key: cord-0002612-qtpd3jj8 authors: Yang, Mei-Lin; Wang, Chung-Teng; Yang, Shiu-Ju; Leu, Chia-Hsing; Chen, Shun-Hua; Wu, Chao-Liang; Shiau, Ai-Li title: IL-6 ameliorates acute lung injury in influenza virus infection date: 2017-03-06 journal: Sci Rep DOI: 10.1038/srep43829 sha: 8de30f4bd800d3e13f4cacdda51ce14e89ccd0dc doc_id: 2612 cord_uid: qtpd3jj8 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. matrix (ECM) turnover and in recovery from lung injury probably through suppressing TGF-β production. Moreover, IL-6 prevents virus-induced apoptosis of lung epithelial cells and enhances phagocytosis of viruses by macrophages. Our findings indicate that IL-6 increases fibroblast apoptosis, macrophage phagocytic activity and epithelial cell survival. We show that IL-6 not only acts as an immune regulator to defend against influenza, but also plays an important role in balancing lung environment. Furthermore, this study sheds some lights on the processes of lung injury and repair during influenza infection. Mice lacking IL-6 are more susceptible to lethal infection with influenza virus. To study the role of endogenous IL-6 in host defense against influenza, we compared the body weight change and survival curves, as well as histological and immunological changes between IL-6-deficient (IL-6 −/− ) and WT C57BL/6 mice after intranasal infection of influenza A/WSN/33 (H1N1) virus (IAV). As shown in Fig. 1a , four out of nine IL-6 −/− mice continued to lose weight and died between 6 and 10 days after infection (middle panel), whereas only one out of 16 WT mice lost weight without weight gain and died at day 10 post-infection (p.i.) (left panel). All of the mice that survived for more than 10 days recovered and survived for at least 16 days. Analysis of the entire body weight curves of the infected mice from day 0 through day 6 while all the mice were still alive reveals that IL-6 −/− mice lost more weight over time on average than WT mice (right panel). Figure 1b shows that deficiency in IL-6 increased the mortality and reduced the survival time in mice after IAV infection. As shown in Fig. 1c , histological examination of the lungs collected at day 6 p.i. revealed that IL-6 −/− mice had higher levels of mononuclear cell accumulation (middle panel) and higher histologic scores (right panel) than WT mice (left and right panels). In the broncoalveolar lavage (BAL) fluid, IL-6 contents were undetectable (left panel) as expected, whereas levels of TGF-β were higher (right panel) in IL-6 −/− mice than in WT mice (Fig. 1d) . Furthermore, IL-6 −/− mice had significantly higher viral loads than WT mice in the BAL fluid at day 7 p.i. (Fig. 1e) . Taken together, these results suggest that IL-6 may be involved in the protection against influenza. IL-6 −/− mice exhibit increased fibroblast accumulation and reduced ECM turnover in the lung following influenza infection. Pneumonia is a common complication of influenza with diffuse alveolar damage, fibroblast proliferation and inflammatory cell infiltration 22 . In some cases, pneumonia finally causes fibrosis accompanied with collagen or ECM deposition and EMT. We therefore compared the levels of fibroblasts, fibronectin, collagen, MMP-9 and MMP-2 in the lungs of IL-6 −/− and WT mice infected with IAV. IL-6 −/− mice had higher amounts of fibroblasts at day 7 p.i. (Fig. 2a) and expressed higher levels of fibronectin at day 10 p.i. (Fig. 2b) than WT mice. However, the amount of fibronectin expressed in lung fibroblasts did not significantly differ between IL-6 −/− and WT mice, as examined by immunofluorescence staining (Supplementary Figure S1a) . TGF-β can induce α -smooth muscle actin (α -SMA) expression, which is relevant to fibrosis formation. Given elevated expression of TGF-β in the BAL fluid of the infected IL-6 −/− mice (Fig. 1d) , we further detected the typical EMT marker α -SMA to assess the potential involvement of IL-6 in the regulation of EMT during influenza infection. We found that expression of α -SMA was similar between WT and IL-6 −/− lungs exhibiting either minor or severe fibroblast accumulation (Supplementary Figure S1b) . Moreover, contents of lung fibroblasts did not differ between IL-6 −/− and WT mice (Supplementary Figure S1c) . To determine whether fibrosis occurred in the mice at a later stage of influenza, collagen deposition in the lung was assessed by picrosirius red staining. IL-6 −/− mice expressed higher levels of collagen in the lung than WT mice detected at day 15 ( Fig. 2c ) and day 28 (Fig. 2d) p.i. Matrix metalloproteinases (MMPs) represent a group of enzymes involved in the degradation of ECM components. We further detected MMP-9 and MMP-2 in the BAL fluid by gelatin zymography. IL-6 −/− mice expressed lower levels of MMP-9 and MMP-2 compared to WT mice (Fig. 2e) , suggesting that deficiency in IL-6 may disturb the degradation and turnover of ECM. Furthermore, the wet-to-dry (wet/dry) ratios of the lungs increased by approximately two-fold in IL-6 −/− mice compared to WT mice (Fig. 2f) , suggesting that deficiency in IL-6 may lead to severe edema and lung injury. Collectively, these results implicate a protective role for IL-6 in influenza infection by reducing fibroblast accumulation and enhancing ECM turnover in the lung. IL-6 deficiency enhances proliferation, migration and survival of lung fibroblasts, as well as increases their production of TGF-β. To further dissect the effect of IL-6 on fibroblast functions, fibroblasts were isolated from the lungs of IL-6 −/− and WT mice and cultured for 72 h. Cell numbers were counted (Fig. 3c) and quantification of its expression (Fig. 3d) . To determine the migratory capability of lung fibroblasts, we used the conditioned medium from IL-6 −/− or WT fibroblasts that had been infected with IAV as the chemoattractant. As shown in Fig. 3e , fibroblasts from IL-6 −/− mice had a higher migratory capability than those from WT mice in response to the conditioned medium of either WT or IL-6 −/− fibroblasts infected with IAV, as determined by the Boyden chamber assay. Notably, IL-6 −/− fibroblasts in response to the conditioned medium from IL-6 −/− fibroblasts had the highest migratory ability among the four treatment conditions. These results indicate that IL-6 −/− lung fibroblasts were more prone to be stimulated to migrate following influenza infection, and that the infected IL-6 −/− fibroblasts secreted more chemoattractant proteins capable of stimulating fibroblast migration, as compared with their WT counterparts. Given that IAV-infected IL-6 −/− mice produced higher levels of TGF-β in the BAL fluid (Fig. 1d) and their lung fibroblasts displayed higher migratory capability in vitro (Fig. 3e ) compared with their WT counterparts, we further examined TGF-β levels in the supernatants of IL-6 −/− and WT fibroblasts with or without infection with IAV. Figure 3f shows that uninfected IL-6 −/− fibroblasts secreted higher levels of TGF-β than their WT counterparts. Notably, levels of TGF-β were further increased when IL-6 −/− fibroblasts were infected with IAV, whereas their contents remained similar in WT fibroblasts regardless of viral infection. These results suggest that deficiency in IL-6 may lead to increases in the migratory capability of lung fibroblasts probably through the elevated production of TGF-β . Fibroblasts from patients with idiopathic pulmonary fibrosis are more active and resistant to apoptosis 23 . To further study whether IL-6 regulated fibroblast survival, we infected lung fibroblasts with IAV and measured the apoptosis of fibroblasts by detection of the cleaved caspase-3. Figure 3g shows that there was a slight reduction in the percentage of cleaved caspase-3 in the infected IL-6 −/− fibroblasts in comparison to their WT counterparts. However, in the absence of viral infection, the levels of cleaved caspase-3 were not significantly different between IL-6 −/− and WT fibroblasts. To evaluate the importance of IL-6 in apoptosis, we treated IL-6 −/− fibroblasts with different doses of recombinant mouse IL-6 and determined their apoptosis. Treatment with IL-6 enhanced the apoptotic potential of IL-6 −/− fibroblasts following IAV infection in a dose-dependent manner ( Fig. 3h ) with concomitant decreases in TGF-β production (Fig. 3i) . Taken together, lacking of IL-6 rendered lung fibroblasts more resistant to virus-induced apoptosis. Thus, IL-6 may be indispensable for reducing fibroblast proliferation, migration and survival through decreasing TGF-β production. Inappropriate activation of epithelial cells and neutrophil apoptosis can lead to tissue injury and diseases, such as ALI and ARDS 24 . As alveolar type II (AT2) cells can differentiate into alveolar type I (AT1) cells for regeneration of damaged lung tissue, AT2 cells are crucial for reducing epithelial cell apoptosis and promoting lung repair. We thus examined whether IL-6 could protect epithelial cells from IAV-induced death. Lung sections obtained from IL-6 −/− and WT mice at day 7 p.i. were double-stained with antibody against the AT2 cell marker surfactant protein C (SP-C) (red) and TUNEL (green). TUNEL-SP-C