated by TLR3 (localized in endosomes) and TLR4. TLR3 binds directly TRIF, whereas TLR4 requires the involvement of the further adaptor TRAM to interact with TRIF. TLR7/9,whose localization has been described in endosomes, activate the signaling pathway through the recruitment of MyD88. The black arrows represent TLR signaling pathway, whereasthe red arrows indicate the effects of TLR signaling activation on DNA transcription. The black thicker lines represent the negative regulation of TLR signaling pathway. Figure is adaptedfrom Ref. [12] and [17].

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of TRAMadaptor (Fig. 1) [26]. TRIF can promote three different signalingpathways [17].

More in detail it can lead, through the involvement of IκB kinasesIKKε and TANK-binding kinase 1 (TBK1), to IFN regulatory factor 3(IRF-3) phophorylation [17] at the C-terminal region [12] and IRF-7activation [3,17]. IRF-3 and IRF-7 translocate into the nucleus, promotingthe expression of genes, such as type I IFN, and especially of IFN-β [17].

Moreover TRIF can induce NF-κB and MAPK activation through thedirect interaction with TRAF6 [17] or, through the presence of a RIPhomotypic interaction motif, can activate NF-κB by recruiting receptor-interacting protein 1 (RIP-1) [3,27].

In human cell lines it has been identified a further adaptor which ischaracterized by the presence of the TIR domain and is called sterile αand armadillo motifs (SARM). SARM is able to inhibit the signalingpathway induced by TRIF [9], but its role has not been yet identified inmammals [17,28]. In nematodes its homolog TIR-1 is fundamental forTLR-independent innate immunity [29].

Upon TLR4 stimulation, the deficiency of TRIF or MyD88 is responsi-ble for a defective NF-κB activation; while TRIF deficiency causes animpaired late activation, deficiency of MyD88 induces a defective earlyactivation [30].

4. The TLR-independent cytosolic pattern-recognition receptors(PRRs) for nucleic acids

In addition to the TLR signaling pathways previously described, re-cent studies have demonstrated a non-TLR system for the recognitionof nucleic acids [31], known as the TLR-independent cytosolic pattern-recognition receptors (PRRs). This was identified for the first time for

the recognition of double-strandedRNA (dsRNA) [32]. In fact in additionto TLR3, dsRNA is recognized by two cytosolic molecules, the retinoicacid-inducible gene I (RIG-I) and the melanoma differentiation-associated protein 5 (Mda-5) (Fig. 1) [17], recently implicated in thepathogenesis of T1D [33]. Unknown cytosolic molecules (representedas “X” in Fig. 1) are involved in the recognition of dsDNA [17,34]. Inboth cases the activation of IFN-β stimulator 1 (IPS-1)-dependentcytosolic signaling pathway occurs, leading to the synthesis of type IIFNs and inflammatory cytokines [17].

5. TLR ligands: bacterial, viral, fungal and protozoan PAMPs

PAMPs are products of metabolic pathways selective of a group ofmicroorganisms [17]; being necessary for their survival, PAMPs arehighly conserved among pathogens, in contrast to the heterogeneityof viral proteins, whose structure is rapidly modified to escape recogni-tion by the host immune system in mammals [10,17].

Several elements of the bacterial cell wall are recognized by TLRs.More in detail TLR4 and TLR2 sense LPS, constituting the wall ofGram-negative bacteria, and peptidoglycan (PG), forming a thick layerin the cell wall of Gram-positive bacteria, respectively. FurthermoreTLR2 recognizes lipoarabinomannan (LAM) of mycobacteria, and bothTLR2/6 or TLR2/1 sense diacyl or triacyl lipopeptides of mycoplasma,mycobacteria and bacteria [3].

Bacterial proteins, such as flagellin, represents PAMPs. Flagellin isthe principal constituent of bacterial flagella; the constant domain D1,relatively conserved among different species [5], is recognized byTLR5. Furthermore unmethylated CpG DNA of bacteria constitutes animmunostimulant sensed by TLR9 which resides in endosomes [3].

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Since TLR9 is present in this intracellular compartment, the recognitionby TLR9 occurs after the transport of bacterial DNA into endosome. HeredsDNA is degraded into single stranded regions containing CpG motifsthrough reducing and acidic conditions. In humans TLR9 expression islimited to B cells and plasmacytoid dendritic cells (pDCs) [5]. Selectivecomponents, not yet identified, from uropathogenic bacteria arerecognized by murine TLR11 [35].

In agreement with the important role played by TLR2 in recognizingPAMPs, it has been demonstrated that TLR2-deficient mice (TLR2−/−)were more susceptible to Streptococcus pneumoniae and Staphylococcusaureus infections [36,37]. After the recognition of bacterial PAMPs byTLRs, the release of inflammatory cytokines, and in particular conditionsof type I IFNs, is induced [3].

Among viral PAMPs recognized by TLRs, there are envelope proteinslike those of respiratory syncytial virus (RSV) and the hemagglutininprotein characterizing Measles virus recognized by TLR4 and TLR2, re-spectively. Also nucleic acids of viruses constitute PAMPs which areidentified by TLR3, TLR7, TLR8 and TLR9. Their DNA, such as the bacterialDNA, presents DNA motifs characterized by unmethylated CpG DNA.This last contains oligonucleotides that are able to strongly induceTNF-I from pDCs [3]. TLR7 and TLR8, whose genes are characterized byelevated homology, reside within the endosomal membrane. In miceTLR8 seems to be nonfunctional [5]. More in detail mouse TLR7 andhuman TLR8 recognize both viral and self guanosine and/or uridine-rich single-stranded RNA (ssRNA) [2,38] and synthetic antiviral mole-cules [38], like Imiquimod and R848 [3], whereas dsRNA produced byviruses during replication constitutes the ligand of TLR3 [3,17]. Accord-ingly TLR3 is able to sense polyinosine-deoxycytidylic acid (poly I:C)which is a synthetic analog of dsRNA and constitutes also a vaccine adju-vant (vide infra) [3]. In pDCs or in natural interferon-α producing cells(NIPCs), characterized by the release of high levels of IFN-α during viralinfections [39], viral PAMPs are recognized mainly by TLR7 [40]. AlsoDCs and fibroblasts secreted type I IFN in response to ssRNA viruses, butthis occurred in a signaling pathway independent from TLR7 [41]. Uponherpes simplex virus 1 and 2 (HSV-1 and HSV-2) infections IFN-α releaseinduced by TLR9 activation is mediated principally by pDCs [42].

Many fungal PAMPs activate TLRs [5], such as glucuronoxylomannanswhich is sensed by TLR4 and cluster of differentiation 14 (CD14),β-glucans and phospholipomannans recognized by TLR2. Accordinglywith the critical role played by MyD88 in TLR signaling pathway,MyD88-deficiency caused a higher susceptibility to develop fungalinfections in mice [3].

Regarding protozoan components recognized by TLRs, TLR2 senselipophosphoglycan (LPG) of Leishmania species and unsaturatedalkylacylglycerol of Trypanosoma species, whereas both TLR2 andTLR4 sense glycosylphosphatidylinositol anchors (GPI-anchors) andglycoinositolphospholipids (GIPLs) of Plasmodium (P.) falciparum,Toxoplasma (T.) gondii and Trypanosoma. Furthermore TLR9 recognizesTrypanosoma species' genomic DNA [3,43]. The human TLR11 is charac-terized by the presence of a stop codon in the gene which renders itnonfunctional, whereas the murine TLR11 recognizes a profilin-likemolecule from T. gondii. It has been hypothesized that more than oneTLR could sense PAMPs from different protozoan parasites, since micedeficient for only one TLR did not show defective responses to liveprotozoan parasites, whereas MyD88 deficiency caused a more generaldefect in TLR signaling [5]. In fact the deficiency of MyD88 influencedprotozoa infections in mice, inducing a defective synthesis of pro-inflammatory cytokines associated with an increased parasitemia andmortality [5,44].

6. The negative regulation of TLR signaling pathway

A strict negative regulation aimed to limit the immune process andinflammation characterizes TLR signaling pathway through severalmolecules with different activities (Fig. 1). Some molecules downregu-late the expression of TLRs, such as TLR4 and TLR9whose degradation is

induced by the E3 ubiquitin ligase Triad3A through a proteasome-dependent pathway (rev. in [17]). Other molecules, like TRAF4, IRF4and STL2, represent negative regulators of TLR signaling by sequestrat-ing molecules involved in this pathway [12,45]. TRAF4 prevents TRAF6recruitment to the adapter complex, although other independentmechanisms, including β-arrestins [46] and the intracellular ubiquitin-editing protein A20 [12,47], are able to negatively regulate TRAF6.

IRF4 and STL2 have the role to bind MyD88 [12]. IRF4 expression isincreased after TLR activation [12] and associated with MyD88 [48].This binding occurs in the same region of IRF5 andprevents IRF5 recruit-ment reducing the IRF5-dependent inflammatory process. ST2L is amember of IL-1 receptor family and, through its binding to MyD88and TIRAP, does not allow their recruitment to TLR4 [12]. Accordingly,IRF4- or ST2L-deficient mice were both characterized by a higherproduction of inflammatory cytokines, associated with hyper-activationof MAPKs and NF-kB (rev. in [12]), and with an altered induction of LPStolerance in case of ST2L deficiency [49].

Some molecules can degrade target proteins [12], such as IRF3whose phosphorylation at Ser339 promotes its ubiquitination anddegradation by proteasome in order to limit IFN responses [50], or thecaspase 8-dependent cleavage of TRAF1 induced by TRIF. This mecha-nism causes the release of a fragment of TRAF1with the ability to inhibitthe TRIF-dependent activation of NF-kB and IRF3 [51]. After TLR2 andTLR4 activation, the downstream signaling pathway is negatively regu-lated through the ubiquitination and degradation of TIRAP promoted bythe E3 ligase suppressor of cytokine signaling-1 (SOCS1) [52].

At last somemolecules inhibit the transcription of gene targets [12],like IL-6 and IL-12β. The access to the promoter region of these genes islimited for NF-kB and transcription factor activator protein 1 (AP-1)through the activity of histone deacetylases, recruited by activatingtranscription factor 3 (ATF3), which alters chromatine structure [53].

7. The possible involvement of nucleic acid PAMPs and endogenousligands in autoimmunity

The involvement of microbial PAMPs and endogenous ligands in au-toimmunity has been hypothesized through the activation of TLR and/ortheir increased expression acting in synergy with the formation ofautoantigen-autoantibody immune complexes (rev. in [2]). Necroticcells can induce danger signal and promote inflammation by activatingTLR4. This activation could be responsible for autoimmune responses[54]. Furthermore TLR4 can bind other endogenous ligands releasedfrom damaged cells, including several extracellular matrix components(ECM), such as hyaluronic acid oligosaccharides and fibronectin extradomain A, and fibrinogen which is able to promote the synthesis ofchemokines by macrophages. Mammals present unmethylated DNA ata very lower level in respect to viruses or bacteria and this may preventautoimmune responses to self-antigens [54].

Also the abnormal internalization and transport of dsDNA fragmentsfrom necrotic cells into endosomes could induce autoimmune responsesafter their binding to TLR3 [54].

Furthermore a correlation between TLRs and endogenous danger-associated molecular patterns, like advanced glycation end productshas been hypothesized. These can be sensed by PPRs that show thesame ligands of TLRs and activate the same intracellular pathway [55].

TLR agonists, such as poly I:C, constitute also vaccine adjuvantswhose aim is the induction of a stronger immune response towardsthe antigen contained in the vaccine and thus they confer a higher pro-tection. Although vaccine adjuvants have been supposed to be impliedin the onset of autoimmune disorders, through the activation of severalendosomal or surface TLRs, no convincing data support thepresence of acorrelation between vaccine adjuvants and autoimmunity [56].

However the host has some mechanisms that permit to avoidautoimmunity; among these, different cell compartments in whichTLRs recognize PAMPs and diverse levels of TLR expression in the vari-ous intracellular structures. In fact the recognition of proteins and lipids

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occurs on the cell membrane, whereas nucleic acids are recognized inendosomes and for this reason the expression of TLR7 is very low incells having high activity of phagocytosis. The recognition of self nucleicacids is avoided by the endosomal expression of TLR7 and TLR9 [57], asdemonstrated by the fact that chimeric TLR9 localized to the cellmembrane was able to respond to CpG DNA [5].

