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Because of localized vascular damage and increased tissue oxygen demand, wound healing occurs in a relatively hypoxic microenvironment. These features are particularly relevant to wound healing and fibrosis in chronic inflammatory conditions, such as Crohn's disease and ulcerative colitis. In these studies, we sought to identify the contribution of hypoxia to mechanisms of wound repair in a model of the intestinal submucosa. Initial studies revealed that hypoxia promotes wound healing, as modeled by an increase in intestinal fibroblast-mediated collagen gel contraction. Guided by results from transcriptional profiling, we identified the selective induction of fibroblast integrin β1 (ITGB1) by hypoxia. Further analysis revealed that hypoxia, as well as pharmacological activators of hypoxia-inducible factor (HIF), induce fibroblast β1 integrin mRNA, protein, and function by as much as 4-fold. Cloning and analysis of the β1 integrin gene promoter revealed a 10 ± 0.8-fold increase in promoter activity in response to hypoxia, and subsequent studies identified a functional DNA binding region for HIF in the ITGB1 gene promoter. Mutational analysis of the HIF binding site within the ITGB1 promoter resulted in a significant loss of ITGB1 hypoxia-inducibility. As proof of principle, studies in a murine model of colitis revealed a correlation between colitic disease severity and tissue ITGBI expression (R²=0.80). Taken together, these results demonstrate that hypoxia induces fibroblast ITGB1 expression and function by transcriptional mechanisms dependent on HIF. [ABSTRACT FROM AUTHOR]

In intact mucosal tissues, epithelial cells are anatomically positioned in proximity to a number of subepithelial cell types, including endothelia. A number of recent studies have suggested that imbalances between energy supply and demand can result in "inflammatory hypoxia." Given these associations, we hypothesized that endothelial-derived, hypoxia-inducible mediators might infuence epithelial function. Guided by cDNA microarray analysis of human microvascular endothelial cells (HMEC-1 line) subjected to hypoxia (pO2 20 torr, 8 h), we identified adrenomedullin (ADM) as a prominent hypoxia-inducible factor (HIF) that acts on epithelial cells through cell surface receptors. We assessed the functional ability for exogenous ADM to signal in human intestinal Caco2 cells in vitro by demonstrating a dose-dependent induction of Erk1/2phosphorylation. Further analysis revealed that ADM deneddylates cullin-2 (Cul2), whose action has been demonstrated to control the activity of HIF. Caco2 cells stably expressing a hypoxic response element (HRE)-driven luciferase promoter confirmed that ADM activates the HIF signaling pathway. Extensions of these studies revealed an increase in canonical HIF-1- dependent genes following stimulation with ADM. To define physiological relevance, we investigated the effect of ADM in a DSS model of murine colitis. Administration of ADMresulted in reduced inflammatory indices and less severe histological inflammation compared to vehicle controls. Analysis of tissue and serum cytokines showed a marked and significant inhibition of colitis-associated TNF-α, IL-1β, and KC. Analysis of circulating ADM demonstrated an increase in serum ADM in murine models of colitis. Taken together, these results identify ADM as an endogenously generated vascular mediator that functions as a mucosal protective factor through fine tuning of HIF activity. [ABSTRACT FROM AUTHOR]

Liver inflammation is a common extraintestinal manifestation in inflammatory bowel disease (IBD), yet, the mechanisms driving gut‐liver axis inflammation remain poorly understood. IBD leads to a breakdown in the integrity of the intestinal barrier causing an increase in portal and systemic gut‐derived antigens, which challenge the liver. Here, we examined the role of platelet activating factor receptor (PAFR) in colitis‐associated liver damage using dextran sulfate sodium (DSS) and anti‐CD40‐induced colitis models. Both DSS and anti‐CD40 models exhibited liver inflammation associated with colitis. Colitis reduced global PAFR protein expression in mouse livers causing an exclusive re‐localization of PAFR to the portal triad. The global decrease in liver PAFR was associated with increased sirtuin 1 while relocalized PAFR expression was limited to Kupffer cells (KCs) and co‐localized with toll‐like receptor 4. DSS activated the NLRP3‐inflammasome and increased interleukin (IL)‐1β in the liver. Antagonism of PAFR amplified the inflammasome response by increasing NLRP3, caspase‐1, and IL‐1β protein levels in the liver. LPS also increased NLRP3 response in human hepatocytes, however, overexpression of PAFR restored the levels of NLPR3 and caspase‐1 proteins. Interestingly, KCs depletion also increased IL‐1β protein in mouse liver after DSS challenge. These data suggest a protective role for PAFR‐expressing KCs during colitis and that regulation of PAFR is important for gut‐liver axis homeostasis. [ABSTRACT FROM AUTHOR]

