Volume 20, Number 4, 2007
© Mary Ann Liebert, Inc.
Type I Interferon Regulates Respiratory Virus Infected
Dendritic Cell Maturation and Cytokine Production
BRIAN D. RUDD,1GARY D. LUKER,2KATHRYN E. LUKER,2RAY S. PEEBLES,3
and NICHOLAS W. LUKACS1
Activation of dendritic cells (DCs) by viruses is critical for both innate and adaptive immune re-
sponses. In this report, we investigated the role of type I interferon (IFN) in the activation of DCs
by respiratory syncytial virus (RSV). Using DCs from type I IFNR?/?mice, these studies indicate
that maturation, including upregulation of co-stimulatory molecules and optimal cytokine produc-
tion, by RSV infection was dependent on type I IFN receptor signaling. Subsequently, studies using
DCs from wild type mice demonstrate that continued production of type I IFN during later stages
of DC maturation could alter their activation profiles. IFN-? and IFN-? were upregulated in DCs
grown from bone marrow of wild type mice after infection with RSV. In order to determine their
function in competent DCs, blocking antibodies were used to specifically inhibit IFN-?/?. The data
demonstrate that production of IFN-?, but not IFN-?, in RSV-infected wild type DCs promotes
chemokine production and toll-like receptor (TLR) expression, while limiting IL-12 production. The
inhibition of IL-12p70 by IFN-? correlated with suppressed IL-12p40 expression levels. Further-
more, the addition of recombinant IFN-? potently inhibited IL-12p40 expression in mature DC sub-
sets during RSV infection, while only the highest dose of IFN-? had any inhibitory effect. Together,
our studies provide insight into the complex regulation of DC maturation and IL-12 production co-
ordinated by type I interferons in RSV-infected dendritic cells, and demonstrate that type I IFN has
specific roles depending upon the stage of DC maturation.
1Department of Pathology, 2Department of Radiology, University of Michigan Medical School, Ann Arbor, Michigan, and
3Vanderbilt University School of Medicine, Department of Medicine, Vanderbilt University, Nashville, Tennessee.
Severe RSV infections early in life have been linked to
the later development of pulmonary problems, even years
after the infection has been resolved (1). The develop-
ment of an appropriate immune response during viral in-
fections relies on early events in innate immune cells to
shape the immediate environment and dictate the course
ESPIRATORY SYNCYTIAL VIRUS (RSV) is the leading
cause of lower respiratory tract infections in infants.
of infection, but may also alter the lung environment and
its reactions to subsequent immune responses.
Dendritic cells (DCs) are antigen-presenting cells lo-
cated at the portals of pathogen entry that link innate
and adaptive immunity. Dendritic cells and phagocytes
rapidly produce type I interferons and IL-12 in response
to viral infections. However, the interaction of type I in-
terferons and IL-12 in virally infected dendritic cells is
still poorly understood. Type I interferons consist of a
single IFN-? gene and a large family of IFN-? genes.
Historically, interferons were known to render cells re-
sistant to some viruses and were considered part of the
first wave of host defense (2). While these cytokines may
play a critical role in antiviral defense, many im-
munoregulatory functions in both innate and adaptive im-
munity have been described. It has recently been shown
that type I interferons may also promote the induction of
interferon-stimulated genes (ISGs), natural killer (NK)
cell cytotoxicity, T-cell polarization, and DC maturation
(3). In this study, we aimed to define the role of type I
interferons in regulating DC activation by RSV.
In response to viruses, a subset of type I interferons
are rapidly secreted and bind the type I interferon recep-
tor in autocrine and paracrine fashion (4). Upon binding
to its receptor, the JAK-STAT pathway is engaged, lead-
ing to the subsequent upregulation of all type I interfer-
ons and ISGs. This positive feedback loop has also been
implicated in the cross-regulation of IL-12 in activated
dendritic cells (5).
In this study, we first aimed to describe the functional
role of type I interferon signaling in the activation of DCs
by RSV, and demonstrate that the type I interferon sig-
naling axis is required for upregulation of co-stimulatory
molecules and production of cytokines and chemokines.
