Non-inflamed Tissue Research Articles

DOP02 Single cell RNA sequencing of Ulcerative Colitis and Crohn's Disease tissue samples informs the selection of Triggering Receptors Expressed on Myeloid Cells 1 (TREM1) as a target for the treatment of Inflammatory Bowel Diseases

Abstract Background Ulcerative colitis (UC) and Crohn’s disease (CD) are complex and heterogenous diseases characterized by chronic, progressive inflammation of the digestive tract. Single cell RNA sequencing combined with computational biology provides unprecedented insights into disease biology, informing novel therapeutic targets and precision medicine approaches. To identify potential mediators of persistent inflammation in inflammatory bowel disease (IBD), we performed single cell RNA sequencing on tissue samples collected from UC and CD patients before and after beginning adalimumab. Methods Forty-one participants, including 21 UC patients, 17 CD patients and 3 healthy individuals, were enrolled at the University of Oxford’s Translational Gastroenterology Unit and Kennedy Institute of Rheumatology. Patients had moderate-to-severe disease based on symptoms and endoscopy, were biologic naïve, and planned to initiate adalimumab. Mucosal biopsies for single cell RNA sequencing were collected for each subject before beginning and after a minimum of 3 months of adalimumab, at which time treatment response was assessed. During the study, clinical response as defined by composite measures was observed in 8 UC and 9 CD patients. Results Differential cell composition and gene expression analysis of single cell data revealed inflammatory monocyte subtypes as highly enriched in inflamed relative to uninflamed samples. This cellular subset remained highly elevated in inflamed samples from adalimumab non-responders. These cells were characterized by expression of multiple cell-surface receptors with potential to drive pathology, including TREM1 (Triggering Receptor Expressed on Myeloid Cells 1), a transmembrane receptor that amplifies anti-bacterial neutrophil and monocyte mediated responses. TREM1+ inflammatory monocytes were highly enriched in UC and CD inflamed samples (Fig. 1), both prior to adalimumab and in adalimumab non-responders. The enrichment of TREM1+ cells was specific to inflamed IBD tissue and was not observed in non-inflamed tissue from IBD patients or healthy subjects; enrichment of TREM1+ cells was also not observed in circulating peripheral blood mononuclear cells. Conclusion TREM1+ neutrophils and monocytes in the gut mucosa amplify innate immune responses to bacteria in the context of epithelial barrier dysfunction and bacterial dysbiosis in IBD. When activated, TREM1 signaling induces multiple soluble mediators relevant for IBD pathology. Our single cell RNA sequencing data provide further evidence that TREM1 is an exciting therapeutic target for UC and CD. We therefore developed a highly potent and selective monoclonal antibody inhibitor of TREM-1, CEL383, with the Phase 1 clinical trial expected to start in 2023.

P042 Distinct gene expression profile associated to inflammation and neoplastic progression in longstanding ulcerative colitis

Abstract Background One of the main drivers of colitis associated colorectal cancer (CA-CRC) is suspected to be long-standing mucosal inflammation, but far from all colitis patients develop cancer. We analyzed mucosal gene expression profiles in ulcerative colitis (UC) patients who have progressed to neoplasia (progressors) compared to those who have not (non-progressors) in order to identify underlying mechanisms for neoplastic progression in UC patients. Methods We performed transcriptome profiling on 143 mucosal samples from non-neoplastic colon segments of 14 UC progressors and 30 UC non-progressors from the prospective CA-CRC cohorts from Lovisenberg Diakonale- and Akershus University Hospitals in Norway. Differentially expressed genes were determined using SurePrint G3 human gene expression 60K microarray (Agilent Tech Inc.). Results 609 genes were differentially expressed between non-progressor- and progressor inflamed tissue, 24 genes between non-progressor- and progressor non-inflamed tissue, 404 genes between non-progressor non-inflamed- and -inflamed tissue and 26 genes between progressor non-inflamed- and inflamed tissue. In inflamed mucosa, genes related to metabolic processes such as xenobiotic- as well as carbohydrate and lipid metabolism were significantly higher expressed in progressors compared to non-progressors. Genes related to innate and adaptive immune responses were expressed at significantly lower levels. These main traits were not influenced by type of inflammation but apparent in both, chronic- and chronic active inflammation. However, chronic inflamed mucosa of progressors were also characterised by dysregulation of processes related to chromatin organization. Treatment with immunomodulators and biologics were equally distributed in the groups. Restriction of the analysis to patients without treatment of these drugs did not significantly influence the main results. In non-inflamed mucosa, the gene expression profile of progressors and non-progressors were remarkably similar. In fact, within the positively and negatively enriched gene ontologies, only the gene S100P was significantly deregulated with a higher expression in non-inflamed mucosa of the progressors. Conclusion Upregulated detoxification as well as dysregulated immune response and chromatin organization may be underlying processes for neoplastic progression in ulcerative colitis. Our findings warrant further evaluation.

