Antiviral Research 119 (2015) 1–7
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Modulation of sterol biosynthesis regulates viral replication and cytokine production in influenza A virus infected human alveolar epithelial cells Kenrie P.Y. Hui, Denise I.T. Kuok, Sara S.R. Kang, Hung-Sing Li, Mandy M.T. Ng, Christine H.T. Bui, J.S. Malik Peiris, Renee W.Y. Chan, Michael C.W. Chan ⇑ Centre of Influenza Research, School of Public Health, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region, China
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Article history: Received 23 January 2015 Revised 18 March 2015 Accepted 8 April 2015 Available online 13 April 2015 Keywords: Influenza A virus Sterol biosynthesis Farnesyl transferase inhibitor Statin Zometa Cytokine
a b s t r a c t Highly pathogenic H5N1 viruses continue to transmit zoonotically, with mortality higher than 60%, and pose a pandemic threat. Antivirals remain the primary choice for treating H5N1 diseases and have their limitations. Encouraging findings highlight the beneficial effects of combined treatment of host targeting agents with immune-modulatory activities. This study evaluated the undefined roles of sterol metabolic pathway in viral replication and cytokine induction by H5N1 virus in human alveolar epithelial cells. The suppression of the sterol biosynthesis by Simvastatin in human alveolar epithelial cells led to reduction of virus replication and cytokine production by H5N1 virus. We further dissected the antiviral role of different regulators of the sterol metabolism, we showed that Zometa, FPT inhibitor III, but not GGTI-2133 had anti-viral activities against both H5N1 and H1N1 viruses. More importantly, FPT inhibitor III treatment significantly suppressed cytokine production by H5N1 virus infected alveolar epithelial cells. Since both viral replication itself and the effects of viral hyper-induction of cytokines contribute to the immunopathology of severe H5N1 disease, our findings highlights the therapeutic potential of FPT inhibitor III for severe human H5N1 diseases. Furthermore, our study is the first to dissect the roles of different steps in the sterol metabolic pathway in H5N1 virus replication and cytokine production. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Highly pathogenic avian influenza (HPAI) H5N1 viruses continue to transmit zoonotically, with mortality higher than 60%, and pose a pandemic threat. Antivirals such as oseltamivir, remain the primary therapy for the treatment of human H5N1 diseases. However, it is effective only when administered in the early stage of the infection and survival of patients cannot be assured even when treated within 4 days of clinical onset (Kandun et al., 2008). Significant improvements in survival rate and the pathogenesis of H5N1-infected mice were observed by combination treatment of antiviral and immune-modulatory drugs (Zheng et al., 2008). There is the need to investigate the effects of host-targeted antiviral agents with immune-modulatory activities for managing zoonotic avian influenza viruses with pandemic potential such as H5N1 and H7N9 viruses. Sterol biosynthesis is linked with antiviral activities of IFNs. IFN treatment reduced sterol metabolism and protects against ⇑ Corresponding author. Tel.: +852 3917 9800; fax: +852 2855 9587. E-mail address: [email protected] (M.C.W. Chan). http://dx.doi.org/10.1016/j.antiviral.2015.04.005 0166-3542/Ó 2015 Elsevier B.V. All rights reserved.
cytomegalovirus infection in mouse macrophages through down regulation of geranylgeraniol (Blanc et al., 2011). Interference on sterol metabolic pathway by drugs such as statins have shown both positive and negative effects on pneumonia and influenza infection in human and animal studies (An et al., 2011; Belser et al., 2013; Vandermeer et al., 2012). One major difference between low pathogenic and HPAI influenza virus is the increased release of cytokine and chemokine that occurs with the lethal infections (Chan et al., 2005; Cheung et al., 2002; Loo and Gale, 2007). Statins have been reported to have the anti-inflammatory properties and can regulate important molecules in vascular biology (van de Garde et al., 2006). However, the cellular responses to statins during influenza infection remain obscure. In addition, there are no reports on the effects of intervention of sterol metabolic pathways on influenza replication or immuno-modulation. We investigated the anti-viral and immuno-modulatory effects that result from pharmacological intervention of the sterol biosynthetic pathway on both seasonal H1N1 and HPAI H5N1 infection in primary human alveolar epithelial cells (AECs). The inhibitors studied and their action are depicted in Fig. 1. They are statin (Simvastatin), Zometa, FPT
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2.2. Reagents and drug treatment
Acetoacetyl-CoA HMGCS1 3-Hydroxy-3-methylglutaryl-CoA HMGCR Simvastan Mevalonate Isopentyl diphosphate (IPP) IDI1
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Simvastatin, and GGTI-2133 were purchased from Sigma Aldrich. Zometa (zoledronate) was purchased from Novartis Pharmaceuticals Ltd, and FPT inhibitor III was purchased from Santa Cruz Biotechnology. Cells were pretreated with inhibitors 60 min before infection and the same concentrations were maintained throughout the infection process.
GGTase I
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Geranylgeranylated proteins Fig. 1. Schematic of the mevalonate-isoprenylation of the sterol biosynthesis pathway. DMAPP, GPP, FPP, and GGPP are key intermediates in the isoprenoid biosynthetic pathway. HMGCR, farnesyl diphosphate synthase (FDPS), farnesyl transferase (FTase) and geranylgeranyl transferase I (GGTase I) are the relevant enzymes and inhibitors (statin – Simvastatin, Zometa, FPT inhibitor III and GGTI2133) are shown in the diagram.
inhibitor III, and GGTI-2133. Thus, we dissect which branch(es) of the sterol biosynthesis involves in virus replication and cytokine induction during influenza infection.
2. Materials and methods 2.1. Cell culture and virus infection Human primary AECs were isolated and cultured as described elsewhere (Chan et al., 2010). These studies were approved by the Institutional Review Board of The University of Hong Kong/ Hospital Authority Hong Kong West Cluster. HPAI A/Hong Kong/ 483/1997 (H5N1) and A/Oklahoma/447/2008 (H1N1) viruses were isolated from patients with fatal H5N1 disease and seasonal flu, respectively. These viruses were cultured and titrated in Madin-Darby canine kidney (MDCK) cells using tissue culture infection dose 50% (TCID50) for quantification. In brief, infection was performed in confluent monolayers of MDCK cells grown in 96-well plate format. Cells were replenished with 115 ll of serum free MEM medium. Viruses were serially diluted with a half log10 increment ranging from 0 log10 (neat) to 7.5 log10. 35 ll of diluted virus inocula were added to four replicates of the 96-well plate and incubated at 37 °C. Cytopathic effect of the cells was recorded after 3 days of incubation. TCID50 was calculated by using the Spearman–Karber method. Virus stocks were aliquoted and stored at 80 °C until use. Cells were infected at a multiplicity of infection (MOI) of 0.01 for replication kinetic analysis and MOI of 2 for cytokine expressions.
