Mucosa Of CD Patients Research Articles

A235 ILEAL MICROBIOME ALPHA DIVERSITY REMAINS LOWER IN ENDOSCOPICALLY AND HISTOLOGICALLY INACTIVE CROHN’S DISEASE COMPARED TO ULCERATIVE COLITIS OR HEALTHY CONTROLS

BackgroundCrohn’s disease (CD) has been associated with a lower alpha diversity when compared to ulcerative colitis (UC) patients and healthy controls (HC), which also depends on disease location and endoscopic activity. However, it is unclear whether the resolution of histologic inflammation may influence mucosal alpha diversity in the ileum.AimsTo characterize the ileal mucosa-associated microbiome diversity in subjects with ileal predominant CD (iCD) in endoscopic (ER) and endo-histologic (EHR) remission, compared to subjects with colonic predominant CD (cCD), UC and HC, respectively.MethodsData from a large cohort of subjects recruited at Mount Sinai Hospital Toronto (2009–2016) was analyzed. ER was defined as a segmental Simple Endoscopic Score for CD of <3 in the ileum. EHR was defined as ER with the absence of active histologic inflammation in ileal biopsies. CD patients were divided according to the Montreal classification into iCD (L1 and L3) and cCD (L2) and compared against UC patients without backwash ileitis and HC. Patients with history of ileocecal resection and/or antibiotic use at baseline were excluded. Microbial 16S rRNA gene was sequenced using the Illumina MiSeq and processed using QIIME2. Alpha diversity was measured using the Shannon index and compared using Kruskal-Wallis test and further pairwise Wilcoxon with Holm correction. An adjusted p-value < 0.05 was considered significant.ResultsWe included 35 CD patients with ileal ER, 81 UC patients and 32 HC. Among CD patients, 20/35 (57.1%) and 15/35 (42.9%) had iCD and cCD, respectively. Ileal mucosal alpha diversity was significantly lower in iCD patients in ER compared with that of UC patients (q=0.004) and HC (q=0.001). No differences in ileal mucosal alpha-diversity were seen between iCD than cCD patients (q=0.12). When histology was included to classify CD and UC patients as EHR, the ileal alpha diversity of iCD patients remained reduced compared to UC patients and HC (q=0.008 and q=0.002, respectively). Again, ileal mucosal alpha-diversity was not significantly lower in iCD than cCD patients among those in ileal EHR (q=0.24).ConclusionsIleal mucosa of CD patients in ER and EHR have lower alpha diversity than UC and HC. These findings suggest that the ileal mucosa of CD patients remains dysbiotic despite achieving endoscopic and histologic remission.Funding AgenciesIBD Genetics Consortium

Identification of Gene Expression Profiles Associated with an Increased Risk of Post-Operative Recurrence in Crohn's Disease.

Ileocolonic resection is frequently needed in the course of Crohn's disease [CD] treatment and post-operative recurrence is extremely common. Our main objective was to analyse gene expression in the mucosa of CD patients at the time of surgery and at post-operative endoscopy, in order to identify predictors and mechanisms of early endoscopic recurrence. We conducted transcriptome analyses on ileal mucosa samples collected from inflamed sections of the surgical specimens [n = 200], from ileal resection margins [n = 149] and in the neo-terminal ileum 6 months after surgery [n = 122]; these were compared with non-inflammatory bowel disease controls [n = 25]. The primary endpoint was post-operative endoscopic recurrence at 6 months. We applied regression models to identify gene signatures predicting endoscopic recurrence. Chronic inflammation was associated with strong expression of inflammatory genes [IL-6, IL-8, IL-1B] and decreased expression of genes involved in metabolic processes, but with a high inter-individual heterogeneity. Gene signatures associated with early endoscopic recurrence were mainly characterized by upregulation of TNFα, IFNγ, IL23A and IL17A. Pathway analyses showed that upregulation of mitochondrial dysfunction within the inflamed sections and JAK/STAT at the ileal margin were predictive of post-operative recurrence. A combined model integrating these top pathway signatures improved the prediction of endoscopic recurrence [area under the curve of 0.79]. STAT3 phosphorylation at the surgical ileal margin was associated with severe recurrence at 6 months. We identified several biological pathways in surgical ileal mucosa specimens associated with an increased risk of disease recurrence. Integration of the JAK/STAT and mitochondrial dysfunction pathways in the clinical model improved the prediction of post-operative recurrence.

