Basic fibroblast growth factor protects against influenza A virus-induced acute lung injury by recruiting neutrophils

Basic fibroblast growth factor protects against influenza A virus-induced acute lung injury by... Abstract Influenza virus (IAV) infection is a major cause of severe respiratory illness that affects almost every country in the world. IAV infections result in respiratory illness and even acute lung injury and death, but the underlying mechanisms responsible for IAV pathogenesis have not yet been fully elucidated. In this study, the basic fibroblast growth factor 2 (FGF2) level was markedly increased in H1N1 virus-infected humans and mice. FGF2, which is predominately derived from epithelial cells, recruits and activates neutrophils via the FGFR2–PI3K–AKT–NFκB signaling pathway. FGF2 depletion or knockout exacerbated influenza-associated disease by impairing neutrophil recruitment and activation. More importantly, administration of the recombinant FGF2 protein significantly alleviated the severity of IAV-induced lung injury and promoted the survival of IAV-infected mice. Based on the results from experiments in which neutrophils were depleted and adoptively transferred, FGF2 protected mice against IAV infection by recruiting neutrophils. Thus, FGF2 plays a critical role in preventing IAV-induced lung injury, and FGF2 is a promising potential therapeutic target during IAV infection. influenza H1N1 virus, recombinant FGF2 protein, neutrophil recruitment, FGFR2–PI3K–AKT–NFκB signaling, therapeutic target Introduction Influenza spreads throughout the world during annual outbreaks, resulting in ~3–5 million cases of severe illness and ~250000–500000 deaths annually; infants and the elderly are particularly vulnerable to influenza. The mechanisms by which influenza virus (IAV) infection cause symptoms in humans have been studied intensively. Some severely infected patients develop acute lung injury (ALI) and even acute respiratory distress syndrome (ARDS), which is the predominant cause of reported influenza-related deaths (Dominguez-Cherit et al., 2009; Louie et al., 2009; Berdal et al., 2011; Ohta et al., 2011). IAV infection may cause inflammation of the airways, epithelial necrosis, edema, hemorrhaging, and respiratory failure (Xu et al., 2006; Rincon, 2012; Ding et al., 2013). Both virus-specific virulence factors and host immunity are associated with exacerbated IAV pathogenesis (Crouser et al., 2009). The currently recognized therapeutic agents against IAV infection include viral m2 channel inhibitors (amantadine and rimantadine), neuraminidase inhibitors (zanamivir, oseltamivir, peramivir, and laninamivir octanoate), and polymerase inhibitors (ribavirin and favipiravir), but IAVs are becoming highly resistant to these drugs, and further evidence is required from clinical trials (Dushianthan et al., 2011; De Clercq and Li, 2016). Basic fibroblast growth factor (bFGF or FGF2), a potent mitogen for many cell types, including airway smooth muscle cells, fibroblasts, and endothelial cells (Redington et al., 2001), is associated with multiple biological processes, including tumor angiogenesis, embryonic development, proliferation, migration, and injury repair (Meyer et al., 1995; Ortega et al., 1998; Fuhrmann-Benzakein et al., 2000; Nugent and Iozzo, 2000; Virag et al., 2007). FGF2 is dysregulated in many inflammatory disorders, such as inflammatory bowel disease (IBD), Crohn’s disease, ulcerative colitis, and rheumatoid arthritis (Byrd et al., 1996; Kanazawa et al., 2001; Song et al., 2015). In immune responses, FGF2 functions to maintain the innate immune homeostasis of antiviral immunity by stabilizing retinoic acid-inducible gene-I (RIG-I) and preventing proteasome-mediated RIG-I degradation (Liu et al., 2015). In a study of the link between FGF2 and ALI by Powers et al. (1994), FGF2 was shown to play a role in the alveolar response to hyperoxic injury via the altered mRNA levels and protein distribution. According to Liebler et al. (1997), FGF2 may participate in directing cell proliferation following pulmonary fibrosis. As shown in the study by Zhao et al. (2015), mesenchymal stem cells (MSCs) and FGF2 synergistically reduced the level of inflammatory cytokines in the treatment of LPS-induced lung injury. Moreover, Guzy et al. (2015) showed that FGF2 is required for epithelial repair and maintaining epithelial integrity after bleomycin-induced lung injury in mice. However, researchers have not determined whether and how FGF2 plays a role in IAV-induced ALI. In this study, we explore the role of FGF2 in host defense against IAV infection using our previous established mouse model (Li et al., 2012). Based on our results, FGF2 plays a pivotal role in IAV-induced lung injury, and the administration of the recombinant FGF2 protein markedly reduces mortality and the severity of lung injury in a preclinical model of IAV infection. The mechanisms underlying these effects of FGF2 include neutrophil activation and recruitment via the PI3K–Akt–NFκB signaling pathway. Results FGF2 is significantly upregulated in patient serum and mouse bronchoalveolar lavage fluid (BALF) following IAV infection Hypercytokinemia has been reported to be an early host response signature in influenza A (H1N1) virus-induced ALI (Tisoncik et al., 2012; Brandes et al., 2013; Liu et al., 2016; Yang and Tang, 2016). However, the roles of individual cytokines in ALI remain largely unclear. We measured FGF2 levels in 156 sera samples from patients confirmed to be infected with the H1N1 virus to investigate the effects of FGF2 on H1N1-induced ALI, and the characteristics reflecting the conditions and outcomes of these patients are described in the Supplementary information (Supplementary Table S1). The FGF2 level was significantly elevated in all IAV-infected patients compared with healthy subjects. Furthermore, levels of the FGF2 protein in H1N1 virus-infected patients were gradually elevated as the fever duration increased and peaked on Day 3 of fever. Correspondingly, levels of the FGF2 protein in all hospitalized patients were markedly higher than the levels in healthy subjects (Figure 1A). Meanwhile, 4-week-old wild-type (WT) B6 mice were intranasally (i.n.) infected with the BJ501 strain at a titer of 105 50% tissue culture infectious dose (TCID50), and we observed a significant increase in pulmonary elastance that represents changes in pressure achieved per unit changes in volume (Supplementary Figure S1A) and decreased arterial partial pressure of oxygen (PaO2) at 3, 5, or 7 days post-infection (DPI) (Supplementary Figure S1B). In addition, the level of the FGF2 protein was increased in the BALF of C57BL/6 mice following challenge with the A/Beijing/501/2009 (BJ501) strain. FGF2 levels began to increase significantly on the fifth day, peaked on the eighth day, and then sharply decreased beginning on the10th day of virus challenge (Figure 1B). Furthermore, FGF2 was expressed at high levels in the lung tissue of IAV BJ501 strain-infected mice at 5 DPI, based on immunohistochemical (IHC) staining (Figure 1C). This observation suggests a potential key role for FGF2 in H1N1 virus-induced ALI. Figure 1 View largeDownload slide FGF2 levels were significantly increased in patients’ sera and mouse BALF. Four-week-old B6 mice were anesthetized with 50 μl of 1% (w/v) pentobarbital sodium and i.n. inoculated with 105 TCID50 of BJ501 H1N1 viruses. All data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. (A) The concentration of FGF2 in the sera from patients infected with influenza A (H1N1) virus strain A/Beijing/501/2009 (BJ501) was determined using a Bio-Plex Human Cytokine Array. (B) The concentration of FGF2 in the BALF of B6 mice infected with 105 TCID50 of the BJ501 strain (n = 5) was measured at 0–14 DPI. (C) FGF2 staining and quantification in the lung tissues from B6 mice infected with 105 TCID50 of the BJ501 strain at 5 DPI. Figure 1 View largeDownload slide FGF2 levels were significantly increased in patients’ sera and mouse BALF. Four-week-old B6 mice were anesthetized with 50 μl of 1% (w/v) pentobarbital sodium and i.n. inoculated with 105 TCID50 of BJ501 H1N1 viruses. All data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. (A) The concentration of FGF2 in the sera from patients infected with influenza A (H1N1) virus strain A/Beijing/501/2009 (BJ501) was determined using a Bio-Plex Human Cytokine Array. (B) The concentration of FGF2 in the BALF of B6 mice infected with 105 TCID50 of the BJ501 strain (n = 5) was measured at 0–14 DPI. (C) FGF2 staining and quantification in the lung tissues from B6 mice infected with 105 TCID50 of the BJ501 strain at 5 DPI. FGF2 deficiency exacerbates IAV-induced lung injury Four-week-old WT B6 mice were i.n. infected with the BJ501 strain at a titer of 103 TCID50 to confirm the exact role of FGF2 in H1N1 virus-induced ALI. Mice were pre-treated or intravenously (i.v.) treated with anti-FGF2 antibodies or isotype control antibodies. After infection with the BJ501 strain, the groups that had been pre-treated or treated with anti-FGF2 antibodies had significantly lower survival rates and showed more severe body weight loss and lung edema than the group treated with the isotype control. Moreover, the pathology of the mice in the anti-FGF2 antibody-pre-treated group was much worse than the mice in the anti-FGF2 antibody-treated group, as determined by weight loss, survival rate, and lung edema (Figure 2A–F). Figure 2 View largeDownload slide Antibody depletion or knockout of FGF2 exacerbates H1N1-induced lung injury. WT B6 mice and FGF2–/– (KO) mice were i.n. inoculated with 103 or 105 TCID50 of the BI501 H1N1 virus. For the antibody pretreatment group, each B6 mouse was sequentially i.v. inoculated with 50 μg of the isotype control or anti-FGF2 antibodies 6 h before infection and at 1 DPI. For the treatment group, each B6 mouse was sequentially injected i.v. with 50 μg of the isotype control or anti-FGF2 antibodies at 3 and 5 DPI. All data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. (A) Changes in the weights of pre-treated B6 mice (n = 10). (B) Survival rates of pre-treated B6 mice (n = 10). (C) Wet-to-dry ratios of lungs from pre-treated B6 mice (n = 6) at 5 DPI. (D) Changes in the weights of treated B6 mice (n = 10). (E) Survival rates of treated B6 mice (n = 10). (F) Wet-to-dry ratios of lungs from treated B6 mice (n = 6) at 5 DPI. (G) Changes in the weights of WT B6 mice and FGF2–/– mice (n = 10). (H) Survival rates of WT B6 mice and FGF2–/– mice (n = 10). (I) Wet-to-dry ratios of lungs from WT B6 mice and FGF2–/– mice (n = 6) at 5 DPI. (J) HE staining of lung tissues from WT B6 mice and FGF2–/– mice at 5 DPI. The mean numbers of infiltrated cells per microscopic field ± SEM are shown (n = 50 fields analyzed for three mice). (K) Virus titers in the lungs of WT B6 mice and FGF2–/– mice (n = 6) at 5 DPI. Similar results were obtained in three independent experiments with 5–10 mice per group. Figure 2 View largeDownload slide Antibody depletion or knockout of FGF2 exacerbates H1N1-induced lung injury. WT B6 mice and FGF2–/– (KO) mice were i.n. inoculated with 103 or 105 TCID50 of the BI501 H1N1 virus. For the antibody pretreatment group, each B6 mouse was sequentially i.v. inoculated with 50 μg of the isotype control or anti-FGF2 antibodies 6 h before infection and at 1 DPI. For the treatment group, each B6 mouse was