Society: AGA

Background: Resolution of intestinal inflammation requires well-tuned communication between the immune compartment and the epithelium, carried out primarily via secreted mediators. The expression of these mediators is predominantly regulated by the transcription factor NF-κB. We have recently demonstrated that NF-κB can drive temporally and functionally distinct transcriptomes in the same cells, leading to divergent cell fate decisions. How these divergent transcriptomes and secretomes would affect resolution of inflammation is not known. Activation of NF-κB in inflammatory bowel diseases (IBD) directly correlates with the stage of inflammation. Although its role in leukocytes has been well characterized, its role remains controversial. Here we aimed to address the controversy by examining the contribution of epithelial NF-κB to disease progression.
Methods: We used transgenic NF-κB activity reporter mice, murine models with activated or repressed NF-κB selectively in intestinal epithelial cells, and human IBD single cell datasets to determine the contribution of epithelial NF-κB to acute inflammation and to resolution.
Results: Although NF-κB was detected in all epithelial cells, it was activated only in a subset of stem and enteroendocrine cells. Transcriptomes of these cells revealed expression of pro-apoptotic mediators. We therefore reasoned that selective suppression of NF-κB in epithelium would be protective in chemically induced colitis. To our surprise, low grade inflammation triggered by activated NF-κB in epithelium was protective in colitis, whereas suppression of NF-κB led to progressive destruction of epithelium and inability to resolve inflammation, concomitant with loss of regenerative capacity of stem cells. Current work focuses on confirmation of these NF-κB signatures in IBD patients.
Conclusion: NF-κB activation supports epithelial regeneration in colitis. The mechanism of regenerative NF-kB activity in epithelium might identify novel targets or protective mediators for IBD therapy.

Neutrophils (PMNs) are the first immune responders to infection/injury playing a critical role in clearing invading microbes and promoting tissue repair. However, dysregulated trafficking of PMNs across mucosal surfaces is a pathological hallmark of diseases characterized by persistent or intermittent bursts of inflammation including inflammatory bowel disease (IBD). The critical final step in PMN trafficking into mucosal lined organs (including the gut) involves transepithelial migration (TEpM). Recent studies have shown that terminal Fucose and GlcNAc glycans on the beta2 integrin CD11b/CD18 can be targeted to reduce PMN intestinal trafficking, highlighting the importance of glycosylation in regulating PMN inflammatory function. The most abundant terminal glycan on human and murine PMN is sialic acid (Sia) and previous studies have demonstrated increased surface sialidase activity in activated PMN. However the role of Sia in regulating PMN intestinal migration and inflammatory function is not well understood. Using human and murine models of PMN intestinal trafficking, we demonstrate that blocking sialidase- mediated removal of a2-3 linked sialic acid from CD11b/CD18 inhibits PMN TEpM in vitro and in vivo. Measurement of SOD inhibitable cytochrome C reduction revealed that sialidase inhibition reduced fMLF stimulated superoxide release by human and murine PMN. Flow cytometric analyses revealed that sialidase inhibition decreased fMLF mediated PMN degranulation responses and fMLF mediated CD11b/CD18 conformational activation. Finally, immunoblotting analyses demonstrated that de-sialylation activates spleen tyrosine kinase (Syk), a key intracellular mediator that signals downstream of CD11b/CD18 in human and murine PMN. Taken together, these data show that upon PMN activation, sialidases cleave Sia from CD11b/CD18 to facilitate conformational integrin activation and subsequent Syk dependent outside in signaling that drives PMN inflammatory effector functions including migration, degranulation, and superoxide release. Therefore, targeting CD11b/CD18 de-sialylation may represent a promising new therapeutic strategy for reducing dysregulated PMN influx and associated mucosal tissue damage in IBD and other chronic inflammatory disorders.

