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.

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. Importance The human gammaherpesviruses Epstein-Barr virus and Kaposi’s sarcoma associated herpesvirus are DNA viruses that cause lifelong infection and are associated with multiple diseases, especially in immunocompromised individuals. Murine gammaherpesvirus 68 (MHV68) is a powerful mouse model that permits close examination of these viruses. Previous studies of MHV68 identified that macrophages are an important in vivo target of infection; how infection within these cells is regulated remains incompletely understood. Here, we demonstrate that MHV68 infection of macrophages is characterized by two divergent outcomes across a population of infected cells: while a small subset of cells undergo lytic replication, to make new virus progeny, the majority of cells are characterized by an atypical, restricted form of infection characterized by a distinct viral gene transcription program not previously reported. These studies highlight important cell-type specific outcomes of gammaherpesvirus infection and identify a potential alternate program by which these viruses usurp macrophages.

A pathological hallmark of mucosal diseases such as inflammatory bowel disease (IBD) is disruption of the intestinal epithelium, a selectively permeable barrier that acts to absorb nutrients and water while simultaneously guarding against pathogens and environmental threats. The intestinal epithelium exists in a metabolic state of low oxygen tension termed “physiologic hypoxia” that is essential for its proper function as an absorptive and barrier organ. An important factor in maintaining hypoxic epithelial homeostasis is the transcription factor family HIF, which is stabilized in hypoxic conditions and mediates epithelial homeostatic response to low oxygen tensions. To identify HIF transcriptional targets in epithelia, chromatin immunoprecipitation (ChIP) was performed in Caco‐2 intestinal epithelial cells (IECs) using HIF‐1α and HIF‐2α specific polyclonal antibodies. ChIP‐enriched DNA was hybridized to a custom promoter microarray (termed ChIP‐chip) comprising a genome‐wide set of predicted transcription start site (TSS) flanking sequences at 50‐bp resolution. This unbiased approach identified autophagy as one of the major HIF‐1 targeted pathways. Binding of HIF‐1 to ATG9A, the only transmembrane autophagy protein, was significantly enriched by exposure of IEC to hypoxia. Validation of the ChIP‐chip revealed prominent induction of ATG9A mRNA and protein. Likewise, using luciferase promoter assays, we identified a functional hypoxia response element upstream of the TSS. Hypoxia‐mediated induction of ATG9A was lost in cells lacking HIF‐1. Strikingly, we found that lentiviral‐mediated knockdown of ATG9A in IECs prevents epithelial barrier formation and results in mislocalization of multiple junctional proteins. Extensions of these findings showed that ATG9A knockdown cells have intrinsic differences in junctional protein expression, suggesting that ATG9A is essential for multiple steps of epithelial junction biogenesis. Our findings have novel applicability for inflammatory disorders that involve a compromised epithelial barrier, such as IBD, and suggest that targeting ATG9A may be a rational strategy for future therapeutic intervention.

SUMMARY: The gammaherpesviruses, including Epstein-Barr Virus and Kaposi’s Sarcoma-associated Herpesvirus, establish lifelong infections by maintaining a balance between lytic and latent infection states. How this balance is regulated in myeloid cells, an important but understudied cell type that can achieve both infection states, remains unclear. Using the murine gammaherpesvirus 68 (MHV68) model, we show that IFNγ and IL-4 reciprocally regulate lytic infection in macrophages in a time- and JAK/STAT signaling dependent-manner, with basal JAK/STAT signaling further restricting lytic infection. Using a combination of MHV68 reporter viruses, cellular and molecular techniques, we demonstrate that IFNγ blocks lytic replication through at least two distinct mechanisms, restricting delivery to or stability of the viral genome in the nucleus and potently restricting lytic cycle progression downstream of the immediate-early viral transactivator, RTA. These findings define specific points during lytic infection where IFNγ to enforce restricted, latent-like infection in myeloid cells. Graphical Highlights: IL-4 and IFNγ reciprocally regulate MHV68 macrophage infection in a time-dependent manner Lytic replication is induced by the JAK1/JAK2 inhibitor Ruxolitinib IFNγ regulates MHV68 infection at two distinct stages IFNγ pre-treatment limits viral nuclear entry