SINGLE CELL RNA SEQUENCING OF ULCERATIVE COLITIS AND CROHN'S DISEASE TISSUE SAMPLES INFORMS THE SELECTION OF TREM1 AS A TARGET FOR THE TREATMENT OF INFLAMMATORY BOWEL DISEASES

Abstract Ulcerative colitis (UC) and Crohn’s disease (CD) are complex and heterogenous diseases characterized by chronic and progressive inflammation of the digestive tract. Single cell RNA sequencing combined with computational biology provides unprecedented insights into disease biology, informing novel therapeutic targets and precision medicine approaches. To identify potential mediators of persistent inflammation in inflammatory bowel disease (IBD), we performed single cell RNA sequencing on tissue samples collected from UC and CD patients before and after beginning adalimumab. In this study, 41 participants, including 21 UC patients, 17 CD patients and 3 healthy individuals, were enrolled at the University of Oxford’s Translational Gastroenterology Unit and Kennedy Institute of Rheumatology. Patients had moderate-to-severe disease based on symptoms and endoscopy, were biologic naïve, and planned to initiate adalimumab. Mucosal biopsies for single cell RNA sequencing were collected from multiple anatomic locations for each subject before beginning and after a minimum of 3 months of adalimumab, at which time treatment response was assessed. During the study, clinical response as defined by composite measures was observed in 8 UC and 9 CD patients. Differential cell composition and gene expression analysis of the single cell data revealed inflammatory monocyte subtypes as highly enriched in inflamed relative to uninflamed samples, and this cellular subset remained highly elevated in inflamed samples from adalimumab non-responders. These cells were characterized by expression of multiple cell-surface receptors with potential to drive pathology, including TREM1 (Triggering Receptor Expressed on Myeloid Cells 1), a transmembrane receptor that amplifies anti-bacterial neutrophil and monocyte mediated responses. TREM1+ inflammatory monocytes were highly enriched in UC and CD inflamed samples (Fig 1), both prior to adalimumab and in adalimumab non-responders. The enrichment of TREM1+ cells was specific to inflamed IBD tissue and was not observed in non-inflamed tissue from IBD patients or healthy subjects; enrichment of TREM1+ cells was also not observed in circulating peripheral blood mononuclear cells. TREM1+ neutrophils and monocytes in the gut mucosa amplify innate immune responses to bacteria in the context of epithelial barrier dysfunction and bacterial dysbiosis in IBD. When activated, TREM1 signaling induces multiple soluble mediators relevant for IBD pathology. Our single cell RNA sequencing data provide further evidence that TREM1 is an exciting therapeutic target for UC and CD, with inhibition of TREM1 signaling expected to address pathology while maintaining an anti-bacterial response. We therefore developed a highly potent and selective monoclonal antibody inhibitor of TREM-1, CEL383, with the Phase 1 clinical trial expected to start in 2023. Figure 1. tSNE plot of myeloid cells from inflamed IBD tissue samples demonstrating high TREM1 expression across inflammatory monocytes

Inflammation and vascular remodeling in COVID-19 hearts

A wide range of cardiac symptoms have been observed in COVID-19 patients, often significantly influencing the clinical outcome. While the pathophysiology of pulmonary COVID-19 manifestation has been substantially unraveled, the underlying pathomechanisms of cardiac involvement in COVID-19 are largely unknown. In this multicentre study, we performed a comprehensive analysis of heart samples from 24 autopsies with confirmed SARS-CoV-2 infection and compared them to samples of age-matched Influenza H1N1 A (n = 16), lymphocytic non-influenza myocarditis cases (n = 8), and non-inflamed heart tissue (n = 9). We employed conventional histopathology, multiplexed immunohistochemistry (MPX), microvascular corrosion casting, scanning electron microscopy, X-ray phase-contrast tomography using synchrotron radiation, and direct multiplexed measurements of gene expression, to assess morphological and molecular changes holistically. Based on histopathology, none of the COVID-19 samples fulfilled the established diagnostic criteria of viral myocarditis. However, quantification via MPX showed a significant increase in perivascular CD11b/TIE2 + —macrophages in COVID-19 over time, which was not observed in influenza or non-SARS-CoV-2 viral myocarditis patients. Ultrastructurally, a significant increase in intussusceptive angiogenesis as well as multifocal thrombi, inapparent in conventional morphological analysis, could be demonstrated. In line with this, on a molecular level, COVID-19 hearts displayed a distinct expression pattern of genes primarily coding for factors involved in angiogenesis and epithelial-mesenchymal transition (EMT), changes not seen in any of the other patient groups. We conclude that cardiac involvement in COVID-19 is an angiocentric macrophage-driven inflammatory process, distinct from classical anti-viral inflammatory responses, and substantially underappreciated by conventional histopathologic analysis. For the first time, we have observed intussusceptive angiogenesis in cardiac tissue, which we previously identified as the linchpin of vascular remodeling in COVID-19 pneumonia, as a pathognomic sign in affected hearts. Moreover, we identified CD11b + /TIE2 + macrophages as the drivers of intussusceptive angiogenesis and set forward a putative model for the molecular regulation of vascular alterations.

A Novel B7-H6-Targeted IgG-Like T Cell-Engaging Antibody for the Treatment of Gastrointestinal Tumors.