Introduction: Intestinal fibrosis is a serious complication of Crohn's disease (CD) and is caused by the excess deposition of extracellular matrix protein. To date, there are no therapies to prevent or treat this and surgical intervention remains the only treatment option. Numerous cell types, including intestinal epithelial cells, are implicated in this but research efforts are impaired by a lack of in vitro models.
Background: We have previously shown that human intestinal organoids (HIOs), derived from induced pluripotent stem cells (iPSCs), can be generated from patients with inflammatory bowel disease (IBD), of which CD is a major subtype. We have also demonstrated that the epithelial component of these organoids (epithelial-only HIOs (eHIOs)) can be purified and a response to the proinflammatory cytokine TNFα, and the profibrotic cytokine TGFβ, can be assayed.
Aim: To determine the role of the intestinal epithelium in intestinal fibrosis using iPSC-derived eHIOs from CD patients.
Methods: The MIRIAD Biobank in Cedars-Sinai contains >12,000 lymphoblastoid cell lines (LCLs) that were generated from IBD patients, and all lines are linked to their corresponding clinical history. LCLs from 19 CD patients (10+/9- intestinal fibrosis) were reprogrammed to iPSCs and directed to eHIOs. A pilot study was carried out in 4 lines (2+/-intestinal fibrosis) and the effects of TNFα and TGFβ were assessed.
Results: eHIOs from all 4 lines were treated with the above cytokines for 8, 24 and 48hrs. A significant increase in the expression of fibrosis-related genes (COL1A1, COL5A1, and MMP9) was observed after 24 hrs in those treated with TGFβ and TNFα/TGFβ. RNA-seq analysis was then carried out at this timepoint in untreated and TNFα/TGFβ-treated eHIOs. From a total of 15,705 genes quantified, 411 genes with an FDR<0.1% and a differential gene expression of ±2-fold change were identified. Notably, MMP9, MMP10, IL11, PDGFB, and TNF were amongst the most upregulated genes and had the highest significance value. Enrichment analysis identified the main pathways affected by the treatment were: ECM organization, focal adhesions, PDFGRB and EMT. Interestingly, a significant increase in the expression of IL11 in resected fibrotic tissue was found.
Conclusion: This study suggests that patient-derived eHIOs can be used to study the role of the intestinal epithelium in intestinal fibrosis. We have identified a number of upregulated genes/pathways that are associated with intestinal fibrosis and confirm that IL11, one of the most upregulated genes in our modeling system, is enriched in resected intestinal fibrotic tissue. RNA-seq analysis will now be carried out in all remaining patient eHIOs to determine the effects of our selected cytokines in a personalized manner and may potentially identify genes/pathways that are preferentially associated with the fibrotic phenotype.

BACKGROUND: Crohn’s disease (CD) is a multifactorial disease involving an abnormal immune response to gut microbiota in genetically-susceptible individuals, resulting in chronic inflammation. The complexity of the immunopathogenesis of CD requires an in-depth study of pathways underlying CD-associated inflammation. We recently reported an increase in MAPKAPK2 (MK2) activity within CD-inflamed intestinal tissues compared to the non-inflamed and healthy controls. An increase in MK2 activity in CD intestinal mucosa was greatly associated with mesenchymal stromal cells, known as myo-/fibroblasts (MFs), suggesting that activation of MK2 is critical to the inflammatory responses in MFs in CD. Herein we expand this study and delineated downstream pathways relevant to the pathological activation of CD-MFs and evaluated the significance of MK2 activation within MFs to the immunopathogenesis of CD in relevant murine models of colitis.
METHODS: CD-tissue-derived MFs, as well MF-specific MK2 conditional KO mice in chronic DSS colitis, were used in this study. Transcriptome and signaling pathways were analyzed by bulk RNAseq and Western blot (WB), respectively.
RESULTS: We confirmed increased activation of MK2 in primary culture of CD-MFs by using immunofluorescence microscopy and WB. Transcriptome analyses demonstrated that MFs derived from inflamed CD tissue regions have high levels of inflammatory cytokines (including CXCL1, CXCL5, TNFSF18) and downregulation of tolerogenic molecules (including IL-33, PD-L1, DLL4) when compared to normal tissue-derived MFs. Inhibition of basal and LPS-inducible MK2 signaling within these CD-MFs by using PF-3644022 (1 µM) resulted in a dramatic decrease of inflammatory responses and upregulation of cytokine and co-stimulatory ligands, including IL-33 and DLL4, that are involved in tolerogenic responses and repair processes. Using a specific siRNA approach, we confirmed these findings with MK2 inhibitor assays and also demonstrated that tristetraprolin (TTP), a direct MK2 substrate, is involved in the LPS-induced response and that the MK2-TTP pathway is engaged in the production of pro-inflammatory factors by MFs. In vivo studies demonstrated that deletion of MK2 within MFs prior to induction of chronic DSS colitis significantly reduced clinical signs of the disease, improved colonic tissue architecture, and downregulated expression of key CD-relevant inflammatory genes Tnf-α, Ccl2, Il-6, Tbx21 in colonic mucosa in mice.
CONCLUSIONS: Our data suggest that MK2-TTP is a key signaling pathway responsible for the inflammatory activation of CD-MFs. Targeting this MK2 pathway within MFs could be a desirable strategy for improving the efficacy of current therapeutic approaches for CD.

Ellen Beswick

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