sequentially injected i.v. with 50 μg of the isotype control or anti-FGF2 antibodies at 3 and 5 DPI. All data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. (A) Changes in the weights of pre-treated B6 mice (n = 10). (B) Survival rates of pre-treated B6 mice (n = 10). (C) Wet-to-dry ratios of lungs from pre-treated B6 mice (n = 6) at 5 DPI. (D) Changes in the weights of treated B6 mice (n = 10). (E) Survival rates of treated B6 mice (n = 10). (F) Wet-to-dry ratios of lungs from treated B6 mice (n = 6) at 5 DPI. (G) Changes in the weights of WT B6 mice and FGF2–/– mice (n = 10). (H) Survival rates of WT B6 mice and FGF2–/– mice (n = 10). (I) Wet-to-dry ratios of lungs from WT B6 mice and FGF2–/– mice (n = 6) at 5 DPI. (J) HE staining of lung tissues from WT B6 mice and FGF2–/– mice at 5 DPI. The mean numbers of infiltrated cells per microscopic field ± SEM are shown (n = 50 fields analyzed for three mice). (K) Virus titers in the lungs of WT B6 mice and FGF2–/– mice (n = 6) at 5 DPI. Similar results were obtained in three independent experiments with 5–10 mice per group. Similarly, 4-week-old FGF2–/– (KO) mice that had been i.n. infected with BJ501 at a titer of 105 TCID50 showed more severe body weight loss than WT B6 mice (Figure 2G). Compared with BJ501-infected WT mice, BJ501-infected FGF2–/– mice showed significantly decreased survival rates (Figure 2H) and significantly increased wet-to-dry ratios of the lung tissue (Figure 2I). Moreover, the lung histopathology was significantly more severe in FGF2–/– mice than in WT mice after BJ501 infection, and the results showed thickened alveolar walls, increased lung interstitium, decreased alveolar space, and leukocyte infiltration (Figure 2J). However, the BJ501 infection did not result in evident histopathology in the brain, liver, kidney or intestine at 5 DPI (Supplementary Figure S2). Additionally, the IAV titers were much higher in the infected lungs of FGF2–/– mice than in WT mice (Figure 2K). In addition, similar alterations in histopathology were observed in mice after challenge with the PR8 strain of IAV (Supplementary Figure S3). Based on these data, FGF2 is required to protect against lethal H1N1 virus infection. Epithelial cells are the major cellular source of FGF2 Because macrophages, neutrophils, and alveolar epithelial cells (AECs) are the main cell populations detected in the inflamed lung tissue after BJ501 infection, we isolated neutrophils, macrophages and AECs from the lungs of BJ501 virus-infected mice at 5 DPI to further determine the source of FGF2 during IAV infection. FGF2 expression was significantly increased in AECs after BJ501 infection, but BJ501 infection did not affect FGF2 expression in neutrophils or macrophages; thus, lung epithelial cells were responsible for the marked IAV infection-induced elevation in the FGF2 level in the total lung tissue (Figure 3A). In addition, FGF2 levels were measured in the A549, BEAS-2B, and MLE-12 epithelial cell lines and in AECs isolated at 0, 24, 48, and 72 h after BJ501 infection (MOI = 1). FGF2 levels were remarkably elevated in all these cells in a time-dependent manner (Figure 3B), but no differences in FGF2 expression were observed in isolated neutrophils or macrophages with or without viral infection at different time points (data not shown). As shown in the confocal microscopy images, FGF2 was primarily expressed in AECs and bronchial epithelial cells of BJ501 virus-infected mice at 5 DPI, as evidenced by the co-localization of FGF2 and E-cadherin in the lung tissue (Figure 3C and D). Figure 3 View largeDownload slide Alveolar epithelial cells are the major source of FGF2. Four-week-old B6 mice were i.n. inoculated with 105 TCID50 of the BI501 H1N1 virus. Neutrophils, macrophages, and AECs were isolated from infected mouse lung tissue at 5 DPI. (A) FGF2 expression in the lung, neutrophils, macrophages, and AECs isolated from infected mice on 5 DPI was assessed using qRT-PCR. (B) FGF2 expression in isolated AECs and infected epithelial cell lines, including A549, BEAS-2B, and MLE-12 cells (MOI = 1). (C and D) Immunohistochemistry for E-cadherin (green), FGF2 (red), and DAPI (blue) in AECs and bronchial epithelial cells from mice infected with 105 TCID50 of the BI501 H1N1 virus at 5 DPI. The panels in Merge 1 are a merge of the FGF2 and E-cadherin images. The panels in Merge 2 are a merge of the FGF2, E-cadherin, and DAPI images. *P < 0.05, **P < 0.01, ***P < 0.001. Figure 3 View largeDownload slide Alveolar epithelial cells are the major source of FGF2. Four-week-old B6 mice were i.n. inoculated with 105 TCID50 of the BI501 H1N1 virus. Neutrophils, macrophages, and AECs were isolated from infected mouse lung tissue at 5 DPI. (A) FGF2 expression in the lung, neutrophils, macrophages, and AECs isolated from infected mice on 5 DPI was assessed using qRT-PCR. (B) FGF2 expression in isolated AECs and infected epithelial cell lines, including A549, BEAS-2B, and MLE-12 cells (MOI = 1). (C and D) Immunohistochemistry for E-cadherin (green), FGF2 (red), and DAPI (blue) in AECs and bronchial epithelial cells from mice infected with 105 TCID50 of the BI501 H1N1 virus at 5 DPI. The panels in Merge 1 are a merge of the FGF2 and E-cadherin images. The panels in Merge 2 are a merge of the FGF2, E-cadherin, and DAPI images. *P < 0.05, **P < 0.01, ***P < 0.001. FGF2 affects the recruitment and activation of neutrophils during IAV infection-induced lung injury Neutrophils, the prototypic cells of the innate immune system, are recruited to infected sites to protect the body from invading pathogens (Koller et al., 2009). In vivo, neutrophils and macrophages constitute the majority of infiltrating cells in inflamed tissues (Perrone et al., 2008) and might exert beneficial anti-pathogenic effects (Perrone et al., 2008; Suzuki et al., 2008; Iwasaki and Pillai, 2014). Neutrophils are rapidly recruited to sites of infection during the innate immune response to influenza A virus (Tumpey et al., 2005; Baskin et al., 2007; Perrone et al., 2008; Wang et al., 2008). Consistent with the findings in the literature, BJ501 infection caused severe lung histopathology and inflammatory cell infiltration in WT mice; notably, FGF2–/– mice showed more severe histopathology but less inflammatory cell infiltration in the lung tissue than the WT mice after infection (Figure 4A). A significant decrease in the number of neutrophils might account for the sharp reduction in the leukocyte count in BJ501-infected lung tissues because the total lymphocyte, macrophage, and NK cell counts were not obviously different between WT and KO mice after BJ501 infection (Figure 4B). Figure 4 View largeDownload slide FGF2 affects neutrophil function in influenza-induced lung injury. (A) HE staining and leukocyte cell counts (n = 50 fields) in lung tissues from B6 mice 3 days after BJ501 infection. FGF2–/– (KO) mice showed reduced leukocyte infiltration and significantly reduced leukocyte counts at 3 DPI. (B) Flow cytometry analysis of leukocyte subsets in mouse BALF obtained from 105 TCID50 of BJ501-infected mice at 3 DPI (n = 5). Similar results were obtained in three independent experiments with five mice per group. (C) Neutrophil chemotaxis assay in vitro. Untreated human neutrophils were allowed to migrate through a migration chamber toward PBS or FGF2 (10−9 M, 1.72 ng/ml). Cells from eight random fields were counted, and migration is expressed relative to the PBS control. (D) Concentrations of neutrophil-related chemokines in vitro. Neutrophils were treated with FGF2 (10−9 M, 1.72 ng/ml) for 6, 12, or 24 h and the levels in cell culture supernatants were determined using a Human ProcartaPlex Panel. *P < 0.05 and **P < 0.01. Figure 4 View largeDownload slide FGF2 affects neutrophil function in influenza-induced lung injury. (A) HE staining and leukocyte cell counts (n = 50 fields) in lung tissues from B6 mice 3 days after BJ501 infection. FGF2–/– (KO) mice showed reduced leukocyte infiltration and significantly reduced leukocyte counts at 3 DPI. (B) Flow cytometry analysis of leukocyte subsets in mouse BALF obtained from 105 TCID50 of BJ501-infected mice at 3 DPI (n = 5). Similar results were obtained in three independent experiments with five mice per group. (C) Neutrophil chemotaxis assay in vitro. Untreated human neutrophils were allowed to migrate through a migration chamber toward PBS or FGF2 (10−9 M, 1.72 ng/ml). Cells from eight random fields were counted, and migration is expressed relative to the PBS control. (D) Concentrations of neutrophil-related chemokines in vitro. Neutrophils were treated with FGF2 (10−9 M, 1.72 ng/ml) for 6, 12, or 24 h and the levels in cell culture supernatants were determined using a Human ProcartaPlex Panel. *P < 0.05 and **P < 0.01. Additionally, we profiled the levels of 23 mouse cytokines and chemokines in mouse BALF samples at 3 DPI. The levels of various cytokines, including IL-1α, IL-1β, IL-6, IL-17, IL-3, IL-5, IL-13, IL-10, IFN-γ, TNF-α, and G-CSF, as well as certain chemokines, including MIP-1α, MIP-1β, RANTES and eotaxin, were significantly increased in WT mice after IAV infection. More importantly, the levels of these cytokines and chemokines were reduced in FGF2–/– mice compared to WT mice (Supplementary Figure S4). Based on these results, neutrophils might contribute to the marked alterations in cytokine and chemokine levels in response to BJ501 infection. FGF2 has been shown to potentiate leukocyte recruitment to sites of inflammation and acts as a chemotactic agent for the recruitment of neutrophils (Zittermann and Issekutz, 2006a; Haddad et al., 2011). In this study, we confirmed that FGF2 promoted neutrophil recruitment in vitro using a neutrophil chemotaxis assay (Figure 4C). Furthermore, FGF2 promoted neutrophil-related chemokine expression in vitro, including the C-X-C chemokines CXCL1, IL-8 (CXCL8), IP10 (CXCL10), and CXCL12, as well as the CC chemokines MCP-1, MIP-1α, MIP-1β, RANTES, and eotaxin (Figure 4D). Thus, an FGF2 deficiency may impair neutrophil function by suppressing neutrophil recruitment and cytokine production. FGF2 enhances NFκB phosphorylation to induce the activation and chemotaxis of neutrophils According to a previous study, FGF receptors (FGFRs) are expressed by neutrophils (Haddad et al., 2011). Therefore, we examined whether H1N1 virus infection alters FGFR expression. Surprisingly, FGFR2 levels were significantly increased concomitantly with reduced FGFR1 and FGFR4 expression in neutrophils in vitro at 6 h after BJ501 infection. However, FGFR3 was expressed at very low levels before or after IAV infection. The viral load (M1 level) was significantly increased after BJ501 infection, indicating the establishment of a model of BJ501 virus-infected neutrophils (Figure 5A). The mammalian FGF family comprises 18 ligands that signal through four conserved tyrosine kinase receptors to activate four key downstream pathways: RAS–RAF–MAPK, PI3K–AKT, signal transducer and activator of transcription (STAT), and phospholipase Cγ (PLCγ) (Turner and Grose, 2010). In this experiment, the application of FGF2 to neutrophils for 6 h did not lead to a significant change in the levels of MAPK pathway members, including P38, JNK, and ERK compared with untreated cells; thus, FGF2-induced activation and neutrophil chemotaxis did not involve MAPK signaling. Moreover, STAT3 levels remained unchanged, and PLCγ expression was not detected (data not shown) after FGF2 protein administration. MAPK and STAT3 signaling were still unchanged following BJ501 infection (Supplementary Figure S5A). In contrast, prominent PI3K and AKT phosphorylation were observed at 6 h after the application of FGF2 with or without BJ501 infection (Figure 5B). NFκB has been confirmed to act downstream of the PI3K/AKT pathway in various cancers (Ghosh-Choudhury et al., 2010; Han et al., 2010; Lin et al., 2010), and AKT activates the NFκB pathway by phosphorylating and activating NFκB pathway intermediates (Ozes et al., 1999; Romashkova and Makarov, 1999). However, the relationships between these two pathways downstream of FGF2 activation have not been fully explored. In this study, inhibitor of NFκB kinase subunit beta (IKKβ), IκB, and NFκB (RELA/p65) were phosphorylated in FGF2-stimulated neutrophils. Meanwhile, the phosphorylation of these signaling molecules was also enhanced following BJ501 infection (Figure 5B). Figure 5 View largeDownload slide FGF2 enhances NFκB phosphorylation and subsequently mediates neutrophil chemotaxis. (A) qRT-PCR analysis of FGFR1–4 and M1 expression in neutrophils infected with BJ501 (MOI = 1) for 0 or 6 h. (B) Western blot analysis of the indicated phosphorylated (p-) and total proteins in neutrophils treated with FGF2 (10−9 M, 1.72 ng/ml) for 6 h. (C) Western blot analysis of signaling pathways in WT and FGF2–/– mice infected with 105 TCID50 of BJ501 or mock infected at 5 DPI. β-Actin was used as an internal control. Data are presented as mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Figure 5 View largeDownload slide FGF2 enhances NFκB phosphorylation and subsequently mediates neutrophil chemotaxis. (A) qRT-PCR analysis of FGFR1–4 and M1 expression in neutrophils infected with BJ501 (MOI = 1) for 0 or 6 h. (B) Western blot analysis of the indicated phosphorylated (p-) and total proteins in neutrophils treated with FGF2 (10−9 M, 1.72 ng/ml) for 6 h. (C) Western blot analysis of signaling pathways in WT and FGF2–/– mice infected with 105 TCID50 of BJ501 or mock infected at 5 DPI. β-Actin was used as an internal control. Data are presented as mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Four-week-old FGF2–/– or WT mice were i.n. infected with BJ501 at a titer of 105 TCID50 or mock infected to further confirm the functional impact of FGFin vivo. The results of western blot analyses revealed a decrease in the levels of phosphorylated PI3K, AKT, and NFκB in FGF2–/– mice infected with BJ501, but no changes were observed in the mock infected mice (Figure 5C). However, the phosphorylation of members of the MAPK and STAT3 pathways were unchanged in the presence or absence of BJ501 infection (Supplementary Figure S5B). Based on these results, activation of the FGFR2–PI3K–AKT–NFκB pathway probably contributes to neutrophil recruitment, chemotaxis, or activation in IAV-infected lung tissues. The recombinant FGF2 protein protects mice from H1N1 infection by recruiting neutrophils Currently, the recombinant FGF2 protein (rFGF2) has been used in various clinical settings, such as wound healing and cornea repair (Fu et al., 1998, 2000; Huang et al., 2011). We treated BJ501-infected mice with rFGF2 and/or neutrophil neutralizing antibodies to further investigate whether FGF2 protected against IAV infection and determine whether the protective effects were associated with neutrophil recruitment. First, 4-week-old WT B6 mice were i.n. infected with 105 TCID50 of the BJ501 virus. Mice were pre-treated or i.v. treated with anti-Ly6G (m1A8) antibodies or isotype control antibodies (m2A3) and FGF2. The survival rates of the BJ501-infected mice were significantly increased, and weight loss was improved by the rFGF2 treatment (Figure 6A and B). Moreover, the lung wet-to-dry weight ratio exhibited a greater improvement in the IAV-infected mice treated with rFGF2 (Figure 6C). Similarly, improved lung histopathology and lung injury scores, as defined by infiltrated leukocyte counts, were observed in the IAV-infected mice treated with rFGF2 (Figure 6D). Furthermore, rFGF2-treated mice showed less viral replication in the infected lung than control mice (Figure 6E). Similar results were obtained in mice challenged with IAV strain PR8 (Supplementary Figure S6). More importantly, more neutrophils were recruited to the lungs of the FGF2 treatment group but not the anti-Ly6G group or anti-Ly6G plus FGF2 group (Figure 6F). In addition, mice that had been pre-treated or treated with m1A8 antibodies showed significantly lower survival rates, more severe body weight loss and lung edema than the isotype control antibody-treated mice. In contrast, pretreatment with FGF2 and m1A8 antibodies did not protect the mice. The histopathological alterations in the lung and viral replication further supported these findings (Figure 6A–D). Thus, rFGF2 protects against H1N1 virus infection in vivo, and the recruited neutrophils were indispensable for the protective effects of FGF2. Figure 6 View largeDownload slide FGF2 alleviates influenza-induced lung injury through a neutrophil-dependent mechanism. (A) Survival rates of B6 mice pre-treated with anti-Ly6G and FGF2 or the isotype control (n = 5). Control vs. control + FGF2: **P < 0.01. (B) Changes in the weights of B6 mice pre-treated with anti-Ly6G and FGF2 or the isotype control (n = 5). (C) Wet-to-dry ratios of lungs from B6 mice pre-treated with anti-Ly6G and FGF2 or the isotype control (n = 5) at 5 DPI. (D) HE staining and quantification of lung tissues from B6 mice at 5 DPI. (E) Virus titers of lungs from treated B6 mice (n = 6) at 5 DPI. (F) Flow cytometry analysis of neutrophil counts in BALF obtained from treated B6 mice at 3 DPI. Similar results were obtained in three independent experiments with 5–6 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001. Figure 6 View largeDownload slide FGF2 alleviates influenza-induced lung injury through a neutrophil-dependent mechanism. (A) Survival rates of B6 mice pre-treated with anti-Ly6G and FGF2 or the isotype control (n = 5). Control vs. control + FGF2: **P < 0.01. (B) Changes in the weights of B6 mice pre-treated with anti-Ly6G and FGF2 or the isotype control (n = 5). (C) Wet-to-dry ratios of lungs from B6 mice pre-treated with anti-Ly6G and FGF2 or the isotype control (n = 5) at 5 DPI. (D) HE staining and quantification of lung tissues from B6 mice at 5 DPI. (E) Virus titers of lungs from treated B6 mice (n = 6) at 5 DPI. (F) Flow cytometry analysis of neutrophil counts in BALF obtained from treated B6 mice at 3 DPI. Similar results were obtained in three independent experiments with 5–6 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001. Neutrophil transfer protects FGF2–/– mice against influenza BJ501 virus infection Neutrophils were purified from the bone marrow of WT mice and adoptively transferred to FGF2–/– mice on Day 1 after infection with 103 TCID50 of BJ501 to investigate whether neutrophils protect FGF2–/– mice from influenza infection. FGF2–/– mice that received an adoptive transfer of WT neutrophils had an increased survival rate and a better outcome of weight change after IAV infection (Figure 7A). The lung edema and virus load of infected neutrophil-transferred mice were significantly decreased compared with FGF2–/– mice (Figure 7B). In addition, the lung histopathology of neutrophil-transferred mice was greatly improved, and the infiltrated leukocyte counts were significantly decreased (Figure 7C and D). The results of the flow cytometry analysis showed the restoration of infiltrating neutrophils in FGF2–/– mice that received WT neutrophils (Figure 7F). These results confirm that mice that received the neutrophil transfer achieved better outcomes upon influenza infection. Figure 7 View largeDownload slide Neutrophil transfer protects FGF2–/– mice from influenza infection. (A) Survival rates of FGF2–/– and neutrophil-transferred FGF2–/– mice (n = 5). (B) Changes in the weights of FGF2–/– and neutrophil-transferred FGF2–/– mice (n = 5). (C) Wet-to-dry ratios of lungs from FGF2–/– and neutrophil-transferred FGF2–/– mice (n = 5) at 5 DPI. (D) HE staining and quantification of lung tissues from B6 mice at 5 DPI. (E) Virus titers in lungs from treated B6 mice (n = 6) at 5 DPI. (F) Flow cytometry analysis of neutrophil counts in BALF obtained from treated B6 mice at 3 DPI. Similar results were obtained in three independent experiments with five mice per group. *P < 0.05, **P < 0.01, ***P < 0.001. Figure 7 View largeDownload slide Neutrophil transfer protects FGF2–/– mice from influenza infection. (A) Survival rates of FGF2–/– and neutrophil-transferred FGF2–/– mice (n = 5). (B) Changes in the weights of FGF2–/– and neutrophil-transferred FGF2–/– mice (n = 5). (C) Wet-to-dry ratios of lungs from FGF2–/– and neutrophil-transferred FGF2–/– mice (n = 5) at 5 DPI. (D) HE staining and quantification of lung tissues from B6 mice at 5 DPI. (E) Virus titers in lungs from treated B6 mice (n = 6) at 5 DPI. (F) Flow cytometry analysis of neutrophil counts in BALF obtained from treated B6 mice at 3 DPI. Similar results were obtained in three independent experiments with five mice per group. *P < 0.05, **P < 0.01, ***P < 0.001. Discussion FGF2 is known to act as a pleiotropic factor in multiple biological processes, including angiogenesis, embryonic development, and wound healing (Ortega et al., 1998; Nugent and Iozzo, 2000; Virag et al., 2007; Han and Gotlieb, 2012; Song et al., 2015). FGF2 accelerates the healing of skin wounds in animal models as well as the healing of eye, retina, and corneal wounds (Bikfalvi et al., 1997). FGF2 also has multiple functions in the central nervous system (Tureyen et al., 2005; Frinchi et al., 2008; Li et al., 2008; Ma et al., 2008; Ma et al., 2009; Mimura et al., 2015). Furthermore, FGF2 promotes the epithelial–mesenchymal transition, invasiveness, and tumor angiogenesis (Marek et al., 2009; Narong and Leelawat, 2011; Wesche et al., 2011; Wang et al., 2015). FGF2 is expressed in normal tissue and is upregulated by chronic inflammatory reactions (Byrd et al., 1996; Kanazawa et al., 2001; Song et al., 2015). The roles of FGF2 in mediating the immune response to IAV infection have not been fully elucidated. Our study is the first to report that FGF2 levels were elevated in serum from IAV-infected patients and BALF of mice infected with H1N1 virus strain BJ501. Mice with an FGF2 deficiency due to either gene knockout or protein inhibition using neutralizing antibodies in vivo were more susceptible to IAV infection. According to a previous report, FGF2 is a potent mitogen for many cell types, including airway smooth muscle cells, fibroblasts, and endothelial cells (Redington et al., 2001). Additionally, FGF2 is secreted by many types of immune cells, such as Treg cells, neutrophils, monocytes, macrophages, and T lymphocytes (Barrios et al., 1997; Byrd et al., 1999; Ohsaka et al., 2001; Haddad et al., 2011; Song et al., 2015). During H1N1 infection, neutrophils represent the majority of infiltrating immune cells in inflamed tissues. In this study, epithelial cells were the major cellular source of FGF2 during H1N1-induced ALI. Humans infected with influenza A virus display a ‘cytokine storm’, which is caused by insufficient control of excessive neutrophil recruitment to the lungs (Wang and Ma, 2008; Tisoncik et al., 2012; Brandes et al., 2013; Liu et al., 2016; Yang and Tang, 