Moreover the DNA and RNA of the host have specific characteristicsin order to prevent autoimmunity (rev. in [17]). More in detail, althoughalso in vertebrates DNA is characterized by the methylation of CpG nu-cleotides, it shows a reduced frequency; furthermore there are specificsequences, identified also in the telomere [2], able to inhibit the signal-ing through TLR9 [2,58]. RNA of vertebrates differs from bacterial RNAfor the presence of many modified nucleotides with a reduced abilityto activate DCs in respect to the RNA of bacteria [59]. Anothermechanism that prevents the autoimmune response is the confinedexpression of nucleic acid-recognizing TLRs to specific cell types, suchas pDCs that express TLR7 and TLR9, but not TLR3. The dual activationof both TCR and B cell receptor (BCR) is essential for B lymphocyteactivation and thus TLR signaling, but under certain conditions, thiscould play a critical role in the pathogenesis of autoimmune disorders[2,17,60], including systemic lupus erythematosus (SLE). This disorderis characterized by the presence of nuclear debris due to the impairedclearance of apoptotic cells that can represent self-antigens able tobind BCRs, induce TLR9 expression and activate both TLR9 and Blymphocytes with the subsequent production of autoantibodies [61].

8. The involvement of TLRs in autoimmune disease onset

Increasing evidence has demonstrated the correlation between thepresence of altered TLR expression, some single nucleotide polymor-phisms (SNPs) or copy number variations (CNV) in TLR genes or inmolecules involved in TLR signaling and the development of severalautoimmune conditions including type I diabetes (T1D) [62–64],autoimmune polyendocrinopathy candidiasis-ectodermal dystrophy(APECED) [65], rheumatoid arthritis (RA) [66–70], SLE [71,72], systemicsclerosis (SSc) [73–76], Behcet's disease (BD) [77,78], Crohn's disease(CD), ulcerative colitis (UC) [79–81], multiple sclerosis (MS) [82,83],vitiligo [84] and myasthenia gravis (MG) [85,86] (Table 1).

8.1. Type I diabetes

Type I diabetes (T1D) represents an organ specific autoimmunedisorder in which pancreatic β cells of the islet of Langherans secerninginsulin are selectively destroyed by T-helper 1 (Th1) lymphocytes[87–89]. These cells begin the infiltration of the pancreas and, throughthe release of cytokines, support cytotoxic T lymphocytes (Tc)which are responsible for the progressive destruction of β cells [62].Although the etiologymust be yet elucidated, a combination of environ-mental, genetic and stochastic factors contributes to its pathogenesis[87,88,90]. The role of TLRs in T1D onset has been analyzed in severalanimal models. C57BL/6 mice co-treated with TLR3 agonist and insulindeveloped insulitis [91]. Furthermore TLR3 and TLR7 engagementconverted β-cell autoreactivity into overt autoimmune disease by up-regulating major histocompatibility complex (MHC) class I moleculeson murine pancreatic β cells [92]. TLR2 activation in APCs contributedto T1D onset in non-obese diabetic (NOD) mice by inducing β-celldeath [93]. In addition TLR activation induced by Kilham rat virus(KRV) infection promoted autoimmune diabetes in bio-breedingdiabetes resistant (BBDR) rats [94]. MyD88 represents another criticalfactor involved in T1D pathogenesis, as MyD88−/− NOD mice shows acomplete prevention from the disease [95,96].

Different expression of several TLRs has been described in T1Dsubjects as compared to healthy individuals. The analysis of TLR9mRNAexpression has been performed on 59 long-term (LT) T1 patients,33 new-onset (NO) T1D patients, 19 subjects at risk (AT) for T1D and 70controls [97]. TLR9 expression was significantly reduced in LT T1D

patients compared to healthy controls, whereas it was increased in theAT group than in controls. No differences have been observed in theNO group [97]. The group of Meyers [98] examined whether alteredTLR signaling was involved in the first stages of T1D. They found anincreased frequency of IL-1β-expressing monocytes and a reduction inmDCs expressing IL-6 in newly diagnosed T1D patients than non-diabetic subjects. In a following study Alkanani and colleagues [99]tested TLR-induced IL-6 and IL-1β release by monocytes and mDCsfrom genetically susceptible subjects positive for islet autoantibodiesand thus characterized by a higher risk to develop T1D. They reporteda slight increase of mDCs, pDCs and monocytes in seropositive individ-uals for autoantibodies than seronegative subjects, although this differ-ence was not statistically significant. They found also a dysregulation ofTLR-induced IL-6 and IL-1β responses in freshly isolated mDCs andmonocytes from seropositive individuals compared with autoantibody-negative subjects. It has beenhypothesized that these immunological ab-normalities observed in at-risk individuals before T1D onset could belinked with the early disease mechanisms by affecting the balance be-tween proinflammatory and regulatory mechanisms in the peripheralblood and/or pancreatic islet β-cells [99]. This alteration may induceislet inflammation and up-regulation of T cells recognizing pancreatic is-lets [100]. We have recently observed [101] that T1D subjects harboringthe protein tyrosine phosphatase non-receptor type 22 (PTPN22)C1858T SNP, which constitutes a genetic variant highly correlated withthe onset of several autoimmune conditions including T1D [90], showedan enhanced TLR response upon stimulationwith CpG that led to alteredB cell phenotype composition. Increasing evidence supports the hypoth-esis that activation of TLRs, including TLR9, could promote autoimmuneconditions, and infections may play a critical role in triggering diseaseonset [101].

Further studies have described the presence of an altered TLR expres-sion not only in T1D subjects, but also in their first-degree relatives. Thewhole-genome expression profile of peripheral bloodmononuclear cells(PBMCs) isolated from 9 T1D patients, their 10 first-degree relatives and10 healthy controls has been analyzed by using the human high-densityexpression microarray chip [62]. T1D subjects showed a significantdifferent expression of 9 genes with respect to controls. First-degree rel-atives of T1D patients, independently by the presence or not of autoanti-bodies, showed up-regulation of TLR2, TLR6 and TIRAP in respect withhealthy controls. This observation supports the idea that autoantibody-negative healthy relatives showed however a different regulation ofseveral immune-related signaling pathways [62], accordingly with thepresence of proinflammatory islet-selective T cell responses observedin these subjects by the group of de Marquesini [102].

Devaraj and colleagues [103] described a significant increase of TLR2and TLR4 ligands in T1D patients, supporting the role played by theproinflammatory environment in T1D onset. Regarding the associationbetween T1D and the presence of SNPs inmolecules involved in TLR sig-naling, the study conducted by the group of Castiblanco [104] on T1Dsubjects from a Colombian population reported no associationbetween the TIRAP (MAL) functional polymorphism S180L and T1D.

T1D was associated with TLR2 SNP (rs3804100) in the Norwegianand Korean populations, but this correlation was not observed in theSpanish population (rev. in [64]). The group of Assmann investigatedwhether TLR3 (rs11721827, rs13126816, rs5743313, rs7668666 andrs3775291) SNPs were involved in T1D onset [105]. 449 T1D patientsand in 507 nondiabetic subjects were analyzed. TLR3 (rs3775291) and(rs13126816) SNPs were associated with risk for T1D. Furthermore anassociation between an early age at diagnosis and poor glycemic controland (rs5743313) and (rs117221827) SNPswas described [105]. Recent-ly 28 SNPs in TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR8 and TLR9, many ofthose even not included in previous genome wide association studies(GWAS), were genotyped in 429 Chinese Han T1D patients and 300healthy controls [64]. The analysis demonstrated for the first time theassociation of T1D with TLR1 ((rs5743612, -626) and (rs4833095, -1017)) and TLR6 (rs3775073, -1329) SNPs. Conversely, in the Caucasian

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T1D population TLR1-626 SNP was not analyzed in GWAS, whereasTLR1-1017 and TLR6-1329 SNPs did not show any correlation with thepathology [64]. It has been hypothesized that this discrepancy betweenthe two studies could be due to different ethnicity. Further investiga-tions on other populations could elucidate the putative association be-tween TLR SNPs and T1D onset [64].

Since in children at risk for T1D IFN-I expression has been reportedbefore the onset of islet autoimmunity and moreover recent studieshave strongly associated T1D with RNA virus infection (rev. in [33]),Lincez and colleagues [33] have evaluated the role of MDA5, encodedby T1D risk gene interferon induced with helicase C domain 1 (IfiH1).This gene selectively recognizes dsRNA virus intermediates. They dem-onstrated that a reduced expression of IfiH1 protected NOD mice fromspontaneous and virus-mediated T1D. Mice heterozygous for the Ifih1gene showed a lower MDA5 protein expression, promoting theexpansion of regulatory T cells (Tregs) with a concomitant reductionof effector CD4+ T cells in the pancreas [33].

8.2. Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy

APECED (OMIM#240300), also called autoimmune polyendocrinesyndrome type I (APS-1) [106], is a rare condition characterized by en-docrine gland destruction andmucocutaneous candidiasis [65]. It repre-sents an autosomal recessive pathology due to mutations in theautoimmune regulator (AIRE) gene [107]. AIRE expression has beenmostly described in the thymus and also in peripheral tissues, such asfetal liver, testis, ovary, peripheral lymphoid organs and in the peripher-al blood, including CD14+, DCs, granulocytes, macrophages and B cells(rev. in [65]). AIRE regulates the negative selection of organ-specific T

Table 1TLRs and molecules of the TLR signaling pathway affected by altered gene expression, CNV and

Gene Altered expression CNV SNP

TLR1 MG [85] nr T1DTLR2 T1D [62];

BD [78];ocular BD [181];MS [197];MG [85]

nr T1DSScvitilocul

TLR3 SSc [76];RA [123–125];ocular BD [181];MG [85]

nr MS (T1DEarlyRA a

TLR4 SSc [76];BD [77,78]; IBD [81];RA [125];ocular BD [181];MS [197];MG [85,86]

nr RA (vitiliIBDCaucSLE (CD (

TLR5 MG [85] nr UC (TLR6 T1D [62];

MG [85]nr T1D

TLR7 RA [124] Childhood SLE onset in Mexican population [71];SLE in Yucatan Mayan [72];BD in Chinese Han population [187]

SLE

TLR8 Ocular BD [181];MG [85]

nr AssopopuSLE[155

TLR9 T1D [97];RA [126];SLE [141];MG [85]

nr MS (RA (SLE

TLR10 MG [85] nr CD [TIRAP (MAL) T1D [62] nr BD (TICAM1 nr nr VitilIRF5 nr nr MS (

RA [tionsSLE iconfiand

cells; its loss caused both an altered negative selection of self-directedT cells in the thymus [65] and the presence of autoantibodies in the pe-ripheral blood.Moreover several tissueswere infiltrated by autoreactiveT lymphocytes [65,108]. Zhu and colleagues [65] observed an over-expression of mRNA and protein induced by the interaction of AIREwith the promoter regions of TLR1, TLR3 and TLR8 genes in a mousemacrophage-like cell line (RAW264.7) stably expressing AIRE. In addi-tion mRNA levels of target gene products, such as TNF-α and IFN-α,were enhanced upon stimulation with TLR1 and TLR3 ligands.

Conversely, the group of Hong [109] did not report any alteration inthe expression of pattern recognition receptors in APECED patients, af-fected or not by Candida infection with respect to healthy controls.

The contrasting results among the two studies could be due to thedifferent cell type or species of origins. However the observations re-ported by Hong et al. [109] regarding the absence of altered TLR2, TLR4and TLR6 expression [109] could not explain the increased susceptibilityof APECED patients to Candida infection [65].

8.3. Rheumatoid arthritis

RA is a chronic inflammatory disease in which joint structure of theaffected subject is destroyed by activated immune cells that infiltrateand accumulate into the synovial joints. Several different cell popula-tions, such as macrophages, which are the principal responsible for sy-novitis, contribute to the release of pro-inflammatory cytokines andother mediators leading to RA onset [110]. Several animal modelswere used to investigate the role of TLR3 and TLR7 in RA pathogenesis.A significant TLR3 up-regulation has been observed in splenocytes inthe rat pristane-induced arthritis (PIA) model upon pristane treatment

SNPs in human autoimmune disorders. nr = not reported.

(rs5743612, -626 and rs4833095, -1017 in Chinese Han [64]).(rs3804100 in Norwegian and Korean populations [64]);(rs5743704, Pro631His in Europeans [74]);igo (rs5743708, Arg753Glu in Turkish population [84]);ar BD (rs2289318 and rs3804099 in Chinese Han population [186]).

rs3775290, c.1377 in Han people from South China [82]);(rs3775291 and rs13126816 [105]);age at T1D diagnosis and poor glycemic control (rs5743313 and rs117221827) [105];nd with sero-negative RA and disease activity (rs3775291 in a Danish population [132]).rs1927911 in Caucasians [70]);go (rs4986790, Asp299Gly in Turkish population [84]);(rs4986790, Asp299Gly in Caucasians [79–81,189–192]; rs4986791, Thr399Ile inasians [79–81,189–192]);D299G in South Indians [152]);rs4986790, Asp299Gly [190]).R392X and N592S in North Indian population [195].(rs3775073, -1329 in Chinese Han population [64]).