P418 Filgotinib decreases molecular markers of JAK1 signal transduction in Crohn’s disease: concordance with endoscopy and histopathology

Abstract Background Filgotinib (FIL) is a JAK1 inhibitor under phase 3 clinical evaluation for treatment of IBD. We conducted a post hoc analysis in a subset of patients with moderately to severely active CD from FITZROY (NCT02048618) to assess the effect of FIL (200 mg QD) on molecular markers of JAK1-related signalling (STAT1 and STAT3 phosphorylation) within epithelium (Ep) and non-Ep regions of intestinal mucosa and explore their correlation to histologic and endoscopic indices. Methods Biopsies were collected at baseline (BL) and week 10 (W10) from the most affected area of each predefined bowel segment (ileum, ascending, transverse, descending colon, and rectum). Within-subject matched biopsies for all segments from FIL (n = 42) and placebo (PBO; n = 18) treated patients were scored for histologic (GHAS) and endoscopic (SES-CD) disease activity totalling to 300 segments. Using specific antibodies and IHC, %pSTAT1 and %pSTAT3 positive nuclei within Ep and non-Ep regions of each biopsy were quantified using machine learning (Visiopharm v.2019.06). Basal pSTAT levels assessed from 182 nondiseased (SES-CD = 0 and GHAS = 0) segments were used to determine a threshold for categorising segments as either low or high molecular disease activity (MDA). Agreement between endoscopy or histology and MDA was evaluated by κ and % agreement by segment and all segments combined. Results Median basal pSTAT1 was similar between colonic Ep and non-Ep regions (1%–2%), but higher in ileal Ep (5%). Median basal pSTAT3 was higher in non-Ep (3%–5%) vs. Ep (1%–2%) regions across all segments. At BL, MDA was elevated in segments with ulceration (~10%, pSTAT1 and pSTAT3). In segments with GHAS activity subscore ≥2 at BL, both Ep MDA (10%–30%) and non-Ep MDA were elevated (25%–35%) and correlated to histologic activity. In segments with low BL MDA, significantly fewer segments with FIL showed MDA worsening at W10 compared with PBO (both pSTAT1 and pSTAT3). In segments with high BL MDA, FIL treatment improved significantly more segments than PBO; this was evident for pSTAT3 only (table). Concordance between MDA and endoscopy was mostly fair to moderate (κ 0.3–0.5), whereas MDA and histology showed moderate to good concordance (κ 0.4–0.8). Conclusion Filgotinib treatment improved JAK1-related MDA within the mucosa of CD patients. Concordance between MDA and clinical indices was highest with histology.

Crohn's Disease Mucosal Transcriptomes Highlight JAK/STAT Pathway Involvement in Postoperative Recurrence

