The bacterium, nontypeable Haemophilus influenzae, enhances host antiviral response by inducing Toll-like receptor expression Evidence for negative regulation of host antiviral response by CYLD Akihiro Sakai1,2*, Tomoaki Koga1*, Jae-Hyang Lim1, Hirofumi Jono1, Kazutsune Harada3, Erika Szymanski1, Haidong Xu1, Hirofumi Kai3 and Jian-Dong Li1 Department of Microbiology & Immunology, University of Rochester Medical Center, NY, USA Gonda Department of Cell & Molecular Biology, House Ear Institute, University of Southern California, Los Angeles, CA, USA Department of Molecular Medicine, Kumamoto University, Japan Keywords cylindromatosis; mixed infection; nontypeable Haemophilus influenzae; signal transduction; Toll-like receptor Correspondence J.-D Li, Department of Microbiology & Immunology, Box 672, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, USA Fax: +1 585 276 2231 Tel: +1 585 275 7195 E-mail: Jian-Dong_Li@urmc.rochester.edu *These authors contributed equally to this work The incidence of mixed viral ⁄ bacterial infections has increased recently because of the dramatic increase in antibiotic-resistant strains, the emergence of new pathogens, and the resurgence of old ones Despite the relatively well-known role of viruses in enhancing bacterial infections, the impact of bacterial infections on viral infections remains unknown In this study, we provide direct evidence that nontypeable Haemophilus influenzae (NTHi), a major respiratory bacterial pathogen, augments the host antiviral response by up-regulating epithelial Toll-like receptor (TLR7) expression in vitro and in vivo Moreover, NTHi induces TLR7 expression via a TLR2-MyD88-IRAK-TRAF6-IKK-NF-jB-dependent signaling pathway Interestingly, CYLD, a novel deubiquitinase, acts as a negative regulator of TLR7 induction by NTHi Our study thus provides new insights into a novel role for bacterial infection in enhancing host antiviral response and further identifies CYLD for the first time as a critical negative regulator of host antiviral response (Received 12 March 2007, revised 23 May 2007, accepted 23 May 2007) doi:10.1111/j.1742-4658.2007.05899.x In the host innate immune system, the surface epithelial cells are situated at host ⁄ environment boundaries and thus act as the first line of host defense against pathogenic bacteria and viruses The principal challenge for the host is to efficiently detect the invading pathogen and mount a rapid defensive response Epithelial cells recognize invading pathogens by directly interacting with pathogen-associated molecular patterns on a variety of pathogens via Toll-like receptors (TLRs) expressed on the host Activation of TLRs, in turn, leads to induction of direct antimicrobial activity which can result in elimination of the invading pathogen before a full adaptive immune response takes effect In addition, activation of TLRs is a prerequisite for the triggering of acquired immunity To date, 11 members of the human TLR family have been identified Of these, TLR2 is critically involved in host response to a variety of Gram-positive bacterial products including peptidoglycan, lipoprotein and lipoarabinomannan [1–5] The importance of TLR2 in host Abbreviations IFNs, interferons; IKKb, IjB kinase b; IL, interleukin; MEF, mouse embryonic fibroblast; NHBE, normal human bronchial epithelial; NTHi, nontypeable Haemophilus influenzae; Q-PCR, quantitative PCR; siRNA, small interfering RNA; TLR, Toll-like receptor; TNF, tumor necrosis factor FEBS Journal 274 (2007) 3655–3668 ª 2007 The Authors Journal compilation ª 2007 FEBS 3655 Regulation of TLR7 by bacterium NTHi A Sakai et al defense was further highlighted by studies with TLR2deficient mice which are susceptible to infection with the Gram-positive bacterium Staphylococcus aureus [6] Furthermore, our recent studies demonstrated that TLR2 also plays a key role in activating host immune and inflammatory response against the Gram-negative bacterium nontypeable Haemophilus influenzae (NTHi), a major cause of exacerbation of chronic obstructive pulmonary disease and otitis media [7–10] Interestingly, TLR2 itself has also been shown to be tightly regulated by bacteria Our recent studies provide evidence that NTHi regulates TLR2 via positive NF-jB and transforming growth factor-b-Smad3 ⁄ signaling pathways and negative epidermal growth factor receptor-dependent Src-MKK3 ⁄ 6-p38 pathways [11–14] In contrast, how virus receptors such as TLR7 or TLR8 are regulated remains largely unknown TLR7 and TLR8 have been identified as receptors for ssRNA and antiviral reagent R848 [15–17] Heil et al [18] have shown that mouse TLR7 recognizes GU-rich ssRNA in a sequence-dependent manner Another study showed that human TLR7 and TLR8 could respond to ssRNA from human parechovirus (HPEV1) [19] Moreover, Chuang & Ulevitch [20] detected expression of TLR7 in lung tissue, implying a potential role for TLR7 in host antiviral response to respiratory pathogens Although most