bluuebunny/crossref_metadata_2025_split huggingface dataset:
P053 Pro-resolution Pathway in Experimental Colitis – A New Possible Therapeutic Approach for Inflammatory Bowel Diseases
Authors: Chaim F H M , Pascoal L B , Palma B B , Rodrigues B L , Castro M M , Fagundes J J , Milanski M , Lopes L R , Velloso L A , Leal R F 
Published: January 2024

Abstract

Background
Inflammatory bowel diseases (IBD) are idiopathic disorders characterized by chronic inflammation of the gastrointestinal tract. New therapeutic strategies involving the inflammatory process are being proposed because the usual therapies indicated for IBD may cause adverse reactions and clinical failure. Recent studies have highlighted the role of specialized pro-resolution lipid mediators (SPMs) in the active resolution of chronic inflammation. Therefore, this study aimed to investigate the SPM Resolvin D2 (RvD2) and its precursor, omega-3 polyunsaturated fatty acid, in experimental colitis induced by dextran sulfate sodium (DSS).

Methods
An experimental model with male mice of the C57BL/6J lineage was carried out. Mice were separated into a control group, and a DSS-induced colitis group (3% dilution in drinking water); both groups were subdivided and received either a standard diet or an experimental diet enriched with 20% of oil rich in omega-3. Another group of mice, after acquiring experimental DSS-induced colitis, was subdivided and treated with RvD2 or anti-TNFα. Throughout the experimental protocols, the body weight, food intake, Disease Activity Index (DAI), metabolic phenotype, and inflammatory profile of these animals were assessed by real-time PCR, ELISA, histology, Western blotting, and lipid analysis. The study was approved by the University of Campinas research ethics committee.

Results
The animals with experimental colitis induced by DSS showed an increase in TNFα and IL22 transcriptional expression besides a reduction in the enzymes involved in the endogenous biosynthesis of RvD2, such as PLA2, 15-LOX, 5-LOX, and its receptor GPR18. Dietary supplementation with omega-3-rich oil increased RvD2 and its precursor besides reducing the DAI, weight loss, colonic shortening, and the histologic colonic inflammation score. Treatment with RvD2 effectively attenuated experimental colitis, reducing TNFα transcriptional levels and p-JNK protein expression. Besides, RvD2 increased the immediate precursor of RvD2 (7,8-epoxy-17-HDHA). All of which indicates that this pathway is responsible for the observed effects.

Conclusion
Our study provided a better understanding of the molecular mechanisms involved in the exacerbated inflammatory response present in colitis. The beneficial effect of RvD2 and its precursor omega-3 may bring about a new therapeutic approach for IBD.
P073 Enhancing the MFSD2A protein for the treatment of colitis-associated cancer: novel insights for pathogenesis and treatment
Authors: Cagliani S , Massimino L , Facoetti A , Spanò S , Errico C , Parigi T L , Colasante G , Danese S , Ungaro F 
Published: January 2024

Abstract

Background
The intrinsic connection between inflammation and tumor promotion is well characterized and is a key pathogenic event in patients with colorectal cancer (CRC). A small fraction of patients with CRC suffers from genetic predisposition. However chronic inflammation, such as ulcerative colitis (UC), increases the risk of intestinal carcinogenesis, thus strengthening the connection between these conditions. A few years ago, our laboratory described that the intestinal endothelium isolated from actively inflamed UC patients displayed defective production of inflammation-resolving DHA-derived metabolites, by comparison with healthy controls and tissues in remission. We also reported that the Major Facilitator Superfamily Domain containing 2A (MFSD2A) participated in the production of the inflammation-resolving DHA-derived metabolites by the intestinal endothelium of patients with UC in remission, ameliorating colonic inflammation. Therefore, we hypothesized that the endothelial MFSD2A also has a role in preventing and/or counteracting cancer-associated inflammation.

Methods
Human Intestinal Microvascular Endothelial Cells (HIMEC) isolated from CRC and healthy samples were transduced with either MFSD2A protein-encoding lentiviral vectors (MFSD2A) or GFP as control, or MFSD2A targeting shRNA or its scramble, and were analyzed by lipidomics. Furthermore, Caco-2 cells co-cultured with HIMEC-MFSD2A were analyzed by FACS, evaluating their proliferation. Moreover, an orthotopic model of CRC was made intrarectally injecting CD1 nude mice with a cell mixture of Caco-2 and HUVEC overexpressing MFSD2A or GFP as control. Mice were gavaged with DHA-ethyl-ester or PBS. FACS analysis was performed on tumor and colonic samples.

