republished original article bacterial proteases in ibd and ibs · 2016-09-08 · bacterial...

Bacterial proteases in IBD and IBSNatalie Steck,1 Kerstin Mueller,2 Michael Schemann,2 Dirk Haller1

ABSTRACTProteases play a decisive role in health and disease. Theyfulfil diverse functions and have been associated with thepathology of gastrointestinal disorders such asinflammatory bowel disease (IBD) and irritable bowelsyndrome (IBS). The current knowledge focuses on host-derived proteases including matrix metalloproteinases,various serine proteases and cathepsins. The possiblecontribution of bacterial proteases has been largelyignored in the pathogenesis of IBD and IBS, althoughthere is increasing evidence, especially demonstrated forproteases from pathogenic bacteria. The underlyingmechanisms extend to proteases from commensalbacteria which may be relevant for disease susceptibility.The intestinal microbiota and its proteolytic capacityexhibit the potential to contribute to the pathogenesis ofIBD and IBS. This review highlights the relevance ofhost- and bacteria-derived proteases and their signallingmechanisms.

INTRODUCTIONInflammatory bowel disease (IBD)dincluding thetwo main distinct pathologies ulcerative colitis(UC) and Crohn’s disease (CD)dand irritablebowel syndrome (IBS) are chronically relapsingdiseases with an accelerating incidence, especiallyin developed countries.1 It is noteworthy that IBS-like symptoms such as diarrhoea or visceral painare frequently observed in some patients with IBDin remission. Although IBD and IBS are twodistinct entities, their complex pathogenesisinvolves common features including alterations inimmune responses, altered microbiota compositionwith an impact on microbiotaehost interactions,impaired intestinal barrier functions, alteredbowel habits and changes in visceral sensitivity.2 3

There is increasing evidence that all of the abovefactors are influenced by proteases, although theparticular protease involved and the pathwaysinfluenced by proteases may be different in IBD andIBS. This review focuses on the involvement ofhost- or bacterial-derived proteases in IBD or IBSand highlights those protease-activated pathwaysthat may be relevant in the pathogenesis of IBDand IBS.

Host-derived proteases in IBD and IBSThe large group of zinc-dependent matrix metal-loproteinases (MMPs) plays a central role in extra-cellular matrix turnover. Furthermore, MMPsproteolytically activate a variety of non-matrixsubstrates such as cytokines, chemokines, growthfactors and junction proteins. The deregulatedexpression or activity of host-derived MMPs hasbeen implicated in several diseases including

arthritis, atherosclerosis and colon cancer.4 5

Increasing evidence suggests that MMPs are thepredominant proteases involved in the pathogen-esis of IBD.6 7 MMPs influence the diseaseprogression in multiple ways involving the func-tion and migration of inflammatory cells as well asmatrix deposition and degradation. The expressionand activity of certain MMPs is increased duringacute inflammation,8 but also an imbalancebetween MMPs and their natural tissue inhibitors(TIMPs) has been reported for IBD.9 Besides MMPs,other proteases including trypsin, neutrophil elas-tase,10 mast cell tryptase,11 cathepsins12 andthrombin13 have been implicated in IBD. Superna-tants of mucosal biopsy specimens from patientswith IBS evoked activation of visceral nociceptiveneurons14 as well as enteric neurons15 and inducedhyperalgesia,16 a key feature of IBS. The responsescould be attributed to serine proteases.

Intestinal microbiota in IBD and IBSChanges in diversity and composition as well asfunctionality of the intestinal microbiota wereassociated with IBD17 18 and IBS.19 Pathogenicinfections are suggested as the starting point forIBD20 and IBS,21 but further work is needed tounderstand the role of commensal bacteria andtheir possible contribution to the pathogenesis ofintestinal disorders. In the context of intestinalinflammatory disease, it has to be considered thatcommensalism changes into a harmful situation forthe host which then becomes a continuousinflammatory trigger.22

Protease activity in the gut lumenExcessive concentrations of proteases have beenfound in the faeces of patients with UC or IBS.23e26

