D A Elphick, Y R Mahida

Transcription

1 1802 Reent advanes in basi siene PANETH CELLS: THEIR ROLE IN INNATE IMMUNITY AND INFLAMMATORY DISEASE THE See end of artile for authors affiliations Correspondene to: Professor Y R Mahida, Institute of Infetion, Immunity, and Inflammation, C Floor, West Blok, Queen s Medial Centre, Nottingham NG7 2UH, UK; Yash.Mahida@ Nottingham.a.uk I D A Elphik, Y R Mahida Gut 2005; 54: doi: /gut n this artile, we disuss the urrent understanding of how the intestinal muosa may exert ontrol over luminal bateria, and how intestinal inflammation ould ensue when this ontrol is lost. We shall review researh showing how Paneth ells, and evolutionarily onserved innate immune mehanisms, are emerging as key mediators of intestinal muosal defene. SMALL BOWEL CHALLENGE Maintenane of a sterile environment in the small intestinal lumen represents a formidable hallenge for the host. The multitude of villi and rypts reate an expansive epithelial surfae of approximately 400 m 2, allowing effiient nutrient absorption but a wealth of potential entry sites for invading mirobes. To heighten the hallenge, the intestinal muosa omprises a single layer of epithelial ells, unlike the multiple layers found at other muosal surfaes. This aids nutrient absorption and water and eletrolyte transport, yet spreads defensive strategies thinly. The nutrient rih luminal ontent would appear to provide an ideal ulture medium, and there is onstant exposure to a large population of miro-organisms, both ingested along with food and from the adjaent olon with its heavy baterial load. In addition, epithelial ells are replaed every 2 5 days from pluripotential stem ells in the base of the rypts 1 and so ontinuous antimirobial protetion for these stem ells is of paramount importane as damage to or parasitisation of stem ells would have severe onsequenes for the maintenane of the normal digestive epithelium. Against all the odds, mirobial density in the healthy proximal small intestine (duodenum, jejunum, proximal ileum) is low. 2 In ontrast, in the distal ileum and olon, there is extensive resident baterial flora (total,10 14 ) onsisting of,400 different speies of anaerobi and aerobi bateria. 3 Muosal defene mehanisms in the proximal small bowel are able to maintain a ruial barrier to mirobial invasion yet allow effiient nutrient absorption. ADAPTIVE VERSUS INNATE IMMUNITY The immune system has many faets, whih an be grouped into adaptive and innate omponents. 4 Adaptive immunity, whih is only found in vertebrates, is mediated by T and B ells whih display struturally unique reeptors that are generated by gene rearrangement. On binding of reeptor to its speifi antigen, lonal expansion of lymphoytes results to eliit a direted immune response. However, it takes three to five days for a suffiient number of lymphoytes to be produed and to differentiate into effetor ells, whih is more than enough time for most pathogens to invade and damage the host. In ontrast, innate immunity refers to inbuilt mehanisms, many of whih are relatively onserved throughout the animal and plant kingdoms, that respond immediately to a wide variety of miro-organisms and an be seen as a first line of defene to ontrol an invasion before lonal lymphoytes an mount a speifi attak. We shall disuss the role of adaptive and innate immune mehanisms in the defene of the gut s epithelial monolayer. Muosal adaptive immunity The adaptive muosal immune system an prinipally be divided into indutive sites, where antigens from muosal surfaes stimulate naïve T and B lymphoytes, and effetor sites, where antigen sensitised ells extravasate and differentiate. Effetor sites inlude the lamina propria and epithelium where synthesis and seretion of seretory IgA (siga) antibodies ours. Whereas systemi immunity depends on antigeni supply to the lymph nodes and spleen via lymph and peripheral blood, the muosal immune system atively samples antigen from muosal surfaes and is tolerant of innouous substanes and ommensal bateria. Indutive sites for muosal immunity onsist of organised lymphati folliles termed muosa assoiated lymphati tissue, whih may our singly or may ongregate in Peyer s pathes (or the appendix), as well as draining lymph nodes 5 Follile assoiated epithelium, whih overs organised lymphoid tissue in the intestine, ontains speialised M ells that transport luminal antigens into the dome area of the follile where antigen presenting dendriti ells (DC) and lymphoytes oexist. DCs also send Gut: first published as /gut on 11 November Downloaded from on 13 August 2018 by guest. Proteted by opyright.

