bluuebunny/crossref_metadata_2025_split huggingface dataset:
A188 ENHANCED MICROBIAL TRYPTOPHAN METABOLISM ACTIVATES THE ARYL HYDROCARBON RECEPTOR AND AMELIORATES INTESTINAL INFLAMMATION
Authors: Rondeau L , Barbosa B , Dang R , Caminero A 
Published: February 2024

Abstract

Background
Intestinal microbiota, diet, and the immune system have been proposed to contribute to the development of inflammatory bowel diseases (IBD). The aryl hydrocarbon receptor (AhR) is a critical regulator of intestinal immunity and mucosal barrier homeostasis that is activated by agonists such as host and microbial tryptophan metabolites. IBD patients have altered microbiota and reduced AhR agonists in intestinal content resulting in downregulation of AhR. Our recent findings indicate that mice harbouring microbiota with impaired tryptophan metabolism and AhR agonist production develop more severe inflammation during colitis.

Aims
To study the influence of diet-microbe interventions on AhR activation and intestinal inflammation in mice with impaired microbial tryptophan metabolism.

Methods
Germ-free C57BL/6 mice from McMaster’s Axenic Gnotobiotic Unit were colonized with cecal content of mice harbouring reduced microbiota with impaired tryptophan metabolism with and without tryptophan metabolizing Clostridium sporogenes. Following three weeks of microbial stabilization, mice were provided a high tryptophan (HT) diet or a low tryptophan (LT) diet as a control. Experimental colitis was induced using dextran sulfate sodium in drinking water (DSS; 2.5% w/v). Briefly, DSS was provided ad libitum for five days, followed by two days of recovery before tissue collection. Activation of AhR was measured in stool and C. sporogenes cultures using an in vitro AhR luciferase reporter assay. AhR agonists were quantified by capillary-electrophoresis-mass spectrometry. Colonic expression of AhR pathway genes was evaluated by RT-qPCR. Susceptibility to colitis was assessed by histologic analysis of distal colon sections, fecal lipocalin-2, weight loss, and clinical scores. Inflammatory gene transcripts were quantified in colon tissue by Nanostring.

Results
C. sporogenes efficiently produces AhR agonists in vitro and in vivo. In mice with impaired tryptophan metabolism, the HT diet increased AhR agonists in intestinal content and colonic AhR pathway gene expression, which was further enhanced in mice colonized with C. sporogenes. Compared to mice fed LT and HT diets, mice colonized with C. sporogenes and fed HT were most protected from weight loss, tissue damage, and immune cell infiltration.

Conclusions
These findings highlight the potential of tryptophan-rich diets and targeted microbe interventions as a novel approach to enhance AhR activation and mitigate inflammation during colitis. Consequently, exploring methods to improve tryptophan metabolism through diet and manipulation of the microbiota could be a promising therapeutic avenue for IBD patients with impaired AhR activation.

Funding Agencies
CCC, CIHR
A191 THE NEURAL SIGNAL, CALCITONIN GENE-RELATED PEPTIDE (CGRG) ENHANCES A REGULATORY PHENOTYPE IN THE HUMAN IL-4-TREATED MACROPHAGE
Authors: Callejas Pina B E , Sousa J A , Flannigan K , Wang A , Li S , Rajeev S , Panaccione R , McKay D 
Published: February 2024

Abstract

Background
Interlukin-4 activated human macrophages (M(IL4) promote epithelial wound healing and exert an anti-colitic effect in a murine model. Blood monocyte-derived M(IL4)s from healthy donors and individuals with Crohn’s disease had increased mRNA expression of the calcitonin gene-related peptide (CGRP) receptor chain, RAMP1, raising the issue of neural modulation of the M(IL4)s reparative function.

Aims
To determine if CGRP-RAMP1 signalling in human IL-4 treated macrophages enhances the anti-colitic phenotype.

Methods
Peripheral blood mononuclear cells from healthy volunteers (HD) and individuals with Crohn’s disease were cultured on plastic (2h, 37°C) and non-adherent cells removed. The adherent cells were cultured with recombinant hM-CSF (10 ng/ml) for 7 days. The resultant macrophages (2.5×105) were differentiated with IL-4 (48h 10 ng/mL). In other cells, CGRP (10 nM) was added 24h after IL-4 and phenotype assessed by qPCR. Rag1-/- mice were treated with M(IL4)±CGRP (1x106 i.p.): 48h later, colitis was induced by oxazolone. FITC dextran was used to evaluate epithelial barrier integrity. A wound healing assay was conducted using CaCo2 epithelial cells and conditioned medium (CM,1:2 dilution) from the different treatments. Prostaglandin D2 production was evaluated by ELISA and cyclooxygenase (COX)-1 and -2 activity inhibited using SC560 and indomethacin.

