Inflammatory Bowel Disease Susceptibility Genes Research Articles

Neutrophils: from IBD to the gut microbiota.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastrointestinal tract that results from dysfunction in innate and/or adaptive immune responses. Impaired innate immunity, which leads to lack of control of an altered intestinal microbiota and to activation of the adaptive immune system, promotes a secondary inflammatory response that is responsible for tissue damage. Neutrophils are key players in innate immunity in IBD, but their roles have been neglected compared with those of other immune cells. The latest studies on neutrophils in IBD have revealed unexpected complexities, with heterogeneous populations and dual functions, both deleterious and protective, for the host. In parallel, interconnections between disease development, intestinal microbiota and neutrophils have been highlighted. Numerous IBD susceptibility genes (such as NOD2, NCF4, LRRK2, CARD9) are involved in neutrophil functions related to defence against microorganisms. Moreover, severe monogenic diseases involving dysfunctional neutrophils, including chronic granulomatous disease, are characterized by intestinal inflammation that mimics IBD and by alterations in the intestinal microbiota. This observation demonstrates the dialogue between neutrophils, gut inflammation and the microbiota. Neutrophils affect microbiota composition and function in several ways. In return, microbial factors, including metabolites, regulate neutrophil production and function directly and indirectly. It is crucial to further investigate the diverse roles played by neutrophils in host-microbiota interactions, both at steady state and in inflammatory conditions, to develop new IBD therapies. In this Review, we discuss the roles of neutrophils in IBD, in light of emerging evidence proving strong interconnections between neutrophils and the gut microbiota, especially in an inflammatory context.

Diet high in linoleic acid dysregulates the intestinal endocannabinoid system and increases susceptibility to colitis in Mice

ABSTRACT Inflammatory bowel disease (IBD) is a multifactorial disease with increasing incidence in the U.S. suggesting that environmental factors, including diet, are involved. It has been suggested that excessive consumption of linoleic acid (LA, C18:2 omega-6), which must be obtained from the diet, may promote the development of IBD in humans. To demonstrate a causal link between LA and IBD, we show that a high fat diet (HFD) based on soybean oil (SO), which is comprised of ~55% LA, increases susceptibility to colitis in several models, including IBD-susceptible IL10 knockout mice. This effect was not observed with low-LA HFDs derived from genetically modified soybean oil or olive oil. The conventional SO HFD causes classical IBD symptoms including immune dysfunction, increased intestinal epithelial barrier permeability, and disruption of the balance of isoforms from the IBD susceptibility gene Hepatocyte Nuclear Factor 4α (HNF4α). The SO HFD causes gut dysbiosis, including increased abundance of an endogenous adherent invasive Escherichia coli (AIEC), which can use LA as a carbon source. Metabolomic analysis shows that in the mouse gut, even in the absence of bacteria, the presence of soybean oil increases levels of LA, oxylipins and prostaglandins. Many compounds in the endocannabinoid system, which are protective against IBD, are decreased by SO both in vivo and in vitro. These results indicate that a high LA diet increases susceptibility to colitis via microbial and host-initiated pathways involving alterations in the balance of bioactive metabolites of omega-6 and omega-3 polyunsaturated fatty acids, as well as HNF4α isoforms.

Dietary Patterns and Gut Microbiota: The Crucial Actors in Inflammatory Bowel Disease.

