Tài liệu Báo cáo khoa học: The bacterium, nontypeable Haemophilus influenzae, enhances host antiviral response by inducing Toll-like receptor 7 expression ppt

In this study, we provide evidence that the bacter-ium, NTHi, enhances host antiviral responses via TLR2-dependent up-regulation of TLR7 expression in human airway epithelial cells in vi

enhances host antiviral response by inducing Toll-like

receptor 7 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 Kai3and Jian-Dong Li1

1 Department of Microbiology & Immunology, University of Rochester Medical Center, NY, USA

2 Gonda Department of Cell & Molecular Biology, House Ear Institute, University of Southern California, Los Angeles, CA, USA

3 Department of Molecular Medicine, Kumamoto University, Japan

In the host innate immune system, the surface

epithe-lial cells are situated at host⁄ environment boundaries

and thus act as the first line of host defense against

pathogenic bacteria and viruses The principal

chal-lenge for the host is to efficiently detect the invading

pathogen and mount a rapid defensive response

Epi-thelial cells recognize invading pathogens by directly

interacting with pathogen-associated molecular

pat-terns 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 patho-gen 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 identi-fied Of these, TLR2 is critically involved in host response to a variety of Gram-positive bacterial prod-ucts including peptidoglycan, lipoprotein and lipoara-binomannan [1–5] The importance of TLR2 in host

Keywords

cylindromatosis; mixed infection;

nontypeable Haemophilus influenzae; signal

transduction; Toll-like receptor 7

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

(Received 12 March 2007, revised 23 May

2007, accepted 23 May 2007)

doi:10.1111/j.1742-4658.2007.05899.x

The incidence of mixed viral⁄ bacterial infections has increased recently because of the dramatic increase in antibiotic-resistant strains, the emer-gence of new pathogens, and the resuremer-gence of old ones Despite the relat-ively 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 anti-viral response by up-regulating epithelial Toll-like receptor 7 (TLR7) expression in vitro and in vivo Moreover, NTHi induces TLR7 expression via a TLR2-MyD88-IRAK-TRAF6-IKK-NF-jB-dependent signaling path-way Interestingly, CYLD, a novel deubiquitinase, acts as a negative regu-lator 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 regula-tor of host antiviral response

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.

defense was further highlighted by studies with

TLR2-deficient 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]

Interest-ingly, TLR2 itself has also been shown to be tightly

regulated by bacteria Our recent studies provide

evi-dence that NTHi regulates TLR2 via positive NF-jB

and transforming growth factor-b-Smad3⁄ 4 signaling

pathways and negative epidermal growth factor

recep-tor-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

patho-gens

Although most exacerbations of chronic obstructive

pulmonary disease are mainly associated with a single

bacterial pathogen, there is a growing body of

evi-dence that a significant proportion of patients

diag-nosed 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

infec-tions in promoting bacterial infecinfec-tions, 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

mul-tiple 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

regula-ting host antiviral response is not known

In this study, we provide evidence that the

bacter-ium, NTHi, enhances host antiviral responses via

TLR2-dependent up-regulation of TLR7 expression

in human airway epithelial cells in vitro and mouse

lung tissue in vivo Moreover, NTHi induces TLR7 expression via a MyD88-IRAK-TRAF6-IKK-NF-jB-dependent mechanism Interestingly, NTHi also indu-ces 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

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 deter-mine 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 cul-tured under air⁄ liquid interface conditions A549 cells transfected with human wild-type TLR7 expression plasmid served as a positive control for TLR7 expres-sion (Fig 1D) Immunofluorescent staining studies were consistent with these findings showing TLR7 up-regulation in NHBE cells 5 h after treatment with NTHi (Fig 1F) Similar results were observed in A549 cells (data not shown) To further confirm whe-ther TLR7 is also up-regulated in vivo, C57BL⁄ 6 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 house-keeping 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

C

G

H

D

F

E B

Fig 1 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 lgÆmL)1NTHi lysate) and time-dependent (15 lgÆmL)1NTHi 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 5 h after treat-ment with NTHi (G) NTHi induced TLR7 expression at the mRNA level in lung tissue from C57BL ⁄ 6 mice (H) TLR7 was up-regulated at the protein level in lung tissue from C57BL ⁄ 6 mice Lung protein was collected 6 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 3 for A, B, C and G) Data shown in (D), (E), (F) and (H) are representative of three or more independent experiments.

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-negat-ive 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 HEK293-pcDNA, HEK293-TLR2 or HEK293-TLR4 cells, stably transfected with pcDNA, TLR2 or TLR4, respectively