exacerbations of chronic obstructive pulmonary disease are mainly associated with a single bacterial pathogen, there is a growing body of evidence that a significant proportion of patients diagnosed with this disease have mixed infections of bacteria and virus [21,22] Moreover, inappropriate antibiotic treatment contributes to the worldwide emergence of antibiotic-resistant strains and leads to increased incidence of polymicrobial infections Despite the relatively well-known role of virus infections in promoting bacterial infections, it is still not clear whether bacterial infection also promotes viral infection in polymicrobial infections nor how TLR7 is regulated The deubiquitinating enzyme, CYLD, loss of which causes the benign human syndrome cylindromatosis, has been identified as a key negative regulator of multiple signaling pathways including NF-jB and p38 in vitro [23–25] Recent in vivo studies have also shown that CYLD plays critical roles in T cell development and tumor cell proliferation [26,27] Its role in regulating host antiviral response is not known In this study, we provide evidence that the bacterium, NTHi, enhances host antiviral responses via TLR2-dependent up-regulation of TLR7 expression in human airway epithelial cells in vitro and mouse 3656 lung tissue in vivo Moreover, NTHi induces TLR7 expression via a MyD88-IRAK-TRAF6-IKK-NF-jBdependent mechanism Interestingly, NTHi also induces the deubiquitinase, CYLD, in a TLR2-dependent manner, which, in turn, acts as a negative regulator of NTHi-induced TLR7 expression This study thus provides new insights into a novel role of bacterial infection in enhancing host antiviral response and also identifies CYLD as a critical negative regulator of host antiviral response Results NTHi up-regulates TLR7 expression in vitro and in vivo We first examined whether NTHi up-regulates TLR7 in human epithelial cells Human lung epithelial A549 cells were treated with NTHi, and then TLR7 mRNA expression was measured by real-time quantitative PCR (Q-PCR) As shown in Fig 1A,B, NTHi up-regulated TLR7 expression at the mRNA level in a dose-dependent and time-dependent manner Similar results were also observed in HeLa cells (human cervix epithelial cells) and primary normal human bronchial epithelial (NHBE) cells (Fig 1C) To determine whether up-regulation of TLR7 mRNA is accompanied by increased TLR7 protein, western blot analysis was carried out with TLR7-specific antibody As shown in Fig 1D,E, up-regulation of TLR7 was also observed at the protein level in a time-dependent manner in A549 cells and primary NHBE cells cultured under air ⁄ liquid interface conditions A549 cells transfected with human wild-type TLR7 expression plasmid served as a positive control for TLR7 expression (Fig 1D) Immunofluorescent staining studies were consistent with these findings showing TLR7 up-regulation in NHBE cells h after treatment with NTHi (Fig 1F) Similar results were observed in A549 cells (data not shown) To further confirm whether TLR7 is also up-regulated in vivo, C57BL ⁄ mice were intratracheally inoculated with NTHi As shown in Fig 1G,H, NTHi up-regulated TLR7 expression at the mRNA and protein levels in the mouse lung in vivo Similar results were also observed in BALB ⁄ c mice (data not shown) It should be noted that no effect of NTHi treatment on the expression of housekeeping genes (e.g human cyclophilin and mouse glyceraldehyde-3-phosphate dehydrogenase) was observed, as assessed by Q-PCR Taken together, these data demonstrate that NTHi up-regulates TLR7 expression at both mRNA and protein levels in vitro and in vivo FEBS Journal 274 (2007) 3655–3668 ª 2007 The Authors Journal compilation ª 2007 FEBS A Sakai et al Regulation of TLR7 by bacterium NTHi A B C D E F G H Fig NTHi induces TLR7 expression in vitro and in vivo (A,B) NTHi-induced TLR7 expression at the mRNA level in human airway epithelial A549 cells in a dose-dependent (0, 0.5, 2.5, 5.0, and 10 lgỈmL)1 NTHi lysate) and time-dependent (15 lgỈmL)1 NTHi lysate) manner, as assessed by real-time Q-PCR analysis (C) Induction of TLR7 by NTHi was also observed in HeLa (human cervix epithelial) and primary NHBE cells at the mRNA level (D) NTHi-induced TLR7 expression at the protein level in A549 cells in a time-dependent manner, as assessed by western blot analysis HeLa cells transfected with wild-type TLR7 expression plasmid were used as positive controls (E) Induction of TLR7 by NTHi was also observed at the protein level in primary NHBE cells cultured under air ⁄ liquid interface conditions (F) NTHi up-regulated TLR7 expression in primary NHBE cells, as assessed by immunofluorescent staining The NHBE cells were fixed and stained h after treatment with NTHi (G) NTHi induced TLR7 expression at the mRNA level in lung tissue from C57BL ⁄ mice (H) TLR7 was up-regulated at the protein level in lung tissue from C57BL ⁄ mice Lung protein was collected h after inoculation with NTHi *P < 0.05, compared