Role for Innate IFNs in Determining Respiratory Syncytial
Teresa R. Johnson,†Sara E. Mertz,* Negin Gitiban,* Sue Hammond,* Robin LeGallo,*
Russell K. Durbin,* and Joan E. Durbin2*
Respiratory syncytial virus (RSV) is the major cause of severe lower airway disease in infants and young children, but no safe and
effective RSV vaccine is yet available. The difficulties involved in RSV vaccine development were recognized in an early vaccine
trial, when children immunized with a formalin-inactivated virus preparation experienced enhanced illness after natural infection.
Subsequent research in animal models has shown that the vaccine-enhanced disease is mediated at least in part by memory cells
producing Th2 cytokines. Previously we had observed enhanced, eosinophilic lung pathology during primary infection of IFN-
deficient STAT1?/?mice that are incapable of generating Th1 CD4?cells. To determine whether these effects depended only on
Th2 cytokine secretion or involved other aspects of IFN signaling, we infected a series of 129SvEv knockout mice lacking the
IFN-??R (IFN-??R?/?), the IFN-?R (IFN-?R?/?), or both receptors (IFN-???R?/?). Although both the IFN-?R?/?and the
IFN-???R?/?animals generated strong Th2 responses to RSV-F protein epitopes, predominantly eosinophilic lung disease was
limited to mice lacking both IFNRs. Although the absolute numbers of eosinophils in BAL fluids were similar between the strains,
very few CD8?T cells could be detected in lungs of IFN-???R?/?animals, leaving eosinophils as the predominant leukocyte.
Thus, although CD4?Th2 cell differentiation is necessary for the development of allergic-type inflammation after infection and
appears to be unaffected by type I IFNs, innate IFNs clearly have an important role in determining the nature and severity of RSV
disease. The Journal of Immunology, 2005, 174: 7234–7241.
able. The difficulties involved in RSV vaccination became evident
in early vaccine trials, when children immunized with a formalin-
inactivated virus preparation experienced enhanced illness after
natural infection (1, 2). Subsequent studies in animal models have
shown that unlike natural infection, formalin-inactivated RSV
priming leads to the development of a Th2 memory response,
which is thought to mediate the immunopathology seen after virus
challenge (3–5). This is of particular concern in light of recent
studies showing a link between severe RSV disease in childhood
and the development of asthma later in life (6). To test this hy-
pothesis, in previous studies we examined whether mice lacking
the ability to develop a Th1 response would develop eosinophilic
airway disease after primary RSV infection (7). For that purpose
we studied mice lacking STAT1 (8), the major mediator of IFN-??
and IFN-? signaling. Th1 differentiation of naive CD4?cells de-
espiratory syncytial virus (RSV)3is the major cause of
severe lower airway disease in infants and young chil-
dren, but no safe and effective RSV vaccine is yet avail-
pends on the presence of the transcription factor T-bet, the induc-
tion of which is STAT1 dependent (9, 10). In fact, RSV-infected
STAT1?/?mice did develop Th2 cytokine responses and severe
eosinophilic lung disease without priming, and this correlated with
high levels of IL-13 production by virus-specific CD4?T cells.
Although these results clearly demonstrated the link between the
development of Th2 cells and enhanced disease, we were struck by
the fact that IFN-??/?animals studied in parallel with STAT1?/?
mice were much less affected. This result was unanticipated, be-
cause we had assumed that mice lacking IFN-? would also default
to a Th2 CD4?T cell differentiation pathway. We concluded that
although CD4?T cells producing Th2 cytokines were necessary
for an allergic-type, eosinophilic response to develop, other factors
could modulate this effect. Because STAT1 mediates signaling
through both the IFN-?? and IFN-? receptors (11), we wondered
whether type I (??) IFNs were acting to diminish the pathology
seen in STAT1 animals. We hypothesized that either IFN-?? was
affecting CD4?T cell differentiation, or other effects of type I IFN
were acting to limit eosinophilic disease.
For a more rigorous approach to these questions, we have com-
pared the innate and adaptive responses to acute RSV infection in
a series of 129SvEv knockout mice lacking the IFN-??R, the IFN-
?R, both receptors, or STAT1?/?. In this study we have observed
that both mouse strains lacking the IFN-?R produce virus-specific
Th2 lymphocytes in response to RSV infection regardless of
IFN-?? signaling. Nonetheless, significant differences remain be-
tween mice lacking both IFNRs and mice that retain the IFN-??R.