As expected, NTHi induced TLR7 mRNA expression in

D

E

G C

F

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

deter-mined 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

expres-sion 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⁄ 2

or TLR2⁄ 6 signaling is involved in TLR7 induction by

using specific TLR1⁄ 2 or TLR2 ⁄ 6 ligands As shown in

Fig 2D, Pam3CSK4 (Pam3), a specific ligand for the

TLR1⁄ 2 heterodimer, and MALP2, a specific ligand for

the TLR2⁄ TLR6 heterodimer, induced TLR7

expres-sion in A549 cells These data suggest that both TLR1⁄ 2

and TLR2⁄ 6, but not TLR4, are involved in TLR7

induction by NTHi We next investigated the

involve-ment of MyD88 in NTHi-induced TLR7 up-regulation

As shown in Fig 2E, overexpression of a

dominant-neg-ative 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

exam-ined 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

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 phos-phorylation of IjBa and subsequent degradation of IjBa Because disruption of the IjBa–NF-jB complex

is required for NF-jB nuclear translocation and acti-vation, we next determined the requirement of IjBa degradation by assessing the effect of the proteasome inhibitor, MG-132, and overexpression of a trans-dominant 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 inves-tigated 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 knock-down 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 2 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 mark-edly 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 mark-edly reduced TLR1 mRNA expression and TLR6 mRNA expression, respectively (upper panels) Both TLR1 and TLR6 knockdown inhibited induced TLR7 expression in A549 cells (lower panels) *P < 0.05, compared with untreated control **P < 0.05, compared with NTHi-treated group transfected with control siRNA (D) Both Pam3CSK4 (Pam3, 250 ngÆmL)1) and MALP2 (1 ngÆmL)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).

NTHi-induced TLR7 expression was markedly reduced

in p65-deficient (p65–⁄ –) mouse embryonic fibroblasts

(MEFs) compared with wild-type MEFs, and its

respon-siveness was rescued in wild-type p65-reconstituted

MEFs (p65+⁄ +) It should be noted that none of the

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-regula-tion in epithelial cells

A

C

E

G

B

F D

CYLD acts as a negative regulator of

NTHi-induced TLR7 up-regulation

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

phos-phorylation 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

whereas CYLD knockdown enhanced it (Fig 4D,E)

These data show that CYLD indeed acts as a

negat-ive regulator of NTHi-induced NF-jB activation We

next sought to determine whether CYLD is a

negat-ive regulator of NTHi-induced TLR7 up-regulation

As shown in Fig 4F,G, overexpression of wild-type

CYLD attenuated NTHi-induced TLR7

up-regula-tion, whereas knockdown of CYLD enhanced it To

further confirm the negative role of CYLD in

induced TLR7 expression, we examined the

NTHi-induced 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

up-regulation 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

CYLD is induced by NTHi via a TLR2-dependent pathway in vitro and in vivo

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 air-way 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 investi-gated the requirement for TLR2 in NTHi-induced CYLD up-regulation in vitro and in vivo Overexpres-sion 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 exam-ined 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 3 NF-jB activation is essential for NTHi-induced TLR7 up-regulation (A) IjBa was phosphorylated and degraded by NTHi in a time-dependent manner in A549 cells, as assessed by western blot analysis (B) MG-132, a proteasome inhibitor that can inhibit NF-jB transloca-tion, 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 l M ) for 2 h and then treated with NTHi for 3 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 reconsti-tution *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 3 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.

G F

Fig 4 CYLD is a negative regulator for NTHi-induced TLR7 up-regulation (A) siRNA of CYLD efficiently reduced CYLD expression at the pro-tein 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 3

in D, E, F, G, H and J) Data shown in (A), (B), (C) and (I) are representative of three or more independent experiments.

first assessed the effect of overexpressing wild-type

TLR7 on expression of type I interferons (IFNs)

inclu-ding IFN-a and IFN-b, tumor necrosis factor (TNF)-a,

interleukin (IL)-1b and IL-8 induced by R848, a

syn-thetic ligand for TLR7 As shown in Fig 6A,

over-expression of wild-type TLR7 enhanced R848-induced

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

Over the past two decades, tremendous efforts have been made towards understanding host defense response to bacteria and viruses Most studies, how-ever, 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 bac-teria and viruses, it is still unclear whether bacbac-teria enhances host antiviral response In this study, we pro-vide epro-vidence 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-IRAK-TRAF6-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 regula-tor of host antiviral response

Of particular interest in this study is the direct evi-dence that NTHi up-regulates TLR7, a host receptor for ssRNA virus, leading to exaggerated TLR7-dependent host antiviral response Our findings may have important implications for host defense and immune response to mixed infections First, the relat-ively low expression of TLR7 observed in

unstimulat-ed epithelial cells is probably an important aspect of TLR7 function, because under limiting conditions, cel-lular responses to pathogen-associated molecular pat-terns can be more stringently regulated by controlling the amount of TLR protein produced Secondly, the increased TLR7 expression contributes to the acceler-ated immune response of epithelial cells as well as resensitization of epithelial cells to invading pathogens

A

C

E

D B

Fig 5 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

Q-PCR analysis (B) NTHi induced CYLD expression at the mRNA

level in the lungs of C57BL⁄ 6 mice (C) Overexpression of a

domin-ant-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 TLR2 cells compared with

HEK293-pcDNA 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 treated wild-type mice ***P < 0.05, compared with

NTHi-treated control in HEK293-pcDNA cells P value was determined by

Student’s t-test Values are the mean ± S.D (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

autoreg-ulatory feedback manner In contrast with the

relat-ively 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 vitroand in vivo, which in turn results in attenuation

of TLR7 expression, leading to inhibition of host

anti-viral responses Thus, the involvement of CYLD may

be essential to ensure the tight control of

NTHi-induced TLR7 up-regulation and the resultant host

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 detri-mental 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 vivousing 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)

A

B

Fig 6 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 5 h and then treated with R848 (10 l M ) for 3 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).