bluuebunny/crossref_metadata_2025 huggingface dataset:
P052 IL-12p70 as a potential driver of pathogenic IL-17+IFNγ+ mucosal memory TIGITneg T cells in a subgroup of severe CD patients
Authors: Heredia M , Barendregt D M H , van den Bogaerdt S , Samsom J N
Published: January 2023

Background

Crohn’s disease (CD) is a chronic disease driven by inflammatory memory CD4+ T cells. Deciphering heterogeneity in underlying immune disease is required for predicting the individual patients’ disease course. We have shown that analysis of circulating CD38+ memory CD4+ T cells, which are enriched for gut-homing, identifies therapy-naïve pediatric CD patients with a more severe disease course. At diagnosis, these patients have increased frequencies of circulating CD38+ memory T cells that do not express TIGIT (TIGITneg). TIGIT (T-cell immunoreceptor with immunoglobulin and ITIM domains), is a coinhibitory receptor selectively induced on CD4+ T cells with a memory phenotype. In healthy controls, TIGIT+CD38+ memory T cells have an inhibitory function while TIGITnegCD38+ memory T cells are enriched in inflammatory cells. In our cohort of CD patients, circulating inflammatory TIGITnegCD38+ memory T cells had increased frequencies of pathogenic IFNγ +IL-17+-double-producing or Th1* cells. We hypothesize that in these severe CD patients, CD-related inflammatory cytokines selectively increase frequencies of Th1* TIGITnegCD38+ memory T cells.

Methods

We cultured peripheral blood memory CD4+ T cells from healthy individuals in vitro with TCR- and co-stimulation together with different cocktails of CD-related inflammatory cytokines during 72h.

Results

Of all cytokines tested, only cocktails containing IL-12p70 increased TIGITneg cell frequencies within gut-homing CD38+ memory T cells, but not CD38neg memory T cells that are enriched in skin-homing. As hypothesized, IL-12p70-stimulated cultures contained higher frequencies of Th1* cells, expressing both hallmark transcription factors T-bet and RORgt, in TIGITnegCD38+ T cells but not within TIGIT+CD38+ T cells. This selective IL-12p70 responsiveness in TIGITneg cells agreed with the higher expression of the IL-12 receptor b2 subunit in TIGITnegCD38+ compared to TIGIT+CD38+ memory T cells. These data argue that IL12p70 initiates reprogramming of the TIGITnegCD38+ memory T cells to the Th1* phenotype. To assess whether IL-12p70 causes expansion of TIGITnegCD38+ cells or downregulates TIGIT expression on TIGIT+CD38+ cells, we traced fluorescently-labelled TIGIT+ cells during culture. TIGIT+ cells did not downregulate TIGIT, indicating that IL-12p70 increased proliferation of TIGITnegCD38+ cells.

Conclusion

The finding that IL-12p70 drives expansion of TIGITnegCD38+ memory T cells with a pathogenic Th1* phenotype argues that this cytokine may enforce chronic inflammation in these severe CD patients. These analyses pave the way toward deciphering key actors involved in CD inflammation at an individual patient level, needed to tailor novel IL-12/IL-23 targeting therapeutic strategies.
P057 Development and evaluation of the i-Tracker Certolizumab and i-Tracker Anti-Certolizumab kits: rapid and innovative tests intended for monitoring patients treated with Certolizumab.
Authors: Khater G , Noguier G , Daviere S , Guilbert V , Montaillier C
Published: January 2023

Background

Certolizumab is a human monoclonal antibody directed against TNFα. This drug is commonly used for the treatment of patients with inflammatory diseases such as rheumatoid arthritis, ankylosing spondylitis or even psoriatic arthritis. Currently, monitoring of Certolizumab plasma levels is available and provides clinicians with relevant information to improve treatment efficacy. Theradiag has just developed new rapid (results in less than 40 minutes) and innovative assays allowing the quantification of Certolizumab: these tests are available on the new innovative i-Track10 chemiluminescence platform and allow the rapid quantification of Certolizumab as well as anti-antibody antibodies. -Certolizumab induced by the patient.

