bluuebunny/crossref_metadata_2025_split huggingface dataset:
P0057 METTL3 potentiates aged CD4+T cell response via programming effector differentiation in elderly ulcerative colitis
Authors: Zhang Y , Xu J , Cao S , Wang J , Lin Y , Liu Y 
Published: January 2025

Abstract

Background
The burden of IBD in the elderly is increasing globally. However, the mechanisms underlying the age-related IBD susceptibility remain unclear. Aging leads to a progressive functional decline of the immune system, rendering the elderly increasingly susceptible to inflammatory and autoimmune disease. As a fundamental mRNA modification, N6-methyladenosine (m6A) participates in various pathological processes. However, the role of METTL3-mediated m6A RNA modification in elderly IBD remains unclear. Herein, we sought to determine the role of immunosenescence in age-dependent colitis and to explore the underlying mechanisms.

Methods
Young (2-3 months), middle-aged (10-12 months), and aged (20-24 months) mice were established models of colitis by 2% dextran sulfate sodium salt (DSS) treatment. We detected the senescence-associated-β-galactosidase (SA-β-Gal) activity in lymphocytes isolated from colon of mice. Single-cell and bulk RNA-seq of colonic immune cells in mice with different ages were performed to investigate the precise molecular mechanisms. CD4+CD25-CD45RBhi T cell adoptive transfer model was used to further analyze the role of senescent CD4+T cells in colitis. Mettl3fl/lfCd4Cre mice were obtained to explicit role of METTL3 in regulating CD4+T cell senescence.

Results
Ageing increased the severity of DSS-induced colitis. CD4+T cell senescence was implicated in the progression of aging-related experimental colitis, exhibiting an increase of SA-β-Gal and accumulation of CD4+ effector memory T (TEM) cells in colon. Aged colonic CD4+ T cells generate higher levels of IFN-γ and IL-17 comparing to young colonic CD4+ T cells. Single-cell analysis revealed that aging increased colonic CD4+ TEM cells, which were associated with T cell-mediated cytotoxicity and cytokine-mediated signaling pathway. Adoptive transfer of aged CD4+CD25-CD45RBhi T cells to Rag-/- mice induced more severe colitis compared with young CD4+CD25-CD45RBhi T cells transfer. Aged CD4+T potentiates Th1/Th17/Tfh differentiation and exacerbates the progression of colitis. METTL3 was increased in aged colonic CD4+T cells. Inhibiting METTL3 in CD4+T cell protects from cellular senescence, effector differentiation and colitis.

Conclusion
These results provide a significant insight into the contribution of senescent CD4+ T cells to age-dependent colitis and provide an attractive possibility that targeting T cell specifically might be a potential strategy to treat elderly IBD patients.
P0058 Modulation of early innate and adaptive immune responses in primary human intestinal tissue ex vivo
Authors: Beneke V , Grieger K , Dehmel S , Neuhaus V , Linge H , Kulik U , Aselmann H , Gundert B , Braun A , Hesse C , Sewald K 
Published: January 2025

Abstract

Background
Intestinal immune cells are organized within specialized tissue niches shaped by the local environment, including dietary components and microbiota. Dysregulation of these interactions can lead to chronic inflammation, a key feature of inflammatory bowel disease. Precision-cut intestinal slices (PCIS) preserve the intestinal microanatomy, including immune cells. However, functional immune responses in PCIS have not been analysed in detail. Here, we aimed to characterise early innate and adaptive immune responses and evaluate the modulatory effects of the bacterial metabolite butyrate in primary human intestinal slices ex vivo.

Methods
PCIS were prepared from ileum or colon tissue of Crohn's disease (CD) patients or patients with other background diseases (non-CD) and stimulated with lipopolysaccharides (LPS), anti-CD3/CD28 antibodies or Concanavalin A (ConA) ± butyrate (10 mM) for 24h. Secretion of 32 immune mediators was analysed in the supernatant by multiplex ELISA. Tissue viability was measured by LDH- and ATP-assay. PCIS were stained with haematoxylin-eosin for histological imaging.