Consistent with these findings, secreted factors ofcolonic biopsy samples from patients with IBD andIBS showed increased proteolytic activity.16

Although the origin of the proteolytic activity wasnot elucidated, it is conceivable that proteasesreleased from biopsy specimens are derived fromthe host. However, increased faecal proteolyticactivity might result from colonic luminal bacteriawhich release serine, cysteine and metal-loproteases.27 Oral antibiotic treatment of miceresulted in reduced numbers of colonic microbiotaand reduced colonic luminal serine proteaseactivity, which provides further evidence for thebacterial origin of the proteases.23 Bacterialproteolytic activity could be demonstrated even inthe absence of inflammation, supporting thehypothesis that bacterial proteases are ubiquitouslypresent in the gut lumen but only have an impactin a susceptible situation.28

1Chair for Biofunctionality,Technische UniversitatMunchen, ZIELeResearchCenter for Nutrition and FoodScience, CDD-Center for Dietand Disease,Freising-Weihenstephan,Germany2Department of Human Biology,Technische UniversitatMunchen,Freising-Weihenstephan,Germany

Correspondence toDirk Haller, Biofunctionality,Technische UniversitatMunchen, Gregor-Mendel-Str.2, 85350Freising-Weihenstephan,Germany; [email protected]

NS and KM are joint firstauthors.

Published Online First9 September 2012

Steck N, et al. Postgrad Med J 2013;89:25–33. doi:10.1136/postgradmedj-2011-300775rep 25

Republished original article

This is a reprint of a paper thatfirst appeared in theGastroenterology andHepatology, 2012, Volume 61,pages 1610–8.

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IMPLICATIONS FOR BACTERIAL PROTEASES INTHE PATHOLOGY OF IBDRole of pathogen-derived proteasesIt has been suggested that alterations in microbialdiversity and composition in disease-susceptiblepopulations may alter innate defence mechanismsleading to chronic immune-mediated activation inactive IBD.29 30 Pathogens can be considered as theinitiation step at the beginning of the diseasedevelopment or during disease progression.18 20 31

Many attempts have been made to identify thebacterial structures or molecules responsible for thepathogenicity of microbes. Besides the mechanismof type-specific secretion systems which allow theinfiltration of bacterial material or whole bacteriainto host cells, proteases have been shown to beinvolved in the infectious process of pathogens.However, the pattern of different types of proteasesand their expression, regulation, activation andsubstrate specificity is very diverse. Host tissueprovides different target points for bacterial prote-ases. In addition to the activation of specific typesof host receptors, which will be discussed later, thedegradation of extracellular matrix and the disrup-tion of epithelial barrier function exhibits the mostfrequently described consequence of bacterialproteases. Targeting host epithelial barrier functionis a central mechanism to be considered in thecomplex pathogenesis of IBD. Impaired intestinalbarrier function has been associated with IBD,32e34

although it is still uncertain whether the loss ofbarrier integrity is the cause or a consequence ofchronic inflammation. Table 1 the proteases from

pathogens targeting epithelial or endothelial barrierfunction in different organs and summarises thegeneral mechanisms attributed to bacterial prote-ases which might be relevant in IBD.

Role of commensal-derived proteasesThe adherence junction protein E-cadherin is thebest described target for bacterial proteases derivedfrom pathogens. This might be surprising asE-cadherin provides cellecell contact on the lateralsite of epithelial cells and it remains unclear howbacterial proteases in the gut lumen get access totheir cleavage sites. We have demonstrated for thefirst time that a commensal-derived protease playsa role in intestinal inflammation. Gelatinase (GelE),a zinc-dependent metalloproteinase from Entero-coccus faecalis, disrupted the integrity of the intes-tinal epithelial barrier by targeting the junctionproteins occludin and E-cadherin.44 The presence ofdisease susceptibility, but not tissue inflammation,is required for E faecalis GelE-mediated disruptionof colonic barrier function (figure 1), supporting thehypothesis that commensal-derived proteasesremain harmless in the healthy host. Similar topathogens, commensal bacteria also possessa variety of virulence genes which are expressedunder certain environmental conditions. E faecalisGelE can be regarded as an example in whicha commensal-derived protease that has been asso-ciated with bacterial virulence plays a role in thedevelopment of intestinal inflammation. Furtheranalysis of the structure/function interplaybetween bacteria and the host, including the

