Innate immune response of human alveolar type II cells infected with severe acute respiratory syndrome-coronavirus

Abstract

Severe acute respiratory syndrome (SARS)-coronavirus (CoV) produces a devastating primary viral pneumonia with diffuse alveolar damage and a marked increase in circulating cytokines. One of the major cell types to be infected is the alveolar type II cell. However, the innate immune response of primary human alveolar epithelial cells infected with SARS-CoV has not been defined. Our objectives included developing a culture system permissive for SARS-CoV infection in primary human type II cells and defining their innate immune response. Culturing primary human alveolar type II cells at an air-liquid interface (A/L) improved their differentiation and greatly increased their susceptibility to infection, allowing us to define their primary interferon and chemokine responses. Viral antigens were detected in the cytoplasm of infected type II cells, electron micrographs demonstrated secretory vesicles filled with virions, virus RNA concentrations increased with time, and infectious virions were released by exocytosis from the apical surface of polarized type II cells. A marked increase was evident in the mRNA concentrations of interferon-β and interferon-λ (IL-29) and in a large number of proinflammatory cytokines and chemokines. A surprising finding involved the variability of expression of angiotensin-converting enzyme-2, the SARS-CoV receptor, in type II cells from different donors. In conclusion, the cultivation of alveolar type II cells at an air-liquid interface provides primary cultures in which to study the pulmonary innate immune responses to infection with SARS-CoV, and to explore possible therapeutic approaches to modulating these innate immune responses.

Figures

Figure 1.

Human alveolar type II cells cultured at an air–liquid interface (A/L). Human alveolar type II cells were cultured under A/L conditions, as described for infection with SARS-CoV. (A) Phase micrograph of the epithelial monolayer with inclusions (lamellar bodies), indicated by arrows, clearly visible within the cytoplasm of type II cells. (B) The effect of A/L culture conditions and treatment with IL-4 and IL-13 on selected proteins by immunoblotting. Lane 1 is a extract of freshly isolated type II cells, lane 2 is empty, lanes 3 and 4 contain cells cultured under A/L conditions with KIAD alone, lanes 5 and 6 contain cells cultured under A/L conditions with KIAD, IL-4, and IL-13, lanes 7 and 8 contain cells cultured under submerged conditions with KIAD, and lanes 9 and 10 contain cells cultured under submerged conditions with KIAD, IL-4, and IL-13. A/L conditions increased the expression of angiotensin-converting enzyme–2 (ACE2), surfactant protein (SP)–A, proSP-B, proSP-C, and ATP-binding cassette sub-family A member 3 (ABCA3), but not fatty acid synthase (FAS). IL-4 and IL-13 significantly increased the expression of ACE2. GAPDH, glyceraldehyde 3–phosphate dehydrogenase.

Figure 2.

Immunofluorescent staining for SARS-CoV nucleocapsid protein and selected alveolar type II cell markers. Cells were grown under A/L conditions as described in Materials and Methods, inoculated with SARS-CoV at an estimated multiplicity of infection of 2, and fixed 24 hours after inoculation. (A–D) Staining for E-cadherin (A), SARS-CoV (B), DAPI (C), and merged (D). (E–H) Staining for SP-A (E), SARS-CoV (F), DAPI (G), and merged (H). (I–L) Staining for TTF-1 (I), SARS-CoV (J), DAPI (K), and merged (L). Cells that are infected with SARS-CoV stain for type II cell markers.

Figure 3.

Ultrastructure of alveolar type II cells infected with SARS-CoV. Cells were cultured as described in Materials and Methods under A/L conditions, inoculated with SARS-CoV on the apical surface, and then fixed and processed for electron microscopy 48 hours after inoculation. (A) A differentiated alveolar type II cell with its characteristic lamellar bodies (LB) and SARS-CoV virions are found in expanded, smooth-walled secretory vesicles in the cytoplasm of the infected cell. Several budding virions are indicated by arrows. (B) Several smooth-walled, double-membrane vesicles (V), which are the site of replication for SARS-CoV RNA, virions in secretory vesicles near the plasma membrane, and virions adsorbed to the apical plasma membrane. The inset depicts a higher magnification of one of these double-membrane vesicles.

Figure 4.

SARS-CoV production by alveolar type II cells. Type II cells were cultured at an A/L and inoculated with SARS-CoV, as described in Materials and Methods. (A) Cells were processed for the extraction of RNA and real-time quantitative RT-PCR at 6 and 24 hours after virus inoculation to measure SARS-CoV–specific viral RNA. For this series of experiments, six different donors were used, and all showed a significant increase in SARS-CoV mRNA at 24 hours after inoculation. (B) The production of infectious virus is shown. In this series of experiments, the yields of virus released from the apical (red) or basolateral (black) surfaces or from cell extracts (blue) at 4, 24, and 48 hours after inoculation were titered by plaque assay in Vero E6 cells and expressed as the total virus yield from each insert. The points represent the means and standard errors from four independent experiments.

Figure 5.

Innate immune responses of alveolar type II cells to SARS-CoV infection. type II cells at an A/L were cultured and inoculated with SARS-CoV, as described in Materials and Methods. After the 4-hour inoculation period, the inoculum was aspirated, and cultures were harvested 6 and 24 hours later. The specific mRNA concentrations of chemokines and cytokines were measured by quantitative RT-PCR, and normalized to the constitutive probe 36B4 (acidic ribosomal phosphoprotein). The white bars represent data from mock-inoculated inserts, and the black bars represent data from SARS-CoV–infected inserts. (A) At 6 hours after virus inoculation, the only significant increase in detected mRNA concentrations involved the interferon-responsive gene ISG56. (B) At 24 hours after virus inoculation, a marked increase in the expression of all selected genes was evident. *P < 0.05. **P < 0.01. ***P < 0.001. The results were derived from six independent donors, with duplicate inserts for each condition and time.