As found previously in the BALB/c mouse, inflammatory infil-
trates from infected mice deficient in IFN-? signaling remained
primarily lymphocytic, whereas bronchoalveolar lavage (BAL)
specimens from IFN-???R?/?(IFN-???R?/?) mice consisted
primarily of eosinophils, with a notable lack of CD8?T cells. The
degree of eosinophilia seen in the lungs of IFN-???R?/?mice
therefore reflects a paucity of responding CTLs as well as a large
*Columbus Children’s Research Institute and Department of Pediatrics, Ohio State
University College of Medicine and Public Health, Columbus, OH 43205; and†Vac-
cine Research Center, National Institutes of Health, Bethesda, MD 20892
Received for publication December 3, 2004. Accepted for publication March
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by Grant AI47226 from the National Institutes of Health
2Address correspondence and reprint requests to Dr. Joan E. Durbin, Department of
Pediatrics, Ohio State University College of Medicine and Public Health, Columbus
Children’s Research Institute, 700 Children’s Drive, Room WA4014, Columbus, OH
43205-2696. E-mail address: firstname.lastname@example.org
3Abbreviations used in this paper: RSV, respiratory syncytial virus; BAL, bronchoal-
veolar lavage; i.n., intranasally; IP-10, IFN-induced protein 10; I-TAC, IFN-induced
T cell ? chemoattractant; MIG, monokine induced by IFN-?; RNAP, RNase protec-
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc.0022-1767/05/$02.00
number of eosinophils presumably elicited by IL-5- and IL-13-
producing CD4?cells. This study demonstrates that although Th2
cytokines are essential for the development of an allergic-type in-
flammatory response, they are not the sole determinant of virally
induced immune pathogenesis.
Materials and Methods
Virus, cells, and virus quantitation
A laboratory stock of RSV A2 (originally obtained from R. Chanock, Na-
tional Institutes of Health, Bethesda, MD) was propagated in HEp-2 cells
(American Type Culture Collection) as previously described (12). Infec-
tivity was assayed using a fibroblast cell line derived from STAT1?/?
mice, designated NY3 (8). For determination of lung titers, lung tissue was
weighed, and samples from individual mice were homogenized in 1 ml of
PBS using a PowerGen125 homogenizer (Fisher Scientific). Dilutions of
lung supernatants were inoculated onto subconfluent monolayers of NY3
cells, and after 1 h, plates were covered with 0.5% methylcellulose in
medium and incubated for 36 h at 37°C. In our experience, NY3 and HEp2
cells give comparable titers. Monolayers were fixed with 2% buffered
formaldehyde and stained with crystal violet.
Mice on the 129SvEv background lacking the type I IFNR subunit 1, if-
nar1?/?(IFN-??R?/?) (13), the type 2 IFNR subunit 1, ifngr1?/?(IFN-
?R?/?) (14), or both genes, ifnnar1?/?/ifngr1?/?(IFN-???R?/?) (15)
were provided by R. Schreiber (Washington University, St. Louis, MO)
and originally derived by M. Aguet (Swiss Institute for Imperial Cancer
Research, Lausanne, Switzerland). Strain-matched controls were purchased
from Taconic Farms. Mice with a targeted disruption of the stat1 gene (8)
were backcrossed onto the 129SvEv strain, and animals of the ninth back-
cross generation were used in these experiments. Cohorts of 6- to 8-wk-old
animals were lightly anesthetized and inoculated intranasally (i.n.) with 107
PFU of RSV A2. Groups of four to six animals were used for each data
Mice were killed, and the tracheas were isolated by blunt dissection. One
milliliter of PBS was injected and slowly withdrawn through a blunt tip
needle inserted and secured in the airway. After the total cell number was
determined, each BAL fluid sample was centrifuged, and the supernatants
were stored at ?70°C until further use. The pellets were resuspended in a
small volume of PBS, and cytospins were prepared using a Shandon Cy-
tospin centrifuge (Thermo Electron). BAL cell counts were made using a
hemocytometer; only cells excluding trypan blue (Invitrogen Life Tech-
nologies) were counted. The numbers of eosinophils and lymphocytes were
calculated by multiplying the total BAL cell numbers by the corresponding
fractions derived from differential counts. The numbers of CD4?T cells,
CD8?T cells, and B cells per sample were calculated using flow cytometry
data to determine the percentage of each cell type within the lymphocyte
gate and total lymphocyte numbers. Cells marking as lymphocytes, taken
together, comprised a total percentage of BAL cells similar to differentials
based on morphology.