Total RNA was extracted using the RNEasy Mini Kit (Qiagen) with DNase treatment and reverse transcribed by one step PrimeScript RT Reagent Kit (Takara). The mRNA expression of viral M gene, cytokines (IFN-b) and chemokines (IP-10, MCP-1 and RANTES) by real-time quantitative PCR analysis using SYBR Premix Ex Taq (Takara) with specific primers on an Applied Biosystems ViiA 7 Real-Time PCR System (Life Technologies). The methods used for quantifying cytokine and beta-actin mRNA have been described previously (Hui et al., 2009). Briefly, the copy number of the target genes was calculated based on the standard curve generated with known number of copies of the target gene. The mRNA expression was normalized with the copy number of betaactin in each sample. The mRNA expression of SREBF2, HMGCS1, HMGCR and IDI1 was measured by using Taqman Primer probe sets purchased from Applied Biosystems and the preparation of reaction mixture followed those previously published (Blanc et al., 2011). The threshold cycle (CT) of target genes was calculated by the software provided by Applied Biosystems. The relative gene expression was calculated by delta-delta-CT method, which is the difference in threshold cycles for the treatment and mock samples after normalized with the CT of beta-actin in each samples. Fold change was presented using mock-treated cells as 1. 2.4. Cytokine assay Cell culture supernatants were harvested at 24 h post-infection (hpi) to measure cytokine and chemokine protein concentrations using Cytometric Bead Array (BD Bioscience) according to the manufacturer’s instructions. The levels of cytokine and chemokine were analysed using FCAP Array Analysis Software (BD Bioscience). Detection limits of IP-10, MCP-1 and RANTES are 34, 32, and 9 pg/ml. 2.5. Statistical analysis The two-tailed Student t test was used between vehicle-treated cells and drug-treated cells with the same virus infection. Results were considered statistically significant with P values < 0.05. Statistical analysis was performed using GraphPad Prism. 3. Results and discussion 3.1. Anti-viral and immuno-modulatory effects of statins in HPAI H5N1 virus infected AECs We tested the intracellular level of cholesterol after HPAI (H5N1) infection and treatment of IFN-b in AECs. The level of cholesterol was significantly reduced by both H5N1 infection and IFN-b treatment when compared to the mock infected cells (data not shown). We then measured the effect of IFN-b or HPAI H5N1 infection on the mRNA expression of the enzymes in the sterol biosynthesis, including 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1), 3-hydroxy-3-methyl-glutarylCoA reductase (HMGCR) and isopentenyl-diphosphate delta
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Fold Change vs Mock
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Fig. 2. Involvement of sterol biosynthesis pathway and Simvastatin in influenza replication and induction of immune responses in AECs. (A) Expression analysis measured by real time-PCR of SREBF2, HMGCS1, HMGCR and IDI1 genes in AECs treated with IFN-b at 25U/ml or infected with HPAI H5N1 at MOI 2 for 24 h. Gene expression in mock infected cells was defined as 1. ⁄⁄⁄P < 0.001 when compared with mock-treated cells. (B) AECs were pretreated with various concentrations (vehicle is indicated by 0 lM) of Simvastatin for 60 min before and after virus adsorption. Cells were infected with HPAI H5N1 at MOI 0.01. Virus production was measured in culture supernatants at 72 hpi by using TCID50. The titer at 0 lM was defined as 100%. Results are means ± SD of at least three separate experiments. ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001 when compared with vehicletreated (0 lM) and influenza-infected cells. (C) AECs were pretreated with vehicle (0 lM) or Simvastatin at 50 lM for 60 min before and after virus adsorption. The cells were infected with mock or H5N1 virus at MOI 2 for 24 h. Protein concentrations of the chemokines were detected by using CBA in culture supernatants. Results are means ± SD of a representative experiment taken from 3 separate experiments. ⁄P < 0.05; ⁄⁄⁄P < 0.001 when compared with vehicle-treated and H5N1-infected cells.
isomerase 1 (IDI1) and the transcription factor – sterol regulatory element binding transcription factor 2 (SREBF2), which regulates the gene expression of this pathway (Fig. 1). The mRNA expression of all four target genes was significantly suppressed by HPAI H5N1 infection or IFN-b by more than 62% (Fig. 2A). These results are in line with the findings that viral infection of mouse cytomegalovirus or IFN-b treatment reduced the sterol biosynthesis in mouse macrophages (Blanc et al., 2011). Using concentrations of Simvastatin without cytotoxicity, we observed that Simvastatin suppressed HPAI H5N1 replication in a dose-dependent manner (Fig. 2B) and the IC50 was 0.74 lM. Simvastatin at 50 lM also suppressed the replication of seasonal H1N1 virus (data not shown). HPAI H5N1 virus induces high level of cytokines and chemokines in patients. We next investigated the cytokine responses of HPAI H5N1 infection in AECs in the presence of Simvastatin. Simvastatin significantly reduced the protein levels of IP-10 and RANTES (Fig. 2C). MCP-1 was measured but the level was below the detection limit. Although there are conflicting results on the therapeutic benefits of statins in influenza models, our findings using the primary human alveolar epithelial cell model are in line with other reports that statins suppressed cytokine production by H5N1 infection (An et al., 2011; Belser et al., 2013).
3.2. Involvement of different regulators of sterol biosynthetic pathway in influenza A virus replication in AECs We next examined the anti-viral effects of various inhibitors of the sterol biosynthetic pathway on HPAI H5N1 infection in AECs by TCID50 assay. Cytotoxicity of all the drugs used in this study was measured at 72 h post-treatment by using LDH cytotoxicity assay. There was no cytotoxicity observed at the concentrations used (data not shown). Zometa significantly reduced HPAI H5N1 replication at 75 and 100 lM, while FPT inhibitor III suppressed H5N1 replication at 50 lM (Fig. 3). In contrast, GGTI-2133 did not affect the H5N1 replication in AECs (Fig. 3). We next tested their anti-viral activities on the low pathogenic human seasonal influenza H1N1 infection in AECs. In the presence of 75 and 100 lM Zometa, seasonal H1N1 replication was significantly suppressed (Fig. 3) and the IC50 was 54.88 lM. Stronger anti-viral effects on the low pathogenic seasonal H1N1 compared to that of HPAI H5N1 were observed when similar concentrations of FPT inhibitor III and GGTI-2133 were used (Fig. 3). GGTI-2133 also suppressed H1N1 influenza replication, although to a less marked degree. Previous reports show that Zometa inhibited hepatitis C virus replication (Agrati et al., 2006). FTI inhibitors suppressed hepatitis
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Fig. 3. Anti-viral effect of interfering sterol biosynthesis pathways on influenza virus replication in AECs. AECs were pretreated with Zometa, FPT inhibitor III (FPTIII), or GGTI-2133, for 60 min before infection. Cells were infected with HPAI H5N1 or a low pathogenic human seasonal H1N1 at MOI 0.01. Virus production was measured in culture supernatants at 72 hpi by using TCID50. (A) Virus titers were presented as percentage and the titer at 0 lM was defined as 100%. (B) Virus titers were presented as TCID50/ml. Results are means ± SD of at least three separate experiments. ⁄P < 0.05 when compared with vehicle-treated (0 lM) and virusinfected cells.