2016). However, neutrophils provide the first line of defense against invading microorganisms and contribute to the fine regulation of inflammatory and immune responses (Futosi et al., 2013; Tecchio et al., 2014). Moreover, neutrophils have a potent anti-microbial armamentarium that includes oxidant-generating systems, powerful proteinases, and cationic peptides contained in granules (Suzuki et al., 2008). In addition, neutrophils function as regulators of inflammatory and immune responses by producing and releasing a large variety of cytokines and chemokines (Tecchio et al., 2014). This variety of cytokines produced by neutrophils enables them to significantly influence not only multiple aspects of the inflammatory and immune responses but also antiviral defense, hematopoiesis, angiogenesis, and fibrogenesis (Cassatella, 1999). FGF2 also enhances the recruitment of monocytes, T cells, and polymorph nuclear leukocytes (PMNs) to inflamed dermal sites (Zittermann and Issekutz, 2006b), and act as a chemotactic agent for the recruitment of neutrophils (Byrd et al., 1996; Wempe et al., 1997; Zittermann and Issekutz, 2006a; Haddad et al., 2011). Consistent with the results from these publications, an FGF2 deficiency reduced leukocyte recruitment, particularly neutrophil recruitment, accompanied by decreases in the levels of cytokines and chemokines in mouse BALF during the early phase of IAV infection in this study. Moreover, FGF2 enhanced neutrophil recruitment in vitro and promoted the expression of neutrophil-related chemokines, such as C-X-C and CC chemokines. Therefore, we speculate that the FGF2 deficiency impaired an effective immune response to IAV infection and FGF2 recruited neutrophils to the infected lung tissue during the early phase of H1N1 infection. Of course, additional mechanistic studies of the protective roles of neutrophils and the ‘cytokine storm’ are warranted. FGFs signal through FGFRs to perform a multitude of physiological functions. FGFRs are expressed on many different cell types and regulate key cell behaviors, such as proliferation, differentiation, and survival (Turner and Grose, 2010). Here, FGFR2 expression was significantly increased in neutrophils after BJ501 infection, whereas the levels of FGFR1 and FGFR4 were decreased, and FGFR3 was weakly expressed before and after infection. Furthermore, FGF2 contributed to leukocyte recruitment or enhanced neutrophil chemotaxis to infected lung tissue mainly through the FGFR2–PI3K–AKT–NFκB signaling pathway (Figure 8). FGF2 interacts with FGFR2, activating the PI3K–AKT–NFκB signaling pathway, which induces cytokine and chemokine production and promotes neutrophil chemotaxis through a feedback mechanism, thus protecting against IAV infection in the early stage. Based on these findings, we further examined the relationship between FGF2 and neutrophils in vivo. The administration of the recombinant murine FGF2 protein protected mice from a lethal H1N1 infection, whereas concomitant treatment with both rFGF2 and anti-Ly6G (m1A8) failed to protect mice against IAV infection, suggesting that the protective role of FGF2 was mainly mediated by neutrophils. Consistent with these findings, infected FGF2–/– mice exhibited a reduced disease severity after receiving neutrophils from WT mice. Thus, the neutrophil-dependent therapeutic roles of FGF2 are to alleviate IAV-induced ALI in the early stage. To the best of our knowledge, we are the first group to systematically show that the FGFR2–PI3K–AKT–NFκB signaling pathway is involved in IAV infection-mediated ALI in mice. Based on this evidence, FGF2 plays a critical role in mediating ALI induced by influenza H1N1 virus infection. Figure 8 View largeDownload slide A schematic explaining the signaling mechanisms by which FGF2 regulates neutrophil chemotaxis and IAV-induced ALI. Figure 8 View largeDownload slide A schematic explaining the signaling mechanisms by which FGF2 regulates neutrophil chemotaxis and IAV-induced ALI. In conclusion, FGF2 performs a critical protective function during the process of IAV infection. The combination of our clinical findings and results from the mouse model has revealed a critical role for FGF2 in the pathogenesis of influenza A (H1N1) virus-induced ALI and has suggested that FGF2 represents a promising potential therapeutic target in IAV-induced lung disease. Materials and methods Viruses and cells The influenza viruses used in this study were influenza A (H1N1) virus strain A/Beijing/501/2009 (BJ501) and influenza A (H1N1) virus strain A/PR/8/1934 (PR8) obtained from the Academy of Military Medical Sciences. The genomic sequence of the BJ501 strain is available in the GenBank database (accession number GQ223415). The strains were propagated in 9- to 11-day-old specific-pathogen-free (SPF) embryonated eggs via the allantoic route. Virus titers were determined based on an assessment of the TCID50 using Madin-Darby canine kidney (MDCK) cells, according to the Reed-Muench method. MDCK, A549 and BEAS-2B cells were purchased from ATCC; MLE-12 cells were kindly provided by Dr Zhuowei Hu (Peking Union Medical College, PUMC). MDCK cells were cultured in DMEM (Gibco); A549, BEAS-2B, and MLE-12 cells were cultured in DMEM/F-12(1:1) basic culture medium (Gibco); and neutrophils were cultured in RPMI-1640 medium (Gibco) supplemented with 10% FBS and 100 U/ml penicillin-streptomycin at 37°C in a humidified atmosphere containing 5% CO2. Mice Four-week-old WT C57BL/6 (abbreviated B6) female mice (Experimental Animal Center of the Academy of Military Medical Sciences, Beijing, China) and 4-week-old FGF2 knockout mice (B6 background, 010698, Jackson Laboratory, USA) were housed in the animal facility at the Beijing Institute of Microbiology and Epidemiology in accordance with institutional guidelines. All experimental protocols were approved by the Institutional Animal Care and Use Committees of the Beijing Institute of Microbiology and Epidemiology (ID: SYXK2015-008) and all experiments were performed in accordance with the approved guidelines. Mouse infections Four-week-old WT B6 mice were anesthetized with 50 μl of 1% (w/v) pentobarbital sodium and then i.n. inoculated with the H1N1 virus or mock infected with PBS as a control. Survival rates, body weight changes, histology, acute pulmonary edema (wet-to-dry ratio), and cytokine levels were evaluated as previously described (Li et al., 2012; Wang et al., 2013; Gu et al., 2016). For the anti-FGF2 antibody treatment, anti-FGF2 antibodies or isotype control antibodies (100 μg/mouse, Abcam, Cat. No. ab33103) (Kujawski et al., 2008) were sequentially administered i.v. at 6 h before and 1 and 3 days after injection (pretreatment group) or at 1, 3, and 5 days after injection (treatment group) with the vehicle control or virus (103 TCID50 of A/Beijing/501/2009). For FGF2–/– mice, 105 TCID50 of A/Beijing/501/2009 was used. For the rescue experiments, each mouse was sequentially i.v. inoculated with 25 μg of the recombinant murine FGF2 protein (Peprotech, Cat. No. 450-33) 12 h before and 1 and 3 days after injection with the vehicle control or virus (105 TCID50 of A/Beijing/501/2009 or 104 TCID50 of A/PR/8/1934). For the neutrophil depletion experiment, a neutrophil-specific antibody, anti-Ly-6G (clone 1A8), and an isotype control antibody (clone 2A3) were purchased from Biox Cell. WT mice were sequentially i.v. administered 50 μg of the anti-mouse Ly6G antibody (clone 1A8) or isotype control antibody (clone 2A3) 6 h before and 1 and 3 days after injection, as previously described (Kawanishi et al., 2016). Viral titration Virus titers were determined in supernatants of mouse lung homogenates from FGF2–/– mice or WT mice on Day 5 after H1N1 infection, as previously described (Li et al., 2012; Wang et al., 2013; Gu et al., 2016). Survival rate and body weight changes Four-week-old WT B6 mice were anesthetized with 50 μl of 1% (w/v) pentobarbital sodium and i.n. inoculated with the virus or vehicle control. The survival rates and body weights of each group of 10 mice were monitored daily for 14 days. Acute pulmonary edema (wet-to-dry ratio) Pulmonary edema was assessed by measuring and recording the lung wet/dry weight ratio as previously described (Li et al., 2012). Adoptive transfer of neutrophils Bone marrow neutrophils were isolated using Percoll (GE Healthcare), as previously described (Mei et al., 2012). Briefly, bone marrow cells were harvested from the femurs of 4-week-old WT B6 mice and suspended in PBS before being layered on a 3-step Percoll (GE Healthcare) gradient (72%, 64%, and 52%), which was centrifuged at 500× g for 30 min. The cells at the interface between 64% and 72% gradients were collected and washed twice with PBS. More than 95% of the neutrophils were viable, as determined using the trypan blue exclusion method. For adoptive transfer, 5 × 106 neutrophils isolated from WT mice were intravenously injected into FGF2–/– mice 24 h before they were i.n. inoculated with the H1N1 virus (103 TCID50 of A/Beijing/501/2009) as described above. Flow cytometry Cells in BALF were stained with anti-CD45-FITC, anti-Ly6G-PE, anti-F4/80-PE, anti-NK1.1-PE and anti-CD3-FITC antibodies (BD Pharmingen) and analyzed using a FACSCalibur flow cytometer (BD Biosciences) to examine leukocyte marker expression. The data were analyzed using FlowJo software (Tree Star). Isotype controls were used for all samples. Statistical analyses Measurements collected at a single time point were analyzed using an ANOVA, and if a significant difference among the groups was observed, the results were further analyzed using a two-tailed t-test. All analyses were performed using GraphPad Prism 5.0 software (GraphPad Software). P < 0.05 was considered statistically significant. All experiments were performed in triplicate. Supplementary material Supplementary material is available at Journal of Molecular Cell Biology online. Acknowledgements We thank Professor Zhuowei Hu (Chinese Academy of Medical Sciences, Peking Union Medical College) for providing the MLE-12 cells. Funding This work was supported in part by funding from the National High Technology Research and Development Program of China (SS2015AA020924), the National Natural Science Foundation of China (81771700), the Ministry of Science and Technology of China (2013ZX10004003 and SS2012AA020905), and the National Major Research and Development Program (2016YFA0502203 and 2017YFC1200800). P.Y. was supported by the Beijing Nova Program (Z141107001814054). Conflict of interest: none declared. Author contributions: K.W., C.L., C.W., Y.D., Z.Z., X.Y., L.X., L.Z., S.Z., and M.X. contributed to the experiments. W.W., H.G., and B.N. analyzed the data. T.L., C.B., S.Z., and L.H. collected the samples. 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Endothelial growth factors VEGF and bFGF differentially enhance monocyte and neutrophil recruitment to inflammation. J. Leukoc. Biol.  80, 247– 257. Google Scholar CrossRef Search ADS PubMed  © The Author (2017). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Molecular Cell Biology Oxford University Press

Basic fibroblast growth factor protects against influenza A virus-induced acute lung injury by recruiting neutrophils

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Oxford University Press
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© The Author (2017). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.