Flow cytometry of BAL cells
BAL samples were incubated with allophycocyanin-conjugated Abs to
CD8, PE-conjugated Abs to CD4, and FITC-conjugated Abs to B220 (BD
Pharmingen) for 30 min at room temperature. Samples were washed and
resuspended in FACS buffer. Data were collected on a FACSCalibur (BD
Biosciences) flow cytometer and analyzed with CellQuest software (BD
Biosciences). Dead cells were excluded on the basis of forward and side
Lungs were harvested from mice 8 days after infection with RSV and were
inflated with 10% buffered formalin. Paraffin sections were stained with
H&E. Differential cell counts were performed on cytospins of BAL cell
pellets harvested from each animal. Cytospins were stained with Wright-
Giemsa, and monocyte/macrophages, lymphocytes, neutrophils, and eosin-
ophils were enumerated based on cell morphology and staining.
Identification of an RSV-F peptide epitope
A series of 57 overlapping peptides corresponding to the 574-aa RSV B
strain, F protein sequence (16) was synthesized by Chiron Mimotopes.
Each peptide was 20 aa in length and overlapped the adjacent peptide by
10 residues. Individual peptides were dissolved in 10% DMSO to a con-
centration of 4 mg/ml and then pooled into sets of 10. As an initial screen,
peptide pools were used to stimulate splenocytes harvested from wild-type
129SvEv mice infected i.n. 28 days previously with 106PFU of RSV. After
7 days of peptide stimulation, splenocytes (0.5 ? 105) from RSV-immune
animals were mixed with 1.5 ? 105gamma-irradiated, peptide-pulsed (10
?g/ml), naive splenocytes in AIM-V medium containing 2.5% heat-inac-
tivated mouse serum (Stellar BioSystems). Each sample was plated in trip-
licate wells of a round-bottom, 96-well plate. Two days after peptide re-
stimulation, 1 ?Ci of [3H]thymidine was added to each well. After an
additional 12-h incubation at 37°C, [3H]thymidine incorporation was mea-
sured using a Wallac Microbeta scintillation counter.
IFN-? ELISPOT assay kits were purchased from U-Cytech. Splenocytes
harvested from RSV-immune 129SvEv mice (wild-type, IFN-??R?/?,
IFN-?R?/?, and IFN-???R?/?) were plated directly or after CD4?cell
depletion. Depletion was performed using mouse CD4 Dynabeads (Dynal
Biotech) according to the manufacturer’s instructions. Splenocytes were
suspended in AIM-V medium containing 2.5% heat-inactivated mouse se-
rum and plated at various dilutions in 96-well plates. Viral or irrelevant
(SIINFEKL) peptides were included at a concentration of 10 ?g/ml. After
24 h at 37°C, the cells were removed, and IFN-?-containing spots were
visualized by sequential incubations with a biotinylated polyclonal anti-
IFN-? Ab, an anti-biotin tertiary Ab, and the detection reagent according to
the manufacturer’s protocol.
Measurement of cytokine transcripts by RNase protection assay
Immune splenocytes were placed in culture and stimulated with 10 ?g/ml
peptide for 1 wk; 6 h after restimulation, RNA was purified using TRIzol
(Invitrogen Life Technologies). RNAPs were performed using the mCK-1
RiboQuant kit purchased from BD Pharmingen. Assays were performed
according to the instructions provided by the manufacturer using 15 ?g of
lymphocyte RNA/sample. Signal intensity over background was quanti-
tated using the Storm PhosphorImager (Molecular Dynamics) and was nor-
malized to an L32 internal control using ImageQuant software.
Cytokine and chemokine assays
BAL supernatants were sent to Linco Research to determine concentrations
of IL-5 and IFN-?. This method uses beads conjugated to analyte-specific
Abs, which are detected by the Luminex100system. IL-13, IFN-?, eotaxin,
and IFN-induced protein 10 (IP-10) concentrations were measured using
ELISA kits purchased from R&D Systems. IFN-? concentrations for in-
dividual samples were consistent between the assay systems.
Data are expressed as the mean ? SEM. Comparisons between groups
were based on ANOVA, calculated using SigmaStat software (SAS Insti-
tute). Dunnett’s procedure, which adjusts for multiple comparisons against
a predefined group, was applied only when there was a significant overall
difference between groups. A value of p ? 0.05 was considered statistically
Control of RSV replication is STAT1 dependent
We have previously observed a strain-dependent increase in virus
production after i.n. infection of STAT1-deficient mice (7). In the
current study we examined the relative RSV susceptibilities of
additional strains of knockout animals (wild-type, IFN-??R?/?,
IFN-?R?/?, IFN-???R?/?, and STAT1?/?), all on a 129SvEv
background. Groups of 10 animals from each genotype were in-
oculated i.n. with 1 ? 107PFU of RSV. The titers shown in Fig.