It is widely believed that diet and the gut microbiota are strongly related to the occurrence and progression of inflammatory bowel disease (IBD), but the effects of the interaction between dietary patterns and the gut microbiota on IBD have not been well elucidated. In this article, we aim to explore the complex relation between dietary patterns, gut microbiota, and IBD. We first comprehensively summarized the dietary patterns associated with IBD and found that dietary patterns can modulate the occurrence and progression of IBD through various signaling pathways, including mammalian target of rapamycin (mTOR), mitogen-activated protein kinases (MAPKs), signal transducer and activator of transcription 3 (STAT3), and NF-κB. Besides, the gut microbiota performs a vital role in the progression of IBD, which can affect the expression of IBD susceptibility genes, such as dual oxidase 2 (DUOX2) and APOA-1 , the intestinal barrier (in particular, the expression of tight junction proteins), immune function (especially the homeostasis between effector and regulatory T cells) and the physiological metabolism, in particular, SCFAs, bile acids (BAs), and tryptophan metabolism. Finally, we reviewed the current knowledge on the interaction between dietary patterns and the gut microbiota in IBD and found that dietary patterns modulate the onset and progression of IBD, which is partly attributed to the regulation of the gut microbiota (especially SCFAs-producing bacteria and Escherichia coli). Faecalibacteria as "microbiomarkers" of IBD could be used as a target for dietary interventions to alleviate IBD. A comprehensive understanding of the interplay between dietary intake, gut microbiota, and IBD will facilitate the development of personalized dietary strategies based on the regulation of the gut microbiota in IBD and expedite the era of precision nutritional interventions for IBD.

Microbial signals, MyD88, and lymphotoxin drive TNF-independent intestinal epithelial tissue damage.

Anti-TNF antibodies are effective for treating patients with inflammatory bowel disease (IBD), but many patients fail to respond to anti-TNF therapy, highlighting the importance of TNF-independent disease. We previously demonstrated that acute deletion of 2 IBD susceptibility genes, A20 (Tnfaip3) and Abin-1 (Tnip1), in intestinal epithelial cells (IECs) sensitized mice to both TNF-dependent and TNF-independent death. Here we show that TNF-independent IEC death after A20 and Abin-1 deletion was rescued by germ-free derivation or deletion of MyD88, while deletion of Trif provided only partial protection. Combined deletion of Ripk3 and Casp8, which inhibits both apoptotic and necroptotic death, completely protected against death after acute deletion of A20 and Abin-1 in IECs. A20- and Abin-1–deficient IECs were sensitized to TNF-independent, TNFR1-mediated death in response to lymphotoxin α (LTα) homotrimers. Blockade of LTα in vivo reduced weight loss and improved survival when combined with partial deletion of MyD88. Biopsies of inflamed colon mucosa from patients with IBD exhibited increased LTA and IL1B expression, including a subset of patients with active colitis on anti-TNF therapy. These data show that microbial signals, MyD88, and LTα all contribute to TNF-independent intestinal injury.

Exploring Links Between Industrialization, Urbanization, and Chinese Inflammatory Bowel Disease.

Background: Evidence is emerging that the incidence of inflammatory bowel diseases (IBD) is dramatically increased in China, but with a geographic variation.Objectives: We performed a review to summarize the link of accelerated industrialization, urbanization to changing trends in the incidence of IBD over the last three decades.Methods: An electronic database search was performed in PubMed, Medline, EMBASE and Google Scholar (for English literature) and the China Science Periodical Database in Wanfang Data (for Chinese literature) from January 1990 to June 2020.Results: By systematically analyzing the changing trends of gross domestic product (GDP) or GDP per capita, population migration from rural areas to cities and increasing incidence of IBD in parallel in different Chinese regions, an association between accelerated industrialization and urbanization and rising rate of IBD was shown. In which, rates of IBD incidence were higher in provinces with a high value of GDP per capita than those provinces with a low value of GDP per capita. Analysis of available epidemiological data revealed that the incidence of IBD was rising in parallel with increasing trends of both gross products of industry and urban population in Yunnan Province in a 14-year interval. Further evidence suggested that industrialization- and urbanization-induced subsequent changes in environmental factors, e.g., Westernized dietary habits and obesity, and work-related stress, might contribute to the increased risk of IBD in China. In addition, the preliminary results showed that urbanization and Westernized dietary habits might induce significant changes in gut microbiota profile that are possibly to increase the risk for IBD in Chinese.Conclusions: Existing evidence to suggest that accelerated industrialization/urbanization is associated with the increasing incidence of IBD in China, which provides novel insights to study the possible mechanisms for the recent increasing incidence of IBD in newly industrialized and urbanized developing countries. In the future, the interaction between relevant environmental factors e.g., air/water pollution and IBD susceptibility genes in Chinese should be examined.