with untreated control P value was determined by Student’s t-test Values are the mean ± SD (n ¼ for A, B, C and G) Data shown in (D), (E), (F) and (H) are representative of three or more independent experiments FEBS Journal 274 (2007) 3655–3668 ª 2007 The Authors Journal compilation ª 2007 FEBS 3657 Regulation of TLR7 by bacterium NTHi A Sakai et al A TLR2-dependent MyD88-IRAK-TRAF6 signaling pathway is required for NTHi-induced TLR7 expression in vitro and in vivo We next sought to determine which surface receptor and downstream adaptors are involved in TLR7 induction by NTHi Because TLR2 is important for mediating NTHi-induced gene transcription, we first investigated the role of TLR2 in NTHi-induced TLR7 up-regulation As shown in Fig 2A, overexpressing a dominant-negative mutant of TLR2 reduced NTHi-induced TLR7 up-regulation, whereas overexpresisng wild-type TLR2 enhanced it To further confirm the requirement of TLR2 in mediating NTHi-induced TLR7 up-regulation, we examined TLR7 induction by NTHi in HEK293pcDNA, HEK293-TLR2 or HEK293-TLR4 cells, stably transfected with pcDNA, TLR2 or TLR4, respectively As expected, NTHi induced TLR7 mRNA expression in A B C D E F G 3658 FEBS Journal 274 (2007) 3655–3668 ª 2007 The Authors Journal compilation ª 2007 FEBS A Sakai et al HEK293-TLR2 cells but not in HEK293-pcDNA or HEK293-TLR4 cells (Fig 2B) As TLR2 is known to form heterodimers with either TLR1 or TLR6, we determined if TLR1 or TLR6 is also involved in mediating TLR7 up-regulation by NTHi by knockdown of TLR1 or TLR6 As shown in Fig 2C, TLR1 small interfering RNA (siRNA) and TLR6 siRNA reduced the expression of TLR1 and TLR6 mRNA, respectively (upper panels) Interestingly, both TLR1 siRNA and TLR6 siRNA inhibited NTHi-induced TLR7 expression (lower panels) We further determined whether TLR1 ⁄ or TLR2 ⁄ signaling is involved in TLR7 induction by using specific TLR1 ⁄ or TLR2 ⁄ ligands As shown in Fig 2D, Pam3CSK4 (Pam3), a specific ligand for the TLR1 ⁄ heterodimer, and MALP2, a specific ligand for the TLR2 ⁄ TLR6 heterodimer, induced TLR7 expression in A549 cells These data suggest that both TLR1 ⁄ and TLR2 ⁄ 6, but not TLR4, are involved in TLR7 induction by NTHi We next investigated the involvement of MyD88 in NTHi-induced TLR7 up-regulation As shown in Fig 2E, overexpression of a dominant-negative mutant form of MyD88 attenuated NTHi-induced TLR7 up-regulation in A549 cells Because activated MyD88 recruits IRAK-1 and subsequently interacts with TRAF6, we investigated if IRAK-1 and TRAF6 are also involved in TLR7 induction As shown in Fig 2F, coexpressing dominant-negative IRAK-1 or TRAF6 but not TRAF2 inhibited NTHi-induced TLR7 expression To further confirm whether TLR2 is also required for TLR7 induction by NTHi in vivo, we examined NTHi-induced TLR7 mRNA expression induced by NTHi in the lungs of wild-type and Tlr2– ⁄ – mice intratracheally inoculated with NTHi Consistent with in vitro data, NTHi-induced TLR7 mRNA expression was much lower in the lungs of Tlr2– ⁄ – mice than in the lungs of wild-type mice (Fig 2G) It should be noted that no effect of any of the above treatments was Regulation of TLR7 by bacterium NTHi observed on the expression of housekeeping genes as assessed by Q-PCR Taken together, these results provide evidence that TLR2 signaling is required for NTHi-induced TLR7 up-regulation in vitro and in vivo NF-jB activation is essential for NTHi-induced TLR7 up-regulation Because of the importance of NF-jB in TLR2-mediated gene transcription, we next sought to determine its involvement in NTHi-induced TLR7 up-regulation We first determined if NTHi activates the NF-jB pathway in A549 cells As shown in Fig 3A, NTHi induced phosphorylation of IjBa and subsequent degradation of IjBa Because disruption of the IjBa–NF-jB complex is required for NF-jB nuclear translocation and activation, we next determined the requirement of IjBa degradation by assessing the effect of the proteasome inhibitor, MG-132, and overexpression of a transdominant mutant of IjBa on NTHi-induced TLR7 up-regulation Figure 3B shows that MG-132 inhibited NTHi-induced nuclear translocation of the NF-jB p65 subunit and up-regulation of TLR7 Consistent with these results, overexpression of a transdominant mutant form of IjBa also reduced NTHi-induced TLR7 up-regulation (Fig 3C) Because IjB kinase b (IKKb) acts as a major upstream kinase of IjBa, we next investigated the role of IKKb in TLR7 induction by NTHi As shown in Fig 3C, a dominant-negative mutant of IKKb inhibited NTHi-induced TLR7 expression We further confirmed the requirement for NF-jB by knockdown of p65 with p65 siRNA As shown in Fig 3D,E, p65 siRNA reduced the expression of p65 protein and inhibited NF-jB activation by NTHi As expected, p65 siRNA markedly inhibited TLR7 induction by NTHi (Fig 3F) The requirement of p65 was further confirmed by using p65-deficient cells As shown in Fig 3G, Fig TLR2 signaling is required for NTHi-induced TLR7 