bluuebunny/crossref_metadata_2025_split huggingface dataset:
P068 Polyunsaturated fatty acid excess fuels pro-inflammatory transcriptional networks in GPX4 deficient intestinal epithelial cells
Authors: Meyer M , Grabherr F , Schwärzler J , Mayr L , Jukic A , Rao Z , Rieder D , Zollner A , Grander C , Trajanoski Z , Koeberle A , Tilg H , Adolph T E 
Published: January 2023

Abstract

Background
There is increasing evidence that the rise in Crohn’s disease (CD) incidence is partially driven by constituents of a Western-style diet such as polyunsaturated fatty acids (PUFA). Recently we could demonstrate that PUFA excess induces a CD-like enteritis in mice lacking antioxidant GPX4 in their intestinal epithelial cells (IECs) and that CD patients clinical disease activity positively correlates with PUFA intake. In this project we investigated the role of epithelial lipid composition and lipid mediators during PUFA induced metabolic gut inflammation. Furthermore, we transcriptionally profiled GPX4 deficient small intestinal cells after stimulation with ω3- and ω6-PUFA.

Methods
Mice lacking one allele of Gpx4 in their IECs (GPX4+/-IEC) and wild-type controls (WT) were fed a PUFA (10% fish oil) enriched Western diet (PUFA-WD) for 12 weeks. Intestinal inflammation was assessed by histopathology. IECs were harvested by scraping of small intestinal sections and lipid profiles and mediators were analysed with LC/MS. GPX4 in murine small intestinal cells (MODE-K) was knocked down by siRNA (siGpx4) scrambled siRNA served as control (siCtrl). MODE-K were stimulated with ω3-, ω6-PUFA, 5,6- and 8,9-DHET or vehicle. Cytokine expression in cell culture supernatant was analysed by ELISA and transcriptome was analysed by bulk RNA sequencing.

Results
Histopathology revealed gut inflammation in GPX4+/-IEC but not WT mice after 12 weeks PUFA-WD feeding. LC/MS analysis of IECs derived from GPX4+/-IEC and WT mice fed the PUFA-WD depicted a significant increase in eicosapentaenoic acid (20:5), docosapentaenoic acid (22:5) and docosahexaenoic acid (22:6) containing phosphatidylethanolamine (PE) and phosphatidylcholine (PC) species (Fig 1a).

All other PE and PC species showed a reduced amount, confirming the enrichment of the specific PUFAs within the IECs by the PUFA-WD. Lipid mediator analysis further revealed an increase in 5,6-DHET and 8,9-DHET only in IECs derived from GPX4+/-IEC but not WT mice (Fig 1b). When stimulated with PUFA, siGpx4 MODE-K produced high amounts of neutrophil attracting chemokine CXCL-1 which is attenuated by the supplementation of anti-inflammatory lipid mediators 5,6-DHET and 8,9-DHET (Fig 2). Transcriptome analysis revealed the upregulation of pro-inflammatory pathways including TNFa, MAPK and NF-κB in siGpx4 vs. siCtrl MODE-K after PUFA stimulation (Fig 3).

Conclusion
By integrating lipidomic and transcriptomic data of a reductionist cell culture system and a model organism, we could show that PUFA excess in a GPX4 deficient individual fuels a pro-inflammatory transcriptional network which outweighs anti-inflammatory ω3-PUFA metabolites influence culminating in gut inflammation.
P071 T-cell, B-cell and monocytic infiltrations of myenteric plexus in IBD
Authors: Wiese J J , Fascì A , Kühl A A , Atreya R , Patankar J , Siegmund B , Prüß M S , Schumann M 
Published: January 2023

Abstract

Background
Patients suffering from IBD frequently suffer from chronic visceral pain, severely affecting quality of life. To better understand factors impacting pain signaling by enteric nervous system (ENS) neuronal cells, we analyzed the infiltration of the myenteric plexus (MP) of the ENS by inflammatory cell populations. In detail, we characterized (i) the immune cell populations infiltrating the MP, (ii) neurotransmitter expression by MP cells and (iii) neuronal cell survival of MP in Crohn’s disease (CD) and ulcerative colitis (UC).

Methods
We included 12 UC and nine CD patients that received colectomies or ileocecal resections, respectively, and 10 control patients with surgeries for non-inflammatory reasons. FFPE material was used for immunohistochemistry analysis of the MP. Immunostaining was performed for the detection of ENS neurons (beta-III tubulin, PGP9.5), for T-cells (CD3, CD4, CD8 and FOXP3), for B-cells (CD20), for monocytes (CD68, CD163) and apoptosis (Annexin V) and the neurotransmitters CGRP and substance P. Intraganglionic and periganglionic immune cell populations were determined by conventional LSM confocal microscopy.

