Recent advances in basic science BACTERIAL INTERACTIONS WITH CELLS OF THE INTESTINAL MUCOSA: TOLL- LIKE RECEPTORS AND NOD2

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1 1182 Reent advanes in basi siene BACTERIAL INTERACTIONS WITH CELLS OF THE INTESTINAL MUCOSA: TOLL- LIKE RECEPTORS AND NOD2 Published online first 19 April 2005 Correspondene to: Dr E Cario, University Hospital of Essen, Division of Gastroenterology and Hepatology, Institutsgruppe I, Virhowstr 171; D Essen, Germany; elke.ario@uni-essen.de ECario Gut 2005; 54: doi: /gut SUMMARY Toll-like reeptors (TLR) and NOD2 are emerging as key mediators of innate host defene in the intestinal muosa, ruially involved in maintaining muosal as well as ommensal homeostasis. Reent observations suggest new (patho-) physiologial mehanisms of how funtional versus dysfuntional TLRx/NOD2 pathways may oppose or favour inflammatory bowel disease (IBD). In health, TLRx signalling protets the intestinal epithelial barrier and onfers ommensal tolerane whereas NOD2 signalling exerts antimirobial ativity and prevents pathogeni invasion. In disease, aberrant TLRx and/or NOD2 signalling may stimulate diverse inflammatory responses leading to aute and hroni intestinal inflammation with many different linial phenotypes. INTRODUCTION The intestinal muosa must rapidly reognise detrimental pathogeni threats to the lumen to initiate ontrolled immune responses but maintain hyporesponsiveness to omnipresent harmless ommensals. Charles Janeway Jr first suggested that so-alled pattern reognition reeptors (PRRs) may play an essential role in allowing innate immune ells to disriminate between self and mirobial non-self based on the reognition of broadly onserved moleular patterns. 1 Tolllike reeptors (TLRs) whih omprise a lass of transmembrane PRRs play a key role in mirobial reognition, indution of antimirobial genes, and the ontrol of adaptive immune responses. NODs (NOD1 and NOD2) are a struturally distint family of intraellular PRRs whih presumably in the ontext of mirobial invasion subserve similar funtions (fig 1). TLRs and NOD2 are widely expressed on various ell types of the gastrointestinal muosa, partiipating in host defene against mirobial pathogens in at least four ways: (1) reognition of moleular patterns present on pathogens; (2) expression at the interfae with the environment of the gastrointestinal lumen; (3) indution of seretion of pro/anti-inflammatory yto- and hemokines that link to the adaptive immune system; and (4) indution of antimirobial effetor pathways. Reent studies have greatly advaned our understanding of these mehanisms through whih the gastrointestinal innate immune system an mediate differential host-mirobial interations in reognition and sorting of the broad luminal spetrum of diverse mirobial produts. Furthermore, related findings propose that mammalian TLRx and NOD2 dysfuntions play a key role in the pathophysiology of IBD. MOLECULAR BASIS OF BACTERIAL-MUCOSAL INTERACTIONS Toll-like reeptors (TLRs) Struture Mammalian TLRs omprise a family of (so far) 11 individual type I transmembrane reeptors whih are haraterised by three ommon strutural features (fig 1A): a divergent ligand binding extraellular domain with leuine rih repeats (LRR), a short transmembrane region, and a highly homologous ytoplasmi Toll/interleukin 1 reeptor (TIR) domain, similar to that of the interleukin 1 reeptor family and essential for initiation of downstream signalling asades. 2 Expression pattern TLRs are differentially (induibly or onstitutively) expressed by many distint ell types throughout the whole gastrointestinal trat in vitro and in vivo, inluding (mature and immature) epithelial ells of the stomah, small intestine, and olon, 3 14 as well as intestinal monoytes/marophages and dendriti ells (Stagg AJ, personal ommuniation, 2005) of the lamina propria and myofibroblasts, 17 endothelial ells, 18 and adipoytes (Siegmund B, personal ommuniation, 2005) of the intestinal submuosa. Gut: first published as on 11 July Downloaded from on 6 Otober 2018 by guest. Proteted by opyright.

2 Some inter ell line and inter laboratory differenes in detetion levels of onstitutive TLRx expression have been desribed for various intestinal epithelial tumour ell lines in vitro It is a ommon, if not expeted, problem that epigeneti drifts from the original progenitor lineage may our in suh ell lines with eah ell ulture passage. Different phenotypes of TLRx expression may also derive from distint ell ulture onditions whih alter differentiation state in vitro. 22 Regarding TLR expression in primary intestinal epithelial ells (IEC), there is field wide onsensus that TLR2 and TLR4 are present only in small amounts on IEC in vivo, thus minimising reognition of lumenal bateria in the healthy intestine. In ontrast, TLR4 is signifiantly inreased in primary IEC throughout the lower gastrointestinal trat in ative disease of both Crohn s disease (CD) and ulerative olitis (UC) 4 and murine olitis Ligand speifiity Different pathogen assoiated moleular patterns seletively ativate different TLRs (that is, eah TLR binds speifi moleular signatures of different lasses of miroorganisms or individual features present on diverse ommensals or A B Reognition Ativation Nod2/CARD15 N CARDs Protein-protein interation NBD Ativation LRR TIR TLR4 LRR Reognition D299G T399I P268S R702W G908R L1007fsinsC Figure 1 Toll-like reeptor 4/nuleotide binding oligomerisation domain 2 (TLR4/NOD2) struture and inflammatory bowel disease assoiated ommon variants. (A) Mammalian TLRs are a family of type I transmembrane reeptors whih are all haraterised by three ommon strutural features, as exemplified for TLR4 here: a divergent ligand binding extraellular domain with multiple leuine rih repeats (LRR), a short transmembrane region and a highly homologous ytoplasmi Toll-interleukin 1 reeptor (TIR) domain. Two ommon osegregating missense mutations (D299G and T399I) that affet the extraellular ligand reognition domain of the TLR4 reeptor are assoiated with deregulated immune responses to lipopolysaharide in humans. (B) NOD1 and NOD2 are members of a family of intraellular proteins that ontain an N terminal aspase reruitment domain (CARD), a entrally loated nuleotide binding domain (NBD), and a C terminal regulatory domain. As shown here, NOD2 protein ontains two N terminal CARDs fused to a entral NBD domain followed by 10 tandem LRRs at the C terminus. Three main NOD2 variants (R702W, G908R, and L1007fsinsC) were onfirmed to be assoiated with suseptibility to some types of Crohn s disease. C pathogens) (fig 2). TLR2, for example, reognises baterial lipopeptides and lipoteihoi aid whih are found abundantly in ell walls of Gram positive bateria. 