Acute Lung Injury Results from Innate Sensing of Viruses by an ER Stress Pathway

Abstract

Incursions of new pathogenic viruses into humans from animal reservoirs are occurring with alarming frequency. The molecular underpinnings of immune recognition, host responses, and pathogenesis in this setting are poorly understood. We studied pandemic influenza viruses to determine the mechanism by which increasing glycosylation during evolution of surface proteins facilitates diminished pathogenicity in adapted viruses. ER stress during infection with poorly glycosylated pandemic strains activated the unfolded protein response, leading to inflammation, acute lung injury, and mortality. Seasonal strains or viruses engineered to mimic adapted viruses displaying excess glycans on the hemagglutinin did not cause ER stress, allowing preservation of the lungs and survival. We propose that ER stress resulting from recognition of non-adapted viruses is utilized to discriminate "non-self" at the level of protein processing and to activate immune responses, with unintended consequences on pathogenesis. Understanding this mechanism should improve strategies for treating acute lung injury from zoonotic viral infections.

Graphical abstract

Figure 1

Lack of HA Globular Head Glycosylation Triggers Lung Inflammation and Pathogenicity Groups of (A and B) ten and (C–J) five BALB/c mice were infected intranasally (A and B) with 10⁴ PFU of pandemic (p) 2009 H1N1 HH04, seasonal (s) H3N2 Pan/99, or seasonal (s) H1N1 Mem/01, (C and D) with 10⁴ PFU of viruses carrying different combinations of pandemic (HK) and seasonal (Pan) HA/NA surface proteins on a six-gene-segment PR8 backbone (HK/HK 6:2, Pan/Pan 6:2, HK/Pan 6:2, and Pan/HK 6:2) and (E–J) with (E, F, G, and J) 5 × 10⁵ and (H and I) 10⁶ PFU of isogenic viruses differing in the number of N-linked glycosylation sites on the HA globular head (HK WT, HK +II, and HK +IV). (A–F) PBS-inoculated mice served as control. (A, C, and E) Lung mRNA levels of Il6 and Ccl2 3 days post infection are depicted as the fold change of levels of the PBS-inoculated mice, and (B, D, and F) body weight (%; compared to starting weight) is shown. (G and H) Body weight changes over 15 days are depicted, and (I) Kaplan-Meier survival curve analysis was performed. (J) Virus lung titers on d2, d3, d5, and d7 post-infection are depicted as PFU/ml. (A–H) Data are represented as mean with SD, (J) each dot represents an individual mouse, and bars show mean. An asterisk (^∗) indicates statistically significant differences (p < 0.05) between (A and B) HH04 pH1N1 and all other groups, (C and D) HK/HK 6:2 and HK/PAN 6:2 and all other groups, (E–I) HK WT and all other groups, and (J) HK +II and HK +IV to HK WT. See also Figures S1 and S2.

Figure 2

Addition of Glycosylation Sites to the Pandemic HA Abolishes Lung Inflammation upon IAV Infection Five (A, C, and D) and four to five (B) BALB/c mice per group were infected with 5 × 10⁵ PFU of isogenic viruses carrying differentially glycosylated HA globular heads (HK WT, HK +II, and HK +IV). PBS-inoculated mice served as control. (A) Total lung mRNA levels of Ifnb1, Il6, Ccl2, Tnf, and Cxcl1 2 days post-infection are shown as n-fold of the PBS-inoculated mice, and (B) total lung protein levels for IL6, CCL2, and TNF in pg/ml 3 and 5 days after infection are depicted. (C and D) Five days after infection, BALF was collected and cellular composition was analyzed. (C) Total cell counts and counts for specific cell types (resident macrophages, exudative macrophages, and neutrophils) are depicted as cells in BALF/mouse. (D) Relative proportions of cell types within the BALF cell composition are shown as parts of whole. (A–C) Data are represented as mean with SD, and (D) mean values for each experimental group were used. An asterisk (^∗) indicates statistically significant differences (p < 0.05) between HK WT and all other groups. In (C) resident macrophages, an asterisk (^∗) indicates statistically significant differences (p < 0.05) between HK WT and PBS and +IV. See also Figures S3 and S4.

Figure 3

Global Lung Transcriptome upon IAV Infection Is Altered due to HA Globular Head Glycosylation Five BALB/c mice per group were infected intranasally with 5 × 10⁵ PFU of isogenic viruses carrying differentially glycosylated HA globular heads (HK WT, HK +II, and HK +IV). PBS-inoculated mice served as controls. Two days after infection, total lung RNA was used for analysis of the global lung transcriptome. (A) Principal-component analysis (PCA) visualization of IAV-infected and PBS control mice is depicted. This visualization captures 40.3% of the variability across samples in the top three components. (B) The Z transformed complete data set shows color-coded expression (heatmap) clustered by gene and displayed by experimental class. (C) Data from a select group (bar indication in B) of genes showing the highest upregulation in HK WT infection was tested for GO enrichment using DAVID. Redundant categories were removed, and the p value of enrichment was −log10 transformed to create a score for visualization in the bar chart. (D) A select group of HK WT-induced genes (ER stress genes) are visualized in this heatmap.

