bluuebunny/crossref_metadata_2025_split huggingface dataset:
A51 MICROBIALLY-MEDIATED IMPAIRMENT OF VAGAL AFFERENT NEURONAL EXCITABILITY IS OREXIN RECEPTOR DEPENDENT
Authors: Sachdev S , Tashtush A , Alward T , Reed D E , Lomax A E 
Published: February 2024

Abstract

Background
An impairment of vagally-mediated satiety signalling has been implicated in the caloric imbalance that leads to weight gain during obesity. Previous studies have suggested that a reduction in the excitability of vagal afferent neurons with cell bodies in nodose ganglia (NG) was responsible, but the cellular mechanisms are unclear. Host and bacterially derived mediators present in the small intestine and stool provide a physiologically relevant model to help elucidate the role luminal mediators play in modulating vagal afferent neuronal excitability.

Aims
We hypothesize that the microbiota of obese individuals and mice produce mediators that impair NG neuron excitability and satiety in mice.

Methods
Perforated patch clamp was used to measure the excitability of NG neurons following exposure to human and mouse fecal supernatants (FS), mouse jejunal supernatants (JS), and mice serum samples. Human FS were from ampersand:003E 5 healthy human donors or FS from ampersand:003E 5 obese donors. Mice FS, JS and serum samples were collected from ampersand:003E 5 obese mice fed a high-fat diet and ampersand:003E 5 control mice fed a normal diet.

Results
NG neurons incubated in FS from obese participants were significantly less excitable (rheobase was 30% higher and action potential discharge at 2x rheobase was 50% lower) than NG neurons exposed to FS from non-obese participants. NG neurons incubated in FS or JS from obese mice were also significantly less excitable (rheobase was 65% higher and action potential discharge at 2x rheobase was 50% lower) than NG neurons incubated with FS or JS from control mice. Lastly, NG neurons incubated with obese mouse serum were significantly less excitable (rheobase was 50% higher and action potential discharge at 2x rheobase was 80% lower) than NG neurons incubated with serum from control mice.
We then attempted to identify mediators that may account for this inhibitory effect by using receptor antagonists that block GABA, ghrelin, and orexin signalling. Ghrelin and GABA receptor antagonists did not block the inhibitory effect of obese patients’ FS on NG neurons but the orexin receptor 1 antagonist (SB-334867;10µM) did. Following this, we incubated the orexin receptor 1 antagonist (SB-334867;10µM) on NG neurons incubated with mouse FS and JS and observed a similar blocking of inhibitory effects back to control values.

Conclusions
These findings suggest that the gut luminal contents of obese mice and humans contain an orexin receptor agonist that inhibits satiety and may contribute to over-eating.

Funding Agencies
CIHRNSERC
A53 MOLECULAR MECHANISMS FOR A ROLE OF SHP-2 IN TLR4 SIGNALING IN INTESTINAL EPITHELIAL CELLS
Authors: Chabot V , Lévesque D , Boisvert F , Rivard N 
Published: February 2024

Abstract

Background
Shp-2 gene polymorphisms are associated with greater susceptibility to ulcerative colitis. How Shp-2 contributes to disease susceptibility is however unclear. Intestinal epithelial cell (IEC)-specific deletion of Shp-2 results in severe colitis in mice (Shp-2IEC-KO). We found important alterations in microbiota composition, namely increased Enterobacteria of the Proteobacteria phylum and decreased Firmicutes levels, that preceded clinical signs of inflammation. Antibiotherapy or epithelial KO of Myd88 inhibits colitis in Shp-2IEC-KO mice. Notably, feces from Shp-2IEC-iKO mice display much more LPS-like bioactivity than do feces from controls (unpublished data). These observations support a role for TLR4/Myd88-bacterial recognition in initiation of colitis in Shp-2IEC-KO mice.

Aims
The aim of the present study was to identify the molecular mechanisms by which Shp-2 may regulate TLR4 signaling in IEC.

Methods
1) The regulation of Shp-2 phosphorylation was analysed in IEC-6 cells stimulated or not with 10 ug/ml of LPS, a TLR4 ligand. 2) We mapped the Shp-2-dependent interactome in LPS-stimulated IEC-6 cells by an APEX2 proximity assay used in combination with mass spectrometry (MS) analysis. Cells stably expressing APEX2-tagged Shp-2 were generated. Addition of doxycycline results in expression of “physiological” levels of Shp-2;APEX2 construct. Proximity biotinylation labeling of Shp-2 by APEX2 was performed after 10 min stimulation with LPS and labeled candidates were analyzed by MS.