23 TLR2 may ooperate with TLR6 and TLR1, suggesting an essential mehanism for diversifying the repertoire of TLR mediated responses. 24 RNA from double stranded and sense single stranded viruses ativates TLR3, whereas RNA from antisense single stranded viruses ativates TLR7 and TLR8. TLR4 is the major reeptor for lipopolysaharide (LPS) ativation 29 whih may require the presene of aessory proteins, suh as MD-2, CD14 and LPS binding protein. Flagellin and flagellated bateria have been identified as speifi ligands for TLR5. 30 Unmethylated CpgDNA found in prokaryoti genomes and DNA viruses modulates TLR9 31 and TLR11 is ativated by uropathogeni bateria, 32 but the speifi ligand has yet to be determined. Importantly, speies speifi differenes in distint TLRx-ligand reognition appear to exist. 33 Furthermore, endogenous mediators may regulate various TLRs, suh as heat shok proteins 34 or fibronetin 35 but it is ontroversial as to whether suh bona fide ligands may diretly ativate TLRs under physiologial onditions, or rather potential ontaminants in the preparations. Nevertheless, TLRs may indeed be ativated by endogenous ligands under pathophysiologial onditions, suh as self - DNA omplexes. CpG sequenes in self -DNA are an important potential trigger for autoantibody seretion in systemi autoimmune disorders. In rheumatoid arthritis, autoantibodies may omplex to hromatin, leading to exaggerated B ell ativation via TLR9. 38 In systemi lupus erythematosus, DNA ontaining immune omplexes within lupus serum stimulate plasmaytoid dendriti ells to produe ytokines and hemokines via TLR9 and CD In primary biliary irrhosis, CpG DNA indued IgM prodution by ativated B ells via TLR9 40 may drive IgG autoantibody responses and omplex formation in a similarly relentless autodysregulatory loop. Thus impaired self versus nonself disrimination by TLRs in autoimmune disease (inluding IBD) may lead to self direted immune responses ontributing to exaggerated prodution of proinflammatory ytokines and subsequent tissue damage. Some reports suggest that TLR4 ligand binding might trigger assembly of a multi-reeptor omplex in whih several omponents apart from the entral ligand binding reeptor ontribute to LPS signalling. It is widely aepted that TLR4 forms a funtional LPS reognition omplex together with MD-2 and CD14 (see Gangloff and Gay 41 for review) but whether several additional moleules (for example, an LPS binding omplex of Hsp70-Hsp90-GDF5-CXCR4 42 or CD55 43 ) whih reside in lipid rafts next to TLR4 play a diret or, if at all, an auxiliary role in LPS binding and downstream signalling is not yet lear. Similarly, it has been proposed that CD36 may at as a failitator or o-reeptor to TLR2/6 for seletive diaylglyeride reognition. 44 Further studies aimed at defining more omplete models of funtional interations between these (and possibly other) moleules and the TLR4/MD-2/CD14 (or TLR1/2/6) ores, respetively, will hopefully soon eluidate potential fine tuning mehanisms of ligand reognition by suh ooperative or ompetitive multi-reeptor omplexes. Subellular distribution Subellular ompartmentalisation of TLRs appears to be a ritial determinant of immune responsiveness. 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3 Lipoproteins ds RNA LPS Flagellin ss RNA CpG DNA Uropathogeni (?) ligand 1184 TLR1 TLR2 on the optimal site of baterial ligand interation, TLRs are strategially loalised on the ell surfae as well as in different subellular ompartments. Dynami redistribution may be regulated by a state of differentiation as well as ligand exposure. TLR2 and TLR4 are mainly expressed at the apial pole of differentiated IEC in vitro, thus well positioned to monitor the lumenal frontline of baterial omponents whereas both reeptors are present mostly in ytoplasmi ompartments in non-differentiated IEC There are several reports suggesting that LPS-TLR4 redistributes between plasma membrane and endosomal strutures whih have been identified as part of the Golgi apparatus Yet the funtional onsequene of reyling and Golgi reruitment of TLR4 on LPS reognition and signalling seems to be ambiguous, possibly refleting ell type or speies speifi differenes. TLR9 is retained in the endoplasmi retiulum 50 but readily yles to sites of CpG DNA after ellular uptake. 51 In ontrast, TLR5 is stably expressed at the basolateral pole of TLR6 TLR3 TLR4 TLR5 Figure 2 TLRx ligand diversity. Different pathogen assoiated moleular patterns (PAMPs) seletively ativate different Toll-like reeptors (TLRs) (that is, eah TLR binds speifi moleular signatures of different lasses of miroorganisms or individual features present on diverse ommensals or pathogens). A short list of the main ligands is presented. Nod2 RIP Caspase Apoptosis TICAM-1 Trif PI3K p38 JNK MKK IKK TLRx MyD88 IRAK TRAF6 TLR7 TLR8 TLR9 TLR11 intestinal epithelia in vitro, 5 the major sene of ation of Salmonella transloated flagellin but in vivo, it seems that TLR5 is expressed on both poles of IEC Signalling Individual TLRs differentially ativate distint signalling events via diverse ofators and adaptor proteins mediating speifi immune responses. To date, at least five different adaptor proteins have been identified in humans: MyD88, Mal/TIRAP, TRIF/TICAM-1, TRAM/Tirp/TICAM-2, and SARM (for review see O Neill and olleagues 53 ). The first identified so-alled lassial pathway mediated by the onserved TIR ytoplasmi domain in TLRs involves reruitment of the adaptor moleule MyD88, ativation of the serine/threonine kinases of the interleukin 1 reeptor assoiated kinase (IRAK) family, subsequently leading to degradation of inhibitor kb (IkB) and transloation of nulear fator kb (NFkB) to the nuleus (fig 3). Downstream, different TIRAP Mal PKR IRF3 NFκB AP-1 CREB Elk-1, et TICAM-1 Trif IP-10 IFN-β TICAM-2 TRAM Tirp STAT1 SRAM TNF-α NO COX-2 SOCS IL-1 IL-6 IL-8 IL-10 IL-12 Gut: first published as on 11 July Downloaded from on 6 Otober 2018 by guest. Proteted by opyright. Figure 3 TLRx/NOD2 signalling pathways. Toll-like reeptor (TLR) signalling is mediated by a omplexity of various seletive pathways. Rip2 is a diret downstream signal transduer of both TLRx and nuleotide binding oligomerisation domain (NOD2), thus possibly allowing regulatory ross talk between these two distint pathways. IFN-, interferon ; IRAK, interleukin 1 reeptor assoiated kinase; IL, interleukin; TNF-a, tumour nerosis fator a; NFkB, nulear fator kb.