2 proesses between gut epithelial ells without disturbing tight juntion integrity and sample ommensal and pathogeni gut bateria. 5 When T and B ells are ativated in Peyer s pathes, they express the a4b7 integrin and migrate to the blood. 6 Gut endothelial ells express MADCAM-1, the ligand for a4b7, whih allows Peyer s path derived ells to migrate from the blood to the lamina propria. 6 The lamina propria is filled with plasma ells that serete 2 5 g of siga into the gut lumen daily. 7 Dimeri siga is one of the most important defene fators on muosal surfaes. It is resistant to proteolysis, and its task is to prevent both the adherene of bateria to muosa and their penetration into the internal environment. Muosal innate immunity Innate immune mehanisms proteting the gut muosa omprise mehanial, ellular, and hemial omponents working at three levels: the extra-epithelial, epithelial, the subepithelial levels. The mehanial element is the physial barrier of the epithelium, along with muus oating, enteroyte shedding, and peristalsis. The hemial element inludes pathogen Figure 1 Shemati diagram of Toll-like reeptor (TLR) and nuleotide binding oligomerisation domain (NOD) protein interations with omponents of the baterial ell wall and subsequent nulear fator kb (NFkB) ativation. Lipopolysaharide (LPS), diaminopimelate (DAP), and muramyl dipeptide (MDP) from the peptidoglyan (PGN) baterial ell wall bind to TLR4, NOD1, and NOD2, respetively. Phosphorylation (P) of IkBa leads to release of NFkB whih migrates to the nuleus to promote transription of speifi genes. Table1 Reeptor TLR2 TLR2+TLR 1 TLR2+TLR6 TLR3 TLR4 TLR5 TLR7 TLR8 TLR9 NOD1 NOD2 Host reognition of mirobial omponents Ligand Lipoteihoi aid from Gram positive bateria Triayl lipoproteins Diayl lipoproteins dsrna Lipopolysaharide Flagellin ssrna reognition on endosomes (mouse) ssrna reognition on endosomes (human) CpG (baterial) DNA D-Glu-meso-DAP (diaminopimelate; Gram negative peptidoglyan motif) Muramyl dipeptide from peptidoglyan TLR, Toll-like reeptor; NOD, nuleotide binding oligomerisation domain. reognition moleules, proteins, or peptides that indue mirobial killing and ytokines that orhestrate an immune response. The ellular element inludes epithelial ells, mast ells, dendriti ells, phagoyti ells, suh as marophages and granuloytes, natural killer ells, and d T ells. The extra-epithelial defene barrier inludes antimirobial proteins and peptides, suh as lysozyme and defensins, whih disrupt mirobial ell walls, muus oating that traps bateria to be removed by peristalsis, and ommensal flora that provides resistane to olonisation. Defene at the level of the epithelial ell inludes the mehanial barrier to penetration of the epithelial monolayer and tight juntions, and pattern reognition reeptors (PRRs) whih reognise highly onserved motifs, designated pathogen assoiated moleular patterns (PAMPs), whih are present in large groups of miro-organisms, but not their hosts, and are usually essential for the pathogeniity or survival of the mirobe. Examples of PAMPs are lipopolysaharide or peptidoglyan, whih are both omponents of baterial, but not host, ell walls. Unlike lymphoyte reeptors in adaptive immunity, reeptors for the innate immune system are germline oded and, therefore, are not altered by prior exposure to pathogens. 8 9 Binding to PRRs triggers the seretion of hemokines that lead to the reruitment of ellular omponents of the innate immune response. Toll-like reeptors (TLRs) and nuleotide binding oligomerisation domain (NOD) proteins are two lasses of PRRs in mammals TLRs are transmembrane moleules whih link the extraellular ompartment, where reognition of mirobial pathogens ours, and the intraellular ompartment, where signalling asades leading to ellular responses are initiated (fig 1). NOD proteins are ytosoli and so reognise mirobial omponents after invasion into the ell. Toll-like reeptors The first disovery of Toll related proteins in mammals in 1998 was quikly followed by demonstration that mammalian TLR4 was the long sought after signalling reeptor in a protein omplex responsible for the reognition of lipopolysaharide leading to the ellular responses that result in endotoxi shok In mammals, there are at least 11 members of the TLR family, and TLR1 9 are onserved in humans and mie. 11 Many of the PAMPs reognised by individual TLRs have been eluidated (see table 1). Ligand binding to the extraellular leuine-rih repeat portion of the TLRs sets up an intraellular asade of reruitment of adaptor moleules and ativation of kinases, 1803 Gut: first published as /gut on 11 November Downloaded from on 13 August 2018 by guest. Proteted by opyright.