Results
Compared to non-treated macrophages (M(0)), M(IL4)s from HD had increased RAMP1 and CLR mRNA. M(IL4)s treated with CGRP showed enhanced expression of CD206, CCL26, RAMP1 and CCL18. When delivered systemically to oxazolone-treated rag1-/- mice, M(IL4,CGRP) had an anti-colitic effect superior to M(IL4)s from the same healthy blood donor. Use of the the human colon CaCo2 cell in vitro wounding model revealed that CM from M(IL4,CGRP) had increased amounts of TGFb and increased wound-healing capacity compared to matched M(IL4)-CM. Moreover, M(IL4,CGRP)s displayed increased COX-1 and PGD2. CM from M(IL4,CGRP)s treated with indomethacin or SC-560 to inhibit COX1 activity failed to promote repair of wounded CaCo2 cell monolayers.

Conclusions
Neuronal input is revealed as an important local modifier capable of boosting the anti-colitic effect of autologous M(IL4) transfer to treat enteric inflammation.

Funding Agencies
CCCHelmsley Charity
A193 DECIPHERING THE INFLUENCE OF CROHN’S DISEASE-ASSOCIATED GENETIC RISK FACTOR, NOD2 IN INTESTINAL FIBRO-STENOSIS
Authors: Mukherjee T , Patel S , Yadav J , Ayyaz A , Tsang D , Narimatsu M , Bayer G , Trcka D , Bader G , Wrana J L , Girardin S E , Philpott D 
Published: February 2024

Abstract

Background
Intestinal fibrosis (IF) is a common debilitating complication associated with Crohn’s disease (CD), an idiopathic inflammatory bowel disease (IBD). IF results in severe strictures that affect the entire gastrointestinal tract, often requiring surgery due to limited or no effective treatment. In general, IF is a consequence of chronic inflammation, excessive extracellular matrix (ECM) deposition, aberrant cellular functions and tissue remodeling involving the innate and adaptive immune system. At the cellular level, fibrosis has been proposed to involve the crosstalk between epithelial cells, mesenchymal stromal cells and immune cells. However, the cascade of events that establish IF is poorly understood as it comprises a dynamic interplay between host genetics, immunity, gut microbiome and aberrant environment insults. Notably, polymorphisms in nucleotide-binding oligomerization domain-containing protein 2 (NOD2; a bonafide cytosolic innate immune receptor), strong genetic risk factors for CD, also increase the incidence of IF. In this context, how NOD2 polymorphisms govern intestinal fibrostenosis in CD remains to be investigated.

Aims
To investigate how CD-associated risk gene, NOD2 contributes to intestinal fibrostenotic disease

Methods
We employed a single-cell RNA sequencing (scRNA-seq) approach to explore the cellular landscape in our CD-mouse model, where we challenged Nod2-deficient mice with a chronic inflammatory insult regime, using dextran sulfate sodium (cDSS) for 3 cycles. Subsequently, changes in inflammatory, metabolic and fibrotic markers in the gut were analyzed by quantitative real-time PCR (qRT-PCR), immunohistochemistry (IHC) or western blot analyses. Further, RNA-In-situ hybridization (RNA-ISH) and flow cytometric analyses were performed to validate cell populations contributing to, and the molecular signatures linked with intestinal fibrostenosis.

Results
We found that Nod2-deficient mice show increased expression of pro-inflammatory cytokines (TNF, IL6) and excessive ECM deposition (fibrosis) in the gut compared to wild-type littermate mice post-cDSS. Curiously, scRNA-seq analysis uncovered altered stromal-immune-cell crosstalk along with the identification of a novel subset of the Dpt+ interstitial stromal population with distinct gene signatures regulated by chronic inflammation. Further, we observed increased senescence (Cdkn2a) mediated trans-differentiation of stroma into fibroblast contributing to IF in Nod2-deficient mice.

Conclusions
The study outlined will deepen our understanding of cellular and molecular mediators as well as inter and intra-cellular networks that drive IF. This will be transformative in devising therapeutic strategies to prevent IF in progressive CD patients.

