Pathogenesis Of Crohn's Disease Research Articles

Functional Implications of MicroRNAs in Crohn's Disease Revealed by Integrating MicroRNA and Messenger RNA Expression Profiling.

Crohn’s disease (CD) is a debilitating inflammatory bowel disease (IBD) that emerges due to the influence of genetic and environmental factors. microRNAs (miRNAs) have been identified in the tissue and sera of IBD patients and may play an important role in the induction of IBD. Our study aimed to identify differentially expressed miRNAs and miRNAs with the ability to alter transcriptome activity by comparing inflamed tissue samples with their non-inflamed counterparts. We studied changes in miRNA–mRNA interactions associated with CD by examining their differential co-expression relative to normal mucosa from the same patients. Correlation changes between the two conditions were incorporated into scores of predefined gene sets to identify biological processes with altered miRNA-mediated control. Our study identified 28 miRNAs differentially expressed (p-values < 0.01), of which 14 are up-regulated. Notably, our differential co-expression analysis highlights microRNAs (i.e., miR-4284, miR-3194 and miR-21) that have known functional interactions with key mechanisms implicated in IBD. Most of these miRNAs cannot be detected by differential expression analysis that do not take into account miRNA–mRNA interactions. The identification of differential miRNA–mRNA co-expression patterns will facilitate the investigation of the miRNA-mediated molecular mechanisms underlying CD pathogenesis and could suggest novel drug targets for validation.

Complementary Roles of Nod2 in Hematopoietic and Nonhematopoietic Cells in Preventing Gut Barrier Dysfunction Dependent on MLCK Activity

Crohn's disease (CD) pathogenesis is multifactorial involving genetic and environmental factors. Loss of function mutations in the nucleotide oligomerization domain 2 (NOD2) gene are the main genetic risk factor for CD. Like patients with CD, Nod2 mice are characterized by an enhanced Th1 immune response and a defective mucosal barrier function evidenced by increased intestinal permeability. We previously showed that the latter is related to hematopoietic Nod2 deficiency. Our aim was to explore the mechanisms by which Nod2 expressed in the hematopoietic and in the nonhematopoietic compartments interplay to control epithelial paracellular permeability. Depletion of CD4 T cells in Nod2 mice and treatments with inhibitors were conducted in chimeric mice transplanted with bone marrow cells from Nod2-deficient donors into Nod2-sufficient recipients or vice versa. Caco-2 cells overexpressing a NOD2 gene which did or did not include a CD-associated polymorphism were treated with inhibitors or siRNAs and cocultured with hematopoietic cells from Peyer's patches. In vivo and in vitro Nod2 in hematopoietic cells regulates epithelial paracellular permeability through cytokine production influencing myosin light chain kinase (MLCK) activity. Indeed, tumor necrosis factor-α and interferon-γ secretion by CD4 T cells upregulated expression and activity of epithelial MLCK leading to increased epithelial tight junction opening. When stimulated by muramyl dipeptide, Nod2 in the nonhematopoietic compartment normalized the permeability and T-cell cytokine secretion and regulated MLCK activity. This MLCK regulation is mediated by TAK1 and RICK-dependent mechanisms. Our study demonstrates how hematopoietic and nonhematopoietic Nod2 regulate intestinal barrier function, improving our knowledge on the mechanisms involved in CD pathogenesis.

Two distinct metacommunities characterize the gut microbiota in Crohn's disease patients.

The inflammatory intestinal disorder Crohn's disease (CD) has become a health challenge worldwide. The gut microbiota closely interacts with the host immune system, but its functional impact in CD is unclear. Except for studies on a small number of CD patients, analyses of the gut microbiota in CD have used 16S rDNA amplicon sequencing. Here we employed metagenomic shotgun sequencing to provide a detailed characterization of the compositional and functional features of the CD microbiota, comprising also unannotated bacteria, and investigated its modulation by exclusive enteral nutrition. Based on signature taxa, CD microbiotas clustered into 2 distinct metacommunities, indicating individual variability in CD microbiome structure. Metacommunity-specific functional shifts in CD showed enrichment in producers of the pro-inflammatory hexa-acylated lipopolysaccharide variant and a reduction in the potential to synthesize short-chain fatty acids. Disruption of ecological networks was evident in CD, coupled with reduction in growth rates of many bacterial species. Short-term exclusive enteral nutrition elicited limited impact on the overall composition of the CD microbiota, although functional changes occurred following treatment. The microbiotas in CD patients can be stratified into 2 distinct metacommunities, with the most severely perturbed metacommunity exhibiting functional potentials that deviate markedly from that of the healthy individuals, with possible implication in relation to CD pathogenesis.