Results
Lipidomic analysis revealed that the loss of MFSD2A in CRC is detrimental because there is a reduced production of pro-resolving lipids confirming that, as for UC, MFSD2A is important for the maintenance of an adequate balance between pro-resolving and pro-inflammatory phenotype. Moreover, enhancing MFSD2A expression at endothelial level limits Caco-2 cell proliferation, supporting its beneficial role. In addition, DHA administration in mice ameliorated clinical symptoms of inflammation upon intestinal endothelial MFSD2A overexpression, supporting the beneficial role of MFSD2A in vivo.

Conclusion
Tumor-associated inflammation remains a crucial hallmark of CRC. Our study seeks to identify the role of MFSD2A in the resolution phase of inflammation, pointing out alternative therapies for CRC treatment. These data suggested that MFSD2A might contrast the tumor-associated inflammation and ensure the correct balance between pro-inflammatory and pro-resolving milieu.
P077 The Immunometabolic Bimodal Mechanism of NLRX1 Agonist NX-13 in a Pig Model of Ulcerative Colitis
Authors: Verstockt B , Mosig R , Dubinsky M , Cataldi F , Danese S 
Published: January 2024

Abstract

Background
In mouse models, NX-13 is an orally active, gut-restricted NLRX1 agonist that has been shown to reduce severity of disease in multiple UC models through a novel immunometabolic, bimodal mechanism. Most preclinical UC studies rely upon rodent models, the human gastrointestinal physiology, microbiome, and immune system bear a greater similarity to that of pigs. Therefore, we sought to expand and validate our preclinical NX-13 results in mice using the dextran sulfate sodium (DSS) pig model of colitis. Here, we describe the disease severity, cellular, and biomarker results, and confirm the immunometabolic MOA observed in other species.

Methods
Pigs were randomized by weight (n=6 per group) and were administered tablets of excipient alone (placebo), NX-13 10 mg, NX-13 50 mg, or NX-13 100mg once daily. Pigs were challenged with 1% DSS in drinking water for 6 days and monitored daily for weight gain (loss). Composite disease activity score (0-12) was taken based on the sum of three subscores (0-4) for rectal bleeding, stool consistency and overall health/activity. Necropsy was conducted on day 7. Feces were collected for fecal calprotectin (FCP) quantification. Colonic tissue was macroscopically scored and collected for analysis by flow cytometry, histopathology and gene expression.

Results
Oral NX-13 treatment protected against weight loss and reduced colitis development with differences as early as day 2, becoming significant between days 4 and 6 as disease worsens in placebo treated pigs (Fig1A). NX-13 yielded a dose-dependent improvement in macroscopic lesion severity and microscopic immune cell infiltration (Fig1B). Specifically, Th1 cells (Fig1C) and TNF producing myeloid cells in the colonic lamina propria were reduced by NX-13, as well as FCP levels in stool (Fig1D) and mRNA expression in the colon (Fig1E). Oral NX-13 treatment reduced inflammatory markers including TNF, monocyte and neutrophil chemo attractants, IL-6, and IL-23 (Fig3F). Additionally, NX-13 treatment affected markers specific to the bimodal immunometabolic MOA of NLRX1 activation, namely upregulation of NLRX1 and mitochondrial metabolism gene COX3 (Fig1G), and reduction of NFkB and NLRP3 (Fig1H).

Conclusion
NX-13 demonstrated fast and clinically meaningful improvement in disease activity, and significantly impacted on key inflammatory markers in a DSS colitis model in pigs. These results align with previous murine results and observations in the phase 1b clinical program. Therefore, they can be used to generate additional mechanistic hypotheses for translational studies in the ongoing NEXUS phase 2 trial in moderate to severe UC.
P095 Accelerated degradation of gut barrier-protecting 3-aminobenzoic acid exacerbate colitis in Inflammatory Bowel Disease
Authors: Tanaka M , Toyonaga T , Nakagawa F , Sumiyoshi N , Shibuya N , Iwamoto T , Minemura A , Ariyoshi T , Matsumoto A , Oka K , Shimoda M , Saruta M 
Published: January 2024

Abstract

Background
Disruption of intestinal epithelial cell (IEC) barrier function is crucial for the development of inflammatory bowel disease (IBD). Decreased diversity and compositional changes in intestinal bacteria, namely dysbiosis, impair IEC barrier function in IBD, but the underlying mechanism is not fully understood. This study aims to identify the impact of dysbiosis-driven changes in intestinal bacterial metabolites on IEC permeability in patients with IBD.