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1674-2788
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1759-4685
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10.1093/jmcb/mjx047
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Abstract

Abstract Influenza virus (IAV) infection is a major cause of severe respiratory illness that affects almost every country in the world. IAV infections result in respiratory illness and even acute lung injury and death, but the underlying mechanisms responsible for IAV pathogenesis have not yet been fully elucidated. In this study, the basic fibroblast growth factor 2 (FGF2) level was markedly increased in H1N1 virus-infected humans and mice. FGF2, which is predominately derived from epithelial cells, recruits and activates neutrophils via the FGFR2–PI3K–AKT–NFκB signaling pathway. FGF2 depletion or knockout exacerbated influenza-associated disease by impairing neutrophil recruitment and activation. More importantly, administration of the recombinant FGF2 protein significantly alleviated the severity of IAV-induced lung injury and promoted the survival of IAV-infected mice. Based on the results from experiments in which neutrophils were depleted and adoptively transferred, FGF2 protected mice against IAV infection by recruiting neutrophils. Thus, FGF2 plays a critical role in preventing IAV-induced lung injury, and FGF2 is a promising potential therapeutic target during IAV infection. influenza H1N1 virus, recombinant FGF2 protein, neutrophil recruitment, FGFR2–PI3K–AKT–NFκB signaling, therapeutic target Introduction Influenza spreads throughout the world during annual outbreaks, resulting in ~3–5 million cases of severe illness and ~250000–500000 deaths annually; infants and the elderly are particularly vulnerable to influenza. The mechanisms by which influenza virus (IAV) infection cause symptoms in humans have been studied intensively. Some severely infected patients develop acute lung injury (ALI) and even acute respiratory distress syndrome (ARDS), which is the predominant cause of reported influenza-related deaths (Dominguez-Cherit et al., 2009; Louie et al., 2009; Berdal et al., 2011; Ohta et al., 2011). IAV infection may cause inflammation of the airways, epithelial necrosis, edema, hemorrhaging, and respiratory failure (Xu et al., 2006; Rincon, 2012; Ding et al., 2013). Both virus-specific virulence factors and host immunity are associated with exacerbated IAV pathogenesis (Crouser et al., 2009). The currently recognized therapeutic agents against IAV infection include viral m2 channel inhibitors (amantadine and rimantadine), neuraminidase inhibitors (zanamivir, oseltamivir, peramivir, and laninamivir octanoate), and polymerase inhibitors (ribavirin and favipiravir), but IAVs are becoming highly resistant to these drugs, and further evidence is required from clinical trials (Dushianthan et al., 2011; De Clercq and Li, 2016). Basic fibroblast growth factor (bFGF or FGF2), a potent mitogen for many cell types, including airway smooth muscle cells, fibroblasts, and endothelial cells (Redington et al., 2001), is associated with multiple biological processes, including tumor angiogenesis, embryonic development, proliferation, migration, and injury repair (Meyer et al., 1995; Ortega et al., 1998; Fuhrmann-Benzakein et al., 2000; Nugent and Iozzo, 2000; Virag et al., 2007). FGF2 is dysregulated in many inflammatory disorders, such as inflammatory bowel disease (IBD), Crohn’s disease, ulcerative colitis, and rheumatoid arthritis (Byrd et al., 1996; Kanazawa et al., 2001; Song et al., 2015). In immune responses, FGF2 functions to maintain the innate immune homeostasis of antiviral immunity by stabilizing retinoic acid-inducible gene-I (RIG-I) and preventing proteasome-mediated RIG-I degradation (Liu et al., 2015). In a study of the link between FGF2 and ALI by Powers et al. (1994), FGF2 was shown to play a role in the alveolar response to hyperoxic injury via the altered mRNA levels and protein distribution. According to Liebler et al. (1997), FGF2 may participate in directing cell proliferation following pulmonary fibrosis. As shown in the study by Zhao et al. (2015), mesenchymal stem cells (MSCs) and FGF2 synergistically reduced the level of inflammatory cytokines in the treatment of LPS-induced lung injury. Moreover, Guzy et al. (2015) showed that FGF2 is required for epithelial repair and maintaining epithelial integrity after bleomycin-induced lung injury in mice. However, researchers have not determined whether and how FGF2 plays a role in IAV-induced ALI. In this study, we explore the role of FGF2 in host defense against IAV infection using our previous established mouse model (Li et al., 2012). Based on our results, FGF2 plays a pivotal role in IAV-induced lung injury, and the administration of the recombinant FGF2 protein markedly reduces mortality and the severity of lung injury in a preclinical model of IAV infection. The mechanisms underlying these effects of FGF2 include neutrophil activation and recruitment via the PI3K–Akt–NFκB signaling pathway. Results FGF2 is significantly upregulated in patient serum and mouse bronchoalveolar lavage fluid (BALF) following IAV infection Hypercytokinemia has been reported to be an early host response signature in influenza A (H1N1) virus-induced ALI (Tisoncik et al., 2012; Brandes et al., 2013; Liu et al., 2016; Yang and Tang, 2016). However, the roles of individual cytokines in ALI remain largely unclear. We measured FGF2 levels in 156 sera samples from patients confirmed to be infected with the H1N1 virus to investigate the effects of FGF2 on H1N1-induced ALI, and the characteristics reflecting the conditions and outcomes of these patients are described in the Supplementary information (Supplementary Table S1). The FGF2 level was significantly elevated in all IAV-infected patients compared with healthy subjects. Furthermore, levels of the FGF2 protein in H1N1 virus-infected patients were gradually elevated as the fever duration increased and peaked on Day 3 of fever. Correspondingly, levels of the FGF2 protein in all hospitalized patients were markedly higher than the levels in healthy subjects (Figure 1A). Meanwhile, 4-week-old wild-type (WT) B6 mice were intranasally (i.n.) infected with the BJ501 strain at a titer of 105 50% tissue culture infectious dose (TCID50), and we observed a significant increase in pulmonary elastance that represents changes in pressure achieved per unit changes in volume (Supplementary Figure S1A) and decreased arterial partial pressure of oxygen (PaO2) at 3, 5, or 7 days post-infection (DPI) (Supplementary Figure S1B). In addition, the level of the FGF2 protein was increased in the BALF of C57BL/6 mice following challenge with the A/Beijing/501/2009 (BJ501) strain. FGF2 levels began to increase significantly on the fifth day, peaked on the eighth day, and then sharply decreased beginning on the10th day of virus challenge (Figure 1B). Furthermore, FGF2 was expressed at high levels in the lung tissue of IAV BJ501 strain-infected mice at 5 DPI, based on immunohistochemical (IHC) staining (Figure 1C). This observation suggests a potential key role for FGF2 in H1N1 virus-induced ALI. Figure 1 View largeDownload slide FGF2 levels were significantly increased in patients’ sera and mouse BALF. Four-week-old B6 mice were anesthetized with 50 μl of 1% (w/v) pentobarbital sodium and i.n. inoculated with 105 TCID50 of BJ501 H1N1 viruses. All data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. (A) The concentration of FGF2 in the sera from patients infected with influenza A (H1N1) virus strain A/Beijing/501/2009 (BJ501) was determined using a Bio-Plex Human Cytokine Array. (B) The concentration of FGF2 in the BALF of B6 mice infected with 105 TCID50 of the BJ501 strain (n = 5) was measured at 0–14 DPI. (C) FGF2 staining and quantification in the lung tissues from B6 mice infected with 105 TCID50 of the BJ501 strain at 5 DPI. Figure 1 View largeDownload slide FGF2 levels were significantly increased in patients’ sera and mouse BALF. Four-week-old B6 mice were anesthetized with 50 μl of 1% (w/v) pentobarbital sodium and i.n. inoculated with 105 TCID50 of BJ501 H1N1 viruses. All data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. (A) The concentration of FGF2 in the sera from patients infected with influenza A (H1N1) virus strain A/Beijing/501/2009 (BJ501) was determined using a Bio-Plex Human Cytokine Array. (B) The concentration of FGF2 in the BALF of B6 mice infected with 105 TCID50 of the BJ501 strain (n = 5) was measured at 0–14 DPI. (C) FGF2 staining and quantification in the lung tissues from B6 mice infected with 105 TCID50 of the BJ501 strain at 5 DPI. FGF2 deficiency exacerbates IAV-induced lung injury Four-week-old WT B6 mice were i.n. infected with the BJ501 strain at a titer of 103 TCID50 to confirm the exact role of FGF2 in H1N1 virus-induced ALI. Mice were pre-treated or intravenously (i.v.) treated with anti-FGF2 antibodies or isotype control antibodies. After infection with the BJ501 strain, the groups that had been pre-treated or treated with anti-FGF2 antibodies had significantly lower survival rates and showed more severe body weight loss and lung edema than the group treated with the isotype control. Moreover, the pathology of the mice in the anti-FGF2 antibody-pre-treated group was much worse than the mice in the anti-FGF2 antibody-treated group, as determined by weight loss, survival rate, and lung edema (Figure 2A–F). Figure 2 View largeDownload slide Antibody depletion or knockout of FGF2 exacerbates H1N1-induced lung injury. WT B6 mice and FGF2–/– (KO) mice were i.n. inoculated with 103 or 105 TCID50 of the BI501 H1N1 virus. For the antibody pretreatment group, each B6 mouse was sequentially i.v. inoculated with 50 μg of the isotype control or anti-FGF2 antibodies 6 h before infection and at 1 DPI. For the treatment group, each B6 mouse was sequentially injected i.v. with 50 μg of the isotype control or anti-FGF2 antibodies at 3 and 5 DPI. All data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. (A) Changes in the weights of pre-treated B6 mice (n = 10). (B) Survival rates of pre-treated B6 mice (n = 10). (C) Wet-to-dry ratios of lungs from pre-treated B6 mice (n = 6) at 5 DPI. (D) Changes in the weights of treated B6 mice (n = 10). (E) Survival rates of treated B6 mice (n = 10). (F) Wet-to-dry ratios of lungs from treated B6 mice (n = 6) at 5 DPI. (G) Changes in the weights of WT B6 mice and FGF2–/– mice (n = 10). (H) Survival rates of WT B6 mice and FGF2–/– mice (n = 10). (I) Wet-to-dry ratios of lungs from WT B6 mice and FGF2–/– mice (n = 6) at 5 DPI. (J) HE staining of lung tissues from WT B6 mice and FGF2–/– mice at 5 DPI. The mean numbers of infiltrated cells per microscopic field ± SEM are shown (n = 50 fields analyzed for three mice). (K) Virus titers in the lungs of WT B6 mice and FGF2–/– mice (n = 6) at 5 DPI. Similar results were obtained in three independent experiments with 5–10 mice per group. Figure 2 View largeDownload slide Antibody depletion or knockout of FGF2 exacerbates H1N1-induced lung injury. WT B6 mice and FGF2–/– (KO) mice were i.n. inoculated with 103 or 105 TCID50 of the BI501 H1N1 virus. For the antibody pretreatment group, each B6 mouse was sequentially