Activation of alveolar epithelial ER stress by b-coronavirus infection disrupts surfactant homeostasis in mice: implications for COVID-19 respiratory failure
Covid-19 syndrome is characterized by an acute lung injury phenotype marked by hypoxemic respiratory failure and high mortality. Alveolar Type 2 (AT2) cells are essential for gas exchange, repair, and regeneration of distal lung epithelium. We have shown that the causative agent, SARS-CoV-2 and other members of the b coronavirus genus induces an ER stress response in vitro however the consequences for host AT2 cell function in vivo are less understood.  To study this, two murine models of coronavirus infection were employed– mouse hepatitis virus-1 (MHV-1) in A/J mice and a C57BL6/j adapted SARS-CoV-2 strain.  MHV-1 infected mice exhibited dose-dependent weight loss through Day 8 (8dpi) with histological evidence of distal lung injury accompanied by elevated BALF cell counts and total protein.  AT2 cells isolated at 4dpi and 8dpi showed evidence of both viral infection and increases in Bip /GRP78 expression, consistent with activation of the unfolded protein response (UPR).  AT2 UPR signaling included elevations in spliced XBP-1 indicating activation of IRE1a signaling as well as a biphasic response in PERK signaling mark by phosphorylated eIF2a protein levels and expression of Atf4, Ppp1r15a, and Ddit3. Viral UPR activation was accompanied by marked reductions in AT2 and BALF surfactant protein (SP-B, SP-C) content, increases in surfactant surface tension, and emergence of a re-programmed epithelial cell population (Krt8+, Cldn4+, and Gdf15+) which was attenuated by treatment with the IRE1a inhibitor OPK711.  As proof-of-concept, C57BL6 mice infected with mouse-adapted SARS-CoV-2 demonstrated similar lung injury and evidence of disrupted surfactant homeostasis.  We conclude that hypoxic respiratory failure from b-coronavirus infection results from an aberrant AT2 cell host response activating multiple ER stress pathways, altering surfactant metabolism / function, and changing AT2 endophenotypes offering a mechanistic link between SARS-CoV-2 infection, AT2 cell biology, and hypoxemic respiratory failure. Overall design: To determine if b-coronavirus infection disrupted functional and phenotypic characteristics of alveolar type 2 (AT2) cells we isolated AT2 and transitional AT2 cells by FACS from lungs of control (n=3)  and 4dpi (n=4) MHV1 infected mice. Lungs were harvested from control and 4 day infected mice, single cells suspension was prepared by mehcanical and enzymatic digetsion.  CD45+ and CD31+ cells were depleted using biotinylated antibodies followed by surface staining with antibody mix to identify At2 and transitional At2 cells. At2 cells were identified as CD45-CD31-Epcam+CD104-MHCII hiCD51- and transitional At2 cells  CD45-CD31-Epcam+CD104-MHCII hiCD51+. Dead cells were excluded by Dapi staining.