1 are derived from lungs taken on day 5 after infection, the peak
of virus production, and on day 8 when virus would normally be
cleared. In vivo, both IFN-?? and IFN-? are induced by virus
infection, albeit with somewhat different kinetics, and we expected
that either cytokine alone would induce an antiviral state (17). This
prediction was borne out; mice lacking either the IFN-??R or the
IFN-?R demonstrated titers similar to those in wild-type mice,
although titers from STAT1?/?animals were increased 100-fold.
7235 The Journal of Immunology
By day 8 after infection, no virus could be detected in lungs of any
strain (data not shown). The surprising finding was that mice un-
able to respond to either type of IFN showed no detectable increase
in virus production relative to wild-type controls. This nonequiva-
lence of the IFN-???R?/?and STAT1?/?mutations was not en-
tirely unexpected, because STAT1 is essential for many, but not
all, IFN signaling pathways (8, 18). What was surprising, and un-
like other viruses, was the relative STAT1 dependence and IFN
independence of RSV replication in vivo (19).
Altered inflammatory responses in lungs of IFN-deficient mice
Along with the increased viral load there was an exacerbated in-
flammatory response in the lungs of STAT1?/?mice (Fig. 2).
There were no significant differences in the numbers of BAL leu-
kocytes recovered from wild-type, IFN-??R?/?, IFN-?R?/?, or
IFN-???R?/?mice on days 5 and 8 after infection. However,
samples from STAT1?/?mice showed 15- and 5-fold greater
numbers of inflammatory cells at 5 and 8 days after infection,
respectively (p ? 0.001 on days 5 and 8). Although we have also
found an enhanced inflammatory response to RSV by STAT1?/?
mice on the BALB/c background, where differences in viral titers
are much smaller (7), the 100-fold increase in virus found in the
STAT1?/?129SvEv animals (Fig. 1) undoubtedly influences
many immune parameters. Because we wanted to determine the
effects of IFN, rather than virus load, on the outcome of RSV
infection, we omitted STAT1?/?mice from the following studies
and focused instead on comparing the responses of mice lacking
either or both IFNRs to those of strain-matched controls.
We had previously described pronounced eosinophilic lung dis-
ease after primary RSV infection of STAT1?/?and IFN-
???R?/?, but not IFN-??R?/?, IFN-?R?/?, or IFN-??/?, mice,
as judged by microscopic examination of infected tissues (7). For
a more quantitative picture, we collected BALs from mice of each
genotype at 5 days (Fig. 3A) and 8 days (Fig. 3B) after i.n. RSV
independent. Cohorts of 4- to 6-wk-old 129SvEv mice of each genotype
were infected i.n. with 107PFU of RSV A2. Titers shown in this figure
(expressed as PFU per gram of lung tissue) derive from plaque assay of
lung homogenates collected on day 5 after inoculation. Only STAT1?/?
mice produced virus at levels greater than those seen in wild-type animals
(p ? 0.001) as determined by one-way ANOVA. By day 8, virus was
below the level of detection in all strains (data not shown).
Control of RSV replication is STAT1 dependent, but IFN
cellular than those from IFN-??R?/?, IFN-?R?/?, IFN-???R?/?mice or
strain-matched controls. BALs were collected from infected animals of
each genotype on days 5 and 8 after RSV infection, and cell counts were
determined for individual animals. Only STAT1?/?mice had a statistically
significant increase in the number of BAL cells (p ? 0.001) at either time
point. We have consistently seen a 2-fold increase in the number of in-
flammatory cells present in BALs from IFN-?R?/?mice on day 8, as has
been reported by others (39), but it was not statistically significant in this
Lung lavages from infected STAT1?/?mice were more
between RSV-infected wild-type and knockout mice. BALs were collected
from RSV-infected animals 5 and 8 days after i.n. inoculation with 107
PFU of RSV, and differential cell counts were performed on Wright-
Giemsa-stained cytospins. At the earlier, day 5, time point (A) neutrophils
were found in mice lacking either or both IFNRs, but not wild-type animals
(p ? 0.05). Differences in percent lymphocytes between IFN-??R?/?and
IFN-???R?/?and controls were also significant (p ? 0.05). By day 8 (B).
BAL from IFN-??R?/?animals were similar to that in wild-type mice.
IFN-?R?/?and IFN-???R?/?mice had elevated percentages of neutro-
phils (p ? 0.05) and eosinophils (p ? 0.001) compared with wild-type
mice. The proportion of lymphocytes in IFN-???R?/?mice was signifi-
cantly decreased (p ? 0.05). C, Day 8 BAL eosinophil composition was
compared in three independent experiments; the percentage of eosinophils
differs significantly (p ? 0.05) between IFN-?R?/?and IFN-???R?/?
mice in all cases.