Funding Agencies
CCC, CIHRThe American Association of Immunologist (AAI)
A194 EARLY LIFE WESTERN-TYPE DIET ACCELERATES THE ONSET OF MURINE IL-10 KO COLITIS
Authors: Ren M , Luchak A , Dang C , Philpott D , Croitoru K 
Published: February 2024

Abstract

Background
Early life is a critical time for gut microbiome and immune development, including the establishment of proper host-microbe interactions. While exposures to western world environmental factors are associated with inflammatory bowel disease (IBD) susceptibility, the link between environment in early life and later disease onset is unclear. One of the most important western world environmental factors is diet, which can distinctly alter the gut microbiome. We believe an early life western-type diet (WD) can affect disease progression in a murine IL-10 KO colitis model through dysregulated T-cell responses against the microbiome.

Aims
We aimed to characterize how an early life WD given to IL-10 KO mice would affect colitis development and the gut microbiome.

Methods
IL-10 KO mice were housed in specific pathogen free conditions and fed normal chow (NC) or WD ad libitum, either between days 10 to 35 (early WD, eWD) or days 35 to 60 (adolescent WD, aWD). Stool was collected from mice at days 35, 56, and 84 for lipocalin-2 (LCN-2) and 16S rDNA sequencing. Mice were sacrificed at day 84, assessing mesenteric lymph node weight, gene expression, colon damage by histopathology, and colon lamina propria leukocyte (LPL) phenotype and cytokine expression by flow cytometry.

Results
Compared to NC and aWD, the eWD fed IL-10 KO mice displayed increased fecal LCN-2 at days 56 and 84, indicating greater inflammation. At sacrifice, eWD fed mice had larger mesenteric lymph nodes and more significant colon damage by histopathological scoring. Colon LPL preparations showed that eWD fed IL-10 KO mice had higher proportions and absolute numbers of effector and regulatory T-cells. Additionally, higher proportions and absolute numbers of those T-cells were IFNγ+, IL-17A+, and IL-22+. qPCR of distal colon tissue revealed increased gene expression of innate and type 3 proinflammatory cytokines such as Il1b, Tnfα, and Il23, while showing no change in Il6 and Il4. Taxa positively associated with WD were able to establish a persistent niche in eWD fed mice but not in aWD fed mice. An increase in only one specific taxon was associated with eWD at all timepoints while also most strongly correlating with inflammatory markers.

Conclusions
This data suggests that eWD feeding during gut microbiome and immune development is uniquely capable of increasing long-term susceptibility to intestinal inflammation in IL-10 KO mice. This appears to be associated with persistent colonization of potentially more inflammatory taxa. This work provides insight into the potential role of early life environmental risk factors, i.e. WD, in the later development of IBD.

Funding Agencies
CAG, CIHRUniversity of Toronto, Taconic Biosciences
A196 CSF2 AUTOANTIBODIES AS A SEROLOGICAL MARKER FOR CROHN'S DISEASE
Authors: Tai S , Del Valle D , Urango R , Croitoru K , Gnjatic S , Korzenik J , Colombel J , Mortha A 
Published: February 2024

Abstract

Background
Crohn’s disease (CD) is a chronic inflammatory disorder of the gastrointestinal tract that severely affects quality of life. Despite technological advances, CD diagnoses remain difficult and invasive, with delayed diagnoses correlated with worse prognosis and complications. CD is multifactorial, involving complex interactions between genetic and environmental risk factors, which leads to difficulty in identifying a cause and cure. However, research alludes to an underlying dysregulation of immune activity that leads to chronic intestinal inflammation. Mononuclear phagocytes (MNP) are essential immune cells that promote intestinal homeostasis through supporting immune tolerance, antimicrobial activity, and barrier integrity. A crucial cytokine that promotes the survival and function of intestinal MNP is Colony Stimulating Factor 2 (CSF2). Intriguingly, previous work in our lab demonstrated that CSF2 autoantibodies (CSF2-Ab) can be detected in the serum of every third CD patient and antedate the onset of CD by up to 6 years. In contrast, these titers are not seen in healthy donors or ulcerative colitis. Moreover, CSF2-Ab were predictive of disease location and severity, and are able to neutralize CSF2 by binding to glycosylations, impacting downstream signaling in MNP. These data suggest a role for CSF2-Ab in promoting intestinal immune dysregulation in CD.

Aims
Our work aims to characterize CSF2-Ab and their role in CD.

Methods
We developed a bead-based flow cytometric assay to screen CD patient serum for CSF2-Ab in several cohorts that contain samples at time points prior to, at, and after diagnosis.

Results
Our preliminary data demonstrate that our assay is rapid and specific for detecting CSF2-Ab of various isotypes in human serum and can be validated using ELISA. Furthermore, we show that this assay can be used to determine the epitope specificity of CSF2-Ab in CD patients in just one single sample.

