REVIEW ARTICLE
Decoding Norovirus Infection in Crohn’s Disease Mathias Chamaillard, PhD,* ,†,‡,§ Annabelle Cesaro, PhD,* ,†,‡,§ Pierre-Emmanuel Lober, PhD,k and Didier Hober, MD, PhDk
Abstract: Although a causing viral infectious agent remains untraceable in Crohn’s disease, most recent genome-wide association studies have linked the FUT2 W143X mutation (resulting in asymptomatic norovirus infection) with the pathogenesis of Crohn’s ileitis and with vitamin B12 deficiency (i.e., a known risk factor for Crohn’s disease with ileal involvement). In line with these findings, host variations in additional genes involved in host response to norovirus infection (such as ATG16L1 and NOD2) predispose humans to Crohn’s ileitis. One may therefore presume that asymptomatic norovirus infection may contribute to disruption of the stability of the gut microbiota leading to Crohn’s ileitis. These paradigms highlight not only the need to revisit the potential transmissibility of Crohn’s disease, but also potential safety issues of forthcoming clinical trials on human probiotic infusions in Crohn’s ileitis by rigorous donors screening program. (Inflamm Bowel Dis 2014;20:767–770) Key Words: Crohn’s ileitis, dysbiosis, fecal transplantation, norovirus, vitamin B12 deficiency
NOT ALL EQUAL AGAINST NOROVIRUS INFECTION Norovirus (originally referred as Norwalk virus after an outbreak of acute gastroenteritis in Norwalk, Ohio) is a positivesense single-stranded RNA, nonenveloped virus that remains the most common cause of nonbacterial foodborne illness outbreaks.1–3 Norovirus is widely disseminated and primarily transmitted through the fecal–oral route resulting in more than 250 million infected individuals worldwide, among which the sub-type GII.4 strain accounts for about 80% of outbreaks. Life’s quality of infected people remains strongly affected by severe watery diarrhea, abdominal pain, low-grade fever, lethargy, malnutrition, and nausea. Morbidity is considerably enhanced among infected infants, immunocompromised hematopoietic stem cell transplantation recipients, and the elderly, as judged by over 200,000 deaths per year worldwide.4,5 Norovirus infects the intestinal mucosa through interactions with oligosaccharide chains of histoblood group antigens.6,7 However, a substantial proportion of infected subjects remain asymptomatic, and not all exposed individuals become infected, despite repeated challenge.8 Notably, about 20% of Europeans are commonly considered as protected against symptomatic norovirus infection,9 because of their inability to express the H type-1 Received for publication October 29, 2013; Accepted November 19, 2013. From the *Institut Pasteur de Lille, Center for Infection and Immunity of Lille, Lille, France; †CNRS, UMR 8204, Lille, France; ‡Institut National de la Santé et de la Recherche Médicale, U1019, Team 7, Equipe FRM, Lille, France; §Univ Lille Nord de France, Lille, France; and kUniversité Lille 2, Faculté de Médecine, CHRU de Lille, Laboratoire de Virologie EA3610, Loos-lez-Lille, France. The authors have no conflicts of interest to disclose. Reprints: Mathias Chamaillard, PhD, Institut Pasteur de Lille, 1, rue Professeur A. Calmette, 59496 Lille, France (e-mail: [email protected]). Copyright © 2013 Crohn’s & Colitis Foundation of America, Inc. DOI 10.1097/01.MIB.0000440613.83703.4a Published online 17 December 2013.
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oligosaccharide ligand that is required for norovirus binding. Mechanistically, bodily secretion of histoblood group antigens is fully inactivated as a result of a nonfunctional enzymatic activity of the a1,2-fucosyltransferase that is encoded by the fucosyltransferase 2 (FUT2) gene.10 Homozygous individuals for the W154X nonsense mutation (referred as rs601338) in the FUT2-encoding gene are considered as nonsecretors and showed enhanced resistance to acute infection by several ribotype of norovirus.11 Consistently, a reduced antibody response to norovirus is commonly observed among nonsecretors.12 Likewise, missense mutations were found to confer partial protection against norovirus infection.13 Notably, individuals bearing a missense A385T mutation within the stem region of FUT2 showed about 5-fold lowered secretion of A-, B- and H- blood group antigens in mucus and body fluids.14 In addition to FUT2, several other mutations (that were maintained by strong selective pressure15,16) result in a nonsecretor phenotype that protect against numerous pathogens, including norovirus.
