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The aim of candidate universal influenza vaccines is to provide broad protection against influenza A and B viruses. Studies have demonstrated that broadly reactive antibodies require Fc–Fc gamma receptor interactions for optimal protection; however, the innate effector cells responsible for mediating this protection remain largely unknown. Here, we examine the roles of alveolar macrophages, natural killer cells, and neutrophils in antibody-mediated protection. We demonstrate that alveolar macrophages play a dominant role in conferring protection provided by both broadly neutralizing and non-neutralizing antibodies in mice. Our data also reveal the potential mechanisms by which alveolar macrophages mediate protection in vivo, namely antibody-induced inflammation and antibody-dependent cellular phagocytosis. This study highlights the importance of innate effector cells in establishing a broad-spectrum antiviral state, as well as providing a better understanding of how multiple arms of the immune system cooperate to achieve an optimal antiviral response following influenza virus infection or immunization. Broadly reactive antibodies that recognize influenza A virus HA can be protective, but the mechanism is not completely understood. Here, He et al. show that the inflammatory response and phagocytosis mediated by the interaction between protective antibodies and macrophages are essential for protection.

Severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is currently causing a worldwide pandemic with high morbidity and mortality. Development of animal models that recapitulate important aspects of coronavirus disease 2019 (COVID-19) is critical for the evaluation of vaccines and antivirals, and understanding disease pathogenesis. SARS-CoV-2 has been shown to use the same entry receptor as SARS-CoV-1, human angiotensin-converting enzyme 2 (hACE2) [1-3]. Due to amino acid differences between murine and hACE2, inbred mouse strains fail to support high titer viral replication of SARS-CoV-2 virus. Therefore, a number of transgenic and knock-in mouse models, as well as viral vector-mediated hACE2 delivery systems have been developed. Here we compared the K18-hACE2 transgenic model to adenovirus-mediated delivery of hACE2 to the mouse lung. We show that K18-hACE2 mice replicate virus to high titers in the nasal turbinates, lung and brain, with high lethality, and cytokine/chemokine production. In contrast, adenovirus-mediated delivery results in viral replication to lower titers limited to the nasal turbinates and lung, and no clinical signs of infection. The K18-hACE2 model provides a stringent model for testing vaccines and antivirals, whereas the adenovirus delivery system has the flexibility to be used across multiple genetic backgrounds and modified mouse strains.

Fc-dependent effector functions are an important determinant of the in vivo potency of therapeutic antibodies. Effector function is determined by the combination of FcRs bound by the antibody and the cell expressing the relevant FcRs, leading to antibody-dependent cellular cytotoxicity (ADCC). A number of ADCC assays have been developed; however, they suffer from limitations in terms of throughput, reproducibility, and in vivo relevance. Existing assays measure NK cell-mediated ADCC activity; however, studies suggest that macrophages mediate the effector function of many antibodies in vivo. Here, we report the development of a macrophage-based ADCC assay that relies on luciferase expression in target cells as a measure of live cell number. In the presence of primary mouse macrophages and specific antibodies, loss of luciferase signal serves as a surrogate for ADCC-dependent killing. We show that the assay functions for a variety of mouse and human isotypes with a model antigen/antibody complex in agreement with the known effector function of the isotypes. We also use this assay to measure the activity of a number of influenza-specific antibodies and show that the assay correlates well with the known in vivo effector functions of these antibodies.

The host tropism of viral infection is determined by a variety of factors, from cell surface receptors to innate immune signaling. Many viruses encode proteins that interfere with host innate immune recognition in order to promote infection. STAT2 is divergent between species and therefore has a role in species restriction of some viruses. To understand the role of STAT2 in human metapneumovirus (HMPV) infection of human and murine tissues, we first infected STAT2−/− mice and found that HMPV could be serially passaged in STAT2−/−, but not WT, mice. We then used in vitro methods to show that HMPV inhibits expression of both STAT1 and STAT2 in human and primate cells, but not in mouse cells. Transfection of the murine form of STAT2 into STAT2-deficient human cells conferred resistance to STAT2 inhibition. Finally, we sought to understand the in vivo role of STAT2 by infecting hSTAT2 knock-in mice with HMPV, and found that mice had increased weight loss, inhibition of type I interferon signaling, and a Th2-polarized cytokine profile compared to WT mice. These results indicate that STAT2 is a target of HMPV in human infection, while the murine version of STAT2 restricts tropism of HMPV for murine cells and tissue.