(rs3853839 in Taiwanese females [154] and in Danish patients [155]).

ciation with rheumatoid factor autoantibody positivity (rs5741883, in Europeanlation [68]);

[156], SLE (rs3764880 in Taiwanese females [154]; rs3764879 in Danish subjects]).rs352140, 2848 in Han people from South China [82]);rs187084, -1486T/C in Turkish population [134]);(rs5743836, -1237 C/T in South Indian Tamils [153]).

193]; CD susceptibility and clinical outcome in a New Zealand population [194].S180L in UK population [185])igo (Caucasians [199])rs4728142 and rs3807306 in Spanish, Swedish and Finnish [83]);67]; RA in European Caucasian [135], Korean [136], Tunisian [137] and Japanese popula-[138];n European Americans [157], African Americans [158], Swedish [159], Chinese [160] andrmed in four ethnicities (European Americans, Hispanic Americans, African-Americans,Asian Americans) [156]; SLE (rs10488631 in an Egyptian cohort [161]).

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and stimulation of TLR3 with poly-I:C. Accordingly, TLR3 inhibition byusing small interfering RNA (siRNA) ameliorated RA in this animalmodel [111]. The treatment with methotrexate (MTX) inhibited RAsymptoms and induced TLR3 over-expression in splenocytes from PIAand collagen-induced arthritis (CIA) rat models [112]. Contrastingdata emerged from the study conducted by Yarilina and colleagues[113], where it was demonstrated that TLR3 activation inhibitedarthritis in CIA and K/BxN serum transfer models. These data supportthe hypothesis that TLR3may play a role in the regulation of inflamma-tion rather than in the promotion of the inflammatory process [113].

Regarding TLR7 role in RA pathogenesis, a brief TLR7 stimulationwith the agonist 1 V136 at low level was not sufficient to inducecytokine release, but onlywas able to establish TLR7 tolerance and ame-liorated disease in the K/BxN serum transfer model [114]. AlthoughTLR7−/−CIAmodel andWTmice developed RAwith a similar frequency,TLR7−/−CIA model was characterized by a decrease in paw swelling,number of paws affected and clinical score than WT mice [115].Accordingly, an amelioration of RA symptoms was observed afterintra-articular TLR7 knockdown in rat CIA model [116].

TLR8 role in RA must be yet fully elucidated, in fact whereas inhumans TLR8, such as TLR7, is functional allowing the recognition ofssRNA, in murine models TLR8 did not activate the downstream signal-ing upon ligation to ssRNA [117]. Furthermore autoimmunity onset wasdescribed in TLR8−/− mice and found associated with enhanced TLR7expression in DCs, whereas autoimmunity did not develop when TLR7and/or TLR8 were knocked down [118]. Thus TLR7 signaling could bemodulated by TLR8 [118].

TLR9 may play an anti-inflammatory role in arthritis through theinduction of tolerance. It has been demonstrated that TLR3, TLR4, TLR7and TLR9 stimulation requires PTPN22, whose variant was associatedwith RA onset, for IFN production [119].

Accordingly arthritis was suppressed after TLR stimulation inPTPN22 knockout mice [120]. PTPN22 has been widely studied both inseveral murine models and in humans since it constitutes one of themost important genes able to influence the susceptibility to autoim-mune disorders by regulating both innate and adaptive immunity [121].

Moreover it has been reported that TLR9 antagonist diminishedarthritis severity in the rat PIA model suggesting a putative role forTLR9 in arthritis onset [122].

TLR expression has been evaluated also in RA synovium whichshowed the expression of TLR3, TLR7, TLR8 and TLR9. Moreover TLR3,TLR7 and TLR9 were up-regulated, especially in RA synovial fibroblasts(RASFs), macrophages and DCs [123–126], as compared with osteoar-thritis or healthy subject tissues. TLR3 and TLR8 ligands promotedcytokine and matrix metalloprotease (MMP) production in RA synovialcells. Among TLRs, the higher amount of TNF released from RA synovialcultures was induced by TLR8 [66].

Several studies have investigated the correlation between TLR SNPsand RA, reporting the presence of several associations in different ethniccohorts. TLR4 Asp299Gly was not associated with RA in an English cohort[127]. Conversely the case-control study conducted in theNetherlands re-ported that the condition of heterozygosity for Asp299Gly was protectivein early untreated RA [128]. Kuuliala and colleagues [129] observed thatAsp299Gly SNP interfered with a quick response to therapy in RA thanAsp299Asp. Regarding TLR2 SNPs, no association has been found betweenTLR2 Arg677Trp (rs number was not reported) and Arg753Gln(rs5743708) SNPs and RA in Spanish patients [130].Moreover no correla-tion was observed between the pathology and TLR1, TLR2, TLR4, TLR6 andTLR9 SNPs in a cohort of French RA patients [131], whereas a recent studydemonstrated that TLR4 (rs1927911) SNPwas associatedwith disease ac-tivity in Caucasian RA patients [70]. Recently it has been demonstratedthe association of TLR3 (rs3775291) SNP with RA in a Danish populationand also with sero-negative RA and disease activity in this subset [132].

TLR9 SNP (rs187084) was modestly associated with RA in a Turkishpopulation, whereas no correlation has been found in French and Dutchcohorts [131,133,134]. A correlation between TLR8 SNP (rs5741883)

and rheumatoid factor autoantibody positivity has been reported in anEuropean population [68]. The case–control study performed by thegroup of Sanchez examined the possible association of TLR4 SNPs(Asp299Gly rs4986790 and Thr399Ile rs4986791) with RA. 224 Spanishpatients affected by RA and 199 healthy subjects were analyzed but anysignificant difference was not observed in TLR4 SNPs genotype or alleledistribution in RA patients with respect to controls [130].

The meta-analysis conducted by the group of Han [67] demonstratedan association between IRF5 SNP and RA. IRF5 confers susceptibility to RAand was able to influence its erosive phenotype in European Caucasians[135]. Moreover this correlation was furtherly confirmed in a Korean[136], Tunisian [137] and Japanese [138] populations. Also MyD88 andMAL/TIRAP are involved in the inflammation and in the process ofdestruction in human RA synovial tissue cultures [139].

8.4. Systemic lupus erythematosus

SLE is a systemic autoimmune condition which occurs morefrequently in women. It is characterized by B cell hyperactivity andthe presence of various circulating autoantibodies [72,140], in particularto dsDNA and RNA-binding proteins [140]. The etiology is multifactori-al: genetic, environmental factors, including hormones, UV light, drugs,infections and immune system alterations are involved in its pathogen-esis (rev. in [72]). Although the role of innate immunity in autoimmunedisorders is not yet fully elucidated, it has been hypothesized that al-tered innate immunity responses may play a critical role in SLE onset[141], as demonstrated by the presence of several immunologicalalterations in individuals affected by SLE. SLE patients showed increasedapoptosis responsible for the release of DNAand RNA that can be sensedby TLR9 and TLR7, respectively [142,143]. PBMCs obtained fromSLE patients with active disease presented an expansion of TLR9-expressing B cells and monocytes in respect to patients with inactivedisease in correlation with the presence of anti-dsDNA antibodies [61].Accordingly, the study performed by the group of Nakano [141] demon-strated an over-expression of TLR9, strictly related to disease activity, onB lymphocytes in SLE subjects. SLE patients are characterized also by anabnormal methylation of DNA. This process leads to the production ofCpG, which could be involved in the activation of TLR9. As a conse-quence of TLR9-CpG interaction, anti-dsDNA antibody and IL-10production are induced [141]. The latter is a regulatory cytokine ableto promote the production of pathogenic antibodies levels in certaindisorders, especially in SLE [141,144].

TLR7 plays a critical role in SLE pathogenesis through the binding toself-antigens containing endogenous RNA followed by the induction oftype I IFN expression [145].

Furthermore increasing evidence observed in murine SLE modelsdemonstrates the role played by TLR7 and TLR9 in autoimmune process-es. Reduction of SLE development was observed after treating (NZB ×NZW)F1 mice with a TLR7 and TLR9 dual inhibitor [146] and C57BL/6(B6)-FasIpr mice carrying the Unc93b1 mutation that affects TLR7 andTLR9 signaling [147].

The study of TLR7-deficient MRL-FasIpr and deficient TLR9-deficientMRL-FasIpr mice revealed a marked suppression of autoimmuneresponses against RNA-related autoantigens [148,149] and the involve-ment of TLR9 in anti-DNA and anti-chromatin autoantibodies production,respectively [148,150].

RNA-selective autoantibody production was enhanced both inFcYRIIB-deficient B6 mice over-expressing TLR7 and in mice presentingthe duplication of TLR7 gene [72].

Several studies have analyzed in different populations whether CNVof TLR7 could represent a risk factor for SLE onset reporting contrastingresults. The study conducted by Kelley and colleagues [151] regardingthe relative copy number of TLR7 in 50 Caucasian and 49 African-American SLE patients and 91 healthy controls, reported that CNV wasnot a risk factor for SLE onset in these populations andwas not associat-ed with the autoantibody profile. Conversely, it has been demonstrated

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that CNV of TLR7was correlated with childhood SLE onset in the Mexi-can population [71]. More in detail SLE women showed a significant in-crease in CNV of TLR7 in respect to controls; furthermore an increasedassociation has been described in female as compared with male pa-tients [71]. A recent work performed by the group of Pacheco [72]highlighted the role of TLR7 CNV in 80 Yucatan Mayan SLE womenand 150 controls, reporting the association between SLE developmentand the increased copy number of this gene to 3 copies observed in10% of SLE patients. However any significant difference in mRNA TLR7expression between patients and controls was not described. It hasbeen hypothesized that the increased copy number of TLR7 could pro-mote autoimmune processes, through the enhanced production ofIFN-α [72].

Several investigations have analyzed the possible association be-tween SLE and TLR SNPs. A recent investigation performed by Rupasree[152] confirmed for the first time that TLR4 D299G SNP increased therisk for SLE among South Indians. Moreover TLR4 (D299G, T399I),TLR9 -1486T N C and TIRAP S180L SNPs contributed towards phenotypicheterogeneity and they were able to influence specific autoantibodyproduction in SLE. Another recent study investigated whether TLR2(R753Q) and TLR9 (-1237C/T) SNPs were associated with lupus suscep-tibility, clinical and autoantibody phenotypes. Samples from 300 SLESouth Indian Tamils and 460 ethnicity matched controls were analyzedby real time PCR [153]; whereas TLR2 gene remained monomorphic inpatients and controls, therefore not conferring susceptibility to SLE,the T allele of TLR9 gene was more frequent and conferred a significantrisk to develop SLE. However both SNPs did not influence the clinical orautoantibody phenotype of SLE and could exert an additive effect in thepresence of other genetic and environmental risk factors increasing thesusceptibility to SLE in South Indian Tamils. Recent investigationsdemonstrated that TLR7 (rs3853839) and TLR8 (rs3764880) increasedrisk of SLE in Taiwanese females [154]. The previously association re-ported between the (rs3853839) SNP of TLR7 and SLE in Asian patientshas also been observed in Danish patients [155]. Furthermore the groupof Armstrong have identified TLR8 as a new SLE-associated gene [156].TLR8 (rs3764879) SNP correlated with SLE in Danish subjects [155].

A diminished susceptibility to SLE onset has been observed in thepresence of a SNP affecting MAL protein which is involved in TLR4 andTLR2 signaling pathways (rev. in [45]). The association between IRF5variants and SLE had been described in several populations, such asEuropean Americans [157], African Americans [158], in a Swedishcohort [159], in Chinese [160] and confirmed in four ethnicities(European Americans, Hispanic Americans, African-Americans, andAsian Americans) by Armstrong and colleagues [156]. A recent studyprovides additional evidence for the association between IRF5rs10488631 variant and lupus susceptibility in anEgyptian cohort [161].

8.5. Systemic sclerosis

SSc is a complex and dynamic connective tissue pathology ofunknown etiology characterized by initial vascular injuries followedby exagerated extracellular matrix (ECM) and collagen production dueto fibroblasts hyperactivation [162,163]. SSc encompasses diffuse cuta-neous SSc (dcSSc) and limited cutaneous SSc (lcSSc). In dSSc, fibrosischaracterized not only the skin but also internal organs causing theirfailure, whereas in lcSSc collagen is deposited prevalently in skin andthere are also vascular complications [73]. No treatment is available tostop the progression of fibrosis [164]. Several studies performed on an-imalmodels and SSc patients have supported the role of TLRs in its path-ogenesis by converting a self-limited tissue repair into an uncontrolledfibrotic process [73,75,76]. Among endogenous TLR ligands involved inSSc onset there are cellular stress proteins, matrix-derived molecules,immune complexes and nucleic acids released from necrotic ordamaged cells [73].