Advanced-stage gastrointestinal cancers represent a high unmet need requiring new effective therapies. We investigated the antitumor activity of a novel T cell-engaging antibody (B7-H6/CD3 ITE) targeting B7-H6, a tumor-associated antigen that is expressed in gastrointestinal tumors. Membrane proteomics and IHC analysis identified B7-H6 as a tumor-associated antigen in gastrointestinal tumor tissues with no to very little expression in normal tissues. The antitumor activity and mode of action of B7-H6/CD3 ITE was evaluated in in vitro coculture assays, in humanized mouse tumor models, and in colorectal cancer precision cut tumor slice cultures. B7-H6 expression was detected in 98% of colorectal cancer, 77% of gastric cancer, and 63% of pancreatic cancer tissue samples. B7-H6/CD3 ITE-mediated redirection of T cells toward B7-H6-positive tumor cells resulted in B7-H6-dependent lysis of tumor cells, activation and proliferation of T cells, and cytokine secretion in in vitro coculture assays, and infiltration of T cells into tumor tissues associated with tumor regression in in vivo colorectal cancer models. In primary patient-derived colorectal cancer precision-cut tumor slice cultures, treatment with B7-H6/CD3 ITE elicited cytokine secretion by endogenous tumor-infiltrating immune cells. Combination with anti-PD-1 further enhanced the activity of the B7-H6/CD3 ITE. These data highlight the potential of the B7-H6/CD3 ITE to induce T cell-redirected lysis of tumor cells and recruitment of T cells into noninflamed tumor tissues, leading to antitumor activity in in vitro, in vivo, and human tumor slice cultures, which supports further evaluation in a clinical study.

Effect of Crohn's disease mesenteric mesenchymal stem cells and their extracellular vesicles on T-cell immunosuppressive capacity.

Crohn's disease (CD) is a chronic inflammatory disease of the gastrointestinal intestinal tract and has characteristic hypertrophic adipose changes observed in the mesentery. To better understand the role of the mesentery in the pathophysiology of Crohn's disease (CD), we evaluated the immunomodulatory potential of mesenchymal stem cells (MSCs) and their secreted extracellular vesicles (EVs) derived from Crohn's patients. MSCs and EVs were isolated from the mesentery and subcutaneous tissues of CD patients and healthy individuals subcutaneous tissues, and were analysed for differentiation, cytokine expression, self‐renewal and proliferation. The varying capacity of these tissue‐derived MSCs and EVs to attenuate T‐cell activation was measured in in vitro and an in vivo murine model. RNA sequencing of inflamed Crohn's disease mesentery tissue revealed an enrichment of T‐cell activation compared to non‐inflamed subcutaneous tissue. MSCs and MSC‐derived EVs isolated from Crohn's mesentery lose their ability to attenuate DSS‐induced colitis compared to subcutaneous tissue‐derived cell or EV therapy. We found that treatment with subcutaneous isolated MSCs and their EV product compared to Crohn's mesentery MSCs or EVs, the inhibition of T‐cell proliferation and IFN‐γ, IL‐17a production increased, suggesting a non‐inflamed microenvironment allows for T‐cell inhibition by MSCs/EVs. Our results demonstrate that Crohn's patient‐derived diseased mesentery tissue MSCs lose their immunosuppressive capacity in the treatment of colitis by distinct regulation of pathogenic T‐cell responses and/or T‐cell infiltration into the colon.

Crohn's disease recurrence updates: first surgery vs. surgical relapse patients display different profiles of ileal microbiota and systemic microbial-associated inflammatory factors.

Background and aimsCrohn’s disease (CD) pathogenesis is still unclear. Remodeling in mucosal microbiota and systemic immunoregulation may represent an important component in tissue injury. Here, we aim to characterize the ileal microbiota in both pathological and healthy settings and to evaluate the correlated systemic microbial-associated inflammatory markers comparing first-time surgery and relapse clinical conditions.MethodsWe enrolled 28 CD patients at surgery; we collected inflamed and non-inflamed mucosa tissues and blood samples from each patient. Bacterial wall adherence was observed histologically, while its composition was assessed through amplicon sequencing of the 16S rRNA gene. In addition, we evaluated the systemic microRNA (miRNA) using quantitative real-time PCR amplification and free fatty acids (FFAs) using gas chromatography–mass spectroscopy.ResultsThe total number of mucosal adherent microbiota was enriched in healthy compared to inflamed mucosa. In contrast, the phylum Tenericutes, the family Ruminococcaceae, and the genera Mesoplasma and Mycoplasma were significantly enriched in the pathological setting. Significant microbiota differences were observed between the relapse and first surgery patients regarding the families Bacillaceae 2 and Brucellaceae and the genera Escherichia/Shigella, Finegoldia, Antrobacter, Gemmatimonas, Moraxella, Anoxibacillus, and Proteus. At the systemic level, we observed a significant downregulation of circulating miR-155 and miR-223, as well as 2-methyl butyric, isobutyric, and hexanoic (caproic) acids in recurrence compared to the first surgery patients. In addition, the level of hexanoic acid seems to act as a predictor of recurrence risk in CD patients (OR 18; 95% confidence interval 1.24–261.81; p = 0.006).ConclusionsWe describe a dissimilarity of ileal microbiota composition comparing CD and healthy settings, as well as systemic microbial-associated inflammatory factors between first surgery and surgical relapse. We suggest that patterns of microbiota, associated with healthy ileal tissue, could be involved in triggering CD recurrence. Our findings may provide insight into the dynamics of the gut microbiota–immunity axis in CD surgical recurrence, paving the way for new diagnostics and therapeutics aimed not only at reducing inflammation but also at maintaining a general state of eubiosis in healthy tissue.