Results
1) Upon stimulation of IEC-6 cells with LPS, Shp-2 protein is rapidly phosphorylated (Y542, Y580) and potentially activated. This increased phosphorylation is abolished by pretreatment with N-acetyl cystein, a commonly used antioxydant. 2) Using Prostar R package, the three replicates of MS data were filtered for the minimum quantification of 66,66% of each protein in at least one condition. They were normalized within conditions using VSN normalization, then the Partially Observed Values (POV) and the Missing in Entire Column (MEC) values were imputed respectively with SLSA and DetQuantile (Quantile 1%, Factor 0,2). A differential analysis between the LPS and non-treated conditions identified 334 proteins enriched with a FC of ± ≥2,5 with a p-value inferior then 0,05. Among the top Shp-2 interactors, we found Tak1 (ampersand:003E3,5X change) and Tab1 (ampersand:003E3,0X change), two proteins involved in NF-kB signaling. Decreased interaction with the TLR effectors Myd88 (ampersand:003C2,5X change) and Irak1 (ampersand:003C3,2X change) was also noted after LPS treatment.

Conclusions
These data suggest for the first time that Shp-2 may regulate NF-kB signaling downstream of TLR4 in IEC. Thus, SHP-2 may contribute to the maintenance of mucosal tolerance to microbiota in the colon.

Funding Agencies
CIHR
A54 SHP2 FUNCTION IN NORMAL AND APC-MUTANT INTESTINAL EPITHELIAL CELLS
Authors: Côté-Biron A , Gagné-Sansfaçon J , Rivard N 
Published: February 2024

Abstract

Background
Somatic SHP2 gain-of-function mutations were found in some colorectal tumours. However, its function in colorectal epithelium remains to be determined. SHP2 is a tyrosine phosphatase necessary for RAS/MAPK pathway activation by most, if not all, tyrosine kinase receptors, as well as by GPCRs and cytokine receptors. SHP2 can also regulate the PI3K, Jak/Stat, RhoA and Hippo pathways in different cell settings. As a result, SHP2 can control cellular processes including growth, migration and differentiation. Previously, we observed that SHP2 levels are enhanced in human colorectal tumours, notably in late adenomas. Likewise, SHP2 silencing inhibited ERK activation and tumour properties of established human CRC cell lines. Hence, these findings support a model in which SHP2 activation promotes tumorigenesis in the intestinal epithelium. But how?

Aims
To analyze the exact cellular and molecular mechanisms underlying SHP2 pro-oncogenic function in intestinal epithelial cells.

Methods
The impact of SHP2 pharmaco-inhibition (RMC-4550 and/or SHP099) on intracellular signaling, cell cycle, proliferation and survival was evaluated in three complementary models: 1) HIEC-6 cells, normal human fetal intestinal crypt-like cells; 2) organoids derived from normal mouse small intestine or ApcMin/+ tumors; 3) Caco-2/15 cells, colorectal cancer cells with APC mutation.

Results
1- Pharmaco-inhibition of SHP2 blocks proliferation of HIEC-6 cells (loss of pRb phosphorylation, decreased EdU staining) and induces cell enlargement and darkening of the outside of perinuclear area. This phenotype was reminiscent of cellular senescence, and we indeed observed increased senescence-associated β-galactosidase activity (SA-β-Gal) and INK4A gene expression. 2- As expected, SHP2 inhibition in normal mouse organoids resulted in a reduction in the number of protrusions and EdU positive cells. RNA sequencing was performed, and gene set enriched revealed significant down regulation of several Ras signaling-dependant genes including cell cycle regulated genes (E2F targets, G2/M checkpoint) and Myc targets 48h after SHP2 inhibition when compared to DMSO treated organoids. Genes associated with PI3K signaling were also diminished in SHP099 treated organoids but no significant modulation of genes associated with Wnt/b-catenin signaling was observed. 3- Notably, SHP2 pharmaco-inhibition in Caco-2/15 cells as well as APC-mutant organoids rapidly induced cell death associated with increased expression of stem cell markers.

Conclusions
These results suggest that SHP2 is required for proliferation of normal IEC and protects against senescence. Notably, its inhibition restrains the proliferative and tumorigenic potential conferred by APC inactivation. Overall, our data suggest that SHP2 could represent a potential target to counter colorectal tumour initiation.