The composition of BAL infiltrates differed significantly
7236 INNATE IFNs AND RSV IMMUNOPATHOLOGY
inoculation and performed differential cell counts on Wright-
Giemsa-stained cytospins made from individual animals (four to
six mice per group). Lavage fluids obtained from wild-type mice
consisted almost entirely of monocytes/macrophages and lympho-
cytes, with the proportion of lymphocytes increasing by day 8
when tissue inflammation was well developed. In IFN-??R?/?,
IFN-?R?/?, and IFN-???R?/?mice, neutrophils were present on
day 5, but decreased to near background levels by day 8. Eosin-
ophils were present only in the lungs of IFN-?R?/?and IFN-
???R?/?animals. In the double-receptor knockout mice, eosino-
phils appeared earlier and consistently made up a significantly
larger proportion of the total infiltrate. These differences were con-
sistent over three similar experiments, one of which is pictured in
Fig. 3, A and B. When these data were analyzed by ANOVA, all
three experiments (Fig. 3C) showed significant differences be-
tween the IFN-?R?/?and IFN-???R?/?strains (p ? 0.05). An-
other significant and consistent difference among the different
genotypes was the relative dearth of lung lymphocytes in IFN-
???R?/?animals on day 8 (p ? 0.05), shown in Fig. 3B.
Photomicrographs corresponding to these BAL data are shown
in Fig. 4. H&E-stained lung sections from infected wild-type (A
and D), IFN-?R?/?(B and E), and IFN-???R?/?(C and F) mice
8 days after inoculation are pictured. As predicted from the equiv-
alent BAL cell numbers (Fig. 2), the overall extent of inflammation
was similar in all four strains. This can be seen at the lower (?200)
magnification. All sections show pronounced perivascular infil-
trates with foci of peribronchial, peribronchiolar, and alveolar in-
flammation. There was little evidence of cytopathic effect or epi-
thelial cell necrosis. Appreciable differences between strains were
apparent only at higher (?400) magnification, where the very dif-
ferent characters of the infiltrates are evident. Consistent with the
data in Fig. 3, the inflammatory process is entirely lymphocytic in
wild-type mice (Fig. 4D) and predominantly lymphocytic in IFN-
?R?/?mice (Fig. 4E), but is overwhelmingly eosinophilic in IFN-
???R?/?animals (Fig. 4F). This relates to both the relative in-
crease in eosinophils and the relative decrease in lymphocytes seen
in the double-knockout animals. Lung sections from IFN-??R?/?
mice (not pictured in this figure) were similar to those from wild-
type mice, but with an overall decrease in lymphocytic
On day 5 after infection, the total number of lymphocytes
present in the lungs of all animals was quite small (data not
shown), but by day 8 we observed a large expansion of T cells in
wild-type and IFN-?R?/?mice. This increase was primarily due
to the expanding CD8?cell population, as calculated from cell
counts, BAL differentials, and flow cytometry data (Fig. 5A). In-
terestingly, this expansion was very limited in mice lacking the
IFN-??R, although the difference in CD8?T cell number was
statistically significant (p ? 0.05) only for the IFN-???R?/?an-
imals. This observation is consistent with data from other labora-
tories showing a role for type I IFNs in the activation and survival
of CTLs (20, 21). There were no significant differences in numbers
of BAL CD4?T cells among strains. Fig. 5B shows the number of
eosinophils present in BALs taken from each strain on day 8
postinfection. These were very similar in IFN-?R?/?and IFN-
???R?/?animals, leading us to conclude that the eosinophilic
predominance in IFN-???R?/?mice reflects primarily a lack of
CD4?T cell differentiation in IFN-deficient knockout mice
Having determined that there were significant qualitative and
quantitative differences between RSV lung disease in IFN-?R?/?
and IFN-???R?/?mice, we sought to determine the role of IFNs
in CD4?T cell differentiation. It has been established that STAT1-
mediated induction of the transcription factor T-bet is required for
the development of Th1 lymphocytes (10). Consistent with this
finding, we have observed Th2 cytokine production by STAT1-
deficient CD4?T cells responding to an RSV-F protein epitope
that stimulates primarily IFN-? in wild-type mice (7). Although a
large body of evidence demonstrates the importance of IFN-? in
Th1 differentiation in vitro, in vivo studies using mice deficient in
IFN-? signaling have not shown a clear Th2 predisposition (22–
24). In light of the uncertainty regarding the role of IFN-? in Th1
differentiation, and with the differing disease patterns observed in
IFN-??/?or IFN-?R?/?and STAT1?/?mice infected with RSV
(7) or influenza (25), it was important to learn whether virus-spe-
cific CD4?T cells from RSV-immune IFN-??R?/?, IFN-?R?/?,
and IFN-???R?/?mice were primarily Th1 or Th2.
