Downloaded from https://academic.oup.com/jmcb/article-abstract/10/6/573/4600201 by Ed 'DeepDyve' Gillespie user on 15 January 2019 doi:10.1093/jmcb/mjx047 Journal of Molecular Cell Biology (2018), 10(6), 573–585 j 573 Published online November 7, 2017 Article Basic ﬁbroblast growth factor protects against inﬂuenza A virus-induced acute lung injury by recruiting neutrophils 1,† 1,† 2 2 3 4 5 Keyu Wang , Chengcai Lai , Tieling Li , Cheng Wang , Wei Wang , Bing Ni , Changqing Bai , 6 2 1 1 1 1 Shaogeng Zhang , Lina Han , Hongjing Gu , Zhongpeng Zhao , Yueqiang Duan , Xiaolan Yang , 1 1 1 1 3, 1, *, Xiliang Wang *, Li Xing , Lingna Zhao , Shanshan Zhou , Min Xia , Chengyu Jiang 1,6, and Penghui Yang * State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China Chinese PLA General Hospital, Beijing 100853, China State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing 100005, China Institute of Immunology, Third Military Medical University, Chongqing 400038, China Beijing 307 Hospital of PLA Afﬁliated with the Chinese Academy of Medical Sciences, Beijing 100070, China Beijing 302 Hospital of PLA, Beijing 100039, China These authors contributed equally to this work. * Correspondence to: Penghui Yang, E-mail: firstname.lastname@example.org; Xiliang Wang, E-mail: email@example.com; Chengyu Jiang, E-mail: firstname.lastname@example.org 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 acti- vates 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. Keywords: inﬂuenza H1N1 virus, recombinant FGF2 protein, neutrophil recruitment, FGFR2–PI3K–AKT–NFκB signaling, therapeutic target Louie et al., 2009; Berdal et al., 2011; Ohta et al., 2011). IAV Introduction infection may cause inﬂammation of the airways, epithelial necro- Inﬂuenza spreads throughout the world during annual out- sis, edema, hemorrhaging, and respiratory failure (Xu et al., 2006; breaks, resulting in ∼3–5 million cases of severe illness and Rincon, 2012; Ding et al., 2013). Both virus-speciﬁc virulence fac- ∼250000–500000 deaths annually; infants and the elderly are tors and host immunity are associated with exacerbated IAV patho- particularly vulnerable to inﬂuenza. The mechanisms by which genesis (Crouser et al., 2009). The currently recognized therapeutic inﬂuenza virus (IAV) infection cause symptoms in humans have agents against IAV infection include viral m2 channel inhibitors been studied intensively. Some severely infected patients (amantadine and rimantadine), neuraminidase inhibitors (zanamivir, develop acute lung injury (ALI) and even acute respiratory dis- oseltamivir, peramivir, and laninamivir octanoate), and polymerase tress syndrome (ARDS), which is the predominant cause of inhibitors (ribavirin and favipiravir), but IAVs are becoming highly reported inﬂuenza-related deaths (Dominguez-Cherit et al., 2009; resistant to these drugs, and further evidence is required from clin- ical trials (Dushianthan et al., 2011; De Clercq and Li, 2016). Received March 23, 2017. Revised October 9, 2017. Accepted November 2, 2017. Basic ﬁbroblast growth factor (bFGF or FGF2), a potent mito- © The Author (2017). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved. gen for many cell types, including airway smooth muscle cells, Downloaded from https://academic.oup.com/jmcb/article-abstract/10/6/573/4600201 by Ed 'DeepDyve' Gillespie user on 15 January 2019 574 j Wang et al. ﬁbroblasts, and endothelial cells (Redington et al., 2001), is for epithelial repair and maintaining epithelial integrity after associated with multiple biological processes, including tumor bleomycin-induced lung injury in mice. However, researchers angiogenesis, embryonic development, proliferation, migration, have not determined whether and how FGF2 plays a role in and injury repair (Meyer et al., 1995; Ortega et al., 1998; IAV-induced ALI. Fuhrmann-Benzakein et al., 2000; Nugent and Iozzo, 2000; In this study, we explore the role of FGF2 in host defense Virag et al., 2007). FGF2 is dysregulated in many inﬂammatory against IAV infection using our previous established mouse disorders, such as inﬂammatory bowel disease (IBD), Crohn’s model (Li et al., 2012). Based on our results, FGF2 plays a piv- disease, ulcerative colitis, and rheumatoid arthritis (Byrd et al., otal role in IAV-induced lung injury, and the