Abstract
Background
The circadian clock is a self-sustained molecular oscillator which drives 24-hour physiological rhythms. It consists of the genes Bmal1 and Clock that positively regulate Cry and ...Per, their negative regulators, resulting in a 24-hour transcription/translation feedback loop. Shift work, which causes disruptions to 24-hour physiological rhythms, has been shown to lead to an increased incidence of inflammatory bowel disease (IBD). We have previously established that mice lacking the non-redundant circadian regulator, Bmal1, exhibit more severe colitis compared to controls.
Purpose
This study aims to investigate the epithelial function of Bmal1 in colonic regeneration during colitis.
Method
In order to assess the cell-specific role of the clock, we tested the regenerative effects of Bmal1 in intestinal epithelial tissue using Vil+/+;Bmal1flox/flox (control) and VilCre/+;Bmal1flox/flox (conditional mutant) mice. Dextran Sulfate Sodium (DSS) was applied to induce acute colitis. Disease progression was evaluated during colitis and during recovery upon removal of DSS treatment. We hypothesized that the absence of a functional circadian clock disrupts effective proliferation and regeneration of intestinal epithelial cells during colitis remission.
Result(s)
Vil
+/+
;Bmal1
flox/flox
control and VilCre/+;Bmal1flox/flox conditional mutant mice exhibit no significant differences in disease severity or tissue histopathology during colitis. However, after the removal of DSS, VilCre/+;Bmal1flox/flox conditional mutants show increased total lesions and overall inflammation, decreased crypt density as well as a higher propensity of hyperplastic crypts in the tissue. Regenerative ability of the colon is decreased in conditional mutants: phosphorylated-histone H3 and Ki67 show persistent increases in mitosis and overall proliferation near ulcerated lesions. This suggests that, while controls complete the regenerative response, conditional mutants fail to recover from colitis and show inefficient regeneration when Bmal1 is disrupted in intestinal epithelial cells.
Conclusion(s)
Our results support a critical role for Bmal1 in intestinal epithelial cells during post-colitis regeneration and recovery.
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CIHR, Other
Please indicate your source of funding;
OGS, Crohn's & Colitis Canada
Disclosure of Interest
None Declared
Abstract
Background
Fecal microbiota transplant (FMT) is the transfer of fecal microbes from a healthy donor to a recipient to normalize gut microbiota. FMT therapy has been effectively used to ...prevent recurrent Clostridium difficile infections. While there are emerging studies applying FMT to other gastrointestinal (GI) disorders including inflammatory bowel disease (IBD), its efficacy in treating IBD, and the mechanisms involved remain unclear. In the GI tract, the mucus barrier plays a critical role in protecting the underlying epithelium against harmful luminal stimuli. The secreted mucin 2 (MUC2) is a glycosylated protein and the major component of the GI mucus layer. Muc2 deficient (-/-) mice are used to model IBD because they develop a leaky gut as well as spontaneous colitis.
Purpose
We investigated the efficacy of FMT in attenuating the development of spontaneous colitis in Muc2-/- mice. This study also evaluated whether FMT using lyophilized stool can successfully alter the gut microbiome of recipient mice.
Method
To achieve successful colonization of transplanted microbiota, Muc2-/- mice were pretreated prior to FMT with the antifungal amphotericin B, followed by an antibiotic mixture of metronidazole, ampicillin, neomycin and vancomycin in their drinking water for ten days. For two consecutive days, FMT was administered to Muc2-/- mice via consumption of lyophilized stool cakes created from donor C57BL/6 mice, while sucrose cakes were given as control. The fecal microbiome was analyzed at baseline, post-antibiotics and biweekly post-FMT using 16S rRNA sequencing. We monitored body weight and signs of spontaneous colitis, including rectal prolapse, until the mice reached their humane endpoint or turned 30 weeks old.
Result(s)
Following antibiotic pretreatment, Muc2-/- mice experienced significant weight loss, with 15-20% requiring euthanization within ten days of receiving antibiotics. Unless FMT was administered post-antibiotics, 95% of the mice developed rectal prolapse or otherwise reached their humane endpoint. In comparison, FMT-treated mice rarely developed rectal prolapse, with 55-80% of the FMT-treated mice surviving until the end of the experiment; some FMT-treated mice had no signs of rectal prolapse when euthanized at 30 weeks. Therefore, FMT significantly improved the survival rate of antibiotic-treated Muc2-/- mice and delayed the onset of rectal prolapse / spontaneous colitis. Microbiome analysis revealed that the lyophilized FMT altered the fecal microbiome as compared to mice receiving the sucrose control.