The cytoplasmic helicase RIG-I is an established sensor for viral 5′-triphosphorylated RNA species. Recently, RIG-I was also implicated in the detection of intracellular bacteria. However, little is known about the host cell specificity of this process and the bacterial pathogen-associated molecular pattern (PAMP) that activates RIG-I. Here we show that RNA of Salmonella enterica serovar Typhimurium activates production of beta interferon in a RIG-I-dependent fashion only in nonphagocytic cells. In phagocytic cells, RIG-I is obsolete for detection of Salmonella infection. We further demonstrate that Salmonella mRNA reaches the cytoplasm during infection and is thus accessible for RIG-I. The results from next-generation sequencing analysis of RIG-I-associated RNA suggest that coding bacterial mRNAs represent the activating PAMP. IMPORTANCE S. Typhimurium is a major food-borne pathogen. After fecal-oral transmission, it can infect epithelial cells in the gut as well as immune cells (mainly macrophages, dendritic cells, and M cells). The innate host immune system relies on a growing number of sensors that detect pathogen-associated molecular patterns (PAMPs) to launch a first broad-spectrum response to invading pathogens. Successful detection of a given pathogen depends on colocalization of host sensors and PAMPs as well as potential countermeasures of the pathogen during infection. RIG-I-like helicases were mainly associated with detection of RNA viruses. Our work shows that S. Typhimurium is detected by RIG-I during infection specifically in nonimmune cells. S. Typhimurium is a major food-borne pathogen. After fecal-oral transmission, it can infect epithelial cells in the gut as well as immune cells (mainly macrophages, dendritic cells, and M cells). The innate host immune system relies on a growing number of sensors that detect pathogen-associated molecular patterns (PAMPs) to launch a first broad-spectrum response to invading pathogens. Successful detection of a given pathogen depends on colocalization of host sensors and PAMPs as well as potential countermeasures of the pathogen during infection. RIG-I-like helicases were mainly associated with detection of RNA viruses. Our work shows that S. Typhimurium is detected by RIG-I during infection specifically in nonimmune cells.

Ly6Chi inflammatory monocytes show high IFN responses, and contribute to both protective and pathogenic functions following influenza virus infection. In order to understand the significance of IFN responses in this subset, we examined monocytes during infection with a lethal H5N1 virus that induces high levels of IFN and a low-pathogenicity H1N1 virus that induces low levels of IFN. We show that H5N1 infection results in early recruitment of high numbers of Ly6Chi monocytes and induction of chemokines and Ifnb1. Using unbiased transcriptomic and proteomic approaches, we also find that monocytes are significantly enriched during H5N1 infection and are associated with chemokine and IFN signatures in mice, and with severity of symptoms after influenza virus infection in humans. Recruited Ly6Chi monocytes subsequently become infected in the lung, produce IFN-β, and mature into FasL+ monocyte-derived cells (FasL+MCs) expressing dendritic cell markers. Both Ccr2-/- and Faslgld mice are protected from lethal infection, indicating that monocytes contribute to pathogenesis. Global loss of type I and type III IFN signaling in Stat2-/- mice results in loss of monocyte recruitment, likely reflecting a requirement for IFN-dependent chemokine induction. Here we show that IFN is not directly required for monocyte recruitment on an IFN-sufficient background, but is required for maturation to FasL+MCs. Loss of IFN signaling skews to a Ly6Clo phenotype associated with tissue repair, suggesting that IFN signaling in monocytes is a critical determinant of influenza virus pathogenesis. Competing Interest Statement The authors have declared no competing interest.

Abstract The Toll/IL-1R domain-containing adaptor protein SARM1 is expressed primarily in the brain, where it mediates axonal degeneration. Additional roles for SARM1 in a number of other processes including TLR-signaling, viral infection, chemokine expression, and expression of the proapoptotic protein XAF1 have also been described. Much of the supporting evidence for SARM1 function has been generated by comparing WT C57BL/6 (B6) mice to SARM1-deficient mice backcrossed to the B6 background. Here we show that the Sarm1 gene lies in a gene-rich region encompassing XAF1, and the MIP and MCP chemokine family loci among other genes. Because gene-targeting of SARM1-deficient strains was done with 129 ES cells and these genes are too close to segregate, they remain 129 in sequence. As this could account for phenotypes attributed to SARM1, we generated new knockout mouse strains on a pure B6 background using CRISPR. Experiments in these new strains confirmed the role of SARM1 in axonal degeneration and susceptibility to WNV infection, but not in susceptibility to VSV or LACV infection, or chemokine or Xaf1 expression. Notably, the Xaf1 gene shows sequence variation between B6 and 129, resulting in coding changes and novel splice variants. Given its known role in apoptosis, XAF1 variants may account for some phenotypes described in previously made SARM1-deficient strains. RNAseq in the new strains reveal changes in the mitochondrial electron transport chain and ribosomal proteins, suggesting possible downstream targets of SARM1. Re-evaluation of described phenotypes in these new strains will be critical for defining the function of SARM1.

Severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is currently causing a worldwide pandemic with high morbidity and mortality. Development of animal models that recapitulate important aspects of coronavirus disease 2019 (COVID-19) is critical for the evaluation of vaccines and antivirals, and understanding disease pathogenesis. SARS-CoV-2 has been shown to use the same entry receptor as SARS-CoV-1, human angiotensin-converting enzyme 2 (hACE2)(1-3). Due to amino acid differences between murine and hACE2, inbred mouse strains fail to support high titer viral replication of SARS-CoV-2 virus. Therefore, a number of transgenic and knock-in mouse models, as well as viral vector-mediated hACE2 delivery systems have been developed. Here we compared the K18-hACE2 transgenic model to adenovirus-mediated delivery of hACE2 to the mouse lung. We show that K18-hACE2 mice replicate virus to high titers in both the lung and brain leading to lethality. In contrast, adenovirus-mediated delivery results in viral replication to lower titers limited to the lung, and no clinical signs of infection with a challenge dose of 10 plaque forming units. The K18-hACE2 model provides a stringent model for testing the ability of vaccines and antivirals to protect against disease, whereas the adenovirus delivery system has the flexibility to be used across multiple genetic backgrounds and modified mouse strains.