Funding Agencies
CIHRFRQS
A56 EVALUATING THE DYNAMICS OF CYP3A11 AND REG3G MODULATION BY IL-22
Authors: Delanne-Cuménal A , Flannigan K , Hirota S 
Published: February 2024

Abstract

Background
IL-22 is a cytokine produced by a number of immune cell populations, including type 3 innate lymphoid cells. We have shown previously that IL-22 suppresses the expression Cyp3a11 in the mouse intestine, a gene that encodes a drug metabolizing enzyme that accounts for roughly 70% of total drug metabolism. In addition, IL-22 induces the expression of Reg3g, the gene that encodes an antimicrobial C-type lectin that has bactericidal activity against Gram+ bacteria in the small intestine. Modulation of these proteins can influence both the oral bioavailability of drugs and microbial composition.

Aims
The aim of this study was to define the existence of reversible modulation of IL-22 on Cyp3a11 and Reg3g expression in the intestinal epithelium.

Methods
In vitro enteroids from each part of the mouse small intestine were cultured in 3D in the presence or absence of IL-22 for 5 days. Cyp3a11 and Reg3g transcript expression was evaluated by qPCR followed by protein analysis to define their modulation at varying timepoints after IL-22 treatment cessation.

Results
IL-22 reduced Cyp3a11 expression, an effect that normalized the day after treatment cessation. However, 4 days after treatment cessation there was rebound hyperexpression of Cyp3a11 in duodenal enteroids, which normalized the following day. IL-22 reduced the expression and activity of Cyp3a11 which normalized soon after stopping IL-22 treatment. In contrast, IL-22 induced the expression of Reg3g, an effect that lasted up to 5 days after treatment cessation. The effect of IL-22 on Cyp3a11 expression and activity was rapidly reversed in contrast to the effect on Reg3g which was more prolonged.

Conclusions
IL-22 suppresses the expression of Cyp3a11 and induces the expression of Reg3g. The reversible nature of these effects is different between both, with the increased expression of Reg3g 5 days after IL-22 treatment cessation. These data suggest that the regulation of gene expression, protein or enzyme activity by IL-22 may exhibit varying temporal effects and should be considered when assessing its role in intestinal mucosal biology.

Funding Agencies
This work was funded by the Dr. Lloyd Sutherland Chair in IBD/GI Research and the Weston Foundation.
A57 LONG-TERM EXPOSURE TO ANTICANCER TREATMENTS INCREASES AUTOPHAGY IN ESOPHAGEAL SQUAMOUS CELL CARCINOMA
Authors: Hamilton M , Douchin J , Gaudette B , Giroux V 
Published: February 2024

Abstract

Background
The most common type of esophageal cancer, esophageal squamous cell carcinoma (ESCC), has a 5-year survival rate of only 15%. This low survival rate is to some extent attributed to a high relapse rate, which is, in part, linked to the presence of cancer stem cells (CSC). CSC are a subpopulation of tumor cells that show reduced sensitivity to conventional anticancer therapies, increased potency and self-renewal capacities. With that in mind, ESCC cell lines were exposed in a prolonged manner to anticancer treatments (radiotherapy, 5-FU chemotherapy, and combined therapy) in our laboratory. As expected, an increase in the proportion of CSC (CD44highCD24high) was observed by flow cytometry following the treatments. In pursuit of innovative therapeutic approaches, we conducted an in-depth investigation of the proteomic profiles of these cell lines by mass spectrometry (MS), seeking to unveil novel biological mechanisms. Interestingly, many proteins associated with metabolism and autophagy were modulated. Several alterations were observed by a metabolomic approach as well, namely an increase in intracellular lactate concentration. Recently, a novel role for lactate has been described: lactylation, which is a post-translational modification. Lactylome of those cell lines revealed alteration in proteins related to autophagy and those results led to my research project.

Aims
Investigate the role of lactylation in autophagy of ESCC.

Methods
Human ESCC cell lines (including the cell lines exposed to anticancer treatments) will be used as cell culture models. Cell and organoid cultures, mass spectrometry, qPCR, WB, and proliferation assay were used.

Results
MS analysis of proteins related to autophagy (i.e. RAB1B, CK2, SNX6 et GABARAP) suggests differential regulation of autophagy in cell lines exposed to prolonged treatments. A strong contribution of the 5-FU treatment to the autophagic modification is observable in the double-treated cells. Thus, we confirmed an increase in the LC3BII/I ratio by WB in the cell lines exposed to anticancer treatments. LC3BII/I WB used in combination with Bafilomycin A1 treatment for inhibition of autophagosome and lysosome fusion is a commonly used read-out for autophagic flux. To further study the link between lactylation, autophagy and CSC, we screened 10 human ESCC cell lines for their LC3BII/I ratio. Four cell lines were selected: TE1, TE2, HCE4 and TE5. Using Bafilomycin A1, we confirmed that the autophagic flux in those cell lines was not blocked. Those cell lines will now be used to perform lactylome analysis of sorted CSC cells.