delta virus (Park et al., 2014) and herpes simplex virus 1 infection (Farassati et al., 2001). GGTI has anti-viral activities against HCV replication (Sagan et al., 2006). However, this is the first report demonstrating their anti-viral effects on influenza A viruses and dissecting which pathways of the sterol metabolism involved in influenza virus infection. We also found that the sensitivity of influenza viruses to these inhibitors is different between the highly pathogenic H5N1 and low pathogenic seasonal H1N1 viruses. The latter is more sensitive to Zometa, GGTI-2133 and FPT inhibitor III than H5N1 virus. 3.3. FPT inhibitor III reduces HPAI H5N1-induced cytokine responses but not Zometa and GGTI-2133 Patients with H5N1 disease have higher serum concentrations of cytokines and chemokines than patients with seasonal influenza. Fatal H5N1 cases have higher viral load and serum cytokine levels than those that survive (de Jong et al., 2006; Peiris et al., 2004). There is in vitro and in vivo studies demonstrating that cytokine dysregulation contributes to the pathogenesis of human H5N1 disease (Chan et al., 2005; Cheung et al., 2002; de Jong et al., 2006; Peiris et al., 2004). Therefore, the immuno-regulatory effects of Zometa, GGTI-2133 and FPT inhibitor III on HPAI H5N1 infection were further investigated. Cytokine and chemokine production was measured in culture supernatants from HPAI H5N1 infected AECs in the presence of various concentrations of the
drugs. The cytokine and chemokine induction by HPAI H5N1 virus was compared with corresponding vehicle-treated and infected cells. H5N1-induced mRNA expression of IFN-b, IP-10 and RANTES was up-regulated in the presence of Zometa in a dose-dependent manner compared to vehicle-treated and H5N1-infected cells (Fig. 4A). However, there was no significant changes on MCP-1 and viral M gene expression. Cytokine and chemokine production in HPAI H5N1 culture supernatants was measured by CBA. In contrast to the gene expression, there were only slight changes of protein levels of RANTES (10%) and MCP-1 (16%) while no difference of IP-10 was observed (Fig. 4B). Therefore, although Zometa up-regulated the transcription level of a number of cytokines and chemokines, there was only subtle difference detected at the protein level. The elevation of cytokine and chemokine expression is in line with findings of others that treatment with Zometa (or also known as zoledronic acid or zoledronate) up-regulated cytokines production in Chlamydia pneumonia-primed human osteoblast-like cells (Rizzo et al., 2014) and lipopolysaccharide-treated macrophages (Muratsu et al., 2013). A small increase (20%) of H5N1-induced IFN-b was observed in the presence of GGTI-2133 while there was no significant effects on the mRNA expression of IP-10, RANTES and viral M gene (Fig. 5A). Treatment of GGTI-2133 up-regulated HPAI H5N1-induced mRNA expression of MCP-1 but without statistical significances (Fig. 5A).The mRNA expression of cytokines and chemokines was consistent with the findings at protein level. The protein production of IP-10 and RANTES by H5N1 virus was not changed while there was a significant increase of H5N1-induced MCP-1 in culture supernatants elicited by the drug treatment (Fig. 5B). So far, GGTI has been shown to stimulate anti-melanoma immune response by up-regulation of membrane FasL and major histocompatibility complex (MHC) class I expression in murine melanoma cells (Sarrabayrouse et al., 2007; Tilkin-Mariame et al., 2005). However, there is no report on the regulation of immune response in terms of cytokine production by GGTI. Here we demonstrate for the first time that GGTI-2133 enhanced the production of H5N1-induced MCP-1 at both mRNA and protein levels. Among the three drugs, FPT inhibitor III has the strongest suppression on HPAI H5N1-induced cytokine responses. Treatment of FPT inhibitor III dramatically reduced the mRNA expression of all tested cytokines and chemokines including IFN-b, IP-10, RANTES, and MCP-1, and viral M gene in a dose-dependent manner at concentration as low as 5 lM (Fig. 6A). The suppressive effects on immune responses were confirmed by measuring the chemokine production in HPAI H5N1 culture supernatants. Treatment of FPT inhibitor III dramatically inhibited the production of IP-10 and RANTES H5N1-infected cultures (Fig. 6B). Protein levels of MCP-1 was too low to be detected in the culture supernatants. FPT inhibitor III has been reported to have anti-cancer properties through inhibition of p38 MAPK and ERK1/2 activation (Sribenja et al., 2013; Zhang et al., 2013) and anti-apoptotic properties in ovarian cancer cells (Hung and Chuang, 1998). Recent studies demonstrated that tipifarnib, a farnesyl transferase inhibitor, inhibited galactosamine/LPS-induced inflammatory cytokine production in mice (Shirozu et al., 2014) and treatment of FTI-277 prior to Streptococcal m1 protein challenge in mice, reduced the accumulation of neutrophils, edema formation, tissue damage and CXC chemokines production in the lung (Zhang et al., 2012). However, there is no previous report on the regulation of immune responses by farnesyl transferase inhibitors during influenza infection. Since cytokine dysregulation contributes, at least in part, to the pathogenesis of severe H5N1 disease, beneficial effects of combination therapies of anti-viral plus immuno-regulatory agent was observed in H5N1-infected mice (Zheng et al., 2008). The above results demonstrated that FPT inhibitor III has the highest potential among the three as a novel therapeutic option because of its strong
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Fig. 4. Immuno-regulatory effects of Zometa on HPAI H5N1 infection in AECs. AECs were pretreated with vehicle (0 lM) or Zometa at 25, 50, 75 and 100 lM for 60 min before infection. Cells were infected with mock or HPAI H5N1 virus at MOI 2 for 24 h. After adsorption with virus, the cells were replenished with the same concentrations of drug as those before infection. (A) mRNA expression of viral M gene, cytokines and chemokines was measured by real-time PCR. (B) Protein concentrations of the chemokines were detected by using CBA in culture supernatants. Results are means ± SD of a representative experiment taken from 3 separate experiments. ⁄P < 0.05; ⁄⁄P < 0.01 when compared with vehicle-treated and H5N1-infected cells.
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Fig. 5. Immuno-regulatory effects of GGTI-2133 on HPAI H5N1 infection in AECs. AECs were pretreated with vehicle (0 lM) or GGTI-2133 (GGTI) at 5, 10, and 25 lM for 60 min before infection. Cells were infected with mock or HPAI H5N1 virus at MOI 2 for 24 h. After adsorption with virus, the cells were replenished with the same concentrations of drug as those before infection. (A) mRNA expression of viral M gene, cytokines and chemokines was measured by real-time PCR. (B) Protein concentrations of the chemokines were detected by using CBA in culture supernatants. Results are means ± SD of a representative experiment taken from 3 separate experiments. ⁄P < 0.05; ⁄⁄ P < 0.01 when compared with vehicle-treated and H5N1-infected cells.
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Fig. 6. Immuno-regulatory effect of FPT inhibitor III on HPAI H5N1 infection in AECs. AECs were pretreated with vehicle (0 lM) or FPT inhibitor III (FPTIII) at 5, 10, 25 and 50 lM for 60 min before infection. Cells were infected with mock or H5N1 virus at MOI 2. After adsorption with virus, the cells were replenished with the same concentrations of drug as those before infection. (A) mRNA expression of viral M gene, cytokines and chemokines was measured at 24 hpi. (B) Protein concentrations of the chemokines were detected by using CBA in culture supernatants collected at 24 hpi. Results are means ± SD of a representative experiment taken from 3 separate experiments. ⁄P < 0.05; ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001 when compared with vehicle-treated and H5N1-infected cells.
immuno-regulatory activities shown in Fig. 6, together with a modest antiviral effect (Fig. 3). By using the above inhibitors, we have evaluated the immunoregulatory role of different branches of sterol metabolic pathway during HPAI H5N1 infection. We demonstrated here that FPT inhibitor III, which is the farnesylation inhibitor, has potent suppression effects on HPAI H5N1-induced immune responses and has modest antiviral activity. These results imply that farnesylation of the sterol biosynthesis pathway is involved in the cytokine and chemokine induction during H5N1 infection. The mechanisms of farnesylation in regulating immune responses in influenza infection have not been reported and further study is needed and this may reveal additional potential targets for therapy of severe influenza disease.