Conclusions
We have developed a rapid and accessible assay to predict CD development years before diagnosis using minimal serum samples. Moreover, this screen will enable subclassification of patients based on their autoantibody reactivity to glycovariants of CSF2. Glycovariants that escape recognition by CD-specific CSF2-Ab could potentially be used as a therapeutic to ameliorate disease. Beyond treatment, understanding how CSF2-Ab epitope specificity and isotypes may change over the course of disease development will serve as a roadmap for elucidating the role of CSF2-Ab in CD.

Funding Agencies
CAG, CIHR
A197 A ROLE FOR NOD2 IN INTESTINAL T CELL MEMORY DEVELOPMENT AND FUNCTION
Authors: Tsankov B K , Luchak A , Nathan N , Philpott D 
Published: February 2024

Abstract

Background
Aberrant resident memory T cell (TRM) responses have been associated with increased intestinal inflammation and Crohn’s disease (CD) pathology in humans. Intestinal TRM cells are not only important for maintaining the integrity of the intestinal epithelial barrier, but also for the rapid clearance of pathogens in the intestine during infection. Understanding the signals received by the intestinal immune system to generate TRM responses is paramount to elucidating treatments for CD. Genetic mutations in NOD2 are associated with the highest risk of CD development. As a host intracellular sensor of bacterial peptidoglycan, NOD2 is critical for initiating both innate and adaptive immune responses. Furthermore, work from our lab as well as those of our collaborators suggest that NOD2 deficiency reduces systemic memory B and T cell responses. However, the role of NOD2 in establishing memory T cell responses in the intestine remains unclear. This work will therefore establish the role of NOD2 signaling in initiating and maintaining optimal TRM responses to achieve intestinal homeostasis and resilience to intestinal inflammation.

Aims
It is the main objective of this project to determine whether NOD2-mediated signalling affects:
1. Antigen-specific intestinal T cell priming in vivo
2. Bona fide intestinal TRM generation
3. Bona fide intestinal TRM function

Methods
Intraperitoneal LCMV-Armstrong infection results in long-lived CD4+ and CD8+ resident-memory T cell generation in the intestinal lamina propria of mice. Wildtype and NOD2 deficient littermate mice were infected with LCMV-Armstrong, and the effects on intestinal T cell priming, effector function, and memory T cell function were examined by flow-cytometry, ex vivo and in vivo antigen-recall experiments, qPCR, and viral plaque assays.

Results
Augmented NOD2 signalling during priming increased LCMV-specific CD4+ T cell numbers in the mesenteric lymph nodes - but not in the spleen, independent of proliferation or cell death. Furthermore, NOD2-deficiency resulted in decreased effector and memory T cell numbers in the small intestinal lamina propria. This effect was associated with decresead memory CD4+ and CD8+ T cell functions in NOD2-deficient mice during ex vivo and in vivo peptide recall experiments.

Conclusions
NOD2 affects T cell priming in the mesenteric lymph nodes, which later affects effector and memory T cell functions in the intestinal lamina propria.

Funding Agencies
CAG, CIHREmerging and Pandemic Infections Consortium
A203 PERORAL ENDOSCOPIC MYOTOMY (POEM) FOR COMPLEX ACHALASIA AND THE POEM DIFFICULTY SCORE: AN UPDATE
Authors: Ching Hui Yee C , Youssef M , Woo M , Bechara R 
Published: February 2024

Abstract

Background
POEM for achalasia can be challenging in patients with complex achalasia (CA) (i.e., type III achalasia, multiple prior treatments, prior myotomy, and sigmoid type). The POEM difficulty score (PDS) identifies factors that contribute to challenging POEM procedures. (Figure 1) Here, we present an update on the PDS in a series of patients with non-CA and CAs.

Aims
Our aim was to determine whether, with the introduction of the TT-J knife with waterjet functionality, the PDS still maintains a strong correlation with procedural efficiency and technical difficulty.

Methods
We retrospectively reviewed patients who underwent POEM for achalasia between May 2018 to July 2023 at the Kingston Health Sciences Center. 139 consecutive POEMs were performed, with 74 CAs. Primary outcomes include correlation of procedural efficiency with the PDS. Secondary outcomes include clinical success at the last followup and adverse events. Demographics and procedural variables were compared with descriptive statistics (mean ± SD; median, min-max range) and the Mann-Whitney U-test. Pre and post-POEM Echkhardt scores were compared using the Wilcoxon signed-rank test.

