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

The bacterium, nontypeable Haemophilus influenzae,
enhances host antiviral response by inducing Toll-like
receptor 7 expression
Evidence for negative regulation of host antiviral response by CYLD
Akihiro Sakai
1,2
*, Tomoaki Koga
1
*, Jae-Hyang Lim
1
, Hirofumi Jono
1
, Kazutsune Harada
3
,
Erika Szymanski
1
, Haidong Xu
1
, Hirofumi Kai
3
and Jian-Dong Li
1
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:
*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 resurgence 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.
FEBS Journal 274 (2007) 3655–3668 ª 2007 The Authors Journal compilation ª 2007 FEBS 3655
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 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 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
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 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.
Regulation of TLR7 by bacterium NTHi A. Sakai et al.
3656 FEBS Journal 274 (2007) 3655–3668 ª 2007 The Authors Journal compilation ª 2007 FEBS
A
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
)1
NTHi lysate) and time-dependent (15 lgÆmL
)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 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. Sakai et al. Regulation of TLR7 by bacterium NTHi
FEBS Journal 274 (2007) 3655–3668 ª 2007 The Authors Journal compilation ª 2007 FEBS 3657
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
AB
D
E
G
C
F
Regulation of TLR7 by bacterium NTHi A. Sakai et al.
3658 FEBS Journal 274 (2007) 3655–3668 ª 2007 The Authors Journal compilation ª 2007 FEBS
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
NTHi-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 ngÆ mL
)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).
A. Sakai et al. Regulation of TLR7 by bacterium NTHi
FEBS Journal 274 (2007) 3655–3668 ª 2007 The Authors Journal compilation ª 2007 FEBS 3659
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
Regulation of TLR7 by bacterium NTHi A. Sakai et al.
3660 FEBS Journal 274 (2007) 3655–3668 ª 2007 The Authors Journal compilation ª 2007 FEBS
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
CYLD attenuated NF-jB activation by NTHi,
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 NTHi-
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.
A. Sakai et al. Regulation of TLR7 by bacterium NTHi
FEBS Journal 274 (2007) 3655–3668 ª 2007 The Authors Journal compilation ª 2007 FEBS 3661
A
DE
G
F
HI J
BC
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.
Regulation of TLR7 by bacterium NTHi A. Sakai et al.
3662 FEBS Journal 274 (2007) 3655–3668 ª 2007 The Authors Journal compilation ª 2007 FEBS
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 bacteria
enhances host antiviral response. In this study, we pro-
vide 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-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 HEK293-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 NTHi-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).
A. Sakai et al. Regulation of TLR7 by bacterium NTHi
FEBS Journal 274 (2007) 3655–3668 ª 2007 The Authors Journal compilation ª 2007 FEBS 3663
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 vitro and 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 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).
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).
Regulation of TLR7 by bacterium NTHi A. Sakai et al.
3664 FEBS Journal 274 (2007) 3655–3668 ª 2007 The Authors Journal compilation ª 2007 FEBS
Bacterial strains and culture conditions
NTHi strain 12, a clinical isolate, was used in this study.
Bacteria were grown on chocolate agar plates at 37 °Cinan
atmosphere of 5% CO
2
. To make NTHi lysate, NTHi were
harvested from a chocolate agar plate after overnight incu-

bation and incubated in 30 mL brain ⁄ heart infusion broth
supplemented with NAD (3.5 lgÆmL
)1
). After overnight
incubation, NTHi were centrifuged at 6000 g for 10 min
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 soni-
cated. Subsequently, the crude extract was collected and
stored at )70 °C [11,12]. NTHi lysates (15 lgÆmL
)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 treat-
ment. Therefore, lysates of NTHi represent a common clin-
ical condition in vivo, especially after antibiotic treatment.
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, HEK293-
TLR2, and HEK293-TLR4, were kindly provided by
D. T. Golenbock [28,29]. All stable cell lines were main-
tained 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 · 10
6

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 see-
ded at 2 · 10
4
cells ⁄ cm
2
on to 24-mm-diameter, 0.4 lm
pore size, semipermeable membrane inserts (Transwell
Permeable Supports; Corning, Corning, NY, USA) in bron-
chial epithelial cell basal medium (Cambrex). The cultures
were grown submerged for the first 7 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 supplemen-
ted 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% CO
2
.

Real-time Q-PCR analysis
Total RNA was isolated with TRIzol reagent (Invitrogen) by
following the manufacturer’s instructions. For the reverse
transcription reaction, TaqMan reverse transcription rea-
gents (Applied Biosystems, Foster City, CA, USA) were
used. In brief, the reverse transcription reaction was per-
formed for 60 min at 37 °C, followed by 60 min at 42 °Cby
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 2 · Universal Master Mix, 1 lL tem-
plate 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-carboxytetramethylrhod-
amine, 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 corres-
ponding software (7000v1.3.1; Applied Biosystems). The rel-
ative quantities of mRNAs were obtained by using the
comparative Ct method and were normalized with predevel-
oped TaqMan assay reagent human cyclophilin or mouse
glyceraldehydes-3-phosphate dehydrogenase as an endog-
enous control (Applied Biosystems). The primers and probes
Fig. 7. Schematic representation of the signaling pathways involved
in the positive and negative regulation of NTHi-induced TLR7
expression.

A. Sakai et al. Regulation of TLR7 by bacterium NTHi
FEBS Journal 274 (2007) 3655–3668 ª 2007 The Authors Journal compilation ª 2007 FEBS 3665
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 des-
cribed [22–24]. The reporter construct NF-jB-luc was as des-
cribed [13,14]. Negative control plasmid and p65 siRNA
plasmid were purchased from Imgenex (San Diego, CA,
USA). The expression plasmid of wild-type TLR7 (pUNO-
hTLR7) 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% conflu-
ent 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). Anti-
bodies 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-
maldehyde solution, permeabilized with 0.5% Triton X-100
solution, and then incubated with anti-(human TLR7) IgG
(diluted 1 : 50) for 1 h at room temperature. Primary anti-
body 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 ⁄ 6 mice and BALB ⁄ c mice were purchased from
Charles River Laboratories, Tlr2
– ⁄ –
mice were kindly provi-
ded by Dr Shizuo Akira (Osaka University), and 7–8-week-

old mice were used in this study. Under anesthesia, mice
were intratracheally inoculated with NTHi or saline as con-
trol. Lung tissues were collected and then stored at ) 80 °C.
Total RNA was isolated from the frozen whole lung tissue
with TRIzol reagent. Protein samples were extracted from
the frozen tissue for western blot analysis. Three mice were
used for each inoculation group. All animal experiments
were approved by the institutional Animal Care and Use
Committee at University of Rochester.
Acknowledgements
We are grateful to Dr S. Akira and Dr D. T. Golen-
bock for kindly providing various reagents. This work
was supported by grants from National Institute of
Health DC005843 and DC004562 (to JDL). We thank
the members of the laboratory of HK in the Graduate
School of Pharmaceutical Sciences at Kumamoto Uni-
versity and the members of the laboratory of JDL at
the University of Rochester for stimulating scientific
discussion.
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Regulation of TLR7 by bacterium NTHi A. Sakai et al.
3668 FEBS Journal 274 (2007) 3655–3668 ª 2007 The Authors Journal compilation ª 2007 FEBS