Table 1 Bacterial proteases target epithelial cell barrier function

Species Classification ProteaseHost target structure/proposedmechanism Reference

Bacillus anthracis Pathogen Metalloproteinase lethal toxin (LT)M4 metalloproteinase neutral protease(Npr599)M6 metalloproteinase immuneinhibitor A metalloproteinase (InhA)

LT impairs barrier function in primaryhuman endothelial cells (alteredVE-cadherin distribution)Npr599 and InhA reduce endothelialbarrier function through increasedsyndecan-1 ectodomain shedding incultivated murine mammary gland cells

35

Citrobacter rodentium Pathogen Lymphostatin: virulence factor consistingof a glycosyltransferase, a protease andan aminotransferase

Disruption of epithelial barrier function viamodulation of the small GTPase Rho andCdc42

36

Clostridium difficile, Clostridium sordellii,Clostridium novyi

Pathogen Large clostridial toxins(glycosyltransferases)

Inactivation of GTPases Rho, Rac andCdc42 in intestinal epithelial cells

37

Clostridium perfringens Opportunistic pathogen Collagenase A Intestinal barrier function, basaltype-IV-collagen, mucus

28

Enterohaemorrhagic Escherichia coli Pathogen Metalloproteinase StcE Cleavage of mucin 7 and glycoprotein340, facilitation of adherence toepithelial-like HEp-2 cells

38

Enterotoxigenic Bacteroidis fragilis Opportunistic pathogen Metalloproteinase fragylisin or B fragilistoxin (BFT)

Induction of g-secretase dependentshedding of E-cadherin ectodomainin HT29 cells

39

Helicobacter pylori Pathogen Serine protease Helicobacter pylori hightemperature requirement A (HpHtrA)

Reduction of epithelial barrier integritythrough targeting E-cadherin

40

Pseudomonas aeroginosa Pathogen Pseudomonas elastase Reduction of barrier function in MDCKcells, altered ZO-1 expression anddisturbed microfilaments

41

Staphylococcus aureus Opportunistic pathogen Serine proteases Modulation of chemokine expressionthrough NF-kB activation

42

Vibrio cholerae Pathogen Metalloproteinase haemagglutinin/protease

Reduction of barrier integrity through actinand tight junction rearrangement

43

26 Steck N, et al. Postgrad Med J 2013;89:25–33. doi:10.1136/postgradmedj-2011-300775rep



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putative role of various bacterial virulence factors incommensal bacteria, will provide importantinsights into the pathogenesis of IBD.

Bacterial mucolytic activity: an underestimatedpotentialThe question of how and when commensal-derivedproteases are involved in IBD is clinically of greatimportance. Patients with IBD have an increasednumber of mucosa-associated bacteria and thethickness of the intestinal mucus layer is dimin-ished.45 46 Mucolytic activities allow bacteria to usemucus as a carbohydrate source and enable them tosurvive in the niche of the intestinal outer mucuslayer. Over 20 years ago, Rhodes and colleagueshypothesised that bacterial mucus degradationcould be associated with IBD, but they could notcorrelate bacterial glycosidase activity with diseaseactivity in patients with IBD.47 However, a recentstudy demonstrated a shift in the mucolyticconsortium of bacteria in IBD patients. The mostabundant mucolytic species in healthy controls,Akkermansia muciniphila, is reduced in IBD whereasRuminococcus species are disproportionally increasedunder conditions of chronic inflammation. Rumi-nococcus a- and b-glycosidases remove terminalsugars from the mucus matrix that subsequentlybecome accessible for other bacteria, providinga possible explanation for the increase in totalmucosa-associated bacteria in IBD.48 Pathogenshave evolved several strategies to disrupt and avoid

mucosal barriers. The enzymatic degradation ofmucins and the downregulation of mucin produc-tion are two of the most common mechanisms.49

The substrate specificity of bacterial proteases isoften not restricted to one molecule. Proteases thathave been demonstrated to digest mucins have alsobeen shown to facilitate adherence to host cells38 orto disrupt epithelial barrier function.28 Alterationsin microbial composition and mucus structureunder disease-susceptible conditions might allowcommensal-derived proteases to gain access to theepithelium, targeting protein substrates that wouldnot be accessible to them under physiologicalconditions.

