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IL-6 Ameliorates Acute Lung Injury in Influenza Virus Infection

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IL-6 Ameliorates Acute Lung Injury in Influenza Virus Infection

Mei-Lin Yang et al. Sci Rep.

Abstract

Interleukin 6 (IL-6) is involved in innate and adaptive immune responses to defend against pathogens. It also participates in the process of influenza infection by affecting viral clearance and immune cell responses. However, whether IL-6 impacts lung repair in influenza pathogenesis remains unclear. Here, we studied the role of IL-6 in acute influenza infection in mice. IL-6-deficient mice infected with influenza virus exhibited higher lethality, lost more body weight and had higher fibroblast accumulation and lower extracellular matrix (ECM) turnover in the lung than their wild-type counterparts. Deficiency in IL-6 enhanced proliferation, migration and survival of lung fibroblasts, as well as increased virus-induced apoptosis of lung epithelial cells. IL-6-deficient lung fibroblasts produced elevated levels of TGF-β, which may contribute to their survival. Furthermore, macrophage recruitment to the lung and phagocytic activities of macrophages during influenza infection were reduced in IL-6-deficient mice. Collectively, our results indicate that IL-6 is crucial for lung repair after influenza-induced lung injury through reducing fibroblast accumulation, promoting epithelial cell survival, increasing macrophage recruitment to the lung and enhancing phagocytosis of viruses by macrophages. This study suggests that IL-6 may be exploited for lung repair during influenza infection.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. IL-6 deficiency increases mouse lethality and promotes lung injury.
IL-6−/− and WT mice were intranasally inoculated with 105 PFU of IAV at day 0. (a) Changes in body weights in the individual WT (left) and IL-6−/− (middle) mice, and mean changes in body weights from day 0 through day 6 while all mice were still alive (right). Body weights were recorded and expressed as a percentage of pre-infection (day 0) body weight. (b) Kaplan-Meier survival curves. The data in a and b were pooled from two independent experiments. (c) Representative H&E-stained lung sections collected at day 6 p.i. (original magnification ×200, scale bar = 200 μm) (left and middle). Br and Alv indicate bronchiole and alveolus, respectively, and *indicates microvessel. Lung histologic scores based on the severity of inflammation and tissue damage (n = 5 for WT, n = 6 for IL-6−/−) (right). (d) Levels of IL-6 and TGF-β in the BAL fluid at day 6 p.i. determined by ELISA (n = 4 for WT, n = 5 for IL-6−/−). N.D., not detectable. (e) Viral loads in the BAL fluid at day 6 p.i. determined by the plaque assay (n = 4).
Figure 2
Figure 2. IL-6 deficiency reduces resolution from severe lung injury during influenza infection.
IL-6−/− and WT mice were intranasally inoculated with 105 PFU of IAV at day 0. (a) Immunohistochemical detection (left) and quantitation (right) of fibroblasts in the lung of mice at day 7 p.i. by monoclonal antibody specific for SFP-1 (original magnification ×200, scale bar = 50 μm). (b) Immunohistochemical detection (left) and quantitation (right) of fibronectin in the lung of mice at day 10 p.i. (original magnification ×200, scale bar = 50 μm). (c,d) Detection of collagen in the lung at day 15 (c) and day 28 (d) p.i. by picrosirius red staining (original magnification ×100, scale bar = 100 μm). Positively stained areas in a-d were quantified by an image analysis software (n = 3 for WT and n = 4 for IL-6−/− in (a); n = 4 in (b); n = 3 in (c); n = 3 in (d). The boxed areas are magnified below each panel. (e) Detection of MMP-2 and -9 in the BAL fluid at day 6 p.i. by gelatin zymography (left). The bands representing MMP-9 (middle) and MMP-2 (right) were quantified by densitometric analysis (n = 5 for WT, n = 4 for IL-6−/−). (f) Wet/dry ratios of the lungs at day 7 p.i. (n = 3).
Figure 3
Figure 3. IL-6 deficiency enhances proliferation, migration and survival of lung fibroblasts, as well as increases their production of TGF-β upon influenza infection.
Primary lung fibroblasts from WT and IL-6−/− mice were collected and cultured. (a) Proliferation rate and (b) doubling time of the fibroblasts were calculated with the Celigo cytometry (n = 3). (c) Immunohistochemical detection and (d) quantitation of viral NP in lung fibroblasts of IL-6−/− and WT mice infected with IAV at an MOI of 1 or mock-infected for 24 h (n = 4). (e) Migratory capability of lung fibroblasts of WT and IL-6−/− mice, as determined by the Boyden chamber assay. The conditioned medium collected from WT and IL-6−/− fibroblasts that had been infected with IAV served as the chemoattractant. The number of migrating cells was the average of the cells counted in three randomly selected fields in each well (n = 3). (f) Levels of TGF-β in the medium of WT and IL-6−/− fibroblasts with or without IAV infection, as determined by ELISA. Values represent the ratio of the TGF-β content to the total cell number (n = 3). (g) Percentages of apoptotic fibroblasts from WT and IL-6−/− mice infected with IAV at an MOI of 1 or mock-infected for 24 h (n = 3). (h) Percentages of apoptotic IL-6−/− fibroblasts infected with IAV at an MOI of 1 in the presence of different concentrations of IL-6 for 24 h. Apoptosis was determined by immunofluorescence staining with anti-cleaved caspase-3 antibody and quantified by the Celigo cytometry (n = 3). (i) Levels of TGF-β in the medium collected from h, as determined by ELISA (n = 3). N.S., non-significant.