administration of 1996; Kanazawa et al., 2001; Song et al., 2015). In immune the recombinant FGF2 protein markedly reduces mortality and the responses, FGF2 functions to maintain the innate immune severity of lung injury in a preclinical model of IAV infection. The homeostasis of antiviral immunity by stabilizing retinoic acid- mechanisms underlying these effects of FGF2 include neutrophil inducible gene-I (RIG-I) and preventing proteasome-mediated activation and recruitment via the PI3K–Akt–NFκB signaling RIG-I degradation (Liu et al., 2015). In a study of the link pathway. between FGF2 and ALI by Powers et al. (1994), FGF2 was shown to play a role in the alveolar response to hyperoxic injury via Results the altered mRNA levels and protein distribution. According to FGF2 is signiﬁcantly upregulated in patient serum and mouse Liebler et al. (1997),FGF2 may participate in directing cell bronchoalveolar lavage ﬂuid (BALF) following IAV infection proliferation following pulmonary ﬁbrosis. As shown in the Hypercytokinemia has been reported to be an early host study by Zhao et al. (2015), mesenchymal stem cells (MSCs) response signature in inﬂuenza A (H1N1) virus-induced ALI and FGF2 synergistically reduced the level of inﬂammatory (Tisoncik et al., 2012; Brandes et al., 2013; Liu et al., 2016; cytokines in the treatment of LPS-induced lung injury. Yang and Tang, 2016). However, the roles of individual cyto- Moreover, Guzy et al. (2015) showed that FGF2 is required kines in ALI remain largely unclear. We measured FGF2 levels in Figure 1 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 10 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 theserafrompatientsinfectedwithinﬂuenzaA(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 10 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 10 TCID50 of the BJ501 strain at 5 DPI. Downloaded from https://academic.oup.com/jmcb/article-abstract/10/6/573/4600201 by Ed 'DeepDyve' Gillespie user on 15 January 2019 FGF2 protects against IAV-induced ALI j 575 –/– 156 sera samples from patients conﬁrmed to be infected with much higher in the infected lungs of FGF2 mice than in WT the H1N1 virus to investigate the effects of FGF2 on H1N1- mice (Figure 2K). In addition, similar alterations in histopath- induced ALI, and the characteristics reﬂecting the conditions ology were observed in mice after challenge with the PR8 and outcomes of these patients are described in the strain of IAV (Supplementary Figure S3). Based on these Supplementary information (Supplementary Table S1). The FGF2 data, FGF2 is required to protect against lethal H1N1 virus level was signiﬁcantly elevated in all IAV-infected patients com- infection. pared with healthy subjects. Furthermore, levels of the FGF2 protein in H1N1 virus-infected patients were gradually elevated Epithelial cells are the major cellular source of FGF2 as the fever duration increased and peaked on Day 3 of fever. Because macrophages, neutrophils, and alveolar epithelial Correspondingly, levels of the FGF2 protein in all hospitalized cells (AECs) are the main cell populations detected in the patients were markedly higher than the levels in healthy sub- inﬂamed lung tissue after BJ501 infection, we isolated neutro- jects (Figure 1A). Meanwhile, 4-week-old wild-type (WT) B6 mice phils, macrophages and AECs from the lungs of BJ501 virus- were intranasally (i.n.) infected with the BJ501 strain at a titer infected mice at 5 DPI to further determine the source of FGF2 of 10 50% tissue culture infectious dose (TCID50), and we during IAV infection. FGF2 expression was signiﬁcantly increased observed a signiﬁcant increase in pulmonary elastance that in AECs after BJ501 infection, but BJ501 infection did not affect represents changes in pressure achieved per unit changes in FGF2 expression in neutrophils or macrophages; thus, lung epi- volume (Supplementary Figure S1A) and decreased arterial par- thelial cells were responsible for the marked IAV infection- tial pressure of oxygen (PaO )at 3, 5,or 7 days post-infection induced elevation in the FGF2 level in the total lung tissue (DPI) (Supplementary Figure S1B). In addition, the level of the (Figure 3A). In addition, FGF2 levels were measured in the A549, FGF2 protein was increased in the BALF of C57BL/6 mice follow- BEAS-2B, and