Differentiated phenotypes of primary murine alveolar epithelial cells and their susceptibility to infection by respiratory viruses

Abstract

Severe respiratory viral infections are associated with spread to the alveoli of the lungs. There are multiple murine models of severe respiratory viral infections that have been used to identify viral and host factors that contribute to disease severity. Primary cultures of murine alveolar epithelial cells provide a robust in vitro model to perform mechanistic studies that can be correlated with in vivo studies to identify cell type-specific factors that contribute to pathology within the alveoli of the lung during viral infection. In this study, we established an in vitro model to compare the responses of type I (ATI) and type II (ATII) alveolar epithelial cells to infection by respiratory viruses used in murine models: mouse-adapted severe acute respiratory syndrome-associated coronavirus (SARS-CoV, v2163), murine coronavirus MHV-1, and influenza A (H1N1) virus, strain PR8. Murine alveolar cells cultured to maintain an ATII cell phenotype, determined by expression of LBP180, were susceptible to infection by all three viruses. In contrast, ATII cells that were cultured to trans-differentiate into an ATI-like cell phenotype were susceptible to MHV-1 and PR8, but not mouse-adapted SARS-CoV. Epithelial cells produce cytokines in response to viral infections, thereby activating immune responses. Thus, virus-induced cytokine expression was quantified in ATI and ATII cells. Both cell types had increased expression of IL-1β mRNA upon viral infection, though at different levels. While MHV-1 and PR8 induced expression of a number of shared cytokines in ATI cells, there were several cytokines whose expression was induced uniquely by MHV-1 infection. In summary, ATI and ATII cells exhibited differential susceptibilities and cytokine responses to infection by respiratory viruses. This in vitro model will be critical for future studies to determine the roles of these specialized cell types in the pathogenesis of respiratory viral infection.

Proteomic analysis reveals down-regulation of surfactant protein B in murine type II pneumocytes infected with influenza A virus

“…The presence of the cleaved HA protein in cell lysates suggests that differentiated ATII cells are capable of post-translational processing of HA, which is required for viral infectivity and correlates with virulence of IAV strains (Blazejewska et al, 2011; Steinhauer, 1999). These data are in agreement with our previous study that demonstrated productive infection of murine ATII cells by PR8 (Kebaabetswe et al, 2013). Infection of primary murine ATII cells provides a robust system for analysis of PR8-regulated changes to the cellular proteome in physiologically relevant target cells.…”

“…Primary cultures of murine ATII cells are not homogenously susceptible to PR8 infection, as can be seen by heterogeneous staining of viral antigens in our cultures (Fig. 1A) (Kebaabetswe et al, 2013). Therefore, it is possible that the IFN-stimulated proteins we are detecting come from either infected or uninfected cells within the culture.…”

“…1A). In a previous study, PR8 replication in primary murine ATII cells peaked at 24 h (Kebaabetswe et al, 2013). This time point is consistent with other proteomic studies using the same strain of influenza A virus in different cell types, including primary cultures (Coombs et al, 2010; Kroeker et al, 2012; Liu et al, 2012).…”

“…Primary ATII cells were isolated from mice and cultured to maintain an ATII phenotype in vitro , as previously described (Kebaabetswe et al, 2013). ATII cultures were inoculated with PR8 or mock-inoculated and infection was analyzed by immunofluorescence assay and immunoblotting 24 h later.…”

“…Primary murine ATII cells thus provide a powerful in vitro model to determine the effects of viral infection on these specialized pulmonary cell types, which corresponds to pathology in the mouse model. We have shown that differentiated cultures of primary murine ATII cells are susceptible to PR8 infection in vitro , resulting in secretion of various pro-and anti-inflammatory cytokines and chemokines (Kebaabetswe et al, 2013). This approach, however, did not offer a comprehensive analysis of viral-host protein interactions that could be important to pathogenesis during infection of ATII cells.…”