DECODING HOST DEFENSE TO NOROVIRUS INFECTION In the gut, norovirus (also commonly referred MNV) infects primarily macrophages and dendritic cells,17 among which conventional dendritic cells play a physiological role in host defense against norovirus by limiting its dissemination to secondary lymphoid tissues18 and antigen presentation to T cells.19 Mechanistically, sensing of MNV-1 infection by dendritic cells requires the expression of a cytosolic surveillance system involving MDA-520 and potentially NOD2.21 Among infected cells, the STAT1-dependent interferon response protects from lethal infection22 by negatively regulating apoptosis of infected cells and systemic dissemination of norovirus.23 Notably, type I interferons have been found to play a key role in restricting MNV replication24 by blocking translation of viral proteins.25 Noteworthy, the aforementioned antiviral response driven by www.ibdjournal.org |
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type I interferons is negatively regulated by the Atg5-Atg12-coupled autophagic machinery,26 while IFN-g–mediated antiviral response of macrophages require expression of the Atg5-Atg12/Atg16L1 expression but not the catabolic activity of authophagy.27 Although B and T lymphocytes were dispensable for resistance to lethal infection, Rag-deficient mice were persistently infected, arguing for a role of adaptive immunity on the outcome of secondary norovirus infection. Noteworthy, norovirus is characterized by the high level of variability of its surface exposed residues. Early secretion of norovirus-specific salivary immunoglobulin A is predictive of an adaptive resistance to infection in a substantial portion of susceptible individuals.8 Long-lasting immunity to norovirus did not require CD8+ cells,28 but rather B and CD4+ T lymphocytes.23,24,29,30
RESISTANCE TO NOROVIRUS INFECTION IS LINKED TO CROHN’S ILEITIS PREDISPOSITION Crohn’s disease (CD) is a public health problem that mostly affects young adults in industrialized countries and, more recently, in emerging countries. The incidence of CD is thought to rise under the influence of many different environmental and genetic factors as judged by a concordance rate of about 50% to 60% among monozygotic twins. In Europe and North America, the socioeconomic impact is increasing with a combined prevalence of about 250 to 300 cases per 100,000 Europeans and North Americans. This translates into as many as 2.2 million patients with CD,31 because the therapeutic management of CD remains far from optimal. Recent genome-wide associations studies unveiled the missense G258S (referred as rs602662) and the noncoding W154X mutations of FUT2 as molecular links between asymptomatic norovirus infection and the pathogenesis of CD.32 A total of 4 noncoding single nucleotide polymorphisms of the FUT2-encoding gene (namely rs504963—30 UTR, rs676388— 30 UTR, 7 rs485186—synonymous exon 2 SNP, and rs492602— synonymous exon 2) were found associated with CD with ileal involvement,33 whereas the secretor status was more frequently found in Crohn’s colitis.34 In contrast to Crohn’s ileitis, none of the patients with CD with colonic involvement were found as nonsecretors.34 This paradigm may account for relapsing CD in some symptomatic patients.35 In line with these indisputable evidences from genetics, one may presume that a substantial proportion of patients with CD remain asymptomatic to a large variety of norovirus infection. Consistently, several serological approaches and stool antigen testing failed to detect any disease-specific patterns of antibodies against several enteric viruses (including norovirus).36,37 Furthermore, reverse transcription polymerase chain reaction analyses failed to document any association between CD and the fecal presence of norovirus.38 More importantly, both G258S and W154X mutations were linked to vitamin B12 deficiency,39,40 i.e., a known risk factor for CD who primarily exhibit transmural inflammatory lesions of the terminal ileum,41 within which Paneth cells are especially numerous at the base of the crypts of Lieberkühn. Paneth cells correspond to one of the 4 main epithelial cell lineages that play