MLE-12 epithelial cell lines and in AECs isolated ing challenge with the A/Beijing/501/2009 (BJ501) strain. FGF2 at 0, 24, 48, and 72 h after BJ501 infection (MOI = 1). FGF2 levels began to increase signiﬁcantly on the ﬁfth day, peaked levels were remarkably elevated in all these cells in a time- on the eighth day, and then sharply decreased beginning on dependent manner (Figure 3B), but no differences in FGF2 the10th day of virus challenge (Figure 1B). Furthermore, FGF2 expression were observed in isolated neutrophils or macro- was expressed at high levels in the lung tissue of IAV BJ501 phages with or without viral infection at different time points strain-infected mice at 5 DPI, based on immunohistochemical (data not shown). As shown in the confocal microscopy images, (IHC) staining (Figure 1C). This observation suggests a potential FGF2 was primarily expressed in AECs and bronchial epithelial key role for FGF2 in H1N1 virus-induced ALI. cells of BJ501 virus-infected mice at 5 DPI, as evidenced by the co-localization of FGF2 and E-cadherin in the lung tissue FGF2 deﬁciency exacerbates IAV-induced lung injury (Figure 3C and D). Four-week-old WT B6 mice were i.n. infected with the BJ501 strain at a titer of 10 TCID50 to conﬁrm the exact role of FGF2 FGF2 affects the recruitment and activation of neutrophils in H1N1 virus-induced ALI. Mice were pre-treated or intraven- during IAV infection-induced lung injury ously (i.v.) treated with anti-FGF2 antibodies or isotype control Neutrophils, the prototypic cells of the innate immune sys- antibodies. After infection with the BJ501 strain, the groups that tem, are recruited to infected sites to protect the body from had been pre-treated or treated with anti-FGF2 antibodies had invading pathogens (Koller et al., 2009). In vivo, neutrophils signiﬁcantly lower survival rates and showed more severe body and macrophages constitute the majority of inﬁltrating cells in weight loss and lung edema than the group treated with the iso- inﬂamed tissues (Perrone et al., 2008) and might exert beneﬁ- type control. Moreover, the pathology of the mice in the anti- cial anti-pathogenic effects (Perrone et al., 2008; Suzuki et al., FGF2 antibody-pre-treated group was much worse than the mice 2008; Iwasaki and Pillai, 2014). Neutrophils are rapidly recruited in the anti-FGF2 antibody-treated group, as determined by to sites of infection during the innate immune response to inﬂu- weight loss, survival rate, and lung edema (Figure 2A–F). enza A virus (Tumpey et al., 2005; Baskin et al., 2007; Perrone –/– Similarly, 4-week-old FGF2 (KO) mice that had been i.n. infected et al., 2008; Wang et al., 2008). Consistent with the ﬁndings in with BJ501 at a titer of 10 TCID50 showed more severe body the literature, BJ501 infection caused severe lung histopath- weight loss than WT B6 mice (Figure 2G). Compared with BJ501- ology and inﬂammatory cell inﬁltration in WT mice; notably, –/– –/– infected WT mice, BJ501-infected FGF2 mice showed signiﬁ- FGF2 mice showed more severe histopathology but less cantly decreased survival rates (Figure 2H) and signiﬁcantly inﬂammatory cell inﬁltration in the lung tissue than the WT mice increased wet-to-dry ratios of the lung tissue (Figure 2I). after infection (Figure 4A). A signiﬁcant decrease in the number Moreover, the lung histopathology was signiﬁcantly more of neutrophils might account for the sharp reduction in the –/– severe in FGF2 mice than in WT mice after BJ501 infection, leukocyte count in BJ501-infected lung tissues because the total and the results showed thickened alveolar walls, increased lung lymphocyte, macrophage, and NK cell counts were not obviously interstitium, decreased alveolar space, and leukocyte inﬁltration different between WT and KO mice after BJ501 infection (Figure 2J). However, the BJ501 infection did not result in evi- (Figure 4B). dent histopathology in the brain, liver, kidney or intestine at 5 Additionally, we proﬁled the levels of 23 mouse cytokines DPI (Supplementary Figure S2). Additionally, the IAV titers were and chemokines in mouse BALF samples at 3 DPI. The levels of Downloaded from https://academic.oup.com/jmcb/article-abstract/10/6/573/4600201 by Ed 'DeepDyve' Gillespie user on 15 January 2019 576 j Wang et al. –/– Figure 2 Antibody depletion or