Mutational analyses of novel rat models with targeted modifications in inflammatory bowel disease susceptibility genes

Mutations and single base pair polymorphisms in various genes have been associated with increased susceptibility to inflammatory bowel disease (IBD). We have created a series of rat strains carrying targeted genetic alterations within three IBD susceptibility genes: Nod2, Atg16l1, and Il23r, using CRISPR/Cas9 genome editing technology. Knock-out alleles and alleles with known human susceptibility polymorphisms were generated on three different genetic backgrounds: Fischer, Lewis and Sprague Dawley. The availability of these rat models will contribute to our understanding of the basic biological roles of these three genes as well as provide new potential IBD animal models.

The autoimmune susceptibility gene, PTPN2, restricts expansion of a novel mouse adherent-invasive E. coli

ABSTRACT Inflammatory bowel disease (IBD) pathogenesis involves significant contributions from genetic and environmental factors. Loss-of-function single-nucleotide polymorphisms (SNPs) in the protein tyrosine phosphatase non-receptor type 2 (PTPN2) gene increase IBD risk and are associated with altered microbiome population dynamics in IBD. Expansion of intestinal pathobionts, such as adherent-invasive E. coli (AIEC), is strongly implicated in IBD pathogenesis as AIEC increases pro-inflammatory cytokine production and alters tight junction protein regulation – suggesting a potential mechanism of pathogen-induced barrier dysfunction and inflammation. We aimed to determine if PTPN2 deficiency alters intestinal microbiome composition to promote expansion of specific bacteria with pathogenic properties. In mice constitutively lacking Ptpn2, we identified increased abundance of a novel mouse AIEC (mAIEC) that showed similar adherence and invasion of intestinal epithelial cells, but greater survival in macrophages, to the IBD-associated AIEC, LF82. Furthermore, mAIEC caused disease when administered to mice lacking segmented-filamentous bacteria (SFB), and in germ-free mice but only when reconstituted with a microbiome, thus supporting its classification as a pathobiont, not a pathogen. Moreover, mAIEC infection increased the severity of, and prevented recovery from, induced colitis. Although mAIEC genome sequence analysis showed >90% similarity to LF82, mAIEC contained putative virulence genes with >50% difference in gene/protein identities from LF82 indicating potentially distinct genetic features of mAIEC. We show for the first time that an IBD susceptibility gene, PTPN2, modulates the gut microbiome to protect against a novel pathobiont. This study generates new insights into gene-environment-microbiome interactions in IBD and identifies a new model to study AIEC-host interactions.