i.v. inoculated with 50 μg of the isotype control or anti-FGF2 antibodies 6 h before infection and at 1 DPI. For the treatment group, each B6 mouse was sequentially injected i.v. with 50 μg of the isotype control or anti-FGF2 antibodies at 3 and 5 DPI. All data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. (A) Changes in the weights of pre-treated B6 mice (n = 10). (B) Survival rates of pre-treated B6 mice (n = 10). (C) Wet-to-dry ratios of lungs from pre-treated B6 mice (n = 6) at 5 DPI. (D) Changes in the weights of treated B6 mice (n = 10). (E) Survival rates of treated B6 mice (n = 10). (F) Wet-to-dry ratios of lungs from treated B6 mice (n = 6) at 5 DPI. (G) Changes in the weights of WT B6 mice and FGF2–/– mice (n = 10). (H) Survival rates of WT B6 mice and FGF2–/– mice (n = 10). (I) Wet-to-dry ratios of lungs from WT B6 mice and FGF2–/– mice (n = 6) at 5 DPI. (J) HE staining of lung tissues from WT B6 mice and FGF2–/– mice at 5 DPI. The mean numbers of infiltrated cells per microscopic field ± SEM are shown (n = 50 fields analyzed for three mice). (K) Virus titers in the lungs of WT B6 mice and FGF2–/– mice (n = 6) at 5 DPI. Similar results were obtained in three independent experiments with 5–10 mice per group. Similarly, 4-week-old FGF2–/– (KO) mice that had been i.n. infected with BJ501 at a titer of 105 TCID50 showed more severe body weight loss than WT B6 mice (Figure 2G). Compared with BJ501-infected WT mice, BJ501-infected FGF2–/– mice showed significantly decreased survival rates (Figure 2H) and significantly increased wet-to-dry ratios of the lung tissue (Figure 2I). Moreover, the lung histopathology was significantly more severe in FGF2–/– mice than in WT mice after BJ501 infection, and the results showed thickened alveolar walls, increased lung interstitium, decreased alveolar space, and leukocyte infiltration (Figure 2J). However, the BJ501 infection did not result in evident histopathology in the brain, liver, kidney or intestine at 5 DPI (Supplementary Figure S2). Additionally, the IAV titers were much higher in the infected lungs of FGF2–/– mice than in WT mice (Figure 2K). In addition, similar alterations in histopathology were observed in mice after challenge with the PR8 strain of IAV (Supplementary Figure S3). Based on these data, FGF2 is required to protect against lethal H1N1 virus infection. Epithelial cells are the major cellular source of FGF2 Because macrophages, neutrophils, and alveolar epithelial cells (AECs) are the main cell populations detected in the inflamed lung tissue after BJ501 infection, we isolated neutrophils, macrophages and AECs from the lungs of BJ501 virus-infected mice at 5 DPI to further determine the source of FGF2 during IAV infection. FGF2 expression was significantly increased in AECs after BJ501 infection, but BJ501 infection did not affect FGF2 expression in neutrophils or macrophages; thus, lung epithelial cells were responsible for the marked IAV infection-induced elevation in the FGF2 level in the total lung tissue (Figure 3A). In addition, FGF2 levels were measured in the A549, BEAS-2B, and MLE-12 epithelial cell lines and in AECs isolated at 0, 24, 48, and 72 h after BJ501 infection (MOI = 1). FGF2 levels were remarkably elevated in all these cells in a time-dependent manner (Figure 3B), but no differences in FGF2 expression were observed in isolated neutrophils or macrophages with or without viral infection at different time points (data not shown). As shown in the confocal microscopy images, FGF2 was primarily expressed in AECs and bronchial epithelial cells of BJ501 virus-infected mice at 5 DPI, as evidenced by the co-localization of FGF2 and E-cadherin in the lung tissue (Figure 3C and D). Figure 3 View largeDownload slide Alveolar epithelial cells are the major source of FGF2. Four-week-old B6 mice were i.n. inoculated with 105 TCID50 of the BI501 H1N1 virus. Neutrophils, macrophages, and AECs were isolated from infected mouse lung tissue at 5 DPI. (A) FGF2 expression in the lung, neutrophils, macrophages, and AECs isolated from infected mice on 5 DPI was assessed using qRT-PCR. (B) FGF2 expression in isolated AECs and infected epithelial cell lines, including A549, BEAS-2B, and MLE-12 cells (MOI = 1). (C and D) Immunohistochemistry for E-cadherin (green), FGF2 (red), and DAPI (blue) in AECs and bronchial epithelial cells from mice infected with 105 TCID50 of the BI501 H1N1 virus at 5 DPI. The panels in Merge 1 are a merge of the FGF2 and E-cadherin images. The panels in Merge 2 are a merge of the FGF2, E-cadherin, and DAPI images. *P < 0.05, **P < 0.01, ***P < 0.001. Figure 3 View largeDownload slide Alveolar epithelial cells are the major source of FGF2. Four-week-old B6 mice were i.n. inoculated with 105 TCID50 of the BI501 H1N1 virus. Neutrophils, macrophages, and AECs were isolated from infected mouse lung tissue at 5 DPI. (A) FGF2 expression in the lung, neutrophils, macrophages, and AECs isolated from infected mice on 5 DPI was assessed using qRT-PCR. (B) FGF2 expression in isolated AECs and infected epithelial cell lines, including A549, BEAS-2B, and MLE-12 cells (MOI = 1). (C and D) Immunohistochemistry for E-cadherin (green), FGF2 (red), and DAPI (blue) in AECs and bronchial epithelial cells from mice infected with 105 TCID50 of the BI501 H1N1 virus at 5 DPI. The panels in Merge 1 are a merge of the FGF2 and E-cadherin images. The panels in Merge 2 are a merge of the FGF2, E-cadherin, and DAPI images. *P < 0.05, **P < 0.01, ***P < 0.001. FGF2 affects the recruitment and activation of neutrophils during IAV infection-induced lung injury Neutrophils, the prototypic cells of the innate immune system, are recruited to infected sites to protect the body from invading pathogens (Koller et al., 2009). In vivo, neutrophils and macrophages constitute the majority of infiltrating cells in inflamed tissues (Perrone et al., 2008) and might exert beneficial anti-pathogenic effects (Perrone et al., 2008; Suzuki et al., 2008; Iwasaki and Pillai, 2014). Neutrophils are rapidly recruited to sites of infection during the innate immune response to influenza A virus (Tumpey et al., 2005; Baskin et al., 2007; Perrone et al., 2008; Wang et al., 2008). Consistent with the findings in the literature, BJ501 infection caused severe lung histopathology and inflammatory cell infiltration in WT mice; notably, FGF2–/– mice showed more severe histopathology but less inflammatory cell infiltration in the lung tissue than the WT mice after infection (Figure 4A). A significant decrease in the number of neutrophils might account for the sharp reduction in the leukocyte count in BJ501-infected lung tissues because the total lymphocyte, macrophage, and NK cell counts were not obviously different between WT and KO mice after BJ501 infection (Figure 4B). Figure 4 View largeDownload slide FGF2 affects neutrophil function in influenza-induced lung injury. (A) HE staining and leukocyte cell counts (n = 50 fields) in lung tissues from B6 mice 3 days after BJ501 infection. FGF2–/– (KO) mice showed reduced leukocyte infiltration and significantly reduced leukocyte counts at 3 DPI. (B) Flow cytometry analysis of leukocyte subsets in mouse BALF obtained from 105 TCID50 of BJ501-infected mice at 3 DPI (n = 5). Similar results were obtained in three independent experiments with five mice per group. (C) Neutrophil chemotaxis assay in vitro. Untreated human neutrophils were allowed to migrate through a migration chamber toward PBS or FGF2 (10−9 M, 1.72 ng/ml). Cells from eight random fields were counted, and migration is expressed relative to the PBS control. (D) Concentrations of neutrophil-related chemokines in vitro. Neutrophils were treated with FGF2 (10−9 M, 1.72 ng/ml) for 6, 12, or 24 h and the levels in cell culture supernatants were determined using a Human ProcartaPlex Panel. *P < 0.05 and **P < 0.01. Figure 4 View largeDownload slide FGF2 affects neutrophil function in influenza-induced lung injury. (A) HE staining and leukocyte cell counts (n = 50 fields) in lung tissues from B6 mice 3 days after BJ501 infection. FGF2–/– (KO) mice showed reduced leukocyte infiltration and significantly reduced leukocyte counts at 3 DPI. (B) Flow cytometry analysis of leukocyte subsets in mouse BALF obtained from 105 TCID50 of BJ501-infected mice at 3 DPI (n = 5). Similar results were obtained in three independent experiments with five mice per group. (C) Neutrophil chemotaxis assay in vitro. Untreated human neutrophils were allowed to migrate through a migration chamber toward PBS or FGF2 (10−9 M, 1.72 ng/ml). Cells from eight random fields were counted, and migration is expressed relative to the PBS control. (D) Concentrations of neutrophil-related chemokines in vitro. Neutrophils were treated with FGF2 (10−9 M, 1.72 ng/ml) for 6, 12, or 24 h and the levels in cell culture supernatants were determined using a Human ProcartaPlex Panel. *P < 0.05 and **P < 0.01. Additionally, we profiled the levels of 23 mouse cytokines and chemokines in mouse BALF samples at 3 DPI. The levels of various cytokines, including IL-1α, IL-1β, IL-6, IL-17, IL-3, IL-5, IL-13, IL-10, IFN-γ, TNF-α, and G-CSF, as well as certain chemokines, including MIP-1α, MIP-1β, RANTES and eotaxin, were significantly increased in WT mice after IAV infection. More importantly, the levels of these cytokines and chemokines were reduced in FGF2–/– mice compared to WT mice (Supplementary Figure S4). Based on these results, neutrophils might contribute to the marked alterations in cytokine and chemokine levels in response to BJ501 infection. FGF2 has been shown to potentiate leukocyte recruitment to sites of inflammation and acts as a chemotactic agent for the recruitment of neutrophils (Zittermann and Issekutz, 2006a; Haddad et al., 2011). In this study, we confirmed that FGF2 promoted neutrophil recruitment in vitro using a neutrophil chemotaxis assay (Figure 4C). Furthermore, FGF2 promoted neutrophil-related chemokine expression in vitro, including the C-X-C chemokines CXCL1, IL-8 (CXCL8), IP10 (CXCL10), and CXCL12, as well as the CC chemokines MCP-1, MIP-1α, MIP-1β, RANTES, and eotaxin (Figure 4D). Thus, an FGF2 deficiency may impair neutrophil function by suppressing neutrophil recruitment and cytokine production. FGF2 enhances NFκB phosphorylation to induce the activation and chemotaxis of neutrophils According to a previous study, FGF receptors (FGFRs) are expressed by neutrophils (Haddad et al., 2011). Therefore, we examined whether H1N1 virus infection alters FGFR expression. Surprisingly, FGFR2 levels were significantly increased concomitantly with reduced FGFR1 and FGFR4 expression in neutrophils in vitro at 6 h after BJ501 infection. However, FGFR3 was expressed at very low levels before or after IAV infection. The viral load (M1 level) was significantly increased after BJ501 infection, indicating the establishment of a model of BJ501 virus-infected neutrophils (Figure 5A). The mammalian FGF family comprises 18 ligands that signal through four conserved tyrosine kinase receptors to activate four key downstream pathways: RAS–RAF–MAPK, PI3K–AKT, signal transducer and activator of transcription (STAT), and phospholipase Cγ (PLCγ) (Turner and Grose, 2010). In this experiment, the application of FGF2 to neutrophils for 6 h did not lead to a significant