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a key role on mucosal immunity, host defense and inflammation resolution.42–44 Furthermore, methyl-deficient diet result in decreased number of Paneth cells45 and mice with hypomorphic expression of ATG16L1 showed an abnormal granule formation in Paneth cells on infection by norovirus.46 Dissemination of the norovirus to secondary lymphoid tissues was increased in infected mice with lowered ATG16L1 expression, which were found more at risk to develop intestinal inflammation afforded by bacterial commensals. Antibiotic treatment was indeed found efficient to improve disease severity in both wild-type and mutant animals. However, norovirus was not detected in the Paneth cells, and its replication was not found impaired within the ileum and the colon of infected mice with lowered ATG16L1 expression,46 suggesting a potential compensatory mechanism for ATG16L1 on the maintenance of the IFN-g– mediated antiviral response.27 However, it remains to be deciphered whether norovirus infection may account for the abnormal granule formation, enhanced necroptosis, and impaired secretion of antimicrobial peptides as what observed in Crohn’s ileitis.47 Recent studies gave some preliminary answers by showing that Norovirus binds to differentiated epithelial cell and Goblet cells before being internalized,48 and that extensive rearrangements of intracellular membranes occur in infected cells as a result of MNV replication.17 Conversely, it remains elusive whether patients with Crohn’s colitis may be protected against norovirus infection as what observed for patients with ulcerative colitis, among which the W154X FUT2 mutation was found to confer a dominant protection.49
“ASYMPTOMATIC NOROVIRAL DRIVER-BACTERIAL PASSENGER” MODEL Host–microbiota interactions are thought to modulate outcome of norovirus infection,50 as evidenced by defects in fucosylating processes within the small intestinal mucosa of germ-free animals.50,51 Consistently, microbiota transition result in an increased in FUT2 expression and enhanced alpha(1,2/3)-fucosyltransferase activity.52 More recently, high-throughput sequencing approaches revealed that most patients infected by norovirus displayed changes of the structure and composition of their fecal microbiota (i.e., referred herein as dysbiosis) independently of age and gender.53 Notably, norovirus-driven dysbiosis was characterized by a decreased abundance of potential anti-inflammatory bacteria related to Faecalibacterium sp. accompanied with an enhanced abundance of Escherichia coli, but all isolated were negative for known virulence factors. One may therefore presume that the aforementioned patients were secretors as they were symptomatic. Recent studies gave preliminary answers whether the primary nonsecretor A allele of rs601338 may indeed drive dysbiosis in caucasians. In CD with colonic involvement, the nonsecretor FUT2 allele was indeed linked to colonic mucosa-associated dysbiosis, including enhanced abundance of Firmicutes and decreased of Proteobacteria.54,55 Likewise, the biliary tract of FUT2 nonsecretor patients suffering from primary sclerosing cholangitis (a known complication of patients with Crohn’s colitis and ulcerative colitis) showed similar changes in the diversity and structure of their bacterial communities.56 As
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what observed in humans, fecal- and colonic mucosa-associated dysbiosis is also observed in mice deficient for either NOD257–59 or FUT2.55 More importantly, recent studies demonstrated that risk for intestinal inflammation in Nod2-deficient mice is dictated by the composition and structure of their fecal microbiota.60 This paradigm argue for the need to revisit the potential communicable nature of CD that was first suggested in the late 1970s. The best evidence of the communicable nature of this condition comes from the observation that inoculation of mucosal homogenates from patients with CD (but not controls) was enough to trigger disease onset in New Zealand white rabbits61 or mice.62 In contrast, no signs of CD-like epithelioid and giant cell granulomas were observed in mice after the injection of autoclaved homogenates from patients with CD. More importantly, exclusion of microorganisms from tissue homogenates by 0.2 mm filtration did not affect the transmissibility of the disease risk. This observation suggests that certain enteric viruses have a role in the pathogenesis of CD. Interestingly, an electron microscopy study confirmed the presence of viral particles in lymphomas after inoculation of nude mice with tissue homogenates from patients with CD but not from controls.63 Collectively, we propose a novel pathophysiological model, whereby asymptomatic norovirus infection may result in Crohn’s ileitis by triggering a selective advantage for outgrowth of intruding, opportunistic bacteria in genetically predisposed hosts.
CONCLUDING REMARKS A role of common viral infection in the pathogenesis of chronic inflammatory diseases is not without precedence, as illustrated by type 1 diabetes.64 It is now strongly suspected that Crohn’s ileitis may result from asymptomatic norovirus infection.65,66 In this context, we propose a novel paradigm in which asymptomatic noroviral infection may drive disease-predisposing dysbiosis in genetically predisposed patients.67 Forthcoming clinical trials on fecal transplantation raise therefore potential clinical safety issues with norovirus gastroenteritis.68