Methods
Caco2 cells were cultured on the trans-well inserts and stimulated with a library of intestinal bacterial metabolites. Proportional changes of trans-epithelial electrical resistance (TEER) were calculated at 48 hours after stimulation (ΔTEER%) and the permeation of 70-kDa FITC-dextran was evaluated using a Qubit fluorometer. Fecal samples were collected from 10 IBD patients with active colonic inflammation (5 ulcerative colitis and 5 Crohn’s disease) and 5 healthy controls (non-IBD, NIBD). Fecal bacteria-derived metabolites were quantified by LC-MS/MS and compared between IBD patients and NIBD controls. 16SrRNA-based metagenomic analysis was performed on the same fecal samples using QIIME2 followed by a PICRUSt2-based functional analysis of microbial communities. Anti-inflammatory effect of 3-aminobenzoic acid (3-ABA) was examined in C57BL/6 mice using a dextran sodium sulfate (DSS)-induced colitis model.

Results
Among 119 intestinal bacterial metabolites, 4-ABA decreased epithelial permeability the most with the highest ΔTEER% value (control vs. 4-ABA, 1.8 vs. 30.9 %, p&lt;0.05). 3-ABA, one of three structural isomers of ABA, also decreased epithelial permeability in a dose dependent manner with higher ΔTEER% value than 4-ABA and reduced permeation of FITC-dextran (control vs. 3-ABA, 8.3 vs 3.4 nM, p&lt;0.05). Fecal level of 3-ABA was significantly lower in IBD patients than in NIBD controls (7.0 vs. 505.0 μM). Functional prediction of intestinal microbiota demonstrated that IBD patients were significantly more enriched with the bacteria involved in the degradation of ABA than NIBD controls. Rectal administration of 3-ABA significantly improved body-weight loss, shortening of colon length, histologic scores, and the expression of inflammatory cytokines in mice with DSS-induced colitis (control vs. 3-ABA; body-weight loss,15.33 vs. 18.91 g; colon length, 6.11 vs. 7.35 cm; histologic score, 2.14 vs 0.60).

Conclusion
Accelerated degradation of luminal 3-ABA by intestinal bacteria may disrupt intestinal epithelial barrier function and exacerbate colitis in patients with IBD.
P105 Mendelian Randomization screening for JAK inhibitor related potential serious adverse events
Authors: Miguel González J A , Pavlidis P , Marigorta U M 
Published: January 2024

Abstract

Background
JAK inhibitors (JAKi) suppress the JAK/STAT signalling pathway through inhibition of the four Janus kinase proteins JAK1, JAK2, JAK3 and TYK2. JAKi are licensed for the treatment of a wide range of immune mediated inflammatory diseases (IMIDs) including inflammatory bowel disease. Concerns have been raised for the potential of this class of medication to associate with serious adverse events, particularly in those older than 65 years of age or with previous cardiovascular comorbidities. Mendelian randomization (MR) uses genetic variants as instrumental variables (IVs) to examine the causal effects of an exposure (e.g. inhibition of a drug target) on an outcome (e.g. the risk of developing a disease and/or potential adverse effects due to alteration of gene activity). We carried out a comprehensive MR evaluation to explore the therapeutic potential of JAKi for a range of IMIDs, and to study the unintended drug effects that might be caused by this therapeutic strategy.

Methods
The basis of MR relies on the use of genetic variants as IVs that are reliably related to the risk factor and that are not susceptible of reverse causation and confounding. We used genetic variants (SNPs) associated with protein levels and gene expression (pQTLs and eQTLs, respectively) as IVs to study the effects of inhibiting the genes in the JAK/STAT signalling pathway. The underlying assumption is that these IVs serve as proxies of the effects of JAKi. As a source of the effect estimates on the selected outcomes, we used summary statistics from a compiled list of genome-wide association studies.

Results
We used the MR approach to chart the range of possible outcomes associated with JAKi treatment. First, we explored the therapeutic potential of JAKi for IMIDs. Second, we evaluated a range of potential adverse events associated with JAKi, focusing particularly on cardiovascular, infectious and cancer-related malignancies. Our MR results confirm a general tendency towards JAK/STAT signalling pathway involvement in IMIDs risk, implying the suitability of JAKi for IMIDs. Regarding potential adverse outcomes, we carried out stratification-based analyses to evaluate whether potential adverse events are enhanced in individuals at particular high risk. The MR analyses highlighted potential risk of JAKi for non-IMIDs traits, particularly when targeting JAK2. This includes already known associations with cardiovascular disease and specific cancer types.