expression in vitro and in vivo (A) Overexpression of a dominant-negative mutant of TLR2 attenuated TLR7 induction by NTHi at the mRNA level, whereas overexpression of wild-type TLR2 enhanced it, in A549 cells *P < 0.05, compared with untreated control **P < 0.05, compared with NTHi-treated group transfected with empty vector (B) NTHi markedly induced TLR7 expression at the mRNA level in HEK293-TLR2 cells, but only weakly in HEK293-pcDNA cells and HEK293-TLR4 cells *P < 0.05, **P > 0.05, respectively, compared with NTHi-treated group in HEK293-pcDNA cells (C) Both TLR1 siRNA and TLR6 siRNA markedly reduced TLR1 mRNA expression and TLR6 mRNA expression, respectively (upper panels) Both TLR1 and TLR6 knockdown inhibited NTHi-induced TLR7 expression in A549 cells (lower panels) *P < 0.05, compared with untreated control **P < 0.05, compared with NTHitreated group transfected with control siRNA (D) Both Pam3CSK4 (Pam3, 250 ngỈmL)1) and MALP2 (1 ngỈmL)1) induced TLR7 expression in A549 cells *P < 0.05, compared with untreated group (E) Overexpression of dominant-negative MyD88 reduced NTHi-induced TLR7 expression at the mRNA level in A549 cells *P < 0.05, compared with untreated control **P < 0.05, compared with NTHi-treated group transfected with empty vector (F) Overexpression of dominant-negative IRAK-1 or TRAF6 but not TRAF2 attenuated TLR7 induction by NTHi in A549 cells *P < 0.05, compared with untreated control **P < 0.05, compared with NTHi-treated group transfected with empty vector (G) NTHi-induced TLR7 expression at the mRNA level was remarkably attenuated in Tlr2– ⁄ – mice compared with wild-type mice *P < 0.05, compared with untreated wild-type control **P < 0.05, compared with NTHi-treated wild-type control P value was determined by Student’s t-test Values are the mean ± SD (n ¼ 3) FEBS Journal 274 (2007) 3655–3668 ª 2007 The Authors Journal compilation ª 2007 FEBS 3659 Regulation of TLR7 by bacterium NTHi A Sakai et al NTHi-induced TLR7 expression was markedly reduced in p65-deficient (p65– ⁄ –) mouse embryonic fibroblasts (MEFs) compared with wild-type MEFs, and its responsiveness was rescued in wild-type p65-reconstituted MEFs (p65+ ⁄ +) It should be noted that none of the A above treatments showed any effect on the expression of housekeeping genes Collectively, these data suggest that IKKb ⁄ IjBa-dependent translocation and activation of NF-jB is required for NTHi-induced TLR7 up-regulation in epithelial cells B C D E F G 3660 FEBS Journal 274 (2007) 3655–3668 ª 2007 The Authors Journal compilation ª 2007 FEBS A Sakai et al Regulation of TLR7 by bacterium NTHi CYLD acts as a negative regulator of NTHi-induced TLR7 up-regulation CYLD is induced by NTHi via a TLR2-dependent pathway in vitro and in vivo We next sought to determine whether CYLD, a recently identified novel deubiquitinase, is involved in NTHi-induced TLR7 expression We first evaluated the efficiency of CYLD siRNA in reducing CYLD expression and inhibiting the NTHi-induced phosphorylation and degradation of IjBa As shown in Fig 4A, CYLD siRNA efficiently reduced CYLD expression in A549 cells transfected with wild-type CYLD Overexpression of wild-type CYLD inhibited IjBa phosphorylation and degradation, whereas CYLD siRNA enhanced it (Fig 4B,C) Next we examined the effect of overexpressing wild-type CYLD or CYLD siRNA on NTHi-induced NF-jB activation As expected, overexpression of wild-type CYLD attenuated NF-jB activation by NTHi, whereas CYLD knockdown enhanced it (Fig 4D,E) These data show that CYLD indeed acts as a negative regulator of NTHi-induced NF-jB activation We next sought to determine whether CYLD is a negative regulator of NTHi-induced TLR7 up-regulation As shown in Fig 4F,G, overexpression of wild-type CYLD attenuated NTHi-induced TLR7 up-regulation, whereas knockdown of CYLD enhanced it To further confirm the negative role of CYLD in NTHiinduced TLR7 expression, we examined the NTHiinduced TLR7 mRNA expression in Cyld– ⁄ – MEFs and lungs As shown in Fig 4H,I, NTHi-induced TLR7 expression at both mRNA and protein levels was much greater in Cyld– ⁄ – MEFs than in wild-type MEFs Similarly, NTHi-induced TLR7 mRNA upregulation was markedly enhanced in Cyld– ⁄ – mouse lung compared with wild-type mouse lung (Fig 4J) It should be noted that none of the above treatments showed any effect on the expression of housekeeping genes Taking these results together, it is evident that CYLD acts as a negative regulator of NTHi-induced TLR7 expression Because a variety of genes involved in the host defense response were induced during the course of infections, we thus examined whether NTHi induces CYLD in airway epithelial A549 and NHBE cells in vitro and in vivo As shown in Fig 5A,B, NTHi up-regulated CYLD in A549 and NHBE cells as well as in the lungs of mice intratracheally inoculated with NTHi We next investigated the requirement for TLR2 in NTHi-induced CYLD up-regulation in vitro and in vivo Overexpression of TLR2 