Conclusions
In brief, our results showed that the autophagic flux is increased in cells exposed to prolonged treatments. This suggests that autophagy could play a role in ESCC response to treatment.

Funding Agencies
CIHRTRIANGLE, Chaires de recherche du Canada, CRCHUS, Université de Sherbrooke
A59 EXPLORING THE ROLE OF ARID1A IN COLONIC HOMEOSTASIS AND REGENERATION
Authors: Lei D , Loe A , Kim T 
Published: February 2024

Abstract

Background
Recent advances in cancer genome analysis have revealed frequently mutated epigenetic regulators as a novel feature in cancer development. Within this context, the Arid1a (AT-rich interactive domain-containing protein 1A) gene, a subunit of the BAF chromatin remodeling complex, has emerged as a frequently mutated gene in many cancers. Additionally, repeated colonic injury caused by diseases such as inflammatory bowel disease (IBD), is also a major risk factor leading to colorectal cancer. Therefore, understanding the connection between colonic epigenetic regulation and its role in response to injury is particularly important for understanding the pathobiology of colorectal cancer.

Aims
Aim 1: Exploring the role of Arid1a during colonic homeostasis.
Mouse model with Arid1a conditionally knocked out in the colon will be analysed at different time points to test whether Arid1a is required during colonic homeostasis.
Aim 2: Determining the role of Arid1a during colonic injury and regeneration.
Mice will be exposed to a known colitis model to induce colonic injury. Arid1a will be deleted after injury to find out its role during regeneration and recovery from the injury.

Methods
To examine the effect of Arid1a loss on colonic homeostasis, mice between 6-10 weeks old were injected with 2mg/20g tamoxifen for three days to induce Arid1a knockout using the VilCreERT2; Arid1afl/fl mousseline. Mice were then euthanised using CO2 chamber and analysed 10 days, 3 weeks, and 8 weeks post Arid1a deletion. Histological analysis was performed to examine morphological changes in the colon. To examine the effect of Arid1a on injury caused by colitis, mice were treated with dextran sulfate sodium (DSS) to cause colitis-like injury. Then,
Arid1a is deleted by injecting 2mg/20g tamoxifen. Changes in histology and specific cell types will be examined both in the short- and long-term.

Results
To test whether Arid1a loss alone causes morphological changes in the colon, I compared the crypt lengths between VilCreERT2; Arid1afl/fl mutant mice with their littermate controls and saw that there was no difference in both morphology and length of the crypts. My results show that
Arid1a loss in the colon after injury resulted in impaired regeneration characterized by the prolonged loss of goblet cells and cryptal structure in the distal colon. In the long term, Arid1a loss before or after DSS treatment both lead to tumor formation, suggesting that Arid1a is required for colonic regeneration and repair while suppressing tumorigenesis.

Conclusions
Based on my results, I conclude that Arid1a deletion alone does not cause significant morphological changes in the colon, but this loss affects colonic regeneration and recovery compared to colons with Arid1a intact. In the long term, the dysregulated colonic regeneration may lead to tumorigenesis.

Funding Agencies
University of Toronto Department of Molecular Genetics
A61 ELUCIDATING INTESTINAL EPITHELIAL INFLAMMASOME MECHANISMS THAT REPAIR THE MUCOSAL BARRIER DURING ENTERIC INFECTION
Authors: Ranger A , Goyal S , Girardin S E 
Published: February 2024

Abstract

Background
To protect the gastrointestinal tract from infection, intestinal epithelial cells (IECs) lining the mucosa possess cytosolic complexes called inflammasomes which detect pathogenic insult and rapidly initiate inflammation to stop infectious spread. Active inflammasomes mediate crucial effector functions such as epithelial cell extrusion, pyroptotic cell death, and the release of interleukin (IL)-18 and prostaglandin E2 (PGE2). Recent literature has shown that IL-18 and PGE2 mediate intestinal repair following epithelial damage from DSS-induced colitis. Exogenous administration of IL-18 in inflammasome deficient mice improves survival and reduces pathology. PGE2 activates intestinal cellular reprogramming to drive intestinal wound healing. Whether inflammasome derived IL-18 and PGE2 mediate epithelial repair during enteric infection is unclear.

Aims
Using murine small intestinal organoids (mSIOs), we will investigate how IL-18 and PGE2 production by intestinal inflammasomes directly impacts IECs at both transcriptional and physiological levels. We hypothesize that inflammasome derived IL-18 and PGE2 will act on the intestinal epithelium resulting in transcriptional reprogramming that promotes tissue regeneration and barrier reinforcement.

