Crohn disease: A current perspective on genetics,autophagy and immunity

Crohn disease (CD) is a chronic and debilitating inflammatory condition of the gastrointestinal tract.¹ Prevalence in western populations is 100–150/100,000 and somewhat higher in Ashkenazi Jews. Peak incidence is in early adult life, although any age can be affected and a majority of affected individuals progress to relapsing and chronic disease. Medical treatments rely significantly on empirical corticosteroid therapy and immunosuppression, and intestinal resectional surgery is frequently required. Thus, 80% of patients with CD come to surgery for refractory disease or complications. It is hoped that an improved understanding of pathogenic mechanisms, for example by studying the genetic basis of CD and other forms of inflammatory bowel diseases (IBD), will lead to improved therapies and possibly preventative strategies in individuals identified as being at risk.     

Distribution of CD4pos -, CD8pos – and Regulatory T Cells in the Upper and Lower Gastrointestinal Tract in Healthy Young Subjects

The gastrointestinal immune system is involved in the development of several autoimmune-mediated diseases, including inflammatory bowel disease, multiple sclerosis, and type 1 diabetes mellitus. Alterations in T-cell populations, especially regulatory T cells (Tregs), are often evident in patients suffering from these diseases. To be able to detect changes in T-cell populations in diseased tissue, it is crucial to investigate T-cell populations in healthy individuals, and to characterize their variation among different regions of the gastrointestinal (GI) tract. While limited data exist, quantitative data on biopsies systematically drawn from various regions of the GI tract are lacking, particularly in healthy young humans. In this report, we present the first systematic assessment of how T cells—including Tregs—are distributed in the gastrointestinal mucosa throughout the GI tract of healthy young humans by means of multi-parameter FACS analysis. Gastroduodenoscopy and colonoscopy were performed on 16 healthy volunteers aged between 18 and 32. Biopsies were drawn from seven GI regions, and were used to determine the frequencies of CD8⁺-, CD4⁺- and Tregs in the gastrointestinal mucosa by means of multi-parameter FACS analysis. Our data show that there is significant variation in the baseline T-cell landscape along the healthy human gastrointestinal tract, and that mucosal T-cell analyses from a single region should not be taken as representative of the entire gastrointestinal tract. We show that certain T-cell subsets in the gastrointestinal mucosa vary significantly among regions; most notably, that Tregs are enriched in the appendiceal orifice region and the ascending colon, and that CD8^pos T cells are enriched in the gastric mucosa.     

Expression of Blimp-1 in Dendritic Cells Modulates the Innate Inflammatory Response in Dextran Sodium Sulfate–Induced Colitis

A single nucleotide polymorphism of PRDM1, the gene encoding Blimp-1, is strongly associated with inflammatory bowel disease. Here, we demonstrate that Blimp-1 in CD103⁺ dendritic cells (DCs) critically contributes to the regulation of macrophage homeostasis in the colon. Dextran sodium sulfate (DSS)-exposed Blimp-1^cko mice with a deletion of Blimp-1 in CD103⁺ DCs and CD11c^hi macrophages exhibited severe inflammatory symptoms, pronounced weight loss, high mortality, robust infiltration of neutrophils in epithelial regions of the colon, an increased expression of proinflammatory cytokines and a significant decrease in CD103⁺ DCs in the colon compared with DSS exposed wild-type (WT) mice. Purified colonic macrophages from Blimp-1^cko mice expressed increased levels of matrix metalloproteinase 8, 9 and 12 mRNA. WT macrophages cocultured with colonic DCs but not bone marrow–derived DCs from Blimp-1^cko produced increased matrix metalloproteinases in an interleukin (IL)-1β– and IL-6–dependent manner. Treatment of Blimp-1^cko mice with anti–IL-1β and anti–IL-6 abrogated the exaggerated clinical response. Overall, these data demonstrate that Blimp-1 expression in DCs can alter an innate inflammatory response by modulating the activation of myeloid cells. This is a novel mechanism of contribution of Blimp-1 for the pathogenesis of inflammatory bowel diseases, implicating another therapeutic target for the development of inflammatory bowel disease.     