dominant-negative mutant attenuated the NTHi-induced CYLD mRNA expression in A549 cells, whereas overexpression of TLR2 wild-type enhanced it (Fig 5C) To confirm the involvement of TLR2 in NTHi-induced CYLD expression, we examined CYLD mRNA induction by NTHi in HEK293-pcDNA and HEK293-TLR2 cells As shown in Fig 5D, CYLD mRNA expression was greatly up-regulated in HEK293-TLR2 cells compared with HEK293-pcDNA cells To further confirm whether TLR2 is required for NTHi-induced CYLD up-regulation in vivo, we examined expression of CYLD mRNA induced by NTHi in the lungs of wild-type and Tlr2– ⁄ – mice Consistent with these in vitro data, NTHi-induced CYLD mRNA expression was much lower in the lungs of Tlr2– ⁄ – mice than in wild-type mice (Fig 5E) It should be noted that none of the above treatments showed any effect on the expression of housekeeping genes These data suggest that CYLD is induced by NTHi via a TLR2-dependent pathway in vitro and in vivo NTHi potentiates TLR7-dependent induction of type I interferons and pro-inflammatory cytokines We have shown that NTHi up-regulates TLR7 in a TLR2-dependent manner We next sought to determine the physiological relevance of TLR7 up-regulation We Fig NF-jB activation is essential for NTHi-induced TLR7 up-regulation (A) IjBa was phosphorylated and degraded by NTHi in a timedependent manner in A549 cells, as assessed by western blot analysis (B) MG-132, a proteasome inhibitor that can inhibit NF-jB translocation, attenuated NTHi-induced translocation of p65 (upper panel) and reduced NTHi-induced TLR7 expression at the mRNA level (lower panel) in A549 cells The cells were pretreated with MG-132 (1 lM) for h and then treated with NTHi for h as assessed by performing real-time Q-PCR analysis (C) Overexpression of IjBa trans-dominant mutant or IKKb dominant-negative mutant attenuated TLR7 induction by NTHi at the mRNA level in A549 cells (D) p65 siRNA efficiently reduced p65 expression in A549 cells, as assessed by western blot analysis (E) p65 siRNA inhibited NF-jB activation by NTHi in A549 cells, as assessed by luciferase assay (F) p65 knockdown using p65 siRNA inhibited NTHi-induced TLR7 expression at the mRNA level *P < 0.05, compared with untreated control **P < 0.05, compared with NTHi-treated group transfected with control siRNA (G) NTHi-induced TLR7 expression was reduced in p65 knockout MEFs and rescued by p65 reconstitution *P < 0.05, compared with untreated control **P < 0.05, compared with NTHi-treated wild-type MEF group ***P > 0.05, compared with NTHi-treated wild-type MEF group P value was determined by Student’s t-test Values are the mean ± SD (n ¼ for B lower panel, C, E, F and G) Data shown in the upper panel of (B) and in (D) are representative of three or more independent experiments FEBS Journal 274 (2007) 3655–3668 ª 2007 The Authors Journal compilation ª 2007 FEBS 3661 Regulation of TLR7 by bacterium NTHi A Sakai et al A B C D E F G H I J Fig CYLD is a negative regulator for NTHi-induced TLR7 up-regulation (A) siRNA of CYLD efficiently reduced CYLD expression at the protein level in A549 cells transfected with wild-type CYLD, as assessed by western blot analysis (B,D) Overexpression of wild-type CYLD attenuated NTHi-induced IjBa phosphorylation (B), IjBa degradation (B) and NF-jB activation (D) in A549 cells, as assessed by western blot analysis (B) and luciferase assay (D) (C,E) siRNA of CYLD enhanced IjBa phosphorylation (C), IjBa degradation (C) and NF-jB activation (E) in A549 cells, as assessed by western blot analysis and luciferase assay (F) Overexpression of wild-type CYLD reduced induction of TLR7 mRNA by NTHi in A549 cells, as assessed by real-time Q-PCR analysis (G) CYLD knockdown using CYLD siRNA enhanced NTHi-induced TLR7 mRNA expression in A549 cells (H) TLR7 induction by NTHi was markedly enhanced at the mRNA level in Cyld– ⁄ – MEFs compared with wild-type MEFs (I) NTHi-induced TLR7 expression was enhanced in Cyld– ⁄ – MEFs compared with wild-type MEFs, as assessed by western blot analysis (J) TLR7 induction by NTHi was enhanced at the mRNA level in Cyld– ⁄ – mouse lung compared with wild-type mouse lung Mouse lungs were treated with NTHi for 15 h *P < 0.05, compared with untreated control **P < 0.05, compared with NTHi-treated group transfected with either empty vector or control siRNA P value was determined by Student’s t-test Values are the mean ± SD (n ¼ in D, E, F, G, H and J) Data shown in (A), (B), (C) and (I) are representative of three or more independent experiments 3662 FEBS Journal 274 (2007) 3655–3668 ª 2007 The Authors Journal compilation ª 2007 FEBS A Sakai et al A C Regulation of TLR7 by bacterium NTHi B D expression of all these genes, indicating that enhanced TLR7 expression indeed enhances host antiviral responses As NTHi treatment markedly up-regulated TLR7 expression, we next sought to evaluate if NTHi pretreatment also enhances R848-induced expression of host antiviral genes As shown in Fig 