3 1804 whih ulminates in the phosphorylation of IkBa, the inhibitory moleule for nulear fator kb (NFkB). 10 NFkB enompasses induible transription fators that are important ativators of genes involved in inflammatory and immunologial responses. 14 The ative form of NFkB onsists of heterogenous dimers that share a homologous region responsible for DNA binding, nulear loalisation, and ytoplasmi interation with IkB proteins. The p60:p50 heterodimer is the predominant form of ative NFkB. It is stored in an inative state in the ytoplasm bound to inhibitory proteins of the IkB family (of whih IkBa is the best haraterised). IkB masks the nulear loalisation signal of NFkB, thereby retaining it in the ytoplasm. Following stimulation (by, for example, ativated TLR, NOD protein, or proinflammatory ytokines suh as interleukin 1 or tumour nerosis fator a), IkB is degraded to release ative NFkB, whih migrates to the nuleus to bind to its sequene reognition motif on promoters of target genes, leading to transriptional upregulation of a number of genes involved in inflammatory responses. NOD proteins The NOD proteins, NOD1 and NOD2, have reently been shown to represent an intraellular pathogen sensing system in mammals, both reognising baterial peptidoglyan, although eah responding to distint motifs of this moleule (see table 1). 15 Like TLRs, NOD1 and NOD2 have a C terminal series of leuine rih repeats that failitate PAMP reognition. At the N terminus, NOD1 has one aspase ativating and reruitment domain (CARD) while NOD2 had two suh CARD domains. NOD1 and NOD2 are also designated CARD4 and CARD15, respetively. Like TLRs, ativation of NOD proteins sets up an intraellular asade of events ulminating in NFkB ativation via IkBa phosphorylation Reently, researh on NOD2 has reeived onsiderable attention as a geneti approah has identified NOD2 as the first suseptibility gene for Crohn s disease, as well as for Blau syndrome, a rare autoinflammatory disease affeting the eye and joints Subepithelial omponents, inluding marophages, dendriti ells, and myofibroblasts, reognise pathogens and their omponents that have breahed the epithelial layer. Ativation of ell assoiated reeptors leads to release of ytokines and hemokines to reruit innate immune effetor ells, and antigen presentation to T ells to eliit an adaptive immune response. Key points: innate immune mehanisms Germline oded mehanisms that respond immediately to mirobes and do not require prior exposure to antigen Pattern reognition reeptors (PRRs) inlude Toll-like reeptors (TLRs) and nuleotide binding oligomerisation domain (NOD) proteins PRRs reognise pathogen assoiated moleular patterns (PAMPs) PAMPs are present on mirobes but not hosts PAMP reognition leads to mirobial killing by various means Antimirobial peptides, suh as defensins, are important effetors Antigen presenting ells failitate an adaptive immune response Figure 2 Small intestinal rypt showing Paneth ells with seretory granules, stem ells, enteroytes, muin sereting goblet ells, and an intermediate ell (features of Paneth and goblet ells). Exposure to baterial produts leads to release of antimirobial peptides and proteins from Paneth and intermediate ells. PANETH CELLS Paneth ells were first desribed over a entury ago as granulated ells at the base of the small intestinal rypts of Lieberkühn. 20 While their funtion remained an enigma until reent years, they are now onsidered to be important in innate intestinal defene as regulators of mirobial density in the small intestine and in the protetion of nearby stem ells. There are on average 5 12 Paneth ells in eah small intestinal rypt and, in ontrast with other epithelial ell types, they migrate down from the stem ell zone to the rypt base where they are relatively long lived (20 days ompared with 3 5 days for enteroytes) Stem ells also give rise to three other ell lineages enteroytes, goblet ells, and enteroendorine ells most of whih migrate upwards and populate the villi. Paneth ells are filled with numerous prominent apial ytoplasmi granules whih, on ell stimulation, an be released into the rypt lumen (fig 2). 