Quantitative Assessment of the Impact of Crohn's Disease on Protein Abundance of Human Intestinal Drug-Metabolising Enzymes and Transporters

Crohn's disease affects the mucosal layer of the intestine, predominantly ileum and colon segments, with the potential to affect the expression of intestinal enzymes and transporters, and consequently, oral drug bioavailability. We carried out a quantitative proteomic analysis of inflamed and non-inflamed ileum and colon tissues from Crohn's disease patients and healthy donors. Homogenates from samples in each group were pooled and protein abundance determined by liquid chromatography–mass spectrometry (LC-MS). In inflamed Crohn's ileum, CYP3A4, CYP20A1, CYP51A1, ADH1B, ALPI, FOM1, SULT1A2, SULT1B1 and ABCB7 showed ≥10-fold reduction in abundance compared with healthy baseline. By contrast, only MGST1 showed ≥10 fold reduction in inflamed colon. Ileal UGT1A1, MGST1, MGST2, and MAOA levels increased by ≥2 fold in Crohn's patients, while only ALPI showed ≥2 fold increase in the colon. Counter-intuitively, non-inflamed ileum had a higher magnitude of fold change than inflamed tissue when compared with healthy tissue. Marked but non-uniform alterations were observed in the expression of various enzymes and transporters in ileum and colon compared with healthy samples. Modelling will allow improved understanding of the variable effects of Crohn's disease on bioavailability of orally administered drugs.

Mucosal Eosinophil Abundance in Non-Inflamed Colonic Tissue Is Associated with Response to Vedolizumab Induction Therapy in Inflammatory Bowel Disease.

Vedolizumab is used as a treatment for patients with inflammatory bowel disease (IBD), but induction therapy leads to clinical response and remission in approximately 55% and 30% of patients with IBD, respectively. In this study, we aimed to explore the predictive value of mucosal eosinophils and serum eotaxin-1 regarding response to vedolizumab induction therapy. Eighty-four (84) patients with IBD (37 Crohn’s disease [CD], 47 ulcerative colitis [UC]) were included. For 24 patients with IBD, histopathology was assessed for eosinophil counts in non-inflamed colonic tissue prior to vedolizumab treatment. For 64 patients with IBD, serum eotaxin-1 levels were quantified prior to (baseline) and during vedolizumab treatment. Serum samples of 100 patients with IBD (34 CD, 66 UC) from the GEMINI 1 and 2 trials were used for external validation. Baseline mucosal eosinophil numbers in non-inflamed colonic tissue were significantly higher in responders to vedolizumab induction therapy when compared to primary non-responders (69 [34–138] vs. 24 [18–28] eosinophils/high-power field, respectively, p < 0.01). Baseline serum eotaxin-1 levels in the discovery cohort were significantly elevated in responders, compared to primary non-responders (0.33 [0.23–0.44] vs. 0.20 [0.16–0.29] ng/mL, p < 0.01). Prediction models based on mucosal eosinophil counts and serum eotaxin-1 showed an area under the curve (AUC) of 0.90 and 0.79, respectively. However, the predictive capacity of baseline serum eotaxin-1 levels could not be validated in the GEMINI cohort. Mucosal eosinophil abundance in non-inflamed colonic tissue was associated with response to vedolizumab induction therapy in patients with IBD. Future studies are warranted to further validate the potential value of mucosal eosinophils and serum eotaxin-1 as biomarkers for response to vedolizumab therapy.

Paroxetine increases delta opioid responsiveness in sensory neurons.

There are currently no Food and Drug Administration (FDA)-approved δ-opioid receptor (DOR)-selective agonists, despite having fewer side effects in rodents and nonhuman primates compared with traditional μ-opioid receptor (MOR) therapeutics (Vanderah, 2010). Targeting peripheral receptors is an attractive strategy to reduce abuse potential. However, peripheral opioid receptors do not readily respond to agonists unless primed by inflammation, which would limit their efficacy in noninflammatory pain patients (Stein et al., 1989). It was recently identified that G-protein-coupled receptor kinase 2 (GRK2) maintains DOR incompetence in noninflamed nociceptors (Brackley et al., 2016, 2017). Here, we report that paroxetine, a selective serotonin reuptake inhibitor (SSRI) and potent GRK2 inhibitor (Thal et al., 2012), reduces chronic GRK2 association with membrane DOR, thereby enhancing peripheral DOR-mediated analgesic competence in the absence of inflammation. Interestingly, paroxetine’s effects on GRK2 in vivo are limited to peripheral tissues in the male rat. The effects of paroxetine on DOR competence are notably antagonized by GRK2 overexpression. This is the first study to suggest that paroxetine induces peripheral DOR analgesic competence through a GRK2-dependent mechanism, improving analgesic efficacy in noninflamed tissue. Because paroxetine targets the protein that governs peripheral opioid receptor responsiveness, and does so in the absence of inflammation, we propose that paroxetine may be suitable as a co-therapy with peripherally-restrictive doses of opioids to improve analgesic efficacy in noninflammatory pain conditions.