6B, NTHi pretreatment potentiated expression of IFN-a, IFN-b, TNF-a, IL-1b and IL-8 induced by R848 It should be noted that none of the above treatments showed any effect on the expression of housekeeping genes Together, these results suggest that NTHi potentiates TLR7-dependent expression of host antiviral genes by inducing TLR7 expression in epithelial cells Discussion E Fig CYLD is induced by NTHi via a TLR2-dependent signaling pathway in vitro and in vivo (A) CYLD was markedly induced by NTHi in A549 and primary NHBE cells as assessed by real-time QPCR analysis (B) NTHi induced CYLD expression at the mRNA level in the lungs of C57BL ⁄ mice (C) Overexpression of a dominant-negative TLR2 attenuated CYLD induction by NTHi at the mRNA level, whereas overexpression of wild-type TLR2 enhanced it, in A549 cells (D) NTHi-induced CYLD expression was enhanced at the mRNA level in HEK293-TLR2 cells compared with HEK293pcDNA cells (E) NTHi-induced CYLD expression was remarkably reduced at the mRNA level in Tlr2– ⁄ – mice compared with wild-type mice *P < 0.05, compared with untreated control **P < 0.05, compared with NTHi-treated group transfected with empty vector or NTHi-treated wild-type mice ***P < 0.05, compared with NTHitreated control in HEK293-pcDNA cells P value was determined by Student’s t-test Values are the mean ± S.D (n ¼ 3) first assessed the effect of overexpressing wild-type TLR7 on expression of type I interferons (IFNs) including IFN-a and IFN-b, tumor necrosis factor (TNF)-a, interleukin (IL)-1b and IL-8 induced by R848, a synthetic ligand for TLR7 As shown in Fig 6A, overexpression of wild-type TLR7 enhanced R848-induced Over the past two decades, tremendous efforts have been made towards understanding host defense response to bacteria and viruses Most studies, however, have focused on investigating bacteria-induced antibacterial response or virus-induced antiviral response Given that under in vivo situations such as polymicrobial infections, mucosal epithelial surfaces are often exposed to multiple pathogens including bacteria and viruses, it is still unclear whether bacteria enhances host antiviral response In this study, we provide evidence that the bacterium, NTHi, enhances the expression of the key genes involved in host antiviral response by up-regulating TLR7 expression in airway epithelial cells in vitro and in vivo Moreover, NTHi induces TLR7 expression via a TLR2-MyD88-IRAKTRAF6-IKK-NF-jB-dependent signaling pathway, and CYLD, a novel deubiquitinase, acts as a negative regulator of TLR7 induction by NTHi (Fig 7) Our study thus provides new insights into a novel role for bacterial infection in enhancing host antiviral response and also identifies CYLD as a critical negative regulator of host antiviral response Of particular interest in this study is the direct evidence that NTHi up-regulates TLR7, a host receptor for ssRNA virus, leading to exaggerated TLR7dependent host antiviral response Our findings may have important implications for host defense and immune response to mixed infections First, the relatively low expression of TLR7 observed in unstimulated epithelial cells is probably an important aspect of TLR7 function, because under limiting conditions, cellular responses to pathogen-associated molecular patterns can be more stringently regulated by controlling the amount of TLR protein produced Secondly, the increased TLR7 expression contributes to the accelerated immune response of epithelial cells as well as resensitization of epithelial cells to invading pathogens FEBS Journal 274 (2007) 3655–3668 ª 2007 The Authors Journal compilation ª 2007 FEBS 3663 Regulation of TLR7 by bacterium NTHi A Sakai et al A B Fig NTHi potentiates TLR7-dependent induction of type I interferons and pro-inflammatory cytokines (A) Overexpression of wild-type TLR7 enhanced R848-induced expression of IFN-a, IFN-b, TNF-a, IL-1b, and IL-8 (from left to right panels) in A549 cells (B) NTHi pretreatment enhanced R848-induced expression of IFN-a, IFN-b, TNF-a, IL-1b, and IL-8 (from left to right panels) in A549 cells The cells were pretreated with (B) or without (A) NTHi for h and then treated with R848 (10 lM) for h Total RNA was collected and analyzed using real-time Q-PCR *P < 0.05, compared with R848-treated mock groups P value was determined by Student’s t-test Values are the mean ± SD (n ¼ 3) Hence, regulation of TLR7 expression may be one of the immune regulatory mechanisms commonly involved in host defense against ssRNA viruses Finally, the observation that TLR7 is up-regulated by NTHi suggests that invading bacteria can not only initiate the host immune response, but also modulate the eventual responsiveness of epithelial cells to the invading virus by regulating the TLR7 expression level Thus, these observations bring new insights to our understanding of the interaction between bacteria and viruses in mixed infections Another major interesting finding of this study is that NTHi-induced