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: 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 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 ª2007 The Authors Journal compilation ª2007 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 ª2007 The Authors Journal compilation ª2007 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. Pvalue was determined by Student’s t-test. Values are the mean ± SD (n¼3 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 ª2007 The Authors Journal compilation ª2007 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 ª2007 The Authors Journal compilation ª2007 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 IjBaand 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 IjBaon 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 IjBaalso 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 IKKbin TLR7 induction by NTHi.
As shown in Fig. 3C, a dominant-negative mutant of
IKKbinhibited 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 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. Pvalue was determined by Student’s
t-test. Values are the mean ± SD (n¼3).
A. Sakai et al.Regulation of TLR7 by bacterium NTHi
FEBS Journal 274 (2007) 3655–3668 ª2007 The Authors Journal compilation ª2007 FEBS 3659