More in detail, C3H/HeJmice carrying a SNP in TLR4 showed a reduc-tion in skin sclerosis induced by bleomycin [165], although endogenous

TLR4 ligands were present at high concentration [76]. Accordinglybleomycin-treated TLR4−/− mice (rev. in [76]) showed a decrease indermal and lung fibrosis than their wild-type counterparts. The groupof Takahashi [76] confirmed the critical role of TLR4 in another SScmurine model called TLR4−/−;TSK/+ mice, characterized by the lossof TLR4, observing a strong decrease in hypodermal fibrosiswith respectto control TSK/+ mice.

It has been described that SSc patients had high levels of highmobil-ity group box 1 (HMGB-1) and hyaluronic acid (HA) both in serum andlesional skin (rev. in [76]). These two molecules stimulate TLR4 and,through the up-regulation of transforming growth factor β (TGF-β)signaling, induced fibroblast activation [165].

Moreover the deletion of TLR4 inhibited the expression of IL-6, apro-fibrotic cytokine involved in SSc pathogenesis [76], in fibroblasts,immune and endothelial cells upon treatment with bleomycin and LPSin vivo and in vitro, respectively. Conversely, enhanced expression ofboth TLR3 and TLR4 were observed in affected skin and lung biopsiesobtained from SSc subjects.

In a recent investigation 14 functional SNPs in TLR2, TLR4, TLR7, TLR8and TLR9 have been genotyped in a discovery cohort of 452 EuropeanSSc patients and 537 geographically-matched healthy controls. The rep-lication cohort was composed of 1,170 SSc patients and 925 controls.The rare Pro631His SNP in TLR2was associated with antitopoisomerasepositivity and with SSc phenotype [74]. Moreover functional studyshowed higher levels of inflammatory mediators produced bymonocyte-derived DCs carrying the TLR2 variant Pro631His after stimu-lation with TLR ligands [74].

8.6. Behcet's disease

BD is an inflammatory disorder whose complex pathogenesis iscaused by genetic and environmental factors [166–168], such as micro-bial factors in genetically susceptible subjects [166,169,170]. It is charac-terized by the presence of recurrent oral aphthous and genital ulcers,uveitis, skin lesions [167,169] and can involve the gastrointestinaltract (intestinal BD) [167,169,171] and the CNS [166,167]. Immunolog-ical defects are present in BD patients, including alterations in the phe-notype and functions of lymphocytes, neutrophil hyper-activation andup-regulation of proinflammatory and Th1-type cytokines [172,173].It has been hypothesized that infectious agents, including Streptococcussanguis [174] or HSV [175,176], could play a role in BD pathogenesis.Furthermore micro-organisms presenting antigens, like heat shockproteins (HSPs), characterized by molecular mimicry could cause auto-immune responses through the activation of selective TCRs and the in-nate immune system by stimulating TLRs. These immunologicalalterations may induce BD onset [77].

The involvement of TLRs in BD pathogenesis has been demonstrated[78]. More in detail TLR2 and TLR4 mRNA expression were observed inileocaecal ulcer lesions, whereas no TLR expression was reported in un-affected sites of the same sample, allowing to hypothesize the existenceof a correlation between TLR expression and intestinal BDmanifestation[78]. Furthermore the co-presence of HSP60, constituting a non-pathogen-derived ligand of TLRs [78,177,178], TLR2 and IL-12 in thelesions could indicate the involvement of TLR2 expressing cells in pro-moting the damaging Th1-type responses [78]. TLR4 mRNAwas consti-tutively increased in PBMCs of BD patients, regardless of diseaseactivity [77]. TLR4 up-regulation might be responsible for the defectiveheme oxygenase (HO-1) expression observed in BD PBMCs, leading tothe enhanced inflammatory response that characterized BD [77]. Infact HO-1 constitutes an inducible heme-degrading enzyme able to sup-press inflammatory processes and whose deficiency was correlated tosevere chronic inflammation in both HO-1 knockout mice [179] andhumans [180]. HO-1 expression was suppressed upon stimulationwith LPS (a TLR4 ligand) or HSP60, whereas LPS inhibitory effect wascompletely blocked by polymyxin B, a LPS neutrizer [77]. A followingstudy confirmed the presence of a marked higher expression of both

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mRNA and protein level not only of TLR4, but also of TLR2, TLR3 andTLR8 in PBMCs from active ocular BD patients as compared with con-trols, leading to hypothesize that TLR up-regulation may be involvedin the pathogenesis of BD [181]. Many studies have investigated thepossible correlation between TLR SNPs and BD.

It has been demonstrated that TLR9 SNPs were not correlated withBD susceptibility in Japanese patients [182]. Dhifallah and colleagues[183] reported no alteration in TLR9 (1237T/C rs5743836) SNP frequen-cy between Tunisian BD patients and healthy controls, whereas differ-ences were observed in the distribution of TLR9 (1486T/C rs187084)SNP genotype frequencies, but they did not reach statistical significance.Furthermore no correlation has been observed between TLR9 SNPs andthe main clinical manifestations. The group of Boiardi [184] investigatedthe association of Asp299Gly (rs4986790) and Thr399Ile (rs4986791)TLR4 SNPs with BD in 189 Italian patients and 210 healthy volunteers,but no statistical significant differences were observed in the distributionof allele and genotype frequencies between BD patients and controls.

TLR2, TLR4 and TIRAP SNPs were analyzed in BD patients fromdifferent geographical areas and only TIRAP S180L was significantlyassociated with the pathology in UK individuals; however this correla-tion was not observed in Middle Eastern patients [185].

In the two-stage control-association study performed by Fang andcolleagues [186], an association between ocular BD and TLR2 SNPs(rs2289318 and rs3804099) was observed in a Han Chinese population,although these two SNPs did not exert any effect on the release ofTNF-α, IL-6, IL-10, and IL-1β. No association was reported between BDand TLR2 SNP (rs13150331), TLR4 SNP (rs7037117), TLR8 SNP(rs3764880) and TLR9 SNPs (rs187084, rs352139, rs352140). A recentinvestigation conducted by the same group [187] provided evidencethat a high copy number of TLR7 conferred risk for BD in a ChineseHan population; however TLR7 CNV did not induce any alteration onthe release of TNF-α, IL-6, IL-1β, and IFN-β.

8.7. Crohn's disease and ulcerative colitis

CD andUC represent two principal forms of idiopathic inflammatorybowel disease (IBD). This is a complex disorder characterized by thepresence of a chronic inflammation affecting the gastrointestinal tractand frequent extra-intestinal manifestations [188]. More in detail aber-rant innate and adaptive immune responses directed towards PAMPsfrom microorganisms constituting the intestinal flora, have beenhypothesized to be responsible for IBD onset in genetically susceptiblesubjects. This hypothesis is supported by the observation that UC andCD patients show an impaired epithelial barrier and an enhancedintestinal permeability [188].

Although its pathogenesis is not fully elucidated, multiple non-genetic and genetic factors could be involved in IBD etiology includingaltered TLR expression and TLR SNPs [81]. TLR4 expression was low inintestinal biopsies from healthy subjects, whereas it was strongly en-hanced in intestinal epithelial cells, local macrophages and DCs presentin the inflamed mucosa of IBD patients (rev. in [81]).

The possible involvement of TLR2 (Arg677Trp and Arg753Glu), TLR4(Asp299Gly and Thr399Ile) and TLR9 (1237T/C) SNPs in IBD pathologyhas been evaluated by Shen and colleagues in Chinese Han andCaucasian populations [80]. They described an association betweenboth TLR4 SNPs and CD and UC only in Caucasians.

A meta-analysis conducted by the same group [81] confirmed thesignificant correlation between TLR4 (Asp299Gly and Thr399Ile) SNPsand CD and UC in the Caucasian population, whereas no associationwas observed between Asp299Gly and CD phenotype, including age atonset. These data were in agreement with the study conducted byFranchimont and colleagues [79] in a prevalent Caucasian populationand with other meta-analyses previously performed [189–192]. Otherstudies have reported contrasting results. In fact whereas some analysisdescribed only a higher frequency of Asp299Gly allele in patients withCD and UC (rev. in [81]), the study conducted by De Jager [190] showed

a significant correlation betweenAsp299Gly and CD, but not UC. Furtheranalysis have to be performed to confirm the association between TLR4SNPs and UC in Caucasians. An association of TLR10 and susceptibility toCD has been described in two cohorts of 284 and 224 CD patients [193].Furthermore genetic variations in TLR10 play a role in interindividualdifferences in CD susceptibility and clinical outcome in a New Zealandpopulation [194].

A recent study investigated whether TLR1-R80T, TLR2-R753Q, TLR3-S258G, TLR5-R392X and -N592S and TLR6-S249P SNPs in UC patientsfrom North Indian [195]. Only TLR5 variants R392X and N592S showedsignificant association with UC. Patients carrying different genotypes ofTLR4 and TLR5 SNPs showed a significant modulation of cytokine level[195].

8.8. Multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory disease affectingthe CNS which is characterized by immune mediated demyelinationand damage of axons. MS constitutes a complex disorder and it hasbeen hypothesized that environmental and genetic factors contributeto its etiology [196]. It is estimated that it affects more than twomillionof people worldwide. The expression of TLR2 and TLR4were assessed byflow-cytometry on PBMCs and a significant high expression of both hasbeen observed inMS patients than in healthy controls [197]. It has beenhypothesized that elevated expression of these genes could be respon-sible for innate immune activation in patients affected by MS [197].TLR3 c.1377 and TLR9 2848 SNPs were related to MS in Han peoplefrom South China, whereas TLR9-1486 did not show any associationwith MS [82]. Two IRF5 SNPs (rs4728142 and rs3807306) and a 5 bpinsertion-deletion in the promoter and first intron of IRF5 genewere as-sociated with MS in Spanish, Swedish and Finnish patients, whereas noassociation was reported between IRF5 rs12539741 SNP and MS [83].

8.9. Vitiligo

Vitiligo represents a complex disease in which the autoimmunedestruction of melanocytes causes the onset of pigmented skinmaculopatches [198]. Although its etiology remains unknown, geneticand environmental factors contribute to its onset. A recent study inves-tigated whether TLR2 SNP (Arg753Glu) and TLR4 SNPs (Asp299Gly andThr399Ile) were associated with this disorder. A total of 100 Turkishpatients affected by vitiligo and 100 controls have been analyzed.They reported a significant association between TLR2 and TLR4Asp299Gly SNPs, while no difference has been observed in TLR4Thr399Ile SNP distribution among vitiligo and healthy subjects [84].Among genes reported to be associatedwith vitiligo, TICAM1 representsa susceptibility loci in Caucasians (rev. in [199]).

8.10. Myasthenia gravis

MG represents a rare T cell-dependent autoimmune syndrome char-acterized by autoantibodies recognizing several components of the neu-romuscular junction (NMJ) and causing muscle weakness and earlyfatigability (rev. in [200,201]). Several forms of MG have been identifiedon the basis of age of disease onset, associated thymus pathologies andthe presence of pathogenic autoantibodies (rev. in [201]). Althoughthe pathogenesis remains unclear, genetic and environmental factors,especially prolonged viral infections, appear to be involved in MGonset [200,201].

Several evidence support the hypothesis that pathogen infectionscould represent triggering factors for MG. This may occur through amechanism of molecular mimicry of microbial antigens that could pro-mote the activation of T lymphocytes against self-antigens. In particularMeasles and Epstein–Barr virus (EBV) infections have been demonstratedto precede this autoimmune condition [200]. Increasing data havehighlighted that thymus, the first organ inwhich occurs autosensitization

980 E. Gianchecchi, A. Fierabracci / Autoimmunity Reviews 14 (2015) 971–983

andmainteinance of the autoimmune response inAChR-positiveMG sub-jects, presents alterations not only in its morphology but also in its func-tions in 80% of patients affected by the generalized form of MG [200,201]. Cavalcante et al. [200] reported the persistence and reactivation ofEBV in the thymus of patients with the early onset MG. EBV infectionmay sustain the autoimmune response in the thymus by activating andimmortalizing thymic self-reactive B lymphocytes. Moreover these cellscan leave the organ and continue autoimmune responses also in the pe-riphery. Here autoimmunity may be sustained by the fact that Tregs aredefective and by the formation of skeletal muscle-derived muscle-typeacetylcholine receptor (AChR)/immune complexes in lymph nodes.Most of the MG cases have autoantibodies towards the AChR (rev. in[201]). The chronic self-perpetuating inflammation may be due to TLR-signaling activation [202]. Few studies have investigated the involvementof TLRs in MG onset. mRNA expression of several TLRs was found to behighly altered in PBMCs obtained from MG patients in respect to non-MG patients: TLR2, TLR3, TLR4, TLR5, TLR8 and TLR9 expression was en-hanced, whereas TLR1, TLR6 and TLR10 was diminished. Furthermore acorrelation between TLR9 mRNA expression and MG clinical severitywas observed; this envisages the potential involvement of TLR signalingpathway in MG pathogenesis [85]. Bernasconi et al. [86] found thatTLR4was over-expressed in hyperplastic thymus fromMGpatients. A po-tential association between viral infection and MG has been supportedalso by the fact that poly (I:C) injections were able to induce the selectiveproliferation of B lymphocytes, the production of serum anti-AChR anti-bodies and MG-like clinical signs in wild-type mice, but not in mice defi-cient for IFN-I receptor [203].