Results
74 (56.7% male) complex and 65 (55.4% male) non-complex POEM procedures were carried out. The mean age is 56.7 ± 16.5 years and 47.3 ± 20.2 years in CA and non-CA respectively. PDS correlates moderately with procedural efficiency with a correlation coefficient of 0.595 (Spearman’s Pampersand:003C0.001). A subanalysis is available in Table 1.
The mean efficiency for non-CA was 3.3min/cm±1.2. The median PDS for non-CA was 1 (0-5). In comparison, the mean efficiency for CA were 3.3min/cm ± 1.3 (type III), 5.3min/cm ± 2.3 (prior myotomy), 4.0min/cm ± 1.7 (≥4 prior procedures), and 5.2min/cm ± 2.4 (sigmoid type). The median PDS for CA was 3 (0-6) (type III), 4 (0-8) (prior myotomy), 3 (0-8) (≥4 prior procedures), and 3 (0-6) (sigmoid type).
CA and non-CA POEM procedures were completed with 97% and 98% clinical success, respectively.

Conclusions
PDS continues to moderately correlate with procedural efficiency using the TT-J knife. However, the presence of spastic contractions correlated poorly with procedural efficiency. Thus, it may be omitted with the use of the TT-J knife. The order of increasing difficulty of POEM in CA appears to be type III, prior myotomy, sigmoid type, and ≥4 prior procedures. Multi-center prospective studies are needed to validate PDS in different clinical settings.
Table 1: Sub-analysis of individual PDS factors and procedural velocity

Figure 1: Peroral Endoscopic Myotomy (POEM) difficulty score (PDS): "FOODS"

Funding Agencies
None
A204 DUPILUMAB IMPROVES HISTOLOGIC, ENDOSCOPIC, AND SYMPTOMATIC ASPECTS OF EOSINOPHILIC ESOPHAGITIS IN PATIENTS AGES 1–ampersand:003C12 YEARS
Authors: Chehade M , Dellon E , Spergel J M , Rothenberg M E , Pesek R D , Collins M H , Hirano I , Liu R , Laws E , Mortensen E , Abdulai R , Maloney J , McCann E , Kosloski M P , Hamilton J D , Samuely C , Glotfelty L , Shabbir A , Chow M 
Published: February 2024

Abstract

Background
Dupilumab is approved in Canada for eosinophilic esophagitis (EoE) in patients (pts) aged ≥12 years who weigh ≥40 kg; no treatments are approved for pts aged ampersand:003C12 years.

Aims
The Phase 3 EoE KIDS trial evaluated dupilumab efficacy and safety vs placebo (PBO) in pts aged 1–ampersand:003C12 years with EoE.

Methods
Part A was a 16-week (W) PBO-controlled study; pts were randomized 2:2:1:1 to weight-tiered dupilumab on a higher-exposure (HE) or lower-exposure (LE) regimen, or PBO (2 groups) to W16. In Part B, pts continued dupilumab, or switched from PBO to dupilumab (HE/LE), to W52.

Results
At W16 (Part A), the HE dupilumab group had an improved proportion of pts achieving peak esophageal intraepithelial eosinophil count (PEC) ≤6 eosinophils/high-power field (hpf) vs PBO (67.6% vs 2.9%, Pampersand:003C0.0001; primary endpoint). At W52 (Part B), 62.9% of HE dupilumab/dupilumab pts, and 52.9% of PBO/HE dupilumab pts, achieved PEC ≤6. At W16, the following improved with HE dupilumab vs PBO: EoE-Histologic Scoring System grade and stage scores (–0.88 vs +0.02 and –0.84 vs +0.05, respectively, both Pampersand:003C0.0001); EoE-Endoscopic Reference Score (–3.5 vs +0.3, Pampersand:003C0.0001); overall caregiver assessed EoE status (global impression of change mean [SD], 2.1 [1.2] vs 3.3 [1.4]; lower scores represent more improvement). Improvements were maintained with continuous dupilumab and improved in PBO/dupilumab pts at W52. LE dupilumab maintained similar improvements achieved through W52; however, effects were generally either comparable or numerically lower with LE dupilumab vs HE dupilumab. At W16, COVID-19, rash, headache, and injection-site erythema occurred more frequently in patients receiving dupilumab, with higher incidence than PBO; AEs were similar through W52.

Conclusions
HE dupilumab met the primary endpoint of PEC ≤6 eos/hpf vs PBO, demonstrated significant changes in histologic and endoscopic aspects of EoE, and improved symptoms with benefits maintained/increased to W52.

Funding Agencies
Research sponsored by Sanofi and Regeneron Pharmaceuticals Inc.