Results
PCIS maintained typical morphology and viability during culture. Of the 32 immune mediators analyzed, 28 were detected in the supernatant and grouped according to their overall secretion levels. Low secretion (≤ 0.1 ng/mL) was observed e.g. for Th2-associated cytokines IL-4, IL-5 and IL-13 as well as epithelium-associated cytokines IL-18 and IL-33. Moderate secretion (≤ 1 ng/mL) was observed for IL-2, TRAIL, IL-17F, GM-CSF, TNF-α and CCL20. High secretion (≤100 ng/mL) was observed for IFN-γ, granzyme A/B, IL-17A, IL-22, CXCL5 and MCP-1. LPS, ConA and anti-CD3/CD28 upregulated the secretion of mediators in tissue from both non-CD and CD patients. Compared to ConA-stimulated tissue from non-CD patients, ConA-stimulated tissue from CD patients showed increased absolute secretion of IL-2 (log2FC: 1.03), TNF-α (log2FC: 3.32), IFN-γ (log2FC: 1.08), IL-17F (log2FC: 3.29), IL-22 (log2FC: 1.36), IL-21 (log2FC: 1.32) and IL-23 (log2FC: 1.50), though relative upregulation was higher in non-CD tissues. Butyrate reduced the ConA-induced release of proinflammatory mediators (e.g. IL-2, IL-17A: 2-3-fold) independent of the disease background.

Conclusion
Our data show robust induction of early innate and adaptive immune responses in primary human intestinal tissue ex vivo, highlighting PCIS as a promising translational model to study pathomechanisms and treatment responses in a patient-specific manner. Further research into immune cell regulation by butyrate and other microbial metabolites directly in patient tissues could support the development of novel therapeutic strategies for IBD.
P0062 Circulating immune cell populations as predictive markers of vedolizumab response in Crohn disease
Authors: Soleto I , González-de Castro C , Arribas E , Ramirez C , Baldan-Martin M , Riestra S , Lucendo A J , García M J , Martín-Arranz E , De Prado Á , Casanova M J , Chaparro M , Bernardo D , Gisbert J P ,   *Authors share co-senior authorship. $ These authors contributed equally to this work
Published: January 2025

Abstract

Background
Crohn's disease (CD) is a chronic inflammatory condition of the gastrointestinal tract, with a globally rising incidence. It may result from a complex interaction among genetic predisposition, environmental influences, and alterations in gut microbiota, leading to dysregulation of both innate and adaptive immune responses. Despite advances in understanding CD, the precise immunological mechanisms underlying this disease remain unclear. Currently, biological therapies, such as vedolizumab, are among the most effective options for inducing clinical remission and controlling inflammation in CD. However, approximately two-thirds of patients do not respond adequately to these treatments. Consequently, it is essential to identify immune cell populations that can predict clinical responses to vedolizumab, facilitating more personalized treatment selection for each patient.

Methods
We characterized peripheral blood mononuclear cells (PBMCs) using spectral flow cytometry with a panel of 40 markers in patients with CD, both before and after 14 weeks of treatment with vedolizumab. Patients were categorized as responders or non-responders based on their SES-CD.

Results
At baseline Hierarchical phenotypic analysis revealed a total of 39 unique subsets of immune cells within PBMCs (Figure 1). The main populations included T cells (66%), monocytes (0.25%), B cells (12%), and natural killer cells (11%). Within the T cell group, CD4+ cells were twice as prevalent as CD8+ cells. In our evaluation of conventional dendritic cells percentages, we noted an increase in this subset within the responder group, contrasting with the trend observed in macrophages. Furthermore, an unsupervised analysis applying dimensionality reduction algorithms identified 59 clusters (Figure 2), with 37 of them showing differences when comparing responder and non-responder patients. Interestingly, no differences were observed between pre-treatment and post-treatment samples.