23 Various histohemial stains, inluding eosin, periodi aid Shiff s stain, and phloxine-tartrazine (fig 3), 24 intensely stain the basi Paneth ell granules whereas more reently preise staining has been ahieved with immunohistohemistry employing antibodies against Paneth ell speifi omponents, inluding lysozyme, 25 defensins (fig 4), 26 and seretory phospholipase A2 (spla2). 27 Sometimes, ells with morphologial features of Paneth ells and goblet ells are observed in the villi and are termed intermediate ells (fig 3). Although the funtion of intermediate ells is not lear, they have been shown to express defensins (figs 3, 4). Paneth ell pattern reognition reeptors Studies in Paneth ell ontaining isolated murine small intestinal rypts have shown seretion of mirobiidal peptides (predominantly ryptdins) following exposure to Gram negative or Gram positive bateria or their produts, lipopolysaharide, lipoteihoi aid, lipid A, and muramyl dipeptide. 29 It is of interest that fungi or protozoa did not stimulate Paneth ell degranulation. Whether the baterial produts interat with Paneth ells diretly or via rypt non-paneth ells remains to be determined. Lipid A is the Gut: first published as /gut on 11 November Downloaded from on 13 August 2018 by guest. Proteted by opyright.

4 Figure 3 Low (A, B) and high (C, D) power views of phloxine-tartrazine stained setions of uninfeted ontrol (A, C) and T spiralis infeted (B, D) murine small intestine. A few Paneth ells (with predominantly yellow granules) are present at the base of uninfeted rypts. In T spiralis infeted intestine, there is a marked inrease in the number of Paneth ells (with red granules). Moreover, intermediate ells (arrowed) are seen in some villi. Two (A, B) of the figures are reprodued from Kamal and olleagues, 28 with permission from Blakwell Publishing Ltd. Figure 4 (A) Setion of human jejunal muosa showing human defensin 5 immunoreative Paneth ells at the base of rypts. (B) Transmission eletron mirograph of human jejunal rypt showing three Paneth ells with eletron dense granules in the apial ytoplasm. biologially ative omponent of lipopolysaharide, and isolated Paneth ells have been shown to respond equivalently to different forms of lipid A and lipoteihoi aid, implying reognition of ommon omponents of these glyoproteins. 30 Surprisingly, murine Paneth ells do not express mrna transripts for TLR4. Lipopolysaharide and lipid A mediated Paneth ell responses, independent of TLR4, were demonstrated in TLR4 defiient C3H/HeJ mie. 30 Murine Paneth ells do express mrna transripts for TLR 1 3 and TLR 5 9. The funtional ativity of TLR9 has reently been demonstrated by Paneth ell degranulation in response to its ligand, CpG (baterial) DNA. 31 NOD2 protein, whih is an intraellular reeptor for muramyl dipeptide, has reently been shown to be expressed in the ytoplasm of Paneth ells. The potential role of Paneth ells in the pathogenesis of Crohn s disease in patients with NOD2 mutations is disussed below. Paneth ell antimirobial peptides Several proteins and peptides, inluding lysozyme, spla2, and enteri a-defensins, ryptdin related sequene peptides, and angiogenin 4, with well doumented antimirobial ativity, have been loalised to Paneth ell granules. On exposure to viable or heat killed bateria or to mirobial produts, suh as lipopolysaharide or lipoteihoi aid, Paneth ells release their granules resulting in inreased onentrations of antimirobial peptides in the intestinal lumen. 29 This is believed to prevent mirobial invasion into the rypt miroenvironment, providing protetion for the stem ell zone, and also ontribute towards the ontrol of mirobial density in the small intestinal lumen. Lysozyme Lysozyme is an antibaterial protein that is found at signifiant onentrations in many human seretions, 1805 Gut: first published as /gut on 11 November Downloaded from on 13 August 2018 by guest. Proteted by opyright.