Conclusion
We performed an outcome-wide exploration based on MR to characterize the therapeutic potential and range of adverse effects expected for JAKi. We recapitulate the known potential of these drugs to treat IMIDs, while uncovering specific severe adverse effects associated with this therapeutic target.
P106 Single-cell transcriptomic analysis reveals insights into the mechanism of action of Granulocyte-monocyte apheresis
Authors: Kioroglou D , Cabriada J L , Martinez Marigorta U , Rodriguez-Lago I 
Published: January 2024

Abstract

Background
Granulocyte-monocyte apheresis (GMA) is a non-pharmacological treatment approved for the management of ulcerative colitis (UC), particularly steroid-dependent cases. The exact mechanism of action and immunological changes associated with GMA remain undescribed. Gene expression analysis at the single-cell level (through scRNA-Seq) has emerged as a key tool of choice to characterize drug response at the molecular level. We used scRNA-Seq to characterize the transcriptomic effects and immune cell population alterations in GMA.

Methods
We generated scRNA-Seq from peripheral blood mononuclear cells (PBMC) of two ulcerative colitis (UC) patients undergoing GMA treatment. We compared the gene expression profile before and after 5 sessions of GMA treatment. The analytical pipeline included quality control and classical filtering steps, cell-type annotation, differential gene expression analysis and pathway enrichment profiling.

Results
We report three main results. First, we observed significant reductions of cell types directly affected by GMA treatment in UC patients. This includes classical CD14+ monocytes and Natural killers, which are central components of the innate system. Of note, we observed a remarkable increase in Double-negative T cells (dnT) after 5 sessions of GMA treatment, suggesting potential expansion of protective populations involved in decreased inflammation. Second, we observe a variety of genes and regulatory pathways altered by GMA treatment. In total, we detect 86 differentially expressed genes (DEGs), which overall are biased towards downregulation (63%). Of note, we detected dnT-s exhibiting upregulation of NEFL that is associated with the MAPK cascade and downregulation of genes related to immune response and signaling pathways. Finally, the effects of GMA treatment extend beyond the above mentioned populations, with particular alterations in CD4+ T cell populations such as CD4+ central memory and CD4+ Naive (7 and 13 DEGs, respectively).

Conclusion
For the first time, we generated single-cell transcriptomic profiles to characterize the effects of GMA treatment in peripheral blood of UC patients. Our preliminary analysis detects important alterations in the gene regulation and cell type composition in samples obtained after 5 sessions of GMA. Through expansion of this dataset to include more time points and profiles for more individuals, we will discuss the longitudinal changes and molecular mechanisms involved in response to GMA treatment.
P114 Role of NLRX1 Agonist NX-13 in Reducing Visceral Hypersensitivity in Preclinical Gastrointestinal Inflammation
Authors: Verstockt B , Vermeire S , Peyrin-Biroulet L , Yarur A , Panaccione R , Mosig R , Cataldi F , Danese S 
Published: January 2024

Abstract

Background
NLRX1 activation reduces inflammation by decreasing oxidative stress and altering cellular metabolism within multiple cell types implicated in ulcerative colitis (UC). Colitis animal models demonstrated reduced disease severity and a phase 1b clinical trial showed signs of rapid symptom and endoscopic improvement in patients with active UC.1, 2 Abdominal pain driven by visceral hypersensitivity may persist in patients even after inflammation has resolved, negatively affecting their quality of life.3, 4 Therapeutic agents which address inflammation, epithelial healing, and visceral hypersensitivity concurrently may provide greater symptomatic relief and improved quality of life as many patients, even in remission, still complain of abdominal pain. We here describe the effects of oral NX-13 in a rat model of visceral hypersensitivity.

Methods
Rats (n=8) were dosed daily for a period of 3 days with NX-13 or vehicle. Under anesthesia, electrodes were positioned to monitor oblique abdominal muscle contraction and a colonic balloon catheter was inserted intra-anally. Visceral pain was assessed at baseline and 3 h post lipopolysaccharide injection (1 mg/kg subcutaneous) through measuring of visceromotor response (VMR) via electromyogram (EMG) recording and visual assessment of abdominal withdrawal reflex (AWR). Data are represented as median (IQR) and statistical significance determined by non-parametric Mann-Whitney U test.