9. Conclusive remarks

The etiopathogenesis of autoimmune disorders is due to the interac-tion of environmental [204] and genetic factors. Increasing evidencesupport the critical role played by TLR pathway in the activation ofimmune-mediated tissue damage that characterizes autoimmune con-ditions. Increasing results obtained from the analysis of several experi-mental models of autoimmunity and the discovery of TLR SNPs andCNV found associated with certain autoimmune conditions in peculiarethnic groups, have highlighted the relevance of the TLR signalingpathway in autoimmunity onset. Molecules involved in TLR pathwaymay represent the targets for novel therapeutics in order to stop theautoimmune process [205]. Pharmacological treatments acting ondifferent targets, such as MyD88, IRAK1, IRAK4, TLR7 and TLR9, havebeen unravelled. Experiments conducted in vitro and in experimentalanimal models of autoimmunity as SLE, IBD, MS and RA, demonstratedthe efficacy of TLRs and TLR signaling cascade inhibitions [205,206].Experimental data however demonstrate that, being TLR pathwaycritically involved in the immune defense of the host against infections,an accurate selection of the target and a strict regulation of its activitywithin the signaling cascade is necessary in order to obtain the desiredtherapeutic effect [205]. Moreover an important contribution to betterelucidate the involvement of TLRs in the pathogenesis of autoimmunitycould be given by whole exome sequencing (WES), representing apowerful tool for the detection of protein coding and splicing variantsassociated with complex inherited pathologies [207].

Acknowledgments

This work was supported by the Italian Ministry of Health RicercaCorrente (201502P0034960).

References

[1] Thwaites R, Chamberlain G, Sacre S. Emerging role of endosomal toll-like receptorsin rheumatoid arthritis. Front Immunol 2014;5:1.

[2] Hurst J, von Landenberg P. Toll-like receptors and autoimmunity. Autoimmun Rev2008;7:204–8.

[3] Kumar H, Kawai T, Akira S. Pathogen recognition in the innate immune response.Biochem J 2009;420:1–16.

[4] Janeway Jr CA, Medzhitov R. Innate immune recognition. Annu Rev Immunol 2002;20:197–216.

[5] Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell2006;124:783–801.

[6] Medzhitov R. Recognition of microorganisms and activation of the immune re-sponse. Nature 2007;449:819–26.

[7] Beutler BA. TLRs and innate immunity. Blood 2009;113:1399–407.[8] Hashimoto C, Hudson KL, Anderson KV. The Toll gene of Drosophila, required for

dorsal–ventral embryonic polarity, appears to encode a transmembrane protein.Cell 1988;52:269–79.

[9] Lemaitre B, Nicolas E, Michaut L, Reichhart J-M, Hoffmann JA. The dorsoventral reg-ulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response inDrosophila adults. Cell 1996;86:973–83.

[10] Takeda K, Akira S. TLR signaling pathways. Semin Immunol 2004;16:3–9.[11] Rock FL, Hardiman G, Timans JC, Kastelein RA, Bazan JF. A family of human recep-

tors structurally related to Drosophila Toll. Proc Natl Acad Sci U S A 1998;95:588–93.

[12] Kawai T, Akira S. TLR signaling. Semin Immunol 2007;19:24–32.[13] Poltorak A, He X, Smirnova I, Liu MY, Huffel CV, Du X, et al. Defective LPS signaling

in C3H/HeJ and C57BL/10ScCr mice: mutation in Tlr4 gene. Science 1998;282:2085–8.

[14] Hoshino K, Takeuchi O, Kawai T, Sanjo H, Ogawa T, Takeda Y, et al. Cutting edge:Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccha-ride: evidence for TLR4 as the Lps gene product. J Immunol 1999;162:3749–52.

[15] Underhill DM, Ozinsky A, Hajjar AM, Stevens A, Wilson CB, Bassetti M, et al. TheToll-like receptor 2 is recruited to macrophage phagosomes and discriminates be-tween pathogens. Nature 1999;401:811–5.

[16] Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol 2004;4:499–511.[17] Kaisho T, Akira S. Toll-like receptor function and signaling. J Allergy Clin Immunol

2006;117:979–87.[18] Hacker H, Vabulas RM, Takeuchi O, Hoshino K, Akira S, Wagner H. Immune cell ac-

tivation by bacterial CpG-DNA through myeloid differentiation marker 88 andtumor necrosis factor receptor-associated factor (TRAF)6. J Exp Med 2000;192:595–600.

[19] SchnareM, Holt AC, Takeda K, Akira S, Medzhitov R. Recognition of CpG DNA isme-diated by signaling pathways dependent on the adaptor protein MyD88. Curr Biol2000;10:1139–42.

[20] Hemmi H, Kaisho T, Takeuchi O, Sato S, Sanjo S, Hoshino K, et al. Small antiviralcompounds activate immune cells via TLR7MyD88-dependent signalling pathway.Nat Immunol 2002;3:196–200.

[21] Hayashi F, Smith KD, Ozinsky A, Hawn TR, Yi EC, Goodlett DR, et al. The innate im-mune response to bacterial flagellin is mediated by Toll-like receptor-5. Nature2001;410:1099–103.

[22] Suzuki N, Suzuki S, Yeh WC. IRAK-4 as the central TIR signaling mediator in innateimmunity. Trends Immunol 2002;23:503–6.

[23] Chen ZJ. Ubiquitin signalling in the NF-kappaB pathway. Nat Cell Biol 2005;7:758–65.

[24] Janssens S, Burns K, Tschopp J, Beyaert R. Regulation of interleukin-1 andlipopolysaccharide-induced NF-κB activation by alternative splicing of MyD88. CurrBiol 2002;12:467–71.

[25] Burns K, Janssens S, Brissoni B, Olivos N, Beyaert R, Tschopp J. Inhibition of IL-1receptor/Toll-like receptor signaling through the alternatively spliced, short formof MyD88 is due to its failure to recruit IRAK-4. J Exp Med 2003;197:263–8.

[26] YamamotoM, Sato S, Hemmi H, Uematsu S, Hoshino K, Kaisho T, et al. TRAM is spe-cifically involved in the Toll-like receptor 4-mediated MyD88-independent signal-ing pathway. Nat Immunol 2003;4:1144–50.

[27] Meylan E, Burns K, Hofmann K, Blancheteau V, Martinon F, Kelliher M, et al. RIP1 isan essential mediator of Toll-like receptor 3-induced NF-kappa B activation. NatImmunol 2004;5:503–7.

[28] Carty M, Goodbody R, Schroder M, Stack J, Moynagh PN, Bowie AG. The humanadaptor SARM negatively regulates adaptor protein TRIF-dependent Toll-like re-ceptor signaling. Nat Immunol 2006;7:1074–81.

[29] Couillault C, Pujol N, Reboul J, Sabatier L, Guichou JF, Kohara Y, et al. TLR-independent control of innate immunity in Caenorhabditis elegans by the TIR do-main adaptor protein TIR-1, an ortholog of human SARM. Nat Immunol 2004;5:488–94.

[30] Covert MW, Leung TH, Gaston JE, Baltimore D. Achieving stability oflipopolysaccharide-induced NF-kappaB activation. Science 2005;309:1854–7.

[31] Kawai T, Akira S. Innate immune recognition of viral infection. Nat Immunol 2006;7:131–7.

[32] Yoneyama M, Kikuchi M, Natsukawa T, Shinobu N, Imaizumi T, Miyagishi M, et al.The RNA helicase RIG-I has an essential function in double-stranded RNA-inducedinnate antiviral responses. Nat Immunol 2004;5:730–7.

[33] Lincez PJ, Shanina I, Horwitz MS. Reduced expression of the MDA5 gene IFIH1 pre-vents autoimmune diabetes. Diabetes 2015;64:2184–93.

[34] Ishii KJ, Coban C, Kato H, Takahashi K, Torii Y, Takeshita F, et al. A Toll-like receptor-independent antiviral response induced by double-stranded B-form DNA. NatImmunol 2006;7:40–8.

[35] Zhang D, Zhang G, Hayden MS, Greenblatt MB, Bussey C, Flavell RA, et al. A toll-likereceptor that prevents infection by uropathogenic bacteria. Science 2004;303:1522–6.

[36] Takeuchi O, Hoshino K, Akira S. Cutting edge: TLR2-deficient and MyD88-deficientmice are highly susceptible to Staphylococcus aureus infection. J Immunol 2000;165:5392–6.

981E. Gianchecchi, A. Fierabracci / Autoimmunity Reviews 14 (2015) 971–983

[37] Echchannaoui H, Frei K, Schnell C, Leib SL, Zimmerli W, Landmann R. Toll-like re-ceptor 2-deficient mice are highly susceptible to Streptococcus pneumoniae menin-gitis because of reduced bacterial clearing and enhanced inflammation. J Infect Dis2002;186:798–806.

[38] Wagner H. The immunobiology of the TLR9 subfamily. Trends Immunol 2004;25:381–6.

[39] Siegal FP, Kadowaki N, Shodell M, Fitzgerald-Bocarsly PA, Shah K, Ho S, et al. The na-ture of the principal type 1 interferon-producing cells in human blood. Science 1999;284:1835–7.

[40] Kato H, Takeuchi O, Sato S, Yoneyama M, Yamamoto M, Matsui K, et al. Differentialroles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature 2006;441:101–5.

[41] Kato H, Sato S, Yoneyama M, Yamamoto M, Uematsu S, Matsui K, et al. Cell type-specific involvement of RIG-I in antiviral response. Immunity 2005;23:19–28.

[42] Krug A, French AR, Barchet W, Fischer JA, Dzionek A, Pingel JT, et al. TLR9-dependent recognition of MCMV by IPC and DC generates coordinated cytokine re-sponses that activate antiviral NK cell function. Immunity 2004;21:107–19.

[43] Kawai T, Akira S. The roles of TLRs, RLRs and NLRs in pathogen recognition. IntImmunol 2009;21:317–37.

[44] Adachi K, Tsutsui H, Kashiwamura S, Seki E, Nakano H, Takeuchi O, et al. Plasmodi-um berghei infection inmice induces liver injury by an IL-12- and toll-like receptor/myeloid differentiation factor 88-dependent mechanism. J Immunol 2001;167:5928–34.

[45] Lee S, Kang D, Ra EA, Lee TA, Ploegh HL, Park B. Negative regulation of TLR9 signal-ing by its N-terminal proteolytic cleavage product. J Immunol 2014;193:3726–35.

[46] Wang Y, Tang Y, Teng L, Wu Y, Zhao X, Pei G. Association of beta-arrestin andTRAF6 negatively regulates Toll-like receptor-interleukin 1 receptor signaling.Nat Immunol 2006;7:139–47.

[47] Boone DL, Turer EE, Lee EG, Ahmad RC, Wheeler MT, Tsui C, et al. The ubiquitin-modifying enzyme A20 is required for termination of Toll-like receptor responses.Nat Immunol 2004;5:1052–60.

[48] Negishi H, Ohba Y, Yanai H, Takaoka A, Honma K, Yui K, et al. Negative regulation ofToll-like-receptor signaling by IRF-4. Proc Natl Acad Sci U S A 2005;102:15989–94.

[49] Brint EK, Xu D, Liu H, Dunne A, McKenzie AN, O'Neill LA, et al. ST2 is an inhibitor ofinterleukin 1 receptor and Toll-like receptor 4 signaling and maintains endotoxintolerance. Nat Immunol 2004;5:373–9.

[50] Saitoh T, Tun-Kyi A, Ryo A, Yamamoto M, Finn G, Fujita T, et al. Negative regulationof interferon-regulatory factor 3-dependent innate antiviral response by the prolylisomerase Pin1. Nat Immunol 2006;7:598–605.

[51] Su X, Li S, Meng M, Qian W, Xie W, Chen D, et al. TNF receptor-associated factor-1(TRAF1) negatively regulates Toll/IL-1 receptor domain-containing adaptor induc-ing IFN-beta (TRIF)-mediated signaling. Eur J Immunol 2006;36:199–206.