Methods

Analytical performance was studied using two types of samples: human serum samples spiked with Certolizumab or anti-Certolizumab antibodies, and human serum samples from patients with inflammatory diseases treated with Certolizumab (n=56 ). For drug assay, Certolizumab was captured using TNFα-coupled magnetic microspheres and detected using an acridinium ester-conjugated anti-Certolizumab polyclonal antibody. For the assay of anti-Certolizumab antibodies, microspheres coupled to Certolizumab were used. The anti-Certolizumab antibodies thus captured were detected by Certolizumab conjugated to the acridinium ester. Light emission from acridinium ester conjugates is directly related to the amount of Certolizumab or anti-Certolizumab antibodies present in the sample.

Results

The quantification of Certolizumab by the i-Tracker Certolizumab kit shows very good accuracy (the percentages of recovery are between 84% and 102%). The intra-test and inter-test reproducibility is very good (CV <4.8% for the Certolizumab assay and CV <10.6% for the anti-Certolizumab antibody assay). No interference from biological agents (bilirubin, hemoglobin, triglycerides, rheumatoid factors) was detected. The assay measurement ranges are 3 µg/mL to 100 µg/mL for Certolizumab and 10 AU/mL to 1500 AU/mL for anti-Certolizumab antibodies. i-Tracker assays were compared to ELISA-based Lisa-Tracker assays. The correlations are excellent: the Certolizumab assay presents an R² = 0.94 and a slope of 0.92, the anti-Certolizumab antibody assay presents a Spearman coefficient of 0.94.

Conclusion

The i-Tracker Certolizumab and anti-Certolizumab kits are rapid tests (results obtained in less than 40 minutes) and highly accurate. Very good correlations were observed with the Lisa-Tracker tests (ELISA method) already used routinely. i-Tracker kits are effective tools for monitoring patients treated with Certolizumab.
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

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

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

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 (< 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

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<0.0001). These results stayed significant, in both CD (3.15±2.08 pg/mL; p<0.05), and UC (2.83±12.7 pg/mL; p <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

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 < 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 < 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

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

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

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<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. ￼
P111 Efficacy of a novel long-acting lipidated interleukin-22, alone and in combination with anti-TNF treatment, in a murine chronic DSS colitis model
Authors: Van De Bunt M , D'Alessio S , Norrild J C , Kjølbye A L , Jorgensen R
Published: January 2023

Background

Most current treatments and new agents in clinical development for Inflammatory Bowel Disease focus on inhibition of excessive inflammation. However, to break the ceiling on current clinical remission rates, new approaches, for example targeting mucosal healing, are needed. The cytokine interleukin-22 (IL-22) plays a key role in epithelial healing and has early proof-of-concept as a potential drug class in a phase 1 trial in Ulcerative Colitis. Here we present results from a mouse model of colitis demonstrating the efficacy of IL-22 agonism and complementarity with existing immunomodulatory treatment options.

Methods

Eight-week-old female C57Bl/6N mice (n=56) were subjected to a DSS-induced model of colitis; mice were given 2% (weight/volume) DSS in the drinking water for three 7-day cycles followed by 7 days of regular drinking water. Mice were treated from day 18 until the end of study with either vehicle, lipidated IL-22, an anti-murine TNF antibody at a suboptimal dose, a combination of the lipidated IL-22 and anti-TNF, and, as positive control, anti-murine TNF at an optimal dose. Colitis severity was monitored during the study and at study end endoscopic scoring was performed. Colon length and weight were measured, and histological analysis on formalin-fixed paraffin sections performed by a blinded pathologist.

Results

At termination, all treatment groups showed reduced body weight loss and improved DAI scores compared to vehicle treated animals, which was significant for all but the suboptimal anti-TNF group. Combination treatment resulted in an additive treatment effect compared to single treatment alone, with mean DAI improving by 13% and 48% for the anti-TNF and IL-22 groups alone versus 68% in combination. Similar effects were observed for the endoscopic and histological scores. The compositive endoscopic score improved by 21% (anti-TNF) and 55% (IL-22) versus 66% for the combination, and histological scores 22% and 52% versus 66%. These combination results likely represent the maximum response that can be achieved in the model, since the improvements obtained are the same as those for the treatment positive control for the model (68% and 62% improvements in endoscopic and histological scores, respectively).