A role for vimentin in Crohn disease

Crohn disease (CD), one of the major chronic inflammatory bowel diseases, occurs anywhere in the gastrointestinal tract with discontinuous transmural inflammation. A number of studies have now demonstrated that genetic predisposition, environmental influences and a dysregulated immune response to the intestinal microflora are involved. Major CD susceptibility pathways uncovered through genome-wide association studies strongly implicate the innate immune response (NOD2), in addition to the more specific acquired T cell response (IL23R, ICOSLG) and autophagy (ATG16L1, IRGM). Examination of the disease-associated microbiome, although complex, has identified several potentially contributory microorganisms, most notably adherent-invasive E.coli strains (AIEC), which have been isolated by independent investigators in both adult and pediatric CD patients. Here we discuss our recent finding that the type-III intermediate filament (IF) protein VIM/vimentin is a novel NOD2 interacting protein that regulates NOD2 activities including inflammatory NFKB1 signaling, autophagy and bacterial handling.     

A role for vimentin in Crohn disease

Crohn disease (CD), one of the major chronic inflammatory bowel diseases, occurs anywhere in the gastrointestinal tract with discontinuous transmural inflammation. A number of studies have now demonstrated that genetic predisposition, environmental influences and a dysregulated immune response to the intestinal microflora are involved. Major CD susceptibility pathways uncovered through genome-wide association studies strongly implicate the innate immune response (NOD2), in addition to the more specific acquired T cell response (IL23R, ICOSLG) and autophagy (ATG16L1, IRGM). Examination of the disease-associated microbiome, although complex, has identified several potentially contributory microorganisms, most notably adherent-invasive E.coli strains (AIEC), which have been isolated by independent investigators in both adult and pediatric CD patients. Here we discuss our recent finding that the type-III intermediate filament (IF) protein VIM/vimentin is a novel NOD2 interacting protein that regulates NOD2 activities including inflammatory NFKB1 signaling, autophagy and bacterial handling.     

Synthetic hookworm-derived peptides are potent modulators of primary human immune cell function that protect against experimental colitis in vivo

The prevalence of autoimmune diseases is on the rise globally. Currently, autoimmunity presents in over 100 different forms and affects around 9% of the world’s population. Current treatments available for autoimmune diseases are inadequate, expensive, and tend to focus on symptom management rather than cure. Clinical trials have shown that live helminthic therapy can decrease chronic inflammation associated with inflammatory bowel disease and other gastrointestinal autoimmune inflammatory conditions. As an alternative and better controlled approach to live infection, we have identified and characterized two peptides, Acan1 and Nak1, from the excretory/secretory component of parasitic hookworms for their therapeutic activity on experimental colitis. We synthesized Acan1 and Nak1 peptides from the Ancylostoma caninum and Necator americanus hookworms and assessed their structures and protective properties in human cell–based assays and in a mouse model of acute colitis. Acan1 and Nak1 displayed anticolitic properties via significantly reducing weight loss and colon atrophy, edema, ulceration, and necrosis in 2,4,6-trinitrobenzene sulfonic acid–exposed mice. These hookworm peptides prevented mucosal loss of goblet cells and preserved intestinal architecture. Acan1 upregulated genes responsible for the repair and restitution of ulcerated epithelium, whereas Nak1 downregulated genes responsible for epithelial cell migration and apoptotic cell signaling within the colon. These peptides were nontoxic and displayed key immunomodulatory functions in human peripheral blood mononuclear cells by suppressing CD4⁺ T cell proliferation and inhibiting IL-2 and TNF production. We conclude that Acan1 and Nak1 warrant further development as therapeutics for the treatment of autoimmunity, particularly gastrointestinal inflammatory conditions.     