after infection. A, Total numbers of lymphocytes were calculated using
total numbers of BAL cells taken from each animal and percentages of total
lymphocytes (cytospin differentials) and lymphocyte subsets (FACS anal-
ysis) of each BAL sample. Difference in numbers of total lymphocytes and
CD8?T cell numbers were statistically significant (p ? 0.05) only for the
IFN-???R?/?animals. Total numbers of eosinophils (B) were similarly
Numbers of BAL lymphocytes and eosinophils on day 8
tions are from wild-type (A, ?200; D, ?400), IFN-? R?/?(B, ?200; E,
?400), and IFN-??? R?/?(C, ?200; F, ?400) animals 8 days after i.n.
RSV inoculation. Eosinophils are large cells filled with bright pink gran-
ules; lymphocytes are small, blue cells with very little cytoplasm. Several
eosinophils are marked with black arrowheads.
Tissue inflammation reflects BAL composition. Tissue sec-
7237The Journal of Immunology
To perform this experiment in the 129SvEv animals, a dominant
viral epitope recognized by RSV-immune CD4?lymphocytes in
this strain was required. Overlapping peptides corresponding to the
RSV-F protein sequence were mixed to make five pools of 10
peptides and one pool of seven peptides, and these peptide pools
were assayed for their ability to stimulate the proliferation of im-
mune splenocytes. Because pool 1 stimulated the most robust pro-
liferation response (data not shown), we next screened peptides
1–10 individually (Fig. 6). From this secondary screen, peptide 9,
corresponding to aa 81–100 (QELDKYKNAVTELQLLMQNT),
was chosen for additional characterization. An IFN-? ELISPOT
assay performed with undepleted or CD4?T cell-depleted im-
mune splenocytes demonstrated that CD4?T cells were required
for an IFN-? response to this peptide. Undepleted cultures pro-
duced an average of 85 spots/106splenocytes compared with an
average of 1 spot/106splenocytes in CD4?T cell-depleted sam-
ples. Depletion of CD8?T cells had no effect on IFN-? production
(data not shown).
Cytokine production by immune CD4?T cells from mice of
each genotype was assessed by quantitation of cytokine gene tran-
scripts after peptide stimulation of cultured splenocytes. Cells were
harvested from two or three mice of each strain that had been
inoculated i.n. with 106PFU of RSV 28 days previously. After 7
days of culture with peptide 9, the splenocytes were washed and
restimulated for a 6-h period before harvesting. Equal amounts of
RNA from each sample were analyzed in a multi-probe RNAP,
and signals were quantitated using the Storm PhosphorImager and
ImageQuant software. Relative levels of cytokine transcripts made
by each strain are shown in Fig. 7. We concluded from this set of
experiments that although RSV does not appear to generate a
strong Th1 response to F protein in the wild-type mouse, mice
lacking the IFN-?R alone or lacking both IFNRs default to a Th2
differentiation pathway in our system.
Role of IFNs in lung cytokine and chemokine production
The earliest response to virus is initiated by infected lung epithelial
cells. A number of cytokines and chemokines are induced by RSV
(26–28), and these inflammatory mediators directly affect the traf-
ficking of lymphocytes and granulocytes to the site of infection.
Chemokines promoting both Th1 and Th2 cell migration are pro-
duced by the infected epithelium in vitro, but in vivo, their syn-
thesis is also affected by the presence of other cytokines. Many of
the chemokines attracting Th1 cells (IFN-inducible T cell ? che-
moattractant (I-TAC), IP-10, monokine induced by IFN-? (MIG))
are induced by IFNs (29–32), and IL-13 is known to increase the
synthesis of eotaxin (33, 34), a major eosinophil attractant. We
wondered whether the altered inflammatory responses in the ab-
sence of IFN-?? and/or IFN-? signaling correlated with an altered
chemokine profile. To answer this question, BAL supernatants
from infected mice were collected 5 days after i.n. inoculation with
107PFU of RSV and were assayed for the presence of IP-10. IP-10
was chosen because it can be induced by either type I or type II
IFNs (29, 32). Fig. 8 shows the data obtained for both eotaxin and
IP-10 assayed at two time points in all four strains of mice. Al-
though no IP-10 could be detected in mice lacking both IFNRs
(IFN-???R?/?), mice retaining either the IFN-??R ((IFN-
??R?/?) or the IFN-?R (IFN-?R?/?) continued to produce IP-10.