Conclusion

A novel long-acting lipidated IL-22, which acts on epithelial repair mechanisms, demonstrated strong and significant improvements on all measured parameters in a chronic DSS model of colitis. Combining the IL-22 mode-of-action with an immunomodulatory agent, exemplified by a murine anti-TNF antibody, demonstrated the complementarity of IL-22 agonism to existing treatment options. Clinical trials to assess safety and tolerability of lipidated IL-22 are expected to commence at the start of 2023.
P115 Trehalose modulates Dextran Sulfate Sodium-induced colitis by regulating macrophage polarization
Authors: Li Y , Law H K W
Published: January 2023

Background

Inflammatory bowel disease (IBD) is a collective term for chronic inflammatory diseases resulting from complex genetic susceptibility, microbial flora, and immune abnormality. Due to the complicated pathogenesis, therapies for IBD are largely limited to the treatment of symptoms. However, these existing drugs sometimes fail or lead to adverse effects in long-term therapies. The development of new drugs and new therapies for IBD are still the top priorities. Trehalose (TRE) is an autophagy inducer through an mTOR-independent pathway. Given its safe and protective nature, TRE has gained significant attention for the potential application in disease treatment. It is a natural product that may be used as a harmless autophagy regulator. In our research, we use the Dextran Sulfate Sodium (DSS)-induced IBD model to explore the therapeutic effects of trehalose on autophagy and inflammation in mice.

Methods

6-8 weeks old C57BL/6J male mice were used for experiments. Mice were given 2% DSS (MP Biomedicals) in autoclaved drinking water for 4 days to induce colitis, Then TRE (2 g/kg body weight, Sigma-Aldrich), rapamycin (1.25mg/kg body weight, MedChemExpress) or normal saline were intraperitoneally injected for 5 days. Mice were weighed daily, and stool consistency, diarrhea, or blood in the stools were monitored to calculate the disease activity index (DAI). At the end of the experiment, the colons were measured, opened longitudinally, and handled according to standard procedures. The tissue samples were collected for RNA, protein extraction, histology, and macrophage isolation respectively. Western Blot was used for autophagy analysis and flow cytometry was used for macrophage subtype and apoptosis analysis. Besides, Real-time quantitative PCR assay was used for examining the cytokine expression.

Results

In TRE-treated mice, the cessation of diarrhea and anal bleeding occurred earlier and hence the DAI scores decreased more rapidly when compared to the other groups. Correspondingly, the colons in TRE-treated mice shortened less than the saline treated control suggesting recovery from DSS-induced colitis. Even though no significant difference was observed in H&E staining and tissue scores for mice in different groups, significant difference in macrophage polarization and cytokine production was observed. DSS-induced intestinal CD86+ macrophages, whereas TRE treatment increased CD206+ macrophages and decreased the expression of pro-inflammatory cytokines, such as IL-6 and TNF-alpha.

Conclusion

Our results demonstrate that TRE decreases the symptoms of experimental colitis by regulating macrophage polarization, probably mediated by macrophage autophagy. These findings indicate the potential of TRE to be an adjuvant therapy for IBD.
P118 Induction of ETS1 and senescence associated secretory phenotype in human intestinal fibroblasts
Authors: Seco-Cervera M , Argilés-Alberola D , Bauset C , Lis-López L , Coll S , Cosín-Roger J , Ortiz-Masià D , Calatayud S , Barrachina M D
Published: January 2023

Background

Fibrosis is a complication commonly present in Crohn’s disease (CD) patients with a structuring (B2) or penetrating (B3) behaviour, with no available treatment. This process is characterized by an excessive extracellular matrix deposition, mainly associated with a dysregulated function of myofibroblasts. We analyse here, the expression of markers of senescence in human intestinal fibroblasts.

