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Microbiome-derived metabolites influence intestinal homeostasis and regulate graft-versus-host disease (GVHD), but the molecular mechanisms remain unknown. Here we show the metabolite sensor G-protein-coupled receptor 43 (GPR43) is important for attenuation of gastrointestinal GVHD in multiple clinically relevant murine models. GPR43 is critical for the protective effects of short-chain fatty acids (SCFAs), butyrate and propionate. Increased severity of GVHD in the absence of GPR43 is not due to baseline differences in the endogenous microbiota of the hosts. We confirm the ability of microbiome-derived metabolites to reduce GVHD by several methods, including co-housing, antibiotic treatment, and administration of exogenous SCFAs. The GVHD protective effect of SCFAs requires GPR43-mediated ERK phosphorylation and activation of the NLRP3 inflammasome in non-hematopoietic target tissues of the host. These data provide insight into mechanisms of microbial metabolite-mediated protection of target tissues from the damage caused allogeneic T cells.

Neurotropic alphaviruses such as western, eastern, and Venezuelan equine encephalitis viruses cause serious and potentially fatal central nervous system infections in humans and are high-priority potential bioterrorism agents. There are currently no widely available vaccines or licensed therapies for these virulent pathogens. To identify potential novel antiviral drugs, we developed a cell-based assay with a western equine encephalitis virus replicon that expresses a luciferase reporter gene and screened a small molecule diversity library of 51,028 compounds. We identified and validated a thieno[3,2-b]pyrrole compound with a half maximal inhibitory concentration of <10 µmol/L, a selectivity index >20, and potent activity against live virus in cultured neuronal cells. Furthermore, a structure-activity relationship analysis with 20 related compounds identified several with enhanced activity profiles, including 6 with submicromolar half maximal inhibitory concentrations. In conclusion, we have identified a novel class of promising inhibitors with potent activity against virulent neurotropic alphaviruses.

Innate cell-autonomous antiviral responses are essential first lines of defense against central nervous system infections but may also contribute to neuropathogenesis. We investigated the relationships between innate immunity and neuronal differentiation using an in vitro culture system with human cell lines to analyze cellular responses to the neurotropic alphavirus western equine encephalitis virus. Human neuronal cells displayed a maturation-dependent reduction in virus-induced cytopathology that was independent of autocrine interferon alpha or beta activity. In addition, maturation was associated with enhanced responsiveness to exogenous stimuli, such that differentiated neurons required five- to ten-fold less type I interferon to suppress viral replication or virus-induced cytopathology compared to immature cells, although this enhanced responsiveness extended to only a subset of unique type I interferons. These results demonstrate that maturation-dependent changes in human neuronal cells may be key determinants in the innate immune response to infections with neurotropic alphaviruses.

Cross-presentation initiates immune responses against tumors and viral infections by presenting extracellular antigen on MHC I to activate CD8+ T cell-mediated cytotoxicity. In vitro studies in dendritic cells (DCs) established SNARE protein SEC22B as a specific regulator of cross-presentation. However, the in vivo contribution of SEC22B to cross-presentation has not been tested. To address this, we generated DC-specific Sec22b knockout (CD11c-Cre Sec22bfl/fl) mice. Contrary to paradigm, SEC22B-deficient DCs efficiently cross-present both in vivo and in vitro. Though in vitro shRNA-mediated Sec22b silencing in bone marrow-derived dendritic cells (BMDCs) reduced cross-presentation, treatment of SEC22B-deficient BMDCs with the same shRNA produced a similar defect, suggesting the Sec22b shRNA modulates cross-presentation through off-target effects. RNAseq of Sec22b shRNA-treated SEC22B-deficient BMDCs demonstrated several changes in the transcriptome. Our data demonstrate that, contrary to the accepted model, that SEC22B is not necessary for cross-presentation, cautioning against extrapolating phenotypes from knockdown studies alone.

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Cell-intrinsic innate immune responses mediated by the transcription factor interferon regulatory factor 3 (IRF-3) are often vital for early pathogen control, and effective responses in neurons may be crucial to prevent the irreversible loss of these critical central nervous system cells after infection with neurotropic pathogens. To investigate this hypothesis, we used targeted molecular and genetic approaches with cultured neurons to study cell-intrinsic host defense pathways primarily using the neurotropic alphavirus western equine encephalitis virus (WEEV). We found that WEEV activated IRF-3-mediated neuronal innate immune pathways in a replicationdependent manner, and abrogation of IRF-3 function enhanced virus-mediated injury by WEEV and the unrelated flavivirus St. Louis encephalitis virus. Furthermore, IRF-3-dependent neuronal protection from virus-mediated cytopathology occurred independently of autocrine or paracrine type I interferon activity. Despite being partially controlled by IRF-3-dependent signals, WEEV also disrupted antiviral responses by inhibiting pattern recognition receptor pathways. This antagonist activity was mapped to the WEEV capsid gene, which disrupted signal transduction downstream of IRF-3 activation and was independent of capsid-mediated inhibition of host macromolecular synthesis. Overall, these results indicate that innate immune pathways have important cytoprotective activity in neurons and contribute to limiting injury associated with infection by neurotropic arboviruses.

Allogeneic stem cell transplantation (allo-HCT) through its graft-versus-tumor (GVT) effects is a curative therapy against many hematological malignancies. However, GVT is linked to harmful graft-versus-host disease (GVHD) after allo-HCT. Both GVT and GVHD require allogeneic T cell responses, which is an energetically costly process that causes oxidative stress. Sirtuin 3 (SIRT3), a mitochondrial histone deacetylases (HDAC), plays an important role in cellular processes through inhibition of reactive oxygen species (ROS). Non-mitochondrial class of HDACs regulate T cell responses; but the role of mitochondrial HDACs, specifically SIRT3, on donor T cell responses after allo-HCT remain unknown. Herein we report that SIRT3 deficient (SIRT3-/-) donor T cells cause reduced GVHD severity in multiple clinically relevant murine models. The GVHD protective effect of allogeneic SIRT3-/- T cells was associated with a reduction in their activation, reduced CXCR3 expression, and no significant impact on cytokine secretion or cytotoxic functions. Intriguingly, the GVHD protective effect of SIRT3-/- T cells was associated with a reduction in ROS production which is contrary to the effect of SIRT3 deficiency on ROS production in other cells/tissues and likely a consequence of their deficient activation. Notably the reduction in GVHD in the gastrointestinal (GI) tract was not associated with a substantial reduction in the graft-versus-tumor (GVT) effect. Collectively these data reveal that SIRT3 activity promotes allogeneic donor T cell responses and ROS production without altering T cell cytokine or cytolytic functions and identify SIRT3 as a novel target on donor T cells to improve outcomes after allo-HCT.