Conclusion(s)
Notably, FMT increased the survival rates of antibiotic-pretreated Muc2-/- mice while delaying the onset of rectal prolapse. This indicates that lyophilized FMT counteracts the typical spontaneous colitis and weight loss seen in Muc2-/- mice. Our findings can offer clinically relevant insight into how FMT induces a shift in the gut microbiome and its potential usage in treating IBD, as well as other gastrointestinal conditions.
Disclosure of Interest
None Declared
Abstract
Background
Pancreatic adenocarcinoma (PAC) is a deadly disease with an overall 5-year survival of less than 8%. The current literature on patient outcomes are limited by small samples sizes ...and patients enrolled in clinical trials. There are no prognostic tools for patients with pancreatic cancer.
Purpose
To develop a prognostic survival model for patients with pancreatic cancer
Method
All patients with a diagnosis of pancreatic cancer cancer from January 2007 to December 2020 were identified through the Ontario Cancer Registry. The primary outcome was survival. The cohort was used to develop a multivariable cox proportional hazards regression model with baseline characteristics under a backward stepwise variable selection process to predict the risk of mortality. Covariates included patient age, sex, tumour location, cancer stage, treatment types, distance to a cancer centre, hospitalizations, comorbidities, access to family physician, and symptoms as captured using the Edmonton Symptom Assessment System datasets.
Result(s)
There was a total of 17,450 pancreatic cancer patients in the cohort, 48% of which were female and the mean age was 72 years. 44% of patients presented with a tumor in the head of the pancreas. Among those with stage data (44%), 24% were stage IV at diagnosis. Mean survival was approximately 0.7 years. Approximately 60% were hospitalized in the 3 months prior to diagnosis. Almost all patients had a family doctor rostered (95%). In multivariate analysis, key predictors of survival assessed at the time of diagnosis were age, sex, tumour location in the pancreas, stage at diagnosis, pain, appetite functional status and treatment choice (all p<0.001). Using these variables, we created a prediction model that can estimate one-year probability of death with high discrimination (area under the curve = 0.82, c-statistic 0.76).
Conclusion(s)
Our model accurately predicts one-year pancreatic cancer survival risk using clinical symptom and performance status data. The model has the potential to be a useful prognostic tool that can be completed by patients and their caregivers in support of patient-centered care.
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CIHR
Disclosure of Interest
None Declared
Abstract
Background
Chronic abdominal pain is the most common complaint of patients with gastrointestinal disorders. Its treatment is of limited efficacy as the pathophysiology is poorly understood. ...The gut microbiome has been shown to affect host physiology, including the neural system function, and growing evidence suggests that it plays an important role in pain perception. Gut microbiota produces a large variety of molecules that can regulate pain perception, such as histamine or bioactive lipids. Phospholipid mediators (PLM) are signaling molecules linked to neurogenic pain as by-products of inflammatory processes in mammals. But it is unknown whether bacteria can produce PLM and whether they play any role in visceral hyperalgesia.
Purpose
To investigate whether PLM are present in stool of patients with chronic abdominal pain, and whether they have potential to induce visceral hypersensitivity.
Method
Stool samples from 27 patients with irritable bowel syndrome (IBS) were collected both during periods of severe and minimal/no pain. The fecal concentration of two PLM (lipid A and lipid B) was determined by ELISA. The passage of lipid A and lipid B through the intestinal barrier was determined ex vivo using Ussing chambers and in vivo by their intracolonic instillation, and their levels assessed in the serosal compartment and the serum, respectively, by ELISA. We used primary cultures of sensory neurons from the dorsal root ganglia (DRG) of conventional mice (SPF) to study the neuronal activation in vitro. Calcium mobilization in DRG sensory neurons was measured by an inverted fluorescence microscope using a fluorescent probe Fluo-4 (1mM) after stimulation with different concentration of lipid A or lipid B. Visceral sensitivity in vivo after intracolonic instillation of combined lipids A and B was evaluated by colorectal distension.