knockout of FGF2 exacerbates H1N1-induced lung injury. WT B6 mice and FGF2 (KO) mice were i.n. inocu- 3 5 lated with 10 or 10 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. various cytokines, including IL-1α, IL-1β, IL-6, IL-17, IL-3, IL-5, the levels of these cytokines and chemokines were reduced in –/– IL-13,IL-10,IFN-γ, TNF-α, and G-CSF, as well as certain chemo- FGF2 mice compared to WT mice (Supplementary Figure S4). kines, including MIP-1α,MIP-1β, RANTES and eotaxin, were signiﬁ- Based on these results, neutrophils might contribute to the cantly increased in WT mice after IAV infection. More importantly, marked alterations in cytokine and chemokine levels in response Downloaded from https://academic.oup.com/jmcb/article-abstract/10/6/573/4600201 by Ed 'DeepDyve' Gillespie user on 15 January 2019 FGF2 protects against IAV-induced ALI j 577 Figure 3 Alveolar epithelial cells are the major source of FGF2. Four-week-old B6 mice were i.n. inoculated with 10 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, macro- phages, 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 10 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 areamerge of theFGF2, E-cadherin, and DAPI images. *P < 0.05,**P < 0.01,***P < 0.001. to BJ501 infection. FGF2 has been shown to potentiate leukocyte 2006a; Haddad et al., 2011). In this study, we conﬁrmed that recruitment to sites of inﬂammation and acts as a chemotactic FGF2 promoted neutrophil recruitment in vitro using a neutro- agent for the recruitment of neutrophils (Zittermann and Issekutz, phil chemotaxis assay (Figure 4C). Furthermore, FGF2 promoted Downloaded from https://academic.oup.com/jmcb/article-abstract/10/6/573/4600201 by Ed 'DeepDyve' Gillespie user on 15 January 2019 578 j Wang et al. Figure 4 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 10 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. −9 Untreated human neutrophils were allowed to migrate through a migration chamber toward PBS or FGF2 (10 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 chemo- −9 kines in vitro. Neutrophils were treated with FGF2 (10 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. neutrophil-related chemokine expression in vitro, including the pathways: RAS–RAF–MAPK, PI3K–AKT, signal transducer and acti- C-X-C chemokines CXCL1, IL-8 (CXCL8), IP10 (CXCL10), and vator of transcription (STAT), and phospholipase Cγ (PLCγ) CXCL12, as well as the CC chemokines MCP-1, MIP-1α, MIP-1β, (Turner and Grose, 2010). In this experiment, the application of RANTES, and eotaxin (Figure 4D). Thus, an FGF2 deﬁciency may FGF2 to neutrophils for 6 h did not lead to a signiﬁcant change impair neutrophil function by suppressing neutrophil recruit- in the levels of MAPK pathway members, including P38, JNK, ment and cytokine production. and ERK compared with untreated cells; thus, FGF2-induced acti- vation and neutrophil chemotaxis did not involve MAPK signal- FGF2 enhances NFκB phosphorylation to induce the activation ing. Moreover, STAT3 levels remained unchanged, and PLCγ and chemotaxis of neutrophils expression was not detected (data not shown) after FGF2 pro- According to a previous study, FGF receptors (FGFRs) are tein administration. MAPK and STAT3 signaling were still unchanged expressed by neutrophils (Haddad et al., 2011). Therefore, we following BJ501 infection (Supplementary Figure S5A). In con- examined whether H1N1 virus infection alters FGFR expression. trast, prominent PI3K and AKT phosphorylation were observed Surprisingly, FGFR2 levels were signiﬁcantly increased concomi- at 6 h after the application of FGF2 with or without BJ501 infec- tantly with reduced FGFR1 and FGFR4 expression in neutrophils tion (Figure 5B). NFκB has been conﬁrmed to act downstream of in vitro at 6 h after BJ501 infection. However, FGFR3 was the PI3K/AKT pathway in various cancers (Ghosh-Choudhury expressed at very low levels before or after IAV infection. The et al., 2010; Han et al., 2010; Lin et al., 2010), and AKT activates viral load (M1 level) was signiﬁcantly increased after BJ501 the NFκB pathway by phosphorylating and activating NFκB path- infection, indicating the establishment of a model of BJ501 way intermediates (Ozes et al., 1999; Romashkova and virus-infected neutrophils (Figure 5A). The mammalian FGF Makarov, 