Systemic and Mucosal Immune Reactivity upon Mycobacterium avium ssp. paratuberculosis Infection in Mice

Mycobacterium avium ssp. paratuberculosis (MAP) is the cause of Johne''s disease, an inflammatory bowel disorder of ruminants. Due to the similar pathology, MAP was also suggested to cause Crohn''s disease (CD). Despite of intensive research, this question is still not settled, possibly due to the lack of versatile mouse models. The aim of this study was to identify basic immunologic mechanisms in response to MAP infection. Immune compromised C57BL/6 Rag2 ^−/− mice were infected with MAP intraperitoneally. Such chronically infected mice were then reconstituted with CD4⁺ and CD8⁺ T cells 28 days after infection. A systemic inflammatory response, detected as enlargement of the spleen and granuloma formation in the liver, was observed in mice infected and reconstituted with CD4⁺ T cells. Whereby inflammation in infected and CD4⁺CD45RB^hi T cell reconstituted animals was always higher than in the other groups. Reconstitution of infected animals with CD8⁺ T cells did not result in any inflammatory signs. Interestingly, various markers of inflammation were strongly up-regulated in the colon of infected mice reconstituted with CD4⁺CD45RB^lo/int T cells. We propose, the usual non-colitogenic CD4⁺CD45RB^lo/int T cells were converted into inflammatory T cells by the interaction with MAP. However, the power of such cells might be not sufficient for a fully established inflammatory response in the colon. Nevertheless, our model system appears to mirror aspects of an inflammatory bowel disease (IBD) like CD and Johne''s diseases. Thus, it will provide an experimental platform on which further knowledge on IBD and the involvement of MAP in the induction of CD could be acquired.     

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4⁺CD45RB^high T cells into T and B cell deficient recipient mice. The CD4⁺CD45RB^high T cell population that largely consists of naïve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.     

Autophagy is decreased in mesenteric fat tissue but not in intestinal mucosae of patients with Crohn’s disease

Crohn??s disease (CD) is a chronic intestinal disease with a multifactorial etiology. Recently, a role for mesenteric fat has been proposed in CD pathophysiology, since fat hypertrophy is detected close to the affected intestinal area; however, there are few studies regarding autophagy and the hypertrophied mesenteric tissue in CD. To evaluate autophagy-related proteins in intestinal mucosae and mesenteric fat of patients with CD and controls, patients with ileocecal CD (CD Group) and with non-inflammatory disease (FC Group) selected for surgery were studied. Expression of LC3-II was determined by immunoblotting of protein extracts. In addition, beclin-1, LC3 and Atg16-L1 RNA levels were measured using RT-PCR. The expression of LC3-II was significantly lower in the mesenteric tissue and higher in intestinal mucosae of CD when compared to controls. However, mRNA expression of autophagy-related proteins was similar when comparing the mesenteric fat groups. These findings suggest a defect in autophagy activation in the mesenteric fat tissue of CD individuals, which could be involved in the maintenance of the inflammatory process.     

Rapid and noninvasive metabonomic characterization of inflammatory bowel disease

Inflammatory bowel diseases (IBD) including Crohn's disease (CD) and ulcerative colitis (UC) have a major impact on the health of individuals and populations. Accurate diagnosis of inflammatory bowel disease (IBD) at an early stage, and correct differentiation between Crohn's disease (CD) and ulcerative colitis (UC), is important for optimum treatment and prognosis. We present here the first characterization of fecal extracts obtained from patients with CD and UC by employing a noninvasive metabonomics approach, which combines high resolution 1H NMR spectroscopy and multivariate pattern recognition techniques. The fecal extracts of both CD and UC patients were characterized by reduced levels of butyrate, acetate, methylamine, and trimethylamine in comparison with a control population, suggesting changes in the gut microbial community. Also, elevated quantities of amino acids were present in the feces from both disease groups, implying malabsorption caused by the inflammatory disease or an element of protein losing enteropathy. Metabolic differences in fecal profiles were more marked in the CD group in comparison with the control group, indicating that the inflammation caused by CD is more extensive in comparison with UC and involves the whole intestine. Furthermore, glycerol resonances were a dominant feature of fecal spectra from patients with CD but were present in much lower intensity in the control and UC groups. This work illustrates the potential of metabonomics to generate novel noninvasive diagnostics for gastrointestinal diseases and may further our understanding of disease mechanisms.     