These studies subsequently defined the relative contri-
bution of IFN-? in RSV-infected DCs. The release of
IFN-? by RSV-infected DCs was not essential for up-
regulation of co-stimulatory molecules, but it played a
critical role in regulating levels of cytokines and
chemokines. Interestingly, our data demonstrate that IFN-
? had a positive impact on CXCL10 and CCL2 levels,
but negatively affects IL-12 production. By specifically
targeting IFN-?, our results define an opposing role for
IFN-? in RSV-infected dendritic cells, as well as demon-
strate that type I IFNs can have divergent effects on DC
activation depending upon the maturation state of the DC.
These data suggest that although activation via the type
I IFN receptor is crucial for maturation of the dendritic
cell, IFN-?, but not IFN-?, plays the primary role in reg-
ulation of bone marrow–derived dendritic cell (BMDC)
MATERIALS AND METHODS
Reagents, virus, and infections
Neutralizing antibodies against IFN-? (RMMA-1),
IFN-?(RMMB-1), and control were purchased from PBL
Biomedical Laboratories (Piscataway, NJ). Recombinant
murine IFN-? and IFN-? and the ELISA kits were also
purchased from PBL Biomedical Laboratories. The A2
strain of RSV was propagated in Hep2 cells. The viral
titer for virus was determined by RSV-specific antibod-
RUDD ET AL.
ies to visualize the plaques in infected cultures and enu-
merate positive plaques in serially diluted Vero cell cul-
tures. The titer for each virus was determined within a
month of performing the studies and the virus stocks were
stored at ?80°C until use. All stocks were shown to be
free of mycoplasma contamination using a detection kit
from Cambrex (East Rutherford, NJ). UV inactivation of
virus was performed in a laminar flow hood for 30 min
with the virus being ?5 inches from the UV light source.
The DCs were incubated with RSV grown and titered
within the previous month and applied to the cultures at
a multiplicity of infection of 1.0 (MOI ? 1.0).
Generation of bone marrow–derived
Mice deficient in type I IFN receptor (IFN-I R?/?) ex-
pression were maintained and provided by Gary and
Kathryn Luker (University of Michigan, Ann Arbor, MI).
129/SvEv mice (wild type) were purchased from Taconic
(Hudson, NY). Anti-type IFN experiments were initially
performed with BMDCs generated from C57BL/6 mice
(Jackson Laboratories, Bar Harbor, ME), but they were
repeated in 129/SvEv DCs with comparable results. Bone
marrow was harvested from mice and seeded in tissue
culture flasks in RPMI 1640–based complete media with
20 ng granulocyte-macrophage colony-stimulating factor
(GM-CSF)/mL (R&D Systems, Minneapolis, MN). The
cells were fed after 3 d and loosely adherent cells were
collected after 6 d and incubated with anti-CD11c anti-
body coupled to magnetic beads (Miltenyi Biotec,
Auburn, CA). The BMDCs were purified using positive
selection for CD11c? cells by running the cell suspen-
sion through a magnetic column. The next day, CD11c?
BMDCs were infected with RSV (MOI ? 1.0) for 2 h,
washed, and resuspended in fresh media for the indicated
times. The cells were then assessed for gene and protein
Quantification of cytokine expression
Protein levels of IL-12p70 were quantitated using a
Bio-Plex bead-based cytokine assay purchased from Bio-
Rad Laboratories (Hercules, CA). RNA was isolated us-
ing Trizol. Levels of IL-12p40 and CCL2 were assessed
using qPCR analysis (TaqMan; Applied Biosystems, Fos-
ter City, CA) with predeveloped primers and probe sets
from PE Biosystems (Foster City, CA). Primers for
CXCL10 and RSV G protein were custom designed as
previously described (6). Primer sequences for IFN-?and
IFN-? have been previously described (7). qPCR analy-
sis of IFN was performed using SYBR green I dye. Re-
sults were normalized to glyceraldehyde-3-phosphate de-
hydrogenase (GAPDH) expression and presented as fold
increase in mRNA expression compared to the level de-
tected at day 0 using the comparative CTmethod (??CT)
established by Applied Biosystems. As outlined by the
literature, this technique provides a standardized method-
ology that is quantitative based upon a standard house-
keeping gene expression for sample comparisons.