Result(s)
The concentration of lipid A (p=0.001) and lipid B (p=0.002) in stool was significantly higher in IBS patients when experiencing severe abdominal pain compared to periods of minimal pain. The concentration of lipid A (p=0.03; p=0.07) and lipid B (p=0.018; p=0.018) in the serosal compartment and in the serum, respectively, was higher compared to the control condition. The percentage of neurons responding to lipid A or lipid B was significantly higher compared to the control condition, at all tested concentrations. Finally, combined lipid A and lipid B induced visceral hypersensitivity within 10 minutes (p=0.0433) and 90 minutes (p=0.0020) after intracolonic instillation.
Conclusion(s)
Our data suggest that PLM can be found in stool of patients with abdominal pain, that they cross the colonic barrier and increase visceral sensitivity. Further studies are needed to ascertain whether gut bacteria produce these PLM and investigate the precise mechanisms by which PLM induce hyperalgesia.
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CIHR, Other
Please indicate your source of funding;
Weston Family Foundation
Disclosure of Interest
None Declared
Abstract
Background
The inflammatory bowel disease questionnaire (IBDQ) is a measure of health-related quality of life (QoL), with higher scores indicating greater QoL. In a prior phase 2 study ...(NCT02589665), mirikizumab, an anti-IL23p19 antibody, demonstrated efficacy and improvement in IBDQ scores in participants with moderately to severely active ulcerative colitis (UC).
Purpose
This analysis evaluated effect of mirikizumab (miri) vs placebo (PBO) on IBDQ scores in patients (pts) with moderately to severely active ulcerative colitis (UC) who had failed prior conventional or biologic therapy in a Phase 3, double-blind, 12-week (W) induction study (LUCENT-1) followed by a 40W maintenance study (LUCENT-2) for a total of 52W continuous therapy.
Method
Pts (N=1162) in LUCENT-1 were randomized 3:1 to receive 300mg miri or PBO intravenously once every four weeks (Q4W). 544 pts who achieved Modified Mayo Score Clinical Response to miri by W12 of induction were rerandomized 2:1 in LUCENT-2 to subcutaneous miri 200mg or PBO Q4W in maintenance period. Randomization was stratified by previous biologic therapy failure, baseline corticosteroid use, and region. LUCENT-1 stratification included baseline (BL) disease activity, and LUCENT-2 included LUCENT-1 clinical remission status. The least squares mean change from BL in IBDQ scores at W12 of induction and W40 of maintenance was determined using analysis of covariance models. BL was W0 of therapy and stratification factors and BL scores were used as covariates. The Minimal Clinically Important Difference (MCID) was defined as an improvement of ≥16 points in total IBDQ score (IBDQ response) and IBDQ remission as a total score ≥170 points. IBDQ response and remission were calculated using non-responder imputations. Treatments were compared using the common risk difference (risk diff).
Result(s)
Miri treatment resulted in significantly greater improvement from BL in IBDQ total and domain scores vs PBO at both W12 of induction and W40 of maintenance (52W treatment) (Table). The proportions of pts who achieved an IBDQ response was significantly greater for miri treated pts vs PBO at W12 (risk diff =17.195%CI:10.7, 23.5) and W40 (29.5 21.0, 37.9). Significantly greater proportions of pts receiving miri achieved IBDQ remission at W12 (18.1 11.8, 24.4) and W40 (28.5 20.1, 37.0) vs PBO (all evaluations and timepoints: p<0.001).
Image
Conclusion(s)
Pts reported significantly greater improvements in IBDQ scores at induction and maintenance with miri compared to PBO. Over 75% of pts achieved a clinically meaningful improvement in QoL, as measured by IBDQ response, at the end of the 52 weeks of miri treatment.