ACKNOWLEDGMENTS
M. Chamaillard was financially supported by grants from the Fondation pour la Recherche Médicale and the European Union’s European Regional Development Fund.
REFERENCES
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6. Hutson AM, Atmar RL, Marcus DM, et al. Norwalk virus-like particle hemagglutination by binding to h histo-blood group antigens. J Virol. 2003;77:405–415. 7. Harrington PR, Vinje J, Moe CL, et al. Norovirus capture with histo-blood group antigens reveals novel virus-ligand interactions. J Virol. 2004;78: 3035–3045. 8. Lindesmith L, Moe C, Marionneau S, et al. Human susceptibility and resistance to Norwalk virus infection. Nat Med. 2003;9:548–553. 9. Kelly RJ, Rouquier S, Giorgi D, et al. Sequence and expression of a candidate for the human Secretor blood group alpha(1,2)fucosyltransferase gene (FUT2). Homozygosity for an enzyme-inactivating nonsense mutation commonly correlates with the non-secretor phenotype. J Biol Chem. 1995;270:4640–4649. 10. Kaneko M, Nishihara S, Shinya N, et al. Wide variety of point mutations in the H gene of Bombay and para-Bombay individuals that inactivate H enzyme. Blood. 1997;90:839–849. 11. Thorven M, Grahn A, Hedlund KO, et al. A homozygous nonsense mutation (428G–.A) in the human secretor (FUT2) gene provides resistance to symptomatic norovirus (GGII) infections. J Virol. 2005; 79:15351–15355. 12. Larsson MM, Rydell GE, Grahn A, et al. Antibody prevalence and titer to norovirus (genogroup II) correlate with secretor (FUT2) but not with ABO phenotype or Lewis (FUT3) genotype. J Infect Dis. 2006;194:1422–1427. 13. Soejima M, Koda Y. Molecular mechanisms of Lewis antigen expression. Leg Med (Tokyo). 2005;7:266–269. 14. Kudo T, Iwasaki H, Nishihara S, et al. Molecular genetic analysis of the human Lewis histo-blood group system. II. Secretor gene inactivation by a novel single missense mutation A385T in Japanese nonsecretor individuals. J Biol Chem. 1996;271:9830–9837. 15. Ferrer-Admetlla A, Sikora M, Laayouni H, et al. A natural history of FUT2 polymorphism in humans. Mol Biol Evol. 2009;26:1993–2003. 16. Koda Y, Tachida H, Soejima M, et al. Ancient origin of the null allele se (428) of the human ABO-secretor locus (FUT2). J Mol Evol. 2000;50: 243–248. 17. Wobus CE, Karst SM, Thackray LB, et al. Replication of Norovirus in cell culture reveals a tropism for dendritic cells and macrophages. PLoS Biol. 2004;2:e432. 18. Elftman MD, Gonzalez-Hernandez MB, Kamada N, et al. Multiple effects of dendritic cell depletion on murine norovirus infection. J Gen Virol. 2013;94:1761–1768. 19. Fang H, Tan M, Xia M, et al. Norovirus P particle efficiently elicits innate, humoral and cellular immunity. PLoS One. 2013;8:e63269. 20. McCartney SA, Thackray LB, Gitlin L, et al. MDA-5 recognition of a murine norovirus. PLoS Pathog. 2008;4:e1000108. 21. Sabbah A, Chang TH, Harnack R, et al. Activation of innate immune antiviral responses by Nod2. Nat Immunol. 2009;10:1073–1080. 22. Karst SM, Wobus CE, Lay M, et al. STAT1-dependent innate immunity to a Norwalk-like virus. Science. 2003;299:1575–1578. 23. Mumphrey SM, Changotra H, Moore TN, et al. Murine norovirus 1 infection is associated with histopathological changes in immunocompetent hosts, but clinical disease is prevented by STAT1-dependent interferon responses. J Virol. 2007;81:3251–3263. 24. Thackray LB, Duan E, Lazear HM, et al. Critical role for interferon regulatory factor 3 (IRF-3) and IRF-7 in type I interferon-mediated control of murine norovirus replication. J Virol. 2012;86:13515–13523. 25. Changotra H, Jia Y, Moore TN, et al. Type I and type II interferons inhibit the translation of murine norovirus proteins. J Virol. 2009;83:5683–5692. 26. Jounai N, Takeshita F, Kobiyama K, et al. The Atg5 Atg12 conjugate associates with innate antiviral immune responses. Proc Natl Acad Sci U S A. 2007;104:14050–14055. 27. Hwang S, Maloney NS, Bruinsma MW, et al. Nondegradative role of Atg5-Atg12/Atg16L1 autophagy protein complex in antiviral activity of interferon gamma. Cell Host Microbe. 2012;11:397–409. 28. Zhu S, Regev D, Watanabe M, et al. Identification of immune and viral correlates of norovirus protective immunity through comparative study of intra-cluster norovirus strains. PLoS Pathog. 2013;9:e1003592. 29. Chachu KA, LoBue AD, Strong DW, et al. Immune mechanisms responsible for vaccination against and clearance of mucosal and lymphatic norovirus infection. PLoS Pathog. 2008;4:e1000236. 30. Chachu KA, Strong DW, LoBue AD, et al. Antibody is critical for the clearance of murine norovirus infection. J Virol. 2008;82:6610–6617. www.ibdjournal.org |