change in the levels of MAPK pathway members, including P38, JNK, and ERK compared with untreated cells; thus, FGF2-induced activation and neutrophil chemotaxis did not involve MAPK signaling. Moreover, STAT3 levels remained unchanged, and PLCγ expression was not detected (data not shown) after FGF2 protein administration. MAPK and STAT3 signaling were still unchanged following BJ501 infection (Supplementary Figure S5A). In contrast, prominent PI3K and AKT phosphorylation were observed at 6 h after the application of FGF2 with or without BJ501 infection (Figure 5B). NFκB has been confirmed to act downstream of the PI3K/AKT pathway in various cancers (Ghosh-Choudhury et al., 2010; Han et al., 2010; Lin et al., 2010), and AKT activates the NFκB pathway by phosphorylating and activating NFκB pathway intermediates (Ozes et al., 1999; Romashkova and Makarov, 1999). However, the relationships between these two pathways downstream of FGF2 activation have not been fully explored. In this study, inhibitor of NFκB kinase subunit beta (IKKβ), IκB, and NFκB (RELA/p65) were phosphorylated in FGF2-stimulated neutrophils. Meanwhile, the phosphorylation of these signaling molecules was also enhanced following BJ501 infection (Figure 5B). Figure 5 View largeDownload slide FGF2 enhances NFκB phosphorylation and subsequently mediates neutrophil chemotaxis. (A) qRT-PCR analysis of FGFR1–4 and M1 expression in neutrophils infected with BJ501 (MOI = 1) for 0 or 6 h. (B) Western blot analysis of the indicated phosphorylated (p-) and total proteins in neutrophils treated with FGF2 (10−9 M, 1.72 ng/ml) for 6 h. (C) Western blot analysis of signaling pathways in WT and FGF2–/– mice infected with 105 TCID50 of BJ501 or mock infected at 5 DPI. β-Actin was used as an internal control. Data are presented as mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Figure 5 View largeDownload slide FGF2 enhances NFκB phosphorylation and subsequently mediates neutrophil chemotaxis. (A) qRT-PCR analysis of FGFR1–4 and M1 expression in neutrophils infected with BJ501 (MOI = 1) for 0 or 6 h. (B) Western blot analysis of the indicated phosphorylated (p-) and total proteins in neutrophils treated with FGF2 (10−9 M, 1.72 ng/ml) for 6 h. (C) Western blot analysis of signaling pathways in WT and FGF2–/– mice infected with 105 TCID50 of BJ501 or mock infected at 5 DPI. β-Actin was used as an internal control. Data are presented as mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. Four-week-old FGF2–/– or WT mice were i.n. infected with BJ501 at a titer of 105 TCID50 or mock infected to further confirm the functional impact of FGFin vivo. The results of western blot analyses revealed a decrease in the levels of phosphorylated PI3K, AKT, and NFκB in FGF2–/– mice infected with BJ501, but no changes were observed in the mock infected mice (Figure 5C). However, the phosphorylation of members of the MAPK and STAT3 pathways were unchanged in the presence or absence of BJ501 infection (Supplementary Figure S5B). Based on these results, activation of the FGFR2–PI3K–AKT–NFκB pathway probably contributes to neutrophil recruitment, chemotaxis, or activation in IAV-infected lung tissues. The recombinant FGF2 protein protects mice from H1N1 infection by recruiting neutrophils Currently, the recombinant FGF2 protein (rFGF2) has been used in various clinical settings, such as wound healing and cornea repair (Fu et al., 1998, 2000; Huang et al., 2011). We treated BJ501-infected mice with rFGF2 and/or neutrophil neutralizing antibodies to further investigate whether FGF2 protected against IAV infection and determine whether the protective effects were associated with neutrophil recruitment. First, 4-week-old WT B6 mice were i.n. infected with 105 TCID50 of the BJ501 virus. Mice were pre-treated or i.v. treated with anti-Ly6G (m1A8) antibodies or isotype control antibodies (m2A3) and FGF2. The survival rates of the BJ501-infected mice were significantly increased, and weight loss was improved by the rFGF2 treatment (Figure 6A and B). Moreover, the lung wet-to-dry weight ratio exhibited a greater improvement in the IAV-infected mice treated with rFGF2 (Figure 6C). Similarly, improved lung histopathology and lung injury scores, as defined by infiltrated leukocyte counts, were observed in the IAV-infected mice treated with rFGF2 (Figure 6D). Furthermore, rFGF2-treated mice showed less viral replication in the infected lung than control mice (Figure 6E). Similar results were obtained in mice challenged with IAV strain PR8 (Supplementary Figure S6). More importantly, more neutrophils were recruited to the lungs of the FGF2 treatment group but not the anti-Ly6G group or anti-Ly6G plus FGF2 group (Figure 6F). In addition, mice that had been pre-treated or treated with m1A8 antibodies showed significantly lower survival rates, more severe body weight loss and lung edema than the isotype control antibody-treated mice. In contrast, pretreatment with FGF2 and m1A8 antibodies did not protect the mice. The histopathological alterations in the lung and viral replication further supported these findings (Figure 6A–D). Thus, rFGF2 protects against H1N1 virus infection in vivo, and the recruited neutrophils were indispensable for the protective effects of FGF2. Figure 6 View largeDownload slide FGF2 alleviates influenza-induced lung injury through a neutrophil-dependent mechanism. (A) Survival rates of B6 mice pre-treated with anti-Ly6G and FGF2 or the isotype control (n = 5). Control vs. control + FGF2: **P < 0.01. (B) Changes in the weights of B6 mice pre-treated with anti-Ly6G and FGF2 or the isotype control (n = 5). (C) Wet-to-dry ratios of lungs from B6 mice pre-treated with anti-Ly6G and FGF2 or the isotype control (n = 5) at 5 DPI. (D) HE staining and quantification of lung tissues from B6 mice at 5 DPI. (E) Virus titers of lungs from treated B6 mice (n = 6) at 5 DPI. (F) Flow cytometry analysis of neutrophil counts in BALF obtained from treated B6 mice at 3 DPI. Similar results were obtained in three independent experiments with 5–6 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001. Figure 6 View largeDownload slide FGF2 alleviates influenza-induced lung injury through a neutrophil-dependent mechanism. (A) Survival rates of B6 mice pre-treated with anti-Ly6G and FGF2 or the isotype control (n = 5). Control vs. control + FGF2: **P < 0.01. (B) Changes in the weights of B6 mice pre-treated with anti-Ly6G and FGF2 or the isotype control (n = 5). (C) Wet-to-dry ratios of lungs from B6 mice pre-treated with anti-Ly6G and FGF2 or the isotype control (n = 5) at 5 DPI. (D) HE staining and quantification of lung tissues from B6 mice at 5 DPI. (E) Virus titers of lungs from treated B6 mice (n = 6) at 5 DPI. (F) Flow cytometry analysis of neutrophil counts in BALF obtained from treated B6 mice at 3 DPI. Similar results were obtained in three independent experiments with 5–6 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001. Neutrophil transfer protects FGF2–/– mice against influenza BJ501 virus infection Neutrophils were purified from the bone marrow of WT mice and adoptively transferred to FGF2–/– mice on Day 1 after infection with 103 TCID50 of BJ501 to investigate whether neutrophils protect FGF2–/– mice from influenza infection. FGF2–/– mice that received an adoptive transfer of WT neutrophils had an increased survival rate and a better outcome of weight change after IAV infection (Figure 7A). The lung edema and virus load of infected neutrophil-transferred mice were significantly decreased compared with FGF2–/– mice (Figure 7B). In addition, the lung histopathology of neutrophil-transferred mice was greatly improved, and the infiltrated leukocyte counts were significantly decreased (Figure 7C and D). The results of the flow cytometry analysis showed the restoration of infiltrating neutrophils in FGF2–/– mice that received WT neutrophils (Figure 7F). These results confirm that mice that received the neutrophil transfer achieved better outcomes upon influenza infection. Figure 7 View largeDownload slide Neutrophil transfer protects FGF2–/– mice from influenza infection. (A) Survival rates of FGF2–/– and neutrophil-transferred FGF2–/– mice (n = 5). (B) Changes in the weights of FGF2–/– and neutrophil-transferred FGF2–/– mice (n = 5). (C) Wet-to-dry ratios of lungs from FGF2–/– and neutrophil-transferred FGF2–/– mice (n = 5) at 5 DPI. (D) HE staining and quantification of lung tissues from B6 mice at 5 DPI. (E) Virus titers in lungs from treated B6 mice (n = 6) at 5 DPI. (F) Flow cytometry analysis of neutrophil counts in BALF obtained from treated B6 mice at 3 DPI. Similar results were obtained in three independent experiments with five mice per group. *P < 0.05, **P < 0.01, ***P < 0.001. Figure 7 View largeDownload slide Neutrophil transfer protects FGF2–/– mice from influenza infection. (A) Survival rates of FGF2–/– and neutrophil-transferred FGF2–/– mice (n = 5). (B) Changes in the weights of FGF2–/– and neutrophil-transferred FGF2–/– mice (n = 5). (C) Wet-to-dry ratios of lungs from FGF2–/– and neutrophil-transferred FGF2–/– mice (n = 5) at 5 DPI. (D) HE staining and quantification of lung tissues from B6 mice at 5 DPI. (E) Virus titers in lungs from treated B6 mice (n = 6) at 5 DPI. (F) Flow cytometry analysis of neutrophil counts in BALF obtained from treated B6 mice at 3 DPI. Similar results were obtained in three independent experiments with five mice per group. *P < 0.05, **P < 0.01, ***P < 0.001. Discussion FGF2 is known to act as a pleiotropic factor in multiple biological processes, including angiogenesis, embryonic development, and wound healing (Ortega et al., 1998; Nugent and Iozzo, 2000; Virag et al., 2007; Han and Gotlieb, 2012; Song et al., 2015). FGF2 accelerates the healing of skin wounds in animal models as well as the healing of eye, retina, and corneal wounds (Bikfalvi et al., 1997). FGF2 also has multiple functions in the central nervous system (Tureyen et al., 2005; Frinchi et al., 2008; Li et al., 2008; Ma et al., 2008; Ma et al., 2009; Mimura et al., 2015). Furthermore, FGF2 promotes the epithelial–mesenchymal transition, invasiveness, and tumor angiogenesis (Marek et al., 2009; Narong and Leelawat, 2011; Wesche et al., 2011; Wang et al., 2015). FGF2 is expressed in normal tissue and is upregulated by chronic inflammatory reactions (Byrd et al., 1996; Kanazawa et al., 2001; Song et al., 2015). The roles of FGF2 in mediating the immune response to IAV infection have not been fully elucidated. Our study is the first to report that FGF2 levels were elevated in serum from IAV-infected patients and BALF of mice infected with H1N1 virus strain BJ501. Mice with an FGF2 deficiency due to either gene knockout or protein inhibition using neutralizing antibodies in vivo were more susceptible to IAV infection. According to a previous report, FGF2 is a potent mitogen for many cell types, including airway smooth muscle cells, fibroblasts, and endothelial cells (Redington et al., 2001). Additionally, FGF2 is secreted by many types of immune cells, such as Treg cells, neutrophils, monocytes, macrophages, and T lymphocytes (Barrios et al., 1997; Byrd et al., 1999; Ohsaka et al., 2001; Haddad et al., 2011; Song et al., 2015). During H1N1 infection, neutrophils represent the majority of infiltrating