Deficiency of HIF-1α enhances influenza A virus replication by promoting autophagy in alveolar type II epithelial cells

Abstract

Infection of influenza A virus (IAV) can trigger exaggerated pulmonary inflammation and induce acute lung injury (ALI). Limiting IAV replication and alleviation of pulmonary inflammation are two important therapeutic strategies for influenza virus infection. Recent studies have shown that hypoxia inducible factor-1α (HIF-1α) is an essential factor for the development and repair of ALI; however, the role and the underlying mechanisms of HIF-1α in IAV-induced ALI remain elusive. Here, we demonstrated that lung epithelial cell-specific Hif1α knockout mice infected with IAV developed more lung IAV replication and severe lung inflammation, which led to increased mortality compared to IAV-infected control mice. Moreover, knockdown of HIF1A in A549 cells (human alveolar type II epithelial cell line) promoted IAV replication in vitro. Mechanistically, knockdown of HIF1A reduced glycolysis by regulating transcription of glycolysis-related enzymes, which subsequently activated the AMPKα-ULK1 signalling pathway. Interestingly, AMPKα-ULK1 signalling promoted autophagy and augmented IAV replication. Taken together, deficiency of HIF-1α in lung epithelial cells reduces glycolysis and enhances AMPKα-ULK1-mediated autophagy, which finally facilitates IAV replication. These findings have deepened our understanding of the role of HIF-1α in regulating IAV replication and provided us novel therapeutic targets for combating influenza infection.

Keywords: AMPKα; HIF-1α; Influenza A virus; ULK1; autophagy; glycolysis.

Figure 1.

HIF-1α deletion in AEC2 leads to increased mortality and weight loss after IAV infection. Survival rate (A) and weight loss (B) of eight to ten weeks old male Hif1α^fl/fl (n = 15) and Spc-Cre⁺.Hif1α^fl/fl (n = 15) mice infected with 5 × 10³ FFU mouse-adapted PR8. The data of survival rate was analysed by Gehan-Breslow-Wilcoxon Test. The data of weight loss was analysed by student’s t-test (two-tailed) and presented as mean ± SD (*p < 0.05, **p < 0.01).

Figure 2.

HIF-1α deletion in AEC2 increases influenza-induced lung inflammation and injury. (A) Hif1α^fl/fl and Spc-Cre⁺.Hif1α^fl/fl mice were infected with 5 × 10³ FFU mouse-adapted PR8. Serial lung sections of representative lungs at day 3 and 6 post IAV infection were stained with haematoxylin and eosin. (B-D) Quantitative mRNA expression of inflammatory genes (Cxcl2, Cxcl10, Ifnb) in the lung of Hif1α^fl/fl and Spc-Cre⁺.Hif1α^fl/fl mice at day 3 post IAV infection with 5 × 10³ FFU mouse-adapted PR8. (E-G) Quantitative mRNA expression of inflammatory genes (Cxcl2, Cxcl10, Ifnb) in the lung of Hif1α^fl/fl and Spc-Cre⁺.Hif1α^fl/fl mice at day 6 post IAV infection with 5 × 10³ FFU mouse-adapted PR8. (H) Quantitative mRNA expression of Hif1α in the lung of Hif1α^fl/fl and Spc-Cre⁺.Hif1α^fl/fl mice at day 6 post IAV infection with 5 × 10³ FFU mouse-adapted PR8. (I-K) Number and flowcytometry analysis of BAL cells isolated from Hif1α^fl/fl and Spc-Cre⁺.Hif1α^fl/fl mice at day 6 post IAV infection with 5 × 10³ FFU mouse-adapted PR8. Monocytes and neutrophils were labelled by surface marker Ly6C and Ly6G. (L) Flow cytometry analysis of pro-SPC⁺ and HIF-1α⁺ cells in digested lung cells from Hif1α^fl/fl and Spc-Cre⁺.Hif1α^fl/fl mice at day 6 post IAV infection with 5 × 10³ FFU mouse-adapted PR8. N=3-8 in each group. The data was analysed by Student’s t-test (two-tailed) and is presented as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 3.

HIF-1α deletion in AEC2 promotes IAV replication in the lung. (A-B) Quantitative mRNA expression of PR8 M gene from the lung of Hif1α^fl/fl and Spc-Cre⁺.Hif1α^fl/fl mice at day 3 (A) and 6 (B) post IAV infection with 5 × 10³ FFU mouse-adapted PR8. (C-D) Virus titres of the lung of Hif1α^fl/fl and Spc-Cre⁺.Hif1α^fl/fl mice at day 3 (C) and 6 (D) post IAV infection with 5 × 10³ FFU mouse-adapted PR8. (E) Flow cytometry analysis of NP⁺ AEC2 cells in digested lung cells of Hif1α^fl/fl and Spc-Cre⁺.Hif1α^fl/fl mice at day 6 post IAV infection with 5 × 10³ FFU mouse-adapted PR8. The AEC2s were labelled with fluorescent anti-pro-SPC and CD326 antibodies. (F) Immunoblot analysis of the supernatant of lung homogenates from Hif1α^fl/fl and Spc-Cre⁺.Hif1α^fl/fl mice at day 3 post IAV infection with 5 × 10³ FFU mouse-adapted PR8(G-J). Intensity analysis of proteins in (F) was performed. (K) Immunoblot analysis of the supernatant of lung homogenates from Hif1α^fl/fl and Spc-Cre⁺.Hif1α^fl/fl mice at day 6 post IAV infection with 5 × 10³ FFU mouse-adapted PR8. (L-O) Intensity analysis of proteins in (K) was performed. β-actin was used as a loading control. The intensities of the protein bands were quantified using Image J software. The data was analysed by Student’s t-test (two-tailed) were presented as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 4.