Methods

Human small intestinal fibroblasts (HSIF; P10760, Innoprot, Spain) were treated during 48h with 2µg/mL of DLL4, 20ng/ml of TNFα, 2ng/ml of IL-1β, 5ng/ml of TGFβ1, and 100ng/ml of. In some cases, fibroblasts were treated with 20nM of siRNA to ETS1 gene (siETS21) or negative control (NC). Gene expression profiles were analysed by RT-qPCR.

Results

Treatment of fibroblasts with IL-1β significantly increased the mRNA expression of a) different senescence-associated secretory phenotype (SASP) factors (IL-1α, IL-1β, IL-8, and Serpine1); b) a metalloprotease ADAM12 and the chitinase CHI3L1 and c) the transcription factor related to senescence ETS proto-oncogene 1 (ETS1), all compared with vehicle treatment. Treatment with PDGF significantly increased the mRNA expression of ADAM12, ETS1, PTEN and the transcription factor, E2F5, while it induced a non-significant increase in the expression of senescence markers such as P16, P21, P53 and MCL1. Treatment with TGFβ1 did not significantly alter the expression of markers reported above and it only significantly increased the expression of IGFBP3 gene, another SASP factor. Both DLL4 and TNFα failed to significantly modify the expression of any of the above markers. Finally, a cellular model of depletion of ETS1 showed that siETS1 fibroblasts in basal conditions exhibited decreased levels of IL-8 and BCL2.

Conclusion

In human primary intestinal fibroblasts, treatment with IL1b increased the expression of senescence associated secretory phenotype while treatment with PDGF increased the expression of markers involved in cell cycle arrest. The induction of ETS1 by both treatments and the involvement of this transcription factor regulating gene expression in basal conditions, suggest a relevant role for ETS1 in the activation of intestinal fibroblasts
P120 Development and validation of a dedicated score to assess transmural response and transmural healing in patients with Crohn’s disease : final results of the DEVISE-CD project
Authors: Buisson A , Junda J , Vignette J , Lecoq E , Bouguen G , Pereira B , Hordonneau C
Published: January 2023

Background

Transmural healing (TH) is associated with better outcomes that endoscopic remission in Crohn’s disease (CD) but is not yet considered as therapeutic target owing to the lack of validated and consensual definition.

In the DEVISE-CD project, we aimed to 1) build and validate a dedicated score to assess transmural response (TR) and TH in CD patients and 2) to confirm that these definitions of TR and TH are associated with improved outcomes.

Methods

DEVISE-CD project included 3 parts: a monocenter cohort of development (N = 274 patients), a monocenter validation cohort (N = 224 patients) and a prospective multicenter validation study (N = 64 patients). Inclusion criteria are given in Figure 1.

Results

In the development cohort, persistence of edema after treatment (HR = 4.78[1.74-13.13], p=0.002), contrast enhancement (HR=4.13[1.50-11.36], p=0.006), diffusion-weighted hyperintensity (HR=6.97 [2.50-19.41], p< 0.001), bowel thickening (HR = 3.73 [1.61- 8.66], p = 0.002), ulcers (HR = 1.90[1.21- 2.97], p=0.005), fat wrapping (HR=1.87 [1.22-2.87], p=0.004) and enlarged lymph nodes (HR=2.31 [1.48-3.63], p < 0.001) were associated with increased risk of bowel damage progression, contrary to the extent of the lesions. The study of collinearity/coexistence of these different lesions showed that edema, bowel thickening, contrast enhancement, and diffusion-weighted hyperintensity coexisted in > 95% of the cases, giving the same information. The bowel thickness (quantitative value) had the best sensitivity to change (mean difference standard = 0.30). C-score was obtained from a multivariable analysis of factors associated to bowel damage progression (Figure 2).

TH (C-score < 0.5), TR50 (> 50%-decrease of C-score in all active segments) or TR25 (>25%-decrease) were associated with a reduced risk of bowel damage progression in the development cohort (HR=0.28[0.13-0.63], p=0.002; HR=0.30[0.15-0.63], p=0.001; HR=0.37[0.19-0.71], p=0.003, respectively) and in validation cohort (HR=0.15[0.04 -0.53], p=0.003; HR=0.36[0.14-0.88], p=0.025; HR=0.46[0.23-0.94], p=0.034; respectively).