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31. Loftus EV Jr. Clinical epidemiology of inflammatory bowel disease: incidence, prevalence, and environmental influences. Gastroenterology. 2004; 126:1504–1517. 32. McGovern DP, Jones MR, Taylor KD, et al. Fucosyltransferase 2 (FUT2) non-secretor status is associated with Crohn’s disease. Hum Mol Genet. 2010;19:3468–3476. 33. Franke A, McGovern DP, Barrett JC, et al. Genome-wide meta-analysis increases to 71 the number of confirmed Crohn’s disease susceptibility loci. Nat Genet. 2010;42:1118–1125. 34. Miyoshi J, Yajima T, Okamoto S, et al. Ectopic expression of blood type antigens in inflamed mucosa with higher incidence of FUT2 secretor status in colonic Crohn’s disease. J Gastroenterol. 2011;46:1056–1063. 35. Gebhard RL, Greenberg HB, Singh N, et al. Acute viral enteritis and exacerbations of inflammatory bowel disease. Gastroenterology. 1982; 83:1207–1209. 36. Van Kruiningen HJ, Mayo DR, Vanopdenbosch E, et al. Virus serology in familial Crohn disease. Scand J Gastroenterol. 2000;35:403–407. 37. Gump D, Caul E, Eade O, et al. Lymphocytotoxic and microbial antibodies in Crohn’s disease and matched controls. Antonie Van Leeuwenhoek. 1981;47:455–464. 38. Kolho KL, Klemola P, Simonen-Tikka ML, et al. Enteric viral pathogens in children with inflammatory bowel disease. J Med Virol. 2012;84: 345–347. 39. Tanaka T, Scheet P, Giusti B, et al. Genome-wide association study of vitamin B6, vitamin B12, folate, and homocysteine blood concentrations. Am J Hum Genet. 2009;84:477–482. 40. Hazra A, Kraft P, Selhub J, et al. Common variants of FUT2 are associated with plasma vitamin B12 levels. Nat Genet. 2008;40:1160–1162. 41. Peyrin-Biroulet L, Rodriguez-Gueant RM, Chamaillard M, et al. Vascular and cellular stress in inflammatory bowel disease: revisiting the role of homocysteine. Am J Gastroenterol. 2007;102:1108–1115. 42. Adolph TE, Tomczak MF, Niederreiter L, et al. Paneth cells as a site of origin for intestinal inflammation. Nature. 2013;503:272–276. 43. Chu H, Pazgier M, Jung G, et al. Human alpha-defensin 6 promotes mucosal innate immunity through self-assembled peptide nanonets. Science. 2012;337:477–481. 44. Salzman NH, Ghosh D, Huttner KM, et al. Protection against enteric salmonellosis in transgenic mice expressing a human intestinal defensin. Nature. 2003;422:522–526. 45. Bressenot A, Pooya S, Bossenmeyer-Pourie C, et al. Methyl donor deficiency affects small-intestinal differentiation and barrier function in rats. Br J Nutr. 2012;1–11. 46. Cadwell K, Patel KK, Maloney NS, et al. Virus-plus-susceptibility gene interaction determines Crohn’s disease gene Atg16L1 phenotypes in intestine. Cell. 2010;141:1135–1145. 47. Chamaillard M, Dessein R. Defensins couple dysbiosis to primary immunodeficiency in Crohn’s disease. World J Gastroenterol. 2011; 17:567–571. 48. Murakami K, Kurihara C, Oka T, et al. Norovirus binding to intestinal epithelial cells is independent of histo-blood group antigens. PLoS One. 2013;8:e66534. 49. Parmar AS, Alakulppi N, Paavola-Sakki P, et al. Association study of FUT2 (rs601338) with celiac disease and inflammatory bowel disease in the Finnish population. Tissue Antigens. 2012;80:488–493.
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50. Bry L, Falk PG, Midtvedt T, et al. A model of host-microbial interactions in an open mammalian ecosystem. Science. 1996;273:1380–1383. 51. Lin B, Hayashi Y, Saito M, et al. GDP-fucose: beta-galactoside alpha1,2fucosyltransferase, MFUT-II, and not MFUT-I or -III, is induced in a restricted region of the digestive tract of germ-free mice by hostmicrobe interactions and cycloheximide. Biochim Biophys Acta. 2000; 1487:275–285. 52. Meng D, Newburg DS, Young C, et al. Bacterial symbionts induce a FUT2-dependent fucosylated niche on colonic epithelium via ERK and JNK signaling. Am J Physiol Gastrointest Liver Physiol. 2007;293: G780–G787. 53. Nelson AM, Walk ST, Taube S, et al. Disruption of the human gut microbiota following Norovirus infection. PLoS One. 2012;7:e48224. 54. Frank DN, Robertson CE, Hamm CM, et al. Disease phenotype and genotype are associated with shifts in intestinal-associated microbiota in inflammatory bowel diseases. Inflamm Bowel Dis. 2011;17:179–184. 55. Rausch P, Rehman A, Kunzel S, et al. Colonic mucosa-associated microbiota is influenced by an interaction of Crohn disease and FUT2 (Secretor) genotype. Proc Natl Acad Sci U S A. 2011;108:19030–19035. 56. Folseraas T, Melum E, Rausch P, et al. Extended analysis of a genomewide association study in primary sclerosing cholangitis detects multiple novel risk loci. J Hepatol. 2012;57:366–375. 57. Rehman A, Sina C, Gavrilova O, et al. Nod2 is essential for temporal development of intestinal microbial communities. Gut. 2011;60:1354–1362. 58. Mondot S, Barreau F, Al Nabhani Z, et al. Altered gut microbiota composition in immune-impaired Nod2(-/-) mice. Gut. 2012;61:634–635. 59. Petnicki-Ocwieja T, Hrncir T, Liu YJ, et al. Nod2 is required for the regulation of commensal microbiota in the intestine. Proc Natl Acad Sci U S A. 2009;106:15813–15818. 