Conclusion
This study presents the first immunophenotyping analysis in CD patients, comparing responders and non-responders to vedolizumab based on SES-CD and highlighting differences between these groups. For the first time, these results reveal circulating immune populations as potential predictors of response to vedolizumab. Although these findings should be validated in a larger cohort to assess their clinical significance in identifying patients most likely to benefit from each therapeutic approach, they open the possibility for changes in clinical practice.
P0066 Enhanced transcription of human endogenous retroviruses and downregulation of TRIM28 in patients with Crohn’s disease and ulcerative colitis
Authors: Ribaldone D G , Bergallo M , Galliano I , Veglio V , Pitoni D , Caviglia G P , Frara S , Anxhela P , Tribocco E , Tovo P A 
Published: January 2025

Abstract

Background
Human endogenous retroviruses (HERVs) represent 8% of the human genome. They originate from ancestral infections of germinal cells. HERVs are no longer infectious, but some retroviral sequences can be activated, and their aberrant expressions have been implicated in a number of diseases, including chronic inflammatory and autoimmune disorders. HERV transcription is regulated by TRIM28 and SETDB1, which are directly involved in epigenetic processes and in the modulation of the immune response. HERVs and TRIM28/SETDB1 expressions have not been investigated in patients affected by inflammatory bowel disease (IBD).

Methods
We assessed, through a PCR real-time Taqman amplification assay, the transcription levels of pol genes of HERV-H, -K, and -W families, of env genes of syncytin 1 (SYN1), SYN2, and HERV-W, as well as of TRIM28 and SETDB1 in whole blood of 48 patients with Crohn’s disease (CD), 20 with ulcerative colitis (UC) and in age-matched 104 healthy controls (HC).

Results
The transcriptional levels of HERV-H-pol (p=0.0003) and HERV-K-pol (p=0.001) were significantly higher in IBD patients as compared with HC, while no significant differences between the two groups were found for the remaining HERVs (Figure 1).
The median mRNA levels of HERV-H-pol in CD was 1.53 (1.22–2.02), in UC was 1.56 (0.96–1.99), in HC was 1.24 (0.63–1.63); median HERV-K-pol in CD was 1.27 (1.00–1.76), in UC was 1.41 (0.99–1.85), in HC was 0.97 (0.71–1.34).
The mRNA levels of TRIM28 were significantly downregulated in IBD patients (median mRNA levels of TRIM28 in CD = 0.73, 0.56–0.92, in UC = 0.76, 0.60–0.92, in HC = 1.01, 0.79–1.25, p &lt; 0.001), while the SETDB1 levels were preserved (p = 0.95) (Figure 2).

Conclusion
Over-expressions of HERVs and impaired transcription of TRM28 in patients affected by CD or UC suggest that they might be main actors in the pathophysiology of IBD and open the way to innovative targeted interventions.
P0068 Distribution of α4β7 integrin and MAdCAM-1 expression according to the location of intestinal inflammation in a murine model of spontaneous chronic enterocolitis
Authors: Martinez Lozano H , Puerto Cantero M , Ruiz-Martín C , Miranda-Bautista J , Marín-Jiménez I , Menchén Viso L 
Published: January 2025

Abstract

Background
The selective blockade of α4β7 integrin is a treatment for inflammatory bowel disease (IBD), with different efficacy according to the location of inflammation along the gastrointestinal tract. A more pronounced effect has been reported in patients with predominant colonic inflammation. It may be attributed to differences in the regional expression of adhesion molecules. This study aimed to investigate the expression of α4β7 integrin and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in colon, ileum, and mesentery, using IL-10 knockout (KO) mice as a model of spontaneous chronic enterocolitis.

Methods
At 20 weeks, the colon, ileum, and mesentery were extracted from IL-10KO and C57BL/6 wild-type (WT) mice. Protein levels of α4β7 integrin and MAdCAM-1 were determined by Western blot in the different tissues. Additionally, pro-inflammatory cytokines (TNF-α and IFN-γ) and epithelial adhesion molecules (Zonula Occludens-1, ZO-1; E-cadherin and Mucin 2, Muc2) expression in the ileum, as well as adiponectin expression in the mesentery, were analyzed using real-time PCR.