In the multicenter study, TH, TR50 and TR25 rates were 28.3%, 41.3%, and 47.8%, respectively, after 12 weeks of anti-TNF and were associated with remission at W52(CDAI < 150, faecal calprotectin < 250 μg/g and no steroid) (OR = 4.6[1.3–15.6], p=0.016; OR=6.9[1.8–26.0], p=0.008; OR=6.0[1.6–22.3], p=0.008; respectively).

Conclusion

The C-score is a validated, reliable and easy-to-use tool that could be implemented in daily practice and clinical trials to assess therapeutic efficacy using news targets such as TH, TR50 and TR25.
P131 Is stress associated with subclinical inflammation in Ulcerative Colitis? A population-based cross-sectional cohort study in southeastern Sweden
Authors: Borghede M , Marsal J , Faresjö T , Hjortswang H
Published: January 2023

Background

Ulcerative colitis (UC) is an immune-mediated chronic inflammatory bowel disease with a relapsing course. Whether perceived stress affects the disease activity remains debated. This study aimed to investigate if clinical and subclinical disease activity, perceived stress, and hair cortisol concentration (HCC) are associated.

Methods

We performed a population-based cross-sectional cohort study with 200 randomly selected adult patients with UC living in the catchment area of Linköping University Hospital in southeastern Sweden. Demographic and disease-related data, bowel symptoms registration, the 14-item Perceived Stress Scale (PSS), the Short Health Scale (SHS), the Inflammatory Bowel Disease Questionnaire (IBDQ), fecal calprotectin (FC), CRP, and hair samples for hair cortisol concentration (HCC) were gathered. HCC has proven a reliable marker of long-term stress, with one centimeter of scalp hair accounting for approximately 4–6 weeks of stress level. A PRO2 score was calculated. Clinical remission was defined as a PRO2 score of zero.

Results

174 patients participated in the study (45.1% females). The median age was 52.0 years (IQR 40.0–65.0) and the median disease duration was 13.0 years (IQR 6.0–24.0). The median PSS score was 21.0 (IQR 16.0–27.0). 124 patients had a PRO2 score of zero. Patients in clinical remission had a significantly lower PSS score (median 21.0 (IQR 15.0–27.0)) compared to those with PRO2≥1 (median 24.0 (IQR 17.5–27.5)) (p<0.01). Patients experiencing more perceived stress (PSS score>21.0) had an impaired quality of life as measured with the SHS (p<0.01) and the IBDQ (p<0.01) compared with patients with lower PSS scores. The median FC was 43.1 µg/g, CRP 1.5 mg/l and HCC 24.7 pg/mg. HCC was lower in patients with clinically active disease (median 21.9 pg/mg) compared with patients in clinical remission (median 26.0 pg/mg) (p<0.05). No significant differences in FC, CRP or HCC were detected comparing patients with high versus low PSS scores. Neither was a correlation between HCC and FC or CRP observed.

Conclusion

Bowel symptoms contribute to the feeling of being stressed. Perceived stress is associated with impaired QoL. Among patients in clinical remission, no association between subclinical inflammation (FC), and perceived stress or long-term stress (HCC) was observed. The issue of whether perceived stress may cause or augment intestinal inflammation, or vice versa, requires further investigation.
P140 Costs of home-monitoring by telemedicine versus standard care management of Inflammatory Bowel Disease – a Danish register-based five-year follow-up study
Authors: Al-Sheikh M , Olsen J , Spangaard M , Egedesø P , Peters-Lehm C , Fjordside E , Bennedsen M , Burisch J , Munkholm P
Published: January 2023

Background

The increasing incidence of Inflammatory bowel diseases (IBD) with subsequent increasing use of costly biological treatments such as well as the need for tight control of IBD places a significant economic direct and indirect cost burden on health care systems. Thus, eCare Constant-Care APP, home-monitoring of Patient Reported Outcome (PROs-SCCAI/HB-I, quality of life QoL, Disability index WEB-DI) and calprotectin (POCT-Point of Care Test) was introduced at North Zealand Hospital (NOH) in Denmark in 2015.