TLR2-dependent up-regulation of TLR7 is negatively regulated by CYLD in an autoregulatory feedback manner In contrast with the relatively well-known role of CYLD in tumorigenesis and T cell development, the role of CYLD in host antiviral response remains largely unknown Our results show for the first time that NTHi induces CYLD expression in vitro and in vivo, which in turn results in attenuation of TLR7 expression, leading to inhibition of host antiviral responses Thus, the involvement of CYLD may be essential to ensure the tight control of NTHiinduced TLR7 up-regulation and the resultant host 3664 antiviral response We can further speculate that the CYLD-dependent autoregulatory feedback loop may represent an important mechanism by which the host can self-limit serious tissue damage caused by detrimental inflammatory responses during polymicrobial infection Our future studies will focus on cloning and identifying the regulatory region of the TLR7 gene that contains the functional NF-jB site (s) in vitro It should be noted that genomic sequence analysis has revealed NF-jB sites within the putative TLR7 promoter region, providing further support for the requirement for NF-jB in TLR7 induction In addition, we will verify the role of CYLD in tightly regulating antiviral response during mixed infection in vivo using CYLD knockout mouse Experimental procedures Reagents MG-132 was purchased from Calbiochem (La Jolla, CA, USA) R848, R837 and Pam3CSK4 were purchased from InvivoGen (San Diego, CA, USA) MALP2 was purchased from Alexis Biochemicals (San Diego, CA, USA) FEBS Journal 274 (2007) 3655–3668 ª 2007 The Authors Journal compilation ª 2007 FEBS A Sakai et al Regulation of TLR7 by bacterium NTHi Fig Schematic representation of the signaling pathways involved in the positive and negative regulation of NTHi-induced TLR7 expression D T Golenbock [28,29] All stable cell lines were maintained as described previously [24] Primary NHBE cells were purchased from Cambrex (La Jolla, CA, USA) NHBE cells were maintained as described previously [23] Various epithelial cells were seeded at 1.2 · 106 cells ⁄ plate on to a 12-well plate and used for each experiments For air ⁄ liquid interface culture, NHBE cells were cultured as described previously [30,31] In brief, NHBE cells were seeded at · 104 cells ⁄ cm2 on to 24-mm-diameter, 0.4 lm pore size, semipermeable membrane inserts (Transwell Permeable Supports; Corning, Corning, NY, USA) in bronchial epithelial cell basal medium (Cambrex) The cultures were grown submerged for the first days and then the air ⁄ liquid interface was created by removing medium from the apical compartment of the cultures The culture medium was changed every other day until the air ⁄ liquid interface was created and changed daily by replacing fresh medium only to the basal compartment during the air ⁄ liquid interface culture NHBE cells were grown in air ⁄ liquid interface for 2–3 weeks before being used for experiments Wild-type and Cyld– ⁄ –MEFs were obtained from E13 embryos and maintained in DMEM supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) p65 knockout and p65 reconstituted MEFs were described previously [32] All cells were maintained at 37 °C in an atmosphere of 5% CO2 Bacterial strains and culture conditions Real-time Q-PCR analysis NTHi strain 12, a clinical isolate, was used in this study Bacteria were grown on chocolate agar plates at 37 °C in an atmosphere of 5% CO2 To make NTHi lysate, NTHi were harvested from a chocolate agar plate after overnight incubation and incubated in 30 mL brain ⁄ heart infusion broth supplemented with NAD (3.5 lgỈmL)1) After overnight incubation, NTHi were centrifuged at 6000 g for 10 using an Avanti J-26XPI, Beckman Coulter, JLA 16, 250, and the supernatant was discarded The pellet of NTHi was resuspended in 10 mL phosphate-buffered saline and sonicated Subsequently, the crude extract was collected and stored at )70 °C [11,12] NTHi lysates (15 lgỈmL)1) were used to treat the cells for all of the other experiments We chose to use NTHi lysates for the following reasons First, NTHi has been shown to be very fragile and undergoes spontaneous autolysis Its autolysis can also be triggered in vivo under various conditions including antibiotic treatment Therefore, lysates of NTHi represent a common clinical condition in vivo, especially after antibiotic treatment Total RNA was isolated with TRIzol reagent (Invitrogen) by following the manufacturer’s instructions For the reverse transcription reaction, TaqMan reverse transcription reagents (Applied Biosystems, Foster City, CA, USA) were used In brief, the reverse transcription reaction was performed for 60 at 37 °C, followed by 60 at 42 °C by using oligo(dT) and random hexamers PCR amplifications were performed by using TaqMan Universal Master Mix, for TNF-a, IL-1b, and IL-8, or SYBR Green Universal Master Mix for human IFN-a, IFN-b, CYLD, TLR7, mouse CYLD and mouse TLR7 In