Results
CD3+ and CD8+ intraganglionic T-cells for CD (CD3+: 1.7 ± 0.6 cells/10.000 µm²; CD8+: 4.8 ± 1.1 cells/10.000 µm²) were found to be increased compared to control patients (CD3+: 0.2 ± 0.1 cells/10.000 µm², p=0.04; CD8+: 0.4 ± 0.2 cells/10.000 µm², p=0.006), Figure 1. CD20+ B-cells and CD68+ monocytes were significantly increased in CD MP as well (CD20+ B-cells in CD: 0.8 cells/10.000 µm², CD20+ B-cells in controls: 0 cells/10.000 µm, p=0.02), CD68+ monocytes in CD: 12.3 cells/10.000 µm², CD68+ monocytes in controls: 3.5 cells/10.000 µm², p=0.001). CD8+ cytotoxic T-cells infiltrating the periganglionic area of the MP were significantly increased for UC. 1.6-fold increased expression of the neurotransmitter CGRP were detected for UC MP, p=0.01. Substance P was found to be 0.7-fold reduced for CD MP, p=0.01. MP-cell survival (Annexin V) was found to be reduced for UC MP, p=0.048. Figure 2 shows representative neurotransmitter immunostainings in two patients per condition.

Conclusion
In conclusion, MP immune cell infiltrations in CD are comprised of CD3+, CD8+ T-cells as well as CD68+ monocytes and CD20+ B-cells. Specifically periganglionically, CD8+ T-cells are found in UC. The expression of the neurotransmitter CGRP is increased in CD-MPs whereas levels of substance P are reduced in UC MP. UC-affected MPs revealed higher levels of apoptosis.
P077 Group 1 innate lymphoid cells in creeping fat of Crohn’s disease
Authors: Mori R , Ogino T , Sekido Y , Hata T , Hamabe A , Takahashi H , Miyoshi N , Uemura M , Mizushima T , Doki Y , Eguchi H 
Published: January 2023

Abstract

Background
About half of patients with Crohn’s Disease (CD) require surgery in the course of treatment, furthermore, reoperation for later recurrence is also performed in high frequency. One of the characteristic findings for CD patients is creeping fat (CF) wrapped around inflamed areas of the intestine, and CF correlates with stenosis of the intestinal tract resulting from changes in the connective tissue of the intestinal wall. Lower postoperative recurrences have been reported among CD patients who underwent extensive CF resection, suggesting that CF would be clinically important. However, the detail mechanisms are still unknown. The aim of this study is to examine the functional mechanism and clinical significance of CF focused on innate lymphoid cells (ILCs) and macrophages.

Methods
Normal mesentery (NM) of ileum was obtained from microscopically and macroscopically intact areas in patients with colorectal cancer. Mesentery was also obtained from surgically resected specimens from patients with CD who were diagnosed based on established clinical, radiologic, and endoscopic criteria. Mesenteric stromal vascular fraction were isolated by enzymatic digestion. Mesentery from CD patients divided CF and areas with no or slight inflammation (non-CF). Early recurrence was defined as Rutgeerts score (≥2) or worsening of clinical symptoms (CDAI≥220).

Results
RNA-seq of CF revealed that expression of immune-related genes and of fibrosis-related genes were elevated compared with NM and non-CF. Both frequency and numbers of ILC1 were increased, and those of ILC2 and ILC3 were decreased in CF compared with NM. Positive associations between ILC1 and inflammatory macrophages (CD14+ CD163low) related to fat fibrosis were observed in frequency and number per unit weight. FACS and RT-PCR showed ILC1 from CF had higher expression of IFNγ than those from non-CF. Co-culture experiment of ILC1 from CF and SVF from NM showed increased expression of fibrosis associated genes (COL1A, COL3A), inflammatory markers of macrophages (iNOS, Mincle), and TGFB1, and these effects were cancelled by IFNγ-neutralizing antibody. Evaluation of risk factors of early recurrence showed high frequency of ILC1 from CF and low preoperative serum albumin level were significantly associated with early recurrence (P = 0.004, P = 0.04, each). There was a significant difference between the ILC1 high (≥ 80 %) and ILC1 low (&lt; 80 %) from CF in terms of early recurrence-free survival (P = 0.02).