4 signalling modules and partially interating omplexes result in ativation of several transription fators, inluding NFkB, AP-1, Elk-1, CREB, STATs, and the subsequent transriptional ativation of genes enoding pro- and anti-inflammatory ytokines and hemokines as well as indution of ostimulatory moleules. All of these various downstream effets are ritially involved in the ontrol of pathogen elimination, ommensal homeostasis, and linkage to the adaptive immunity. The mehanisms of agonist reognition and engagement with ell signalling may show onsiderable variation between TLRs and for eah TLR perhaps also show variations between ell types and organ origin. Thus analysis of ytokine gene expression to distint ligands in marophages has identified important differenes in biologial responses indued by individual TLRs. Signalling through different TLRs an result in onsiderable qualitative differenes in TH dependent immune responses by differential modulation of MAPKs and the transription fator -FOS. 54 However, the preise moleular mehanisms that differentially influene ell type dependent outome of TLRx speifi signalling effets are not yet fully understood. NODs Struture The NOD (nuleotide binding oligomerisation domain) family (also alled the CATERPILLAR family) omprises at present more than 20 different mammalian NOD-LRR proteins whih mostly ontain three distint funtional domains (fig 1B): a arboxy terminal ligand reognition (LRR) domain, a entrally loated nuleotide binding domain (NBD), and a struturally variable amino terminal effetor binding domain whih onsists of protein-protein interation domains, suh as aspase reruitment domains (CARDs) or pyrin domains (reviewed by Inohara and Nunez 55 and Chamaillard and olleagues 56 ). Reent researh has mostly foused on two ytosoli reeptors of this family, NOD1 (also designated CARD4) and NOD2 (CARD15), whih both play a major role in intestinal regulation of proinflammatory signalling through NFkB in response to distint baterial ligands. NOD2 shares signifiant homology with NOD1, but ontains two, instead of one, CARD domains at its N terminus. Toll-like reeptors (TLRs) 11 mammalian, type I transmembrane reeptors with divergent LRR-extraellular domain, homologous TIR. Eah TLR binds distint moleular signatures present on diverse ommensals/pathogens. NODs 20 mammalian intraellular proteins with C terminal LRR, entral NBD, and N terminal CARD(s). PGN derived ligands: NOD2 muramyl dipeptide; NOD1 ie-dap Both Constitutively or induibly expressed by many different ells throughout the gastrointestinal trat. Downstream ativation of pro/anti-inflammatory ytokine seretion and/or apoptoti asades. Expression pattern NOD2 has been shown to be onstitutively or induibly expressed in monoytes, marophages, T and B ells, dendriti ells, 57 as well as IEC, inluding Paneth ells. NOD1 is ubiquitously expressed in many tissues and ells. Ligand speifiity A speifi motif of peptidoglyan (PGN), muramyl dipeptide (MDP), has been identified as (so far) the sole ligand of NOD2, whereas NOD1 senses a Gram negative PGN derivative, y-d-glutamyl-meso-diamino-pimeli aid (ie- DAP) Based on these studies, it has beome evident that neither NOD1 nor NOD2 sense LPS diretly but rather deteted ontaminations of these PGN motifs within nonpurified LPS preparations in earlier studies. Initially it was also believed that PGN is o-reognised, independently of its MDP or ie-dap omponents, by TLR2. But TLR2 mediated ell ativation indued by ommerially available Staphyloous aureus PGN preparations results from signifiant impurities. 71 It remains to be determined whether MDP or ie-dap diretly bind to NODs through LRRs or rather through as yet unidentified interim o-mediators. Another essential question is how muropeptides from non-invasive bateria gain entry into the ytosol and thus aess to intraellular NODs. A reent study provided the first answer by demonstrating that non-invasive Heliobater pylori delivers PGN to intraellular NOD1 in epithelial ells only in the presene of a funtional ag pathogeniity island enoding a baterial type IV seretion system. 72 In addition, enzymes present in the lumen or produed from phagoyti ells in the lamina propria may possibly digest baterial ell wall PGN, resulting in release and subsequent ellular uptake of muropeptides. Subellular distribution NOD2 protein has been deteted throughout the ytoplasm of IEC in vivo 59 but the exat subellular organelle loalisation of NOD1/NOD2 as well as the plae of interation with PGN fragments have not yet been identified. Signalling Despite enormous researh developments and advanes in this field during the last few years, urrent knowledge and understanding regarding detailed NOD1/2 signalling pathways is still limited (fig 3). On ligand stimulation, both NOD1 and NOD2 enter into CARD-CARD interations with the serine-threonine kinase Rip2/RICK/CARDIAK whih leads to NFkB ativation 73 and augmentation of aspase indued apoptoti asades. An intermediate region that is loated between CARD and the kinase domain of Rip2 interats with IKK (also alled NEMO), thereby linking NOD1 and NOD2 to the regulatory subunit of the IKK omplex whih initiates its ubiquitinylation. Funtional onsequenes deriving from NOD2 (and mutant variants) signalling events are disussed below. PHYSIOLOGICAL IMPACT OF BACTERIAL-MUCOSAL INTERACTIONS: BENEFICIAL EFFECTS FOR THE HOST LINK TO MUCOSAL HOMEOSTASIS Tolerane In the intestine, tolerane is an essential muosal defene mehanism maintaining hyporesponsiveness to harmless lumenal ommensals and their produts. Exaggerated 1185 Gut: first published as on 11 July Downloaded from on 6 Otober 2018 by guest. Proteted by opyright.