1999). However, the relationships between these two family comprises 18 ligands that signal through four conserved pathways downstream of FGF2 activation have not been fully tyrosine kinase receptors to activate four key downstream explored. In this study, inhibitor of NFκB kinase subunit beta Downloaded from https://academic.oup.com/jmcb/article-abstract/10/6/573/4600201 by Ed 'DeepDyve' Gillespie user on 15 January 2019 FGF2 protects against IAV-induced ALI j 579 Figure 5 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-) −9 and total proteins in neutrophils treated with FGF2 (10 M, 1.72 ng/ml) for 6 h. (C) Western blot analysis of signaling pathways in WT and –/– 5 FGF2 mice infected with 10 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. (IKKβ), IκB, and NFκB (RELA/p65) were phosphorylated in FGF2- absence of BJ501 infection (Supplementary Figure S5B). Based stimulated neutrophils. Meanwhile, the phosphorylation of on these results, activation of the FGFR2–PI3K–AKT–NFκB path- these signaling molecules was also enhanced following BJ501 way probably contributes to neutrophil recruitment, chemotaxis, infection (Figure 5B). or activation in IAV-infected lung tissues. –/– Four-week-old FGF2 or WT mice were i.n. infected with BJ501 at a titer of 10 TCID50 or mock infected to further con- The recombinant FGF2 protein protects mice from H1N1 ﬁrm the functional impact of FGF in vivo. The results of western infection by recruiting neutrophils blot analyses revealed a decrease in the levels of phosphory- Currently, the recombinant FGF2 protein (rFGF2) has been –/– lated PI3K, AKT, and NFκB in FGF2 mice infected with BJ501, used in various clinical settings, such as wound healing and but no changes were observed in the mock infected mice cornea repair (Fu et al., 1998, 2000; Huang et al., 2011). We (Figure 5C). However, the phosphorylation of members of the treated BJ501-infected mice with rFGF2 and/or neutrophil MAPK and STAT3 pathways were unchanged in the presence or neutralizing antibodies to further investigate whether FGF2 Downloaded from https://academic.oup.com/jmcb/article-abstract/10/6/573/4600201 by Ed 'DeepDyve' Gillespie user on 15 January 2019 580 j Wang et al. protected against IAV infection and determine whether the pro- lung histopathology and lung injury scores, as deﬁned by inﬁl- tective effects were associated with neutrophil recruitment. trated leukocyte counts, were observed in the IAV-infected mice First, 4-week-old WT B6 mice were i.n. infected with 10 TCID50 treated with rFGF2 (Figure 6D). Furthermore, rFGF2-treated mice of the BJ501 virus. Mice were pre-treated or i.v. treated with showed less viral replication in the infected lung than control anti-Ly6G(m1A8) antibodies or isotype control antibodies mice (Figure 6E). Similar results were obtained in mice chal- (m2A3) and FGF2. The survival rates of the BJ501-infected mice lenged with IAV strain PR8 (Supplementary Figure S6). More were signiﬁcantly increased, and weight loss was improved by importantly, more neutrophils were recruited to the lungs of the the rFGF2 treatment (Figure 6A and B). Moreover, the lung wet- FGF2 treatment group but not the anti-Ly6G group or anti-Ly6G to-dry weight ratio exhibited a greater improvement in the IAV- plus FGF2 group (Figure 6F). In addition, mice that had been pre- infected mice treated with rFGF2 (Figure 6C). Similarly, improved treated or treated with m1A8 antibodies showed signiﬁcantly Figure 6 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. Downloaded from https://academic.oup.com/jmcb/article-abstract/10/6/573/4600201 by Ed 'DeepDyve' Gillespie user on 15 January 2019 FGF2 protects against IAV-induced ALI j 581 lower survival rates, more severe body weight loss and lung ede- (Figure 7C and D). The results of the ﬂow cytometry analysis –/– ma than the isotype control antibody-treated mice. In contrast, showed the restoration of inﬁltrating neutrophils in FGF2 mice pretreatment with FGF2 and m1A8 antibodies did not protect the that received WT neutrophils (Figure 7F). These results conﬁrm mice. The histopathological alterations in the lung and viral repli- that mice that received the neutrophil transfer achieved better cation further supported these ﬁndings (Figure 6A–D). Thus, outcomes upon inﬂuenza infection. rFGF2 