Defective Lymphatics in Crohn’s Disease: Tertiary Lymphoid Follicles Plug the Gap

Defective Lymphatics in Crohn’s Disease: Tertiary Lymphoid Follicles Plug the Gap

The gut microbiota of siblings offers insights into microbial pathogenesis of inflammatory bowel disease

ABSTRACTSiblings of patients with Crohn's disease (CD) have elevated risk of developing CD and display aspects of disease phenotype, including faecal dysbiosis. In our recent article we have used 16S rRNA gene targeted high-throughput sequencing to comprehensively characterize the mucosal microbiota in healthy siblings of CD patients, and determine the influence of genotypic and phenotypic factors on the gut microbiota (dysbiosis). We have demonstrated that the core microbiota of both patients with CD and healthy siblings is significantly less diverse than controls. Faecalibacterium prausnitzii contributed most to core metacommunity dissimilarity between both patients and controls and between siblings and controls. Phenotype/genotype markers of CD risk significantly influenced microbiota variation between and within groups, of which genotype had the largest effect. Individuals with elevated CD-risk display mucosal dysbiosis characterized by reduced diversity of core microbiota and lower abundance of F. prausnitzii. The presence of this dysbiosis in healthy people at-risk of CD implicates microbiological processes in CD pathogenesis.

Dysbiosis in Crohn's disease - Joint action of stochastic injuries and focal inflammation in the gut

ABSTRACTGut homeostasis involves interrelated biological networks that include the immune system, specialized cells of the epithelium, such as Paneth and goblet cells, as well as triggers derived from the microbiota. Disruption of these homeostatic interactions may lead to the pathogenesis of inflammatory bowel diseases (IBD). To develop more targeted and individual treatments in Crohn's disease and ulcerative colitis, it becomes more and more important to link key mechanisms of the disease pathogenesis to distinct IBD subsets. For the first time, our laboratory demonstrated a causal role of the microbiota for the development of Crohn's disease (CD)-like ileitis, supporting the hypothesis that a non-infectious, dysbiotic microbial ecosystem harbors aggressive traits relevant for the induction of chronic inflammation in the disease-susceptible host (i.e. TNFΔARE mouse model). Despite a growing body of evidence claiming a primary role for Paneth cells in the pathogenesis of ileal CD, we showed in the TNFΔARE mouse model that Paneth cell failure or exhaustion is a secondary event to inflammation. Therefore, additional mechanisms may act synergistically to initialize the development of CD-like pathology. Hereby, we propose a novel hypothesis suggesting that individual development of dysbiotic communities is based on stochastic injury and focal inflammation of the epithelial lining that propagate radially, finally leading to an aggressive microbial milieu.

Brain changes detected by functional magnetic resonance imaging and spectroscopy in patients with Crohn's disease.

Crohn’s disease (CD) is a chronic, non-specific granulomatous inflammatory disorder that commonly affects the small intestine and is a phenotype of inflammatory bowel disease (IBD). CD is prone to relapse, and its incidence displays a persistent increase in developing countries. However, the pathogenesis of CD is poorly understood, with some studies emphasizing the link between CD and the intestinal microbiota. Specifically, studies point to the brain-gut-enteric microbiota axis as a key player in the occurrence and development of CD. Furthermore, investigations have shown white-matter lesions and neurologic deficits in patients with IBD. Based on these findings, brain activity changes in CD patients have been detected by blood oxygenation level dependent functional magnetic resonance imaging (BOLD-fMRI). BOLD-fMRI functions by detecting a local increase in relative blood oxygenation that results from neurotransmitter activity and thus reflects local neuronal firing rates. Therefore, biochemical concentrations of neurotransmitters or metabolites may change in corresponding brain regions of CD patients. To further study this phenomenon, brain changes of CD patients can be detected non-invasively, effectively and accurately by BOLD-fMRI combined with magnetic resonance spectroscopy (MRS). This approach can further shed light on the mechanisms of the occurrence and development of neurological CD. Overall, this paper reviews the current status and prospects on fMRI and MRS for evaluation of patients with CD based on the brain-gut-enteric microbiota axis.