5 1186 inflammatory responses in the absene of pathogeni bateria would be otherwise deleterious. Several moleular immune mehanisms that ensure tolerane via TLRs in IEC have reently been desribed (see detailed review by Cario and Podolsky 76 ): dereased surfae reeptor expression whih limits frontline reognition, 4 19 high expression levels of the downstream signalling suppressor Tollip whih inhibits IRAK ativation, 11 ligand indued ativation of peroxisome proliferator ativated reeptor (PPAR) whih unouples NFkB dependent target genes in a negative feedbak loop, and external regulators whih may suppress TLR mediated signalling pathways. Additional tolerising mediators negatively modulating immune responses by interferene with the TLR signalling omplex have reently been identified in other ell systems, suh as SIGIRR (single immunoglobulin interleukin 1R related moleule; also known as TIR8), 79 TRIAD3A, 80 the zin finger protein A20, 81 and ST2. 82 Interestingly, TIR8 defiient mie are more suseptible to developing intestinal inflammation, suggesting a ruial role for TIR8 in tuning muosal tolerane towards ommensals. 83 The ubiquitin ligase TRIAD3A diretly enhanes proteolyti degradation of TLR4 and TLR9. 80 A20 also downregulates the TLR4 pathway by deubiquitination of TRAF6 and bloking NFkB ativation 84 as well as the TLR3 pathway by inhibition of interferon (IFN)-b gene expression via TRIF. 85 Commensal bateria may assist the host in maintaining muosal homeostasis by suppressing inflammatory responses and inhibiting speifi intraellular signal transdution pathways, 86 potentially diretly via TLR4 through elevation of PPAR expression, unoupling NFkB dependent target genes in a negative feedbak loop 77 whih may lead to attenuation of oloni inflammation. 78 Administration of CpG-DNA ameliorates the severity of dextran sodium sulphate (DSS) indued olitis via TLR9 89 and limits ytokine derived intestinal epithelial proinflammatory immune responses. 90 Commensals may also play a key role in preventing allergi sensitisation via TLR ativation (reviewed by Prioult and Nagler-Anderson 91 ). TLR4 dependent signals by the intestinal miroflora protet the host by inhibiting allergi responses to food antigens, while TLR4 mutant/knokout mie are highly suseptible to develop food allergy, whih orrelates with high levels of TH2 ytokines, interleukin (IL)-4 and IL Thus LPS stimulation may prevent allergen indued TH2-type inflammation by upregulation of TH1 responses via TLR4 in regulatory T ells. 93 Intestinal muosal intolerane (that is, exaggerated immune responsiveness towards ommensals) may our as a onsequene of endogenously or exogenously indued disturbane of any of the TLR dependent signalling mehanisms of tolerane desribed above. Further mehanisti understanding of suh host benefiial TLR/NOD signalling mehanisms of ommensal mediated suppression of intestinal inflammation and how imbalane in suh signalling events may lead to intestinal disease ould potentially provide an effiient immunoadjuvant therapeuti approah in IBD (and atopi diseases). Muosal barrier protetion Commensals are able to not only suppress but also atively indue expression of host genes that partiipate in important physiologial funtions, inluding ell differentiation and maturation. 94 In order to maintain muosal homeostasis against tissue damage, ommensal indued host modulatory effets seem to require funtional TLRx and NOD. Healthy gut onditions appear to demand onstant exposure of the intestinal surfae to ommensal derived TLRx ligands and basal state of ativation of downstream signalling pathways, thus ensuring rapid restitution and limited inflammatory responses. 95 Defiient TLRx or NOD signalling may imbalane ommensal dependent muosal homeostasis, failitating injury and leading to disease. Emerging evidene exists that ommensals diretly assist the host to strengthen intestinal epithelial barrier resistane. Our group has reently desribed an important role for ommensal indued TLR2 signalling in enhanement of intestinal epithelial barrier funtion, whih orrelated with distint tight juntion assoiated morphologial hanges. 99 However, our results also suggested that impaired funtion of TLR2 itself did not lead to inreased intestinal epithelial permeability. In this ontext, further studies are needed to investigate whether lak of host protetive TLR2 (or any other TLRx) ligands in the lumen that may be present in the resident miroflora ould lead to loss of barrier protetion, failitating invasion of pathogeni bateria in disease. Baterial learane and antimirobial ativity NOD2 has been found to exert antibaterial ativity in intestinal epithelial ells limiting survival of enteri bateria after invasion. Baterial learane of Salmonella typhimurium is strongly aelerated in IEC expressing a funtional NOD2 protein, whereas L1007fsinsC mutant expressing IEC are virtually unable to lear the pathogen in vitro. 61 NOD2, as well as RIP2 (as downstream omponent of the NOD2 signalling pathway) knokout mie, exhibit a profoundly dereased ability to lear intraellular Listeria monoytogenes, induing persistent immune ativation by ombined loss of antibaterial ativity, dysregulation of ytokine prodution, and imbalane of T ell ativation. Although the NOD2 indued gene targets that lead to elimination of invasive intraellular bateria are not yet identified in detail, reent reports suggest that TLR as well as NOD signalling pathways may ritially be involved in ommensal indued antimirobial peptide prodution, suh as defensins. These natural antibioti peptides represent an important mehanism of host defene in innate immunity by effiiently killing phagoytosed mirobes, thus helping to prevent pathogeni bateria from rossing the muosal barrier (reviewed by Ganz 103 ). Interestingly, b-defensin 2 may diretly at as an endogenous ligand for TLR4, 104 thus possibly exponentiating bateriidal ativity in a reverse autorine loop. However, the full spetrum of host benefiial signalling pathways ativated by ommensal derived TLR/NOD ligands whih may balane responsiveness and survival and onfer PhysiologialeffetsofTLRsandNODsinthe healthy gastrointestinal trat Tolerane (that is, maintaining hyporesponsiveness to harmless lumenal ommensals). Inhibition of allergi responses to food antigens. Protetion of intestinal epithelial barrier funtion. Baterial learane by indution of antimirobial peptide prodution. Maintenane of ommensal and muosal homeostasis. 