Role of defective autophagia and the intestinal flora in Crohn disease.
ABSTRACT: The precise mechanisms underlying the development of Crohn disease (CD) remain controversial, but sufficient data have been collected to suggest that an uncontrolled immune response within the intestinal mucosa leads to inflammation in a genetically susceptible host. Although lack of mucosal regulatory T cells causes colitis in humans and experimental rodents, patients with CD have more rather than less regulatory activity in the intestine, apparently excluding defects in tolerance as the cause of CD. Genome-wide association studies have identified many gene variants that confer susceptibility and which seem associated to diminished functioning of especially innate immunity. In apparent agreement, CD patients are impaired with respect to innate immune responses and controlling bacterial flora in the intestine. Furthermore, severe genetic deficiencies in innate immunity, like e.g., lack of NADP oxidase activity or diminished function of the Wiskott Aldrich syndrome protein are associated with colitis in mice and men, and are often mistakenly diagnosed as CD. Thus we favor the view that the primary defect in CD is a lack in innate immunity, causing second tier immunological defenses to combat otherwise easily controlled bacterial breaches of the mucosal barrier.
Project description:Intestinal macrophages play a central role in regulation of immune responses against commensal bacteria. In general, intestinal macrophages lack the expression of innate-immune receptor CD14 and do not produce proinflammatory cytokines against commensal bacteria. In this study, we identified what we believe to be a unique macrophage subset in human intestine. This subset expressed both macrophage (CD14, CD33, CD68) and DC markers (CD205, CD209) and produced larger amounts of proinflammatory cytokines, such as IL-23, TNF-alpha, and IL-6, than typical intestinal resident macrophages (CD14-CD33+ macrophages). In patients with Crohn disease (CD), the number of these CD14+ macrophages were significantly increased compared with normal control subjects. In addition to increased numbers of cells, these cells also produced larger amounts of IL-23 and TNF-alpha compared with those in normal controls or patients with ulcerative colitis. In addition, the CD14+ macrophages contributed to IFN-gamma production rather than IL-17 production by lamina propria mononuclear cells (LPMCs) dependent on IL-23 and TNF-alpha. Furthermore, the IFN-gamma produced by LPMCs triggered further abnormal macrophage differentiation with an IL-23-hyperproducing phenotype. Collectively, these data suggest that this IL-23/IFN-gamma-positive feedback loop induced by abnormal intestinal macrophages contributes to the pathogenesis of chronic intestinal inflammation in patients with CD.
Project description:Chronic intestinal inflammation, as seen in inflammatory bowel disease (IBD), results from an aberrant and poorly understood mucosal immune response to the microbiota of the gastrointestinal tract in genetically susceptible individuals. Here we used serological expression cloning to identify commensal bacterial proteins that could contribute to the pathogenesis of IBD. The dominant antigens identified were flagellins, molecules known to activate innate immunity via Toll-like receptor 5 (TLR5), and critical targets of the acquired immune system in host defense. Multiple strains of colitic mice had elevated serum anti-flagellin IgG2a responses and Th1 T cell responses to flagellin. In addition, flagellin-specific CD4(+) T cells induced severe colitis when adoptively transferred into naive SCID mice. Serum IgG to these flagellins, but not to the dissimilar Salmonella muenchen flagellin, was elevated in patients with Crohn disease, but not in patients with ulcerative colitis or in controls. These results identify flagellins as a class of immunodominant antigens that stimulate pathogenic intestinal immune reactions in genetically diverse hosts and suggest new avenues for the diagnosis and antigen-directed therapy of patients with IBD.
Project description:TNF-alpha and IL-1 have been associated with mucosal inflammation in both Crohn's disease (CD) and ulcerative colitis (UC). Innate immune defects have been associated with CD, specifically CARD15/NOD2. Recently, Toll-like receptor 8 (TLR8) signaling has been shown to enhance generation of both cytokines. Interestingly, TLR8 is located on the X chromosome and inflammatory bowel disease (IBD) has been associated with abnormalities of the X chromosome. The aim was to test whether TLR8 haplotypes are associated with IBD.Subjects (735 CD, 343 UC, 245 controls) were genotyped. Single nucleotide polymorphisms (SNPs) were chosen to tag common Caucasian haplotypes.Both "risk (H4)" and "protective (H1)" TLR8 haplotypes were observed associated with CD in females. Eighteen percent of CD females had H4 compared with 9% of controls (P = 0.02). Fifty-nine percent of CD females had H1 compared with 72% of controls (P = 0.01). H1 was also negatively associated with UC in females (59% of UC, 72% of controls P = 0.03). Diplotype analysis of CD, UC, and all IBD in females revealed that 2 protective haplotypes (H1/H1) had a markedly diminished odds ratio, 0.4-0.5. The presence of a risk haplotype (H4 / not H1) had a significantly increased odds ratio, 2.0-2.2. Thus, the risk for IBD was 4-5 times higher in females with 1 risk haplotype than with the protective/protective diplotype.TLR8 is an X-linked IBD susceptibility gene with both common predisposing and protecting haplotypes. These associations further emphasize the importance of genetic variation in innate immunity as determinants, not only of CD, but of UC as well.