Eotaxin was induced in all strains, but eosinophilia occurred only
in those lacking virus-specific Th1 cells, that is, the IFN-?R?/?
and IFN-???R?/?mice. In addition, eotaxin levels remained sig-
nificantly elevated on day 8 only in mice lacking both IFNRs (p ?
These same BAL supernatants from each animal were also as-
sayed for the presence of Th2 cytokines. Levels of IFN-?, IL-5,
and IL-13 in lavage samples collected at 5 and 8 days after inoc-
ulation were determined by ELISA or by the Luminex100system
(Fig. 9). Although there were increased levels of IL-5 and IL-13 in
lungs of IFN-?R?/?and IFN-???R?/?mice, these differences
mune 129SvEv splenocytes. Activation of immune cells by individual pep-
tides was assayed by measuring [3H]thymidine incorporation after in vitro
peptide stimulation. Peptides derived from the F protein sequence of RSV
B were combined in pools. Because pool 1 (no. 1–10) induced the highest
level of proliferation, the peptides making up this pool were assayed in-
dividually. Peptide 9, from pool 1, corresponds to aa 81–100 of the RSV-F
Identification of a viral peptide recognized by RSV-im-
IFN-???R?/?mice produced high levels of Th2 cytokines. Splenocytes
from RSV-immune animals of each genotype were expanded in culture.
RNA was harvested from each culture after peptide stimulation, and RNAP
was used to determine relative levels of cytokine transcripts. Signal inten-
sity was quantitated by PhosphorImager and normalized to levels of ribo-
somal protein L32.
Memory CD4?T cells from RSV-immune IFN-?R?/?and
ELISA on day 5 (f) and day 8 (?) after primary RSV infection.
BAL chemokines IP-10 and eotaxin were assayed by
7238 INNATE IFNs AND RSV IMMUNOPATHOLOGY
were statistically significant only for IL-5. IFN-? levels were sim-
ilarly high for all strains on day 5, probably reflecting production
by NK as well as T cells. By day 8, these levels had dropped, most
precipitously in the IFN-??R?/?animals. Thus, it appears that
chemokine synthesis is affected by early IFN production, and this
suggests that chemokine levels may be influenced by the presence
of Th2 lymphocytes.
Although many childhood diseases have been largely eradicated or
attenuated by successful vaccination programs, RSV remains a
major pediatric problem. One of the obstacles in the development
of a safe and effective RSV vaccine is the danger that the domi-
nance of a Th2 memory compartment will lead to enhanced dis-
ease, rather than protection, after immunization. We had previ-
ously observed that in mice lacking a functional STAT1 gene,
primary infection with RSV led to severe eosinophilic lung disease
that appeared to be mediated by Th2 lymphocytes. In retrospect,
this is not surprising, because T-bet, the transcription factor shown
to be essential for Th1 development of naive CD4 T cells (35, 36),
is itself STAT1 dependent (37, 38). Therefore, even in the setting
of primary RSV infection, it appears that host predisposition to
Th2 differentiation determines whether an eosinophilic response
This conclusion was inconsistent with the absence of this pa-
thology in infected mice lacking IFN-?, a cytokine known to ac-
tivate STAT1 and drive Th1 differentiation of naive CD4?T cells.
In vivo experiments to determine whether IFN-? itself is essential
for Th1 development have been complicated by the importance of
IFN-? in establishing IL-12 responsiveness. Current models of na-
ive CD4?cell activation postulate that STAT1 activation is re-
quired for Th1 commitment, but that the STAT4-mediated IL-12
signal is still necessary for IFN-? production in response to Ag
(10, 38). Because IFN-? up-regulates IL-12R expression, a robust
Th1 response should require IFN-?, and we would expect mice
lacking this cytokine to mount a Th2 response to virus infection.
Nonetheless, in our experiments with either IFN-??/?BALB/c or
129SvEv IFN-?R?/?mice, eosinophils were present in infiltrates
surrounding vessels and airways of infected lungs, but only at low
levels. This observation has been made by others (24, 39), who
concluded, as we did, that RSV pathogenesis in IFN-?-deficient
animals is similar to that in wild-type animals. We therefore won-
dered whether, in the absence of IFN-?, IFN-?? could act in a
compensatory manner to inhibit Th2 lymphocyte development and
eosinophilic disease (7). To test this hypothesis we needed to de-
termine 1) whether Th2 differentiation was blunted in IFN-?R?/?
mice, and 2) whether mice lacking both IFNRs (IFN-???R?/?)
responded differently from those lacking one or the other.