Altered intestinal microbiota-host mitochondria crosstalk in new onset Crohn's disease.

Intestinal microbial dysbiosis is associated with Crohn's disease (CD). However, the mechanisms leading to the chronic mucosal inflammation that characterizes this disease remain unclear. In this report, we use systems-level approaches to study the interactions between the gut microbiota and host in new-onset paediatric patients to evaluate causality and mechanisms of disease. We report an altered host proteome in CD patients indicative of impaired mitochondrial functions. In particular, mitochondrial proteins implicated in H2S detoxification are downregulated, while the relative abundance of H2S microbial producers is increased. Network correlation analysis reveals that Atopobium parvulum controls the central hub of H2S producers. A. parvulum induces pancolitis in colitis-susceptible interleukin-10-deficient mice and this phenotype requires the presence of the intestinal microbiota. Administrating the H2S scavenger bismuth mitigates A. parvulum-induced colitis in vivo. This study reveals that host–microbiota interactions are disturbed in CD and thus provides mechanistic insights into CD pathogenesis.

Colonic cytokines for severe ulcerative colitis

Aim of investigation. To assess colonic cytokines profile at severe ulcerative colitis (UC). Material and methods. Overall 48 biopsy specimens of colonic mucosa obtained at resection operations in 7 patients with total form of UC were investigated. All patients underwent total colectomy for severe treatment-resistant UC. Activity of inflammatory process was determined by severity of erosive and ulcerative lesions of the colon. Cytokine concentration was evaluated in supernatant fluid after centrifugal separation of 10 mg biopsy specimen homogenate by the protein analyzer «Bio-Plex» («Bio-Rad», USA). Colonic mucosa biopsy specimens of unaffected parts of the large intestine resected in 10 patients with colorectal cancer were used as controls. Results. Tissue levels of IL-1β, IL-8, IL-6, TNF-α (tumor necrosis factor-α), IFN-γ (interferon-γ), IL-17 and IL-12 in biopsy specimens the large intestine of UC patients were significantly elevated. The most significant elevation was found for IL-8 and IL-1β, with the ratio of 3:1 (in the control group 0,35:1). The cytokine content in biopsy specimens of the large intestine has been increased in comparison to terminal portion of the small intestine, and the cytokine level in the left and transverse part of the colon was higher, than in cecum and ascending colon. At severe inflammation as compared to moderate inflammation the most significant increase was found for IL-8, IL-6 and TNF-α. Cytokine profile with the prevalence of IL-1β (IL-1β>IL-8>IL-6 (IFN-γ)>TNF-α) was observed in 14 (77,7%) of 18 biopsy specimens with moderate inflammation while similar cytokine pattern was observed in 9 (47,3%) of 19 biopsy specimens with severe inflammation (p=0,05). Domination of IL-8 (IL-8> IL-1β> IL-6 (TNF-α)> TNF-α (IL-6)) in cytokine profile was found in 3 of 18 (16,6%) biopsy specimens with moderate and in 9 of 19 (47,3%) biopsy specimens with a severe inflammation (p<0,05). Cytokines IL-2, IL-4, IL-5, IL-7, IL-10 and IL-13 when undetected neither in UC patients, nor in control group. Conclusions. Significant increase in proinflammatory cytokine level with prevalence of IL-1β and IL-8 at moderate and severe erosive - ulcerative involvement respectively was observed in colonic mucosa biopsies in comparison to terminal ileum of UC patients and control group; in the left part and transverse colon versus ascending colon and caecum of UC patients. Elevation of IFN-γ and IL-17 content at severe inflammation indicates presence of common mechanisms in ulcerative colitis disease and Crohn's disease pathogenesis.

An overview of the bacterial contribution to Crohn disease pathogenesis.

Crohn disease (CD) is a chronic inflammatory condition primarily affecting the gastro-intestinal tract and is characterized by reduced bacterial diversity. The exact cause of disease is unknown; however, evidence suggests that several components, including microbiota, may contribute to the underlying pathology and disease development. Perturbation of the host-microbe commensal relationship is considered the main driving force of tissue destruction and pathological changes seen in CD. Several putative bacterial pathogens including species from Mycobacterium, Campylobacter and Helicobacter are postulated in the aetiology of CD. However, to date, no strong evidence supports a single bacterium contributing overall to CD pathogenesis. Alternatively, dysbiosis or bacterial imbalance is more widely accepted as a leading factor in the disrupted host-immune system cross-talk resulting in subsequent intestinal inflammation. Depletion of symbiont microbes including Firmicutes, Bifidobacterium and Clostridia, in conjunction with an increase in pathobiont microbes from Bacteroidetes and Enterobacteria, is a striking feature observed in CD. No single factor has been identified as driving this dysbiosis, although diet, antibiotic exposure and possible early life events in presence of underlying genetic susceptibility may contribute. The aim of this review is to highlight the current accumulating literature on the proposed role of bacteria in the pathogenesis of CD.