5 1806 inluding tears, breast milk, saliva, and gastri and small intestinal seretions. It is expressed in the intestinal trat by gastri and pylori glands, duodenal Brunner s glands, small intestinal Paneth ells, marophages, and granuloytes, but not in the normal olon. 36 Lysozyme is predominantly ative against Gram positive bateria. It leaves glyosidi bonds whih stabilise peptidoglyan, resulting in baterial lysis. Gram negative bateria have an outer membrane whih protets the underlying peptidoglyan ell wall and so are relatively resistant to lysozyme. The likely importane of lysozyme in intestinal innate defene has been suggested by its lak in Paneth ells in newborn infants with nerotising enteroolitis. 37 The lak of lysozyme may render these infants suseptible to baterial transloation and subsequent sepsis syndrome. Seretory phospholipase A2 spla2 is a omponent of Paneth ell granules 27 and is released into the intestinal lumen on stimulation by baterial produts, inluding lipopolysaharide. 38 spla2 purified from murine small intestinal Paneth ells has been shown to have bateriidal ativity against Salmonella typhimurium and Listeria monoytogenes, indiating its role in small intestinal muosal defene. 39 Enteri a-defensins of mie and men Defensins are small (29 45 amino aids in length) ationi peptides that have been divided into two main families, the a- and b-defensins, on the basis of the disulphide bond pairing pattern. The defensins are synthesised as preursor polypeptides that are post-translationally proessed into mature ative peptides. The antimirobial ativity of these peptides is believed to be due to the formation of a membrane spanning pore that eventually leads to lysis of the baterium. In humans, four neutrophil defensins (HNP-1, -2, -3, -4) were identified first, followed by two enteri a-defensins (HD-5, -6), expression of whih is normally restrited to Paneth ells in the small intestine. Enteri a-defensins have been well haraterised in murine Paneth ells and are termed ryptdins ( rypt defensins ). In ontrast with enteri a-defensins, human b-defensin 1 (HBD-1) and other members of the b-defensin family appear to be expressed by most epithelial ells of the small and large intestine. HBD-1 is expressed onstitutively while HBD-2 is indued by stimuli that ativate the transriptional fator NFkB. 41 Defensins may have multiple roles, dependent on onentration. Murine ryptdins reah onentrations of mg/ml in the rypt miroenvironment. 29 These onentrations are at least 1000 times higher than minimal bateriidal onentrations in vitro, indiating the potential for a-defensins to maintain sterility in the rypt and protet the stem ells. It is plausible that lower onentrations of a-defensins that reah the small intestinal lumen have an appreiable effet on resident miroflora. In keeping with this, mie defiient in the proessing enzyme matrilysin that do not express ative ryptdins are suseptible to small intestinal olonisation by non-invasive Esherihia oli speies. 42 Cryptdin 2 and 3 have also been shown to form pores in intestinal epithelial ells and to lead to a hloride seretory response, 43 whih would be expeted to flush the epithelial surfae of bateria. More reently, ryptdin 3 (but not the non-pore forming ryptdin 4) has been reported to indue expression of the potent polymorphonulear hemoattratant interleukin 8 via alium dependent ativation of p38 mitogen ativated protein kinase (MAPK) and NFkB signalling pathways. 44 These studies suggest that in addition to antimirobial ativity, some enteri a-defensins are apable of induing biologial effets in epithelial ells to enhane muosal protetion against luminal miro-organisms. Cryptdin related sequene (CRS) peptides CRS peptides have reently been desribed as a group of highly potent antimirobial peptides in mie. 34 They are a family of ovalently linked dimeri antimirobial moleules, a feature that might ontribute to their diversity and potentiate effiient protetion of the intestinal muosa. They are proessed in a similar way to ryptdins, with highly similar pro-regions of the peptide, with two possible proessing sites for matrilysin (enzyme that proessed ryptdin preursors, see below). CRS peptides have also been shown to bind and redue the immunostimulatory ativity of LPS. 34 Angiogenins Angiogenins have reently been established as a family of endogenous antimirobial proteins. 