60. Couturier-Maillard A, Secher T, Rehman A, et al. NOD2-mediated dysbiosis predisposes mice to transmissible colitis and colorectal cancer. J Clin Invest. 2013;123:700–711. 61. Cave DR, Mitchell DN, Brooke BN. Experimental animal studies of the etiology and pathogenesis of Crohn’s disease. Gastroenterology. 1975;69: 618–624. 62. Mitchell DN, Rees RJ. Further observations on the transmissibility of Crohn’s disease. Ann N Y Acad Sci. 1976;278:546–559. 63. Das KM, Valenzuela I, Williams SE, et al. Studies of the etiology of Crohn’s disease using athymic nude mice. Gastroenterology. 1983;84: 364–374. 64. Hober D, Sauter P. Pathogenesis of type 1 diabetes mellitus: interplay between enterovirus and host. Nat Rev Endocrinol. 2010;6:279–289. 65. Lidar M, Langevitz P, Shoenfeld Y. The role of infection in inflammatory bowel disease: initiation, exacerbation and protection. Isr Med Assoc J. 2009;11:558–563. 66. Hubbard VM, Cadwell K. Viruses, autophagy genes, and Crohn’s disease. Viruses. 2011;3:1281–1311. 67. Foxman EF, Iwasaki A. Genome-virome interactions: examining the role of common viral infections in complex disease. Nat Rev Microbiol. 2011; 9:254–264. 68. Schwartz M, Gluck M, Koon S. Norovirus gastroenteritis after fecal microbiota transplantation for treatment of Clostridium difficile infection despite asymptomatic donors and lack of sick contacts. Am J Gastroenterol. 2013;108:1367.
Decoding Norovirus Infection in Crohn’s Disease Mathias Chamaillard, PhD,* ,†,‡,§ Annabelle Cesaro, PhD,* ,†,‡,§ Pierre-Emmanuel Lober, PhD,k and Didier Hober, MD, PhDk
Abstract: Although a causing viral infectious agent remains untraceable in Crohn’s disease, most recent genome-wide association studies have linked the FUT2 W143X mutation (resulting in asymptomatic norovirus infection) with the pathogenesis of Crohn’s ileitis and with vitamin B12 deficiency (i.e., a known risk factor for Crohn’s disease with ileal involvement). In line with these findings, host variations in additional genes involved in host response to norovirus infection (such as ATG16L1 and NOD2) predispose humans to Crohn’s ileitis. One may therefore presume that asymptomatic norovirus infection may contribute to disruption of the stability of the gut microbiota leading to Crohn’s ileitis. These paradigms highlight not only the need to revisit the potential transmissibility of Crohn’s disease, but also potential safety issues of forthcoming clinical trials on human probiotic infusions in Crohn’s ileitis by rigorous donors screening program. (Inflamm Bowel Dis 2014;20:767–770) Key Words: Crohn’s ileitis, dysbiosis, fecal transplantation, norovirus, vitamin B12 deficiency
NOT ALL EQUAL AGAINST NOROVIRUS INFECTION Norovirus (originally referred as Norwalk virus after an outbreak of acute gastroenteritis in Norwalk, Ohio) is a positivesense single-stranded RNA, nonenveloped virus that remains the most common cause of nonbacterial foodborne illness outbreaks.1–3 Norovirus is widely disseminated and primarily transmitted through the fecal–oral route resulting in more than 250 million infected individuals worldwide, among which the sub-type GII.4 strain accounts for about 80% of outbreaks. Life’s quality of infected people remains strongly affected by severe watery diarrhea, abdominal pain, low-grade fever, lethargy, malnutrition, and nausea. Morbidity is considerably enhanced among infected infants, immunocompromised hematopoietic stem cell transplantation recipients, and the elderly, as judged by over 200,000 deaths per year worldwide.4,5 Norovirus infects the intestinal mucosa through interactions with oligosaccharide chains of histoblood group antigens.6,7 However, a substantial proportion of infected subjects remain asymptomatic, and not all exposed individuals become infected, despite repeated challenge.8 Notably, about 20% of Europeans are commonly considered as protected against symptomatic norovirus infection,9 because of their inability to express the H type-1 Received for publication October 29, 2013; Accepted November 19, 2013. From the *Institut Pasteur de Lille, Center for Infection and Immunity of Lille, Lille, France; †CNRS, UMR 8204, Lille, France; ‡Institut National de la Santé et de la Recherche Médicale, U1019, Team 7, Equipe FRM, Lille, France; §Univ Lille Nord de France, Lille, France; and kUniversité Lille 2, Faculté de Médecine, CHRU de Lille, Laboratoire de Virologie EA3610, Loos-lez-Lille, France. The authors have no conflicts of interest to disclose. Reprints: Mathias Chamaillard, PhD, Institut Pasteur de Lille, 1, rue Professeur A. Calmette, 59496 Lille, France (e-mail: [email protected]). Copyright © 2013 Crohn’s & Colitis Foundation of America, Inc. DOI 10.1097/01.MIB.0000440613.83703.4a Published online 17 December 2013.