doubly stained cells were evident in the IL-6 −/− lungs, whereas AT2 cells undergoing apoptosis were hardly detectable in the WT lungs (Fig. 4a) . Moreover, the numbers of double-positive cells were approximately doubled in the lungs of IL-6 −/− mice compared with those of WT mice (Fig. 4b) . We further used human bronchial epithelial cells (BEAS-2B) and mouse lung epithelial cells (MLE-12) to assess whether IL-6 reduced IAV-induced epithelial cell apoptosis. Addition of IL-6 decreased the percentage of cleaved caspase-3 in IAV-infected BEAS-2B and MLE-12 cells (Fig. 4c) in a dose-dependent manner. Blocking of IL-6 activity with anti-IL-6 neutralizing antibody in MLE-12 cells infected with IAV increased TGF-β production in the culture medium (Fig. 4d) . Taken together, these results indicate that in addition to promoting fibroblast apoptosis, IL-6 improves epithelial cell survival with concomitant inhibition of TGF-β production. To determine whether IL-6 played a role in macrophage generation and function, we first examined the differentiation potential of bone marrow cells of WT and IL-6 −/− mice into macrophages. Bone marrow cells from both strains of mice were isolated and treated with macrophage colony-stimulating factor (M-CSF) for 7 days to generate bone marrow-derived macrophages (BMDMs). We found that numbers of BMDMs obtained from IL-6 −/− and WT mice were not significantly different (Supplementary Figure S2) . We also examined whether BMDMs were infectable with IAV. Only less than 4% of BMDMs expressed viral NP, indicative of productive infection with IAV, at 48 h p.i. in either WT or IL-6 −/− mice, suggesting low infectability of mouse BMDMs with IAV (Supplementary Figure S3) . We next examined whether thioglycollate-elicited peritoneal macrophages, which are convenient sources for mouse macrophages, were permissive for productive infection with IAV. Viral NP was clearly detectable in IAV-infected peritoneal macrophages at 24 and 48 h p.i. by immunohistochemical examination (Fig. 5a) . Notably, about 40-50% of the macrophages became productively infected with IAV at 24 h or 48 h p.i. (Fig. 5b) , producing 3 × 10 4 -6 × 10 4 plaque-forming units (PFU)/ml of virus particles (Fig. 5c) . These results identified productive replication of IAV in mouse peritoneal macrophages. Following IAV infection, infiltration of macrophages in the lung (Fig. 5d ) and BAL fluid (Fig. 5e ) was markedly decreased in IL-6 −/− mice at day 7 p.i., as detected by staining with antibodies against macrophage antigens Mac3 and F4/80, respectively. Given that peritoneal macrophages were much more susceptible to productive IAV infection than BMDMs, we used peritoneal macrophages for further studies. Notably, numbers of thioglycollate-elicited peritoneal macrophages isolated from IL-6 −/− mice were reduced by 75% compared with those from WT mice (Fig. 5f) . Furthermore, lack of IL-6 hampered the migratory response of these macrophages toward fetal bovine serum (FBS) that served as the chemoattractant (Fig. 5g) . To investigate whether IL-6 provided survival signals to macrophages, peritoneal macrophages were infected with or without IAV for 48 h, and cell death was analyzed by the lactate dehydrogenase (LDH) release assay. Compared with WT macrophages, more cell death occurred in IL-6 −/− macrophages in the absence of IAV infection, which were further increased after viral infection (Fig. 5h) . Collectively, these results suggest that presence of IL-6 may promote the migration and recruitment of macrophages to the lung and reduce their death during influenza infection. Phagocytic activities of macrophages are decreased in IL-6 −/− mice. Clearance of apoptotic epithelial cells and neutrophils leads to resolution of inflammation and repair 25 . In the infection process, macrophage infiltration as well as ingestion of particles and dead cells by macrophages are important for pathogen clearance and removal of dead cells. To investigate whether IL-6 impacted macrophage phagocytosis, we examined phagocytosis of viral particles and dead infected cells by macrophages from IL-6 −/− and WT mice. Flow cytometric analysis shows that FITC-labeled IAV particles were more efficiently ingested by the peritoneal macrophages isolated from WT mice than from IL-6 −/− mice (Fig. 6a) . Furthermore, similar results were observed when QD649 quantum dots were used for assessing the phagocytic activity of macrophages (Fig. 6b) . To mimic real virus-induced cell death, we infected MDCK cells with IAV for 24 h, and then mixed these infected cells