Mucosa-Associated Ileal Microbiota in New-Onset Pediatric Crohn's Disease.

The composition of the intestinal microbiome seems relevant to the pathogenesis of Crohn's disease (CD), with differences in both diversity and composition of the gut microbiota in patients with CD compared with healthy individuals. However, there are still conflicting reports on the importance of various bacterial taxa in the pathogenesis of CD. The aim of this study was to characterize the composition of mucosa-associated intestinal microbiota in newly diagnosed pediatric patients with CD. Mucosa-associated bacteria were identified from ileal biopsy specimens obtained at colonoscopy of 10 patients with either ileal or ileocolonic new-onset CD and 15 controls without mucosal inflammation. Microbial composition was performed by profiling the 16S rDNA V6 region using Illumina sequencing. Samples were analyzed for differences in alpha/beta diversity and also for differentially abundant taxa. Alpha diversity did not differ between the controls and CD cases or between CD subjects with localized ileal disease compared with those with more extensive disease. Controls also did not clearly separate from patients with CD by principal coordinate analyses; however, 117 operational taxonomic units were found to be differentially abundant between the two groups. In particular, numerous operational taxonomic units associated with Faecalibacterium prausnitzii species were increased in children with CD. These findings contribute to emerging evidence regarding dysbiosis in pediatric CD, and provide additional evidence challenging the protective role of F. prausnitzii in CD.

Management of Crohn's Disease in the New Era of Gut Rehabilitation and Intestinal Transplantation.

Despite recent therapeutic advances, patients with Crohn's disease (CD) continue to experience high recurrence with cumulative structural damage and ultimate loss of nutritional autonomy. With short bowel syndrome, strictures, and enteric fistulae being the underlying pathology, CD is the second common indication for home parenteral nutrition (HPN). With development of intestinal failure, nutritional management including HPN is required as a rescue therapy. Unfortunately, some patients do not escape the HPN-associated complications. Therefore, the concept of gut rehabilitation has evolved as part of the algorithmic management of these patients, with transplantation being the ultimate life-saving therapy. With type 2 intestinal failure, comprehensive rehabilitative measures including nutritional care, pharmacologic manipulation, autologous reconstruction, and bowel lengthening is often successful, particularly in patients with quiescent disease. With type 3 intestinal failure, transplantation is the only life-saving treatment for patients with HPN failure and intractable disease. With CD being the second common indication for transplantation in adults, survival outcome continues to improve because of surgical innovation, novel immunosuppression, and better postoperative care. Despite being a rescue therapy, the procedure has achieved survival rates similar to other solid organs, and comparable to those who continue to receive HPN therapy. With similar technical, immunologic, and infectious complications, survival is similar in the CD and non-CD recipients. Full nutritional autonomy is achievable in most survivors with better quality of life and long-term cost-effectiveness. CD recurrence is rare with no impact on graft function. Further progress is anticipated with new insights into the pathogenesis of CD and mechanisms of transplant tolerance.