Flow cytometric analysis
Cells were stained with the indicated antibodies (BD
Pharmingen, San Diego, CA) that were specific for co-
stimulatory molecules and analyzed using a fluorescence-
activated cell sorter (FACS) Calibur and Cell Quest soft-
ware (BD Biosciences, San Jose, CA). Isotype control
antibodies were used to demonstrate specificity of our
staining and to establish the criteria for our flow cytom-
All results are expressed as mean ? SE. Statistical sig-
nificance was calculated by ANOVA followed by the stu-
dent’s Neuman Kühl post-test to calculate the p value.
Significance was determined at the level of p ? 0.05.
Type I interferon receptor is essential for
RSV-induced dendritic cell maturation
To examine the kinetics of IFN-?/? production in
RSV-infected dendritic cells from wild type mice, we pu-
rified CD11c? BMDCs and infected (MOI ? 1.0) these
ROLE OF TYPE I INTERFERON IN RSV-INFECTED DENDRITIC CELLS
cells for 8 and 24 h post-infection (hrpi). RNA was iso-
lated at the given time points and levels of IFN-? and
IFN-? were examined by qPCR. Following RSV infec-
tion, IFN-? and IFN-? were highly induced at 8 hrpi and
were lower by 24 hrpi (Fig. 1). UV-inactivated RSV was
incapable of driving type I IFN production, indicating
live RSV was again required for type I interferon syn-
thesis in RSV-infected dendritic cells (Fig. 1A). Inter-
estingly, IFN-?was induced at ?4-fold higher levels than
IFN-?, and IFN-? expression continued to be detectable
at 24 hrpi. We also examined the level of type I IFN pro-
tein expressed after 24 hrpi from wild type DCs (Fig.
1B). Both IFN-? and IFN-? levels were undetectable un-
less infected with RSV. IFN-? was produced at 10-fold
higher levels than IFN-? based on the specific protein
levels (Fig. 1B).
Upon maturation, dendritic cells upregulate co-stimu-
latory molecules and activate T cells in the adaptive im-
mune response. Since a number of reports have shown a
correlation between type I interferon and the maturation
of dendritic cells by viruses (8,9), we first asked whether
type I interferon was required for upregulation of co-stim-
ulatory molecules in RSV-infected dendritic cells. To this
end, we infected bone marrow–derived dendritic cells
(BMDCs) from wild type and IFN-I R?/?mice with
RSV. After 24 hours, the DCs were analyzed by flow cy-
tometry for levels of CD40 and CD86 to determine the
maturation status. RSV significantly upregulated both
CD40 and CD86 expression in wild type DCs (Fig. 2).
However, upregulation of co-stimulatory molecules was
severely impaired in IFN-I R?/?DCs (Fig. 2), demon-
8 16 24
Time post-infection (hr)
vated RSV, or incubated with media alone for 8, 24, or 48 h. (A) At the indicated time points, expression levels of IFN-?4 or
IFN-? were measured by qPCR and expressed as the mean fold change relative to media controls. (B) To further assess the rel-
ative production of type I IFNs protein levels were assessed in DCs after 24 h of RSV infection. Data are representative of three
repeat experiments, with n ? 3 per treatment per experiment.
Type I IFNs are upregulated in DCs infected with RSV. Wild type DCs were infected with live RSV, UV-inacti-
strating that type I interferon is essential for virus-induced
maturation in RSV-infected dendritic cells. UV-inactivated
RSV was incapable of upregulating CD40 and CD86 in
wild type DCs, indicating that replication-competent RSV
was required to promote DC maturation (data not shown).
RUDD ET AL.