Role of NOD2 and hepcidin in inflammatory periapical periodontitis

The immunological response occurring during periapical inflammation includes expression of nucleotide binding oligomerization domain containing 2 and hepcidin. Nucleotide binding oligomerization domain containing 2 deficiency increases infiltration of inflammatory cells close to alveolar bone. Hepcidin has an important role in iron metabolism affecting bone metabolism.We investigated the role of nucleotide binding oligomerization domain containing 2 and hepcidin in inflammatory periapical periodontitis. Periapical periodontitis was induced in rats and confirmed by micro-computed tomography. Nucleotide binding oligomerization domain 2 and hepcidin were evaluated through immunohistochemistry. Bioinformatics analysis was undertaken usingthe Kyoto Encyclopedia of Genes and Genomes and Gene Ontology databases. Micro-computer tomography revealed alveolar bone resorption in the periapical region and furcation area of mandibular molars in rats of the periapical periodontitis group. Immunohistochemistry showed increased expressionof nucleotide binding oligomerization domain containing 2 and hepcidin around root apices in rats of the periapical periodontitis group. Bioinformatics analysis of differentially expressed genes in inflamed and non-inflamed tissues revealed enrichment in the NOD-like receptor signaling pathway. Our data suggest that nucleotide binding oligomization domain contain2 and hepcidin have important roles in periapical periodontitis severity because they can reduce alveolar bone loss.They could elicit new perspectives for development of novel strategies for periapical periodontitis treatment.

PRDX6 alleviates lipopolysaccharide-induced inflammation and ferroptosis in periodontitis

Objective Periodontitis is a progressive and inflammatory oral disease and results in the damage of the supporting tissues of teeth. Peroxiredoxin 6 (PRDX6) is an antioxidant enzyme identified as a regulator in ferroptosis. This study aimed to investigate whether PRDX6 could protect human gingival fibroblasts (HGFs) from lipopolysaccharide (LPS)-induced inflammation and its mechanisms. Material and methods Both inflamed and non-inflamed human gingival tissues were collected to assess the expression of PRDX6 and nuclear factor erythropoietin 2-related factor 2 (NRF2) by Immunohistochemistry and Western blotting. Furthermore, the molecular mechanisms of PRDX6 have been clarified in PRDX6 silenced cells. The inflammatory cytokines in HGFs were measured by RT-qPCR and ELISA. The lipid hydroperoxide (LOOH) was detected by C11-BODIPY. Results The expression of PRDX6 and NRF2 were decreased in gingival tissues of severe periodontitis patients. The increased LPS-induced LOOH and inflammatory cytokines were found in PRDX6 knockdown HGFs. Besides, the inhibition of ferroptosis or PRDX6 phospholipase A2 activity (PLA2) alleviated LPS-induced inflammatory cytokines and LOOH. However, inhibiting NRF2 signalling upregulated those in HGFs. Conclusions Therefore, this study provided a new mechanistic insight that PRDX6, regulated by the NRF2 signalling, alleviates LPS-induced inflammation and ferroptosis in human gingival fibroblasts.

HIF1α-Dependent Induction of TFRC by a Combination of Intestinal Inflammation and Systemic Iron Deficiency in Inflammatory Bowel Disease.

Background and Aims: Iron deficiency (ID) is a frequent extra-intestinal manifestation in patients with Inflammatory Bowel Disease (IBD), who often do not respond to iron supplementation. Iron is a cofactor for hydroxylases that suppress the hypoxia-inducible factor-1α (HIF1α), a transcription factor regulating iron homeostasis. We hypothesized that iron deficiency affects mucosal HIF1α activity in IBD. Methods: IBD patients (n = 101) were subdivided based on iron status (ferritin levels or transferrin saturation) and systemic inflammation (C-reactive protein levels). 154 corresponding ileal and colonic biopsies were analyzed for differential expression of 20 HIF1α pathway-associated genes and related to iron and inflammation status. In vitro expression of selected HIF1α pathway genes were analyzed in wild-type and HIF1A-null Caco-2 cells. Results: Gene expression of the mucosal HIF1α pathway was most affected by intestinal location and inflammatory status. Especially, ileal mucosal TFRC expression, encoding the transferrin receptor TFR1, was increased in inflamed tissue (p < 0.001), and further enhanced in ID. Accordingly, TFRC expression in inflamed tissue associated negatively with serum iron levels, which was not observed in the non-inflamed mucosa. The HIF1α pathway agonist DMOG increased TFRC expression in Caco-2 cells, which was blunted in HIF1A-null cells. Conclusion: We demonstrate that inflammation and anatomical location primarily determine HIF1α pathway activation and downstream TFRC expression in the intestinal mucosa. IBD patients with ID may benefit from treatment with HIF1α-agonists by 1) increasing TFRC-mediated iron absorption in non-inflamed tissue and 2) decreasing mucosal inflammation, thereby improving their responsiveness to oral iron supplementation.