4. Conclusion Host-targeting strategies are a new direction in treating severe human influenza disease. There are controversies on the use of statins against influenza infection in in vitro cell and, animal models. However, there is not much information on in vitro study of statins and influenza infection. We first demonstrated that the gene expression of the sterol metabolic pathway was suppressed by both influenza infection and by treatment with IFN-b, which is in line with the previous report showing that suppression of sterol biosynthesis is one of the anti-viral activities of IFNs (Blanc et al., 2011). We next showed that a number of inhibitors of the sterol biosynthetic pathway possess anti-viral effects against both HPAI H5N1 and low pathogenic human seasonal H1N1 virus infection in AECs. By using these inhibitors to block different branches of the sterol metabolic pathway, we found that Simvastatin has the strongest anti-viral effects against H5N1. This may be due to the
fact that Simvastatin inhibits HMGCR and leads to the blockage of the whole mevalonate pathway. While the other inhibitors are downstream of the mevalonate pathway, we found that each branch of the sterol pathway partially contributes to the viral replication. We also showed that the low pathogenic human seasonal H1N1 (447/08) was more sensitive to the inhibitors than HPAI H5N1 (483/97). There may be strain differences for the dependence on sterol biosynthetic pathway. HPAI H5N1 viruses significantly induce more cytokines and chemokines in patients when compared to the low pathogenic seasonal influenza viruses. Cytokine storm is believed to be one of the mechanisms of pathogenesis of severe H5N1 disease in humans. Beneficial effects were observed when combination of antiviral drugs and immuno-modulator were administered in HPAI influenza infected mice (Zheng et al., 2008). We have shown here that FPT inhibitor III has potent suppressive effects on HPAI H5N1-induced immune responses, and significant inhibition of viral replication at mRNA expression and progeny virus production. This study aims at understanding the anti-viral and immuno-regulatory roles of the sterol biosynthesis pathways during influenza infection by using a number of pharmacological inhibitors. Taken all together, our data demonstrate a previously undefined regulatory role of farnesylation of the sterol biosynthetic pathway on H5N1induced cytokine responses and suggest that farnesyl transferase inhibitors may represent novel therapeutic options for immunemodulation in severe influenza diseases.
5. Funding We acknowledge research funding from National Institute of Allergy and Infectious Diseases (NIAID), under CEIRS contract No. HHSN272201400006C, Area of Excellence Scheme of the
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University Grants Committee (Grant AoE/M-12/96), Research Grants Council of the Hong Kong Special Administrative Region, China (Project No. T11-705/14N) and Seed Funding for Basic Research (reference no: 201309159004), The University of Hong Kong. 6. Author contributions KPYH: Study design, analysis and interpretation of results, perform experiments and writing of the manuscript; SSRK, DITK, HSL, MMTN, CHTB: Perform experiments, analysis and interpretation of results; JSMP: Study design, analysis and interpretation of results and critical review of the manuscript; RWYC: Study design, perform experiments, analysis and interpretation of results; MCWC: Overall coordination, study design, analysis and interpretation of results, perform experiments and writing of the manuscript. All authors declare no competing financial interests. Acknowledgments Dr. Alan DL Sihoe and staff from the Division of Cardiothoracic Surgery, Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong and Queen Mary Hospital provided the human lung tissues. A/Oklahoma/447/08 virus was provided by Gillian Air (University of Oklahoma Health Sciences Center). Dr. Rachel Ching, Ms. Joanne Fong, Mr. Man Chun Cheung from the Centre of Influenza Research, School of Public Health, Li Ka Shing Faculty of Medicine, The University of Hong Kong and Mr. Kevin Fung in the Department of Pathology, The University of Hong Kong provided technical support. References Agrati, C., Alonzi, T., De Santis, R., Castilletti, C., Abbate, I., Capobianchi, M.R., D’Offizi, G., Siepi, F., Fimia, G.M., Tripodi, M., Poccia, F., 2006. Activation of Vgamma9Vdelta2 T cells by non-peptidic antigens induces the inhibition of subgenomic HCV replication. Int. Immunol. 18, 11–18. An, S.C., Xu, L.L., Li, F.D., Bao, L.L., Qin, C., Gao, Z.C., 2011. Triple combinations of neuraminidase inhibitors, statins and fibrates benefit the survival of patients with lethal avian influenza pandemic. Med. Hypotheses 77, 1054–1057. Belser, J.A., Szretter, K.J., Katz, J.M., Tumpey, T.M., 2013. Simvastatin and oseltamivir combination therapy does not improve the effectiveness of oseltamivir alone following highly pathogenic avian H5N1 influenza virus infection in mice. Virology 439, 42–46. Blanc, M., Hsieh, W.Y., Robertson, K.A., Watterson, S., Shui, G., Lacaze, P., Khondoker, M., Dickinson, P., Sing, G., Rodriguez-Martin, S., Phelan, P., Forster, T., Strobl, B., Muller, M., Riemersma, R., Osborne, T., Wenk, M.R., Angulo, A., Ghazal, P., 2011. Host defense against viral infection involves interferon mediated downregulation of sterol biosynthesis. PLoS Biol. 9, e1000598. Chan, M.C., Cheung, C.Y., Chui, W.H., Tsao, S.W., Nicholls, J.M., Chan, Y.O., Chan, R.W., Long, H.T., Poon, L.L., Guan, Y., Peiris, J.S., 2005. Proinflammatory cytokine responses induced by influenza A (H5N1) viruses in primary human alveolar and bronchial epithelial cells. Respir. Res. 6, 135. Chan, M.C., Chan, R.W., Yu, W.C., Ho, C.C., Yuen, K.M., Fong, J.H., Tang, L.L., Lai, W.W., Lo, A.C., Chui, W.H., Sihoe, A.D., Kwong, D.L., Wong, D.S., Tsao, G.S., Poon, L.L., Guan, Y., Nicholls, J.M., Peiris, J.S., 2010. Tropism and innate host responses of the 2009 pandemic H1N1 influenza virus in ex vivo and in vitro cultures of human conjunctiva and respiratory tract. Am. J. Pathol. 176, 1828–1840. Cheung, C.Y., Poon, L.L., Lau, A.S., Luk, W., Lau, Y.L., Shortridge, K.F., Gordon, S., Guan, Y., Peiris, J.S., 2002. Induction of proinflammatory cytokines in human macrophages by influenza A (H5N1) viruses: a mechanism for the unusual severity of human disease? Lancet 360, 1831–1837. de Jong, M.D., Simmons, C.P., Thanh, T.T., Hien, V.M., Smith, G.J., Chau, T.N., Hoang, D.M., Chau, N.V., Khanh, T.H., Dong, V.C., Qui, P.T., Cam, B.V., Ha do, Q., Guan, Y.,
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Modulation of sterol biosynthesis regulates viral replication and cytokine production in influenza A virus infected human alveolar epithelial cells Kenrie P.Y. Hui, Denise I.T. Kuok, Sara S.R. Kang, Hung-Sing Li, Mandy M.T. Ng, Christine H.T. Bui, J.S. Malik Peiris, Renee W.Y. Chan, Michael C.W. Chan ⇑ Centre of Influenza Research, School of Public Health, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region, China