Italian cohort of patients affected by inflammatory bowel disease is characterised by variation in glycerophospholipid,free fatty acids and amino acid levels

Background

Inflammatory bowel disease is a group of pathologies characterised by chronic inflammation of the intestine and an unclear aetiology. Its main manifestations are Crohn’s disease and ulcerative colitis. Currently, biopsies are the most used diagnostic tests for these diseases and metabolomics could represent a less invasive approach to identify biomarkers of disease presence and progression.

Objectives

The lipid and the polar metabolite profile of plasma samples of patients affected by inflammatory bowel disease have been compared with healthy individuals with the aim to find their metabolomic differences. Also, a selected sub-set of samples was analysed following solid phase extraction to further characterise differences between pathological samples.

Methods

A total of 200 plasma samples were analysed using drift tube ion mobility coupled with time of flight mass spectrometry and liquid chromatography for the lipid metabolite profile analysis, while liquid chromatography coupled with triple quadrupole mass spectrometry was used for the polar metabolite profile analysis.

Results

Variations in the lipid profile between inflammatory bowel disease and healthy individuals were highlighted. Phosphatidylcholines, lyso-phosphatidylcholines and fatty acids were significantly changed among pathological samples suggesting changes in phospholipase A₂ and arachidonic acid metabolic pathways. Variations in the levels of cholesteryl esters and glycerophospholipids were also found. Furthermore, a decrease in amino acids levels suggests mucosal damage in inflammatory bowel disease.

Conclusions

Given good statistical results and predictive power of the model produced in our study, metabolomics can be considered as a valid tool to investigate inflammatory bowel disease.

     

High-throughput clone library analysis of the mucosa-associated microbiota reveals dysbiosis and differences between inflamed and non-inflamed regions of the intestine in inflammatory bowel disease

Background  

The gut microbiota is thought to play a key role in the development of the inflammatory bowel diseases Crohn's disease (CD) and ulcerative colitis (UC). Shifts in the composition of resident bacteria have been postulated to drive the chronic inflammation seen in both diseases (the "dysbiosis" hypothesis). We therefore specifically sought to compare the mucosa-associated microbiota from both inflamed and non-inflamed sites of the colon in CD and UC patients to that from non-IBD controls and to detect disease-specific profiles.     

IL-21/IL-21R信号在IBD病变形成机制中作用的研究进展

炎症性肠病(Inflammatory bowel disease,IBD)的发病机制至今尚不明确,普遍认为是由肠黏膜免疫调节异常、持续性肠道感染、肠黏膜屏障缺损、遗传和环境等多种因素相互作用导致的。近年来,研究发现IBD患者血清中IL-21水平异常升高,提示IL-21/IL-21R信号可能在IBD的病变形成中发挥重要作用。IL-21是一种重要的具有多重生物学功能的细胞因子,通过对CD4+T细胞、CD8+T细胞、Th17细胞、B细胞、巨噬细胞、树突状细胞等多种细胞产生影响,从而参与IBD的发生发展。本文就IL-21/IL-21R信号在炎症性肠病发病机制中的研究进展进行综述。     

Macrophages and Dendritic Cells Emerge in the Liver during Intestinal Inflammation and Predispose the Liver to Inflammation