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6 Health Disease Disease APC Healing Barrier protetion Tolerane Homeostasis Antimirobial ativity Baterial invasion Equilibrium Funtional TLRx NOD2 Injury Barrier destrution Intolerane Antimirobial ativity Baterial invasion integrity of the healthy intestinal muosa (fig 4) remains to be identified in depth. PATHOPHYSIOLOGICAL IMPACT OF BACTERIAL- MUCOSAL INTERACTIONS: GENETIC ABERRATIONS LINK TO MUCOSAL DISEASE Toll-like reeptor polymorphisms assoiated with IBD Chroni reurrent intestinal inflammation in IBD may result from undue stimulation of the muosal immune system by the resident miroflora. Healthy intestinal muosa expresses low onentrations of TLR2 or TLR4 protein in vivo. However, TLR4 expression is signifiantly inreased in IEC and lamina propria mononulear ells throughout the lower gastrointestinal trat in assoiation with IBD 4 and murine olitis TLR4 upregulation ould also result from ligands other than lumenal LPS. T ell derived ytokines, suh as IFN- and tumour nerosis fator a (TNF-a), whih play signifiant pathophysiologial roles in triggering IBD, have been found to upregulate intestinal epithelial TLR4 expression in vitro It remains to be shown whether upregulated TLR4 onfers funtional hyperresponsiveness of the intestinal epithelium to LPS or rather reflets a loss of response. The TLR4 gene is loalised on hromosome 9 (q32 33), 105 a genomi region in whih a CD suseptibility gene has been impliated. 106 In ative IBD, variant alleles in the TLR4 gene ould indue funtional dysregulation of the LPS reeptor. Gain of funtion mutations ould funtionally exhibit proinflammatory effets in response to physiologial onentrations of LPS. Two ommon mutations in the human TLR4 gene, D299G and T399I, have been observed to our at a general frequeny of between 6% and 10% in Cauasian Dysfuntional Commensal Muosal Inflammation Disequilibrium Figure 4 TLRx/NOD2 physiology and pathophysiology. In the healthy gut, both Toll-like reeptors (TLRx) and nuleotide binding oligomerisation domain 2 (NOD2) are majorly involved in host defene and tissue repair responses, thus maintaining muosal as well as ommensal homeostasis. TLRx signalling protets the intestinal epithelial barrier, onfers tolerane, and promotes healing. NOD2 exerts antimirobial ativity through defensin prodution and prevents intraellular baterial invasion. When ommensal and/or muosal homeostasis are impaired due to geneti and/or environmental triggers, disease may develop: baterial dysreognition and intolerane through aberrant TLR and/or NOD signalling stimulates exaggerated proinflammatory responses leading to hroni inflammation via ytokine and hemokine prodution. TLR dysfuntion indues tissue damage and barrier destrution by loss of ommensal mediated oloni epithelial progenitor responses. NOD2 dysfuntion leads to defetive sensing of mirobial threats and loss of bateriidal responses, thus allowing baterial invasion into the host. Commensals Epithelium TLRx Cytokines Invasion NOD2 NFκB Gain of dysfuntion polymorphisms TH1-type Apoptosis Figure 5 Current onept of how TLRx/NOD2 dysfuntions may ontribute to the pathophysiology of Crohn s disease. Toll-like reeptor (TLRs) and nuleotide binding oligomerisation domains (NODs) are present in the intestinal epithelium, the frontline of the muosal immune system. Gain of dysfuntion reeptor variants may allow ommensals to ross the intestinal epithelial barrier and gain aess to the antigen presenting ells (APC) of the underlying muosa. Subsequent ommensal mediated stimulation of APC signalling may indue nulear fator kb (NFkB) dysregulation leading to exaggerated TH1 responses and perpetuation of apoptosis. populations. 107 D299G polymorphism has been assoiated with CD as well as UC in a Belgian population, 108 but no assoiation was found in Sottish 109 or German 110 populations. Inreased suseptibility to IBD has been assoiated with oexistene of TLR4 and/or CD14 and NOD2 mutated alleles in a Greek population. 111 Although the D299G mutation (but not the T399I mutation) has been shown to interrupt TLR4 mediated LPS signalling in vitro, 112 the funtional phenotypi onsequene remains unresolved in IBD. Highly variable TLR4 gene mutations have been identified in various mie strains whih exhibit a broad distribution of different phenotypi responses to LPS, ranging from hyper- to hyposensitivity, 113 suggesting that additional gene interations of the diverse strain bakgrounds were involved. TLR4 knokout mie exhibit diminished TH1 driven immune responses and are therefore highly resistant to develop hroni nematode infetions of the gut. 114 Spontaneous olitis ourring in STAT3 knokout mie does not develop when these mie are rossed with TLR4 knokout mie, suggesting that aberrant TLR4 signalling in response to the indigenous intestinal flora ontributes to the development of intestinal inflammation through the TH1 pathway. 115 The role of TLR4 in TH1 dominant TNBS olitis has not yet been examined. These studies so far suggest that ommensal mediated TLR4 signalling of muosal T ells an be detrimental, leading to some forms of murine muosal inflammation assoiated with exess TH1 responses (fig 5). Of note, TH1 type shifts appear to have a signifiant pathogeneti role in CD. However, other data from a different murine olitis model also support the fat that TLR4 signalling may exert ytoprotetive harateristis, at least against hemially indued tissue injury in the intestinal muosa. TLR4 mutant (C3H/HeJBir) and TLR4 defiient mie show inreased suseptibility to DSS olitis in omparison with wild-type mie, suggesting that ommensal mediated protetion of the intestinal epithelial barrier (together with other muosal ells) might be signifiantly impaired in TLR4 dysfuntion after toxi DSS damage. Muosa 1187 Gut: first published as on 11 July Downloaded from on 6 Otober 2018 by guest. Proteted by opyright.