a r t i c l e
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Article history: Received 23 January 2015 Revised 18 March 2015 Accepted 8 April 2015 Available online 13 April 2015 Keywords: Influenza A virus Sterol biosynthesis Farnesyl transferase inhibitor Statin Zometa Cytokine
a b s t r a c t Highly pathogenic H5N1 viruses continue to transmit zoonotically, with mortality higher than 60%, and pose a pandemic threat. Antivirals remain the primary choice for treating H5N1 diseases and have their limitations. Encouraging findings highlight the beneficial effects of combined treatment of host targeting agents with immune-modulatory activities. This study evaluated the undefined roles of sterol metabolic pathway in viral replication and cytokine induction by H5N1 virus in human alveolar epithelial cells. The suppression of the sterol biosynthesis by Simvastatin in human alveolar epithelial cells led to reduction of virus replication and cytokine production by H5N1 virus. We further dissected the antiviral role of different regulators of the sterol metabolism, we showed that Zometa, FPT inhibitor III, but not GGTI-2133 had anti-viral activities against both H5N1 and H1N1 viruses. More importantly, FPT inhibitor III treatment significantly suppressed cytokine production by H5N1 virus infected alveolar epithelial cells. Since both viral replication itself and the effects of viral hyper-induction of cytokines contribute to the immunopathology of severe H5N1 disease, our findings highlights the therapeutic potential of FPT inhibitor III for severe human H5N1 diseases. Furthermore, our study is the first to dissect the roles of different steps in the sterol metabolic pathway in H5N1 virus replication and cytokine production. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Highly pathogenic avian influenza (HPAI) H5N1 viruses continue to transmit zoonotically, with mortality higher than 60%, and pose a pandemic threat. Antivirals such as oseltamivir, remain the primary therapy for the treatment of human H5N1 diseases. However, it is effective only when administered in the early stage of the infection and survival of patients cannot be assured even when treated within 4 days of clinical onset (Kandun et al., 2008). Significant improvements in survival rate and the pathogenesis of H5N1-infected mice were observed by combination treatment of antiviral and immune-modulatory drugs (Zheng et al., 2008). There is the need to investigate the effects of host-targeted antiviral agents with immune-modulatory activities for managing zoonotic avian influenza viruses with pandemic potential such as H5N1 and H7N9 viruses. Sterol biosynthesis is linked with antiviral activities of IFNs. IFN treatment reduced sterol metabolism and protects against ⇑ Corresponding author. Tel.: +852 3917 9800; fax: +852 2855 9587. E-mail address: [email protected] (M.C.W. Chan). http://dx.doi.org/10.1016/j.antiviral.2015.04.005 0166-3542/Ó 2015 Elsevier B.V. All rights reserved.
cytomegalovirus infection in mouse macrophages through down regulation of geranylgeraniol (Blanc et al., 2011). Interference on sterol metabolic pathway by drugs such as statins have shown both positive and negative effects on pneumonia and influenza infection in human and animal studies (An et al., 2011; Belser et al., 2013; Vandermeer et al., 2012). One major difference between low pathogenic and HPAI influenza virus is the increased release of cytokine and chemokine that occurs with the lethal infections (Chan et al., 2005; Cheung et al., 2002; Loo and Gale, 2007). Statins have been reported to have the anti-inflammatory properties and can regulate important molecules in vascular biology (van de Garde et al., 2006). However, the cellular responses to statins during influenza infection remain obscure. In addition, there are no reports on the effects of intervention of sterol metabolic pathways on influenza replication or immuno-modulation. We investigated the anti-viral and immuno-modulatory effects that result from pharmacological intervention of the sterol biosynthetic pathway on both seasonal H1N1 and HPAI H5N1 infection in primary human alveolar epithelial cells (AECs). The inhibitors studied and their action are depicted in Fig. 1. They are statin (Simvastatin), Zometa, FPT
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2.2. Reagents and drug treatment
Acetoacetyl-CoA HMGCS1 3-Hydroxy-3-methylglutaryl-CoA HMGCR Simvastan Mevalonate Isopentyl diphosphate (IPP) IDI1
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+IPP FPT inhibitor III GGPP GGTase II
Simvastatin, and GGTI-2133 were purchased from Sigma Aldrich. Zometa (zoledronate) was purchased from Novartis Pharmaceuticals Ltd, and FPT inhibitor III was purchased from Santa Cruz Biotechnology. Cells were pretreated with inhibitors 60 min before infection and the same concentrations were maintained throughout the infection process.
GGTase I
GGTI2133
Geranylgeranylated proteins Fig. 1. Schematic of the mevalonate-isoprenylation of the sterol biosynthesis pathway. DMAPP, GPP, FPP, and GGPP are key intermediates in the isoprenoid biosynthetic pathway. HMGCR, farnesyl diphosphate synthase (FDPS), farnesyl transferase (FTase) and geranylgeranyl transferase I (GGTase I) are the relevant enzymes and inhibitors (statin – Simvastatin, Zometa, FPT inhibitor III and GGTI2133) are shown in the diagram.
inhibitor III, and GGTI-2133. Thus, we dissect which branch(es) of the sterol biosynthesis involves in virus replication and cytokine induction during influenza infection.
2. Materials and methods 2.1. Cell culture and virus infection Human primary AECs were isolated and cultured as described elsewhere (Chan et al., 2010). These studies were approved by the Institutional Review Board of The University of Hong Kong/ Hospital Authority Hong Kong West Cluster. HPAI A/Hong Kong/ 483/1997 (H5N1) and A/Oklahoma/447/2008 (H1N1) viruses were isolated from patients with fatal H5N1 disease and seasonal flu, respectively. These viruses were cultured and titrated in Madin-Darby canine kidney (MDCK) cells using tissue culture infection dose 50% (TCID50) for quantification. In brief, infection was performed in confluent monolayers of MDCK cells grown in 96-well plate format. Cells were replenished with 115 ll of serum free MEM medium. Viruses were serially diluted with a half log10 increment ranging from 0 log10 (neat) to 7.5 log10. 35 ll of diluted virus inocula were added to four replicates of the 96-well plate and incubated at 37 °C. Cytopathic effect of the cells was recorded after 3 days of incubation. TCID50 was calculated by using the Spearman–Karber method. Virus stocks were aliquoted and stored at 80 °C until use. Cells were infected at a multiplicity of infection (MOI) of 0.01 for replication kinetic analysis and MOI of 2 for cytokine expressions.