Previous reports demonstrated that type I interferon
signaling in the early phase of DC maturation was es-
sential to sustain inflammatory cytokine production in the
later phases (9). Given the profound defect in CD40 and
CD86 expression in IFN-I R?/?DCs, we next examined
R?/?were infected with RSV for 24 h. Expression of co-stimulatory molecules CD40 and CD86 were analyzed by FACS. Open
curves depict unstimulated DCs; shaded histograms represent RSV-infected DCs. The mean fluorescence intensity (MFI) for each
condition is indicated in the upper corner. Data are representative of three repeat experiments with similar results.
Type I interferon signaling is required for RSV-induced maturation of DCs. DCs derived from wild type and IFN-I
wild type Neg
IFN-I R?I? Neg
rived from wild type and IFN-I R?/?mice were infected with RSV. (A) At 8 and 24 h, protein levels of IL-12p70 in the super-
natant were determined. (B) Expression levels of CXCL10, CCL2, TLR3, and RSV-G were measured using qPCR and the rela-
tive fold increase compared to RSV-infected wild-type DCs. Data are representative of three repeat experiments, with n ? 3 per
treatment per experiment. Each time point represents the mean ? SE (*p ? 0.05).
Type I interferon signaling is required for optimal cytokine/chemokine production in RSV-infected DCs. DCs de-
the role of type I interferon receptor for IL-12 produc-
tion in RSV-infected BMDCs obtained from wild type
and IFN-I R?/?mice. Interestingly, IFN-I R?/?DCs in-
fected with RSV had significantly reduced levels of IL-
12p70 protein compared to wild type DCs (Fig. 3A).
These results indicate a partial requirement for type I in-
terferon signaling in optimal IL-12 production in RSV-
Interferons have also been shown to induce specific
chemokines involved in antiviral immunity (10). Next we
compared mRNA levels of CXCL10 and CCL2 in wild
type and IFN-I R?/?DCs. Induction of both CXCL10
and CCL2 were almost entirely abrogated in IFN-I R?/?
DCs infected with RSV (Fig. 3B). In addition, when we
examined TLR3 expression of the IFN-I R?/?dendritic
cells, they were deficient in their ability to increase TLR3
expression (Fig. 3B). While one possible interpretation
of these results might be that the IFN-I R?/?DCs were
not infected, our assessment of RSV-G protein expres-
sion demonstrated that there was actually an increase in
RSV gene expression in the DCs from IFN-I R?/?mice.
These results indicate that the induction of chemokines
and important innate sensing molecules such as TLR3 in
ROLE OF TYPE I INTERFERON IN RSV-INFECTED DENDRITIC CELLS
RSV-infected DCs is dependent on activation by the type
I interferon receptor.
Type I interferon differentially regulates cytokine
production during dendritic cell maturation
Since type I interferon receptor signaling was essen-
tial for the induction of interferon-dependent chemo-
kines, we asked whether RSV-induced chemokine pro-
duction was affected by the release of IFN-? or IFN-?
by infected DCs. In particular, CXCL10 and CCL2 have
previously been shown to be type I IFN inducible during
viral infection as was recently reviewed (11). We com-
pared mRNA levels of CXCL10 and CCL2 from RSV-
infected cells (MOI ? 1.0) pretreated with a pan-in-
Pretreatment of DCs with anti-IFN suppressed levels of
IFN activity ?60% (data not shown). qPCR analysis of
CXCL10 and CCL2 from cells treated with anti-IFN-?
showed a dramatic reduction in those chemokines com-
pared to untreated and isotype-control treated cells, indi-
cating that IFN-? mediates chemokine production in
RSV-infected dendritic cells (Fig. 4). Antibodies to IFN-
a specific anti-IFN-?.