brief, reactions were performed in duplicate containing · Universal Master Mix, lL template cDNA, 100 nm primers, and 100 nm probe in a final volume of 12.5 lL, and analyzed in a 96-well optical reaction plate (Applied Biosystems) Probes for TaqMan include a fluorescent reporter dye, 6-carboxyfluorescein, at the 5¢ end and a fluorescent quencher dye, 6-carboxytetramethylrhodamine, at the 3¢ end to allow direct detection of the PCR product Reactions were amplified and quantified using an ABI 7500 sequence detector and the manufacturer’s corresponding software (7000v1.3.1; Applied Biosystems) The relative quantities of mRNAs were obtained by using the comparative Ct method and were normalized with predeveloped TaqMan assay reagent human cyclophilin or mouse glyceraldehydes-3-phosphate dehydrogenase as an endogenous control (Applied Biosystems) The primers and probes Cell culture Human lung epithelial cell line, A549, and human cervix epithelial cell line, HeLa, were maintained as described [13,14,23] Stable cell lines, HEK293-pcDNA, HEK293TLR2, and HEK293-TLR4, were kindly provided by FEBS Journal 274 (2007) 3655–3668 ª 2007 The Authors Journal compilation ª 2007 FEBS 3665 Regulation of TLR7 by bacterium NTHi A Sakai et al for human and mouse CYLD, human TNF-a, IL-1b and IL-8 were described previously [23,24] The primers for human TLR7 were as follows: forward primer, 5¢-TTAACC TGGATGGAAACCAGCTA-3¢; reverse primer, 5¢-TCAA GGCTGAGAAGCTGTAAGCTA-3¢ The primers for mouse TLR7 were as follows: forward primer, 5¢-TTGGCT TTTGTCCTAATGCTCAA-3¢; reverse primer, 5¢-TATCG GAAATAGTGTAAGGCCTCAA-3¢ The primers for IFNs were as follows: IFN-a forward primer, 5¢-GGCCTCGCCC TTTGCTT-3¢; IFN-a reverse primer, 5¢-AGCCCAGAGAG CAGCTTGACT-3¢; IFN-b forward primer, 5¢-TCCCTGA GGAGATTAAGCAGCT-3¢; IFNb reverse primer, 5¢-GGA GCATCTCATAGATGGTCAATG-3¢ Plasmids, transfections and luciferase assay The expression plasmids of TLR2 dominant negative (DN), TLR2 wild-type, MyD88 DN, IRAK-1 DN, TRAF2 DN, TRAF6 DN, IjBa DN (S32 ⁄ 36 A), IKKb DN (K49A), wild-type CYLD, and siRNA CYLD were as previously described [22–24] The reporter construct NF-jB-luc was as described [13,14] Negative control plasmid and p65 siRNA plasmid were purchased from Imgenex (San Diego, CA, USA) The expression plasmid of wild-type TLR7 (pUNOhTLR7) was purchased from InvivoGen All of the transient transfections were carried out in duplicate for Q-PCR and triplicate for the luciferase assay, using TransIT-LT1 reagent (Mirus, Madison, WI, USA) following the manufacturer’s instructions In all cotransfections, an empty vector was used as a control siRNA The TLR1 siRNA and TLR6 siRNA were purchased from Dharmacon (Lafayette, CO, USA) A549 cells were cultured on 12-well plates A final concentration of 33 nm TLR1 siRNA or TLR6 siRNA was transfected into 40–50% confluent cells using Lipofectamine 2000 (Invitrogen) At 40 h after transfection, cells were used for each experiment Western blot analysis Antibodies against phospho-IjBa and IjBa were purchased from Cell Signaling Technology (Beverly, MA, USA) Antibodies against TLR7 were purchased from Imgenex and Santa Cruz Biotechnology (Santa Cruz, CA, USA) An antibody against p65 was purchased from Santa Cruz Biotechnology Monoclonal antibody against b-actin was purchased from Sigma (St Louis, MO, USA) Immunofluorescent staining Cells were cultured on four-chamber microscope slides After NTHi treatment, the cells were fixed in 4% parafor- 3666 maldehyde solution, permeabilized with 0.5% Triton X-100 solution, and then incubated with anti-(human TLR7) IgG (diluted : 50) for h at room temperature Primary antibody was detected with Rhodamine-conjugated anti-rabbit IgG (Santa Cruz Biotechnology) Samples were visualized with an Axiovert 40 CFL microscope (Carl Zeiss) In vivo study C57BL ⁄ mice and BALB ⁄ c mice were purchased from Charles River Laboratories, Tlr2– ⁄ – mice were kindly provided by Dr Shizuo Akira (Osaka University), and 7–8-weekold 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Physiol Lung Cell Mol Physiol 289, L1049–L1060 32 Ishinaga H, Jono H, Lim JH, Kweon SM, Xu H, Ha UH, Xu H, Koga T, Yan C, Feng XH, et al (2007) TGF-beta induces p65 acetylation to enhance bacteriainduced NF-kappaB activation EMBO J 26, 1150–1162 FEBS Journal 274 (2007) 3655–3668 ª 2007 The Authors Journal compilation ª 2007 FEBS ... effect on the expression of housekeeping genes Together, these results suggest that NTHi potentiates TLR7-dependent expression of host antiviral genes by inducing TLR7 expression in epithelial... we provide evidence that the bacterium, NTHi, enhances the expression of the key genes involved in host antiviral response by up-regulating TLR7 expression in airway epithelial cells in vitro and... Inhibition of p38 MAPK by glucocorticoids via induction of MAPK phosphatase-1 enhances nontypeable Haemophilus influenzaeinduced expression of toll-like receptor J Biol Chem 277 , 474 44– 474 50 Lund JM, Alexopoulou