Total RNA was extracted using the RNEasy Mini Kit (Qiagen) with DNase treatment and reverse transcribed by one step PrimeScript RT Reagent Kit (Takara). The mRNA expression of viral M gene, cytokines (IFN-b) and chemokines (IP-10, MCP-1 and RANTES) by real-time quantitative PCR analysis using SYBR Premix Ex Taq (Takara) with specific primers on an Applied Biosystems ViiA 7 Real-Time PCR System (Life Technologies). The methods used for quantifying cytokine and beta-actin mRNA have been described previously (Hui et al., 2009). Briefly, the copy number of the target genes was calculated based on the standard curve generated with known number of copies of the target gene. The mRNA expression was normalized with the copy number of betaactin in each sample. The mRNA expression of SREBF2, HMGCS1, HMGCR and IDI1 was measured by using Taqman Primer probe sets purchased from Applied Biosystems and the preparation of reaction mixture followed those previously published (Blanc et al., 2011). The threshold cycle (CT) of target genes was calculated by the software provided by Applied Biosystems. The relative gene expression was calculated by delta-delta-CT method, which is the difference in threshold cycles for the treatment and mock samples after normalized with the CT of beta-actin in each samples. Fold change was presented using mock-treated cells as 1. 2.4. Cytokine assay Cell culture supernatants were harvested at 24 h post-infection (hpi) to measure cytokine and chemokine protein concentrations using Cytometric Bead Array (BD Bioscience) according to the manufacturer’s instructions. The levels of cytokine and chemokine were analysed using FCAP Array Analysis Software (BD Bioscience). Detection limits of IP-10, MCP-1 and RANTES are 34, 32, and 9 pg/ml. 2.5. Statistical analysis The two-tailed Student t test was used between vehicle-treated cells and drug-treated cells with the same virus infection. Results were considered statistically significant with P values < 0.05. Statistical analysis was performed using GraphPad Prism. 3. Results and discussion 3.1. Anti-viral and immuno-modulatory effects of statins in HPAI H5N1 virus infected AECs We tested the intracellular level of cholesterol after HPAI (H5N1) infection and treatment of IFN-b in AECs. The level of cholesterol was significantly reduced by both H5N1 infection and IFN-b treatment when compared to the mock infected cells (data not shown). We then measured the effect of IFN-b or HPAI H5N1 infection on the mRNA expression of the enzymes in the sterol biosynthesis, including 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1), 3-hydroxy-3-methyl-glutarylCoA reductase (HMGCR) and isopentenyl-diphosphate delta
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Fold Change vs Mock
A Mock H5N1 IFN-β (25U/ml)
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Fig. 2. Involvement of sterol biosynthesis pathway and Simvastatin in influenza replication and induction of immune responses in AECs. (A) Expression analysis measured by real time-PCR of SREBF2, HMGCS1, HMGCR and IDI1 genes in AECs treated with IFN-b at 25U/ml or infected with HPAI H5N1 at MOI 2 for 24 h. Gene expression in mock infected cells was defined as 1. ⁄⁄⁄P < 0.001 when compared with mock-treated cells. (B) AECs were pretreated with various concentrations (vehicle is indicated by 0 lM) of Simvastatin for 60 min before and after virus adsorption. Cells were infected with HPAI H5N1 at MOI 0.01. Virus production was measured in culture supernatants at 72 hpi by using TCID50. The titer at 0 lM was defined as 100%. Results are means ± SD of at least three separate experiments. ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001 when compared with vehicletreated (0 lM) and influenza-infected cells. (C) AECs were pretreated with vehicle (0 lM) or Simvastatin at 50 lM for 60 min before and after virus adsorption. The cells were infected with mock or H5N1 virus at MOI 2 for 24 h. Protein concentrations of the chemokines were detected by using CBA in culture supernatants. Results are means ± SD of a representative experiment taken from 3 separate experiments. ⁄P < 0.05; ⁄⁄⁄P < 0.001 when compared with vehicle-treated and H5N1-infected cells.
isomerase 1 (IDI1) and the transcription factor – sterol regulatory element binding transcription factor 2 (SREBF2), which regulates the gene expression of this pathway (Fig. 1). The mRNA expression of all four target genes was significantly suppressed by HPAI H5N1 infection or IFN-b by more than 62% (Fig. 2A). These results are in line with the findings that viral infection of mouse cytomegalovirus or IFN-b treatment reduced the sterol biosynthesis in mouse macrophages (Blanc et al., 2011). Using concentrations of Simvastatin without cytotoxicity, we observed that Simvastatin suppressed HPAI H5N1 replication in a dose-dependent manner (Fig. 2B) and the IC50 was 0.74 lM. Simvastatin at 50 lM also suppressed the replication of seasonal H1N1 virus (data not shown). HPAI H5N1 virus induces high level of cytokines and chemokines in patients. We next investigated the cytokine responses of HPAI H5N1 infection in AECs in the presence of Simvastatin. Simvastatin significantly reduced the protein levels of IP-10 and RANTES (Fig. 2C). MCP-1 was measured but the level was below the detection limit. Although there are conflicting results on the therapeutic benefits of statins in influenza models, our findings using the primary human alveolar epithelial cell model are in line with other reports that statins suppressed cytokine production by H5N1 infection (An et al., 2011; Belser et al., 2013).
3.2. Involvement of different regulators of sterol biosynthetic pathway in influenza A virus replication in AECs We next examined the anti-viral effects of various inhibitors of the sterol biosynthetic pathway on HPAI H5N1 infection in AECs by TCID50 assay. Cytotoxicity of all the drugs used in this study was measured at 72 h post-treatment by using LDH cytotoxicity assay. There was no cytotoxicity observed at the concentrations used (data not shown). Zometa significantly reduced HPAI H5N1 replication at 75 and 100 lM, while FPT inhibitor III suppressed H5N1 replication at 50 lM (Fig. 3). In contrast, GGTI-2133 did not affect the H5N1 replication in AECs (Fig. 3). We next tested their anti-viral activities on the low pathogenic human seasonal influenza H1N1 infection in AECs. In the presence of 75 and 100 lM Zometa, seasonal H1N1 replication was significantly suppressed (Fig. 3) and the IC50 was 54.88 lM. Stronger anti-viral effects on the low pathogenic seasonal H1N1 compared to that of HPAI H5N1 were observed when similar concentrations of FPT inhibitor III and GGTI-2133 were used (Fig. 3). GGTI-2133 also suppressed H1N1 influenza replication, although to a less marked degree. Previous reports show that Zometa inhibited hepatitis C virus replication (Agrati et al., 2006). FTI inhibitors suppressed hepatitis
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B
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Fig. 3. Anti-viral effect of interfering sterol biosynthesis pathways on influenza virus replication in AECs. AECs were pretreated with Zometa, FPT inhibitor III (FPTIII), or GGTI-2133, for 60 min before infection. Cells were infected with HPAI H5N1 or a low pathogenic human seasonal H1N1 at MOI 0.01. Virus production was measured in culture supernatants at 72 hpi by using TCID50. (A) Virus titers were presented as percentage and the titer at 0 lM was defined as 100%. (B) Virus titers were presented as TCID50/ml. Results are means ± SD of at least three separate experiments. ⁄P < 0.05 when compared with vehicle-treated (0 lM) and virusinfected cells.