RSV ? alFN-alpha
RSV ? alFN-beta
RSV ? Isotype
IFN-? (20 ?g/mL), isotype control (20 ?g/mL), or medium prior to RSV infection. The antibodies were left in the media and
cells were infected with RSV. At 24 hrpi, RNA levels of CXCL10 (A), CCL2 (B), and TNF (C) were analyzed by qPCR and
expressed as the fold change relative to media controls. Similarly, the mRNA was also examined for expression of TLRs (D),
which are upregulated after RSV activation. Data are representative of three repeat experiments, with n ? 3 per treatment per
experiment. Each time point represents the mean ? SE. (*p ? 0.05)
IFN-? is essential for chemokine production in RSV-infected DCs. DCs were pretreated with either anti-IFN-?, anti-
? did not reduce any of the same responses observed
by blocking IFN-?. The ability of the antibody against
IFN-? to neutralize was verified using recombinant
IFN-? on uninfected DCs (data not shown). In contrast,
when we examined another often-produced DC-derived
cytokine, tumor necrosis factor (TNF), no alteration
was detected after blockade of IFN-? (Fig. 4C). In ad-
dition to cytokine production, the activation and mat-
uration of DCs by type I IFN also can regulate the ex-
pression of additional innate molecules such as toll-like
receptors (TLRs) that recognize viral nucleic acid and
further allow maturation of DCs. In the present studies
we examined the expression of TLR3, that recognizes
dsRNA, and TLR9, that recognizes unmethylated CpG,
and were both upregulated (Fig. 4D). The data illus-
trate that by neutralizing IFN-?, but not IFN-?, the ex-
pression of these TLRs are both significantly reduced
after RSV infection.
The data in the IFN-I R?/?DCs described above in-
dicate dependence on type I IFN for IL-12 production.
To determine the specific role of type I IFN in IL-12 pro-
duction, we also measured IL-12 levels in RSV-infected
RUDD ET AL.
DCs treated with anti-IFN-?/?. Only neutralization of
IFN-? resulted in significant upregulation of IL-12p70
protein, indicating that the release of IFN-? in RSV-in-
fected BMDCs profoundly impairs the ability of RSV to
synthesize IL-12 (Fig. 5A). The mechanism of IL-12 in-
hibition by IFN-? has been attributed to the transcrip-
tional downregulation of the IL-12p40 chain (12). To test
this possibility, we next measured the upregulation of IL-
12p40 mRNA in RSV-infected DCs treated with anti-
type I IFN antibodies. The accumulation of IL-12p40
mRNA in anti-IFN-?–treated DCs correlated with the el-
evated levels of IL-12p70 protein (Fig. 5A and 5B). These
findings were also verified when we infected DCs in the
presence of increasing amounts of IFN-?or IFN-?. When
the recombinant cytokines were used, although IFN-?
was able to reduce IL-12p40 expression at the highest
dose, it was not as effective as IFN-?, which induced a
dose-dependent reduction in IL-12p40 mRNA (Fig. 5C
and 5D). When IL-12p35 subunit mRNA was examined
in these same studies, no alteration after any of the treat-
ments compared to control treated cells was observed
(data not shown).
Units of IFN?
Units of IFN?
?g/mL), isotype control (20 ?g/mL), or medium, and infected with RSV for 24 h. Protein levels of IL-12p70 (A) and RNA lev-
els of IL-12p40 (B) were determined as previously described. Increasing concentrations of recombinant murine IFN-? (C) or
IFN-? (D) were also added to DCs with RSV, and expression levels of IL-12p40 were determined at 24 h. Data are representa-
tive of three repeat experiments, with n ? 3 per treatment per experiment. Each time point represents the mean ? SE (*p ? 0.05).
Neutralization of IFN-? upregulates IL-12 in DCs infected with RSV. DCs were pretreated with anti-IFN-? (20
Blocking IFN-? does not alter co-stimulatory
molecules on RSV-infected dendritic cells
Our data with IFN-I R?/?mice also suggest that DC
maturation does not occur by direct signaling by RSV
infection, but requires the autocrine/paracrine type I in-
terferon-signaling pathway. To examine the relative
contribution of IFN-?, we next evaluated the ability of
RSV-infected DCs pretreated with anti-IFN-? to up-
regulate CD40 and CD86. However, neutralization of
IFN-? did not significantly impair DC expression of
these two co-stimulatory molecules by itself, since they
were similar to control DCs infected with RSV (Fig. 6).