[52] Mansell A, Smith R, Doyle SL, Gray P, Fenner JE, Crack PJ, et al. Suppressor of cyto-kine signaling 1 negatively regulates Toll-like receptor signaling by mediating Maldegradation. Nat Immunol 2006;7:148–55.

[53] Gilchrist M, Thorsson V, Li B, Rust AG, Korb M, Kennedy K, et al. Systems biologyapproaches identify ATF3 as a negative regulator of Toll-like receptor 4. Nature2006;441:173–8.

[54] Richez C, Blanco P, Rifkin I, Moreau JF, Schaeverbeke T. Role for toll-like receptors inautoimmune disease: the example of systemic lupus erythematosus. Joint BoneSpine 2011;78:124–30.

[55] Bot A. In this issue: autoimmunity and innate immunity. Int Rev Immunol 2014;33:1–2.

[56] van der Laan JW, Gould S, Tanir JY, Vaccines ILSIHESI, Committee Adjuvants SafetyProject. Safety of vaccine adjuvants: focus on autoimmunity. Vaccine 2015;33:1507–14.

[57] Edwards AD, Diebold SS, Slack EM, Tomizawa H, Hemmi H, Kaisho T, et al. Toll-likereceptor expression in murine DC subsets: lack of TLR7 expression byCD8alpha + DC correlates with unresponsiveness to imidazoquinolines. Eur JImmunol 2003;33:827–33.

[58] Gursel I, Gursel M, Yamada H, Ishii KJ, Takeshita F, Klinman DM. Elements in mam-malian telomeres suppress bacterial DNA-induced immune activation. J Immunol2003;171:1393–400.

[59] Kariko K, Buckstein M, Ni H, Weissman D. Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin ofRNA. Immunity 2005;23:165–75.

[60] Rifkin IR, Leadbetter EA, Busconi L, Viglianti G, Marshak-Rothstein A. Toll-like re-ceptors, endogenous ligands, and systemic autoimmune disease. Immunol Rev2005;204:27–42.

[61] Papadimitraki ED, Choulaki C, Koutala E, Bertsias G, Tsatsanis C, Gergianaki I, et al.Expansion of toll-like receptor 9-expressing B cells in active systemic lupus erythe-matosus: implications for the induction and maintenance of the autoimmune pro-cess. Arthritis Rheum 2006;54:3601–11.

[62] Stechova K, Kolar M, Blatny R, Halbhuber Z, Vcelakova J, Hubackova M, et al.Healthy first degree relatives of patients with type 1 diabetes exhibit significantdifferences in basal gene expression pattern of immunocompetent cells comparedto controls: expression pattern as predeterminant of autoimmune diabetes. Scand JImmunol 2011;75:210–9.

[63] LeeMS. Treatment of autoimmune diabetes by inhibiting the initial event. ImmuneNetw 2013;13:194–8.

[64] Sun C, Zhi D, Shen S, Luo F, Sanjeevi CB. SNPs in the exons of Toll-like receptors areassociated with susceptibility to type 1 diabetes in Chinese population. HumImmunol 2014;75:1084–8.

[65] Zhu W, Yang W, He Z, Liao X, Wu J, Sun J, et al. Overexpressing autoimmune regu-lator regulates the expression of toll-like receptors by interacting with their pro-moters in RAW264.7 cells. Cell Immunol 2011;270:156–63.

[66] Sacre SM, Lo A, Gregory B, Simmonds RE, Williams L, Feldmann M, et al. Inhibitorsof TLR8 reduce TNF production from human rheumatoid synovial membrane cul-tures. J Immunol 2008;181:8002–9.

[67] Han SW, Lee WK, Kwon KT, Lee BK, Nam EJ, Kim GW. Association of polymor-phisms in interferon regulatory factor 5 gene with rheumatoid arthritis: ametaanalysis. J Rheumatol 2009;36:693–7.

[68] Enevold C, Radstake TR, Coenen MJ, Fransen J, Toonen EJ, Bendtzen K, et al. Multi-plex screening of 22 single-nucleotide polymorphisms in 7 Toll-like receptors: anassociation study in rheumatoid arthritis. J Rheumatol 2010;37:905–10.

[69] Lin YT, Verma A, Hodgkinson CP. Toll-like receptors and human disease: lessonsfrom single nucleotide polymorphisms. Curr Genomics 2012;13:633–45.

[70] Davis ML, LeVan TD, Yu F, Sayles H, Sokolove J, Robinson W, et al. Associations oftoll-like receptor (TLR)-4 single nucleotide polymorphisms and rheumatoid arthri-tis disease progression: an observational cohort study. Int Immunopharmacol2015;24:346–52.

[71] García-Ortiz H, Velázquez-Cruz R, Espinosa-Rosales F, Jiménez-Morales S, Baca V,Orozco L. Association of TLR7 copy number variation with susceptibility tochildhood-onset systemic lupus erythematosus in Mexican population. AnnRheum Dis 2010;69:1861–5.

[72] Pacheco GV, Cruz DC, González Herrera LJ, Pérez Mendoza GJ, Adrián Amaro GI,Nakazawa Ueji YE, et al. Copy number variation of TLR-7 gene and its associationwith the development of systemic lupus erythematosus in female patients fromYucatan Mexico. Genet Epigenet 2014;6:31–6.

[73] Wei J, Bhattacharyya S, Tourtellotte WG, Varga J. Fibrosis in systemic sclerosis:emerging concepts and implications for targeted therapy. Autoimmun Rev 2011;10:267–75.

[74] Broen JC, Bossini-Castillo L, van Bon L, Vonk MC, Knaapen H, Beretta L, et al. A rarepolymorphism in the gene for Toll-like receptor 2 is associated with systemic scle-rosis phenotype and increases the production of inflammatory mediators. ArthritisRheum 2012;64:264–71.

[75] Bhattacharyya S, Kelley K, Melichian DS, Tamaki Z, Fang F, Su Y, et al. Toll-like re-ceptor 4 signaling augments transforming growth factor-β responses: a novelmechanism for maintaining and amplifying fibrosis in scleroderma. Am J Pathol2013;182:192–205.

[76] Takahashi T, Asano Y, Ichimura Y, Toyama T, Taniguchi T, Noda S, et al. Ameliora-tion of tissue fibrosis by toll-like receptor 4 knockout inmurinemodels of systemicsclerosis. Arthritis Rheum 2015;67:254–65.

[77] Kirino Y, Takeno M,Watanabe R, Murakami S, Kobayashi M, Ideguchi H, et al. Asso-ciation of reduced heme oxygenase-1 with excessive Toll-like receptor 4 expres-sion in peripheral blood mononuclear cells in Behçet's disease. Arthritis Res Ther2008;10:R16.

[78] Nara K, Kurokawa MS, Chiba S, Yoshikawa H, Tsukikawa S, Matsuda T, et al. In-volvement of innate immunity in the pathogenesis of intestinal Behçet's disease.Clin Exp Immunol 2008;152:245–51.

[79] Franchimont D, Vermeire S, El Housni H, Pierik M, Van Steen K, Gustot T, et al. De-ficient host-bacteria interactions in inflammatory bowel disease? The toll-like re-ceptor (TLR)-4 Asp299gly polymorphism is associated with Crohn's disease andulcerative colitis. Gut 2004;53:987–92.

[80] Shen XY, Shi RH, Wang Y, Zhang HJ, Zhou XQ, Shen FC, et al. Toll-like receptorgene polymorphisms and susceptibility to inflammatory bowel disease in Chi-nese Han and Caucasian populations. Zhonghua Yi Xue Za Zhi 2010;90:1416–20.

[81] Shen X, Shi R, Zhang H, Li K, Zhao Y, Zhang R. The Toll-like receptor 4 D299G andT399I polymorphisms are associated with Crohn's disease and ulcerative colitis: ameta-analysis. Digestion 2010;81:69–77.

[82] Ji X, Wang Y, Huang G, ZhouW. TLR3c.1377, TLR9-1486, and TLR9 2848 gene poly-morphisms andmultiple sclerosis. Zhong Nan Da Xue Xue Bao Yi Xue Ban 2010;35:116–22.

[83] Kristjansdottir G, Sandling JK, Bonetti A, Roos IM, Milani L, Wang C, et al. Interferonregulatory factor 5 (IRF5) gene variants are associated with multiple sclerosis inthree distinct populations. J Med Genet 2008;45:362–9.

[84] Karaca N, Ozturk G, Gerceker BT, Turkmen M, Berdeli A. TLR2 and TLR4 gene poly-morphisms in Turkish vitiligo patients. J Eur Acad Dermatol Venereol 2013;27:e85–90.

[85] Wang YZ, Yan M, Tian FF, Zhang JM, Liu Q, Yang H, et al. Possible involvement ofToll-like receptors in the pathogenesis of myasthenia gravis. Inflammation 2013;36:121–30.

[86] Bernasconi P, Barberis M, Baggi F, Passerini L, CannoneM, Arnoldi E, et al. Increasedtoll-like receptor 4 expression in thymus of myasthenic patients with thymitis andthymic involution. Am J Pathol 2005;167:129–39.

[87] Silveira PA, Grey ST. B cells in the spotlight: innocent bystanders or majorplayers in the pathogenesis of type 1 diabetes. Trends Endocrinol Metab2006;17:128–35.

[88] Bluestone JA, Herold K, Eisenbarth G. Genetics, pathogenesis and clinical interven-tions in type 1 diabetes. Nature 2010;464:1293–300.

[89] Fierabracci A. The potential of multimer technologies in type 1 diabetes predictionstrategies. Diabetes Metab Res Rev 2011;27:216–29.

[90] Gianchecchi E, Palombi M, Fierabracci A. The putative role of the C1858T polymor-phism of protein tyrosine phosphatase PTPN22 gene in autoimmunity. AutoimmunRev 2013;12:717–25.

[91] Wen L, Peng J, Li Z, Wong FS. The effect of innate immunity on autoimmune diabe-tes and the expression of Toll-like receptors on pancreatic islets. J Immunol 2004;172:3173–80.

[92] Lang KS, RecherM, Junt T, Navarini AA, Harris NL, Freigang S, et al. Toll-like receptorengagement converts T-cell autoreactivity into overt autoimmune disease. NatMed 2005;11:138–45.

982 E. Gianchecchi, A. Fierabracci / Autoimmunity Reviews 14 (2015) 971–983

[93] Kim HS, HanMS, Chung KW, Kim S, Kim E, KimMJ, et al. Toll-like receptor 2 sensesbeta-cell death and contributes to the initiation of autoimmune diabetes. Immunity2007;27:321–33.

[94] Zipris D, Lien E, Xie JX, Greiner DL, Mordes JP, Rossini AA. TLR activation synergizeswith Kilham rat virus infection to induce diabetes in BBDR rats. J Immunol 2005;174:131–42.

[95] Wen L, Ley RE, Volchkov PY, Stranges PB, Avanesyan L, et al. Innate immunity andintestinal microbiota in the development of Type 1 diabetes. Nature 2008;455:1109-3.

[96] Aumeunier A, Grela F, Ramadan A, Pham Van L, Bardel E, Gomez Alcala A, et al. Sys-temic Toll-like receptor stimulation suppresses experimental allergic asthma andautoimmune diabetes in NOD mice. PLoS One 2010;5 e11484.

[97] Han D, Cai X, Wen J, Matheson D, Skyler JS, Kenyon NS, et al. Innate and adaptiveimmune gene expression profiles as biomarkers in human type 1 diabetes. ClinExp Immunol 2012;170:131–8.

[98] Meyers A, Shah R, Gottlieb P, Zipris D. Altered toll-like receptor signaling pathwaysin human type 1 diabetes. J Mol Med 2010;88:1221–31.

[99] Alkanani AK, Rewers M, Dong F, Waugh K, Gottlieb PA, Zipris D. Dysregulated Toll-like receptor-induced interleukin-1β and interleukin-6 responses in subjects at riskfor the development of type 1 diabetes. Diabetes 2012;61:2525–33.

[100] Kristiansen OP, Mandrup-Poulsen T. Interleukin-6 and diabetes: the good, the bad,or the indifferent? Diabetes 2005;54:S114–24.

[101] Gianchecchi E, Crinò A, Giorda E, Luciano R, Perri V, Russo AL, et al. Altered B cellhomeostasis and toll-like receptor 9-driven response in type 1 diabetes carriersof the C1858T PTPN22 allelic variant: implications in the disease pathogenesis.PLoS One 2014;9 e110755.

[102] Petrich de Marquesini LG, Fu J, Connor KJ, Bishop AJ, McLintock NE, Pope C, et al.IFN-gamma and IL-10 islet-antigen-specific T cell responses in autoantibody-negative first-degree relatives of patients with type 1 diabetes. Diabetologia2010;53:1451–60.

