Rachael Kostelecky

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

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.

Mucosal acidification is an often‐underappreciated aspect of mucosal inflammation. Like other chronic inflammatory diseases, tissue acidification has been observed in inflammatory bowel disease (IBD) patients, with reports of colonic pH as low as 4. We recently demonstrated that neutrophil (PMN) transepithelial migration rapidly acidifies the microenvironment resulting in inflammatory acidification of the mucosa. Furthermore, we identified an adaptive tissue response in IEC which buffered extracellular acidification during PMN transepithelial migration, suggesting pH homeostasis is important during acute inflammation. Very little is known in regard to the impact of extracellular acidification on the intestinal epithelial cell (IEC) function. To better understand the impact of extracellular pH on IEC cell function, we investigated the impact of extracellular acidosis on IEC gene expression. Guided by an unbiased RNAseq of T84 IEC exposed to low pH, we identified significant induction of several CREB associated genes. Based on this observation, we examined the phosphorylation of CREB under acidic conditions. Under acidic conditions, CREB is rapidly phosphorylated. We observed CREB phosphorylation within 5 minutes of exposure to acidic conditions and peaks at 30 min. Extending these studies we investigated the mechanism(s) involved in acidosis induced CREB phosphorylation. Interestingly, cAMP signaling, a common signaling pathway for CREB phosphorylation, appears to not be involved in acidosis‐induced CREB phosphorylation. Under acidic conditions, there was no significant increase in cAMP levels in IEC and acidosis‐induced CREB phosphorylation was still observed in T84 IEC treated with cAMP inhibitors. KEGG analysis of the RNAseq data revealed the induction of several MAPK regulatory genes, including members of the NR4A and DUSP families. Based on this observation we examined several key proteins involved in MAPK signaling, ERK1/2 and MSK1. Under acidic conditions ERK1/2 and MSK1 are rapidly phosphorylation and treatment with a MEK1/2 or MSK1 inhibitor significant attenuated CREB phosphorylation under acidic conditions. Further examination revealed CREB phosphorylation signals through the Ga _I ‐linked GPCR, GRP31. Finally, we extended our studies in vivo and examined mucosal acidification in a murine model of ileitis, TNFΔARE mice. Using a novel pH reporting E. coli strain, we identified significant mucosal acidification in the distal ileum of TNFΔARE mice when compared to WT mice. Mucosal acidification was positively correlated with the expression of several acidification associated genes; FosB, NR4A1, and CXCL1. These results identify extracellular acidification as a significant signaling mechanism during tissue inflammation. Thus, regulation of GPR31 or MSK1 may serve as new therapeutic targets to limit acidosis signaling during mucosal inflammation.

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.

Background & Objective: Primary Sclerosing Cholangitis (PSC) is a progressive cholestatic liver disease of unknown etiology that carries a strong association with inflammatory bowel disease (IBD). PSC‐associated IBD (PSC‐IBD) displays a unique colitis phenotype characterized by right side predominant inflammation and increased risk of colorectal cancer compared to non‐PSC‐IBD. Although the initiation/progression PSC‐IBD remains unclear, some evidence supports an integral role for the gut‐specific mucosal vascular addressin cell adhesion molecule (MAdCAM‐1) in modulating both hepatic and intestinal inflammation. Multidrug resistance protein 2 (Mdr2) knockout mice develop spontaneous cholestatic liver injury and fibrosis mirroring human PSC. To further elucidate disease mechanisms of PSC‐IBD, we combined Mdr2−/− mice with the chemically inducible model of DSS colitis (Mdr2−/−/DSS). We hypothesize that alterations in bile acids promote mucosal inflammation in this murine model of combined cholestasis/colitis. There are currently no good models for PSC‐IBD, so our objective is to develop a murine model mimicking PSC‐IBD for studying the gut‐liver axis and proinflammatory crosstalk. Methods & Results: Mice were fed 1.5% DSS for 5 days and recovered for 4 days before being euthanized. Transformed sinusoidal endothelial cells (TSECs) were treated with 5 ng/ml of TNF‐αand/or 100μM of ursodeoxycholic acid (UDCA). UDCA treated mice were fed 0.5% UDCA fortified chow for 2 weeks and throughout the previously described DSS course. In this model, Mdr2−/− mice displayed marked alterations in intestinal bile acid composition and increased susceptibility to DSS colitis based on weight loss, disease activity index, histology, and inflammatory cytokines, etc. Mdr2−/− mice also displayed an upregulation of colonic MAdCAM‐1 expression as compared to C57BL/6 colitis controls. In vitro work utilizing transformed sinusoidal endothelial cells (TSECs) and bEnd.3 cells (brain endothelial cells) revealed that MAdCAM‐1 was potently induced by TNF‐α and that this induction was significantly attenuated by co‐treatment with ursodeoxycholic acid (UDCA). In vivo , DSS‐treated Mdr2−/− mice fed with UCDA fortified chow demonstrated significant attenuation of colitis susceptibility and decreased colonic MAdCAM‐1 expression as compared to standard chow controls. Conclusions: These findings suggest a unique interplay between alterations in bile acid homeostasis and intestinal MAdCAM‐1 expression in modulating pro‐inflammatory cross‐talk along the gut‐liver axis and identify intestinal bile acid signaling as a potential therapeutic target for the treatment of PSC‐IBD. Support or Funding Information: University of Colorado GI and Liver Innate Immune Program (GALIIP)

Tissue acidification is commonly associated with a number of inflammatory states. The primary mechanism of extracellular acidification is thought to be increased accumulation of lactic acid resulting from enhanced glycolysis. Like other chronic inflammatory diseases, tissue acidification has been observed in inflammatory bowel disease patients, with reports of colonic pH as low as 4. We have recently demonstrated that neutrophil (PMN) transepithelial migration rapidly acidifies the microenvironment resulting in inflammatory acidification in the mucosa. Very little is known with regard to the mechanism(s) involved in this acidification or the impact of such acidification on the intestinal epithelial cell (IEC) function. Initial observations showed that physical interaction between IEC and PMN was required for inflammatory acidification; however, when transmigration was blocked using anti‐CD11b antibodies, we observed significant extracellular acidification in the absence of PMN transepithelial migration. This observation suggests that binding of the CD11b receptor plays a crucial role promoting extracellular acidification. Expanding upon these observations, we analyzed extracellular lactate secretion. We observed that lactic acid was released by IEC 4 to 5 hours after the addition of PMN and in quantities not high enough to account for the extracellular acidification observed. Additionally, the concentration of lactic acid released in the anti‐CD11b treated cells was similar to control IEC in the absence of PMN. When combined, these data indicate lactate is not a significant mediator of extracellular acidification following PMN transmigration. Given the large number of PMNs present and chronic tissue acidification observed during active inflammation, we investigated the impact of extracellular acidification on the regulation of epithelial derived pro‐inflammatory markers. When T84 IEC were cultured in pH 6.0 media for 3 or 6 hours we observed increases in pro‐inflammatory markers, including IL‐8. To better understand how extracellular acidification alters IEC gene regulation, we utilized a transcription factor array to examine changes in transcription factor expression under acidic conditions. This screen identified CREB as one of the major transcription factors controlling responses to acidification. Further examination revealed that CREB phosphorylation occurs rapidly, is cAMP independent, and requires MSK1. Inhibition of MSK1 prevents the induction of IL‐8 and other acidification associated gene transcription. Theses result identified extracellular acidification as a significant signaling mechanism during tissue inflammation. Targeted regulation of MSK1 may serve as a new therapeutic approach to limit pro‐inflammatory signaling during mucosal inflammation. Support or Funding Information: R01 DK095491, NIH