Results
Compared to the vehicle control, oral NX-13 delayed the onset of muscle contraction in response to colonic distension in LPS-treated rats. Further, NX-13 treated rats experienced reduced contraction intensity and reduced sustained abdominal muscle contraction period compared to the vehicle control. Specifically, NX-13 decreased the number of AWR during colonic expansion compared to the control group (p=0.01, Fig1A). Moreover, NX-13 desensitized the VMR response by numerically increasing the minimum volume of the colonic distension balloon required to induce significant VMR. The mean minimum volume of water injected required to induce significant VMR increased 23%, from 650 μL in the vehicle group to 800 μL in the NX-13 treated rats (p=0.16, Fig1B). Lastly, NX-13 visually reduced the maximum abdominal EMG amplitude during colonic distention (p=0.19, Fig1C).

Conclusion
Adequate management of persistent pain in IBD patients with or without active bowel inflammation remains an unmet need in the treatment of IBD. The potential for NX-13 to specifically reduce visceral hypersensitivity and abdominal pain will be evaluated further in the ongoing phase 2 human NEXUS trial in patients with UC.
1Leber et al. J Immunol 203(12)
2Peyrin-Biroulet et al. JCC (17)Supp
3Abreu et al. JCC (14)Supp
4Wils et al. J Clin Med 11(15)
P117 Transglutaminase 2 is elevated in Crohn’s disease associated strictures and exerts profibrotic activities in myofibroblasts and experimental intestinal fibrosis
Authors: Chandra J , Czarnecki D , Mukherjee P K , Mao R , Bergin M , Huang L , Johnson T , West G , Kessler S , Fiocchi C , De La Motte C , Rieder F 
Published: January 2024

Abstract

Background
Intestinal fibrosis is a significant clinical problem in Inflammatory bowel disease (IBD). This may lead to intestinal strictures induced by excessive accumulation of extracellular matrix (ECM) produced by human intestinal myofibroblasts (HIMF). Transglutaminase (TG)2 covalently cross-links ECM-associated proteins, increases ECM stiffness and has direct pro-fibrotic effects. We investigated the role of TG2 in intestinal fibrosis in human tissues, HIMF and transgenic mouse models.

Methods
TG2 was detected in human tissues from Crohn’s disease (CD) strictures (CDs; n=10), CD non-strictured (CDns; n=20), ulcerative colitis (UC; n=10) and non-IBD controls (NL; n=14) by immunofluorescence (IF), qPCR, in-situ hybridization (ISH), and biotinylated cadaverin incorporation (BCI). Relative contribution of TG2 was measured by BCI using a TG2-selective blocking antibody (TAb). The cellular source of TG2 was evaluated by IF. HIMF ECM production was measured using a novel ECM deposition assay and TG2-inhibitors. HIMF were exposed to anti-TG2 antibody or isotype control and bulk RNA-sequencing (RNAseq) analysis was performed. Tamoxifen-inducible global Cre/TG2 floxed conditional KO (cKO) mice were used in the DSS and TNBS fibrosis models.

Results
Total TG activity was increased in CDs and UC vs. NL in the submucosa (SM) and muscularis propria (MP; P≤ 0.04). Strikingly 90-99% of TG activity were attributable to TG2 in all groups. TG2 activity correlated with fibrosis scores (P≤0.02), but not inflammation scores. TG2 amount was elevated in CDs patients vs. NL in qPCR and ISH (P≤0.05). IF revealed HIMF as the major source of TG2 in the intestine as indicated by colocalization with alpha-SMA. HIMF, invitro, produced 82–97% of active TG2. Inhibition of TG2 by cell-permeable inhibitor or Tab reduced fibronectin (FN) and collagen (COL)1 and 3 deposition by HIMF. RNAseq showed TAb-treated cells expressed 432 downregulated and 402 upregulated genes. Functional enrichment analysis showed pathways associated with fibroblast activation/proliferation, ECM, and fibrosis-associated signaling (e.g. AKT) pathways to be downregulated in TAb-treated HIMF. Associated genes included nuclear protein (NUPR1) and sphingolipid signaling pathway proteins (SMPD1 and CTSD), which may be potential targets for TG2-mediated activity in HIMF. Deletion of TGM2 prior to inducing murine DSS or TNBS experimental fibrosis did not lead to any difference in inflammation, but decreased fibrosis (picrosirius red intensity, COL1/FN expression, wall thickness) in TG2 cKO mice vs. wild type (P≤0.05).

Conclusion
TG2 is involved in inflammation-independent progression of intestinal fibrosis and may represent a future anti-fibrotic target.