35 Although impliated in the growth of human tumour ells in mie, human angiogenin has been shown to exhibit mirobiidal ativity against systemi baterial and fungal pathogens. A likely role for this lass of mirobiidal proteins in the defene of the intestinal muosa has been demonstrated by expression of angiogenin 4 by mouse Paneth ells. 35 Baterial produts stimulate the release of the mirobiidal angiogenin 4 from Paneth ell granules into the gut lumen. Paneth ell: baterial interations Paneth ells respond to baterial stimulation by releasing antimirobial peptides/proteins stored within granules into the rypt lumen. Of these antimirobial peptides, a-defensins have been the most thoroughly studied. Mouse a-defensins Enteri a-defensins (ryptdins) from mouse small intestinal Paneth ells were the first non-leuoyte defensins to be identified, 45 and six have now been haraterised and found to exhibit broad spetrum antimirobial ativity. They are ative against E oli, Listeria monoytogenes, Staphyloous aureus, and Giardia lamblia, although individual ryptdins exhibit antimirobial ativity of varying range and poteny. 46 Antibody to ryptdins 1, 2, 3, and 6 has been shown in vitro to neutralise 70% of the bateriidal ativity sereted by mouse rypts, indiating the importane of a-defensins in muosal defene. 29 The mehanisms by whih preursor forms of ryptdins are proessed into mature ative peptides have reently been haraterised. Paneth ell granules in the mouse have been found to ontain matrilysin (MMP-7), a matrix metalloproteinase enzyme whih leaves the propeptide so that Paneth ell degranulation releases ative defensin into the rypt lumen. 42 Prosegment and mature ryptdins have been demonstrated in Paneth ell granules, and analyses suggest that the majority of proryptdin is proessed by MMP-7 within the granules. 29 The in vivo importane of ryptdin ativation has been shown by MMP-7 null mie, whih aumulate proryptdin in their Paneth ell granules and are more suseptible to oral administration of the enteri pathogen Salmonella typhimurium. 42 Gut: first published as /gut on 11 November Downloaded from on 13 August 2018 by guest. Proteted by opyright.

6 Inhibition of Paneth ell ryptdin and lysozyme expression has been reported after oral inoulation of mie with wildtype Salmonella typhimurium. 47 Studies using heat killed and mutant S typhimurium showed that diret interations between live wild-type bateria and epithelial ells are required to inhibit ryptdin expression. Moreover, it was demonstrated that the epithelial ell responses are mediated via the p38 MAPK pathway. The effet of S typhimurium on ryptdin and lysozyme expression appears to be pathogen speifi as Listeria monoytogenes, whih also invades murine (and human) small intestine, did not have any effet. Human enteri a-defensins Whereas six enteri a-defensins have been haraterised in mie, only two (HD-5 and HD-6) have been identified in humans. 48 These, like ryptdins, are predominantly expressed in Paneth ells of the small intestine, and HD-5 has been isolated from ileal tissue and haraterised Reombinant HD-5 has been shown to have a wide spetrum of killing ativity, being ative against both Gram positive and Gram negative bateria as well as fungi (Listeria monoytogenes, Esherihia oli, Salmonella typhimurium, and Candida albians) The important ontribution of human a-defensins to enteri muosal immunity has been demonstrated by a reent in vivo study in whih transgeni mie expressing HD-5 in small intestinal Paneth ells profoundly influened muosal protetion against virulent Salmonella typhimurium. 52 This was manifest by a redution in the baterial burden in the intestinal lumen and faees, dereased baterial transloation to the spleen, and higher survival rates after lethal Salmonella hallenge. 