PROTEOLYTIC ACTIVATION OF NEURONSThe functional relevance of the elevated proteaselevels in patients with IBD and IBS has beendemonstrated. In a rodent model, faecal superna-tants of diarrhoea-predominant IBS patients withelevated serine protease contents induced visceralhypersensitivity and increased paracellular perme-ability via the protease-activated receptor (PAR)2.26 However, faecal supernatants of patients withUC generated hyposensitivity to rectal distension,which was a result of PAR4 activation by cathepsinG. This anti-nociceptive action overpowered thepro-nociceptive effects of PAR2 activating proteasesin the faecal supernatants.24 This study suggestedthat even though patients with IBD and IBS haveincreased faecal protease levels, the functionallyactive proteases must be different and, in addition,signal through distinct mechanisms. Supernatantsof colonic biopsy samples of patients with IBSreleased proteolytic mediators that directly sensi-tised murine sensory neurons and generated visceraland somatic hypersensitivity through the activa-tion of PAR2.16 Further studies showed thatmucosal biopsy supernatants from patients withIBS also activated human enteric neurons15 as wellas rat visceral nociceptive neurons.14 In bothstudies proteases have been identified as the mainmediator responsible for neural excitation. Serineproteases such as thrombin, trypsin or mast celltryptase, which are released by epithelial cells,immune cells, blood cells or even neurons, activateenteric and visceral sensory neurons as well asenteric glia by targeting PARs.50 51 There are fourPARs (PAR1, PAR2, PAR3, PAR4), all of which areG-protein coupled tethered ligand receptors. Theyare activated by the proteolytic cleavage of the N-terminus which allows the tethered ligand to bindto the receptor. PAR1, PAR2 and PAR4 activateenteric neurons (see below for further details).However, PAR4 inhibits excitability of dorsal rootganglion neurons which supply the sensory inner-vation of the gut.52 This effect may explain thefinding that PAR4 exerts analgesic effects bysuppressing somatic and visceral hyperalgesia andpain.53 54 The functional expression of PARs indifferent cells may have a clinical impact in that itmay open new ways to specifically target proteaseactions. In this context, it is important to realise

Figure 1 Enterococcus faecalis GelE impairs barrier function in the susceptible host. Toassess the impact of GelE on colonic barrier function, different susceptibility modelswere used for intestinal inflammation. Interleukin-10-deficient (IL-10�/�) and TNFΔARE/Wt

mice are genetically-driven spontaneous mouse models for the development of chronicinflammation in the colon and ileum, respectively. Rag2-deficient (Rag2�/�) mice lack Band T cells and develop colitis after transfer of CD4+ T cells. NOD2-deficient (NOD2�/�)mice lack the intracellular pattern recognition receptor NOD2 (nucleotide-bindingoligomerisation domain 2) and are susceptible to certain bacterial infections. Appropriatewild type (Wt) counterparts 129SvEv and C57B6 were used as controls. Except for T celltransferred Rag2�/� mice, all other models did not develop histological changes withrespect to inflammation in the colon after 8 weeks. Distal colon segments of the micewere apically stimulated with purified GelE (10 mg/ml) for 5 h in Ussing chambersystems. The results are expressed as the percentage change in transepithelial electricalresistance (TER) compared with the initial value of the tissue. Data are partiallypublished44 and represent median with 25th and 75th percentiles from five animals pergroup; *p#0.05.