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Other
Please indicate your source of funding;
Eli Lilly and Company
Disclosure of Interest
B. Sands Consultant of: Abivax, Amgen, Arena Pharmaceuticals, Artugen Therapeutics, AstraZeneca, Bacainn Therapeutics, Boehringer Ingelheim, Boston Scientific, Bristol Myers Squibb, Calibr, Celltrion, ClostraBio, Eli Lilly and Company, Enthera, Evommune, Galapagos NV, Genentech, Gilead Sciences, GlaxoSmithKline, Gossamer Bio, InDex Pharmaceuticals, Innovation Pharmaceuticals, Inotrem, Ironwood Pharmaceuticals, Janssen, Kaleido Biosciences, Kallyope, MiroBio, Morphic Therapeutic, MRM Health, Pfizer, Progenity, Prometheus Therapeutics and Diagnostics, Protagonist Therapeutics, Q32 Bio, Surrozen, Takeda, Teva, TLL Pharmaceutical, USWM Enterprises, and Viela Bio, B. Feagan Shareholder of: Gossamer Bio, Consultant of: AbbVie, AdMIRx, AgomAb Therapeutics, Akebia Therapeutics, Alivio Therapeutics, Allakos, Amgen, Applied Molecular Transport, Arena Pharmaceuticals, Avir Pharma, Azora Therapeutics, Boehringer Ingelheim, Boston Scientific, Celgene/Bristol Myers Squibb, Connect BioPharma, Cytoki Pharma, Disc Medicine, Ecor1 Capital, Eli Lilly and Company, Equillium, Everest Clinical Research, F. Hoffmann-La Roche, Ferring Pharmaceuticals, Galapagos NV, Galen/Atlantica, Genentech/Roche, Gilead Sciences, GlaxoSmithKline, Glenmark Pharmaceuticals, Gossamer Bio, HotSpot Therapeutics, Imhotex, ImmuNext, InDex Pharmaceuticals, Intact Therapeutics, Janssen, Japan Tobacco, Kaleido Biosciences, Leadiant Biosciences, Millennium Pharmaceuticals, MiroBio, Morphic Therapeutics, Mylan, Novartis, OM Pharma, Origo Biopharma, Otsuka, Pandion Therapeutics, Pfizer, Progenity, Prometheus Therapeutics and Diagnostics, PTM Therapeutics, Q32 Bio, Rebiotix, RedHill, Biopharma, Redx Pharma, Sandoz, Sanofi, Seres Therapeutics, Surrozen, Takeda, Teva, Thelium Therapeutics, Theravance Biopharma, TiGenix, Tillotts Pharma AG, UCB Pharma, VHsquared, Viatris, Ysios Capital, and Zealand Pharma, T. Gibble Employee of: Eli Lilly and Company, K. Traxler Employee of: Eli Lilly and Company, N. Morris Employee of: Eli Lilly and Company, X. Li Employee of: Eli Lilly and Company, S. Schreiber Grant / Research support from: personal fees and/or travel support from: AbbVie, Amgen, Arena Pharmaceuticals, Biogen, Bristol Myers Squibb, Celgene, Celltrion, Eli Lilly and Company, Dr. Falk Pharma, Ferring Pharmaceuticals, Fresenius Kabi, Galapagos NV, Gilead Sciences, I-MAB Biopharma, Janssen, Merck Sharp & Dohme, Mylan, Novartis, Pfizer, Protagonist Therapeutics, Provention Bio, Roche, Sandoz/Hexal, Shire, Takeda, Theravance Biopharma, and UCB Pharma, V. Jairath Consultant of: AbbVie, Alimentiv, Arena Pharmaceuticals, Asahi Kasei Pharma, Asieris Pharmaceuticals, AstraZeneca, Bristol Myers Squibb, Celltrion, Eli Lilly and Company, Ferring Pharmaceuticals, Flagship Pioneering, Fresenius Kabi, Galapagos NV, Genentech, Gilead Sciences, GlaxoSmithKline, Janssen, Merck, Mylan, Pandion Therapeutics, Pendopharm, Pfizer, Protagonist Therapeutics, Reistone Biopharma, Roche, Sandoz, Second Genome, Shire, Takeda, Teva, Topivert, Ventyx Biosciences, and Vividion Therapeutics, A. Armuzzi Consultant of: AbbVie, Allergan, Amgen, Arena Pharmaceuticals, Biogen, Bristol Myers Squibb, Celgene, Celltrion, Eli Lilly and Company, Ferring Pharmaceuticals, Galapagos NV, Gilead Sciences, Janssen, Merck Sharp & Dohme, Mylan, Novartis, Pfizer, Protagonist Therapeutics, Roche, Samsung Bioepis, Sandoz, Takeda, and TiGenix, J. Jones: None Declared
Abstract
Background
Opportunistic pathogens have been postulated to drive dysregulated inflammation in inflammatory bowel disease (IBD). Indeed, adherent-invasive Escherichia coli (AIEC) isolated ...from IBD patients have pathobiont and pro-inflammatory characteristics. Current treatments for IBD suppress the immune response and do not target key microbial drivers, therefore novel strategies are required.