A Novel Role of SLC26A3 in Maintenance of Intestinal Barrier Function

Inflammatory Bowel Disease (IBD) is a global health burden currently affecting around 3 million people in the United States with increasing incidence worldwide. The pathogenesis of IBD is still unclear due to the multifactorial nature of the disease. Compromised intestinal barrier is one of the most critical early events linked to the onset of intestinal inflammation. In this regard, SLC26A3 or DRA (Down Regulated in Adenoma) is an essential transporter for chloride absorption in the mammalian intestine and has been identified as an IBD susceptibility gene in certain cohorts. Further, DRA levels are markedly decreased in colonic mucosa of IBD patients and in mouse models of gut inflammation. Also, DRA KO mice are more susceptible to DSS colitis. However, the mechanisms underlying increased susceptibility to inflammation by loss of DRA are not fully understood.AimsTo examine the impact of loss of DRA (in DRA KO mice) on epithelial barrier function and expression of key tight junction and adherens junction proteins and elucidate underlying mechanisms.MethodsWild type and DRA KO mice (8–10 weeks old, M/F) were used. Protein expression was measured by immunoblotting and immunofluorescence. FITC‐dextran flux was examined utilizing Ussing chamber.ResultsDRA KO mice exhibited an increase in colonic paracellular permeability (~ 5 fold, p<0.05) indicating impaired barrier function. This was associated with a decrease in levels of TJ protein ZO‐1 and occludin (~ 65%, p<0.05) and major AJ protein, E‐cadherin (~ 60%, p<0.05). Also, the expression of pore forming, Claudin 2 was significantly upregulated in DRA KO mouse colon (~ 3 fold, p<0.05). No significant changes were observed in claudin 1 or 3. Since mouse intestine is a complex physiological system, we next examined if DRA silencing in a model epithelial Caco‐2 cell line, affects barrier function and tight junction protein expression. shRNA Knock‐down of DRA in Caco‐2 cells resulted in a marked decrease in TEER (~ 40%, p<0.05) and occludin expression (~ 60%, p<0.05). Similarly, enteroids derived from DRA KO mice exhibited decreased expression of occludin and E‐cadherin proteins (~ 50%, p<0.05). There was no alteration in mRNA levels of TJ/AJ proteins in DRA KO mice indicating role of post‐transcriptional mechanisms. To understand the mechanisms underlying these alterations, we next examined the expression of RNA‐binding proteins that are known to regulate the translation of occludin and E‐cadherin. Our results showed a marked increase in expression of CUGBP1 (a negative regulator of TJ/AJ proteins) and a marked decrease in expression of intact HuR (a positive regulator).ConclusionOur studies suggest a novel role of a membrane chloride transporter DRA in maintenance of intestinal barrier function and thus a down regulation of this protein may play a key role in pathogenesis of IBD.Support or Funding InformationSupported by NIDDK and Department of Veterans AffairsThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cellular and Molecular Therapeutic Targets in Inflammatory Bowel Disease-Focusing on Intestinal Barrier Function.

The human gut relies on several cellular and molecular mechanisms to allow for an intact and dynamical intestinal barrier. Normally, only small amounts of luminal content pass the mucosa, however, if the control is broken it can lead to enhanced passage, which might damage the mucosa, leading to pathological conditions, such as inflammatory bowel disease (IBD). It is well established that genetic, environmental, and immunological factors all contribute in the pathogenesis of IBD, and a disturbed intestinal barrier function has become a hallmark of the disease. Genetical studies support the involvement of intestinal barrier as several susceptibility genes for IBD encode proteins with key functions in gut barrier and homeostasis. IBD patients are associated with loss in bacterial diversity and shifts in the microbiota, with a possible link to local inflammation. Furthermore, alterations of immune cells and several neuro-immune signaling pathways in the lamina propria have been demonstrated. An inappropriate immune activation might lead to mucosal inflammation, with elevated secretion of pro-inflammatory cytokines that can affect the epithelium and promote a leakier barrier. This review will focus on the main cells and molecular mechanisms in IBD and how these can be targeted in order to improve intestinal barrier function and reduce inflammation.

Clinical and genetic risk factors for decreased bone mineral density in Japanese patients with inflammatory bowel disease.

Patients with inflammatory bowel disease (IBD) are at a high risk of low bone mineral density (BMD). Reportedly, clinical and genetic factors cause low BMD in Caucasians; however, studies in non-Caucasian populations remain scarce. Clinical risk factors for low BMD were investigated in 266 Japanese patients with IBD, and a genome-wide association analysis (GWAS) was performed using linear regression with associated clinical factors as covariates. Genotyping was performed using a population-optimized genotyping array (Japonica array® ). After quality control, the genotype data of 4384682 single-nucleotide polymorphisms (SNPs) from 254 patients with IBD were used for GWAS. Body mass index, age, and disease duration were independently associated with the BMD of the femoral neck (P=1.41E-13, 1.04E-5, and 1.58E-3, respectively), and body mass index and sex were associated with the BMD of the lumbar spine (P=6.90E-10 and 6.84E-3, respectively). In GWAS, 118 and 42 candidate SNPs of the femoral neck and lumbar spine, respectively, were identified. Among 118, 111 candidate SNPs of the femoral neck were located within the SLC22A23 gene, which is a known IBD susceptibility gene (minimum P=1.42E-07). Among 42, 18 candidate SNPs of the lumbar spine were located within the MECOM gene, which is associated with osteopenia (minimum P=5.86E-07). Interestingly, none of the known loci showed a significant association with BMD. Although clinical risk factors for low BMD in IBD were similar to those in the general population, genetic risk factors were rather different.