PTH-055 ABO Blood Group-Specific Immunoglobulins in Crohn’s Disease

Introduction The pathogenesis of Crohn’s disease (CD) is complex and believed to be interplayed by genetic susceptibility, enteric microflora and tissue injury mediated by abnormal immune response. Several studies have described the association between ABO blood group, enteric microflora composition and predisposition to gastrointestinal (GI) infections. The mucosal layer of the GI tract forms the first layer of immune defenses with mucus abundant in secretory immunoglobulins, and studies have shown that non-O blood group and FUT2-determined ABO antigen non-secretor were linked to more severe CD. It was suggested that ABO antibodies are induced via an enteric route by cross-reactivity from microflora-derived molecules . However, little is known about the role of ABO antibodies in the context of CD. Therefore we assessed whether blood group-specific antibodies are implicated in CD pathogenesis. Methods We compared the immunoglobulin class profile of blood group-specific antibodies in blood group A, B and O individuals. Plasma samples were collected from CD cohort (n = 45, recruited from the FaMIsHED study) with age- and gender-matched healthy donors (n = 89 data from service development study). Medications of CD cohort include none (n = 15), 5 ASA (n = 21), immunosuppressant (n = 12) and biologics (n = 10). A1rr and Brr cells were used, anti-A1 and anti-B IgG, IgA and IgM levels were measured by Relative Median Fluorescence using flow cytometry. Mann-Whitney U test was used to assess statistical significance of different antibody levels between CD and healthy cohorts. Results Blood group O CD patients (n = 20) have significantly lower anti-A1 IgG, IgA and IgM compared to the healthy blood group O cohort with p value of 0.0001, 0.0009 and Conclusion CD patients were found to have lower than baseline ABO antibodies. However, it is not known whether these findings represent the cause or effect of disturbed immune modulations, or their roles in modulating intestinal microflora. Further research into the difference in ABO antibodies class profile in a larger cohort, and comparison of ABO antibodies class profile of CD patients in remission and relapse are required. Disclosure of Interest None Declared

The Role of the Mesentery in Crohn's Disease: The Contributions of Nerves, Vessels, Lymphatics, and Fat to the Pathogenesis and Disease Course.

Crohn's disease (CD) is a complex gastrointestinal disorder involving multiple levels of cross talk between the immunological, neural, vascular, and endocrine systems. The current dominant theory in CD is based on the unidirectional axis of dysbiosis-innate immunity-adaptive immunity-mesentery-body system. Emerging clinical evidence strongly suggests that the axis be bidirectional. The morphologic and/or functional abnormalities in the mesenteric structures likely contribute to the disease progression of CD, to a less extent the disease initiation. In addition to adipocytes, mesentery contains nerves, blood vessels, lymphatics, stromal cells, and fibroblasts. By the secretion of adipokines that have endocrine functions, the mesenteric fat tissue exerts its activity in immunomodulation mainly through response to afferent signals, neuropeptides, and functional cytokines. Mesenteric nerves are involved in the pathogenesis and prognosis of CD mainly through neuropeptides. In addition to angiogenesis observed in CD, lymphatic obstruction, remodeling, and impaired contraction maybe a cause and consequence of CD. Lymphangiogenesis and angiogenesis play a concomitant role in the progress of chronic intestinal inflammation. Finally, the interaction between neuropeptides, adipokines, and vascular and lymphatic endothelia leads to adipose tissue remodeling, which makes the mesentery an active participator, not a bystander, in the disease initiation and precipitation CD. The identification of the role of mesentery, including the structure and function of mesenteric nerves, vessels, lymphatics, and fat, in the intestinal inflammation in CD has important implications in understanding its pathogenesis and clinical management.

The arachidonic acid metabolite 11β-ProstaglandinF2α controls intestinal epithelial healing: deficiency in patients with Crohn's disease.

In healthy gut enteric glial cells (EGC) are essential to intestinal epithelial barrier (IEB) functions. In Crohn’s Disease (CD), both EGC phenotype and IEB functions are altered, but putative involvement of EGC in CD pathogenesis remains unknown and study of human EGC are lacking. EGC isolated from CD and control patients showed similar expression of glial markers and EGC-derived soluble factors (IL6, TGF-β, proEGF, GSH) but CD EGC failed to increase IEB resistance and healing. Lipid profiling showed that CD EGC produced decreased amounts of 15-HETE, 18-HEPE, 15dPGJ2 and 11βPGF2α as compared to healthy EGC. They also had reduced expression of the L-PGDS and AKR1C3 enzymes. Produced by healthy EGC, the 11βPGF2 activated PPARγ receptor of intestinal epithelial cells to induce cell spreading and IEB wound repair. In addition to this novel healing mechanism our data show that CD EGC presented impaired ability to promote IEB functions through defect in L-PGDS-AKR1C3-11βPGF2α dependent pathway.