protects against H1N1 virus infection in vivo, and the recruited neutrophils were indispensable for the protective Discussion effects of FGF2. FGF2 is known to act as a pleiotropic factor in multiple bio- logical processes, including angiogenesis, embryonic develop- –/– Neutrophil transfer protects FGF2 mice against inﬂuenza ment, and wound healing (Ortega et al., 1998; Nugent and BJ501 virus infection Iozzo, 2000; Virag et al., 2007; Han and Gotlieb, 2012; Song Neutrophils were puriﬁed from the bone marrow of WT mice et al., 2015). FGF2 accelerates the healing of skin wounds in ani- –/– and adoptively transferred to FGF2 mice on Day 1 after infec- mal models as well as the healing of eye, retina, and corneal tion with 10 TCID50 of BJ501 to investigate whether neutro- wounds (Bikfalvi et al., 1997). FGF2 also has multiple functions –/– –/– phils protect FGF2 mice from inﬂuenza infection. FGF2 mice in the central nervous system (Tureyen et al., 2005; Frinchi that received an adoptive transfer of WT neutrophils had an et al., 2008; Li et al., 2008; Ma et al., 2008; Ma et al., 2009; increased survival rate and a better outcome of weight change Mimura et al., 2015). Furthermore, FGF2 promotes the epithe- after IAV infection (Figure 7A). The lung edema and virus load of lial–mesenchymal transition, invasiveness, and tumor angiogen- infected neutrophil-transferred mice were signiﬁcantly decreased esis (Marek et al., 2009; Narong and Leelawat, 2011; Wesche –/– compared with FGF2 mice (Figure 7B). In addition, the lung et al., 2011; Wang et al., 2015). FGF2 is expressed in normal tis- histopathology of neutrophil-transferred mice was greatly improved, sue and is upregulated by chronic inﬂammatory reactions (Byrd and the inﬁltrated leukocyte counts were signiﬁcantly decreased et al., 1996; Kanazawa et al., 2001; Song et al., 2015). The roles –/– –/– –/– Figure 7 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 trea- ted 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. Downloaded from https://academic.oup.com/jmcb/article-abstract/10/6/573/4600201 by Ed 'DeepDyve' Gillespie user on 15 January 2019 582 j Wang et al. of FGF2 in mediating the immune response to IAV infection have inﬂuence not only multiple aspects of the inﬂammatory and not been fully elucidated. immune responses but also antiviral defense, hematopoiesis, Our study is the ﬁrst to report that FGF2 levels were elevated angiogenesis, and ﬁbrogenesis (Cassatella, 1999). FGF2 also in serum from IAV-infected patients and BALF of mice infected enhances the recruitment of monocytes, T cells, and polymorph with H1N1 virus strain BJ501. Mice with an FGF2 deﬁciency due nuclear leukocytes (PMNs) to inﬂamed dermal sites (Zittermann to either gene knockout or protein inhibition using neutralizing and Issekutz, 2006b), and act as a chemotactic agent for the antibodies in vivo were more susceptible to IAV infection. recruitment of neutrophils (Byrd et al., 1996; Wempe et al., According to a previous report, FGF2 is a potent mitogen for 1997; Zittermann and Issekutz, 2006a; Haddad et al., 2011). many cell types, including airway smooth muscle cells, ﬁbroblasts, Consistent with the results from these publications, an FGF2 and endothelial cells (Redington et al., 2001). Additionally, FGF2 deﬁciency reduced leukocyte recruitment, particularly neutrophil is secreted by many types of immune cells, such as Treg cells, recruitment, accompanied by decreases in the levels of cyto- neutrophils, monocytes, macrophages, and T lymphocytes kines and chemokines in mouse BALF during the early phase of (Barrios et al., 1997; Byrd et al., 1999; Ohsaka et al., 2001; IAV infection in this study. Moreover, FGF2 enhanced neutrophil Haddad et al., 2011; Song et al., 2015). During H1N1 infection, recruitment in vitro and promoted the expression of neutrophil- neutrophils represent the majority of inﬁltrating immune cells in related chemokines, such as C-X-C and CC chemokines. inﬂamed tissues. In this study, epithelial cells were the major cel- Therefore, we speculate that the FGF2 deﬁciency impaired an lular source of FGF2 during H1N1-induced ALI. effective immune response to IAV infection and FGF2 recruited Humans infected with inﬂuenza A virus display a ‘cytokine neutrophils to the infected lung tissue during the