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The SP-R210 Isoforms of the Myosin 18A SP-A Receptor Mediate Influenza A Virus Infection with Distinct Biological Outcomes in Macrophages
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A4718 - The SP-R210 Isoforms of the Myosin 18A SP-A Receptor Mediate Influenza A Virus Infection with Distinct Biological Outcomes in Macrophages
Author Block: Y. Chen1, S. Hu1, T. Umstead1, E. Yau2, Z. C. Chroneos1; 1Pediatrics, Pennsylvania State University College of Medicine, Hershey, PA, United States, 2Pennsylvania State University College of Medicine, Hershey, PA, United States.
Rationale: Influenza A viruses (IAVs) cause highly contagious respiratory infections and widespread lung inflammation by mechanisms that are poorly understood. Alveolar macrophages (AMs) and surfactant protein A (SP-A) are critical for resolution of IAV infection. SP-A modulates macrophage activation and clearance of bacterial pathogens through the Myosin 18A (Myo18A) receptor, which of two alternatively spliced cell-surface isoforms in macrophages named previously as SP-R210L and SP-R210S that differ by an amino-terminal PDZ domain. SP-R210 isoforms regulate the threshold of macrophage activation and innate recognition of pathogens. Based on these findings we tested the hypothesis whether the SP-A/SP-r210 isoform pathways mediate IAV infection and inflammatory responses using SP-R210L-deficient (SP-R210L (DN)) macrophages. Methods: We generated SP-R210L(DN) macrophages either by dominant negative disruption or conditional knockout in alveolar macrophages, and used native and deglycosylated SP-A, SP-R210 antibodies, flow cytometry, confocal microscopy, siRNA, RNA sequencing, and ELISA assays to determine the role of SP-R210 isoforms in IAV infection of macrophages. Results: Competitive inhibition assays using native SP-A, deglycosylated SP-A, or SP-R210 antibodies demonstrate that SP-R210 is a common receptor for both SP-A and IAV. Flow cytometry and microscopy assays, however, revealed that SP-R210L is required for nuclear transport and replication of the viral genome in macrophages. SP-R210S mediates sorting of IAV to a restrictive endosomal compartment that mediates IAV degradation and anti-viral signaling as demonstrated by enhanced secretion of TNFα and IFNβ and expression of interferon inducible genes. Furthermore, we show that SP-R210S-mediated restriction of IAV replication and endosomal signaling depends on recruitment of the interferon inducible trans-membrane protein 3 (IFITM3). Conclusions: Our study revealed that SP-R210 isoforms mediate IAV uptake in macrophages, but SPR210L and SP-R210S play differential non-redundant roles in intracellular trafficking pathways that mediate IAV replication and antiviral recognition, respectively. These findings support the notion that IAV co-opts different SP-R210 isoforms to dysregulate inflammatory responses by lung macrophages.
Author Block: Y. Chen1, S. Hu1, T. Umstead1, E. Yau2, Z. C. Chroneos1; 1Pediatrics, Pennsylvania State University College of Medicine, Hershey, PA, United States, 2Pennsylvania State University College of Medicine, Hershey, PA, United States.
Rationale: Influenza A viruses (IAVs) cause highly contagious respiratory infections and widespread lung inflammation by mechanisms that are poorly understood. Alveolar macrophages (AMs) and surfactant protein A (SP-A) are critical for resolution of IAV infection. SP-A modulates macrophage activation and clearance of bacterial pathogens through the Myosin 18A (Myo18A) receptor, which of two alternatively spliced cell-surface isoforms in macrophages named previously as SP-R210L and SP-R210S that differ by an amino-terminal PDZ domain. SP-R210 isoforms regulate the threshold of macrophage activation and innate recognition of pathogens. Based on these findings we tested the hypothesis whether the SP-A/SP-r210 isoform pathways mediate IAV infection and inflammatory responses using SP-R210L-deficient (SP-R210L (DN)) macrophages. Methods: We generated SP-R210L(DN) macrophages either by dominant negative disruption or conditional knockout in alveolar macrophages, and used native and deglycosylated SP-A, SP-R210 antibodies, flow cytometry, confocal microscopy, siRNA, RNA sequencing, and ELISA assays to determine the role of SP-R210 isoforms in IAV infection of macrophages. Results: Competitive inhibition assays using native SP-A, deglycosylated SP-A, or SP-R210 antibodies demonstrate that SP-R210 is a common receptor for both SP-A and IAV. Flow cytometry and microscopy assays, however, revealed that SP-R210L is required for nuclear transport and replication of the viral genome in macrophages. SP-R210S mediates sorting of IAV to a restrictive endosomal compartment that mediates IAV degradation and anti-viral signaling as demonstrated by enhanced secretion of TNFα and IFNβ and expression of interferon inducible genes. Furthermore, we show that SP-R210S-mediated restriction of IAV replication and endosomal signaling depends on recruitment of the interferon inducible trans-membrane protein 3 (IFITM3). Conclusions: Our study revealed that SP-R210 isoforms mediate IAV uptake in macrophages, but SPR210L and SP-R210S play differential non-redundant roles in intracellular trafficking pathways that mediate IAV replication and antiviral recognition, respectively. These findings support the notion that IAV co-opts different SP-R210 isoforms to dysregulate inflammatory responses by lung macrophages.
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