The aim of this study was to assess long-term cost effectiveness of remote telemedicine services (eCare) compared to standard care (sCare) for IBD patients during 5 years follow-up.

Methods

A retrospective register-based study was carried out among patients with ulcerative colitis (UC) or Crohn’s disease (CD) continuously included in the telemedicine service Constant Care (CC) in 2015-2020. Direct and indirect healthcare costs over a 5-year period were obtained from Danish registers and compared to a control group of IBD patients from all five regions in Denmark who were 1:5 matched on year of birth, gender, education, income, disease, use of biologics, duration of disease and Charlson Comorbidity index. The IBD patients (cases and controls) were identified in the National Patient Register (NPR) via registered ICD-10 diagnosis codes. Direct and indirect costs were estimated on a yearly basis from two years before inclusion in CC (i.e., the index year) and five years after using a linear regression model.

Results

A total of 572 IBD patients were included in the study. Average annual costs of IBD-related outpatient visits were significantly (P<.005) higher among patients enrolled in CC compared to controls before and after index date whereas costs of hospital admissions were non-significant (ns). However, a slight decrease was observed in year 2 and 5 from index year (ns). The total direct costs were higher among CC patients compared to controls in the year prior to enrollment in CC and the five years after (Figure 1) primarily driven by the costs of outpatient visits.

Significantly (P<.05) fewer CC patients were on early retirement compared to controls and CC patients received less public transfer payments compared to controls (ns).

Conclusion

Telemedicine care was associated with higher cost than standard care. One potential confounder is inefficient out-patient registration in NRP which highlights the need for continuous validation of clinical registration approaches in Denmark. A prospective RCT is warranted in future.
P151 Assessing comprehensive remission for Ulcerative Colitis in clinical practice: International consensus recommendations
Authors: Schreiber S W , Danese S , Dignass A , Domènech E , Fantini M , Ferrante M , Halfvarson J , Hart A , Magro F , Lees C , Leone S , Pierik M , Field P , Schofield H , Peyrin-Biroulet L
Published: January 2023

Background

Real-world treatment of ulcerative colitis (UC) requires a patient-centric, holistic definition of ‘comprehensive remission' that goes beyond standard regulatory definitions. In an international initiative, we reviewed aspects of UC that affect patients and attempted to achieve consensus on which aspects should be considered in a definition of ‘comprehensive remission’, using a modified Delphi process. This initiative summarized expert consensus recommendations on aspects of UC that are important to patients and that affect their lives, how to measure and score these aspects, and objective markers of mucosal inflammation that are relevant to evaluate in remission, such as endoscopic and histological activity and inflammatory biomarkers.

Methods

The Delphi consensus panel comprised 10 physicians with expertise in UC and one patient advocate, and two additional physicians chaired the process. To inform the Delphi statements, we asked 18 patients about their experiences of living with UC in remission, asked panel members about their experience of treating patients and systematically reviewed associations between outcome measures in UC. Panel members voted on a 6-point Likert scale in three rounds of anonymous voting, the final round of which was preceded by a live discussion. Consensus was met if ≥ 67% of the panel agreed with the statement (4–6 on the 6-point Likert scale). Statements not meeting consensus in voting rounds 1 and 2 were revised and any not meeting consensus by the end of voting round 3 were discarded (Figure 1).

Results

The panel agreed that a definition of comprehensive remission should include aspects of UC that are currently measured in clinical trials (blood in stools, stool frequency, disease-related quality of life [QoL], endoscopic remission, histological inflammatory activity, inflammatory biomarkers and discontinuation of corticosteroids), but also additional patient-reported symptoms should be added. These were bowel urgency, abdominal pain, extra-intestinal manifestations, fatigue and sleep disturbance (Table 1). Suggestions for how the severity could be measured and thresholds for remission were agreed for each aspect, with the exception of extra-intestinal manifestations. Disease activity evaluated using ultrasound was voted as important to consider but was not recommended to be included in a measure of comprehensive remission at this stage.

Conclusion

We used a robust methodology to reach agreement and provide recommendations on the aspects of UC that can be used to define comprehensive remission and assessed in clinical practice. We plan to develop this as a combined measure which may also have applicability in clinical trials.