early phase of storm’, which is caused by insufﬁcient control of excessive neu- H1N1 infection. Of course, additional mechanistic studies of the trophil recruitment to the lungs (Wang and Ma, 2008; Tisoncik protective roles of neutrophils and the ‘cytokine storm’ are et al., 2012; Brandes et al., 2013; Liu et al., 2016; Yang and warranted. Tang, 2016). However, neutrophils provide the ﬁrst line of FGFs signal through FGFRs to perform a multitude of physio- defense against invading microorganisms and contribute to the logical functions. FGFRs are expressed on many different cell ﬁne regulation of inﬂammatory and immune responses (Futosi types and regulate key cell behaviors, such as proliferation, dif- et al., 2013; Tecchio et al., 2014). Moreover, neutrophils have a ferentiation, and survival (Turner and Grose, 2010). Here, FGFR2 potent anti-microbial armamentarium that includes oxidant- expression was signiﬁcantly increased in neutrophils after BJ501 generating systems, powerful proteinases, and cationic peptides infection, whereas the levels of FGFR1 and FGFR4 were decreased, contained in granules (Suzuki et al., 2008). In addition, neutro- and FGFR3 was weakly expressed before and after infection. phils function as regulators of inﬂammatory and immune Furthermore, FGF2 contributed to leukocyte recruitment or responses by producing and releasing a large variety of cyto- enhanced neutrophil chemotaxis to infected lung tissue mainly kines and chemokines (Tecchio et al., 2014). This variety of through the FGFR2–PI3K–AKT–NFκB signaling pathway (Figure 8). cytokines produced by neutrophils enables them to signiﬁcantly FGF2 interacts with FGFR2, activating the PI3K–AKT–NFκB signal- ing pathway, which induces cytokine and chemokine production and promotes neutrophil chemotaxis through a feedback mech- anism, 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 infec- tion, 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 thera- peutic 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. In conclusion, FGF2 performs a critical protective function dur- ing the process of IAV infection. The combination of our clinical ﬁndings and results from the mouse model has revealed a crit- Figure 8 A schematic explaining the signaling mechanisms by which ical role for FGF2 in thepathogenesisofinﬂuenzaA(H1N1) FGF2 regulates neutrophil chemotaxis and IAV-induced ALI. virus-induced ALI and has suggested that FGF2 represents a Downloaded from https://academic.oup.com/jmcb/article-abstract/10/6/573/4600201 by Ed 'DeepDyve' Gillespie user on 15 January 2019 FGF2 protects against IAV-induced ALI j 583 5 4 promising potential therapeutic target in IAV-induced lung control or virus (10 TCID50 of A/Beijing/501/2009 or 10 TCID50 disease. of A/PR/8/1934). For the neutrophil depletion experiment, a neutrophil-speciﬁc antibody, anti-Ly-6G (clone 1A8), and an iso- Materials and methods type control antibody (clone 2A3) were purchased from Biox Viruses and cells Cell. WT mice were sequentially i.v. administered 50 μg of the The inﬂuenza viruses used in this study were inﬂuenza A anti-mouse Ly6G antibody (clone 1A8) or isotype control anti- (H1N1) virus strain A/Beijing/501/2009 (BJ501) and inﬂuenza A body (clone 2A3) 6 h before and 1 and 3 days after injection, as (H1N1) virus strain A/PR/8/1934 (PR8) obtained from the previously described (Kawanishi et al., 2016). Academy of Military Medical Sciences. The genomic sequence of the BJ501 strain is available in the GenBank database (acces- Viral titration sion number GQ223415). The strains were propagated in 9-to Virus titers were determined in supernatants of mouse lung –/– 11-day-old speciﬁc-pathogen-free (SPF) embryonated eggs via homogenates from FGF2 mice or WT mice on Day 5 after the allantoic route. Virus titers were determined based on an H1N1 infection, as previously described (Li et al., 2012; Wang assessment of the TCID50 using Madin-Darby canine kidney et al., 2013; Gu et al., 2016). (MDCK) cells, according to the Reed-Muench method. MDCK, A549 and BEAS-2B cells were purchased from ATCC; MLE-12 Survival rate and body weight changes cells were kindly provided by Dr Zhuowei Hu (Peking Union Four-week-old WT B6 mice were anesthetized with 50 μlof Medical College, PUMC). MDCK cells were cultured in DMEM 1%(w/v) pentobarbital