with macrophages from IL-6 −/− and WT mice at a ratio of 2 : 1 for 2 h. Figure 6c shows that more virus-infected cells were engulfed by WT macrophages than by IL-6 −/− macrophages, as observed by fluorescence microscopy and quantified by the Celigo cytometer. Taken together, these results suggest that deficiency in IL-6 may impair phagocytic clearance of influenza virus and virus-infected cells by macrophages. Tissue remodeling is crucial for lung repair and regeneration after influenza-induced tissue injury. In the present study, we demonstrate that IL-6 plays a critical role in promoting lung repair in mice with influenza infection through participating in the interplay of macrophages, fibroblasts and lung epithelial cells, as well as through inhibiting TGF-β production. When normal tissue is damaged, tissue regeneration contains several complicated steps, including inflammation, proliferation and remodeling. Fibroblasts are involved in the proliferation and remodeling phases. The roles of TGF-β in the interplay between fibroblasts and epithelial cells have been studied in much detail. TGF-β has a contrast role in epithelial cells. It induces apoptosis in bronchiolar epithelial cells and diminishes lung epithelial regeneration 26 . Moreover, TGF-β promotes epithelial cells undergoing EMT to become myofibroblasts 27 . Targeting TGF-β activity and its downstream signaling pathways effectively attenuates fibrosis formation 28, 29 . Previous studies have indicated that IL-6 and TGF-β participate in the pathogenesis of lung diseases, such as ARDS 30 , lung fibrosis, asthma 31 and chronic obstructive pulmonary disease 32 . Influenza virus induces activation of latent TGF-β , which can determine viral pathogenesis and is associated with virus-induced cell apoptosis 4, 12 . Influenza virus-infected mice with heterozygous mutation in the cystic fibrosis transmembrane conductance regulator had higher levels of IL-6 and alveolar macrophages in the BAL fluid, and did not develop ALI 33 . Such effects were associated to TGF-β -dependent production of IL-6 33 . Recently, it was shown that integrin β 6 subunit gene knockout mice had increased survival after influenza infection and reduced ALI, which were attributed to the loss of β 6-activated TGF-β and increases in activated CD11b + alveolar macrophages and type I interferon signaling in the lung 34 . These results are in accordance with our findings. We show that IL-6 is essential for the survival and the recovery from severe lung injury in mice after influenza infection, which is associated with reduced TGF-β production. Loss of IL-6 interferes with the functions of lung fibroblasts, including increases in proliferation rate and migratory capability, as well as resistance to virus-induced apoptosis, which may be mediated by increased TGF-β production. Notably, accumulation of fibroblasts in the lung causes deposition of collagen and fibronectin. Furthermore, addition of recombinant IL-6 to the lung epithelial cells infected with influenza virus decreased caspase-3 activation, indicative of enhanced survival, and reduced TGF-β production. Collectively, we identify a novel role for IL-6 in the lung repair process. Depletion of alveolar macrophages, which are critical for host defense against influenza, leads to increased susceptibility to influenza virus infection and massive pathology in pigs and mice 35, 36 . Effects of IL-6 on immune cells have been extensively studied. IL-6 controls monocyte differentiation into macrophages 37 . Migration and infiltration of macrophage are also dependent on the IL-6 and Stat3 signaling pathway 38 . A recent report has shown that bone-marrow derived dendritic cells from IL-6-deficient mice displayed defects of phagocytosis of fluorescent carboxylate-modified polystyrene latex beads 31 . These findings are in agreement with our results that IL-6 is essential for alleviating influenza symptoms and subsequent lung injury by promoting macrophage recruitment to the lung and by phagocytosing virus-infected cells. Whether these activities of macrophages serve to enhance virus clearance or reduce lung inflammation is currently not clear, but warrants further investigation. Several lines of evidence have suggested that TGF-β and IL-6 can regulate each other in different circumstances. IL-6 increases trafficking of TGF-β receptor to non-lipid raft-associated pools, resulting in augmented TGF-β 1/Smad signaling 39 . Moreover, TGF-β 1-induced IL-6 expression participates in trans-differentiation of fibroblasts to myofibroblasts 40 . By contrast, TGF-β inhibits IL-6 signaling by reducing Stat activity in the intestinal epithelial cells, and serves as a negative regulator in uncontrolled inflammation 41 . We show that TGF-β production is upregulated in the lung of IL-6 knockout mice. Furthermore, addition of recombinant IL-6 to IL-6 −/− fibroblasts reduces virus-induced TGF-β production, whereas addition of neutralizing IL-6 antibody in lung epithelial cells increases TGF-β production. Our results are partially consistent with a previous study showing that hepatocyte growth factor and IL-6 inhibit TGF-β -mediated fibroblast-myofibroblast transition through reduction of α -SMA expression 42 . These findings suggest that IL-6 and TGF-β may positively or negatively regulate each other under different conditions. Not only does IL-6 promote host defense to pathogen invasion, it also resolves disease onset. We identify a novel role for IL-6 produced by fibroblasts during influenza infection in promoting apoptosis and reducing proliferation of fibroblasts, as well as balancing fibroblast migration through downregulating TGF-β production. Moreover, IL-6 is essential for macrophages to phagocytose virus-infected cells. In influenza infection, mice deficient in IL-6 had decreased survival and more severe lung injury. Therefore, we demonstrate that IL-6 not only acts as an immune regulator for defending against influenza, but also plays an important role in balancing lung environment. Female C57BL/6 mice were purchased from the Laboratory Animal Center of National Cheng Kung University (NCKU) or National Laboratory Animal Center (Taipei, Taiwan). IL-6 −/− (B6.129S2-Il6 tm1kopf /J) mice with C57BL/6 background were purchased from Jackson Laboratory and maintained in the Laboratory Animal Center of NCKU. MDCK cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% cosmic calf serum (Hyclone, Logan, UT), 2 mM L-glutamine and 50 μ g/ml gentamicin. Primary lung fibroblasts were isolated from WT and IL-6 −/− mice, maintained in DMEM with 10% cosmic calf serum, 2 mM L-glutamine and 50 μ g/ml gentamicin, and used between the fourth and seventh passages. Human bronchial BEAS-2B epithelial cells were maintained in BEBM medium (Lonza, Rockland, ME). Mouse MLE-12 epithelial cells were maintained in F12 medium with 4% FBS, 2 mM L-glutamine, 0.1 mM non-essential amino acid, 50 μ g/ml gentamicin, 5 μ g/ml insulin, 10 ng/ml epidermal growth factor, 1 μ g/ml transferrin and 500 ng/ml hydrocortisone. Influenza A/WSN/33 (H1N1) virus was propagated and titrated in MDCK cells as described previously 43 . All in vitro work on influenza virus was carried out in biosafety level 2 laboratories. All animal work was conducted in animal biosafety level 2 facilities at NCKU. The experimental protocols adhered to the rules of the Animal Protection Act of Taiwan and were approved by the Animal Care and Use Committee of NCKU (IACUC number: 104088). Animal studies. Groups of female C57BL/6 mice and IL-6 −/− mice at 4-6 weeks of age were intranasally inoculated with 10 5 PFU of IAV which corresponded to 1.5 × lethal dose (LD 50 ) at day 0. The mice were monitored daily for illness, weight loss and death for 16 days after viral infection. IAV-infected mice that had received different treatments were killed at day 7 or 10 p.i. The lungs were removed, formalin-fixed and paraffin-embedded for hematoxylin and eosin (H&E) staining using standard methods. Inflammatory changes on the basis of numbers of inflammatory cells and tissue damage in the lungs were determined by histology from H&E-stained longitudinal cross sections and scored on a 0-3 scale (0 = no change, 1 = mild, 2 = moderate, 3 = severe) 43 . For immunohistochemical staining, tissue sections were deparaffinized, antigen-retrieved using protease K (100 μ g/ml, Life Technologies, Carlsbad, CA) digestion for 10 min at room temperature and incubated with rabbit anti-human SFP-1 (S100A4) antibody (1:400, Abcam, Cambridge, UK), rabbit anti-human fibronectin antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA), rat anti-mouse Mac3 antibody (1:50; M3/84, BD Biosciences PharMingen, San Diego, CA) and monoclonal mouse anti-α -SMA antibody (1:400, Sigma-Aldrich, St. Louis, MO). After sequential incubation with appropriate horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature and 3-amino-9-ethyl carbazole (AEC) as the substrate chromogen, the slides were counterstained with hematoxylin. The signal intensity of immunohistochemical staining was further quantified using the Image J software (https://imagej.nih.gov/ij/). To