Innate immune response to influenza A virus in differentiated human alveolar type II cells

Abstract

Alveolar Type II (ATII) cells are important targets for seasonal and pandemic influenza. To investigate the influenza-induced innate immune response in those cells, we measured the global gene expression profile of highly differentiated ATII cells infected with the influenza A virus at a multiplicity of infection of 0.5 at 4 hours and 24 hours after inoculation. Infection with influenza stimulated a significant increase in the mRNA concentrations of many host defense-related genes, including pattern/pathogen recognition receptors, IFN, and IFN-induced genes, chemokines, and suppressors of cytokine signaling. We verified these changes by quantitative real-time RT-PCR. At the protein level, we detected a robust virus-induced secretion of the three glutamic acid-leucine-arginine (ELR)-negative chemokines CXCL9, CXCL10, and CXCL11, according to ELISA. The ultraviolet inactivation of virus abolished the chemokine and cytokine response. Viral infection did not appear to alter the differentiation of ATII cells, as measured by cellular mRNA and concentrations of surfactant proteins. However, viral infection significantly reduced the secretion of surfactant protein (SP)-A and SP-D. In addition, influenza A virus triggered a time-dependent activation of phosphatidylinositol 3-kinase signaling in ATII cells. The inhibition of this pathway significantly decreased the release of infectious virus and the chemokine response, but did not alter virus-induced cell death. This study provides insights into influenza-induced innate immunity in differentiated human ATII cells, and demonstrates that the alveolar epithelium is a critical part of the initial innate immune response to influenza.

Figure 1.

Primary cultured human alveolar Type II (ATII) cells develop a differentiated phenotype in vitro. ATII cells were suspended in Dulbecco's minimum essential medium with 10% FBS and antibiotics, plated on inserts coated with 20% Matrigel and 80% rat-tail collagen for 2 days, and cultured with keratinocyte growth factor (K) for 2 days and KIAD for an additional 2 days. Cells were processed for phase microscopy (A, ×20) and immunofluorescent staining with surfactant protein (SP)–A (B, red, ×40), proSP-B (C, green, ×40), and E-cadherin (D, red, ×40). (E) The expression of ATII cell differentiation proteins were evaluated by Western blotting, and compared with freshly isolated ATII cells and cells cultured with 1% charcoal-stripped (CS)-FBS alone. Lane 1, freshly isolated ATII cells; lane 2, cells cultured with 1% CS-FBS; lane 3, cells cultured with K for 2 days, and KIAD for an additional 2 days. SP-B was run under nonreduced conditions. Protein loading was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Figure 2.

Heat maps of influenza-induced cytokine and IFN-related genes in human ATII cells from microarray experiments. ATII cells isolated from lungs of three patients were cultured, and infected with influenza A/PR/8/34 (PR/8) virus. RNA from virus-infected and noninfected cells at 4 hours and 24 hours post inoculation (PI) were extracted and subjected to Affymetrix HG-U133 Plus 2.0 gene chip analyses. Responses of innate immune genes were clustered using the “heat map” function from R statistical software (R Foundation, Vienna, Austria). (A) Cytokine-related genes at 4 hours PI. (B) Cytokine-related genes at 24 hours PI. (C) IFN-related genes at 4 hours PI. (D) IFN-related genes at 24 hours PI.

Figure 3.

Verification of influenza A virus (IAV)–induced innate immune response in ATII cells at the mRNA and protein levels. ATII cells were cultured and infected with or without PR/8 virus. Cells were harvested at 4 hours and 24 hours PI for evaluation of the mRNA expression of innate immune response genes by real-time RT-PCR and the secretion of ELR-negative chemokine at 24 hours PI by ELISA. (A–E) Real-time RT-PCR. (A) ELR-negative CXC chemokines. (B) Suppressors of cytokine signaling (SOCSs). (C) Pathogen recognition receptors (PRRs). (D) IFN genes. (E) IFN-stimulated genes. The data show the expression level of each gene relative to the constitutive probe 36B4 (5). (F) Protein secretion of CXCL9–11 by ELISA. Open bars, noninfected cells; solid bars, virus-infected cells. *P < 0.05 and **P < 0.01 versus noninfected cells; n = 8–17.

Figure 4.

Ultraviolet (UV) inactivation of virus eliminates the IAV-induced response in ATII cells. ATII cells were infected with live PR/8 or UV-inactivated PR/8, and were harvested for the evaluation of mRNA changes of innate immune response–related genes at 4 hours and 24 hours by real-time RT-PCR, and for the evaluation of the secretion of IL-29 and CXCL10 at 24 hours PI by ELISA . (A) Real-time RT-PCR. Open bars, noninfected cells; solid bars, live virus–infected cells; striped bars, UV-inactivated virus–infected cells. (B) ELISA. *P < 0.05, **P < 0.01, and ***P < 0.001, versus noninfected cells; n = 4.

Figure 5.

Infection with IAV stimulates the phosphorylation (p) of Akt and ERK in human ATII cells. Cultured human Type II cells were infected with PR/8 virus at MOI of 5. At designated time points, the cellular protein was harvested and evaluated for the expression of phosphorylated and total Akt and ERK signaling by Western blotting. (A) Representative results from one of four patients. Lane 1, mock (no virus) or time 0; lane 2, 15 minutes after addition of virus; lane 3, 30 minutes after addition of virus; lane 4, 1 hour after addition of virus; lane 5, 4 hours after addition of virus; lane 6, 24 hours after addition of virus. (B) Quantification of relative expression of signal proteins in virus-infected ATII cells. **P < 0.01, versus noninfected cells; ***P < 0.001, versus noninfected cells; n = 4.

Figure 6.

Inhibition of phosphatidylinositol 3–kinase (PI3k) decreases the release of influenza viral particles and the virus-induced chemokine response, but does not impair viral infection in ATII cells. Cultured human Type II cells were treated with PI3k inhibitor LY294002 and ERK inhibitor PD98059, 1 hour before inoculation with PR/8 virus. After infection, cells were continually cultured with inhibitors. At 24 hours PI, cells were fixed for fluorescent staining with murine anti-nucleoprotein of IAV. Cell supernatants were evaluated for the release of infectious virus by standard plaque assay and secretion of CXCL9, CXCL10, CXCL11, and IL-29 by ELISA. (A) Secretion of chemokines. Data are expressed as percentage of secretion with virus alone. (B) Release of infectious virus. (C) Immunofluorescent stain with influenza A nucleoprotein. *P < 0.05, versus virus treatment alone. ***P < 0.001, versus virus treatment alone.

Figure 7.

UV inactivation of virus, but not PI3k inhibitor, abolishes IAV-induced activation of caspase-3 and poly (ADP-ribose) polymerase (PARP). Cultured human Type II cells were infected with live and UV-inactivated PR/8 virus. The cellular protein from attached and detached cells from the same well was harvested and evaluated for the expression of viral nucleoprotein (NP), caspase-3 (Casp3), and PARP at 24 hours PI. Some cells were treated with PI3k inhibitor LY294002, 1 hour before inoculation, and were incubated with the inhibitor throughout the infection. (A) Representative results from one of three patients. Lane 1, control cells; lane 2, live PR/8 virus–infected cells; lane 3, UV-inactivated PR/8 virus–infected cells; lane 4, PI3k inhibitor and live PR/8 virus–treated cells. (B) Quantification of relative expression of proteins after they were normalized to expression of GAPDH in each condition (n = 3).