[103] Devaraj S, DasuMR, Park SH, Jialal I. Increased levels of ligands of Toll-like receptors2 and 4 in type 1 diabetes. Diabetologia 2009;52:1665–8.

[104] Castiblanco J, Varela DC, Castaño-Rodríguez N, Rojas-Villarraga A, Hincapié ME,Anaya JM. TIRAP (MAL) S180L polymorphism is a common protective factoragainst developing tuberculosis and systemic lupus erythematosus. Infect GenetEvol 2008;8:541–4.

[105] Assmann TS, Brondani Lde A, Bauer AC, Canani LH, Crispim D. Polymorphisms inthe TLR3 gene are associatedwith risk for type 1 diabetesmellitus. Eur J Endocrinol2014;170:519–27.

[106] Proust-Lemoine E, Saugier-Veber P, Wémeau JL. Polyglandular autoimmune syn-drome type I. Presse Med 2012;41:e651–62.

[107] Fierabracci A. Recent insights into the role and molecular mechanisms of the auto-immune regulator (AIRE) gene in autoimmunity. Autoimmun Rev 2011;10:137–43.

[108] Sedivá A, Ciháková D, Lebl J. Immunological findings in patients with autoimmunepolyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) and their familymembers: are heterozygotes subclinically affected? J Pediatr Endocrinol Metab2002;15:1491–6.

[109] Hong M, Ryan KR, Arkwright PD, Gennery AR, Costigan C, Dominguez M, et al. Pat-tern recognition receptor expression is not impaired in patients with chronicmucocutanous candidiasis with or without autoimmune polyendocrinopathy can-didiasis ectodermal dystrophy. Clin Exp Immunol 2009;156:40–51.

[110] Boissier MC, Semerano L, Challal S, Saidenberg-Kermanac'h N, Falgarone G. Rheu-matoid arthritis: from autoimmunity to synovitis and joint destruction. JAutoimmun 2012;39:222–8.

[111] Meng L, Zhu W, Jiang C, He X, Hou W, Zheng F, et al. Toll-like receptor 3 upregula-tion in macrophages participates in the initiation and maintenance of pristane-induced arthritis in rats. Arthritis Res Ther 2010;12:R103.

[112] ZhuW,Meng L, Jiang C, Xu J,Wang B, Han Y, et al. Overexpression of toll-like recep-tor 3 in spleen is associated with experimental arthritis in rats. Scand J Immunol2012;76:263–70.

[113] Yarilina A, DiCarlo E, Ivashkiv LB. Suppression of the effector phase of inflammatoryarthritis by double-stranded RNA is mediated by type I IFNs. J Immunol 2007;178:2204–11.

[114] Hayashi T, Gray CS, Chan M, Tawatao RI, Ronacher L, McGargill MA, et al. Preven-tion of autoimmune disease by induction of tolerance to Toll-like receptor 7. ProcNatl Acad Sci U S A 2009;106:2764–9.

[115] Alzabin S, Kong P, Medghalchi M, Palfreeman A, Williams R, Sacre S. Investigationof the role of endosomal Toll-like receptors inmurine collagen-induced arthritis re-veals a potential role for TLR7 in disease maintenance. Arthritis Res Ther 2012;14:R142.

[116] Chen SY, Shiau AL, Li YT, Lin YS, Lee CH, Wu CL, et al. Suppression of collagen-induced arthritis by intra-articular lentiviral vector-mediated delivery of Toll-likereceptor 7 short hairpin RNA gene. Gene Ther 2012;19:752–60.

[117] Heil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C, Akira S, et al. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science2004;303:1526–9.

[118] Demaria O, Pagni PP, Traub S, de Gassart A, Branzk N, Murphy AJ, et al. TLR8 defi-ciency leads to autoimmunity in mice. J Clin Invest 2010;120:3651–62.

[119] Begovich AB, Carlton VE, Honigberg LA, Schrodi SJ, Chokkalingam AP, AlexanderHC, et al. A missense single-nucleotide polymorphism in a gene encoding a proteintyrosine phosphatase (PTPN22) is associated with rheumatoid arthritis. Am J HumGenet 2004;75:330–7.

[120] Wang Y, Stanford S, ZhouW, Auger Jennifer L, Cheng G, Campbell A, et al. Rheuma-toid arthritis-associated PTPN22 modulates toll-like receptor-mediated, type 1interferon-dependent innate immunoregulation. Arthritis Rheum 2012;64:2452.

[121] Zheng J, Petersen F, Yu X. The role of PTPN22 in autoimmunity: learning frommice.Autoimmun Rev 2014;13:266–71.

[122] Herman S, Fischer A, Pfatschbacher J, HoffmannM, Steiner G. A TLR 9 antagonist di-minishes arthritis severity in a rat model of rheumatoid arthritis. Ann Rheum Dis2011;70:A39.

[123] Brentano F, Schorr O, Gay RE, Gay S, Kyburz D. RNA released from necrotic synovialfluid cells activates rheumatoid arthritis synovial fibroblasts via Toll-like receptor 3.Arthritis Rheum 2005;52:2656–65.

[124] Roelofs MF, Joosten LA, Abdollahi-Roodsaz S, van Lieshout AW, Sprong T, van denHoogen FH, et al. The expression of toll-like receptors 3 and 7 in rheumatoid arthri-tis synovium is increased and costimulation of toll-like receptors 3, 4, and 7/8 re-sults in synergistic cytokine production by dendritic cells. Arthritis Rheum 2005;52:2313–22.

[125] Ospelt C, Brentano F, Rengel Y, Stanczyk J, Kolling C, Tak PP, et al. Overexpression oftoll-like receptors 3 and 4 in synovial tissue from patients with early rheumatoidarthritis: toll-like receptor expression in early and longstanding arthritis. ArthritisRheum 2008;58:3684–92.

[126] Tamaki Y, Takakubo Y, Hirayama T, Konttinen YT, Goodman SB, YamakawaM, et al.Expression of Toll-like receptors and their signaling pathways in rheumatoid syno-vitis. J Rheumatol 2011;38:810–20.

[127] Kilding R, Akil M, Till S, Amos R, Winfield J, Iles MM, et al. A biologicallyimportant single nucleotide polymorphism within the toll-like receptor-4gene is not associated with rheumatoid arthritis. Clin Exp Rheumatol 2003;21:340–2.

[128] Radstake TR, Franke B, Hanssen S, Netea MG, Welsing P, Barrera P, et al. The Toll-like receptor 4 Asp299Gly functional variant is associated with decreased rheuma-toid arthritis disease susceptibility but does not influence disease severity and/oroutcome. Arthritis Rheum 2004;50:999–1001.

[129] Kuuliala K, Orpana A, Leirisalo-Repo M, Kautiainen H, Hurme M, Hannonen P, et al.Polymorphism at position +896 of the Toll-like receptor 4 gene interferes withrapid response to therapy in rheumatoid arthritis. Ann Rheum Dis 2006;65:1241–3.

[130] Sanchez E, Orozco G, Lopez-Nevot MA, Jimenez-Alonso J, Martin J. Polymorphismsof toll-like receptor 2 and 4 genes in rheumatoid arthritis and systemic lupus ery-thematosus. Tissue Antigens 2004;63:54–7.

[131] Jaen O, Petit-Teixeira E, Kirsten H, Ahnert P, Semerano L, Pierlot C, et al. No evi-dence of major effects in several Toll-like receptor gene polymorphisms in rheu-matoid arthritis. Falgarone G; European Consortium on Rheumatoid ArthritisFamilies. Arthritis Res Ther 2009;11:R5.

[132] Laska MJ, Hansen B, Troldborg A, Lorenzen T, Stengaard-Pedersen K, Junker P, et al.A non-synonymous single-nucleotide polymorphism in the gene encoding Toll-likeReceptor 3 (TLR3) is associated with sero-negative rheumatoid arthritis (RA) in aDanish population. BMC Res Notes 2014;7:716.

[133] Coenen MJ, Enevold C, Barrera P, Schijvenaars MM, Toonen EJ, Scheffer H, et al. Ge-netic variants in toll-like receptors are not associated with rheumatoid arthritissusceptibility or anti-tumour necrosis factor treatment outcome. PLoS One 2010;5 e14326.

[134] Etem EO, Elyas H, Ozgocmen S, Yıldırım A, Godekmerdan A. The investigation oftoll-like receptor 3, 9 and 10 gene polymorphisms in Turkish rheumatoid arthritispatients. Rheumatol Int 2011;31:1369–74.

[135] Dawidowicz K, Allanore Y, Guedj M, Pierlot C, Bombardieri S, Balsa A, et al. The in-terferon regulatory factor 5 gene confers susceptibility to rheumatoid arthritis andinfluences its erosive phenotype. Ann Rheum Dis 2011;70:117–21.

[136] Kim YJ, Park JH, Kim I, Kim JO, Bae JS, Shin HD, et al. Putative role of functional in-terferon regulatory factor 5 (IRF5) polymorphism in rheumatoid arthritis in a Ko-rean population. J Rheumatol 2008;35:2106–18.

[137] Maalej A, Hamad MB, Rebaï A, Teixeira VH, Bahloul Z, Marzouk S, et al. Associationof IRF5 gene polymorphisms with rheumatoid arthritis in a Tunisian population.Scand J Rheumatol 2008;37:414–8.

[138] Shimane K, Kochi Y, Yamada R, Okada Y, Suzuki A, Miyatake A, et al. A single nucle-otide polymorphism in the IRF5 promoter region is associated with susceptibilityto rheumatoid arthritis in the Japanese population. Ann Rheum Dis 2009;68:377–83.

[139] Sacre SM, Andreakos E, Kiriakidis S, Amjadi P, Lundberg A, Giddins G, et al. The Toll-like receptor adaptor proteins MyD88 and Mal/TIRAP contribute to the inflamma-tory and destructive processes in a human model of rheumatoid arthritis. Am JPathol 2007;170:518–25.

[140] Kotzin BL. Systemic lupus erythematosus. Cell 1996;85:303–6.[141] Nakano S, Morimoto S, Suzuki J, Nozawa K, Amano H, Tokano Y, et al. Role of path-

ogenic auto-antibody production by Toll-like receptor 9 of B cells in active systemiclupus erythematosus. Rheumatology (Oxford) 2008;47:145–9.

[142] Rahman A, Isenberg D. Systemic lupus erythematosus. N Engl J Med 2008;358:929–39.

[143] Rönnblom L, Pascual V. The innate immune system in SLE: type I interferons anddendritic cells. Lupus 2008;17:394–9.

[144] Robinson ES, Werth VP. The role of cytokines in the pathogenesis of cutaneouslupus erythematosus. Cytokine 2014;73:326–34.

[145] Eichler E, Nickerson D, Altshuler D, Bowcock A, et al. Completing the map of humangenetics variation. Nature 2007;447:161–5.

[146] Barrat FJ, Meeker T, Chan JH, Guiducci C, Coffman RL. Treatment of lupus-pronemice with a dual inhibitor of TLR7 and TLR9 leads to reduction of autoantibodyproduction and amelioration of disease symptoms. Eur J Immunol 2007;37:3582–6.

[147] Tabeta K, Hoebe K, Janssen EM, Du X, Georgel P, Crozat K, et al. The Unc93b1 mu-tation 3d disrupts exogenous antigen presentation and signaling via Toll-like re-ceptors 3, 7 and 9. Nat Immunol 2006;7:156–64.

983E. Gianchecchi, A. Fierabracci / Autoimmunity Reviews 14 (2015) 971–983

[148] Christensen SR, Shupe J, Nickerson K, Kashgarian M, Flavell RA, Shlomchik MJ. Toll-like receptor 7 and TLR9 dictate autoantibody specificity and have opposing in-flammatory and regulatory roles in a murine model of lupus. Immunol 2006;25:417–28.

[149] Santiago-Raber ML, Kikuchi S, Borel P, Uematsu S, Akira S, Kotzin BL, et al. Evidencefor genes in addition to Tlr7 in the Yaa translocation linked with acceleration ofsystemic lupus erythematosus. J Immunol 2008;181:1556–62.

[150] Christensen SR, Kashgarian M, Alexopoulou L, Flavell RA, Akira S, Shlomchik MJ.Toll-like receptor 9 controls anti-DNA autoantibody production in murine lupus. JExp Med 2005;202:321–31.

[151] Kelley J, Johnson M, Alarcón G, Kimberly R, Edberg J. Variation in the relative copynumber of the TLR-7 gene in patients with systemic lupus erythematosus andhealthy control subjects. Arthritis Rheum 2007;56:3375–8.

[152] Rupasree Y, Naushad SM, Rajasekhar L, Uma A, Kutala VK. Association of TLR4(D299G, T399I), TLR9–1486T N C, TIRAP S180L and TNF-α promoter (-1031,-863, -857) polymorphisms with risk for systemic lupus erythematosus amongSouth Indians. Lupus 2015;24:50–7.