detect apoptotic epithelial cells in the lungs, paraffin-embedded lung tissue sections were subjected to co-stain with goat anti- ELISA. BAL was performed as described previously 43 . Lung fibroblasts collected from WT and IL-6 −/− mice or MLE-12 were infected with IAV at MOI of 1 and 2, respectively, in the presence of rabbit anti-IL-6 neutralizing antibody (1:400, Abcam) or isotype-matched control IgG for 24 h. The levels of TGF-β and IL-6 cytokines in the BAL fluid and culture medium were quantified using DuoSet ELISA kits (R&D, Minneapolis, MN). Lung sections collected from mice infected with IAV at days 15 and 28 p.i. were stained with picrosirius red to determine the degree of collagen deposition. To assess lung edema, the wet/dry ratio of the infected lungs was assessed at day 7 p.i. The lungs were dissected, weighed, and dried at 60 °C for 2 days. The wet/dry ratio was then calculated by dividing the wet weight by the final dry weight. Fibroblast functional assay. To assess fibroblast functions, we analyzed the proliferation and migration capabilities of lung fibroblasts collected from WT and IL-6 −/− mice. Fibroblasts were cultured in DMEM containing 10% FBS for 24, 48 and 72 h. The proliferation rate and doubling time were calculated by the Celigo cytometer. Migratory capabilities of fibroblasts were analyzed using the Boyden chamber assay. The cells were placed in the upper compartment and allowed to migrate through the pores of the membrane into the lower compartment, in which the conditioned medium from each cell type after infection with IAV for 24 h served as the chemoattractant, and incubated for 24 h. The migrated cells were fixed by methanol and stained with Giemsa. The number of migrating cells was the average of the cells counted in three randomly selected fields in each well. Macrophage functional assays. Macrophages collected from WT and IL-6 −/− mice after peritoneal injection with thioglycollate (3%) for 3 days were used to analyze the migratory capability of macrophages by the Boyden chamber assay. The macrophages were placed in the upper compartment and allowed to migrate through the pores of the membrane into the lower compartment, in which FBS served as the chemoattractant, and incubated for 24 h. The migrating cells were fixed by methanol and stained with Giemsa. The number of migrating cells was the average of the cells counted in three randomly selected fields in each section. To assess the phagocytosis of viruses or nanoparticles by macrophages, peritoneal macrophages (10 6 cells) were incubated with IAV that had been labeled with FITC (NHS-Fluorescein; Pierce, Rockford, Ill) at an MOI of 1 as described previously 44 or with 5 × 10 10 QD649 quantum dot particles for 30 min at 37 °C and then treated with 40 μ l of 0.1% trypan blue to quench extracellular florescence. Macrophages were then stained with DAPI. After being washed with phosphate-buffered saline (PBS), florescence was analyzed by flow cytometry (BD Biosciences, San Diego, CA) or the Celigo cytometer, and photographed with fluorescence microscopy. Furthermore, the phagocytosis of virus-infected cells by macrophages was also assessed. MDCK cells that had been infected with IAV at an MOI of 1 and then labeled with biotin (NHS-LS-Biotin; Pierce) were mixed with macrophages (at a ratio of two virus-infected cells to one macrophage), and incubated at 37 °C for 2 h. The cell mixture was washed with PBS, fixed with 3.7% formaldehyde, permeabilized with 0.01% Triton X-100 and then added with Dylight488-conjugated streptavidin (1:200, Jackson ImmunoResearch, West Grove, PA). Macrophages were detected by rat anti-mouse F4/80 antibody (1:50, Serotec, Oxford, UK). The number of macrophages containing engulfed cells was determined using fluorescence microscopy and the Celigo cytometer. The percentage of phagocytosis was calculated as the number of engulfing macrophages relative to the total number of macrophages. For the survival assay, macrophages (10 4 cells) were infected with IAV for 48 h, and the cytotoxicity was measured by CytoTox 96 non-radioactive cytotoxicity assay (Promega). Statistical analysis. Data are expressed as mean ± standard deviation (SD). Differences in body weights between two groups were compared by repeated-measures analysis of variance (ANOVA). The survival analysis was performed using the Kaplan-Meier survival curve and log-rank test. For the remaining data, statistical differences were compared by Student's t-test between two groups and by one-way ANOVA with Bonferroni post hoc test