IBD-associated G protein-coupled receptor 65 variant compromises signalling and impairs key functions involved in inflammation

Background and aimsInflammatory bowel diseases (IBD) result in chronic inflammation of the gastrointestinal tract. Genetic studies have shown that the GPR65 gene, as well as its missense coding variant, GPR65*Ile231Leu, is associated with IBD. We aimed to define the signalling and biological pathways downstream of GPR65 activation and evaluate the impact of GPR65*231Leu on these. MethodsWe used HEK 293 cells stably expressing GPR65 and deficient for either Gαs, Gαq/11 or Gα12/13, to define GPR65 signalling pathways, IBD patient biopsies and a panel of human tissues, primary immune cells and cell lines to determine biologic context, and genetic modulation of human THP-1-derived macrophages to examine the impact of GPR65 in bacterial phagocytosis and NLRP3 inflammasome activation. ResultsWe confirmed that GPR65 signals via the Gαs pathway, leading to cAMP accumulation. GPR65 can also signal via the Gα12/13 pathway leading to formation of stress fibers, actin remodeling and RhoA activation; all impaired by the IBD-associated GPR65*231Leu allele. Gene expression profiling revealed greater expression of GPR65 in biopsies from inflamed compared to non-inflamed tissues from IBD patients or control individuals, potentially explained by infiltration of inflammatory immune cells. Decreased GPR65 expression in THP-1-derived macrophages leads to impaired bacterial phagocytosis, increased NLRP3 inflammasome activation and IL-1β secretion in response to an inflammatory stimulus. ConclusionsWe demonstrate that GPR65 exerts its effects through Gαs- and Gα12/13-mediated pathways, that the IBD-associated GPR65*231Leu allele has compromised interactions with Gα12/13 and that KD of GPR65 leads to impaired bacterial phagocytosis and increased inflammatory signalling via the NLRP3 inflammasome. This work identifies a target for development of small molecule therapies.

INCREASE IN MK2 ACTIVITY IN CROHN’S DISEASE: ROLE IN THE INFLAMMATORY ACTIVATION OF FIBROBLASTS

Abstract INTRODUCTION AND HYPOTHESIS Mesenchymal cells known as myo-/fibroblasts (MFs) are critical immunosuppressors under gut mucosal homeostasis. Expression of immune checkpoint PD-L1 by MFs plays a key role in the control of T cell inflammatory responses. In Crohn’s disease (CD), MFs switch their activity from immunosuppressive to pro-inflammatory, where they are also known as Inflammatory Fibroblasts. However, the mechanisms responsible for these pathological changes in MF activity are unknown. Map-kinase-activated protein kinase 2 (MK2) is a major regulator of inflammation in the gut. MK2 is downstream of p38 signaling, it evokes a sub-pathway that directly regulates the production of key inflammatory cytokines implicated in CD (such as TNF-α, IL-1, and IL-6). Thus, we hypothesized that activation of MK2 signaling is critical to the pathological changes in MFs during the immunopathogenesis of CD. METHODS Human normal and CD tissues and derived MFs, as well as animal models relevant to CD, were used in this study. MF signaling/activity was analyzed using RNAseq, qRT-PCR, western blot (WB), cytokine/chemokine multiplex arrays, and confocal microscopy. RESULTS In situ analysis demonstrated an increase in MK2 activity within the inflamed compared to the non-inflamed CD and healthy control intestinal tissues, which was confirmed by WB and multiplex signaling array analysis. In situ increase in MK2 activity in CD intestinal mucosa was greatly associated with mesenchymal stromal cells that bear a “myofibroblast” phenotype (positive for α-SMA expression). An increase in MK2 activity was also observed in primary MF cultures isolated from CD (CD-MFs) when compared to normal (N-) MFs. MK2 activity within CD-MFs was also associated with a significant decrease in the expression of the immunosuppressive checkpoint PD-L1 and an increase in the expression of inflammatory CCL2 and IL-6. Inhibition of MK2 activity within CD-MFs through using the MK2-specific inhibitor PF-3644022 (10 μM) reversed the inflammatory activity of MFs. Remarkably, we observed a differential role of p38 and MK2 in the regulation of PD-L1 expression in MFs: while p38 was required for basal expression of PD-L1, activation of MK2 downregulates PD-L1 expression. These data indicate a unique role of MK2 activation in pathological reprogramming of MFs in CD. Use of MK2 inhibitor in a therapeutic modality in chronic DSS and IL-10 KO murine models of CD also significantly reduce MF-linked inflammatory responses in vivo. CONCLUSION Our data suggest that an increase in MK2 activity in CD is critical to the reprogramming of the MF from immunosuppressive toward pathological Inflammatory fibroblasts. Targeting MK2 activity within MFs could be a desirable strategy for improving the efficacy of current IBD therapeutic approaches.