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oligosaccharide ligand that is required for norovirus binding. Mechanistically, bodily secretion of histoblood group antigens is fully inactivated as a result of a nonfunctional enzymatic activity of the a1,2-fucosyltransferase that is encoded by the fucosyltransferase 2 (FUT2) gene.10 Homozygous individuals for the W154X nonsense mutation (referred as rs601338) in the FUT2-encoding gene are considered as nonsecretors and showed enhanced resistance to acute infection by several ribotype of norovirus.11 Consistently, a reduced antibody response to norovirus is commonly observed among nonsecretors.12 Likewise, missense mutations were found to confer partial protection against norovirus infection.13 Notably, individuals bearing a missense A385T mutation within the stem region of FUT2 showed about 5-fold lowered secretion of A-, B- and H- blood group antigens in mucus and body fluids.14 In addition to FUT2, several other mutations (that were maintained by strong selective pressure15,16) result in a nonsecretor phenotype that protect against numerous pathogens, including norovirus.
DECODING HOST DEFENSE TO NOROVIRUS INFECTION In the gut, norovirus (also commonly referred MNV) infects primarily macrophages and dendritic cells,17 among which conventional dendritic cells play a physiological role in host defense against norovirus by limiting its dissemination to secondary lymphoid tissues18 and antigen presentation to T cells.19 Mechanistically, sensing of MNV-1 infection by dendritic cells requires the expression of a cytosolic surveillance system involving MDA-520 and potentially NOD2.21 Among infected cells, the STAT1-dependent interferon response protects from lethal infection22 by negatively regulating apoptosis of infected cells and systemic dissemination of norovirus.23 Notably, type I interferons have been found to play a key role in restricting MNV replication24 by blocking translation of viral proteins.25 Noteworthy, the aforementioned antiviral response driven by www.ibdjournal.org |
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type I interferons is negatively regulated by the Atg5-Atg12-coupled autophagic machinery,26 while IFN-g–mediated antiviral response of macrophages require expression of the Atg5-Atg12/Atg16L1 expression but not the catabolic activity of authophagy.27 Although B and T lymphocytes were dispensable for resistance to lethal infection, Rag-deficient mice were persistently infected, arguing for a role of adaptive immunity on the outcome of secondary norovirus infection. Noteworthy, norovirus is characterized by the high level of variability of its surface exposed residues. Early secretion of norovirus-specific salivary immunoglobulin A is predictive of an adaptive resistance to infection in a substantial portion of susceptible individuals.8 Long-lasting immunity to norovirus did not require CD8+ cells,28 but rather B and CD4+ T lymphocytes.23,24,29,30
RESISTANCE TO NOROVIRUS INFECTION IS LINKED TO CROHN’S ILEITIS PREDISPOSITION Crohn’s disease (CD) is a public health problem that mostly affects young adults in industrialized countries and, more recently, in emerging countries. The incidence of CD is thought to rise under the influence of many different environmental and genetic factors as judged by a concordance rate of about 50% to 60% among monozygotic twins. In Europe and North America, the socioeconomic impact is increasing with a combined prevalence of about 250 to 300 cases per 100,000 Europeans and North Americans. This translates into as many as 2.2 million patients with CD,31 because the therapeutic management of CD remains far from optimal. Recent genome-wide associations studies unveiled the missense G258S (referred as rs602662) and the noncoding W154X mutations of FUT2 as molecular links between asymptomatic norovirus infection and the pathogenesis of CD.32 A total of 4 noncoding single nucleotide polymorphisms of the FUT2-encoding gene (namely rs504963—30 UTR, rs676388— 30 UTR, 7 rs485186—synonymous exon 2 SNP, and rs492602— synonymous exon 2) were found associated with CD with ileal involvement,33 whereas the secretor status was more frequently found in Crohn’s colitis.34 In contrast to Crohn’s ileitis, none of the patients with CD with colonic involvement were found as nonsecretors.34 This paradigm may account for relapsing CD in some symptomatic patients.35 In line with these indisputable evidences from genetics, one may presume that a substantial proportion of patients with CD remain asymptomatic to a large variety of norovirus infection. Consistently, several serological approaches and stool antigen testing failed to detect any disease-specific patterns of antibodies against several enteric viruses (including norovirus).36,37 Furthermore, reverse transcription polymerase chain reaction analyses failed to document any association between CD and the fecal presence of norovirus.38 More importantly, both G258S and W154X mutations were linked to vitamin B12 deficiency,39,40 i.e., a known risk factor for CD who primarily exhibit transmural inflammatory lesions of the terminal ileum,41 within which Paneth cells are especially numerous at the base of the crypts of Lieberkühn. Paneth cells correspond to one of the 4 main epithelial cell lineages that play