In It for the Long Haul: Managing the Complexity of Crohn’s Disease

The challenges of, and opportunities for optimal long-term management of Crohn’s disease (CD) and real-world experience of managing CD and its application in clinical practice were discussed at this symposium. CD is a complex disease, which requires effective treatment options to improve the quality of life for patients, both in terms of intestinal and extraintestinal manifestations (EIMs). Increased gut permeability of luminal antigens may play a primary role in the pathogenesis of CD, leading to dysregulation of the host’s immune response, and resulting in increased levels of tumour necrosis factor (TNF)-α and interferon (IFN)-γ in the inflamed mucosa of patients. Appropriate management goals need to be established by the physician and patient together. Anti-TNF therapy is not suitable for all patients, and a significant proportion of patients will be primary non-responders. Safety must also be considered as part of a patient-tailored assessment. Vedolizumab is a gut-selective antibody to α4β7 integrin for the treatment of ulcerative colitis (UC) and CD. An integrated Phase II and III safety analysis showed that vedolizumab exposure was not associated with increased risk of any infection or serious infection, or any cases of progressive multifocal leukoencephalopathy (PML), a rare and usually fatal viral disease characterised by progressive damage of the white matter of the brain at multiple locations. Data from the GEMINI trials with vedolizumab showed it to be effective versus placebo, in terms of eliciting both initial and sustained responses, and inducing remission in CD. The real-world studies with vedolizumab in &gt;800 CD patients, most of whom failed ≥1 anti-TNF therapy, confirmed the efficacy and safety reported in clinical trials. Up to 30% of CD patients are receiving vedolizumab as a first biologic in the real-world setting.

Recent insights into the molecular pathogenesis of Crohn's disease: a review of emerging therapeutic targets.

Chronic inflammatory bowel diseases (IBDs) are a subject of great interest in gastroenterology, due to a pathological mechanism that is difficult to explain and an optimal therapeutic approach still undiscovered. Crohn’s disease (CD) is one of the main entities in IBD, characterized by clinical polymorphism and great variability in the treatment response. Modern theories on the pathogenesis of CD have proven that gut microbiome and environmental factors lead to an abnormal immune response in a genetically predisposed patient. Genome-wide association studies in patients with CD worldwide revealed several genetic mutations that increase the risk of IBD and that predispose to a more severe course of disease. Gut microbiota is considered a compulsory and an essential part in the pathogenesis of CD. Intestinal dysmicrobism with excessive amounts of different bacterial strains can be found in all patients with IBD. The discovery of Escherichia coli entero-invasive on resection pieces in patients with CD now increases the likelihood of antimicrobial or vaccine-type treatments. Recent studies targeting intestinal immunology and its molecular activation pathways provide new possibilities for therapeutics. In addition to antitumor necrosis factor molecules, which were a breakthrough in IBD, improving mucosal healing and resection-free survival rate, other classes of therapeutic agents come to focus. Leukocyte adhesion inhibitors block the leukocyte homing mechanism and prevent cellular immune response. In addition to anti-integrin antibodies, chemokine receptor antagonists and SMAD7 antisense oligonucleotides have shown encouraging results in clinical trials. Micro-RNAs have demonstrated their role as disease biomarkers but it could also become useful for the treatment of IBD. Moreover, cellular therapy is another therapeutic approach under development, aimed for severe refractory CD. Other experimental treatments include intravenous immunoglobulins, exclusive enteral nutrition, and granulocyte colony-stimulating factors.

P-175 Pleiotropic Effects of Novel Functional LRRK2 Variation on Crohnʼs Disease and Parkinsonʼs Disease Risk