Purpose
Our aim was to determine whether bacteriophage therapy targeted against AIEC could reduce the severity of E. coli-driven colitis in gnotobiotic mice.
Method
Adult germ-free C57BL/6 mice were colonized with altered Schaedler-like flora (ASF) and E. coli NRG857c, a Crohn’s disease-associated bacterial isolate. Three weeks later, mice were treated with daily phage (selected by killing curves bioassays against E. coli NRG857c) or PBS for 2 weeks (n=6/group). Mice were then exposed to low-dose dextran sulfate sodium (2%; DSS) in drinking water for 5 days, followed by 2 days of water. PBS-treated mice (n=6) that received no DSS were used as additional negative controls. Mice were monitored daily for weight, stool consistency, and occult blood. At sacrifice, colon tissue was collected for histological analysis and fecal contents were cultured to determine bacterial load. In separate experiments, C57BL/6NTac-Il10em8Tac (IL-10-/-) mice were colonized with ASF-like microbiota and E. coli NRG857c. Three weeks later, mice (n=5) were treated with weekly phage or PBS (n=5) for 7 weeks. Mice were monitored weekly as described above.
Result(s)
Daily phage treatment reduced the severity of clinical symptoms induced by acute DSS administration (p < 0.001 vs. DSS-PBS treated mice). At endpoint, phage treatment was associated with lower histological scores as compared with DSS-PBS controls (p < 0.0001). A 1-log reduction in AIEC bacterial load was observed in phage treated mice as compared with DSS-PBS controls (p < 0.001). In IL-10-/- mice, weekly phage treatment delayed the spontaneous onset of colitis (p < 0.0001 vs. PBS-treated mice). At endpoint, mice treated with phage had lower colitis scores. Reduced weekly AIEC bacterial load was observed in phage-treated mice.
Conclusion(s)
Lytic phages, targeting a known AIEC pathobiont isolated from Crohn’s disease patients, ameliorate acute intestinal injury and delay onset of spontaneous colitis. Future work will investigate the mechanisms by which phage therapy prevents and treats colitis, to better inform clinical trial design.
Disclosure of Interest
None Declared
Abstract
Background
Canada is one of the most prolific users of the herbicide glyphosate (tradename Roundup®), with over 25 million kilograms purchased annually. Glyphosate is commonly applied ...pre-harvest as a desiccant, leading to higher residues in many foods consumed within Canada, including wheat, cereals, legumes, and soy products. Glyphosate prevents the synthesis of aromatic amino acids by inhibiting the Shikimate pathway, which, besides killing weeds, can also inhibit bacterial growth and promote bacterial dysbiosis. Dysbiosis has been associated with multiple disease states, including intestinal inflammation and metabolic dysfunction, which both include anxiety, depression, and cognitive dysfunction as co-morbidities.
Purpose
My goal is to clarify if there are harmful glyphosate effects on the gut bacteriome, resulting in altered metabolic health, damaging intestinal inflammation, and behaviour changes through the gut-brain axis. We explored two environmentally relevant doses throughout our experiments: (1) the acceptable daily intake currently set by the Environmental Protection Agency (EPA) (1.75mg/kg body weight/day); (2) the North American dose calculated with a registered dietician based on literature values of glyphosate within items that make up a typical Canadian diet (0.01mg/kg body weight/day.)
Method
Breeding pairs consisting of Muc2-/- (colitis susceptible) and Muc2+/- (littermate control) mice were exposed to glyphosate during pregnancy. A subset of each generation underwent behavioural testing to characterize anxiety, memory, and locomotor activity. Animals also underwent metabolic testing, including oral glucose and insulin tolerance tests. Only the parental generation of animals received glyphosate. In total, three generations of animals were raised.
Result(s)
Our results show that healthy mice whose great-grandparents were exposed to glyphosate at levels currently deemed safe by the EPA exhibited decreased locomotor activity. Furthermore, mice whose parents were exposed to either North American or EPA glyphosate levels exhibit symptoms of metabolic dysfunction in healthy and colitis-susceptible mice, including higher fasting blood glucose, an inability to efficiently clear glucose and impaired insulin response. Additionally, Muc2-/- mice whose parents were exposed to levels of glyphosate found within the North American diet during pregnancy showed significant impairments in working memory.