The mRNA and protein levels of HIF-1α are upregulated after IAV infection in vitro. (A) Heatmap of the innate immune response related-genes in A549 cells mock-infected or infected with 2 MOI PR8 virus by FPKM value of RNAseq at 24 h post infection (hpi). (B) FPKM value analysis of HIF1A gene expression in A549 cells mock-infected or infected with 2 MOI PR8 virus at 24 hpi. (C) A549 cells were mock-infected or infected with the PR8 virus at MOI of 2. Cells were collected at 24 hpi and were subjected to flow cytometry analysis. Mean fluorescence intensity (MFI) of HIF-1α expression analysis showed. (D) A549 cells were infected with the PR8 virus at MOI of 2. Cell lysates were collected at 0, 3, 6, 12, 24, and 48 hpi and subjected to Western blot analysis. The data was analysed by Student’s t-test (two-tailed) and is presented as mean ± SD (*p < 0.05).

Figure 5.

HIF-1α knockdown enhances IAV replication through autophagy. (A) Effect of HIF-1α knockdown on IAV replication in A549 cells. A549 were infected with pLKO.1 scrambled or shRNA-HIF1A lentivirus, infected with PR8 at MOI of 2 and harvested at 24 hpi to analyse M gene and HIF1A expression by RT-qPCR. (B) Western blot analysis of samples treated and harvested as in (A). (C) Intensity analysis of proteins, p-AMPKα^Thr172 and p-ULK1^Ser555, in (B) was performed. (D) A549 cells were infected with GFP-LC3B reporter lentivirus and then transfected with pLKO.1 scrambled or shRNA-HIF1A lentivirus and treated with rapamycin. Representative puncta images of A549-GFP-LC3B cells mock-infected or infected with PR8 at MOI of 2 at 24 hpi. (E) Quantification of GFP-LC3B puncta in (D). (F) Effect of HIF-1α knockdown on transcription of inflammatory cytokines genes (IFNB, MCP1, IL6, CCL20) of A549 cells mock-infected or infected with 2 MOI PR8 at 24 hpi. The data was analysed by Student’s t-test (two-tailed) and is presented as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 6.

Autophagy inhibition reduces replication of Influenza A Virus in vitro. (A-D) A549 cells were pretreated with an increasing dose of LY294002 (A) or 3-MA (B) or chloroquine (C) or Rapamycin (D) for 30 min and then mock-infected or infected with PR8 at MOI of 5. Cell lysates were collected at 24 hpi and were subjected to Western blot analysis. (E-F) Effect of BECN1 and p62 knockdown on IAV replication in A549 cells. A549 cells were infected with pLKO.1 scrambled or shRNA-BECN1 (E) or shRNA-p62 (F) lentiviruses, infected with PR8 and harvested at 24 hpi for Western blot analysis. Results are representative of three independent experiments.

Figure 7.

HIF-1α knockdown downregulates glycolysis and activates the AMPKα-ULK1 signaling pathway during IAV infection. (A) Effect of HIF-1α knockdown on the expression of glycolysis genes (LDHA, HK2, GLUT-1, PKM2) during IAV infection in A549 cells mock-infected or infected with PR8 at 24 hpi by RT-qPCR. (B) A549 cells were pretreated with 2-DOG or vehicle for 30 min and then were mock-infected or infected with the PR8 virus for 24 h. Cells were harvested and analysed by Western blotting. (C) Intensity analysis of proteins, p-AMPKα^Thr172 and p-ULK1^Ser555, in (B) was performed. The data were analysed by Student’s t-test (two-tailed) and is presented as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 8.

The AMPKα signalling pathway is essential autophagic control of IAV replication. (A) A549 cells were mock-infected or infected with PR8 at MOIs of 5. At 1, 3, and 6 hpi, cells were collected, and cell lysates subjected to Western blot analysis. (B) A549 cells were infected with PR8 at MOIs of 0, 1, and 10, respectively. The cells were collected, and cell lysates were subjected to Western blot analysis at 24 hpi. (C-D) Effect of AMPKα and ULK1 knockdown on IAV replication in A549 cells. A549 cells were infected with pLKO.1 scrambled or shRNA-AMPKα or shRNA-ULK1 lentiviruses, mock-infected or infected with PR8 and then harvested at 24 hpi for Western blot analysis. Results are representative of three independent experiments.