52 However, HD-5 transgeni mie did not have a survival advantage when intraperitoneally inoulated with Salmonella, indiating that the effet of HD-5 is mediated in the intestinal lumen. The mehanism of inreased intestinal defene against Salmonella in the HD-5- transgeni mie is not lear, as HD-5 and mouse ryptdins have similar in vitro ativity against this pathogen. In ontrast with mie, human Paneth ell granules ontain only the proform of HD-5 (amino aids 20 94; fig 5), and they do not ontain matrilysin In vitro, pro-hd-5 an be proessed to the mature form (amino aids 63 94) by trypsin, whih together with a 1 -antitrypsin and panreati seretory trypsin inhibitor (Kazal-type trypsin inhibitor) are expressed in Paneth ells Analysis of terminal ileal fluid taken at endosopy has onfirmed that the predominant in vivo form of luminal HD-5 is indeed the mature (amino aids 63 94) peptide predited by in vitro trypsin leavage. 51 Luminal aspirates also yielded a slightly longer HD-5 form (amino aids 56 94). Of note, both of these isoforms are C terminal to an arginine residue, whih is typial of trypsin leavage. It has therefore been proposed that following seretion by Paneth ells, enzymatially ative trypsin proesses pro-hd-5 to the mature form in vivo. 51 Key points: enteri a-defensins However, a further luminal form of HD-5 has been isolated from ileal neobladder urine. 49 In these subjets, after radial ystetomy for transitional ell arinoma of the bladder, an ileal neobladder was fashioned. Enteri defensins were then isolated from urine. The and mature HD-5 forms found in ileal aspirates were also found in neobladder urine, but a longer form, 36 94, was also present. This form has been shortened from pro-hd-5 (20 94) but not at a trypsin leavage site. This same form (36 94) was also found in vitro when isolated small intestinal rypts were stimulated with either the holinergi agonist arbamyl holine or baterial lipopolysaharide. 26 It may therefore be that alternative mehanisms, or additional steps, for the proessing of pro- HD-5 to the mature form exist. Paneth ell: parasite interations Paneth ell granule depletion has been observed in subjets with human immunodefiieny virus related ryptosporidiosis 55 and mie infeted with the nematode Trihinella spiralis 28 but the mehanism remains to be determined. In parasite infeted mie, there is also an inrease in the number of Paneth ells (fig 3), whih is maximal around the time of worm expulsion Transfer experiments in T ell defiient mie suggest that muosal T ells play an important role in induing an inrease in the number of Paneth ells in parasite infeted mie. 28 Expression profiling studies in the small intestine of T spiralis infeted mie have shown that Paneth ell speifi ryptdins are strongly represented. 56 Paneth ells: a link with inflammatory bowel disease Metaplasti Paneth ells in olitis Although in the normal gastrointestinal trat Paneth ells are onfined to the small intestine, they an appear in aberrant sites in various disease states. Expression of small intestinal epithelium elsewhere in the gastrointestinal trat is termed intestinal metaplasia, and the term omplete metaplasia is used if Paneth ells are present. 57 Formation of intestinal metaplasia is usually preeded by hroni inflammation suh as in Barrett s oesophagus or in the stomah following hroni Heliobater pylori infetion. 58 Paneth ell metaplasia ours in hroni inflammatory onditions of the olon, most notably ulerative olitis and oloni Crohn s disease, 26 but also divertiulitis 59 and radiation olitis. 60 Metaplasti Paneth ells seen in inflammatory bowel disease olitis are usually Signal Propeptide Mature peptide MRTIAILAAILLVALQAQA ESLQERADEATTQKQSGEDNQDLAISFAGNGLSALRTSGSQAR ATCYCRTGRCATRESLSGVCEISGRLYRLCCR Figure 5 Amino aid sequene of human defensin (HD)-5. Preursor form of HD-5 (amino aids 20 94) is stored in Paneth ell granules, and has been shown to be proessed to three predominant forms (amino aids 63 94, 56 94, 36 94) during and/or after release into the lumen (see text for details). Two forms exist in humans: HD-5 and HD-6 Sereted into the rypt lumen by Paneth ells Seletively attak mirobial ell membranes Mouse defensin isoforms provide 70% of rypt antimirobial ativity Transgeni mie expressing HD-5 are proteted against orally administered virulent Salmonella typhimurium Gut: first published as /gut on 11 November Downloaded from on 13 August 2018 by guest. Proteted by opyright.