P090 Unraveling the rewiring of cytokine signalling in Inflammatory Bowel Disease using a novel network biology approach

Abstract Background Intercellular communication mediated by cytokines is critical to the development of immune responses, particularly in the context of chronic immune-mediated disorders such as inflammatory bowel disease (IBD). By releasing these small molecular weight peptides, the source cells can influence numerous intracellular processes in the target cells, including the secretion of other cytokines downstream. However, there are no readily available resources and studies modelling cytokine-cytokine interactions that would be important for better understanding the dysregulation of cytokine signalling in IBD, and revealing potential new therapeutic targets for patients. Methods Based on the workflow of a cytokine signalling resource, CytokineLink, we developed previously, in this effort we built novel interaction networks using single-cell expression data from patients with ulcerative colitis (UC) and Crohn’s disease (CD). We defined two meta-networks for each disease: a cell–cell communication network connected by cytokines; and a cytokine–cytokine interaction network depicting the potential ways in which cytokines can affect the activity of each other. Results The rewiring of cell–cell and cytokine–cytokine interaction networks between healthy, non-inflamed and inflamed intestinal tissues in IBD was measured using the generated meta-networks. Cytokine-cytokine interaction networks revealed that in active UC, the Th1 cytokine IFNg and the chemokines CCL28 and CCL14 were central to cytokine-mediated communication networks. In CD, IL12B and IL13 formed the central cytokines in inflamed ileal tissues. Cell–cell interaction networks identified likely main cell types mediating the signalling of these key cytokines in non-inflamed and inflamed intestinal tissues: mast cells being the main source of IL13 in both IBD patients and healthy individuals. Conclusion We generated interaction networks aimed at capturing the most prevalent cell–cell and cytokine–cytokine interactions using disease specific single-cell expression data. Using the generated interaction networks we identified the condition-specific rewiring of cytokine mediated signalling in IBD, and key cytokines that could be targeted for ameliorating inflammatory responses. The data can be used by the community as a platform for hypothesis-generation. Potential use cases are deconvoluting the complexities of cytokine signalling in IBD to reveal potential cytokine drug targets, or predicting the downstream effects of drugs on cytokine responses.

P577 Mucosal eosinophil abundance in non-inflamed colonic tissue predict response to vedolizumab induction therapy in inflammatory bowel disease

Abstract Background Vedolizumab has shown efficacy, safety and tolerability as treatment for patients with inflammatory bowel disease (IBD). However, vedolizumab induction therapy only shows clinical response and remission in roughly 55% and 30% of IBD patients, respectively. Vedolizumab binds and blocks migration of T-lymphocytes and eosinophils. In this study, we aimed to explore the predictive value of mucosal eosinophils and serum eotaxin-1, an eosinophil chemoattractant, regarding response to vedolizumab induction therapy. Methods 84 IBD patients treated within the University Medical Center Groningen (UMCG) (37 Crohn’s disease [CD], 47 ulcerative colitis [UC]) were included. In a subset of 24 IBD patients (9 CD, 15 UC) histopathological data were analyzed for eosinophil counts in high power fields (hpf) in non-inflamed colon ascendens tissue prior to vedolizumab treatment. In another subset of 64 IBD patients, (28 CD, 36 UC) baseline serum eotaxin-1 was quantified prior to vedolizumab treatment. Clinical response or remission was defined as a decrease of the Harvey Bradshaw Index (HBI) for CD or Simple Clinical Colitis Activity Index (SCCAI) for UC together with physician’s global assessment (PGA). Serum eotaxin-1 was externally assessed as a biomarker for response to vedolizumab induction therapy in 100 IBD patients derived from the GEMINI 1 &amp; 2 trials. Results Baseline eosinophil mucosal count was significantly higher in vedolizumab induction therapy responders, compared to primary non responders (69[34–138] vs. 24[18–28] eosinophils/hpf respectively, P&amp;lt;0.01). Baseline serum eotaxin-1 levels in the UMCG cohort were significantly elevated in therapy responders, compared to primary non-responders (0.33 vs. 0.20 ng/mL, P&amp;lt;0.01). The final prediction model based on mucosal eosinophil count showed an area under the curve (AUC) of 0.90 and serum eotaxin-1 an adjusted AUC of 0.79 The optimal with balanced cut-off value for eosinophil count was &amp;gt; 30 eosinophils/hpf with a sensitivity of 90.9% and specificity of 92.3% (Youden’s index 0.83). Results derived from the GEMINI I &amp; II cohorts did not show any associations between eotaxin-1 levels and therapy response. Conclusion Mucosal eosinophil abundance in non-inflamed colon ascendens biopsies can predict vedolizumab induction therapy response in IBD patients. More studies are warranted to confirm these preliminary results and further investigate the additional value of eotaxin-1 regarding predicting vedolizumab therapy response.

P067 Human intestinal CD103+SIRPα+ conventional dendritic cells from patients with Crohn′s disease prime IL-17+ T-cells