Conclusion
In CF, ILC1 plays a certain role in macrophage function related to fat fibrosis via IFNγ production. CD patients with high ILC1 (≥80%) in the CF have tendency of early recurrence.
P087 Calcitonin gene related peptide beta (CGRPβ): a new player in inflammatory bowel disease pathophysiology
Authors: Pascual M , Gárate G , Serrano M S , García García M J , Castro B , González Quintanilla V , Crespo J , Pascual J , Rivero M 
Published: January 2023

Abstract

Background
Calcitonin gene related peptide (CGRP) is mainly present in the central nervous system (α isoform) and enteric nervous system (β isoform). CGRP is known to be the most potent vasodilator of the body, and it promotes migration of inflammatory cells.
Although its function in the gut is not well established yet, CGRPβ is postulated to have a protective role against inflammation in the gut, promoting migration of inflammatory cells, regulating nociception, motility and gut microbioma.
The aim of the study is to analyse serum levels of CGRPβ in patients newly diagnosed with inflammatory bowel disease (IBD), to unravel its potential role in the disease.

Methods
CGRPβ levels were measured by ELISA (CUSABIO, China) in early morning samples of newly diagnosed patients with IBD. Classification of the disease (ulcerative colitis- UC, Crohn’s disease- CD or unclassified inflammatory bowel disease- U-IBD), demographic data and treatment during sample collection were also collected. The results were compared with healthy controls (HC) stratified by age and sex. Statistical analysis was performed using SPSS and CGRPβ levels were compared with Mann-Whitney and Dunn’s tests.

Results
Seventy-one patients with newly diagnosed IBD (mean age 48.3±16.5 years, 63.4% females) were matched with 71 HC (mean age= 48.2±16.7 years-64.8% females). Samples were collected between 0 and 249 days after IBD diagnosis was established (median 49 days, interquartile range 29-66 days).
Thirty patients were diagnosed as CD, 36 as UC and 5 as U-IBD. Most patients with CD had ileal location and inflammatory behaviour. One of these patients was on methotrexate due to a prior diagnosis of rheumatoid arthritis; the rest were either with no treatment, aminosalicylates, steroids or budesonide, or some combination of these. Half of the patients with UC had proctitis. Six had no treatment, and the rest were prescribed aminosalicylates, seven of them in combination with steroids. These characteristics are shown in table 1.
Serum CGRPβ levels in patients with IBD were significantly decreased (2.95±1.68 pg/mL) compared to HC (4.65±2.59 pg/mL), (p&lt;0.0001). These results stayed significant, in both CD (3.15±2.08 pg/mL; p&lt;0.05), and UC (2.83±12.7 pg/mL; p &lt;0.01), but not in IBD-U (2.56±1.73 pg/mL), though the number of U-IBD cases was low. CGRPβ concentrations did not differ significantly in patients with CD and UC. The results are illustrated in figures 1 and 2.

Conclusion
The consistent decrease in CGRPβ levels in IBD shown here strongly supports a protective role of this peptide in the homeostasis of the intestinal mucosa. This work was supported by a grant from the Instituto de Salud Carlos III (PI20/01358).
P088 Restoration of gut microbiota and changes in inflammatory susceptibility by dextran sulfate sodium after antibiotic treatment in mice
Authors: Jeong E S , Bae S , Song E M , Byeon J R , Lee A , Choe A R , Hong J T , Park Y H , Tae C H , Moon C M , Jung H K , Shim K N , Jung S A , Kim S E 
Published: January 2023

Abstract

Background
It has been suggested that changes in gut microbiota have an important effect on the development of inflammatory bowel disease (IBD). In studies using germ-free mice, it has been reported that the absence of microbiota rarely causes dextran sulfate sodium (DSS)-colitis. This study was to investigate whether the changes in DSS-colonic inflammation in mice treated with antibiotics were comparable to the results in germ-free mice.

Methods
Ampicillin + enrofloxacin were treated in six 9-week-old C57BL/6 mice for initial 3 days and stools were sampled daily to measure the total amount of bacterial 16S rRNA. The time of loss and restoration of gut microbiota was investigated after repeated antibiotics. The severity of colitis and barrier damage were compared between the following groups: (i) control group, (ii) DSS group, and, and (iii) DSS + antibiotics group. The DSS group was allowed to drink 3% DSS from day 5 and maintained for 7 days, and colon tissues were obtained from all groups on day 12. The inflammatory markers (IL-1α, IL-6, IL-17, tumor necrosis factor (TNF)-α) and gut barrier markers (Zonular occludens (ZO)-1, occludin, claudin-1, claudin-4) in colon tissues were comparatively analyzed between groups.