The genomic region on chromosome 12q12 containing leucine-rich repeat kinase 2 (LRRK2) includes major association signals for both Crohn's disease (CD) and Parkinson's disease (PD). Numerous functional roles have been reported for LRRK2 including autophagy, an important process believed to be involved in pathogenesis of CD. Importantly, relatively uncommon, non-synonymous variation in this region demonstrates major allele frequency differences between Ashkenazi Jewish (AJ) and non-Jewish European ancestry (NJ) populations. Thus far, no systematic analysis of this locus in association with CD has been conducted. Therefore, we sought to parse potential causal variants underlying CD association, to compare the roles of such variants against those implicated in PD, and to contrast the results in the AJ and NJ populations. Using an exome sequencing-based semi-custom array, we first conducted genotyping in 1477 independent AJ CD cases and 2614 AJ controls. We then performed imputation using sequencing and genotype data from 20,479 CD cases, PD cases, and healthy controls of AJ and NJ ancestry to identify additional variants within the LRRK2 locus associated with a shared risk of CD and PD. To examine the role of the top LRRK2 association signals in the process of autophagy we performed in vitro studies on monocyte-derived macrophages from 13 AJ CD patients (3 N2081D carriers, 4 N551K carriers, 5 non-carriers, and 1 carrier of both coding changes, who was treated as a non-carrier due to the presumed opposing effects of the 2 mutations). We detected 2 independent novel CD association signals at the coding non-synonymous mutations N2081D, conferring CD risk (OR = 1.7, P = 4.3 × 10−8), and N551K, a protective variant (OR = 0.72, P = 1.4 × 10−6). Similar results were also observed in an independent NJ cohort (OR = 1.6, P = 2.1 × 10−6 and OR = 0.89, P = 5.1 × 10−2, respectively). Importantly, both mutations were previously linked to PD, with the same magnitude and direction of effect. The G2019S coding change, strongly associated with PD, showed no evidence of association with CD. Further analyses of the imputed data showed a broad CD-association peak tagged by N2081D and additional extensive genetic pleiotropy between CD and PD within the extended LRRK2 locus, with higher minor allele frequencies in the AJ compared to NJ individuals. To test whether the novel CD-associated LRRK2 mutations affect the process of autophagy we compared the mean fluorescent intensity between genotype-specific human monocyte-derived macrophages following a pulse of lysosensor (PBS-treated versus media control). We observed an attenuated sensitivity to stress in N2081D-carrier cells and an increased sensitivity in carriers of the protective N551K mutation (P = 0.04, order-constrained ANOVA), suggesting impaired lysosomal acidification in N2081 carriers, which could impact protein degradation and turnover of the destroyed cell organelles in immune cells. We identified 2 non-synonymous mutations in the LRRK2 gene significantly associated with CD risk that may play a functional role in the process of autophagy. In addition, we demonstrated extensive pleiotropy between CD and PD risk within the LRRK2 locus. These findings have significant implications for molecular targeting of LRRK2 for the treatment of CD and PD.

MicroRNA-124 Promotes Intestinal Inflammation by Targeting Aryl Hydrocarbon Receptor in Crohn's Disease.

Dysregulation of microRNAs (miRNAs) is associated with a variety of diseases, including Crohn's disease (CD), but the essential biological functions and crucial targets of miRNAs remain largely unknown. The present study investigated the aberrant colonic mucosal miRNAs in active CD patients. miRNA levels were assayed in inflamed colon of active CD patients by quantitative real-time polymerase chain reaction. The influence of differential expressed miR-124 on its putative target, the aryl hydrocarbon receptor (AHR), was investigated in CD patients, intestinal epithelial cells (IECs) and 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis mice. The role of miR-124 was further studied in experimental colitis mice by intracolonic administration of miR-124 inhibitors or precursors. We found an inverse correlation between miR-124 and AHR protein levels in colon tissues and IECs of active CD patients. Further results demonstrated that miR-124 suppressed AHR expression by directly targeting the AHR 3'-untranslated region (3'-UTR) in Caco-2 cells and HT-29 cells. MiR-124 mediated the inflammatory response in lipopolysaccharide-stimulated cells through retroregulation of AHR in vitro. Downregulation or upregulation of miR-124 in TNBS-induced colitic colon alleviated or aggravated experimental colitis, respectively. These findings suggest that miR-124 induces intestinal inflammation by inhibiting AHR to modulate pro-inflammatory cytokine production and thereby promotes the pathogenesis of CD.

Serum Cytokine Profiles in Children with Crohn's Disease.

Crohn's disease (CD) is a chronic inflammatory bowel disease that can be diagnosed at any age. There are two major patient groups based on diagnosis of this disease, before or after the age of 20 (juvenile/adolescent or adult), with disease progression in adults usually milder than in juvenile CD patients. Immune mechanisms have been suggested to play an important role in CD pathogenesis, with cytokines governing the development of the immune response. Upregulation of inflammatory cytokines in serum of juvenile and adult CD patients has been documented; still little is known about age-dependent differences in serum cytokine profiles of CD patients. We applied multiplex technology to analyze serum levels of 12 cytokines in juveniles and adults. We show that during the acute stage of the disease all CD patients have high serum levels of CXCL10, which remains upregulated during remission. Increased serum levels of TNF-α and IL-6 during the acute stage was characteristic of juvenile CD patients, whereas adult CD patients had upregulated levels of GM-CSF and IFN-γ. Taken together, these results demonstrate age-dependent differences in cytokine profiles, which may affect the pathogenesis of CD in patients at different ages of disease onset.