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that the contribution of PARs is different betweenrodent and human enteric neurons. While submu-cous neurons in the guinea pig have strongresponses to PAR2 activation, PAR1 seems to be thedominant receptor in human submucousneurons.55 It needs to be considered that PARs arepresent on muscle cells, epithelial cells and immunecells as well as on neurons. For example, humantissue resident macrophages are activated by PAR2ligands.55 Therefore, the disease relevant PARsignalling is a combination of several neuronal andnon-neuronal pathways. It is not known whetherthe neural- and glia-mediated actions of proteasesare part of the cascades initiating or maintaininginflammatory processes or whether they serveprotective functions. In principle, bacterial prote-ases may also be able to activate nerves in thegastrointestinal tract but this has not yet beendemonstrated. For the purpose of this review, wetherefore addressed this open issue and found thatthe metalloproteinase GelE from commensalE faecalis activated about 20% of guinea-pig entericneurons (figure 2). Interestingly, pretreatment ofthe tissue with GelE negatively influenced theability of a selective PAR2-activating peptide toexcite neurons. This suggests that GelE may signalthrough PAR2-expressing or dependent pathways,but further evidence for such a route is needed.

HOST RECEPTORS FOR BACTERIAL PROTEASESProtease-activated receptorsThe potential of different bacterial proteases totarget PAR as well as other host receptors has beenshown for respiratory and oral epithelial cells as

well as for platelets and neutrophils (table 2).However, the mode of action of bacterial proteasesin the gastrointestinal tract, except for E faecalisGelE,44 is still largely unknown. In general, prote-ases target the G-protein coupled PAR receptorsand induce activation by proteolytic cleavage of theN-terminal ligand or inactivation by disarmingthe receptor due to alternative cleavage of theN-terminus. The bacterial arginine-specific cysteineprotease gingipain-R of the periodontitis pathogenPorphyromonas gingivalis has been shown to cleaveand activate PAR2 on human neutrophils, PAR1and PAR4 on platelets to induce platelet aggrega-tion and PAR1 and PAR2 on an oral epithelial cellline to induce secretion of the pro-inflammatorycytokine interleukin 6 (IL-6).56e58 In addition toPARs, several other cell surface proteins such asoccludin and E-cadherin were targets of gingipain inMadin-Darby canine kidney cells65 and CD14human monocytes.66 The lung opportunisticpathogen Pseudomonas aeruginosa secreted the elas-tolytic metalloproteinase P aeruginosa elastase/LasBwhich disarmed PAR2 by proteolysis, made thereceptor unresponsive to activating proteases andavoided receptor internalisation and mobilisation ofintracellular pools. However, P aeruginosa elastasehas also been shown to increase respiratoryepithelial permeability by cleavage of tight junctionproteins zonula occludens 1 and 2. FurthermoreP aeruginosa released the exoprotease LepA whichinduced the activation of the transcription factorNF-kB via PAR1, PAR2 and PAR4 activation.60 61 67

Serratia marcescens, which is an opportunisticpathogen of the respiratory and urinary tract,

Figure 2 Effect of the bacterial protease GelE from Enterococcus faecalis on guinea-pig submucous neurons. (A) Asubmucous ganglion. Individual neurons were labelled with the voltage sensitive dye Di-8-ANEPPS which allowed directdetection of action potentials. (B) Application of GelE (100 nM) for 30 s induced action potential discharge (sharpupward deflections) whereas the control buffer solution alone had no effect (C).The traces are from the neuron markedby a black cross in (A). GelE and buffer were applied shortly before the recording period as indicated by the horizontalbars. GelE significantly increased action potential discharge (D). The ability of the PAR2-AP SLIGRL-NH2 (100 mM) toevoke action potential discharge was significantly impaired by predisposing the neurons to GelE (100 nM). Symbols marksignificant differences as explained in the figure. The numbers indicate numbers of tissues/ganglia/neurons studied.




