Allopurinol Disrupts Purine Metabolism to Increase Damage in Experimental Colitis.

Inflammatory bowel disease (IBD) is marked by a state of chronic energy deficiency that limits gut tissue wound healing. This energy shortfall is partially due to microbiota dysbiosis, resulting in the loss of microbiota-derived metabolites, which the epithelium relies on for energy procurement. The role of microbiota-sourced purines, such as hypoxanthine, as substrates salvaged by the colonic epithelium for nucleotide biogenesis and energy balance, has recently been appreciated for homeostasis and wound healing. Allopurinol, a synthetic hypoxanthine isomer commonly prescribed to treat excess uric acid in the blood, inhibits the degradation of hypoxanthine by xanthine oxidase, but also inhibits purine salvage. Although the use of allopurinol is common, studies regarding how allopurinol influences the gastrointestinal tract during colitis are largely nonexistent. In this work, a series of in vitro and in vivo experiments were performed to dissect the relationship between allopurinol, allopurinol metabolites, and colonic epithelial metabolism and function in health and during disease. Of particular significance, the in vivo investigation identified that a therapeutically relevant allopurinol dose shifts adenylate and creatine metabolism, leading to AMPK dysregulation and disrupted proliferation to attenuate wound healing and increased tissue damage in murine experimental colitis. Collectively, these findings underscore the importance of purine salvage on cellular metabolism and gut health in the context of IBD and provide insight regarding the use of allopurinol in patients with IBD.

Murine Gammaherpesvirus 68 Efficiently Infects Myeloid Cells Resulting In An Atypical, Restricted Form Of Infection.

The gammaherpesviruses (γHVs) establish a lifelong infection in their hosts, with the cellular outcome of infection intimately regulated by target cell type. Murine gammaherpesvirus 68 (MHV68), a small animal model of γHV infection, infects macrophages in vivo, resulting in a range of outcomes, from lytic replication to latent infection. Here, we have further investigated the nature of MHV68 macrophage infection using reductionist and primary in vivo infection studies. While MHV68 readily infected the J774 macrophage cell line, viral gene expression and replication were significantly impaired relative to a fully permissive fibroblast cell line. Lytic replication only occurred in a small subset of MHV68-infected J774 cells, despite the fact that these cells were fully competent to support lytic replication following pre-treatment with interleukin-4, a known potentiator of replication in macrophages. In parallel, we harvested virally-infected macrophages at 16 hours after MHV68 infection in vivo and analyzed gene expression by single cell RNA-sequencing. Among virally infected macrophages, only rare (0.25%) cells had lytic cycle gene expression, characterized by detection of multiple lytic cycle RNAs. In contrast, ~50% of virally-infected macrophages were characterized by expression of ORF75A, ORF75B and/or ORF75C, in the absence of other detectable viral RNAs. Selective transcription of the ORF75 locus also occurred in MHV68-infected J774 cells. In total, these studies indicate that MHV68 efficiently infects macrophages, with the majority of cells characterized by an atypical state of restricted viral transcription, and only rare cells undergoing lytic replication.

Essential role for epithelial HIF-mediated xenophagy in control of Salmonella infection and dissemination.

The intestinal mucosa exists in a state of "physiologic hypoxia," where oxygen tensions are markedly lower than those in other tissues. Intestinal epithelial cells (IECs) have evolved to maintain homeostasis in this austere environment through oxygen-sensitive transcription factors, including hypoxia-inducible factors (HIFs). Using an unbiased chromatin immunoprecipitation (ChIP) screen for HIF-1 targets, we identify autophagy as a major pathway induced by hypoxia in IECs. One important function of autophagy is to defend against intracellular pathogens, termed "xenophagy." Analysis reveals that HIF is a central regulator of autophagy and that in vitro infection of IECs with Salmonella Typhimurium results in induction of HIF transcriptional activity that tracks with the clearance of intracellular Salmonella. Work in vivo demonstrates that IEC-specific deletion of HIF compromises xenophagy and exacerbates bacterial dissemination. These results reveal that the interaction between hypoxia, HIF, and xenophagy is an essential innate immune component for the control of intracellular pathogens.

Contact-dependent, polarized acidification response during neutrophil-epithelial interactions.