Conclusion(s)
This study is the first to highlight the transgenerational effects of glyphosate at levels previously deemed safe for human exposure on locomotor activity, working memory and metabolism. Additionally, this study highlights how environmental toxins within our food system, such as glyphosate, may play a vital role in the etiology of many diseases in healthy and colitis-susceptible populations by promoting a sedentary lifestyle, metabolic dysfunction, and behavioural deficits.
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CAG, CCC, Other
Please indicate your source of funding;
NSERC
Disclosure of Interest
None Declared
Abstract
Background
Inflammatory bowel disease (IBD) affects an estimated 270,000 people in Canada and is rapidly increasing in prevalence. All patients have relapsing disease, and a subset of ...individuals do not respond to current treatments. Further, there are no approved treatment options in Canada that reverse IBD-induced intestinal fibrosis. We have previously shown type 1 regulatory cells (Tr1s) are capable of suppressing inflammatory macrophages, promote barrier function of human intestinal epithelial cells, and induce differentiation of mucin-producing goblet cells. We hypothesize that Tr1 cells can prevent inflammatory damage and fibrosis in an mouse model of acute gut damage.
Purpose
We hypothesize that Tr1 cells can prevent inflammatory damage and fibrosis in an mouse model of gut damage. Here we evalute the therapeutic potential of Tr1 cells in an model of acute intestinal epithelial damage.
Method
Tr1 cells were isolated and expanded from CD4+ CD44high FOXP3- cells. Their phenotype was characterized by flow cytometry and cytokine secretion was measured via ELISA. WT B6 mice were given 2% DSS in H2O for 7 days, followed by H2O alone for 7 days. Prior to DSS treatment, mice were sub-lethally irradiated to facilitate engraftment, and given I.P. injections of PBS or 0.5 – 2 x 106 Tr1 cells. Mice weights and health scores were recorded daily. At the endpoint, blood, spleen, and mesenteric lymph nodes were analyzed for Tr1 cell engraftment (or lack thereof) for each mouse. Complete white blood counts were performed for each mouse. Additionally, proximal, medial, and distal portions of the ileum were processed for histologic scoring.
Result(s)
Tr1 cells isolated from CD4+ CD44high FOXP3- cells produce high levels of IL-10 following stimulation (>35,000 pg/ml/1 x 105 cells). Additionally, these cells express high levels of Tr1 markers CD49b and Lag-3. Optimization experiments indicated no significant differences between mice irradiated and given DSS and mice only given DSS (no irradiation). Our results suggest no significant differences in inflammatory cell infiltrate scores between control and Tr1 treated mice. However, gut architecture scores appeared to improve with increasing Tr1 doses. Further, weight change improved with Tr1 treatment, as compared to PBS controls. Interestingly, Tr1 treatment appeared to decrease total eosinophil and neutrophil counts from peripheral blood.
Conclusion(s)
Our initial findings indicate Tr1 adoptive transfer prior to acute damage via DSS improves gut damage and weight loss.
Please acknowledge all funding agencies by checking the applicable boxes below
CIHR
Disclosure of Interest
None Declared
Abstract
Background
Patients with IBD have an altered microbiome and gut microenvironment leading to changes in their Short-Chain Fatty Acid (SCFA) profile. Pathobionts are commensal organisms that ...become pathogenic under specific conditions, likely related to microenvironmental gut changes. This is especially relevant to children given that early life exposures are critical to microbiome development and impact immune response.
Purpose
The objectives of our study were to: 1) determine if microbes from IBD patients are more proinflammatory compared to non-IBD microbes; and 2) define how SCFAs affect host-response to potential pathobionts.
Method
Fructooligosaccharide (FOS) fermentation and SCFA production were assessed by HPLC-RID using whole-intestinal microbe culture collected as mucosal washes from pIBD (n=10) and non-IBD patients (n=8). Individual anaerobic bacteria were isolated from individual patients and screened for proinflammatory response interleukin (IL)-1ß using THP-1 macrophages. Caco-2 (epithelial) or THP-1 cells were pre-exposed to individual SCFAs (50 mM acetate or formate; 10 mM succinate; or 1 mM butyrate or propionate), then infected with Bacteroides fragilis, isolated from patients. Microscopy (bacterial staining with HEMA 3), qPCR, ELISA, TransEpithelial Electrical Resistance (TEER), and culturing were used to determine invasion potential, cytokine expression, barrier, and secretion.