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produced the zinc metalloproteinase, serralysin,which has been shown to induce IL-6 and IL-8mRNA expression in a respiratory cell line andtransactivation of AP-1, C/EBP- and NF-kB-drivenpromotors via PAR2.62 A house dust mite cysteineprotease allergen (Der p1) has been shown to targetPAR2 but to inactivate PAR1 in respiratoryepithelial cells and to induce release of pro-inflam-matory cytokines.63 The mite allergens Der p3and Der p9, which are serine proteases, activatedlung epithelial cells by interaction with PAR2and induced the release of pro-inflammatorycytokines.64

Pattern recognition receptorsToll-like receptors (TLR) are part of the innateimmune system and belong to the pattern recog-nition receptor family that identifies microbialpathogens through the recognition of pathogen- ormicrobial-associated molecular patterns (carbohy-drates, nucleic acids, peptidoglycans, lipoteichoicacids, lipoproteins). Direct proteolytic activation ofa full-length TLR has recently been described forthe avian TLR15. Virulence-associated microbial-derived proteases from the fungi Candida guillier-mondii, Trichosporon spp., Penecillium spp. and Mucorspp. and the Gram-negative opportunistic pathogenP aeruginosa have been shown to activate TLR15,which is a unique type of receptor that combinesTLR characteristics with an activation mechanismtypical of the evolutionary distinct PARs.68

Although it is still unknown which proteases acti-vate TLR15, one could speculate that these areserine proteases as the receptor activation could beinhibited by the serine protease inhibitor phenyl-methanesulfonylfluoride (PMSF). Mammaliansseem to lack a TLR that is able to sense proteolyticactivity, although mammalian TLR4 can be acti-vated by elastase-activated compounds from theextracellular matrix.69 Furthermore, lipopolysac-charide (LPS) recognition by TLR4 was indirectlyinfluenced by trypsin which is augmented in ilealinflammation and cleaved MD-2, an accessoryglycoprotein essential for TLR4 signalling. Theproteolysis of MD-2 provided a mechanism for

intestinal epithelial LPS tolerance that helped toregulate immune responses to commensal bacteria-derived ligands.70 Recent work connected TLR andPAR signalling and described distinct features of theTLRePAR interaction which contribute to signaldiversity in inflammation and host antimicrobialresponses.71 The cross-talk between differentreceptor signalling cascades and the recognition ofdiverse microbial signals provide homeostasis, butfurther investigations are needed to understand thiscomplex network more fully.

E-cadherinThe adherence junction protein E-cadherin isa calcium-dependent single-pass transmembraneprotein generally expressed in the lateral plasmamembrane of epithelial cells. E-cadherin providescontact to adjacent cells and plays a major role inepithelial cell differentiation. The intracellulardomain is highly conserved and signals throughcytoplasmic proteins from the catenin family.72

The extracellular domain could act as a receptor forbacterial or fungal entry into epithelial cells, whichso far has only been demonstrated for surfaceproteins. Internalin A from Listeria monocytogenes73

and invasion Als3 from Candida albicans74 75

interact with E-cadherin and mediate the internal-isation of the respective microorganisms.E-cadherin is also targeted by bacterial proteases;the metalloproteinase toxin BFT from Bacteroidisfragilis was shown to induce the shedding of theE-cadherin ectodomain through an unknownintestinal epithelial cell receptor-mediated induc-tion of g-secretase.39 A direct E-cadherin cleavagecould be demonstrated for the trypsin-like serineprotease HpHtrA from Helicobacter pylori.40 Wedemonstrated that a commensal-derived metal-loproteinase, GelE from E faecalis, was able todegrade the extracellular domain of E-cadherin inthe susceptible host, suggesting that proteases fromthe commensal gut microbiota might play a role inthe disease pathogenesis of chronic intestinalinflammation, but only if they get access to thetarget tissue under predisposed conditions.44 Theproteolytic cleavage of E-cadherin, either direct or

Table 2 Protease-activated receptors (PARs) as targets for bacterial and mite proteases

Species Classification Protease Host target structure/proposed mechanism Reference

Porphyromonas gingivalis Pathogen in periodontitis Cysteine protease gingipain-R Activation of PAR2 on neutrophils 56

Activation of PAR1 and PAR2 induced release of pro-inflammatorycytokine IL-6 from oral epithelial cells