Figure 4

ER-Stress-Mediated Sensing of Pandemic HA as Mechanism of Immune Recognition A549 lung epithelial cells (A–C and F–I) or primary NHBE cells (D and E) were infected with isogenic viruses carrying differentially glycosylated HA globular heads (HK WT and HK +II) or (A, D, and F–I) mock infected. The MOI used was (A, B, F, and G) MOI = 5 and (C–I) MOI = 10. (A, D, and I) Cellular mRNA levels of (A, D, and I) IFNB1, (A, D, and I) IL6, and (A and I) CCL2 are shown as n-fold of the mock infected cells (A and D) 24 hr and (I) 12 hr after infection, and (B and E) virus titers are depicted in PFU/ml. (H) Cellular mRNA level of the depicted ER stress genes 12 hr after infection are shown as n-fold of the mock infected cells. (C) qPCR (RT² Profiler)-based analysis of inflammatory gene expression of HK WT- and HK +II-infected cells is shown. (C, upper panel) Heatmap visualization of fold increase of inflammatory genes after HK WT infection compared to HK +II infection and (C, lower panel) list of genes upregulated in HK WT compared to HK +II infection are depicted. (F and G) Cells were harvested 12 hr after infection for western blot analysis. Detection of the indicated proteins was done with specific antibodies. (A, B, D, E, H, and I) Data are represented as mean with SD. (A, B, D, E, and I) Representative data of three independent experiments each consisting of three biological samples are shown. (F and G) Representative blots of three independent experiments are shown and (H) mean values from three independent experiments, each consisting of three biological samples, are shown. (C) cDNA of three different biological samples were pooled and analyzed. An asterisk (^∗) indicates statistically significant differences (p < 0.05) between HK WT and all other groups. See also Figure S5.

Figure 5

ER Stress as a Central Regulator for Inflammatory Responses upon Viral Infections A549 lung epithelial cells were infected with (A–C) isogenic IAV carrying differentially glycosylated HA globular heads (HK WT and HK +II; MOI = 10) and (D) two RSV A2 virus variants differing in surface protein glycosylation (RSV HEp2 and RSV Vero; MOI = 5). Mock infected cells served as control. After infection, cells were treated with (A and B) 10 mM PBA or (C) 500 μg/ml TUDCA. (A) Cells were harvested 12 hr after infection for western blot analysis, and indicated proteins were detected with specific antibodies. (B–D) Cellular mRNA levels of (B–D) IFNB1, IL6, CCL2, and (D) DDIT3 are shown as n-fold of the mock-infected cells (B and C) 12 hr and (D) 24 hr after infection. (B–D) Data are represented as mean with SD. (A) Representative blots of three independent experiments; (B and C) representative data of three independent experiments, each consisting of three biological samples; and (D) representative data of two independent experiments, each consisting of four biological samples, are shown. An asterisk (^∗) indicates statistically significant differences (p < 0.05) between (B and C) HK WT and all other groups and (D) RSV HEp2 to all other groups. See also Figure S5.

Figure 6

ER-Stress-Mediated Inflammation Triggers Pandemic IAV Pathogenicity (A) Five BALB/c mice per group were intranasally infected with 10⁶ PFU of isogenic viruses carrying differentially glycosylated HA globular heads (HK WT and HK +II) or PBS for control. At d2 post-infection, different groups were either treated with 100 μg LTA or PBS intranasally. (B) Ten C57BL/6 WT and eleven Ccl2^−/− mice per group were infected intranasally with 10⁵ PFU HK WT virus. (C and D) Five BALB/c mice per group were infected intranasally with 10⁶ PFU HK WT virus. (C) Animals were treated i.p. with 25 or 50 mg/kg/day imatinib and (D) 200 mg/kg/day PBA or solvent control. Survival was monitored for 15 days, and Kaplan-Meier survival curve analysis was performed. An asterisk (^∗) indicates statistically significant differences (p < 0.05) between (A) HK +II PBS d2 and HK +II LTA d2 treated mice, (B) C57BL/6 WT and Ccl2^−/− mice, and (C and D) between HK WT-infected mice and all treatment groups.

Figure 7

Addition of Glycosylation Sites to the 2009 Pandemic Virus HA Abrogates Lung Inflammation and Pathogenicity (A and B) A549 lung epithelial cells were infected with the pandemic 2009 H1N1 (HH04) virus (MOI = 5) and treated with 10 mM PBA or left untreated. Mock infected cells served as control, and cells were harvested 12 hr after infection. (A) Western blot analysis was conducted, and detection of the indicated proteins was done with specific antibodies. (B) IL6 mRNA level is shown as n-fold of the mock infected cells. (C–E) Five BALB/c mice per group were infected intranasally with (C) 10³ PFU or (D and E) 10⁴ of isogenic viruses carrying different glycosylated HA globular heads (HH04 and HH04 HA +II), (C) with PBS-infected animals serving as control. (C) Total lung mRNA levels of Ifnb1, Il6, Ccl2, and Ddit3 3 days after infection are shown as n-fold of the PBS-inoculated mice. (D) Body weight changes over the course of infection (15 days) are depicted, and (E) Kaplan-Meier survival curve analysis was performed. (B–D) Data are represented as mean with SD. (A) Representative blots of three independent experiments and (B) representative data of three independent experiments, each consisting of three biological samples, are shown. An asterisk (^∗) indicates statistically significant differences (p < 0.05) between HH04 and the other groups and (C) between HH04 and PBS for Ddit3 mRNA.