Project description:Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) causes an acute respiratory distress syndrome (ARDS) that resembles neonatal RDS – a condition attributable to the deficiency of the surface tension reducing factor surfactant. Using a novel multi-cell type, human-induced pluripotent stem cell (hiPSC)-derived lung organoid (LO) system, validated against primary lung cells, we found that surfactant proteins (SP) A, B, C and D are dynamically regulated by SARS-CoV-2 infection. In the absence of SP-B (including through the study of LOs from SP-B deficient patients), resistance to infection was impaired and inflammatory cytokine/chemokine production and interferon (IFN) responses were modulated in lung cells of non-hematopoietic origin and without a circulatory system, suggesting specialization of viral inflammatory responses by human lung cells. Such cells can be infected independent of ACE2 and TMPRSS2 expression via what we propose is a non-canonical endocytotic route. Single cell RNA sequencing revealed broad susceptibility of many lung cell types with differential expression of BSG, cathepsins, and ACE2 that all facilitate viral entry. SARS-CoV-2 triggers a rapid loss of lung cell viability via apoptosis that impairs viral dissemination evident in the presence of Bcl-xL antagonism. Treatment with exogenous surfactant, recombinant SP-B, or genomic correction of the SP-B deletion mutation restored resistance to SARS-CoV-2 infection and improved cell viability. These findings suggest an unrecognized specialized intrinsic intra-pulmonary inflammation-based feedback loop of developmental origin that autonomously, without the intercession of hematopoietic derivatives, attempts to restore lung homeostasis after infection by inhibiting viral dissemination, averting lung epithelial demise, and dampening inflammatory cascades -- an additional inherent host-pathogen defense system. Given that SP-B is central to this response, an antiviral role may now be ascribed to this structural component that heretofore had been regarded as necessary only for surface tension reduction.

Project description: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.

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An autonomous intrapulmonary inflammatory feedback response, mediated by surfactants, operates as an inherent viral defense mechanism: evidence from acute SARS-CoV-2 infection [Combined Proximal, Distal, and Whole Lung Organoid scRNA-seq]

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