7 1188 Appropriate regeneration and restitution following epithelial wounding with DSS may require ommensal mediated amplifiation of oloni epithelial progenitor responses whih are speifially impaired in mie laking MyD88, 117 downstream of TLR4. Loss of funtion TLR4 mutations diminish sensitivity to LPS in C3H/HeJ mie 29 but paradoxially predispose to Gram negative infetions, suh as live Salmonella typhimurium. 118 Thus D299G mutation ould onfer hypersensitivity to Esherihia oli or any other Gram negative ommensal or pathogeni ligand ausing unwanted inflammation in IBD. Flagellin, the strutural omponent of baterial flagella, is sereted by several ommensal and pathogeni bateria, inluding Salmonella typhimurium, and indues intestinal epithelial hemokine seretion via TLR5 whih will, in turn, initiate dendriti ell migration and reruitment to the muosal site of inflammation. 5 Interestingly, dominant antigens in sera from oliti C3H/HeJBir (TLR4 mutant) mie seem to be flagellins. 119 Similar hyperreativity to flagellins was also observed in sera from patients with CD. Although it is unlear whether there was any signifiant orrelation with TLR4 (or NOD2) mutations in these CD patients, one may speulate that TLR4 mutations (similar to NOD2) ould lead to impaired baterial learane and thus greater presene of baterial antigens in the lumen, inluding flagellins. It is also possible that TLR4 mutation may indue geneti disequilibrium of TLR5 (or other TLRx) leading to unwanted inflammation through flagellin (or other TLRx speifi ligand) dysreognition. Results from a preliminary report suggest that CD is also assoiated with TLR9 promoter polymorphisms in a single German ohort. 121 Any assoiation between IBD and TLR2 R753Q polymorphism whih has reently been identified in septi patients has not yet been investigated. Although MyD88 defiient mie exhibit inreased suseptibility to DSS indued olitis and tissue damage, mutations within TLR efferent signalling proteins have not yet been desribed for IBD (or other intestinal diseases). NOD2 a major suseptibility gene for Crohn s disease In 2001, NOD2 was mapped via linkage disequilibrium to hromosome 16q12 as an exellent andidate gene for CD (IBD1). Consequently, ertain geneti variations have been assoiated with inreased suseptibility to some types of CD Three major variants of the LRR region (within or nearby) inlude one frameshift mutation (L1007fsinsC) and two missense mutations (R702W and G908R), suggesting that a defet in baterial reognition may be assoiated with CD. Initial studies demonstrated that up to 8% of Cauasian CD patients possessed at least one allele of the most ommon mutation, L1007fsinsC, ausing a trunated NOD2 protein TLR4 alteration assoiated with IBD Upregulation of muosal TLR4 expression in ative human IBD. Assoiation of TLR4 D299G -polymorphism with IBD in seletive populations. Murine TLR4 gain of funtion : spontaneous intestinal inflammation with TH1 exess. Murine TLR4 loss of funtion : inreased suseptibility to DSS olitis and Gram infetions. laking the last 33 amino aids. In CD, NOD2 expression in Paneth ells is inreased, possibly seondarily indued by proinflammatory ytokines. Previous studies have started to reveal the moleular mehanisms involved by whih NOD2 may influene innate immune responses in the intestinal muosa. It seems that different NOD2 mutations may span a spetrum of diverse phenotypes, ranging from omplete loss of funtion to maximal gain of funtion. NOD2 mutations within NBD lead to onstitutive ligand independent NFkB ativation, ausing a hroni systemi inflammatory disorder known as Blau syndrome. 55 Conversely, it has been suggested that CD assoiated NOD2 mutants whih are predominantly found in the mirobial ligand dependent LRR domain rather reflet loss of funtion phenotypes. Several in vitro transfetion studies showed that human CD assoiated NOD2 mutants signifiantly abolish NFkB ativation in response to MDP. However, paradoxially, marophages within the intestinal lamina propria of CD patients overprodue NFkB targets, inluding exaggerated prodution of proinflammatory ytokines, suh as TNF-a and IL-1b (reviewed by Podolsky 127 ). Aordingly, a reent in vivo study now demonstrates that MDP stimulated marophages isolated from mie generated with a murine NOD2 2932iC variant, homologous to the human NOD2 3020insC ( = L1007fsinsC) variant, exhibit enhaned NFkB ativation, inreased apoptosis, and elevated IL-1b seretion, 128 possibly implying an important mehanism of how dysfuntional NOD2 may trigger intestinal inflammation in some types of CD (fig 5). Thus this murine gain of funtion NOD2 frameshift mutation in the LRR region may imbalane funtions of both terminal parts of the whole protein: baterial dysreognition through the impaired LRR domain, ligand independent NFkB ativation, as well as unontrolled apoptosis and subsequent indution of IL-1b proessing and release through the hyperative CARD domains. In another in vivo study whih was simultaneously published by a different group, it was shown that mie laking full length NOD2 protein (NOD2 knokout) are more suseptible to oral infetion with the baterial pathogen Listeria monoytogenes. 96 The NOD2 gene produt is most abundant in ileal Paneth ells whih express a diverse population of mirobiidal defensins restriting olonisation or invasion of small intestinal epithelium by bateria. 129 Stimulation with the NOD2 ligand MDP eliits ryptidin seretion from murine NOD2 expressing Paneth ells. 130 Prodution of a subgroup of muosal antimirobial peptides, known as ryptidins, is signifiantly diminished in NOD2 knokout animals, 96 potentially supporting the reently NOD2 mutations assoiated with some forms of Crohn s disease or olitis Upregulation of NOD2 expression in Paneth ells in ative CD. Worldwide genotypi and phenotypi heterogeneity (major mutations: L1007fsinsC, R702W, G908R). CD assoiated mutant transfetants abolish NFkB ativation in response to MDP in vitro. Murine NOD2 2932iC : gain of funtion (indution of NFkB and IL-1b, unontrolled apoptosis). Murine NOD22/2: loss of funtion (dereased baterial learane due to lak of ryptidin prodution). Gut: first published as on 11 July Downloaded from on 6 Otober 2018 by guest. Proteted by opyright.