R4272 --> 865.5 --> Background: Emerging evidence suggests there is a specific duodenal mucosal associated microbiota (MAM) signature for functional dyspepsia (FD), although study numbers are low and consensus on biomarkers have not been reached. Additionally, we now know that FD is associated with high duodenal eosinophil counts and this site‐specific immune response is likely interacting or responding to the MAM. As duodenal biopsies for research can be difficult to obtain, we investigated formalin‐fixed paraffin‐embedded (FFPE) biopsies, routinely collected for pathology, to sequence the MAM and compared them to the MAM profiles of matched RNAlater preserved biopsies. Methods: Duodenal biopsies (n=4) stored in RNAlater were homogenized for 3 mins at 5,0000 rpm using the Precellys 24 homogenizer after removal from RNAlater solution. The total DNA was then extracted and purified with the Promega Maxwell automated DNA recovery system following manufacturer's instructions. DNA from matched FFPE sections (40 µm) were extracted with Qiagen deparaffinization solution and FFPE DNA extraction kit following manufacturer's instructions. 16S amplicon sequencing was completed targeting the V6‐V8 region. Reads were processed with QIIME2/DADA2 and referenced against the SILVA SSU r138 database. Analysis of microbiota diversity was performed in R packages and Calypso (http://cgenome.net). Data was transformed using Cumulative Sum Scaling + log2. Results: The mean sequencing depth of RNAlater biopsies was 4115 and FFPE was 20170. The alpha diversity was significantly greater for FFPE sequenced samples when compared to whole biopsies (Chao1 P=0.029, Figure 1). Additionally, RDA multivariate analysis demonstrated significant clustering (P=0.024) of FFPE communities compared to the patient matched whole biopsy extracts. Permutational multivariate analysis of variance (adonis function) and ANOSIM Bray‐Curtis were also both significant when comparing the MAM of each extraction type (P=0.0297 and 0.023 (R =1), respectively). There were also fewer "unknown" taxonomic assignments for FFPE samples compared to the matched biopsies. Conclusions: Here we have shown that MAM 16S sequencing from FFPE tissue sections (tested 2‐4yrs old) is a viable option and means that single biopsies can be multi‐functional (histopathology and viable DNA recovery). The viability of MAM sequence amplification from FFPE provides a tantalising opportunity as the historical catalogue of samples where there were previously no specific biopsies collected for microbial analysis is extensive and would substantially contribute to the integration of FD associated MAM profiles. [ABSTRACT FROM AUTHOR]