Results
Analysis demonstrated a significant increase in gene expression of the pro-inflammatory cytokines TNF-α and IFN-γ in the ileum of IL-10 KO mice compared with WT mice (p&lt;0.01). In addition, IL-10KO mice exhibited reduced expression of ZO-1, E-cadherin, and Muc2 in the ileum relative to WT mice (p&lt;0.001). IL-10 deficiency also led to a marked decrease in adiponectin expression in the mesentery (p&lt;0.05). We then analyzed the protein level of MAdCAM-1 and α4β7 integrin according to anatomic region. Remarkably, the protein analysis revealed significantly higher levels of MAdCAM-1 (p&lt;0.01) and α4β7 integrin (p&lt;0.05) in the colon of IL-10 KO mice compared to WT. In the ileum, while MAdCAM-1 levels remained unchanged, α4β7 integrin expression was increased in IL-10KO mice (p&lt;0.01). No significant differences in MAdCAM-1 or α4β7 integrin levels were observed in the mesentery between IL-10KO and WT mice

Conclusion
IL-10-deficient mice develop inflammation in the ileum, accompanied by a reduction in adiponectin production in the mesentery. In this setting, both α4β7 integrin and MAdCAM-1 showed increased expression in the colon, which was not observed in the ileum or mesentery, despite the inflammation. This differential regional expression pattern of adhesion molecules may explain the variation in the efficacy of anti-integrin therapies. Characterizing these patterns in patients with IBD could help identify those who would benefit most from these treatments.
P0072 Unveiling mesenteric and intestinal changes in Crohn’s disease: insights into fibrosis from surgical specimens
Authors: Joo Y H , Park Y , Song E M , Choe A R , Byeon J R , Noh G T , Kim H S , Choi E , Byun J , Park J , Ahn Y H , Jung S A 
Published: January 2025

Abstract

Background
Recent studies have emphasized the role of mesenteric changes, including creeping fat, alongside intestinal alterations in the progression of Crohn's disease (CD). This study aims to investigate intestinal fibrosis and mesenteric changes using tissues obtained from CD patients who underwent surgery for complications such as strictures and perforations.

Methods
From September 2023 to September 2024, intestinal (CD-Inflamed-Bowel/CD-Uninflamed-Bowel) and mesenteric tissues (CD-Inflamed-Mes/CD-Uninflamed-Mes/ CD-Uninvolved-Mes) were collected from nine CD patients who underwent surgery. Additionally, intestinal (Normal-Bowel) and mesenteric tissues (Normal-Mes) were obtained from one patient who underwent anterior resection for adenoma. Specimens were obtained from the small intestine (4 patients) and the colon (5 patients). Samples were analyzed for morphological changes (H&E staining), collagen deposition (Masson’s trichrome), and fibrosis marker expression (qPCR).

Results
Preoperative CT or MR enterography revealed the presence of creeping fat in all nine CD patients, with five showing strictures accompanied by upstream bowel dilatation. In intestinal tissues, increased collagen fiber deposition was observed in CD-Inflamed-Bowel compared to CD-Uninflamed-Bowel, whereas mesenteric tissues showed no significant differences in collagen deposition among Uninvolved, Uninflamed, and Inflamed groups. qPCR analysis of intestinal tissues revealed significantly higher expression of the fibrosis marker COL1A1 in CD-Inflamed-Bowel compared to Normal-Bowel and CD-Uninflamed-Bowel (119.9 ± 260 vs. 1.0 ± 0.0 vs. 1.0 ± 0.7, P = 0.041). In mesenteric tissues, COL1A1 expression was significantly elevated in CD patients compared to controls (28.0 ± 7.7 vs. 1.0 ± 0.0, P = 0.002), though no significant differences in COL1A1 expression were found among Uninvolved, Uninflamed, and Inflamed mesenteric tissues within the same patient (17.7 ± 3.7, 25.1 ± 9.2, 41.0 ± 21.2, P = 0.9372). Other fibrosis markers (Vimentin, TGF-β, MMP-1, CHI3L1, CXCL1, and PDPN) showed similar trends.