8 proposed onept of a defensin defiieny defet in human IBD. 131 Expression of defensin related ryptidin 4, whih is mostly sereted from Paneth ells in the distal small intestine, 132 is partiularly dereased in NOD2 knokout mie 96 whereas ileal expression of two human a-defensins (HD-5 and HD-6) is signifiantly diminished in NOD2 mutant CD patients. 102 Interestingly, human mutations in the NOD2 gene are strongly assoiated with distint phenotypi expressions of ileal disease (orresponding to the loation of Paneth ells), mainly involving fibrostenosing ompliations, but not formation of epitheloid granulomas. 137 However, in heterogeneous CD, it is likely that Paneth ell dysfuntion is only one pathophysiologial mehanism whih is speifially assoiated with a ertain phenotypial subtype of inflammatory disease with severe hyperproliferative hanges in the lower ileum in response to, yet unidentified, pathogeni or ommensal organisms. It is important to note that neither NOD2 knokout nor NOD2 frameshift mutant mie develop intestinal inflammation spontaneously However, in ontrast with NOD2 2932iC mutant mie, NOD2 knokout mie do not demonstrate signifiantly enhaned suseptibility to olitis in the DSS indued model, implying possibly variable mehanisti phenotypes of innate host defene. Genotypi heterogeneity at the single NOD2 lous ould not only lead to intraspeies but also interspeies variability of phenotypes. Thus diret proof is needed of whether the desribed signalling targets and effets of murine NOD2 dysfuntions indeed represent phenotypi onsequenes of NOD2 L1007fsinsC (and other human NOD2 mutants (in assoiation with or without TLRx mutations)) whih diretly ontribute to the omplex pathophysiology of IBD in humans. TLRx/NOD2 synergy? From a physiologial point of view, it is oneivable that TLR and NOD pathways may ooperate in (positive and/or negative) regulatory feedbak loops to modulate immunologial responses. Synergisti effets between different sensing events ould be advantageous for the host by exhibiting more ontrolled and immediate innate immune responses to potential threats. On the other hand, non-synergisti states of suh interations ould lead to disease by unontrolled ations of the innate immune system. Ligand dysreognition and subsequent disequilibrium of signalling events ould thus indue imbalane of pro- versus antiapoptosis and disturbane of baterial learane in the intestinal muosa. TAK and Rip have previously been identified as ommon regulatory hekpoints of NOD2 and TLRx signalling pathways (fig 3) whih ould ombine multiple signalling pathways (inluding TNFR/IL1R/IL18R) of both the innate and adaptive immune systems. In addition, negative ross talk between TLR2 and NOD2 has reently been proposed. Stimulation with the NOD2 ligand MDP inhibited TLR2 driven TH1 ytokine prodution by takling NFkB. Conversely, in NOD2 knokout mie, stimulation with syntheti lipopeptide was observed to result in NFkB dysregulation and subsequent imbalane of ytokine prodution (inrease in IL-12, derease in IL-10) via TLR Thus NOD2 may exhibit anti-inflammatory ativities limiting NFkB ativation after baterial stimulation of the TLR2 pathway, suggesting that the hyperinflammatory muosal response in CD may be due to lak of the inhibitory funtion of NOD2 on proinflammatory TLR2 signalling to baterial ligands in the lumen. However, two other reent studies failed to observe a similar negative regulatory effet of NOD2 on TLR These ontrasting results may imply ambiguous interrelations between these two pathways under different experimental onditions whih remain to be larified. In this ontext, one may also onsider that PGN was used as a presumed TLR2 ligand in some of these studies but, when highly purified, PGN sensing does not seem to our via TLR2. 71 Taken together, disussion of potential TLRx-NOD2 interrelations and their funtional impat remains unresolved at this point of the investigation and requires further study. Geneti and phenotypi heterogeneity of TLR4/NOD2 arriers Mutations of PRRs may modify detetion of pathogens, leading to altered suseptibility to disease. As outlined above, ertain mutations of TLR4 and NOD2 have been strongly assoiated with CD, implying that PRR dysfuntion may be involved in the pathogenesis of some types of CD (fig 5). NOD2 mutants have been assoiated with fibrostenosing ileal disease in CD but at this point in the investigation it remains elusive whether NOD2 genotyping would be helpful in linial pratie to predit the severity of the disease ourse or treatment response. 143 Compelling evidene emerges that geneti heterogeneity between ethni populations exists for TLR4 and NOD2 variant alleles, whih may reflet phenotypi heterogeneity in IBD worldwide Interestingly, no ommon NOD2 variant was found in Japanese CD patients. 146 Therefore, absene of a mutation in a healthy individual may not exlude development of intestinal disease later on, while on the other hand, a healthy individual may arry NOD2 variants, without ever developing intestinal disease. But protetive gene-environment or gene-gene interations at this lous remain to be identified. Therefore, reognition that the ontribution of NOD2 mutations to disease suseptibility may signifiantly vary in different raial and ethni populations in geographial areas has led to inreased efforts towards identifying potential geneti, environmental, and immunologial ofators that may determine manifestation or resistane to disease in suseptible individuals. Phenotypi alterations of the innate immune response in different individuals may be aused by diverse mutational pressure and seletivity within imbalaned interplays of several gene ombinations, but not all genes that may modulate innate immune responsiveness have yet been funtionally identified. Thus future studies will need to extend forward geneti approahes in this field (that is, starting with the immunologial differene of healthy versus diseased NOD2/TLR4- mutant arriers and then proeed from the top down to identify the ausative gene omplex(es)). It will be important to disset the multi-fator auses of the detrimental shift from host-benefiial to host-threatening innate immune reognition of ommensals via TLRx/NOD2 in human relevant studies in vivo. Role of TLRx/NOD2 in gastrointestinal muosal diseases other than IBD? Coelia disease is a HLA linked inflammatory disorder of the small intestine that is triggered by gluten peptides and strongly assoiated with irulating muosal IgA autoantibodies to tissue transglutaminase. Twin studies led to the onlusion that oelia disease is strongly linked to geneti fators but it does not neessarily strike both twins of an idential pair, 147 suggesting that environmental fators may 1189 Gut: first published as on 11 July Downloaded from on 6 Otober 2018 by guest. Proteted by opyright.