R4338 --> 865.6 --> Background: There is comorbidity between anxiety disorders and gastrointestinal disorders, with both linked to adverse early life events. The microbiome gut‐brain‐axis (MGBA), a bidirectional communication system, is plastic throughout the neonatal period and we hypothesised that disruption of the MGBA development through inflammation and infection mediates this relationship. Here, we used a well‐established neonatal rodent immune activation model to investigate the long‐term effect of neonatal lipopolysaccharide (LPS) exposure on adult behaviour and the relationship to microbiome composition. Methods: Wistar rats were injected with LPS (0.05mg/kg) or saline (equivolume) on postnatal days 3 and 5. In adulthood, behavioural tests were performed to assess anxiety‐like behaviour, and microbiota sequencing was performed on stool samples. Results: There were distinctly different behavioural phenotypes for LPS‐exposed males and females. LPS‐exposed males displayed typical anxiety‐like behaviours with significantly decreased social interaction and increased defecation relative to controls. LPS‐exposed females displayed a different behavioural phenotype with significantly increased social interaction and exploration compared to controls. With respect to microbiota profiling data, there were distinct beta diversity clusters for each group which were distinct for each sex. Bacteroidota was significantly increased for LPS‐exposed females and Proteobacteria was decreased for LPS‐exposed rats of both sexes versus controls. Additionally, alterations in predicted functional pathways for neurotransmitters were observed based on LPS‐ exposure and sex. A significant decrease in the relative abundance of D‐glutamine and D‐glutamate metabolism was found in LPS exposed females compared to control females, while no change was observed in males. Conclusions: These results indicate that neonatal immune activation alters both adult behaviour and microbiota communities in sex‐specific ways. These findings highlight the importance of sex in determining the impact of early life immune activation events on the development of anxiety‐like behaviours and suggests a potential moderating effect of the gut brain axis. These findings may begin to explain the underlying biology to sex prevalence in anxiety disorders and disorders of the gut. [ABSTRACT FROM AUTHOR]

R4289 --> 867.3 --> BACKGROUND: Human colonic spirochetosis (CS) is an infection caused by the Brachyspira genus. Although Brachyspira have been considered commensal, recent evidence suggests they are associated with low grade inflammation and irritable bowel syndrome. Increased eosinophils, mast cells and lymphocyte aggregates have been reported in CS, however, little is known about immune cell localization. We hypotheses that CS infection is associated with a distinct mucosal immune signature and we aim to characterize the distribution of these immune cells during spirochete infection. METHOD: Paraffin‐embedded colonic biopsy samples from 20 CS patients (mean age 45.7, 70% male), 20 age/sex‐matched control patients (mean age 45.6, 70% male) without CS, and 13 healthy controls (mean age 39.8, 31% male) were examined. CS positive and negative areas of CS patient biopsies were identified by Treponema pallidum IHC staining. Intra‐epithelial lymphocyte (IEL) was identified by H&E staining, eosinophil was identified by congo‐red staining, mast cell was identified by CD117 IHC staining. Tissue sections were visualised using Aperio AT2 digital scanner. Peak numbers of eosinophil and mast cell were counted in 3 fields of intra‐epithelial and lamina propria area. RESULTS: Intra‐epithelial eosinophils and mast cells were significantly higher in CS patients compared to matched controls (p=0.043, p=0.022) and healthy controls (p=0.023, p=0.032) Lamina propria mast cells were significantly higher in CS patients compared to matched controls (p=0.009) and healthy controls (p=0.004). In addition, IELs were significantly higher in CS patients compared to matched controls (p=0.002) and healthy controls (p<0.0001). Upon examining the localisation of these immune cells we found that, CS positive areas of CS patient biopsies had significantly higher intra‐epithelial eosinophils, compared to healthy controls (p=0.049), but not matched controls, or CS negative areas of CS patient biopsies. For intra‐epithelial mast cells, there were no differences between CS positive areas and CS negative areas compared to healthy controls, while CS negative areas had significantly higher intra‐epithelial mast cells compared to matched controls (p=0.009). Lamina propria mast cells were significantly higher in CS positive and negative areas compared to matched controls (CS+ p=0.02, CS‐ p=0.03) and healthy controls (CS+ p=0.01, CS‐ p=0.01), while there were no differences between these areas. Finally, IELs were significantly higher in CS positive areas compared with CS negative areas (p=0.0171), while there were no differences between CS negative areas, matched and healthy controls. CONCLUSION: Increased IELs are associated with spirochete positive areas, suggests that IELs actively respond to spirochete infection. Increased intra‐epithelial eosinophils and mast cells in CS patients compared to controls suggests that these cells actively migrate towards spirochetes colonizing the mucosal epithelium. These data suggest that intestinal spirochetes provoke mucosal immune responses and may be an underappreciated intestinal pathogen. [ABSTRACT FROM AUTHOR]