Together, neutralization of IFN-? had no discernable af-
fect on DC co-stimulation in RSV-infected DCs, but it
ROLE OF TYPE I INTERFERON IN RSV-INFECTED DENDRITIC CELLS
is necessary for optimal production of specific cy-
tokines, chemokines, and TLRs.
The above-described experiments establish that type I
interferon receptor signaling is essential for the upregu-
lation of co-stimulatory molecules and optimal cy-
tokine/chemokine production in RSV-infected DCs early
in the maturation process. When type I interferon pro-
duction was assessed it was observed that a significant
upregulation of both IFN-? and IFN-? was induced by
RSV, with IFN-? expressed at eight- to 10-fold higher
levels when we assessed the protein. The inability of RSV
?g/mL), isotype control (20 ?g/mL), or medium, and infected with RSV for 24 h. Expression levels of the co-stimulatory mol-
ecules CD40 and CD86 were analyzed by FACS. Open curves depict isotype controls; shaded histograms represent each treat-
ment group. The MFI for each condition is indicated in the upper corner. Data are representative of three repeat experiments with
IFN-? is not required for RSV-induced maturation of wild type DCs. DCs were pretreated with anti-IFN-? (20
to upregulate CD40 and CD86, chemokines, and opti-
mize IL-12p70 levels, in DCs from the type I IFN re-
ceptor knockout mice we demonstrated the importance
of this signaling molecule during the initial stages of RSV
infection. Given the global impairments in the activation
of IFN-I R?/?DCs infected with RSV, the function of
type I IFNs was further assessed in DCs from wild type
mice using neutralizing antibodies specific for IFN-?
or IFN-?. Similar to RSV-infected IFN-I R?/?DCs,
chemokine production was significantly downregulated
in anti-IFN-?–treated cells. The most interesting aspect
of these studies was that blocking IFN-? has no effect
on the expression of co-stimulatory molecules, but it aug-
ments IL-12p70 production. Thus, while the expression
of co-stimulatory molecules and IL-12p70 production is
severely impaired in IFN-I R?/?DCs, DCs treated with
anti-IFN-? expressed similar levels of co-stimulatory
molecules, but enhanced IL-12p70 production. These ob-
servations can be explained by at least two possible mech-
anisms: First, type I interferon signaling is important in
maturing RSV-infected DCs to the point of being able to
produce IL-12, but continued production of IFN-? regu-
lates IL-12 in maturing DCs. Alternatively, RSV might
require type I interferon signaling to initiate the induc-
tion of IL-12p35 (5), but later regulate IL-12p40 pro-
duction in activated DCs via a negative feedback loop in-
volving type I IFN (12,13). Thus, these data demonstrate
that DCs have the ability to modify their own responses
by continued production of IFN-? during later stages of
Our recognition that IL-12 regulation is linked to DC
maturation is based on the observation that optimal IL-
12 levels are only produced in DCs that are capable of
undergoing maturation. While the type I interferon re-
ceptor is required for optimal IL-12p70 production in
DCs stimulated with a combination of TLR ligands that
are able to induce IL-12p70 (5), IFN-I R?/?DCs stim-
ulated with dsRNA or lipopolysaccharide do not mature
(14). When IFN-? was specifically targeted in RSV-in-
fected DCs, the ability to upregulate co-stimulatory mol-
ecules is not impaired. The key finding is that IFN-? be-
ing produced in DCs that are capable of undergoing
maturation is an important regulator of cytokine produc-
tion by both limiting IL-12p70 and by augmenting
chemokine production. While the implications of these
observations are not completely clear, they may have sig-
nificant impact on T-lymphocyte recruitment and differ-
entiation during antigen presentation. Of considerable in-
terest is the fact that when IFN-? was blocked a similar
effect was not observed. This may be due to the fact that
the majority of type I IFN expressed in these BMDC stud-
ies was IFN-?. However, rIFN-? was not able to regu-
late IL-12p40 expression in a similar manner as IFN-?
upon RSV infection. This result might explain how pDCs,
RUDD ET AL.
which are the primary producers of IFN-?, may partici-
pate in the maturation of mDCs within the lymph nodes
without altering IL-12 production, and therefore aid in
the overall antiviral response (15).