Allele-specific DNA methylation of disease susceptibility genes in Japanese patients with inflammatory bowel disease.

BackgroundInflammatory bowel disease (IBD) has an unknown etiology; however, accumulating evidence suggests that IBD is a multifactorial disease influenced by a combination of genetic and environmental factors. The influence of genetic variants on DNA methylation in cis and cis effects on expression have been demonstrated. We hypothesized that IBD susceptibility single-nucleotide polymorphisms (SNPs) regulate susceptibility gene expressions in cis by regulating DNA methylation around SNPs. For this, we determined cis-regulated allele-specific DNA methylation (ASM) around IBD susceptibility genes in CD4+ effector/memory T cells (Tem) in lamina propria mononuclear cells (LPMCs) in patients with IBD and examined the association between the ASM SNP genotype and neighboring susceptibility gene expressions.MethodsCD4+ effector/memory T cells (Tem) were isolated from LPMCs in 15 Japanese IBD patients (ten Crohn's disease [CD] and five ulcerative colitis [UC] patients). ASM analysis was performed by methylation-sensitive SNP array analysis. We defined ASM as a changing average relative allele score () >0.1 after digestion by methylation-sensitive restriction enzymes. Among SNPs showing >0.1, we extracted the probes located on tag-SNPs of 200 IBD susceptibility loci and around IBD susceptibility genes as candidate ASM SNPs. To validate ASM, bisulfite-pyrosequencing was performed. Transcriptome analysis was examined in 11 IBD patients (seven CD and four UC patients). The relation between rs36221701 genotype and neighboring gene expressions were analyzed.ResultsWe extracted six candidate ASM SNPs around IBD susceptibility genes. The top of (0.23) was rs1130368 located on HLA-DQB1. ASM around rs36221701 ( = 0.14) located near SMAD3 was validated using bisulfite pyrosequencing. The SMAD3 expression was significantly associated with the rs36221701 genotype (p = 0.016).ConclusionsWe confirmed the existence of cis-regulated ASM around IBD susceptibility genes and the association between ASM SNP (rs36221701) genotype and SMAD3 expression, a susceptibility gene for IBD. These results give us supporting evidence that DNA methylation mediates genetic effects on disease susceptibility.

Intestinal Epithelial Cell Autophagy Is Required to Protect against TNF-Induced Apoptosis during Chronic Colitis in Mice

Genome-wide association studies have linked polymorphisms in the autophagy gene ATG16L1 with susceptibility to inflammatory bowel disease (IBD). However, the cell-type-specific effects of autophagy on the regulation of chronic intestinal inflammation have not been investigated. Here, we assessed theeffect of myeloid-specific or intestinal epithelial cell (IEC)-specific deletion of Atg16l1 on chronic colitis triggered by the intestinal opportunistic pathogen Helicobacter hepaticus in mice. Although Atg16l1 deficiency in myeloid cells had little effect on disease, mice selectively lacking Atg16l1 in IECs (Atg16l1VC) developed severely exacerbated pathology, accompanied by elevated pro-inflammatory cytokine secretion and increased IEC apoptosis. Using exvivo IEC organoids, we demonstrate that autophagy intrinsically controls TNF-induced apoptosis and invivo blockade of TNF attenuated the exacerbated pathology in Atg16l1VC mice. These findings suggest that the IBD susceptibility gene ATG16L1 and the process of autophagy within the epithelium control inflammation-induced apoptosis and barrier integrity to limit chronic intestinal inflammation.