The liver is a physiological site of immune tolerance, the breakdown of which induces immunity. Liver antigen-presenting cells may be involved in both immune tolerance and activation. Although inflammatory diseases of the liver are frequently associated with inflammatory bowel diseases, the underlying immunological mechanisms remain to be elucidated. Here we report two murine models of inflammatory bowel disease: RAG-2^−/− mice adoptively transferred with CD4⁺CD45RB^high T cells; and IL-10^−/− mice, accompanied by the infiltration of mononuclear cells in the liver. Notably, CD11b⁻CD11c^lowPDCA-1⁺ plasmacytoid dendritic cells (DCs) abundantly residing in the liver of normal wild-type mice disappeared in colitic CD4⁺CD45RB^high T cell-transferred RAG-2^−/− mice and IL-10^−/− mice in parallel with the emergence of macrophages (Mφs) and conventional DCs (cDCs). Furthermore, liver Mφ/cDCs emerging during intestinal inflammation not only promote the proliferation of naïve CD4⁺ T cells, but also instruct them to differentiate into IFN-γ-producing Th1 cells in vitro. The emergence of pathological Mφ/cDCs in the liver also occurred in a model of acute dextran sulfate sodium (DSS)-induced colitis under specific pathogen-free conditions, but was canceled in germ-free conditions. Last, the Mφ/cDCs that emerged in acute DSS colitis significantly exacerbated Fas-mediated hepatitis. Collectively, intestinal inflammation skews the composition of antigen-presenting cells in the liver through signaling from commensal bacteria and predisposes the liver to inflammation.     

An Important Role of the SDF-1/CXCR4 Axis in Chronic Skin Inflammation

Inflammatory angiogenesis and vascular remodeling play key roles in the chronic inflammatory skin disease psoriasis, but little is known about the molecular mediators of vascular activation. Based on the reported elevated mRNA levels of the angiogenic chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 in psoriasis, we investigated the relevance of the SDF-1/CXCR4 axis in two experimental models of chronic psoriasis-like skin inflammation. The cutaneous expression of both SDF-1 and CXCR4 was upregulated in the inflamed skin of K14-VEGF-A transgenic mice and in imiquimod-induced skin inflammation, with expression of CXCR4 by blood vessels and macrophages. Treatment with the CXCR4 antagonist AMD3100 potently inhibited skin inflammation in both models, associated with reduced inflammatory angiogenesis and inflammatory cell accumulation, including dermal CD4⁺ cells and intraepidermal CD8⁺ T cells. Similar anti-inflammatory effects were seen after treatment with a neutralizing anti-SDF-1 antibody. In vitro, inhibition of CXCR4 blocked SDF-1-induced chemotaxis of CD11b⁺ splenocytes, in agreement with the reduced number of macrophages after in vivo CXCR4 blockade. Our results reveal an important role of the SDF-1/CXCR4 axis in skin inflammation and inflammatory angiogenesis, and they indicate that inhibition of the SDF-1/CXCR4 axis might serve as a novel therapeutic strategy for chronic inflammatory skin diseases.     

A chromosome 8 gene-cluster polymorphism with low human beta-defensin 2 gene copy number predisposes to Crohn disease of the colon

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.     

Whole genome and exome sequencing of monozygotic twins discordant for Crohn’s disease

Background

Crohn’s disease (CD) is an inflammatory bowel disease caused by genetic and environmental factors. More than 160 susceptibility loci have been identified for IBD, yet a large part of the genetic variance remains unexplained. Recent studies have demonstrated genetic differences between monozygotic twins, who were long thought to be genetically completely identical.

Results

We aimed to test if somatic mutations play a role in CD etiology by sequencing the genomes and exomes of directly affected tissue from the bowel and blood samples of one and the blood-derived exomes of two further monozygotic discordant twin pairs. Our goal was the identification of mutations present only in the affected twins, pointing to novel candidates for CD susceptibility loci. We present a thorough genetic characterization of the sequenced individuals but detected no consistent differences within the twin pairs. An estimate of the CD susceptibility based on known CD loci however hinted at a higher mutational load in all three twin pairs compared to 1,920 healthy individuals.

Conclusion

Somatic mosaicism does not seem to play a role in the discordance of monozygotic CD twins. Our study constitutes the first to perform whole genome sequencing for CD twins and therefore provides a valuable reference dataset for future studies. We present an example framework for mosaicism detection and point to the challenges in these types of analyses.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-564) contains supplementary material, which is available to authorized users.