Neutrophil (PMN) infiltration during active inflammation imprints changes in the local tissue environment. Such responses are often accompanied by significant extracellular acidosis that result in predictable transcriptional responses. In this study, we explore the mechanisms involved in inflammatory acidification as a result of PMN-intestinal epithelial cell (IEC) interactions. Using recently developed tools, we revealed that PMN transepithelial migration (TEM)-associated inflammatory acidosis is dependent on the total number of PMNs present during TEM and is polarized toward the apical surface. Extending these studies, we demonstrate that physical separation of the PMNs and IECs prevented acidification, whereas inhibition of PMN TEM using neutralizing antibodies enhanced extracellular acidification. Utilizing pharmaceutical inhibitors, we demonstrate that the acidification response is independent of myeloperoxidase and dependent on reactive oxygen species generated during PMN TEM. In conclusion, inflammatory acidosis represents a polarized PMN-IEC-dependent response by an as yet to be fully determined mechanism.

Mucosal acidosis elicits a unique molecular signature in epithelia and intestinal tissue mediated by GPR31-induced CREB phosphorylation.

Metabolic changes associated with tissue inflammation result in significant extracellular acidosis (EA). Within mucosal tissues, intestinal epithelial cells (IEC) have evolved adaptive strategies to cope with EA through the up-regulation of SLC26A3 to promote pH homeostasis. We hypothesized that EA significantly alters IEC gene expression as an adaptive mechanism to counteract inflammation. Using an unbiased RNA sequencing approach, we defined the impact of EA on IEC gene expression to define molecular mechanisms by which IEC respond to EA. This approach identified a unique gene signature enriched in cyclic AMP response element-binding protein (CREB)-regulated gene targets. Utilizing loss- and gain-of-function approaches in cultured epithelia and murine colonoids, we demonstrate that EA elicits prominent CREB phosphorylation through cyclic AMP-independent mechanisms that requires elements of the mitogen-activated protein kinase signaling pathway. Further analysis revealed that EA signals through the G protein-coupled receptor GPR31 to promote induction of FosB, NR4A1, and DUSP1. These studies were extended to an in vivo murine model in conjunction with colonization of a pH reporter Escherichia coli strain that demonstrated significant mucosal acidification in the TNFΔARE model of murine ileitis. Herein, we observed a strong correlation between the expression of acidosis-associated genes with bacterial reporter sfGFP intensity in the distal ileum. Finally, the expression of this unique EA-associated gene signature was increased during active inflammation in patients with Crohn's disease but not in the patient control samples. These findings establish a mechanism for EA-induced signals during inflammation-associated acidosis in both murine and human ileitis.

Bile acids modulate colonic MAdCAM-1 expression in a murine model of combined cholestasis and colitis.

Primary sclerosing cholangitis (PSC) is a progressive fibrosing cholestatic liver disease that is strongly associated with inflammatory bowel disease (IBD). PSC-associated IBD (PSC-IBD) displays a unique phenotype characterized by right-side predominant colon inflammation and increased risk of colorectal cancer compared to non-PSC-IBD. The frequent association and unique phenotype of PSC-IBD suggest distinctive underlying disease mechanisms from other chronic liver diseases or IBD alone. Multidrug resistance protein 2 knockout (Mdr2-/-) mice develop spontaneous cholestatic liver injury and fibrosis mirroring human PSC. As a novel model of PSC-IBD, we treated Mdr2-/- mice with dextran sulfate sodium (DSS) to chemically induce colitis (Mdr2-/-/DSS). Mdr2-/- mice demonstrate alterations in fecal bile acid composition and enhanced colitis susceptibility with increased colonic adhesion molecule expression, particularly mucosal addressin-cell adhesion molecule 1 (MAdCAM-1). In vitro, ursodeoxycholic acid (UDCA) co-treatment resulted in a dose dependent attenuation of TNF-α-induced endothelial MAdCAM-1 expression. In the combined Mdr2-/-/DSS model, UDCA supplementation attenuated colitis severity and downregulated intestinal MAdCAM-1 expression. These findings suggest a potential mechanistic role for alterations in bile acid signaling in modulating MAdCAM-1 expression and colitis susceptibility in cholestasis-associated colitis. Together, our findings provide a novel model and new insight into the pathogenesis and potential treatment of PSC-IBD.