7 1808 seen in the rypt region, and are morphologially idential to small intestinal Paneth ells. 26 Immunohistohemial studies indiate that they express the antimirobial proteins lysozyme, 61 spla2, 62 and a-defensins. 26 It is likely that in the olon affeted by inflammatory bowel disease, metaplasti Paneth ells help protet the damaged oloni epithelium against baterial invasion. NOD2 protein is expressed in Paneth ells NOD2 protein is a ytosoli PRR of the innate immune system that reognises muramyl dipeptide, a onstituent of peptidoglyan whih is present in baterial ell walls. 63 Mutations in the NOD2 gene, on hromosome 16, have been assoiated with Crohn s disease They lead to a defiieny in the gene produt to sense muramyl dipeptide. 64 Three main mutations in this gene aount for 80% of the total mutations assoiated with Crohn s disease, and 25 43% of Cauasian Crohn s patients arry at least one of these mutations. Two of these single nuleotide polymorphisms result in single amino aid hanges (R702W, G908R), and the third a frameshift mutation leading to a trunated protein (1007 fs). All mutations affet the C terminal leuine-rih repeat, or muramyl dipeptide reeptor, domain. Genotype-phenotype orrelations have demonstrated that NOD2 mutations are assoiated with ileal disease, a tendeny to develop stritures, and with a younger age at onset. 67 As the NOD2 gene has reently been shown to be expressed in the ytoplasm of Paneth ells, there is onsiderable urrent interest in a possible link between the presene of defetive NOD2 protein in Paneth ells and the development of small bowel Crohn s disease. It may be that defetive reognition of baterial produts via NOD2 leads to dysregulation of Paneth ell mediated responses against intestinal bateria in Crohn s disease. This redued baterial sensing may lead to an inreased suseptibility of the intestinal muosa to invasion by luminal bateria, and to the development of hroni inflammation. NOD2 protein-antimirobial peptides: is there a link within Paneth ells? Altered expression of antimirobial peptides seondary to expression of the mutant NOD2 gene in Paneth ells ould lead to abnormal olonisation of the small bowel with mirobes, whih may provoke hroni inflammation via adaptive immune mehanisms. There is some reent evidene of a link between the NOD2 gene and expression of a-defensins in Paneth ells. NOD2 defiient (NOD2 2/2 ) mie have been found to show redued Paneth ell expression of transripts of the enteri a-defensins ryptdin 4 and ryptdin 10 sequene and there was further redution in expression of these transripts after intragastri infetion with the Gram positive intraellular baterium Listeria monoytogenes. 68 Interestingly, NOD2 2/2 mie are outwardly healthy and display no overt symptoms or histologial evidene of intestinal inflammation when Key points: NOD2 protein Mutations in NOD2 are assoiated with Crohn s ileitis NOD2 is found in Paneth ells, whih are prominent in the ileum Paneth ells release a-defensins to maintain rypt sterility a-defensin expression may be redued in Crohn s ileitis observed for up to six months However, when hallenged with L monoytogenes via the intragastri route, NOD2 2/2 mie had greater suseptibility to infetion, as shown by signifiantly greater numbers of bateria reovered from the liver and spleen ompared with wild-type ontrols. In ontrast, NOD2 2/2 mie showed no differene to wild-type mie when inoulated with L monoytogenes, either by intravenous or intraperitoneal injetion, indiating that NOD2 may play a key role in mediating protetion of the intestinal muosa against baterial infetion. Expression of enteri a-defensin mrna has reently been shown to be redued in terminal ileal biopsies from patients with Crohn s disease, with redued expression being more pronouned in the presene of NOD2 mutations. 70 Although this result adds further redene to the hypothesis that Crohn s disease may be assoiated with a redution in muosal innate immunity, there is muh yet to be explained. For example, the mehanism by whih NOD2 may regulate expression of Paneth ell a-defensins remains to be determined. A number of reent studies have reported the effet of NOD2 mutations in responses by ells other than Paneth ells but are not onsidered further as they are beyond the sope of this artile. SUMMARY A variety of mehanisms are needed to mediate host protetion against miro-organisms in the intestine prior to indution of highly speifi adaptive immune responses. Reent studies suggest that Paneth ells play an important role in innate host defene via their ability to serete antimirobial peptides and proteins. There is onsiderable urrent interest in host and mirobial fators that may regulate Paneth ell funtion. As they are able to respond to omponents of the ommensal mirobial flora, there is also signifiant urrent interest in the ontribution of these ells to the pathogenesis of inflammatory bowel disease.... Authors affiliations D A Elphik, Y R Mahida, Institute of Infetion, Immunity, and Inflammation and Division of Gastroenterology, University of Nottingham and University Hospital, Nottingham, UK Conflit of interest: None delared. REFERENCES 1 Booth C, Potten CS. Gut instints: thoughts on intestinal epithelial stem ells. 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