Background: Crohn's disease displays heterogeneity in terms of disease location, progression and response to treatment. Most patients require an intestinal resection due to complicated disease, and a majority of them will experience disease recurrence. We aimed to characterize biological pathways driving postoperative recurrence and to identify predictors which could guide therapeutic management. Methods: In this multicentre prospective study, we conducted transcriptome analysis on ileal mucosa of CD patients undergoing ileocolonic resection. Samples were collected from the most inflamed part of the ileum (n=200), from the ileal resection margin (n=149) and in the neo-terminal ileum 6 months after surgery (n=122). The primary endpoint was postoperative endoscopic recurrence at month 6. We applied a regression model to identify gene signatures predicting for endoscopic recurrence. Findings: Molecular patterns of early endoscopic recurrence significantly differed from those observed in chronic inflammation. Gene expression analysis of the inflamed area of the surgical specimens identified clusters of CD patients. However, pathway gene signatures at the ileal margin were superior to those of the inflamed area to predict postoperative outcome. Several pathways, including JAK/STAT signalling, cell adhesion molecules and extracellular matrix protein were associated with a higher risk of endoscopic recurrence. JAK/STAT pathway activation at the ileal margin predicted early postoperative endoscopic recurrence (AUC of 0*74) and was associated with a higher of clinical relapse on the long term. A significant increase of p-STAT3 levels at the ileal margin was associated with severe endoscopic recurrence (i3, i4) compared to no recurrence (i0) (p=0*02) and moderate recurrence (i1, i2) (p=0*04). Interpretation: Gene expression analysis at the ileal margin of the surgical specimen may strongly predict postoperative outcome. The inflammatory processes associated with postoperative recurrence differ from those engaged in chronic inflammation. Activation of JAK/STAT signalling at the ileal margin of the surgical specimen is predictive of postoperative recurrence and should be specifically targeted. Trial Registration: ClinicalTrials.gov (NCT03458195). Funding Statement: This work was supported by Helmsley Charitable Trust and the Institut national de sante et de la recherche biomedical (INSERM). Declaration of Interests: SN received honoraria from MSD, Abbvie, Takeda, Janssen, HAC Pharma, Tillots, Ferring, and Novartis. BP received honoraria from AbbVie, MSD, Takeda, Janssen, Bioagaran, and Ferring. AB received honoraria from MSD, Abbvie, Ferring, Takeda, Vifor Pharma, Sanofi-Aventis, Hospira, and Janssen. XT received honoraria from Abbvie, MSD, Takeda, Ferring, Norgine, and Janssen. MF received honoraria from AbbVie, MSD, Takeda, Janssen, Pfizer, Ferring, and Boehringer. PS received honoraria from Takeda, MSD, Biocodex, Ferring and Abbvie and financial support from Takeda. MA received honoraria from Janssen, Takeda, Pfizer, MSD, Abbvie, Ferring, Amgen, Biogen, Celgene, and Genentech/Roche. All other authors have no conflict of interest regarding this study. Ethics Approval Statement: All patients provided an informed written consent. The study was approved by AFFSAPS (IDRCB: 2009-A00205-52) and the French Ethic Committee (CPP 2009/17).

Acupuncture and Immunity

As a traditional therapy applied for thousands of years, acupuncture has recently been attracting more and more investigators throughout the world. In the theory of traditional Chinese medicine, it is proposed that acupuncture can strengthen the human body to resist diseases by puncturing needles at certain points. The characteristic that acupuncture enhances resistance is closely related with the immune system, which functions in defense, homeostasis, and surveillance. More and more research has revealed that acupuncture can regulate immunity, for example, to enhance anticancer and antistress immune function and exert anti-inflammation effects. This may be the basis of acupuncture in preventing and treating later diseases. This special issue was developed to stimulate the continuing efforts in promoting the research on acupuncture and immunity. The acupuncture point ST36 (Zusanli) is widely applied in immune-related diseases. In “Electroacupuncture at Bilateral Zusanli Points (ST36) Protects Intestinal Mucosal Immune Barrier in Sepsis” M. Zhu et al. reported that EA preconditioning at ST36 obviously ameliorated CLP-induced intestinal injury and high permeability and exerted protective effects on intestinal mucosal immune barrier by increasing the concentration of sIgA and the percentage of CD3+, γ/δ, and CD4+ T cells and the ratio of CD4+/CD8+ T cells, which eventually decreased the mortality of sepsis. “Moxibustion and Acupuncture Ameliorate Crohn's Disease by Regulating the Balance between Th17 and Treg Cells in the Intestinal Mucosa” by C. Zhao et al. provided evidence from a randomized controlled clinical trial that moxibustion and acupuncture regulated the ratio of Th17 and Treg cells in the intestinal mucosa of CD patients and restored the balance between these immune cell subsets, providing the basis for clinical application of treatment for CD. “Herb-Partitioned Moxibustion Regulates the TLR2/NF-κB Signaling Pathway in a Rat Model of Ulcerative Colitis” by X. Wang et al. assessed the regulation of the TLR2/NF-κB signaling pathway by herb-partitioned moxibustion in the intestinal mucosa of rats with ulcerative colitis (UC). It was reported that herb-partitioned moxibustion modulated the excessive local immune response by inhibiting TLR2 signaling, thereby promoting the repair of damaged colonic mucosa. “Mediators, Receptors, and Signalling Pathways in the Anti-Inflammatory and Antihyperalgesic Effects of Acupuncture” by J. L. McDonald et al. reviewed both demonstrated and proposed anti-inflammatory effects of acupuncture and analyzed the complex crosstalk between receptors during inflammation to elucidate the mediators and signaling pathways activated by acupuncture. Researches with new advances which demonstrated that acupuncture activated a novel cholinergic anti-inflammatory pathway and chemokine-mediated proliferation of opioid-containing macrophages in inflamed tissues were also included. “Immunoregulation on Mice of Low Immunity and Effects on Five Kinds of Human Cancer Cells of Panax japonicus Polysaccharide” by Z. Jie et al. investigated the immunoregulative effects of Panax japonicus polysaccharide (PJPS) on mice of low immunity. The results indicated that PJPS significantly improved the immune function of mice processed by cyclophosphamide and PJPS did not work on the five kinds of human cancer cells. In summary, this issue provides different evidence presented by diverse authors covering several topics related to advances in acupuncture for inflammation or immune diseases. As inflammation is the coherent pathophysiologic progress in many kinds of diseases, immune system and response of the human body are influenced in diseases such as cancer; the anti-inflammation effect of acupuncture may be a very important underlying mechanism of acupuncture in treating diseases. Moreover, TCM focuses on the theory that prevention before the onset of the diseases or intervening in an early stage of diseases is much better than treating after the onset. Preconditioning by acupuncture which means stimulation with acupuncture before the onset of diseases has been widely applied from ancient time to present clinical practice, proposing a potential field in preventing diseases. Acupoints selection, combination of the acupoints, or different methods of stimulation of acupuncture may result in quite different effects on immune system. Further research is urgently essential to elucidate the relation between acupuncture and immune responses and what kind of stimulation might induce the best effect. Of course, the selected topics and papers are not a comprehensive representation of the area of this special issue. Nonetheless, they represent many-faceted evidence that we have the pleasure of sharing with the readers.