Numerous signaling mediators in the type I interferon
loop that regulate IL-12p70 production have been de-
scribed and include STAT1 and IRF3 (5,16). The de-
creases in IL-12p70 in IFN-I R?/?, STAT1?/?, and
IRF3?/?DCs was largely attributed to impaired IL-
12p35 production. Recent evidence suggests that IRF3 is
a key transcription factor in IL-12p35 production (16).
While induction of IRF3 occurs through direct TLR sig-
naling pathways, it may be further amplified by signal-
ing through the type I interferon receptor. Thus, lower
levels of IL-12p70 in RSV-infected IFN-I R?/?DCs may
represent only the direct IRF3 pathway via TLR signal-
ing, and be missing the amplified signal that occurs in
wild type DCs through the type I interferon receptor.
However, high levels of type I inteferons have also been
demonstrated to inhibit IL-12p70 by limiting IL-12p40
production in both human and murine dendritic cells
(12,13,17,18). While IL-12p40 is inducible, it is com-
monly produced in large excess. This may explain why
high levels of interferons are required to downregulate
IL-12p40 to quantities that would begin to limit IL-12p70
levels. While we see little or no induced p19 mRNA lev-
els or IL-23 production after RSV infection (data not
shown), the regulation of IL-12p40 by type I IFN during
other responses could also affect IL-23 production as
well. Interestingly, while the anti-IFN-? treatment al-
lowed enhanced production of IL-12p70, there was a re-
duction in TLR3 and TLR7 expression by the RSV-in-
fected DCs. The implications of this opposing regulation
of TLRs and IL-12 are also unclear, but may relate to the
overall maturation state of the DC as it becomes less re-
sponsive to external stimuli by downregulating TLRs at
the same time that is producing more IL-12 to skew the
ensuing virus-specific adaptive responses. Our data sug-
gest that sufficient amounts of IFN-? are produced in
RSV-infected DCs to limit IL-12p70 production. The in-
hibition of IL-12p70 by IFN-? correlated with suppres-
sion of only IL-12p40, since IL-12p35 levels were sim-
ilar among all treatment groups. These latter studies are
consistent with previous observations that demonstrated
that endogenous type I IFN produced in LCMV- and
MCMV-infected mice profoundly inhibited IL-12 pro-
duced by DCs (19,20). Consistent with the data that show
the ability of IFN-? to regulate IL-12 were studies ex-
amining production of IFN-? by pDCs in MCMV-in-
fected mice, that showed inhibited IL-12 levels in non-
pDC subsets (19,21).
Studies indicate that effective viral responses correlate
with sufficient IL-12 production by immune cells (22,23).
In studies more specific to RSV infection, investigators
used IL-12p40?/?mice, and demonstrated a role of IL-
12p40 in limiting viral replication, inflammation, mucus
production, and Th2 cytokine production (24,25). Previ-
ous results indicated that treatment of RSV-infected mice
with anti-IL-12 resulted in increased AHR and mucus ex-
pression mediated by IL-13 (26,27). These studies fur-
ther support seminal studies linking type I IFNs with im-
mune responses in RSV infection (28). The current study
suggests that excessive IFN-? might contribute to pul-
monary pathology by altering IL-12 levels during severe
RSV responses, and limit development of effective an-
tiviral responses. This latter suggestion may be intrigu-
ing and testable during a normal RSV infection in wild
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Address reprint requests to:
Dr. Nicholas W. Lukacs
Department of Pathology
University of Michigan Medical School
1301 Catherine, 5214 Medical Science 1
Ann Arbor, MI 48109
Received June 4, 2007; accepted September 4, 2007
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