9 1190 be involved in the onset of the disease. Commensals or pathogens ould potentially provide suh required ofators for the development of this T ell mediated disease. 148 A reent study provided first evidene of attahment of rod shaped (not further haraterised) bateria to the intestinal epithelium in the upper small bowel of some untreated and treated oelia disease patients, but not in healthy ontrols, suggesting that bateria may be involved in the pathogenesis of oelia disease. 149 It is possible that baterial produts may sensitise CD4+ T ells to gluten via TLRx. 150 Interestingly, ertain gluten fragments eliit a diret innate response, while others preferentially drive adaptive responses. 151 Thus one may also speulate that dysfuntional TLRx (or other PRRs) ould diretly reognise dietary gluten peptides as non-self leading to release of IL-15 (see omment by Shuppan and olleagues 152 ). But no study has yet learly examined the potential role of TLRs and their speifi ligands in the pathophysiology of oelia disease. Heliobater pylori indues NFkB ativation in gastri epithelial and monoyti ells but it is ontroversial as to whih speifi TLR(s) may be involved in this immune response TLR2, 153 TLR4, or TLR5, whereas others have suggested that NOD1 may at as a sensor. 72 Further evaluation of potential involvement of TLRx/NODx in other pathogeni infetions of the intestine will be of linial importane. CONCLUSIONS AND PERSPECTIVES Reent studies have begun to define the mehanisms through whih ruial PRRs may regulate intestinal innate immunity. Cooperative as well as ompetitive interations may our between different baterial and non-baterial ligands via TLRs and NODs or other omponents of the innate immune system leading to differential pro- as well as anti-inflammatory immune responses in different ell types. Both TLRx and NOD2 are signifiantly involved in host defene and tissue repair responses, thus ruially maintaining muosal homeostasis. TLRx signalling protets intestinal epithelial barrier and maintains tolerane while NOD2 signalling exerts antimirobial ativity and prevents baterial invasion. Thus both reeptors olletively exhibit distint features that ensure ommensal as well as muosal homeostasis. Imbalane of the omplex interrelations between ommensals and PRRs may result in tissue injury and subsequent inflammation of the intestinal muosa (fig 4). Aberrant TLR and/or NOD signalling may stimulate diverse inflammatory responses leading to hroni intestinal inflammation with many different linial phenotypes. Perspetives of urrent and future studies Charaterisation of the distint phenotypi onsequenes of NOD2 L1007fsinsC and TLR4 D299G mutations, possibly resulting in speifi subforms of IBD. Identifiation of geneti, environmental, and immunologial ofators that determine manifestation or resistane to IBD in suseptible individuals. Clarifiation of potential positive/negative TLRx-NOD2 synergy. Investigation of PRR involvement in other gastrointestinal disorders, suh as oelia disease or enteri infetions. Evaluation of the potential value of PRRs (ativation versus inativation) as therapeuti targets. Further studies of the physiologial and pathophysiologial mehanisms within this network of possible ell-ell, ligandligand, and PRR-PRR signalling interations that may favour or prevent inflammatory disease ould lead to promising novel approahes that may differentially exploit the TLR/NOD pathways as a means of induing salutary immune responses for treatment of IBD. It is likely that differential therapeuti strategies will need to inlude agonists as well as antagonists of PRRs, taking into aount differenes in PRR pathophysiology at different stages of disease as well as phenotypi and genotypi heterogeneity between distint subgroups of IBD patients. Prophylati appliation of seletive TLR/NOD2 ligands ould enhane desired ommensal mediated tissue protetive proesses in order to prevent disease. One an aute inflammatory episode has broken out, some of the untoward effets of intestinal inflammation ould be abrogated by bloking unontrolled signal transdution by speifi TLR/NOD2 inhibitors, thus dampening tissue destrutive effets. One key element to this disease modifying approah might be to blunt, rather than entirely eliminate, the dysregulated innate responses in aute IBD. In this ontext, areful assessment of adverse effets will be ritial when modulating suh fundamental host defene pathways of innate immunity. Given the rapid and exiting advanements of researh in this field over the last few years, it is reasonable to presume that more immunologial evidene and onrete diretions for the potential value of these PRRs as therapeuti targets in IBD (and possibly other intestinal diseases) will emerge in the near future. ACKNOWLEDGEMENTS Supported by grants from the Deutshe Forshungsgemeinshaft (Ca226/4-1; Ca226/5-1/SPP Innate Immunity) and the Medial Faulty, University Hospital of Essen (IFORES). I thank Drs Andrew Gewirtz, Cathy Nagler-Anderson, Dana Philpott, Dan Podolsky, and Eduard Stange for interesting omments and disussions. Conflit of interest: None delared. REFERENCES 1 Medzhitov R, Janeway CA Jr. Deoding the patterns of self and nonself by the innate immune system. Siene 2002;296: Xu Y, Tao X, Shen B, et al. Strutural basis for signal transdution by the Toll/ interleukin-1 reeptor domains. Nature 2000;408: Cario E, Rosenberg IM, Brandwein SL, et al. 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