Epithelial Cell Inflammasomes in Intestinal Immunity and Inflammation.

Pattern recognition receptors (PRR), such as NOD-like receptors (NLRs), sense conserved microbial signatures, and host danger signals leading to the coordination of appropriate immune responses. Upon activation, a subset of NLR initiate the assembly of a multimeric protein complex known as the inflammasome, which processes pro-inflammatory cytokines and mediates a specialized form of cell death known as pyroptosis. The identification of inflammasome-associated genes as inflammatory bowel disease susceptibility genes implicates a role for the inflammasome in intestinal inflammation. Despite the fact that the functional importance of inflammasomes within immune cells has been well established, the contribution of inflammasome expression in non-hematopoietic cells remains comparatively understudied. Given that intestinal epithelial cells (IEC) act as a barrier between the host and the intestinal microbiota, inflammasome expression by these cells is likely important for intestinal immune homeostasis. Accumulating evidence suggests that the inflammasome plays a key role in shaping epithelial responses at the host–lumen interface with many inflammasome components highly expressed by IEC. Recent studies have exposed functional roles of IEC inflammasomes in mucosal immune defense, inflammation, and tumorigenesis. In this review, we present the main features of the predominant inflammasomes and their effector mechanisms contributing to intestinal homeostasis and inflammation. We also discuss existing controversies in the field and open questions related to their implications in disease. A comprehensive understanding of the molecular basis of intestinal inflammasome signaling could hold therapeutic potential for clinical translation.

Caspase recruitment domain 9, microbiota, and tryptophan metabolism: dangerous liaisons in inflammatory bowel diseases.

Inflammatory bowel diseases (IBDs) develop as a result of a combination of genetic predisposition, dysbiosis of the gut microbiota, and environmental influences. Here, we describe an example of how caspase recruitment domain 9 (CARD9), one of the numerous IBD susceptibility genes, participate to colitis susceptibility by shaping gut microbiota to produce tryptophan metabolites. Recent study showed that CARD9 mice are more susceptible to colitis as a result of impaired interleukin 22 signaling pathway. Furthermore, aryl hydrocarbon receptor (AhR) ligands from tryptophan metabolism by the gut microbiota participate to intestinal homeostasis by inducing production of interleukin 22 by intestinal immune cells. These data suggest an interaction between CARD9 and the ability of gut microbiota to produce AhR ligands. The microbiota from CARD9 mice fails to metabolize tryptophan leading to defective AhR activation which contributes to the susceptibility of mice to colitis by decreased interleukin 22 production. These effects were abrogated in the presence of AhR agonist. Reduced production of AhR ligands is also observed in the microbiota from individuals with IBD, particularly in those with CARD9 risk alleles associated with IBD. Correcting impaired microbiota functions, such as ability to produce AhR ligands, is an attractive strategy in IBD.

NOD2 Suppresses Colorectal Tumorigenesis via Downregulation of the TLR Pathways

Although NOD2 is the major inflammatory bowel disease susceptibility gene, its role in colorectal tumorigenesis is poorly defined. Here, we show that Nod2-deficient mice are highly susceptible to experimental colorectal tumorigenesis independent of gut microbial dysbiosis. Interestingly, the expression of inflammatory genes and the activation of inflammatory pathways, including NF-κB, ERK, and STAT3 are significantly higher in Nod2-/- mouse colons during colitis and colorectal tumorigenesis, but not at homeostasis. Consistent with higher inflammation, there is greater proliferation of epithelial cells in hyperplastic regions of Nod2-/- colons. Invitro studies demonstrate that, while NOD2 activates the NF-κB and MAPK pathways in response to MDP, it inhibits TLR-mediated activation of NF-κB and MAPK. Notably, NOD2-mediated downregulation of NF-κB and MAPK is associated with the induction of IRF4. Taken together, NOD2 plays a critical role in the suppression of inflammation and tumorigenesis in the colon via downregulation of the TLR signaling pathways.