immune cells in inflamed tissues. In this study, epithelial cells were the major cellular source of FGF2 during H1N1-induced ALI. Humans infected with influenza A virus display a ‘cytokine storm’, which is caused by insufficient control of excessive neutrophil recruitment to the lungs (Wang and Ma, 2008; Tisoncik et al., 2012; Brandes et al., 2013; Liu et al., 2016; Yang and Tang, 2016). However, neutrophils provide the first line of defense against invading microorganisms and contribute to the fine regulation of inflammatory and immune responses (Futosi et al., 2013; Tecchio et al., 2014). Moreover, neutrophils have a potent anti-microbial armamentarium that includes oxidant-generating systems, powerful proteinases, and cationic peptides contained in granules (Suzuki et al., 2008). In addition, neutrophils function as regulators of inflammatory and immune responses by producing and releasing a large variety of cytokines and chemokines (Tecchio et al., 2014). This variety of cytokines produced by neutrophils enables them to significantly influence not only multiple aspects of the inflammatory and immune responses but also antiviral defense, hematopoiesis, angiogenesis, and fibrogenesis (Cassatella, 1999). FGF2 also enhances the recruitment of monocytes, T cells, and polymorph nuclear leukocytes (PMNs) to inflamed dermal sites (Zittermann and Issekutz, 2006b), and act as a chemotactic agent for the recruitment of neutrophils (Byrd et al., 1996; Wempe et al., 1997; Zittermann and Issekutz, 2006a; Haddad et al., 2011). Consistent with the results from these publications, an FGF2 deficiency reduced leukocyte recruitment, particularly neutrophil recruitment, accompanied by decreases in the levels of cytokines and chemokines in mouse BALF during the early phase of IAV infection in this study. Moreover, FGF2 enhanced neutrophil recruitment in vitro and promoted the expression of neutrophil-related chemokines, such as C-X-C and CC chemokines. Therefore, we speculate that the FGF2 deficiency impaired an effective immune response to IAV infection and FGF2 recruited neutrophils to the infected lung tissue during the early phase of H1N1 infection. Of course, additional mechanistic studies of the protective roles of neutrophils and the ‘cytokine storm’ are warranted. FGFs signal through FGFRs to perform a multitude of physiological functions. FGFRs are expressed on many different cell types and regulate key cell behaviors, such as proliferation, differentiation, and survival (Turner and Grose, 2010). Here, FGFR2 expression was significantly increased in neutrophils after BJ501 infection, whereas the levels of FGFR1 and FGFR4 were decreased, and FGFR3 was weakly expressed before and after infection. Furthermore, FGF2 contributed to leukocyte recruitment or enhanced neutrophil chemotaxis to infected lung tissue mainly through the FGFR2–PI3K–AKT–NFκB signaling pathway (Figure 8). FGF2 interacts with FGFR2, activating the PI3K–AKT–NFκB signaling pathway, which induces cytokine and chemokine production and promotes neutrophil chemotaxis through a feedback mechanism, thus protecting against IAV infection in the early stage. Based on these findings, we further examined the relationship between FGF2 and neutrophils in vivo. The administration of the recombinant murine FGF2 protein protected mice from a lethal H1N1 infection, whereas concomitant treatment with both rFGF2 and anti-Ly6G (m1A8) failed to protect mice against IAV infection, suggesting that the protective role of FGF2 was mainly mediated by neutrophils. Consistent with these findings, infected FGF2–/– mice exhibited a reduced disease severity after receiving neutrophils from WT mice. Thus, the neutrophil-dependent therapeutic roles of FGF2 are to alleviate IAV-induced ALI in the early stage. To the best of our knowledge, we are the first group to systematically show that the FGFR2–PI3K–AKT–NFκB signaling pathway is involved in IAV infection-mediated ALI in mice. Based on this evidence, FGF2 plays a critical role in mediating ALI induced by influenza H1N1 virus infection. Figure 8 View largeDownload slide A schematic explaining the signaling mechanisms by which FGF2 regulates neutrophil chemotaxis and IAV-induced ALI. Figure 8 View largeDownload slide A schematic explaining the signaling mechanisms by which FGF2 regulates neutrophil chemotaxis and IAV-induced ALI. In conclusion, FGF2 performs a critical protective function during the process of IAV infection. The combination of our clinical findings and results from the mouse model has revealed a critical role for FGF2 in the pathogenesis of influenza A (H1N1) virus-induced ALI and has suggested that FGF2 represents a promising potential therapeutic target in IAV-induced lung disease. Materials and methods Viruses and cells The influenza viruses used in this study were influenza A (H1N1) virus strain A/Beijing/501/2009 (BJ501) and influenza A (H1N1) virus strain A/PR/8/1934 (PR8) obtained from the Academy of Military Medical Sciences. The genomic sequence of the BJ501 strain is available in the GenBank database (accession number GQ223415). The strains were propagated in 9- to 11-day-old specific-pathogen-free (SPF) embryonated eggs via the allantoic route. Virus titers were determined based on an assessment of the TCID50 using Madin-Darby canine kidney (MDCK) cells, according to the Reed-Muench method. MDCK, A549 and BEAS-2B cells were purchased from ATCC; MLE-12 cells were kindly provided by Dr Zhuowei Hu (Peking Union Medical College, PUMC). MDCK cells were cultured in DMEM (Gibco); A549, BEAS-2B, and MLE-12 cells were cultured in DMEM/F-12(1:1) basic culture medium (Gibco); and neutrophils were cultured in RPMI-1640 medium (Gibco) supplemented with 10% FBS and 100 U/ml penicillin-streptomycin at 37°C in a humidified atmosphere containing 5% CO2. Mice Four-week-old WT C57BL/6 (abbreviated B6) female mice (Experimental Animal Center of the Academy of Military Medical Sciences, Beijing, China) and 4-week-old FGF2 knockout mice (B6 background, 010698, Jackson Laboratory, USA) were housed in the animal facility at the Beijing Institute of Microbiology and Epidemiology in accordance with institutional guidelines. All experimental protocols were approved by the Institutional Animal Care and Use Committees of the Beijing Institute of Microbiology and Epidemiology (ID: SYXK2015-008) and all experiments were performed in accordance with the approved guidelines. Mouse infections Four-week-old WT B6 mice were anesthetized with 50 μl of 1% (w/v) pentobarbital sodium and then i.n. inoculated with the H1N1 virus or mock infected with PBS as a control. Survival rates, body weight changes, histology, acute pulmonary edema (wet-to-dry ratio), and cytokine levels were evaluated as previously described (Li et al., 2012; Wang et al., 2013; Gu et al., 2016). For the anti-FGF2 antibody treatment, anti-FGF2 antibodies or isotype control antibodies (100 μg/mouse, Abcam, Cat. No. ab33103) (Kujawski et al., 2008) were sequentially administered i.v. at 6 h before and 1 and 3 days after injection (pretreatment group) or at 1, 3, and 5 days after injection (treatment group) with the vehicle control or virus (103 TCID50 of A/Beijing/501/2009). For FGF2–/– mice, 105 TCID50 of A/Beijing/501/2009 was used. For the rescue experiments, each mouse was sequentially i.v. inoculated with 25 μg of the recombinant murine FGF2 protein (Peprotech, Cat. No. 450-33) 12 h before and 1 and 3 days after injection with the vehicle control or virus (105 TCID50 of A/Beijing/501/2009 or 104 TCID50 of A/PR/8/1934). For the neutrophil depletion experiment, a neutrophil-specific antibody, anti-Ly-6G (clone 1A8), and an isotype control antibody (clone 2A3) were purchased from Biox Cell. WT mice were sequentially i.v. administered 50 μg of the anti-mouse Ly6G antibody (clone 1A8) or isotype control antibody (clone 2A3) 6 h before and 1 and 3 days after injection, as previously described (Kawanishi et al., 2016). Viral titration Virus titers were determined in supernatants of mouse lung homogenates from FGF2–/– mice or WT mice on Day 5 after H1N1 infection, as previously described (Li et al., 2012; Wang et al., 2013; Gu et al., 2016). Survival rate and body weight changes Four-week-old WT B6 mice were anesthetized with 50 μl of 1% (w/v) pentobarbital sodium and i.n. inoculated with the virus or vehicle control. The survival rates and body weights of each group of 10 mice were monitored daily for 14 days. Acute pulmonary edema (wet-to-dry ratio) Pulmonary edema was assessed by measuring and recording the lung wet/dry weight ratio as previously described (Li et al., 2012). Adoptive transfer of neutrophils Bone marrow neutrophils were isolated using Percoll (GE Healthcare), as previously described (Mei et al., 2012). Briefly, bone marrow cells were harvested from the femurs of 4-week-old WT B6 mice and suspended in PBS before being layered on a 3-step Percoll (GE Healthcare) gradient (72%, 64%, and 52%), which was centrifuged at 500× g for 30 min. The cells at the interface between 64% and 72% gradients were collected and washed twice with PBS. More than 95% of the neutrophils were viable, as determined using the trypan blue exclusion method. For adoptive transfer, 5 × 106 neutrophils isolated from WT mice were intravenously injected into FGF2–/– mice 24 h before they were i.n. inoculated with the H1N1 virus (103 TCID50 of A/Beijing/501/2009) as described above. Flow cytometry Cells in BALF were stained with anti-CD45-FITC, anti-Ly6G-PE, anti-F4/80-PE, anti-NK1.1-PE and anti-CD3-FITC antibodies (BD Pharmingen) and analyzed using a FACSCalibur flow cytometer (BD Biosciences) to examine leukocyte marker expression. The data were analyzed using FlowJo software (Tree Star). Isotype controls were used for all samples. Statistical analyses Measurements collected at a single time point were analyzed using an ANOVA, and if a significant difference among the groups was observed, the results were further analyzed using a two-tailed t-test. All analyses were performed using GraphPad Prism 5.0 software (GraphPad Software). P < 0.05 was considered statistically significant. All experiments were performed in triplicate. Supplementary material Supplementary material is available at Journal of Molecular Cell Biology online. Acknowledgements We thank Professor Zhuowei Hu (Chinese Academy of Medical Sciences, Peking Union Medical College) for providing the MLE-12 cells. Funding This work was supported in part by funding from the National High Technology Research and Development Program of China (SS2015AA020924), the National Natural Science Foundation of China (81771700), the Ministry of Science and Technology of China (2013ZX10004003 and SS2012AA020905), and the National Major Research and Development Program (2016YFA0502203 and 2017YFC1200800). P.Y. was supported by the Beijing Nova Program (Z141107001814054). Conflict of interest: none declared. Author contributions: K.W., C.L., C.W., Y.D., Z.Z., X.Y., L.X., L.Z., S.Z., and M.X. contributed to the experiments. W.W., H.G., and B.N. analyzed the data. T.L., C.B., S.Z., and L.H. collected the samples. 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Journal of Molecular Cell BiologyOxford University Press

Published: Nov 7, 2017

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