Results
Bacterial 16S rRNA was not detected from day 4 after the antibiotics, and restoration started from day 11. After repeated antibiotics, microbial depletion was re-confirmed on day 14, but restoration appeared on day 18, confirming faster recovery. The expression levels of IL-1α, IL-17, and TNF-α in the DSS + antibiotics group did not differ from the control but were significantly lower than the DSS group (all p &lt; 0.01). However, the expression of ZO-1 and claudin-4 was significantly lower in the DSS + antibiotics treatment group than in the control group (p &lt; 0.05).

Conclusion
The antibiotic cocktail could dramatically reduce gut microbiota over a period, but the effect would decrease as it is repeated. Depletion of gut microbiota after antibiotics would dramatically reduce DSS-colonic inflammation but could damage the gut barrier or at least not prevent barrier dysfunction.
P099 IL-33 activates CD73-expressing cells promoting tumorigenesis during colitis-associated colorectal cancer
Authors: Lopetuso L R , Privitera G , Di Martino L , Scaldaferri F , Gasbarrini A , Cominelli F , De Salvo C , Pizarro T 
Published: January 2023

Abstract

Background
Colorectal cancer associated to chronic colitis (CAC) has a different pathogenesis compared to sporadic or familial colorectal cancer and represents the major complication of IBD. IL-33 and its receptor, ST2, are important factors in the pathogenesis of IBD. Emerging evidence suggests the potential contribution to inflammation-driven tumorigenesis that can lead to CAC. The aim of our study was to characterize the precise contribution of IL-33/ST2 axis in the azoxymethane (AOM)/dextran sodium sulfate (DSS) model of CAC.

Methods
C57/BL6 wild-type (WT), Il33-/-, T1/St2-/- and Nt5e-/- mice were given a single dose of AOM followed by two cycles of 3% DSS for 7d in drinking water. Vehicle-treated WT mice served as controls and were sacrificed at the same time points. Another group of WT followed the same protocol and received CD73 inhibitor i.p. treatment or vehicle. Disease Activity Index, endoscopic, stereomicroscopic and histological evaluations of colons were performed. IHC, immunofluorescence (IF), qPCR, and ELISA were done on full-thickness colons. RNA-Seq was performed on whole tissues from AOM/DSS treated WT, Il33-/- and T1/St2-/-. qPCR analysis was done on isolated polyps from WT, Il33-/- and T1/St2-/- for Nt5e (CD73) and adenosine pathway targets.

Results
Il33, Ilrl1(ST2L), and Ilrl1(sST2) mRNA transcripts, as well as IL-33 and total ST2 proteins were dramatically elevated in AOM/DSS-treated WT mice vs. controls. IHC and IF of treated WT mice revealed localization of IL-33 to the colonic epithelium and to cells within the polyp LP morphologically consistent with stromal and mast cells. Using IF, IL-33 co-localized with sub-epithelial myofibroblast markers Actin and Vimentin, or with mast cell markers Tryptase and MCPT1. AOM/DSS treatment in Il33-/- and T1/St2-/- mice resulted in a significant decreased polyp number and size vs. WT, with colonoscopy revealing the development of protruding lesions with abnormal vascular patterns, suggesting tumorous lesions in WT mice, while all deficient mice showed their absence with a more impressive mucosal inflammation, likely due to reduced epithelial proliferation and repair caused by the deficiency of IL-33 signaling. RNA-Seq identified a significant reduction of Nt5e and adenosine pathway targets in Il33-/- and T1/St2-/-vs. WT. qPCR on isolated polyps confirmed this observation. AOM/DSS-treated Nt5e-/- showed a significant decreased polyp number and size vs. controls. Therapeutic inhibition of CD73 produced similar results.

Conclusion
Our results suggest that the IL-33/ST2 axis promotes tumorigenesis in CAC through the activation of CD73. Further studies are underway to determine mechanisms of action that support these findings.
P102 A small molecule selective integrin α4β7 inhibitor demonstrates efficacy in a chronic model of Inflammatory Bowel Disease
Authors: Redhu N S , Rowe M , Lee D , Jain D , Sang A , Granata D , Harrison B , Cui D , Bursavich M , Ray A S , Lippa B , Rogers B N , Wong J 
Published: January 2023

Abstract

Background
Integrin α4β7 regulates the recruitment of T cells to intestinal mucosa through its interaction with mucosal addressin cell adhesion molecule (MAdCAM)-1. Disruption of this interaction has been clinically validated for the treatment of inflammatory bowel diseases (IBD) by the anti-α4β7 antibody vedolizumab. The current study was aimed at elucidating the preclinical efficacy of MT-103, a potent and selective small molecule α4β7 inhibitor in the clinically relevant CD4+CD45RBhi T cell transfer (TCT) colitis mouse model.