Conclusion
This study highlights inflammatory and fibrotic changes in the intestine and mesentery during CD progression. Notably, mesenteric fibrosis was evident even in uninvolved intestinal segments without active inflammation, suggesting mesenteric inflammation and fibrosis may precede intestinal involvement. These findings support the mesentery as a novel potential target for early treatment for disease progression of CD.
P0074 Colonic goblet cell dysfunction and gastric intestinal metaplasia in patients with anterior gradient 2 defects and infantile Inflammatory bowel disease
Authors: Azabdaftari A , Al-Shaibi A , Thomas T , Jainarayanan A , Capitani M , Verleur N , Agarwal D , Dooley K , Rich-Griffin C , Provine N , Klenerman P , Elawad M , Travis S , Buckley C , Dendrou C , Lo B , Uhlig H 
Published: January 2025

Abstract

Background
Biallelic anterior gradient 2 (AGR2) loss-of-function variants cause infantile-onset inflammatory bowel disease, alongside histological changes of the gastrointestinal epithelium. The underlying mechanisms on epithelial development and function are not understood.

Methods
We conducted single-cell RNA-sequencing of gastrointestinal biopsies and organoids from AGR2-deficient patients. Organoids were used for functional investigation of the epithelium and mucus layer by flow cytometry, confocal microscopy, and using a microfluidic organ-chip. Multiplexed imaging was used to characterise intestinal metaplasia in the antrum.

Results
AGR2-deficiency caused region-specific changes in epithelial cell populations. In the gastric antrum, AGR2-deficiency led to defective epithelial cell differentiation into premalignant metaplastic enterocytes associated with an up-regulation of gastric cancer markers. In the colon, despite a histological depletion of goblet cells, single-cell analysis demonstrated an over-abundance of goblet cells in AGR2-deficiency, exhibiting high ER-stress. We identified defective processing of MUC2 and reduced MUC2 storage in goblet cells without signs of crinophagy, suggesting a stress-induced secretion. Similar to AGR2-deficiency, in ulcerative colitis, goblet cells revealed high ER-stress and no reduction in cell numbers.

Conclusion
Our data highlight AGR2-deficiency as a genetic model of precancerous intestinal metaplasia in the gastric antrum. Apparent colonic goblet cell depletion in AGR2-deficiency and in ulcerative colitis is a histological artefact, representing a reduction of stored mucins in goblet cells via stress-induced mucus secretion.
P0079 Serum calprotectin [cpa9-hne], a biomarker of neutrophil activity, is associated with histological inflammation and fibrosis burden
Authors: Sorokina Alexdóttir M , Poulsen A , Tsapanou Katranara T , Buhl Riis L , Pehrsson M , Rasmussen J , Gögeneur I , Burisch J , Karsdal M A , Bay-Jensen A C , Seidelin J B , Mortensen J H 
Published: January 2025

Abstract

Background
Infiltration of neutrophils into the intestinal tissue is a crucial component of the inflammatory response in Crohn‘s disease (CD). CPa9-HNE, a calprotectin fragment released by human neutrophil elastase during NETosis, has been shown to strongly correlate with endoscopic disease activity in inflammatory bowel disease (IBD) and serves as an indicator for pharmacodynamic response. Furthermore, emerging data suggests that neutrophil extracellular traps (NETs) significantly contribute to the pathogenesis of fibrosis. For the first time, we investigated the association of CPa9-HNE with histological grading of both inflammation and fibrosis.

