Advanced non-alcoholic fatty liver disease (NAFLD) is a rapidly emerging global health problem associated with pre-disposing genetic polymorphisms, most strikingly an isoleucine to methionine substitution in patatin-like phospholipase domain-containing protein 3 (PNPLA3-I148M). Here, we study how human hepatocytes with PNPLA3 148I and 148M variants engrafted in the livers of chimeric mice respond to a hypercaloric Western-style diet. As early as 4 weeks, mice developed dyslipidemia, impaired glucose tolerance, and steatohepatitis selectively in the human graft, followed by pericellular fibrosis after 8 weeks of hypercaloric feeding. The PNPLA3 148M variant, either from a homozygous 148M human donor or overexpressed in a homozygous 148I donor background, caused widespread microvesicular steatosis and even more severe steatohepatitis. We conclude that PNPLA3 148M in human hepatocytes exacerbates NAFLD. These models will facilitate mechanistic studies into human genetic variants associated with advanced fatty liver diseases.

SummaryIntratumoral (IT) delivery of immune-activating viruses can serve as an important strategy to turn “cold” tumors into “hot” tumors, resulting in overcoming resistance to immune checkpoint blockade (ICB). Modified vaccinia virus Ankara (MVA) is a highly attenuated, non-replicative vaccinia virus that has a long history of human use. Here we report that IT recombinant MVA (rMVA), lacking E5R encoding an inhibitor of the DNA sensor cyclic GMP-AMP synthase (cGAS), expressing a dendritic cell growth factor, Fms-like tyrosine kinase 3 ligand (Flt3L), and a T cell co-stimulator, OX40L, generates strong antitumor immunity, which is dependent on CD8+ T cells, the cGAS/STING-mediated cytosolic DNA-sensing pathway, and STAT1/STAT2-mediated type I IFN signaling. Remarkably, IT rMVA depletes OX40hi regulatory T cells via OX40L/OX40 interaction and IFNAR signaling. Taken together, our study provides a proof-of-concept for improving MVA-based cancer immunotherapy, through modulation of both innate and adaptive immunity.One Sentence SummaryIntratumoral delivery of recombinant MVA for cancer immunotherapy

3The pulmonary immune system consists of a network of tissue-resident cells as well as immune 4 cells that are recruited to the lungs during infection and/or inflammation. How the two immune 5 components cross-talk during an acute viral infection is not well understood. Intranasal infection 6 of mice with vaccinia virus causes lethal pneumonia and systemic dissemination. Here we report 7 that vaccinia host range protein C7 is a critical virulence factor. Vaccinia virus with deletion of C7 8 (VACV∆C7L) is non-pathogenic in wild-type C57BL/6J mice, but it gains virulence in mice 9 lacking STAT2, or IFNAR1, or MDA5/STING. We provide evidence that lung type II alveolar 10 epithelial cells (AECs) provide first-line of defense against VACV∆C7L infection by inducing 11 IFN-b and IFN-stimulated genes via the activation of the MDA5 and STING-mediated nucleic 12 acid-sensing pathways. This leads to recruitment of CCR2 + inflammatory monocytes into the lungs 13 to fight against viral dissemination. 14 In this study, we first demonstrated that vaccinia host range protein C7 is a virulence factor. 45 VACV∆C7L is non-pathogenic in WT C57BL/6J mice at a high dose of infection intranasally, but 46 gained virulence in STAT2, IFNAR1, or MDA5/STING-deficient mice. VACV∆C7L is non-47 pathogenic in RAG1-deficient mice, which lack T and B cells. Thus, this attenuated VACV mutant 48provides us with a model to evaluate the lung innate immune responses to acute pulmonary 49 infection with a DNA virus. We found that VACV∆C7L infection triggers the release of IFN-b, 50 CCL2, CXCL9, and CXCL10 into bronchioalveolar (BAL), whereas WT VACV infection fails to 51 do so. Infection of primary lung AECII with VACV∆C7L in vitro leads to the induction of IFNB 52 and IFN-stimulated genes (ISGs), which is dependent on the MDA5-dependent cytosolic dsRNA-53 sensing pathway as well as the STING-dependent cytosolic DNA-sensing pathway. 54 55 Given that both IFNAR1 and STAT2-deficient mice are more susceptible to VACV∆C7L 56 infection, we hypothesize that type I IFN signaling plays an important role in restricting 57 VACV∆C7L, either through stimulating IFNAR on the lung AEC and/or hematopoietic cell 58 populations. To probe the relative contributions of lung non-hematopoietic resident cells versus 59 hematopoietic cells in host defense against VACV∆C7L, we generated bone marrow chimeric 60 mice. Our results indicate that the IFNAR signaling on the non-hematopoietic cells (likely the 61 AECII) plays a critical role in host defense, and IFNAR signaling on hematopoietic cells also 62 contributes. Using IFNAR1 fl/fl -Sftpc cre-ERT2 mice, we showed that type I IFN signaling on lung 63AECIIs is important for host defense against VACV∆C7L infection. 64 65To probe whether myeloid cells play a role in host defense against VACV∆C7L infection, we 66 transiently depleted CCR2 + inflammatory monocytes in CCR2-DTR mice through administration 67 of diphtheria toxin (DT), and found that depletion of CCR2 + inflammatory monocytes renders the 68 mice susceptible to VACV∆C7...