57

Activation of PAR1 and PAR4 on platelets mediated induction ofplatelet aggregation

58

Treponema denticola Pathogen in periodontitis Released peptidases Inhibitory effect on proteolysis-mediated PAR2 activation 59

Pseudomonas aeroginosa Opportunistic lung pathogen Metalloproteinase P aeroginosaelastase/LasB

Inactivation of PAR2 in respiratory epithelial cells led tounresponsiveness to activating proteases

60

ExoproteaseLepA Activation of PAR1, PAR2 and PAR4 induced activation of NF-kB 61

Serratia marcescens Opportunistic pathogen of therespiratory and urinary tract

Metalloproteinase serralysin Activation of PAR2 induced expression of IL-6 and IL-8 transcripts 62

House dust mite Cysteine protease allergen Der p1 Inactivation of PAR1, activation of PAR2 mediated release ofpro-inflammatory cytokines in respiratory epithelial cells

63

Serine protease Der p3 and Der p9 Activation of PAR2 induced release of pro-inflammatorycytokines in lung epithelial cells

64

IL, interleukin.




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indirect, reflects an important mechanism forbacteriadespecially pathogensdto reach theintercellular space and to translocate across theepithelium. Furthermore, E-cadherin is targeted bya number of endogenous metalloproteinasesincluding stromelysin-1, matrilysin,76 ADAM-1077

and meprin-b.78

Protease-dependent receptor activationThe release of tumour necrosis factor a (TNFa) isassociated with an increased permeability of thegut epithelial barrier. The blockade of TNF withanti-TNF antibodies is an established strategy inthe treatment of IBD. The increase of soluble bio-logically active TNF arises from the conversion ofmembrane-bound TNF by TNF-converting enzyme(TACE).79 80 TACE or ADAM17 belong to theADAM (a disintegrin and a metalloprotease) familyof metal-dependent proteases and has been addi-tionally implicated in the shedding of othermembrane-bound precursors of cytokines andgrowth factors.81 One of these factors is trans-forming growth factor-a which in turn activatesthe epidermal growth factor receptor (EGFR).Phosphorylated EGFR induces the activation of

mitogen activated protein kinases and mediateschanges in intestinal permeability.82 Many of theseprocesses and receptor activation mechanisms aredescribed in the context of carcinogenesis,83

a frequent IBD-associated complication. In partic-ular, the fact that numerous cytokines and growthfactors are membrane-associated as precursors andrequire proteolytic conversion may representa novel mechanism for bacterial-derived proteases.

CLINICAL IMPLICATIONSThe inhibition of host-derived proteases has beendiscussed as a therapeutic option in IBD but thereare only limited reports with inconsistent results.Studies in animal models showed that inhibition ofcathepsins12 and tryptase84 ameliorates chemical-induced colitis. Furthermore, the serine proteaseinhibitor camostat has been reported to induce andmaintain remission in patients with UC.85 VariousMMP inhibitors have been shown to improveinflammation, particularly in rat models ofIBD86e88 but also in dextran sodium sulfate (DSS)-induced colitis in mice. Despite the fact that MMPinhibitors showed beneficial therapeutic effects in

Figure 3 Protease activity in the gut. Illustration of possible involvements of proteases in disease-related mechanismsof inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). The role of PAR-dependent pathways and thecontribution of PARs are based on data from human tissue. PAR2 would play a significant role in rodent enteric neurons.The PAR-related pathways shown in the top part may be primarily relevant in IBS while the bottom pathways prevail inIBD; however, an overlap of some mechanisms is likely. Ca, calcium; ECM, extracellular matrix; GelE, gelatinase fromEnterococcus faecalis; IEC, intestinal epithelial cells; PAR, protease-activated receptor.