Abstract Background Crohn’s disease (CD) is a chronic inflammation of the human gastrointestinal tract. Intestinal conventional dendritic cells (cDC) maintain the balance between immunity against pathogens and tolerance towards nutrients and commensals. However, there is not much information about DC composition, phenotype and function in the human gut in CD. Methods Human intestinal resections were obtained from patients with ileocolonic CD and proximal colonic cancer. Both inflamed and non-inflamed tissue was obtained from the ileum and colon from CD patients. The non-affected tissue from the proximal (terminal ileum) and distal (proximal colon) sides of the colonic cancer resections (minimum 10cm distance from the tumor) constituted the reference tissue. Tissue was disaggregated and cDC and macrophages enriched by flow-sorting. Human intestinal cDC were identified within singlet viable leukocytes as CD14-CD64-HLA-DR+CD11c+. Type 1 cDC (cDC1) were defined as CD103+SIRPα- while type 2 cDC (cDC2) and identified as SIRPα+ and further divided into subsets based on the expression of CD103. Macrophages were identified within singlet viable leukocytes as CD14+CD64+HLA-DR+. All three cDC subsets and total macrophages were used to stimulate allogeneic CD4+ naïve T-cells which were further characterized by flow cytometry. Results All 3 human intestinal cDC subsets from non-CD controls were able to stimulate naïve T-cells as opposed to intestinal macrophages. Indeed, stimulated T-cells produced IL-10 and co-expressed FoxP3, being this capacity increased in both CD103+ subsets (CD103+SIRPα- and CD103+SIRPα+). Moreover, all three ileal cDC subsets stimulated T-cells more efficiently than their paired colonic counterparts, although the acquired T-cell profile did not differ among tissues. None of the cDC subets, on the contrary, induced the expression of IFNγ or IL-17 on stimulated T-cells. Referred to CD patients, all three colonic cDC subsets were more stimulatory than their counterparts from the control tissue, while CD103+SIRPα+ cDC from the inflamed ileum primed the generation of IL-17+ responding T-cells. Conclusion All human intestinal cDC subsets prime the generation of regulatory FoxP3+ and IL-10+ T-cells. This capacity was increased in both CD103+SIRPα- and CD103+SIRPα+, in agreement with the regulatory functions attributed to CD103+ cDC. All ileal cDC subsets were also more stimulatory than their colonic counterparts. Nevertheless, colonic cDC from CD patients were more stimulatory that non-CD controls, rendering CD103+SIRPα+ cDC from the inflamed tissue in CD with a higher capacity to generate Th17 cells, which suggests that their regulatory functions can be reshaped by the inflamed tissue microenvironment.

Biochemical and rheological analysis of human colonic culture mucus reveals similarity to gut mucus

The goal of this project was to validate the functional relevance and utility of mucus produced by an in vitro intestinal cell culture model. This is facilitated by the need to physiologically replicate both healthy and abnormal mucus conditions from native intestinal tissue, where mucus properties have been connected to intestinal disease models. Mucus harvested from colonic cell cultures derived from healthy donors was compared to mucus collected from surgically resected, noninflamed transverse colon tissue. The rheological and biochemical properties of these mucus samples were compared using oscillational rheometry, particle-tracking microrheology, multiangle laser light scattering, refractometry, and immunohistochemical imaging. An air-liquid interface culture of primary human colonic epithelial cells generated a continuous monolayer with an attached mucus layer that displayed increasing weight percent (wt%) of solids over 1 week (1.3 ± 0.5% at 2 days vs. 2.4 ± 0.3% at 7 days). The full range of mucus concentrations (0.9–3.3%) observed during culture was comparable to that displayed by ex vivo mucus (1.3–1.9%). Bulk rheological measurements displayed similar wt%-based complex viscosities between in vitro and ex vivo mucus, with the complex viscosity of both systems increasing with wt% of solids. Particle-tracking microrheology showed higher complex viscosities for ex vivo mucus samples than in vitro mucus which was explained by a greater fraction of water present in in vitro mucus than ex vivo, i.e., in vitro mucus is more heterogeneous than ex vivo. Refractometry, multiangle laser light scattering, and immunostaining showed increased mucus complex size in ex vivo mucus compared with in vitro mucus, which may have been due to the admixture of mucus and cellular debris during ex vivo mucus collection. The air-liquid interface culture system produced intestinal mucus with similar composition and rheology to native human gut mucus, providing a platform to analyze pathological differences in intestinal mucus.

A Serological Biomarker of Laminin Gamma 1 Chain Degradation Reflects Altered Basement Membrane Remodeling in Crohn's Disease and DSS Colitis.

The laminin gamma 1 chain (LMγ1) is abundant along the crypt-villus axis in the intestinal basement membrane. We investigated whether a serological biomarker of laminin degradation was associated with disease activity in patients with Crohn's disease (CD) and in rats with dextran sulfate sodium (DSS)-induced colitis. Serum samples from CD patients (n = 43), healthy subjects (n = 19), and Sprague Dawley rats receiving 5-6% DSS water for five days and regular drinking water for 11days were included in this study. The LG1M biomarker, a neo-epitope degradation fragment of the LMγ1 chain generated by matrix metalloproteinases-9 (MMP-9), was measured in serum to estimate the level of laminin degradation. Serum LG1M was elevated in CD patients with active and inactive disease compared to healthy subjects (p < 0.0001). LG1M distinguished CD patients from healthy subjects, with an area under the curve (AUC) of 0.81 (p < 0.0001). Serum LG1M was decreased in DSS rats compared to controls 2days after DSS withdrawal, and increased upon reversal of the disease. Increased serum LG1M in active and inactive CD patients supports the evidence of altered LM expression in both inflamed and non-inflamed tissue. Moreover, lower LG1M levels in the early healing phase of DSS-induced colitis may reflect ongoing mucosal repair.