Methods
Mice were administered either MT-103 via subcutaneously implanted minipumps across 3 dose cohorts (3, 10, or 30 mg/ml), or an antibody specific for IL12p40 (25 mg/kg) or α4β7 (30 mg/kg), through intraperitoneal injections for 7 weeks. Colitis development was evaluated via readouts including body weights, gross colon weight by length ratios, colonic tissue gene expression, and histopathological scores.

Results
MT-103 significantly inhibited the development of colitis in mice at all 3 doses equivalently, as evident from significantly improved gross colon weight/length ratios in comparison to vehicle controls which presented with moderate-severe colitis (Figure 1A). MT-103 treatment also led to a significant protection from weight loss that occurred in vehicle-treated mice. Importantly, mice treated with MT-103 exhibited remarkably improved histopathology scores, including inflammation, hyperplasia, and gland loss compared to vehicle controls (Figure 1B). Furthermore, broad gene transcriptional analysis showed that MT-103 broadly suppressed pro-inflammatory pathways and processes within colonic tissues. Specifically, MT-103-induced downregulation of Ifng, Il17a, and Il1b, besides reduced Cd3 as a surrogate metric of T cell trafficking inhibition to the colon, and conversely enabled upregulation of anti-inflammatory cytokine Il10. The colitis-protective effects of MT-103 were equivalent, if not superior, to the saturating doses of α4β7 mAb or anti-IL12p40 mAb when compared to their respective vehicle controls.

Conclusion
Treatment with MT-103 protects from colitis by restoring colonic tissue homeostasis as measured by body fitness and an array of immunological and histopathological assessments. These proof-of-concept data demonstrate an α4β7-specific small molecule, MT-103, can occlude pathogenic T cells from initiating disease in a chronic IBD model, equivalent to an α4β7 blocking antibody.
P109 Using baseline Peripheral blood mononuclear cell (PBMC) transcriptomics of Crohn Disease patients receiving Ustekinumab treatment as a tool to assess potential response at 1 year
Authors: Granot M , Braun T , Efroni G , Fudim E , Yavzori M , Haj O , Ben-Horin S , Kopylov U , Haberman Ziv Y 
Published: January 2023

Abstract

Background
Ustekinumab, a monoclonal antibody to the p40 subunit of interleukin-12 and interleukin-23, is used to treat Crohn Disease (CD) and clinical remission after one year was observed in about 50% of patients. We aimed to identify predictors for clinical response using PBMC transcriptomics obtained from Crohn Disease patients just prior to Ustekinumab treatment initiation.

Methods
RNA extraction from leukocytes was performed using Qiagen AllPrep RNA/DNA Mini Kit, followed by PolyA-RNA selection, fragmentation, cDNA synthesis, adaptor ligation, TruSeq RNA sample library preparation (Illumina, San Diego, CA), and paired-end 75bp sequencing. mRNA genes with transcripts per million (TPM) values above 1 in at least 20% of the samples were used. Differential gene expression was performed using DESeq2.

Results
We processed blood samples from 36 CD patients (13 males, [36%], median age 35 [IQR 29-41] years). Samples were obtained at baseline just before starting Ustekinumab treatment. 22/36 (61%) were define as responders and 14/36 (39%) as non-responders after 1 year based on Physician Global Assessment (PGA). Small bowel disease was significantly more common among responders 82% versus 36%, p=0.004. Principal component analysis (PCA) using the 12773 genes that passed expression filtering showed partial separation (Fig. 1).
Differential gene expression between responders (n=22) and non-responders (n=14), did not show gene expression signature that passed FDR correction, however the analyses identified 68 genes including CXCL1/2/3 that were induced in non-responders vs. responders with p&lt;0.05 and fold change above 1.5. Functional annotation enrichments of these 68 genes using ToppGene indicated enrichment (Fig. 2) for cytokine activity (FDR=1.98E-05), CXCR chemokine receptor binding (FDR=2.11E-05), Interleukin-10 signaling (FDR=5.03E-07), Genes encoding secreted soluble factors (FDR=1.73E-05), Myeloid Cells, granulocytes CD11b+ (FDR=6.64E-12), and Myeloid Cells Dendritic cells (FDR=1.80E-08).

Conclusion
No strong differences were found in the blood transcriptomics between responders and non-responders. However, among the non-responders we noted an increased inflammatory response enriched for pathways linked with cytokine activity and chemokine receptor binding and innate granulocytes and Myeloid signature. A larger cohort is required to validate and farther explore these findings.
Fig. 1: Principal component analysis (PCA) using the 12773 genes that passed expression filtering showed partial separation. ￼

Fig. 2: Functional annotation enrichments of the 68 genes induced in PBMC. ￼