sodium and i.n. inoculated with the (Gibco); A549, BEAS-2B, and MLE-12 cells were cultured in virus or vehicle control. The survival rates and body weights of DMEM/F-12(1:1) basic culture medium (Gibco); and neutrophils each group of 10 mice were monitored daily for 14 days. were cultured in RPMI-1640 medium (Gibco) supplemented with 10% FBS and 100 U/ml penicillin-streptomycin at 37°Cina Acute pulmonary edema (wet-to-dry ratio) humidiﬁed atmosphere containing 5%CO . Pulmonary edema was assessed by measuring and recording the lung wet/dry weight ratio as previously described (Li et al., Mice 2012). Four-week-old WT C57BL/6 (abbreviated B6) female mice (Experimental Animal Center of the Academy of Military Medical Adoptive transfer of neutrophils Sciences, Beijing, China) and 4-week-old FGF2 knockout mice (B6 Bone marrow neutrophils were isolated using Percoll (GE background, 010698, Jackson Laboratory, USA) were housed in Healthcare), as previously described (Mei et al., 2012). Brieﬂy, the animal facility at the Beijing Institute of Microbiology and bone marrow cells were harvested from the femurs of 4-week- Epidemiology in accordance with institutional guidelines. All old WT B6 mice and suspended in PBS before being layered on experimental protocols were approved by the Institutional Animal a 3-step Percoll (GE Healthcare) gradient (72%, 64%, and 52%), Care and Use Committees of the Beijing Institute of Microbiology which was centrifuged at 500× g for 30 min. The cells at the and Epidemiology (ID: SYXK2015-008) and all experiments were interface between 64% and 72% gradients were collected and performed in accordance with the approved guidelines. washed twice with PBS. More than 95% of the neutrophils were viable, as determined using the trypan blue exclusion method. Mouse infections For adoptive transfer, 5 × 10 neutrophils isolated from WT –/– Four-week-old WT B6 mice were anesthetized with 50 μlof mice were intravenously injected into FGF2 mice 24 h before 1%(w/v) pentobarbital sodium and then i.n. inoculated with the they were i.n. inoculated with the H1N1 virus (10 TCID50 of H1N1 virus or mock infected with PBS as a control. Survival A/Beijing/501/2009) as described above. rates, body weight changes, histology, acute pulmonary edema (wet-to-dry ratio), and cytokine levels were evaluated as previ- Flow cytometry ously described (Li et al., 2012; Wang et al., 2013; Gu et al., Cells in BALF were stained with anti-CD45-FITC, anti-Ly6G-PE, 2016). For the anti-FGF2 antibody treatment, anti-FGF2 anti- anti-F4/80-PE, anti-NK1.1-PE and anti-CD3-FITC antibodies (BD bodies or isotype control antibodies (100 μg/mouse, Abcam, Pharmingen) and analyzed using a FACSCalibur ﬂow cytometer Cat. No. ab33103)(Kujawski et al., 2008) were sequentially (BD Biosciences) to examine leukocyte marker expression. The administered i.v. at 6 h before and 1 and 3 days after injection data were analyzed using FlowJo software (Tree Star). Isotype (pretreatment group) or at 1, 3, and 5 days after injection (treat- controls were used for all samples. ment group) with the vehicle control or virus (10 TCID50 –/– 5 of A/Beijing/501/2009). For FGF2 mice, 10 TCID50 of Statistical analyses A/Beijing/501/2009 was used. For the rescue experiments, Measurements collected at a single time point were analyzed each mouse was sequentially i.v. inoculated with 25 μg of the using an ANOVA, and if a signiﬁcant difference among the recombinant murine FGF2 protein (Peprotech, Cat. No. 450-33) groups was observed, the results were further analyzed using a 12 h before and 1 and 3 days after injection with the vehicle two-tailed t-test. All analyses were performed using GraphPad Downloaded from https://academic.oup.com/jmcb/article-abstract/10/6/573/4600201 by Ed 'DeepDyve' Gillespie user on 15 January 2019 584 j Wang et al. Crouser, E.D., Shao, G., Julian, M.W., et al. (2009). Monocyte activation by Prism 5.0 software (GraphPad Software). P < 0.05 was con- necrotic cells is promoted by mitochondrial proteins and formyl peptide sidered statistically signiﬁcant. All experiments were per- receptors. Crit. 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Journal of Molecular Cell Biology – Oxford University Press
Published: Dec 1, 2018
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