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experimental colitis models, a clinical trial investi-gating the beneficial potential of the MMP inhib-itor prinomastat in lung cancer failed because ofunacceptable adverse effects.89 In an animal model,serine protease inhibitors have been shown to beeffective in inhibiting the pronociceptive effectsinduced by the supernatants of mucosal biopsiesand faeces samples from patients with IBS.16 26

These results suggest the use of serine proteaseinhibitors in the treatment of IBS symptoms. Todate, studies that use inhibitors of host-derivedproteases have shown rather poor efficacy in thetreatment of human IBD. Adverse side effects,presumably resulting from non-specific actionsand/or broad spectrum inhibition of host proteasesalso affecting functions of other organs, need to beaddressed before protease inhibitors become a ther-apeutic option. In addition, the use of endogenousprotease inhibitors could be considered as a treat-ment option in IBD to avoid adverse side effectsand to reconstitute the proteolytic balance in thegut. One example is elafin, a serine protease inhib-itor, the expression of which is reduced in the gutmucosa of IBD patients.90 Furthermore, it has beendemonstrated that elafin protects against DSS-induced colitis through the inhibition of pro-inflammatory mediators and the strengthening ofepithelial barrier function.91

The inhibition of PARs by PAR antagonists couldbe an effective strategy to suppress protease-medi-ated action, particularly if they specifically targeta particular PAR. The use of PAR antagonists asa treatment option for IBD and IBS is supportedby their involvement in the generation of pain,inflammation, increased permeability, as well asmotor and secretory disorders in the intestine.In this respect it may even be beneficial todevelop PAR agonists which would provide thePAR4-mediated anti-nociceptive action in humans.A few clinical trials have shown that the

administration of probiotics such as the probioticmixture VSL#3 improved IBS symptoms includingpain/discomfort, distension/bloating and defeca-tion92 93 and induced and maintained remission inpatients with UC.94 95 Furthermore, the feedingstudy conducted by Kunze et al provided evidencefor the interaction between the probiotic Lactoba-cillus reuteri and colonic enteric neurons.96 Althoughprobiotic efficacy could be demonstrated in IBDand IBS, the active functional components/struc-

tures, the molecular mechanisms or the primaryprobiotic target still remain to be clarified. It maybe speculated that the protective role of probioticsmay be related to the release of proteases and/orprotease inhibitors. Such a mechanism has recentlybeen reported for the serine protease lactocepinfrom Lactobacillus paracasei which inhibited therecruitment of pro-inflammatory cells to the site ofinflammation through degradation of chemo-kines.97 The anti-inflammatory effect of lactocepinsuggested that bacterial proteases play an impor-tant role in the regulation of intestinal homeostasis.

SUMMARYWe are at the very beginning of understanding thecomplex intestinal microbial proteolytic capacitywhich shows significant potential to be involved inthe pathogenesis of IBD, IBS and other gastroin-testinal disorders. The mechanisms so far describedare mostly restricted to proteases from pathogens,whereas proteases from commensal gut bacteriahave generally been ignored. As shown in figure 3,the proteolytic activity in the gut is a result of host-derived proteases expressed by a variety of differentcell types, as well as bacterial proteases produced bydifferent species. The coordinated interactions ofhost and bacterial-derived proteases maintainhomeostasis but become relevant to disease ifderegulated. GelE from E faecalis is the first exampleof a bacteriaehost interaction involvinga commensal bacterial protease that is implicated inthe pathogenesis of IBD and other gut diseasesassociated with inflammation.

Funding This work was supported by the German ResearchFoundation (Deutsche Forschungsgemeinschaft) grant GRK 1482 andthe European Union grant FP7 Intestinal Proteases: Opportunity forDrug Discovery (IPODD).

Competing interests None.

Contributors All authors wrote the manuscript. NS and KMprepared the figures and tables.

Provenance and peer review Not commissioned; externally peerreviewed.

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Key messages

< Host-derived proteases are involved in the pathogenesis of IBD and IBS< Protease-activated receptors are specific targets for host-derived and

bacteria-derived proteases< Proteases belong to virulence factors of intestinal pathogens and commensals< Mechanistically, bacterial proteases target and impair barrier function of

epithelial cells and activates enteric neurons< Certain susceptibility is necessary for the involvement of commensal-derived

proteases in IBD2009;155:257e65.




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