Lymphoepithelial Interactions

Intestinal lymphoepithelial interactions occur in the epithelium and subepithelial space. We asked whether or not lamina propria lymphocytes (LPL) could promote intestinal epithelial cell (IEC) differentiation. In contrast to epithelial cells in UC mucosa, which do not differentiate because of rapid turnover, differentiation of epithelial cells in CD mucosa occurs in the crypts. This dysregulation is driven by alterations in the crosstalk between CD LPL and CD IECs, leading to an acceleration of their differentiation. This alteration seems to involve the transcription factor CDX2 via the activation of the PI3K and MAPK pathways and provides new insights into the dysfunction of the epithelial barrier in CD versus UC. The absence of lymphocytes in Rag1-deficient mice was associated with a defect in colonic IEC differentiation, restored by co-transfer of naïve and regulatory T cells. Interestingly, the transfer of naïve T cells alone induced an acceleration of IEC differentiation similar to what was seen in the colonic mucosa of CD patients. Thus, there is a crosstalk between LPL and IECs that is altered in CD, which leads to an absorptive phenotype of IEC differentiation.

HLA restriction patterns of gliadin- and astrovirus-specific CD4+ T cells isolated in parallel from the small intestine of celiac disease patients.

Celiac disease is a common HLA-DQ2-associated enteropathy caused by an abnormal T-cell-mediated immune response to ingested wheat gliadin proteins. We have previously isolated in situ activated mucosal T cells from celiac disease patients and demonstrated that these T cells were gliadin specific and predominantly DQ2 restricted. In contrast to this, gliadin-specific T cells isolated from peripheral blood display a variable HLA restriction pattern, thereby indicating that the skewed DQ restriction of T cells resident in the celiac lesions could be dictated by a preference for DQ-mediated antigen presentation in the mucosa of CD patients. To address this, we analyzed the HLA restriction of T cells recognizing astrovirus, a common gastroentetitis virus, isolated from intestinal mucosa of six celiac disease patients. As an internal control, gliadin-specific T cells were isolated and analyzed in parallel. The gliadin-specific mucosal T cells were marked in their DQ2 restriction, whereas the parallel astrovirus-specific T cells were predominantly restricted by DR molecules. Our data indicate that the repertoire of T cells present in celiac lesions is determined by the priming antigen(s) and not by a skewing in the expression of functional HLA class II isotypes in the disease affected small intestinal mucosa.