Methods
The cohort included 62 patients with stenotic Crohn‘s disease. Inclusion criterion was the presence of stenotic disease requiring bowel resection, as confirmed by radiographic assessment. Upon surgery, the resected specimens were collected and histologically graded according to transmural fibrosis burden, and inflammation (D'Haens score). Serum was drawn at baseline (before resection). CPa9-HNE was measured, reflecting a human neutrophil elastase-derived fragment of calprotectin. Spearman’s rank correlation was applied.

Results
CPa9-HNE showed an overall positive correlation to the D'Haens score (ρ=0.279, p&lt;0.005). Significant positive correlation was observed between CPa9-HNE and the presence of neutrophils, both in the epithelium and the lamina propria (ρ=0.309, ρ=0.349, p&lt;0.005 both), as well as with epithelial damage (ρ=0.262, p&lt;0.005). We also observed a positive correlation between CPa9-HNE and transmural fibrosis burden, specifically fibrosis in the submucosa (ρ=0.294, p&lt;0.005)

Conclusion
This study showed that CPa9-HNE, a biomarker of neutrophil activity, correlated with the presence of neutrophils in the intestinal tissue, as well as epithelial damage. Interestingly, CPa9-HNE also showed association to submucosal fibrosis, emphasizing the importance of considering neutrophil activity when targeting the fibro-inflammatory axis. These findings highlight the potential use of CPa9-HNE as a complementary tool for histological grading of inflammation and fibrosis burden, and could be used to further explore the role of neutrophil extracellular traps in intestinal fibrosis.
P0081 Novel in vitro protocol to isolate and expand murine polyclonal Treg cells for efficacy testing in murine models of IBD
Authors: Bretin A , González-King Garibotti H , Benonisson H , Preti M , Peris E , Burgueno J , Vaithilingam V , Häll M , Brännud I , Kurhe Y , Aldridge J , Jennbacken K , Nilsson G , Piras A , Dazzi F , Kozlowska A , Borde A , Birrell M , Preite S 
Published: January 2025

Abstract

Background
Regulatory T cells (Treg) are a subset of CD4+ T cells that play a crucial role in maintaining immune tolerance and preventing autoimmunity. In the context of Inflammatory Bowel Disease (IBD), dysregulation of Treg function has been implicated in the pathogenesis of the disease. Studies have shown that polyclonal Treg have the potential to suppress inflammatory responses and ameliorate colitis in preclinical models. This has led to a growing interest in harnessing the therapeutic potential of polyclonal Treg cells as a potential treatment for IBD. To support this new research and effectively produce Treg, we prompted the in-house development of a new platform to isolate, in vitro expand, and evaluate murine regulatory T cells in mouse models of IBD.

Methods
We used a two-step purification method using a pre-enrichment of CD4/CD25/CD62L-postive cells followed by FACS sorting and alternative cell culture methods to establish a protocol for the scalable isolation and expansion of naïve murine polyclonal Treg (muTreg). We then assessed the efficacy of the muTreg in the CD45RBhigh naïve CD4+ T cell transfer model of colitis.

Results
Quality check of the isolated muTreg showed a high purity that was sustained over a 7-day in vitro expansion period (over 96% FOXP3+) with an over 10-fold expansion. muTreg also conserved their functional activity and showed a 70% suppression of naïve T cells in vitro. Upon administration into the T cell transfer model, muTreg led to a dose-dependent improvement of colitis, as demonstrated by a decrease of inflammation markers such as colon weight, pro-inflammatory cytokine/chemokines, and colon histopathological score. muTreg also showed a strong, dose dependent suppression of effector CD4+ T cells in vivo despite a significant loss of FOXP3 expression under inflammatory conditions (over 60% loss of FOXP3 expression).

Conclusion
In summary, we successfully developed a novel platform for isolating and expanding muTreg and demonstrated their ability to alleviate T cell-induced colitis in a dose-dependent manner. Thus, our preclinical data lay the foundations for the investigation of the effect of Treg therapies in pre-clinical models and pave the way to study antigen specific CAR Treg cells or CRISPR-edited Treg in murine in vivo IBD models.