Project description:The pathogenesis of Crohn's disease (CD), an idiopathic inflammatory bowel disease, is attributed, in part, to intestinal bacteria that may initiate and perpetuate mucosal inflammation in genetically susceptible individuals. Paneth cells (PC) are the major source of antimicrobial peptides in the small intestine, including human alpha-defensins HD5 and HD6. We tested the hypothesis that reduced expression of PC alpha-defensins compromises mucosal host defenses and predisposes patients to CD of the ileum. We report that patients with CD of the ileum have reduced antibacterial activity in their intestinal mucosal extracts. These specimens also showed decreased expression of PC alpha-defensins, whereas the expression of eight other PC products either remained unchanged or increased when compared with controls. The specific decrease of alpha-defensins was independent of the degree of inflammation in the specimens and was not observed in either CD of the colon, ulcerative colitis, or pouchitis. The functional consequence of alpha-defensin expression levels was examined by using a transgenic mouse model, where we found changes in HD5 expression levels, comparable to those observed in CD, had a pronounced impact on the luminal microbiota. Thus, the specific deficiency of PC defensins that characterizes ileal CD may compromise innate immune defenses of the ileal mucosa and initiate and/or perpetuate this disease.
Project description:Defensins are endogenous antimicrobial peptides that protect the intestinal mucosa against bacterial invasion. It has been suggested that deficient defensin expression may underlie the chronic inflammation of Crohn disease (CD). The DNA copy number of the beta-defensin gene cluster on chromosome 8p23.1 is highly polymorphic within the healthy population, which suggests that the defective beta-defensin induction in colonic CD could be due to low beta-defensin-gene copy number. Here, we tested this hypothesis, using genomewide DNA copy number profiling by array-based comparative genomic hybridization and quantitative polymerase-chain-reaction analysis of the human beta-defensin 2 (HBD-2) gene. We showed that healthy individuals, as well as patients with ulcerative colitis, have a median of 4 (range 2-10) HBD-2 gene copies per genome. In a surgical cohort with ileal or colonic CD and in a second large cohort with inflammatory bowel diseases, those with ileal resections/disease exhibited a normal median HBD-2 copy number of 4, whereas those with colonic CD had a median of only 3 copies per genome (P=.008 for the surgical cohort; P=.032 for the second cohort). Overall, the copy number distribution in colonic CD was shifted to lower numbers compared with controls (P=.002 for both the surgical cohort and the cohort with inflammatory bowel diseases). Individuals with < or = 3 copies have a significantly higher risk of developing colonic CD than did individuals with > or = 4 copies (odds ratio 3.06; 95% confidence interval 1.46-6.45). An HBD-2 gene copy number of < 4 was associated with diminished mucosal HBD-2 mRNA expression (P=.033). In conclusion, a lower HBD-2 gene copy number in the beta-defensin locus predisposes to colonic CD, most likely through diminished beta-defensin expression.
Project description:The role of the innate immunity in the pathogenesis of Crohn's disease (CD), an inflammatory bowel disease, is a subject of increasing interest. Neutrophils (PMN) are key members of the innate immune system which migrate to sites of bacterial infection and initiate the defence against microbes by producing reactive oxygen species (ROS), before undergoing apoptosis. It is believed that impaired innate immune responses contribute to CD, but it is as yet unclear whether intrinsic defects in PMN signal transduction and corresponding function are present in patients with quiescent disease. We isolated peripheral blood PMN from CD patients in remission and healthy controls (HC), and characterised migration, bacterial uptake and killing, ROS production and cell death signalling. Whereas IL8-induced migration and signalling were normal in CD, trans-epithelial migration was significantly impaired. Uptake and killing of E. coli were normal. However, an increased ROS production was observed in CD PMN after stimulation with the bacterial peptide analogue fMLP, which was mirrored by an increased fMLP-triggered ERK and AKT signal activation. Interestingly, cleavage of caspase-3 and caspase-8 during GMCSF-induced rescue from cell-death was decreased in CD neutrophils, but a reduced survival signal emanating from STAT3 and AKT pathways was concomitantly observed, resulting in a similar percentage of end stage apoptotic PMN in CD patients and HC. In toto, these data show a disturbed signal transduction activation and functionality in peripheral blood PMN from patients with quiescent CD, which point toward an intrinsic defect in innate immunity in these patients.