delta virus (Park et al., 2014) and herpes simplex virus 1 infection (Farassati et al., 2001). GGTI has anti-viral activities against HCV replication (Sagan et al., 2006). However, this is the first report demonstrating their anti-viral effects on influenza A viruses and dissecting which pathways of the sterol metabolism involved in influenza virus infection. We also found that the sensitivity of influenza viruses to these inhibitors is different between the highly pathogenic H5N1 and low pathogenic seasonal H1N1 viruses. The latter is more sensitive to Zometa, GGTI-2133 and FPT inhibitor III than H5N1 virus. 3.3. FPT inhibitor III reduces HPAI H5N1-induced cytokine responses but not Zometa and GGTI-2133 Patients with H5N1 disease have higher serum concentrations of cytokines and chemokines than patients with seasonal influenza. Fatal H5N1 cases have higher viral load and serum cytokine levels than those that survive (de Jong et al., 2006; Peiris et al., 2004). There is in vitro and in vivo studies demonstrating that cytokine dysregulation contributes to the pathogenesis of human H5N1 disease (Chan et al., 2005; Cheung et al., 2002; de Jong et al., 2006; Peiris et al., 2004). Therefore, the immuno-regulatory effects of Zometa, GGTI-2133 and FPT inhibitor III on HPAI H5N1 infection were further investigated. Cytokine and chemokine production was measured in culture supernatants from HPAI H5N1 infected AECs in the presence of various concentrations of the
drugs. The cytokine and chemokine induction by HPAI H5N1 virus was compared with corresponding vehicle-treated and infected cells. H5N1-induced mRNA expression of IFN-b, IP-10 and RANTES was up-regulated in the presence of Zometa in a dose-dependent manner compared to vehicle-treated and H5N1-infected cells (Fig. 4A). However, there was no significant changes on MCP-1 and viral M gene expression. Cytokine and chemokine production in HPAI H5N1 culture supernatants was measured by CBA. In contrast to the gene expression, there were only slight changes of protein levels of RANTES (10%) and MCP-1 (16%) while no difference of IP-10 was observed (Fig. 4B). Therefore, although Zometa up-regulated the transcription level of a number of cytokines and chemokines, there was only subtle difference detected at the protein level. The elevation of cytokine and chemokine expression is in line with findings of others that treatment with Zometa (or also known as zoledronic acid or zoledronate) up-regulated cytokines production in Chlamydia pneumonia-primed human osteoblast-like cells (Rizzo et al., 2014) and lipopolysaccharide-treated macrophages (Muratsu et al., 2013). A small increase (20%) of H5N1-induced IFN-b was observed in the presence of GGTI-2133 while there was no significant effects on the mRNA expression of IP-10, RANTES and viral M gene (Fig. 5A). Treatment of GGTI-2133 up-regulated HPAI H5N1-induced mRNA expression of MCP-1 but without statistical significances (Fig. 5A).The mRNA expression of cytokines and chemokines was consistent with the findings at protein level. The protein production of IP-10 and RANTES by H5N1 virus was not changed while there was a significant increase of H5N1-induced MCP-1 in culture supernatants elicited by the drug treatment (Fig. 5B). So far, GGTI has been shown to stimulate anti-melanoma immune response by up-regulation of membrane FasL and major histocompatibility complex (MHC) class I expression in murine melanoma cells (Sarrabayrouse et al., 2007; Tilkin-Mariame et al., 2005). However, there is no report on the regulation of immune response in terms of cytokine production by GGTI. Here we demonstrate for the first time that GGTI-2133 enhanced the production of H5N1-induced MCP-1 at both mRNA and protein levels. Among the three drugs, FPT inhibitor III has the strongest suppression on HPAI H5N1-induced cytokine responses. Treatment of FPT inhibitor III dramatically reduced the mRNA expression of all tested cytokines and chemokines including IFN-b, IP-10, RANTES, and MCP-1, and viral M gene in a dose-dependent manner at concentration as low as 5 lM (Fig. 6A). The suppressive effects on immune responses were confirmed by measuring the chemokine production in HPAI H5N1 culture supernatants. Treatment of FPT inhibitor III dramatically inhibited the production of IP-10 and RANTES H5N1-infected cultures (Fig. 6B). Protein levels of MCP-1 was too low to be detected in the culture supernatants. FPT inhibitor III has been reported to have anti-cancer properties through inhibition of p38 MAPK and ERK1/2 activation (Sribenja et al., 2013; Zhang et al., 2013) and anti-apoptotic properties in ovarian cancer cells (Hung and Chuang, 1998). Recent studies demonstrated that tipifarnib, a farnesyl transferase inhibitor, inhibited galactosamine/LPS-induced inflammatory cytokine production in mice (Shirozu et al., 2014) and treatment of FTI-277 prior to Streptococcal m1 protein challenge in mice, reduced the accumulation of neutrophils, edema formation, tissue damage and CXC chemokines production in the lung (Zhang et al., 2012). However, there is no previous report on the regulation of immune responses by farnesyl transferase inhibitors during influenza infection. Since cytokine dysregulation contributes, at least in part, to the pathogenesis of severe H5N1 disease, beneficial effects of combination therapies of anti-viral plus immuno-regulatory agent was observed in H5N1-infected mice (Zheng et al., 2008). The above results demonstrated that FPT inhibitor III has the highest potential among the three as a novel therapeutic option because of its strong
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Fig. 4. Immuno-regulatory effects of Zometa on HPAI H5N1 infection in AECs. AECs were pretreated with vehicle (0 lM) or Zometa at 25, 50, 75 and 100 lM for 60 min before infection. Cells were infected with mock or HPAI H5N1 virus at MOI 2 for 24 h. After adsorption with virus, the cells were replenished with the same concentrations of drug as those before infection. (A) mRNA expression of viral M gene, cytokines and chemokines was measured by real-time PCR. (B) Protein concentrations of the chemokines were detected by using CBA in culture supernatants. Results are means ± SD of a representative experiment taken from 3 separate experiments. ⁄P < 0.05; ⁄⁄P < 0.01 when compared with vehicle-treated and H5N1-infected cells.
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Fig. 5. Immuno-regulatory effects of GGTI-2133 on HPAI H5N1 infection in AECs. AECs were pretreated with vehicle (0 lM) or GGTI-2133 (GGTI) at 5, 10, and 25 lM for 60 min before infection. Cells were infected with mock or HPAI H5N1 virus at MOI 2 for 24 h. After adsorption with virus, the cells were replenished with the same concentrations of drug as those before infection. (A) mRNA expression of viral M gene, cytokines and chemokines was measured by real-time PCR. (B) Protein concentrations of the chemokines were detected by using CBA in culture supernatants. Results are means ± SD of a representative experiment taken from 3 separate experiments. ⁄P < 0.05; ⁄⁄ P < 0.01 when compared with vehicle-treated and H5N1-infected cells.
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Fig. 6. Immuno-regulatory effect of FPT inhibitor III on HPAI H5N1 infection in AECs. AECs were pretreated with vehicle (0 lM) or FPT inhibitor III (FPTIII) at 5, 10, 25 and 50 lM for 60 min before infection. Cells were infected with mock or H5N1 virus at MOI 2. After adsorption with virus, the cells were replenished with the same concentrations of drug as those before infection. (A) mRNA expression of viral M gene, cytokines and chemokines was measured at 24 hpi. (B) Protein concentrations of the chemokines were detected by using CBA in culture supernatants collected at 24 hpi. Results are means ± SD of a representative experiment taken from 3 separate experiments. ⁄P < 0.05; ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001 when compared with vehicle-treated and H5N1-infected cells.
immuno-regulatory activities shown in Fig. 6, together with a modest antiviral effect (Fig. 3). By using the above inhibitors, we have evaluated the immunoregulatory role of different branches of sterol metabolic pathway during HPAI H5N1 infection. We demonstrated here that FPT inhibitor III, which is the farnesylation inhibitor, has potent suppression effects on HPAI H5N1-induced immune responses and has modest antiviral activity. These results imply that farnesylation of the sterol biosynthesis pathway is involved in the cytokine and chemokine induction during H5N1 infection. The mechanisms of farnesylation in regulating immune responses in influenza infection have not been reported and further study is needed and this may reveal additional potential targets for therapy of severe influenza disease.