Result(s)
FOS fermentation by microbes from IBD patients led to increased acetate and decreased butyrate production. Microbes from IBD patients were more proinflammatory, with increased IL-1ß secretion and reactive oxygen species generation, compared to those from non-IBD patients. B. fragilis isolated from an IBD patient was found to be a potential pathobiont with increased invasion and cytokine production in both epithelial and macrophage cells. Invasion was further increased when cells were exposed to SCFAs, particularly acetate and butyrate. Furthermore, the strain isolated from the IBD patient was observed (by microscopy) to be more adherent to the epithelial barrier, causing a loss of membrane integrity (TEER). The inflammasome pathway were significantly upregulated in Caco-2 cells infected with IBD-isolated B. fragilis and incubated with acetate or butyrate.
Conclusion(s)
The altered microbiome in IBD patients leads to an increased acetate and decreased butyrate, which likely affects host response to microbes and promotes inflammation. This changed microenvironment promotes the development of pathobionts, such as the identified B. fragilis strain we isolated from an IBD patient. The observation that acetate and butyrate increased host susceptibility to this strain indicates that understanding diet and SCFA production are instrumental in treating chronic conditions such as IBD. The role of diet as both a treatment and potential cause of inflammation in IBD is becoming more apparent; therefore, it is crucial that we understand the complex role diet has in host-microbe interactions.
Please acknowledge all funding agencies by checking the applicable boxes below
CIHR, Other
Please indicate your source of funding;
Mitacs- Weston Foundation
Disclosure of Interest
None Declared
Abstract
Background
Intestinal epithelial cells (IECs) provide an essential physical barrier between harsh luminal contents and underlying host tissue. The maintenance of intestinal homeostasis in ...this rapidly renewing tissue must be intricately regulated through the proliferation and differentiation of intestinal stem cells (ISCs). Dysregulation of this system results in the loss of barrier function, causing pathologies in both intestinal and extra-intestinal diseases. While Parkinson’s Disease (PD) is primarily a neurodegenerative disorder, there is increasing evidence linking PD progression and gastrointestinal dysfunction. For instance, constipation and increased bowel permeability are frequently observed years prior to development of motor dysfunction in PD, people with inflammatory bowel disease are more likely to develop PD, and a positive correlation exists between gastrointestinal infections and PD incidence. Our group recently developed a model to investigate the role of the gut in PD, demonstrating that mice with genetic ablation of the PD-associated gene Pink1 exhibited motor phenotypes only when previously infected with Gram-negative Citrobacter rodentium intestinal bacteria. As Pink1 and other PD-associated genes are expressed in IECs, we hypothesize that PD-associated gene mutations directly affect the epithelium and impact early PD pathophysiology.
Purpose
Investigate the impact of Pink1 and other PD-associated genes in IECs under steady state and infection.
Method
Single-cell RNA sequencing was performed on IECs isolated from Pink1 WT and KO mice, at steady state and following in vivo C. rodentium infection. Mice were sacrificed at an early timepoint of infection (day 6) to elucidate transcriptional differences between epithelial lineages of each genotype. Additionally, ex vivo colonoids were derived from primary mouse tissue and treated with lipopolysaccharide (LPS) to determine how PINK1 loss-of-function affects the inflammatory response of the epithelium.
Result(s)
Our data revealed that loss-of-function of PINK1 profoundly affected the ISC compartment and several epithelial lineages. Specifically, ISCs from infected Pink1 KO mice demonstrated differentially regulated proliferative and cell cycle genes, while transit amplifying cells showed dysregulated expression of tight junction genes, and enterocytes displayed differentially expressed oxidative damage and apoptotic genes. Preliminary data from colonoids showed that Pink1 KO mice, when stimulated with LPS, had increased pro-inflammatory cytokine gene expression.
Conclusion(s)
In Pink1 KO intestinal epithelial cells, there is indeed an altered cellular response upon infection in vivo and LPS treatment ex vivo. However, more information is needed to decern the mechanistic role of IECs in PD. By investigating the role of PD genes in the gastrointestinal tract, these studies carry important implications for understanding the initiation and progression of PD.
Disclosure of Interest
None Declared