Project description:Interactions between the host and gut microbial community likely contribute to Crohn disease (CD) pathogenesis; however, direct evidence for these interactions at the onset of disease is lacking. Here, we characterized the global pattern of ileal gene expression and the ileal microbial community in 359 treatment-naive pediatric patients with CD, patients with ulcerative colitis (UC), and control individuals. We identified core gene expression profiles and microbial communities in the affected CD ilea that are preserved in the unaffected ilea of patients with colon-only CD but not present in those with UC or control individuals; therefore, this signature is specific to CD and independent of clinical inflammation. An abnormal increase of antimicrobial dual oxidase (DUOX2) expression was detected in association with an expansion of Proteobacteria in both UC and CD, while expression of lipoprotein APOA1 gene was downregulated and associated with CD-specific alterations in Firmicutes. The increased DUOX2 and decreased APOA1 gene expression signature favored oxidative stress and Th1 polarization and was maximally altered in patients with more severe mucosal injury. A regression model that included APOA1 gene expression and microbial abundance more accurately predicted month 6 steroid-free remission than a model using clinical factors alone. These CD-specific host and microbe profiles identify the ileum as the primary inductive site for all forms of CD and may direct prognostic and therapeutic approaches.
Project description:Skeletal abnormalities are common comorbidities of inflammatory bowel disease (IBD). Patients suffering from IBD, including ulcerative colitis and Crohn's disease, present with skeletal complications. However, the mechanism underpinning IBD-associated bone loss remains vague. Intestinal inflammation generates an inflammatory milieu at the intestinal epithelium that leads to dysregulation of mucosal immunity through gut-residing innate lymphoid cells (ILCs) and other cell types. ILCs are recently identified mucosal cells considered as the gatekeeper of gut immunity and their function is regulated by intestinal epithelial cell (IEC)-secreted cytokines in response to the inflammatory microenvironment. We first demonstrate that serum as well as IECs collected from the intestine of dextran sulfate sodium (DSS)-induced colitis mice contain high levels of inflammatory and osteoclastogenic cytokines. Mechanistically, heightened inflammatory response of IECs was associated with significant intrinsic activation of NF-?B (nuclear factor kappa-light-chain-enhancer of activated B cells) in IECs and increased frequency of ILC1, ILC3, and myeloid osteoclast progenitors. Validating the central role of IEC-specific NF-?B activation in this phenomenon, conditional expression of constitutively active inhibitor kappa B kinase 2 (IKK2) in IECs in mice recapitulates the majority of the cellular, inflammatory, and osteolytic phenotypes observed in the chemically induced colitis. Furthermore, conditional deletion of IKK2 from IECs significantly attenuated inflammation and bone loss in DSS-induced colitis. Finally, using the DSS-induced colitis model, pharmacologic inhibition of IKK2 was effective in reducing frequency of ILC1 and ILC3 cells, attenuated circulating levels of inflammatory cytokines, and halted colitis-associated bone loss. Our findings identify IKK2 in IECs as viable therapeutic target for colitis-associated osteopenia.
Project description:Innate lymphoid cells (ILCs) play an important role in regulating immune responses at mucosal surfaces. The transcription factor T-bet is crucial for the function of ILC1s and NCR<sup>+</sup> ILC3s and constitutive deletion of T-bet prevents the development of these subsets. Lack of T-bet in the absence of an adaptive immune system causes microbiota-dependent colitis to occur due to aberrant ILC3 responses. Thus, T-bet expression in the innate immune system has been considered to dampen pathogenic immune responses. Here, we show that T-bet plays an unexpected role in negatively regulating innate type 2 responses, in the context of an otherwise intact immune system. Selective loss of T-bet in ILCs leads to the expansion and increased activity of ILC2s, which has a functionally important impact on mucosal immunity, including enhanced protection from Trichinella spiralis infection and inflammatory colitis. Mechanistically, we show that T-bet controls the intestinal ILC pool through regulation of IL-7 receptor signalling. These data demonstrate that T-bet expression in ILCs acts as the key transcriptional checkpoint in regulating pathogenic vs. protective mucosal immune responses, which has significant implications for the understanding of the pathogenesis of inflammatory bowel diseases and intestinal infections.
Project description:Celiac disease (CD) is a common autoimmune disorder triggered by the ingestion of gluten in genetically susceptible individuals. Although the mechanisms underlying gliadin-mediated activation of adaptive immunity in CD have been well-characterized, regulation of innate immune responses and the functions of certain immune cell populations within the epithelium and lamina propria are not well-understood at present. Innate lymphoid cells (ILCs) are types of innate immune cells that have lymphoid morphology, lack antigen-specific receptors, and play important roles in tissue homeostasis, inflammation, and protective immune responses against pathogens. Information regarding the diversity and functions of ILCs in lymphoid organs and at mucosal sites has grown over the past decade, and roles of different ILC subsets in the pathogenesis of some inflammatory intestinal diseases have been proposed. However, our understanding of the contribution of ILCs toward the initiation and progression of CD is still limited. In this review, we discuss current pathophysiological aspects of ILCs within the gastrointestinal tract, findings of recent investigations characterizing ILC alterations in CD and refractory CD, and suggest avenues for future research.