Figure 4
Figure 4. IL-6 deficiency increases influenza virus-induced apoptosis of lung epithelial cells.
(a) Double staining of lung sections with TUNEL and anti-SP-C antibody (original magnification ×100, scale bar = 50 μm). WT and IL-6−/− mice were infected with 105 PFU of IAV and their lungs were collected at day 7 p.i. for immunohistochemical examination. Arrows indicate TUNEL/SP-C-double positive cells. The boxed areas in the merged panels are magnified and shown in the rightmost panels. (b) TUNEL/SP-C-double positive cells were quantified by averaging the number of doubly stained cells in three randomly selected fields in each section (n = 3). (c) Percentages of apoptotic BEAS-2B (left) and MLE-12 (right) epithelial cells infected with IAV at an MOI of 2 in the presence of different concentrations of IL-6 for 24 h. Apoptosis was determined by immunofluorescence staining with anti-cleaved caspase-3 antibody and quantified by the Celigo cytometry (n = 5). (d) Levels of TGF-β in the medium collected from MLE-12 cells infected with IAV at an MOI of 2 in the presence of anti-IL-6 neutralizing antibody (αIL-6) or isotype-matched control IgG for 24 h (n = 3).
Figure 5
Figure 5. IL-6 deficiency hampers macrophage recruitment to the lung and increases virus-induced cell death during influenza infection.
(a) Immunohistochemical examination of NP-positive cells in thioglycollate-elicited macrophages infected with IAV at an MOI of 1 for 24 h. Representative images are shown (original magnification ×100, scale bar = 200 μm). (b) NP-positive macrophages were quantified by averaging the number of positively stained cells in four randomly selected fields (n = 3). (c) Titers of IAV produced from the infected macrophages were quantified by the plaque assay (n = 3). (d) Immunohistochemical detection of macrophages in the lung with anti-Mac-3 antibody at day 7 p.i. in WT and IL-6−/− mice infected with 105 PFU of IAV. Macrophages in the lung were quantified by averaging the number of Mac-3-positive cells in three randomly selected fields in each section (n = 4). (e) Immunohistochemical detection of macrophages in the BAL fluid with anti-F4/80 antibody at day 7 p.i. in WT and IL-6−/− mice infected with 105 PFU of IAV. Total numbers of macrophages in the BAL fluid were quantified by counting F4/80-positive cells (n = 4). (f). Total numbers of thioglycollate-elicited peritoneal macrophages collected from WT and IL-6−/− mice were counted using a hemocytometer (n = 3 for WT, n = 5 for IL-6−/−). (g) Migratory capability of thioglycollate-elicited peritoneal macrophages from WT and IL-6−/− mice determined by the Boyden chamber assay. The number of migrating cells was the average of the cells counted from four randomly selected fields in each section (n = 6). (h) Cytotoxicity of thioglycollate-elicited peritoneal macrophages from WT and IL-6−/− mice infected with IAV at an MOI of 1 for 24 h, as assessed by the LDH release assay (n = 4).
Figure 6
Figure 6. IL-6 deficiency reduces phagocytic activities of macrophages.
The phagocytic activities of elicited macrophages collected from WT and IL-6−/− mice were assessed for their uptakes of FITC-labeled IAV (a), QD649 quantum dot particles (b) and IAV-infected MDCK cells (c). (a) Phagocytosis of FITC-labeled IAV was measured by flow cytometry and expressed as mean fluorescent intensity (MFI) (n = 3). (b) The macrophages treated with 5 × 1010 QD649 particles for 30 min were stained with DAPI, and their phagocytic activity was determined by the Celigo cytometer. Representative images are shown (original magnification ×200, scale bar = 100 μm). The percentage of phagocytosis was identified as the proportion of macrophages (blue) engulfing QD649 (red) (n = 4). (c) The macrophages were cultured with biotin labeled-MDCK cells that had been infected with IAV at an MOI of 1 for 2 h, permeabilized, and then fixed for immunofluorescence staining. The cell mixtures were stained with anti-F4/80 antibody and Dylight-488 for detection of macrophages and biotin-labeled MDCK cells, respectively. The phagocytic activity was measured with the Celigo cytometer. Representative images are shown (original magnification ×200, scale bar = 100 μm). The percentage of phagocytosis was identified as the proportion of macrophages (red) engulfing MDCK cells (green) (n = 4). Arrows indicate macrophages that engulfed virus infected-MDCK cells (yellow).

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