Methods
WT and Nlrc4-/- mSIOs were stimulated with flagellin or FlaTox to activate the NAIP-NLRC4 inflammasome. The transcriptomic landscape was assessed with bulk-RNA sequencing and inflammasome activation was assessed by Western blot. IL-18 and PGE2 dependent gene targets were identified using Il-18-/- and indomethacin treated mSIOs by RT-qPCR.

Results
Bulk-RNA sequencing revealed an inflammasome activation dependent transcriptional signature in murine IECs upon NAIP-NLRC4 inflammasome activation. GO term analysis showed these genes to be involved in actin cytoskeleton rearrangement, epidermal growth factor signaling, and regulation of cell-cell adhesion. The expression of PGE2 gene targets were significantly reduced by indomethacin treatment while IL-18 gene targets remained unaffected in Il-18-/- IECs. FlaTox induced the same transcriptional changes but substantially amplified their expression. FlaTox but not flagellin led to a significant drop in Lgr5 (stem cells) and Lyz1 (Paneth cell) expression and an increase in Ly6a, a marker of fetal-like stem cells.

Conclusions
This data demonstrates that inflammasome signaling causes transcriptional reprogramming of IECs that activates pathways involved in maintaining epithelial barrier integrity. While IL-18 had no effects on gene expression at 4 hours, PGE2 paracrine signaling contributed to the expression of wound healing genes. Stronger inflammasome mediated damage with FlaTox amplified the repair response at the transcript level and we observed transient reprogramming of IECs into a fetal-like state suggesting active tissue regeneration.

Funding Agencies
CCC, CIHREPIC: Emerging & Pandemic Infections Consortium
A62 EXAMINING THE ACTIVATION OF XENOBIOTIC RECEPTORS PXR AND AHR IN COLONOIDS USING MICROBIAL METABOLITES AND CHEMICAL LIGANDS
Authors: Shenoda Eva I , Hirota Simon A 
Published: February 2024

Abstract

Background
The Aryl hydrocarbon receptor (AhR) and pregnane X receptor (PXR) are vital xenobiotic receptors activated by foreign substances, playing pivotal roles in regulating chemical metabolism. Traditionally, these receptors have been closely linked to their roles in mediating responses to toxic compounds. However, recent findings have revealed their newfound significance in maintaining gut homeostasis and regulating inflammatory processes.

Aims
We investigated the dual roles of AhR and PXR activation. We started with dosage response experiments to determine optimal treatment conditions, such as treatment concentration and exposure time. Subsequently, we compared transcriptomic responses induced by chemical ligands with those from microbial metabolites, aiming to understand how AhR and PXR activation can yield both adverse and beneficial effects.

Methods
Using mouse 3D colonoids and 2D monolayer cultures, PXR and AhR were activated using the microbial metabolites indole-3-propionic acid (IPA) and indole-3-pyruvic acid (IPyA), respectively. Concentrations were determined through dosage response experiments. Changes to PXR and AhR target gene expression were measured using qPCR. We compared the gene induction by IPA and IPyA to responses driven by the chemical ligands pregnenolone 16α-carbonitrile (PCN) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), respectively.

Results
In 3D cell culture, PCN induced PXR target genes Cyp2c55 and Abcb1a, while IPA had no significant effect. Cyp3a11 was absent. Both IPyA and TCDD induced AhR genes, Cyp1a1 and Cyp1b1, with varying induction, and Cyp1a2 showed no response. In 2D cell culture, PCN significantly increased Cyp2c55 gene expression, but neither PCN nor IPA significantly induced Abcb1a. In 3D culture, TCDD significantly induced Cyp1a1 and Cyp1b1, while IPyA only induced Cyp1a1, revealing differences in AhR gene induction between 2D and 3D cell culture models.

Conclusions
Our research reveals contrasting gene induction patterns following AhR and PXR activation through microbial metabolites and chemical ligands. These varied responses highlight the multifaceted roles these receptors play within the gut environment. This study enhances our understanding of xenobiotic receptors' contributions to maintaining gut homeostasis and regulating inflammatory processes, shedding light on the interplay between ligands and receptors.

Methods
First, we isolate the crypts of mice to create 3D organoids, were we have access to the basolateral side. To capture the true biological system, we convert the 3D culture to 2D. This enables us to explore both the apical and basolateral sides, retaining the crucial polarity of the cells.

Funding Agencies
CIHR