MDR1 deficiency impairs mitochondrial homeostasis and promotes intestinal inflammation.

The multidrug-resistance-1 (MDR1) gene encodes an ATP-dependent efflux transporter that is highly expressed in the colon. In mice, loss of MDR1 function results in colitis with similarities to human inflammatory bowel diseases (IBD). Here, we show that MDR1 has an unexpected protective role for the mitochondria where MDR1-deficiency results in mitochondrial dysfunction with increased mitochondrial ROS (mROS) driving the development of colitis. Exogenous induction of mROS accelerates, whilst inhibition attenuates colitis in vivo; these effects are amplified in MDR1-deficiency. In human IBD, MDR1 is negatively correlated to SOD2 gene expression required for mROS detoxification. To provide direct evidential support, we deleted intestinal SOD2 gene in mice and showed an increased susceptibility to colitis. We exploited the GWA datasets and found many (~5%) of IBD susceptibility genes with direct roles in regulating mitochondria homeostasis. As MDR1 primarily protects against xenotoxins via its efflux function, our findings implicate a distinct mitochondrial toxin + genetic susceptibility interaction leading to mitochondrial dysfunction, a novel pathogenic mechanism that could offer many new therapeutic opportunities for IBD.

Characterization and Genetic Analyses of New Genes Coding for NOD2 Interacting Proteins

NOD2 contributes to the innate immune response and to the homeostasis of the intestinal mucosa. In response to its bacterial ligand, NOD2 interacts with RICK and activates the NF-κB and MAPK pathways, inducing gene transcription and synthesis of proteins required to initiate a balanced immune response. Mutations in NOD2 have been associated with an increased risk of Crohn’s Disease (CD), a disabling inflammatory bowel disease (IBD). Because NOD2 signaling plays a key role in CD, it is important to further characterize the network of protein interacting with NOD2. Using yeast two hybrid (Y2H) screens, we identified new NOD2 interacting proteins (NIP). The primary interaction was confirmed by coimmunoprecipitation and/or bioluminescence resonance energy transfer (BRET) experiments for 11 of these proteins (ANKHD1, CHMP5, SDCCAG3, TRIM41, LDOC1, PPP1R12C, DOCK7, VIM, KRT15, PPP2R3B, and C10Orf67). These proteins are involved in diverse functions, including endosomal sorting complexes required for transport (ESCRT), cytoskeletal architecture and signaling regulation. Additionally, we show that the interaction of 8 NIPs is compromised with the 3 main CD associated NOD2 mutants (R702W, G908R and 1007fs). Furthermore, to determine whether these NOD2 protein partners could be encoded by IBD susceptibility genes, a transmission disequilibrium test (TDT) was performed on 101 single nucleotide polymorphisms (SNPs) and the main corresponding haplotypes in genes coding for 15 NIPs using a set of 343 IBD families with 556 patients. Overall this work did not increase the number of IBD susceptibility genes but extends the NOD2 protein interaction network and suggests that NOD2 interactome and signaling depend upon the NOD2 mutation profile in CD.

R-Spondins Are Expressed by the Intestinal Stroma and are Differentially Regulated during Citrobacter rodentium- and DSS-Induced Colitis in Mice.

