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.

Fig. 1

Trans-differentiation of murine ATII cells to an ATI-like cell phenotype. (A) ATII cells were cultured on fibronectin-coated coverslips for the indicated times and immunofluorescence assay was used to detect expression of phenotypic marker proteins of ATII cells, LBP180, or ATI cells, T1α. Nuclei were stained with DAPI, inset panels. (B) ATII cells were lysed on the day of isolation (day 0) or cultured on fibronectin and lysed on the indicated days. Cell lysates were analyzed by Western blot analysis using antibody against T1α or β-actin, as a protein loading control. The images shown are representative of three replicate experiments.

Fig. 2

Maintenance of ATII cell phenotype in cultured murine cells. ATII cells were cultured on 70% collagen/30% matrigel (CM) matrix for 5 or 7 days with or without keratinocyte growth factor (KGF) in the medium. Immunofluorescence assay was used to detect the expression of (A) ATII marker protein, LBP180, or (B) ATI marker protein, T1α. Cells cultured on fibronectin were used as a positive control for T1α expression.

Fig. 3

Susceptibility of primary murine alveolar epithelial cells to infection by mouse-adapted SARS-CoV. (A) Murine cells were cultured as either an ATI or ATII cell phenotype for 5 days, then were inoculated with mouse-adapted SARS-CoV, isolate v2163 for 24 or 48 h. Infection was analyzed by immunofluorescence assay of viral nucleocapsid protein (red) and nuclei were stained with DAPI (blue). (B) Cells cultured for 5 days with an ATI cell phenotype were analyzed by immunofluorescence using rabbit antibodies against ACE2 or TMPRSS2 (green) and nuclei were stained with DAPI (blue). A fluorescently labeled antibody against rabbit IgG was used as a negative control.

Fig. 4

Susceptibility of primary murine alveolar epithelial cells to infection by murine coronavirus, MHV-1. (A) Murine cells were cultured as either an ATI or ATII cell phenotype for 5 days, then were inoculated with MHV-1 for 12 or 24 h. Infection was analyzed by immunofluorescence assay of viral nucleocapsid protein (green) and nuclei were stained with DAPI (blue). (B) Cells were cultured on fibronectin (ATI phenotype) and inoculated with MHV-1 or mock-inoculated. Cells were photographed on phase contrast at 24 h p.i. (C) Infection of MHV-1-inoculated ATI and ATII cells was analyzed by plaque assay of supernatant medium at the indicated times. The mean virus titers and standard errors from four replicate experiments are shown.

Fig. 5

Susceptibility of primary murine alveolar epithelial cells to infection by influenza A virus, PR8. (A) Murine cells were cultured as either an ATI or ATII cell phenotype for 5 days, then were inoculated with PR8. Infection was analyzed by immunofluorescence assay of viral hemagglutinin protein (red) and nuclei were stained with DAPI (blue) at the indicated times post infection (p.i.). (B) Co-localization of ATI (T1α) or ATII (LBP180) phenotypic proteins (green) and PR8 antigen (red) was analyzed by dual IFA 24 h p.i. Nuclei were stained with DAPI (blue). (C) Infection of PR8-inoculated ATI and ATII cells was analyzed by plaque assay of supernatant medium at the indicated times. The mean virus titers and standard errors from five replicates are shown.

Fig. 6

Expression of IL-1β by primary murine alveolar epithelial cells in response to respiratory viral infection. Murine cells were cultured as an ATI (A) or ATII (B) cell phenotype, infected by the indicated viruses, and IL-1β mRNA was quantified by RT-qPCR. All ATII cell samples (B) were infected for 24 h. Mock-inoculated and LPS-treated cells were used as negative and positive controls, respectively. Expression of IL-1β was normalized to that of β-actin and the means and standard errors from 3 to 5 replicate experiments are shown. Statistically significant differences in expression compared to mock samples were determined by unpaired t test. *p < 0.05, **p < 0.005.