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a key role on mucosal immunity, host defense and inflammation resolution.42–44 Furthermore, methyl-deficient diet result in decreased number of Paneth cells45 and mice with hypomorphic expression of ATG16L1 showed an abnormal granule formation in Paneth cells on infection by norovirus.46 Dissemination of the norovirus to secondary lymphoid tissues was increased in infected mice with lowered ATG16L1 expression, which were found more at risk to develop intestinal inflammation afforded by bacterial commensals. Antibiotic treatment was indeed found efficient to improve disease severity in both wild-type and mutant animals. However, norovirus was not detected in the Paneth cells, and its replication was not found impaired within the ileum and the colon of infected mice with lowered ATG16L1 expression,46 suggesting a potential compensatory mechanism for ATG16L1 on the maintenance of the IFN-g– mediated antiviral response.27 However, it remains to be deciphered whether norovirus infection may account for the abnormal granule formation, enhanced necroptosis, and impaired secretion of antimicrobial peptides as what observed in Crohn’s ileitis.47 Recent studies gave some preliminary answers by showing that Norovirus binds to differentiated epithelial cell and Goblet cells before being internalized,48 and that extensive rearrangements of intracellular membranes occur in infected cells as a result of MNV replication.17 Conversely, it remains elusive whether patients with Crohn’s colitis may be protected against norovirus infection as what observed for patients with ulcerative colitis, among which the W154X FUT2 mutation was found to confer a dominant protection.49
“ASYMPTOMATIC NOROVIRAL DRIVER-BACTERIAL PASSENGER” MODEL Host–microbiota interactions are thought to modulate outcome of norovirus infection,50 as evidenced by defects in fucosylating processes within the small intestinal mucosa of germ-free animals.50,51 Consistently, microbiota transition result in an increased in FUT2 expression and enhanced alpha(1,2/3)-fucosyltransferase activity.52 More recently, high-throughput sequencing approaches revealed that most patients infected by norovirus displayed changes of the structure and composition of their fecal microbiota (i.e., referred herein as dysbiosis) independently of age and gender.53 Notably, norovirus-driven dysbiosis was characterized by a decreased abundance of potential anti-inflammatory bacteria related to Faecalibacterium sp. accompanied with an enhanced abundance of Escherichia coli, but all isolated were negative for known virulence factors. One may therefore presume that the aforementioned patients were secretors as they were symptomatic. Recent studies gave preliminary answers whether the primary nonsecretor A allele of rs601338 may indeed drive dysbiosis in caucasians. In CD with colonic involvement, the nonsecretor FUT2 allele was indeed linked to colonic mucosa-associated dysbiosis, including enhanced abundance of Firmicutes and decreased of Proteobacteria.54,55 Likewise, the biliary tract of FUT2 nonsecretor patients suffering from primary sclerosing cholangitis (a known complication of patients with Crohn’s colitis and ulcerative colitis) showed similar changes in the diversity and structure of their bacterial communities.56 As
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what observed in humans, fecal- and colonic mucosa-associated dysbiosis is also observed in mice deficient for either NOD257–59 or FUT2.55 More importantly, recent studies demonstrated that risk for intestinal inflammation in Nod2-deficient mice is dictated by the composition and structure of their fecal microbiota.60 This paradigm argue for the need to revisit the potential communicable nature of CD that was first suggested in the late 1970s. The best evidence of the communicable nature of this condition comes from the observation that inoculation of mucosal homogenates from patients with CD (but not controls) was enough to trigger disease onset in New Zealand white rabbits61 or mice.62 In contrast, no signs of CD-like epithelioid and giant cell granulomas were observed in mice after the injection of autoclaved homogenates from patients with CD. More importantly, exclusion of microorganisms from tissue homogenates by 0.2 mm filtration did not affect the transmissibility of the disease risk. This observation suggests that certain enteric viruses have a role in the pathogenesis of CD. Interestingly, an electron microscopy study confirmed the presence of viral particles in lymphomas after inoculation of nude mice with tissue homogenates from patients with CD but not from controls.63 Collectively, we propose a novel pathophysiological model, whereby asymptomatic norovirus infection may result in Crohn’s ileitis by triggering a selective advantage for outgrowth of intruding, opportunistic bacteria in genetically predisposed hosts.
CONCLUDING REMARKS A role of common viral infection in the pathogenesis of chronic inflammatory diseases is not without precedence, as illustrated by type 1 diabetes.64 It is now strongly suspected that Crohn’s ileitis may result from asymptomatic norovirus infection.65,66 In this context, we propose a novel paradigm in which asymptomatic noroviral infection may drive disease-predisposing dysbiosis in genetically predisposed patients.67 Forthcoming clinical trials on fecal transplantation raise therefore potential clinical safety issues with norovirus gastroenteritis.68
ACKNOWLEDGMENTS
M. Chamaillard was financially supported by grants from the Fondation pour la Recherche Médicale and the European Union’s European Regional Development Fund.
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