The R-spondin family of proteins has recently been described as secreted enhancers of β-catenin activation through the canonical Wnt signaling pathway. We previously reported that Rspo2 is a major determinant of susceptibility to Citrobacter rodentium-mediated colitis in mice and recent genome-wide association studies have revealed RSPO3 as a candidate Crohn’s disease-specific inflammatory bowel disease susceptibility gene in humans. However, there is little information on the endogenous expression and cellular source of R-spondins in the colon at steady state and during intestinal inflammation. RNA sequencing and qRT-PCR were used to assess the expression of R-spondins at steady state and in two mouse models of colonic inflammation. The cellular source of R-spondins was assessed in specific colonic cell populations isolated by cell sorting. Data mining from publicly available datasets was used to assess the expression of R-spondins in the human colon. At steady state, colonic expression of R-spondins was found to be exclusive to non-epithelial CD45- lamina propria cells, and Rspo3/RSPO3 was the most highly expressed R-spondin in both mouse and human colon. R-spondin expression was found to be highly dynamic and differentially regulated during C. rodentium infection and dextran sodium sulfate (DSS) colitis, with notably high levels of Rspo3 expression during DSS colitis, and high levels of Rspo2 expression during C. rodentium infection, specifically in susceptible mice. Our data are consistent with the hypothesis that in the colon, R-spondins are expressed by subepithelial stromal cells, and that Rspo3/RSPO3 is the family member most implicated in colonic homeostasis. The differential regulation of the R-spondins in different models of intestinal inflammation indicate they respond to specific pathogenic and inflammatory signals that differ in the two models and provides further evidence that this family of proteins plays a key role in linking intestinal inflammation and homeostasis.

Mice lacking myosin IXb, an inflammatory bowel disease susceptibility gene, have impaired intestinal barrier function and superficial ulceration in the ileum.

Genetic studies have implicated MYO9B, which encodes myosin IXb (Myo9b), a motor protein with a Rho GTPase activating domain (RhoGAP), as a susceptibility gene for inflammatory bowel disease (IBD). Moreover, we have recently shown that knockdown of Myo9b in an intestinal epithelial cell line impairs wound healing and barrier function. Here, we investigated whether mice lacking Myo9b have impaired intestinal barrier function and features of IBD. Myo9b knock out (KO) mice exhibit impaired weight gain and fecal occult blood (indicator of gastrointestinal bleeding), and increased intestinal epithelial cell apoptosis could be detected along the entire intestinal axis. Histologic analysis revealed intestinal mucosal damage, most consistently observed in the ileum, which included superficial ulceration and neutrophil infiltration. Focal lesions contained neutrophils and ultrastructural examination confirmed epithelial discontinuity and the deposition of extracellular matrix. We also observed impaired mucosal barrier function in KO mice. Transepithelial electrical resistance of KO ileum is >3 fold less than WT ileum. The intestinal mucosa is also permeable to high molecular weight dextran, presumably due to the presence of mucosal surface ulcerations. There is loss of tight junction-associated ZO-1, decreased lateral membrane associated E-cadherin, and loss of terminal web associated cytokeratin filaments. Consistent with increased Rho activity in the KO, there is increased subapical expression of activated myosin II (Myo2) based on localization of phosphorylated Myo2 regulatory light chain. Except for a delay in disease onset in the KO, no difference in dextran sulfate sodium-induced colitis and lethality was observed between wild-type and Myo9b KO mice.

Genetically engineered mouse models for studying inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic intestinal inflammatory condition that is mediated by very complex mechanisms controlled by genetic, immune, and environmental factors. More than 74 kinds of genetically engineered mouse strains have been established since 1993 for studying IBD. Although mouse models cannot fully reflect human IBD, they have provided significant contributions for not only understanding the mechanism, but also developing new therapeutic means for IBD. Indeed, 20 kinds of genetically engineered mouse models carry the susceptibility genes identified in human IBD, and the functions of some other IBD susceptibility genes have also been dissected out using mouse models. Cutting-edge technologies such as cell-specific and inducible knockout systems, which were recently employed to mouse IBD models, have further enhanced the ability of investigators to provide important and unexpected rationales for developing new therapeutic strategies for IBD. In this review article, we briefly introduce 74 kinds of genetically engineered mouse models that spontaneously develop intestinal inflammation.