4. Conclusion Host-targeting strategies are a new direction in treating severe human influenza disease. There are controversies on the use of statins against influenza infection in in vitro cell and, animal models. However, there is not much information on in vitro study of statins and influenza infection. We first demonstrated that the gene expression of the sterol metabolic pathway was suppressed by both influenza infection and by treatment with IFN-b, which is in line with the previous report showing that suppression of sterol biosynthesis is one of the anti-viral activities of IFNs (Blanc et al., 2011). We next showed that a number of inhibitors of the sterol biosynthetic pathway possess anti-viral effects against both HPAI H5N1 and low pathogenic human seasonal H1N1 virus infection in AECs. By using these inhibitors to block different branches of the sterol metabolic pathway, we found that Simvastatin has the strongest anti-viral effects against H5N1. This may be due to the
fact that Simvastatin inhibits HMGCR and leads to the blockage of the whole mevalonate pathway. While the other inhibitors are downstream of the mevalonate pathway, we found that each branch of the sterol pathway partially contributes to the viral replication. We also showed that the low pathogenic human seasonal H1N1 (447/08) was more sensitive to the inhibitors than HPAI H5N1 (483/97). There may be strain differences for the dependence on sterol biosynthetic pathway. HPAI H5N1 viruses significantly induce more cytokines and chemokines in patients when compared to the low pathogenic seasonal influenza viruses. Cytokine storm is believed to be one of the mechanisms of pathogenesis of severe H5N1 disease in humans. Beneficial effects were observed when combination of antiviral drugs and immuno-modulator were administered in HPAI influenza infected mice (Zheng et al., 2008). We have shown here that FPT inhibitor III has potent suppressive effects on HPAI H5N1-induced immune responses, and significant inhibition of viral replication at mRNA expression and progeny virus production. This study aims at understanding the anti-viral and immuno-regulatory roles of the sterol biosynthesis pathways during influenza infection by using a number of pharmacological inhibitors. Taken all together, our data demonstrate a previously undefined regulatory role of farnesylation of the sterol biosynthetic pathway on H5N1induced cytokine responses and suggest that farnesyl transferase inhibitors may represent novel therapeutic options for immunemodulation in severe influenza diseases.
5. Funding We acknowledge research funding from National Institute of Allergy and Infectious Diseases (NIAID), under CEIRS contract No. HHSN272201400006C, Area of Excellence Scheme of the
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University Grants Committee (Grant AoE/M-12/96), Research Grants Council of the Hong Kong Special Administrative Region, China (Project No. T11-705/14N) and Seed Funding for Basic Research (reference no: 201309159004), The University of Hong Kong. 6. Author contributions KPYH: Study design, analysis and interpretation of results, perform experiments and writing of the manuscript; SSRK, DITK, HSL, MMTN, CHTB: Perform experiments, analysis and interpretation of results; JSMP: Study design, analysis and interpretation of results and critical review of the manuscript; RWYC: Study design, perform experiments, analysis and interpretation of results; MCWC: Overall coordination, study design, analysis and interpretation of results, perform experiments and writing of the manuscript. All authors declare no competing financial interests. Acknowledgments Dr. Alan DL Sihoe and staff from the Division of Cardiothoracic Surgery, Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong and Queen Mary Hospital provided the human lung tissues. A/Oklahoma/447/08 virus was provided by Gillian Air (University of Oklahoma Health Sciences Center). Dr. Rachel Ching, Ms. Joanne Fong, Mr. Man Chun Cheung from the Centre of Influenza Research, School of Public Health, Li Ka Shing Faculty of Medicine, The University of Hong Kong and Mr. Kevin Fung in the Department of Pathology, The University of Hong Kong provided technical support. References Agrati, C., Alonzi, T., De Santis, R., Castilletti, C., Abbate, I., Capobianchi, M.R., D’Offizi, G., Siepi, F., Fimia, G.M., Tripodi, M., Poccia, F., 2006. Activation of Vgamma9Vdelta2 T cells by non-peptidic antigens induces the inhibition of subgenomic HCV replication. Int. Immunol. 18, 11–18. An, S.C., Xu, L.L., Li, F.D., Bao, L.L., Qin, C., Gao, Z.C., 2011. Triple combinations of neuraminidase inhibitors, statins and fibrates benefit the survival of patients with lethal avian influenza pandemic. Med. Hypotheses 77, 1054–1057. Belser, J.A., Szretter, K.J., Katz, J.M., Tumpey, T.M., 2013. Simvastatin and oseltamivir combination therapy does not improve the effectiveness of oseltamivir alone following highly pathogenic avian H5N1 influenza virus infection in mice. Virology 439, 42–46. Blanc, M., Hsieh, W.Y., Robertson, K.A., Watterson, S., Shui, G., Lacaze, P., Khondoker, M., Dickinson, P., Sing, G., Rodriguez-Martin, S., Phelan, P., Forster, T., Strobl, B., Muller, M., Riemersma, R., Osborne, T., Wenk, M.R., Angulo, A., Ghazal, P., 2011. Host defense against viral infection involves interferon mediated downregulation of sterol biosynthesis. PLoS Biol. 9, e1000598. Chan, M.C., Cheung, C.Y., Chui, W.H., Tsao, S.W., Nicholls, J.M., Chan, Y.O., Chan, R.W., Long, H.T., Poon, L.L., Guan, Y., Peiris, J.S., 2005. Proinflammatory cytokine responses induced by influenza A (H5N1) viruses in primary human alveolar and bronchial epithelial cells. Respir. Res. 6, 135. Chan, M.C., Chan, R.W., Yu, W.C., Ho, C.C., Yuen, K.M., Fong, J.H., Tang, L.L., Lai, W.W., Lo, A.C., Chui, W.H., Sihoe, A.D., Kwong, D.L., Wong, D.S., Tsao, G.S., Poon, L.L., Guan, Y., Nicholls, J.M., Peiris, J.S., 2010. Tropism and innate host responses of the 2009 pandemic H1N1 influenza virus in ex vivo and in vitro cultures of human conjunctiva and respiratory tract. Am. J. Pathol. 176, 1828–1840. Cheung, C.Y., Poon, L.L., Lau, A.S., Luk, W., Lau, Y.L., Shortridge, K.F., Gordon, S., Guan, Y., Peiris, J.S., 2002. Induction of proinflammatory cytokines in human macrophages by influenza A (H5N1) viruses: a mechanism for the unusual severity of human disease? Lancet 360, 1831–1837. de Jong, M.D., Simmons, C.P., Thanh, T.T., Hien, V.M., Smith, G.J., Chau, T.N., Hoang, D.M., Chau, N.V., Khanh, T.H., Dong, V.C., Qui, P.T., Cam, B.V., Ha do, Q., Guan, Y.,
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