[153] Devaraju P, Gulati R, Antony PT, Mithun CB, Negi VS. Susceptibility to SLE in SouthIndian Tamils may be influenced by genetic selection pressure on TLR2 and TLR9genes. Mol Immunol 2015;64:123–6.

[154] Wang CM, Chang SW, Wu YJ, Lin JC, Ho HH, Chou TC, et al. Genetic variations inToll-like receptors (TLRs 3/7/8) are associated with systemic lupus erythematosusin a Taiwanese population. Sci Rep 2014;4:3792.

[155] Enevold C, Nielsen CH, Jacobsen RS, HermansenML, Molbo D, Avlund K, et al. Singlenucleotide polymorphisms in genes encoding toll-like receptors 7, 8 and 9 in Dan-ish patients with systemic lupus erythematosus. Mol Biol Rep 2014;41:5755–63.

[156] Armstrong DL, Reiff A, Myones BL, Quismorio FP, Klein-Gitelman M, McCurdy D,et al. Identification of new SLE-associated genes with a two-step Bayesian studydesign. Genes Immun 2009;10:446–56.

[157] Graham RR, Kozyrev SV, Baechler EC, ReddyMV, Plenge RM, Bauer JW, et al. A com-mon haplotype of interferon regulatory factor 5 (IRF5) regulates splicing and ex-pression and is associated with increased risk of systemic lupus erythematosus.Nat Genet 2006;38:550–5.

[158] Kelly JA, Kelley JM, Kaufman KM, Kilpatrick J, Bruner GR, Merrill JT, et al. Interferonregulatory factor-5 is genetically associated with systemic lupus erythematosus inAfrican Americans. Genes Immun 2008;9:187–94.

[159] Sigurdsson S, Göring HH, Kristjansdottir G, Milani L, Nordmark G, Sandling JK, et al.Comprehensive evaluation of the genetic variants of interferon regulatory factor 5(IRF5) reveals a novel 5 bp length polymorphism as strong risk factor for systemiclupus erythematosus. Hum Mol Genet 2008;17:872–81.

[160] Siu HO, YangW, Lau CS, Chan TM,Wong RW,WongWH, et al. Association of a hap-lotype of IRF5 gene with systemic lupus erythematosus in Chinese. J Rheumatol2008;35:360–2.

[161] Elghzaly AA, Metwally SS, El-Chennawi FA, Elgayaar MA, Mosaad YM, El-Toraby EE,et al. IRF5, PTPN22, CD28, IL2RA, KIF5A, BLK and TNFAIP3 genes polymorphismsand lupus susceptibility in a cohort from the Egypt Delta; relation to other ethnicgroups. Hum Immunol 2015 Jun 17. http://dx.doi.org/10.1016/j.humimm.2015.06.001.

[162] Gabrielli A, Avvedimento EV, Krieg T. Scleroderma. N Engl J Med 2009;360:1989–2003.

[163] Asano Y. Future treatments in systemic sclerosis. J Dermatol 2010;37:54–70.[164] Varga J, AbrahamD. Systemic sclerosis: a prototypic multisystem fibrotic disorder. J

Clin Invest 2007;117:557–67.[165] Yamamoto T, Takagawa S, Katayama I, Yamazaki K, Hamazaki Y, Shinkai H, et al.

Animal model of sclerotic skin. I: Local injections of bleomycin induce scleroticskin mimicking scleroderma. J Invest Dermatol 1999;112:456–62.

[166] Lehner T. Immunopathogenesis of Behçet's disease. Ann Med Interne 1999;150:483–7.

[167] Yurdakul S, Hamuryudan V, Yazici H. Behçet syndrome. Curr Opin Rheumatol2004;16:38–42.

[168] Karasneh J, Gül A, Ollier WE, Silman AJ, Worthington J. Whole-genome screeningfor susceptibility genes in multicase families with Behçet's disease. ArthritisRheum 2005;52:1836–42.

[169] Sakane T, Takeno M, Suzuki N, Inaba G. Behçet's disease. N Engl J Med 1999;341:1284–91.

[170] MelikogluM, Uysal S, Krueger JG, Kaplan G, Gogus F, Yazici H, et al. Characterizationof the divergent wound-healing responses occurring in the pathergy reaction andnormal healthy volunteers. J Immunol 2006;177:6415–21.

[171] Takada Y, Fujita Y, Igarashi M, Katsumata T, Okabe H, Saigenji K, et al. IntestinalBehçet's disease-pathognomonic changes in intramucosal lymphoid tissues and ef-fect of a “rest cure” on intestinal lesions. J Gastroenterol 1997;32:598–604.

[172] Gül A. Behçet's disease: an update on the pathogenesis. Clin Exp Rheumatol 2001;19:S6–S12.

[173] Zierhut M, Mizuki N, Ohno S, Inoko H, Gül A, Onoé K, et al. Immunology and func-tional genomics of Behçet's disease. Cell Mol Life Sci 2003;60:1903–22.

[174] Skin Anonymous, hypersensitivity to streptococcal antigens and the induction ofsystemic symptoms by the antigens in Behcet's disease: a multicenter study,. TheBehcet's Disease Research Committee of Japan. J Rheumatol 1989;16:506–11.

[175] Sezer FN. The isolation of a virus as the cause of Behcet's diseases. Am J Ophthalmol1953;36:301–15.

[176] Lee S, Bang D, Cho YH, Lee ES, Sohn S. Polymerase chain reaction reveals herpessimplex virus DNA in saliva of patients with Behcet's disease. Arch Dermatol Res1996;288:179–83.

[177] Ohashi K, Burkart V, Flohé S, Kolb H. Cutting edge: heat shock protein 60 is a puta-tive endogenous ligand of the toll-like receptor-4 complex. J Immunol 2000;164:558–61.

[178] Vabulas RM, Ahmad-Nejad P, da Costa C, Miethke T, Kirschning CJ, Häcker H, et al.Endocytosed HSP60s use toll-like receptor 2 (TLR2) and TLR4 to activate the toll/interleukin-1 receptor signaling pathway in innate immune cells. J Biol Chem2001;276:31332–9.

[179] Poss KD, Tonegawa S. Reduced stress defense in heme oxygenase 1-deficient cells.Proc Natl Acad Sci U S A 1997;94:10925–30.

[180] Yachie A, Niida Y, Wada T, Igarashi N, Kaneda H, Toma T, et al. Oxidative stresscauses enhanced endothelial cell injury in human heme oxygenase-1 deficiency.J Clin Invest 1999;103:129–35.

[181] Liu X, Wang C, Ye Z, Kijlstra A, Yang P. Higher expression of Toll-like receptors 2, 3,4, and 8 in ocular Behcet's disease. Invest Ophthalmol Vis Sci 2013;54:6012–7.

[182] Ito A, Ota M, Katsuyama Y, Inoko H, Ohno S, Mizuki N. Lack of association of Toll-like receptor 9 gene polymorphism with Behçet's disease in Japanese patients. Tis-sue Antigens 2007;70:423–6.

[183] Ben Dhifallah I, Lachheb J, Houman H, Hamzaoui K. Toll-like-receptor gene poly-morphisms in a Tunisian population with Behçet's disease. Clin Exp Rheumatol2009;27:S58–62.

[184] Boiardi L, Atzeni F, Casali B, Farnetti E, Nicoli D, Pipitone N, et al. Toll-like receptor 4(TLR4) gene polymorphisms in Italian patients with Behçet's disease. Clin ExpRheumatol 2009;27:S43–7.

[185] Durrani O, Banahan K, Sheedy FJ, McBride L, Ben-Chetrit E, Greiner K, et al. TIRAPSer180Leu polymorphism is associated with Behcet's disease. Rheumatology (Ox-ford) 2011;50:1760–5.

[186] Fang J, Hu R, Hou S, Xiang Q, Qi J, Zhou Y, et al. Association of TLR2 gene polymor-phisms with ocular Behcet's disease in a Chinese Han population. InvestOphthalmol Vis Sci 2013;54:8384–92.

[187] Fang J, Chen L, Tang J, Hou S, Liao D, Ye Z, et al. Association between copy numbervariations of TLR7 and ocular Behçet's disease in a Chinese Han population. InvestOphthalmol Vis Sci 2015;56:1517–23.

[188] Geremia A, Biancheri P, Allan P, Corazza GR, Di Sabatino A. Innate and adaptive im-munity in inflammatory bowel disease. Autoimmun Rev 2014;13:3–10.

[189] Browning BL, Huebner C, Petermann I, Gearry RB, BarclayML, Shelling AN, et al. Hastoll-like receptor 4 been prematurely dismissed as an inflammatory bowel diseasegene? Association study combined with meta-analysis shows strong evidence forassociation. Am J Gastroenterol 2007;102:2504–12.

[190] De Jager PL, Franchimont D,Waliszewska A, Bitton A, Cohen A, Langelier D. The roleof the Toll receptor pathway in susceptibility to inflammatory bowel diseases.Genes Immun 2007;8:387–97.

[191] Hong J, Leung E, Fraser AG, Merriman TR, Vishnu P, Krissansen GW. TLR2, TLR4 andTLR9 polymorphisms and Crohn's disease in a New Zealand Caucasian cohort. JGastroenterol Hepatol 2007;22:1760–6.

[192] Hume GE, Fowler EV, Doecke J, Simms LA, Huang N, Palmieri O, et al. Novel NOD2haplotype strengthens the association between TLR4 Asp299Gly and Crohn's dis-ease in an Australian population. Inflamm Bowel Dis 2008;14:585–90.

[193] Abad C, González-Escribano MF, Diaz-Gallo LM, Lucena-Soto JM, Márquez JL, Leo E,et al. Association of Toll-like receptor 10 and susceptibility to Crohn's disease inde-pendent of NOD2. Genes Immun 2011;12:635–42.

[194] Morgan AR, LamWJ, Han DY, Fraser AG, Ferguson LR. Genetic variationwithin TLR10is associatedwith Crohn's disease in aNewZealandpopulation. Hum Immunol 2012;7:416–20.

[195] Meena NK, Ahuja V, Meena K, Paul J. Association of TLR5 gene polymorphisms inulcerative colitis patients of north India and their role in cytokine homeostasis.PLoS One 2015;10:e0120697.

[196] Trapp BD, Nave KA. Multiple sclerosis: an immune or neurodegenerative disorder?Annu Rev Neurosci 2008;31:247–69.

[197] Hasheminia SJ, Zarkesh-Esfahani SH, Tolouei S, Shaygannejad V, Shirzad H,Hashemzadeh Chaleshtory M. Toll like receptor 2 and 4 expression in peripheralblood mononuclear cells of multiple sclerosis patients. Iran J Immunol 2014;11:74–83.

[198] Kovacs SO. Vitiligo. J Am Acad Dermatol 1998;38:647–66.[199] Spritz RA. Modern vitiligo genetics sheds new light on an ancient disease. J

Dermatol 2013;40:310–8.[200] Cavalcante P, Cufi P, Mantegazza R, Berrih-Aknin S, Bernasconi P, Le Panse R. Etiol-

ogy of myasthenia gravis: innate immunity signature in pathological thymus.Autoimmun Rev 2013;12:863–74.

[201] Marx A, Pfister F, Schalke B, Saruhan-Direskeneli G, Melms A, Ströbel P. The differ-ent roles of the thymus in the pathogenesis of the various myasthenia gravis sub-types. Autoimmun Rev 2013;12:875–84.

[202] Ning S. Innate immune modulation in EBV infection. Herpesviridae 2011;2:1.[203] Cufi P, Dragin N, Weiss JM, Martinez-Martinez P, Roussin R, Berrih Aknin S, et al.

Implication of dsRNA signaling in the etiology of autoimmune myasthenia gravis.Ann Neurol 2013;73:281–93.

[204] Temajo NO, Howard N. The mosaic of environment involvement in autoimmunity:the abrogation of viral latency by stress, a non-infectious environmental agent, isan intrinsic prerequisite prelude before viruses can rank as infectious environmen-tal agents that trigger autoimmune diseases. Autoimmun Rev 2014;13:635–40.

[205] Zhu J, Mohan C. Toll-like receptor signaling pathways-therapeutic opportunities.Mediators Inflamm 2010;2010:781235.

[206] Horton CG, Pan ZJ, Farris AD. Targeting Toll-like receptors for treatment of SLE. Me-diators Inflamm 2010;2010.

[207] Johar AS, Anaya JM, Andrews D, Patel HR, FieldM, Goodnow C, et al. Candidate genediscovery in autoimmunity by using extreme phenotypes, next generation se-quencing and whole exome capture. Autoimmun Rev 2015;14:204–9.