among three or more groups. The differences were considered significant if P values were < 0.05. Statistical tests were performed using GraphPad Prism (version 6.0, GraphPad software, San Diego, CA). Serial evaluation of high-resolution computed tomography findings in patients with pneumonia in novel swine-origin influenza A (H1N1) virus infection Lung histopathological findings in fatal pandemic influenza A (H1N1) Pneumonia and respiratory failure from swine-origin influenza A (H1N1) in Mexico Influenza promotes collagen deposition via α vβ 6 integrin-mediated transforming growth factor β activation Transforming growth factor-β : activation by neuraminidase and role in highly pathogenic H5N1 influenza pathogenesis IL-22 is essential for lung epithelial repair following influenza infection Timed action of IL-27 protects from immunopathology while preserving defense in influenza TGF-β -induced EMT: mechanisms and implications for fibrotic lung disease Active transforming growth factor-β 1 activates the procollagen I promoter in patients with acute lung injury The acute respiratory distress syndrome TGF-β directs trafficking of the epithelial sodium channel ENaC which has implications for ion and fluid transport in acute lung injury Influenza virus neuraminidase activates latent transforming growth factor β Influenza induces endoplasmic reticulum stress, caspase-12-dependent apoptosis, and c-Jun N-terminal kinase-mediated transforming growth factor-β release in lung epithelial cells Interleukin-6 is crucial for recall of influenza-specific memory CD4 + T cells Essential role of IL-6 in protection against H1N1 influenza virus by promoting neutrophil survival in the lung IL-6 induces long-term protective immunity against a lethal challenge of influenza virus The induction of antibody production by IL-6 is indirectly mediated by IL-21 produced by CD4 + T cells Symptom pathogenesis during acute influenza: Interleukin-6 and Other cytokine responses Clinical aspects and cytokine response in severe H1N1 influenza A virus infection Inhibition of the cytokine response does not protect against lethal H5N1 influenza infection Role of host cytokine responses in the pathogenesis of avian H5N1 influenza viruses in mice Viral pneumonias in adults: Radiologic and pathologic findings Comparison of the morphological and biochemical changes in normal human lung fibroblasts and fibroblasts derived from lungs of patients with idiopathic pulmonary fibrosis during FasL-induced apoptosis The acute respiratory distress syndrome Macrophage engulfment of apoptotic neutrophils contributes to the resolution of acute pulmonary inflammation in vivo TGF-β 1 as an enhancer of Fas-mediated apoptosis of lung epithelial cells Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix Therapeutic effect of a peptide inhibitor of TGF-β on pulmonary fibrosis Gene transfer of soluble transforming growth factor type II receptor by in vivo electroporation attenuates lung injury and fibrosis TGF-β is a critical mediator of acute lung injury Critical role of IL-6 in dendritic cell-induced allergic inflammation of asthma Interleukin-6 neutralization alleviates pulmonary inflammation in mice exposed to cigarette smoke and poly(I:C) TGF-β -induced IL-6 prevents development of acute lung injury in influenza A virus-infected F508del CFTR-heterozygous mice An epithelial integrin regulates the amplitude of protective lung interferon responses against multiple respiratory pathogens Alveolar macrophages are indispensable for controlling influenza viruses in lungs of pigs Transient ablation of alveolar macrophages leads to massive pathology of influenza infection without affecting cellular adaptive immunity IL-6 switches the differentiation of monocytes from dendritic cells to macrophages Interleukin-6/STAT3 pathway is essential for macrophage infiltration and myoblast proliferation during muscle regeneration Interleukin-6 regulation of transforming growth factor (TGF)-β receptor compartmentalization and turnover enhances TGF-β 1 signaling TGF-β -induced interleukin-6 participates in transdifferentiation of human Tenon's fibroblasts to myofibroblasts TGF-β down-regulates IL-6 signaling in intestinal epithelial cells: Critical role of SMAD-2 Inhibitory effects of HGF and IL-6 on TGF-β 1 mediated vocal fibroblastmyofibroblast differentiation Galectin-1 binds to influenza virus and ameliorates influenza virus pathogenesis Use of FITC-labeled influenza virus and flow cytometry to assess binding and internalization of virus by monocytes-macrophages and lymphocytes