We approached the question of T cell differentiation in infected
IFN-?-deficient mice by comparing cytokine responses of four
mouse strains to a single MHC class II restricted viral peptide. The
peptide used (F9), corresponding to aa 81–100 of the RSV-F pro-
tein, specifically stimulated the proliferation of and IFN-? produc-
tion by CD4?T cells from RSV-immune wild-type 129SvEv
mice. Using an RNAP to survey simultaneously for Th1 and Th2
cytokine production, we observed that although peptide-stimulated
T cells from IFN-?R?/?and IFN-???R?/?animals produced lev-
els of IFN-? transcript similar to those in wild-type animals, IL-13
production was increased 10- to 20-fold (Fig. 7). The elevated
amounts of IL-13 as well as IL-5 induced by peptide F9 in the
absence of the IFN-?R demonstrated that the IFN-? pathway is
essential for inhibiting the Th2 response to RSV infection.
Having found that both the IFN-?R?/?and the IFN-???R?/?
animals default to a Th2 lymphocyte response after infection, an
alternate explanation for the significant differences in lung pathol-
ogy between these animals was required. Characterization of BAL
samples from mice of each genotype at two time points suggested
that the major difference between these strains was not an increase
in eosinophils, but, rather, an absence of CD8?lymphocytes in
both strains of mice lacking the IFN-??R?/?. In wild-type and
IFN-?R?/?mice, there was a large expansion in the number of
lymphocytes present in the lungs by day 8 after inoculation. In
IFN-?R?/?animals, this expansion occurred in the presence of
eosinophils and neutrophils (Fig. 3). In contrast, mice lacking both
IFNRs showed little increase in lymphocyte numbers as the infec-
tion progressed, and eosinophils were the predominant cell type on
day 8. This pattern was seen consistently in three replicate exper-
iments and was significant in each (p ? 0.05). Evaluation of tissue
sections showed a similar picture (Fig. 4), with largely lympho-
cytic infiltrates in infected IFN-?R?/?mice and predominantly
eosinophilic inflammation in lungs of IFN-???R?/?animals.
We have observed that Th2 cytokine production by CD4?T
cells is necessary, but not sufficient, for the development of eosin-
ophilic lung inflammation after RSV infection. Other factors con-
tribute to pulmonary eosinophilia during primary infection in mice.
It has been shown that a large number of genes are induced by
RSV infection of respiratory epithelial cells; among these are che-
mokines that regulate inflammatory cell trafficking (26, 27, 40, 41).
A number of innate immune mediators will impact relative levels
of chemokine production by these cells (31), and among these are
the IFNs. Both IFN-?? and IFN-? are produced early in infection:
IFN-?? by the virus-infected cells and innate IFN-? by activated
ELISA, and IL-5 (C) was assayed by LINCOplex on day 5 (f) and day 8
(?) after primary RSV infection.
BAL cytokines IFN-? (A) and IL-13 (B) were assayed by
7239The Journal of Immunology
NK cells (17). A number of the chemokines responsible for Th1
and CTL trafficking, among them the CXCR3 ligands I-TAC,
MIG, and IP-10, are IFN-inducible genes (31, 32, 41). We won-
dered whether IP-10, which is known to be induced by both type
I and type II IFNs, would be detectable in mice lacking either or
both IFNRs and whether differences in chemokine production
could be contributing to the differences in pathology. As shown in
Fig. 9, IP-10 was detectable in BALs from all animals on day 5,
except in those lacking both IFNRs. Eotaxin, another RSV-in-
duced chemokine, is a chemoattractant for activated Th2 cells and
eosinophils (42). Although eotaxin was present in BALs from mice
of all genotypes at early times, levels were significantly increased
only in IFN-???R?/?mice on day 8 (p ? 0.001; Fig. 9A). Al-
lergic inflammation is a self-amplifying process, and the ele-
vated eotaxin levels in animals with eosinophilic disease may
reflect this situation. If activated Th2 cells and eosinophils se-
creting IL-13 (43) traffic to the lung, increasing IL-13 concen-
trations will up-regulate eotaxin production by infected epithelium
(34) (see Fig. 10).
In summary, our data suggest that although Th2 lymphocytes
are necessary for the development of an eosinophilic response to
primary RSV infection, they are not sufficient. In this system it is
the IFN-?? and IFN-? produced during the innate immune re-
sponse, rather than lymphocyte IFN-?, that determines disease out-
come. RSV lung pathology in IFN-?R?/?animals with an intact
type I IFN signaling pathway is similar to that seen in wild-type
controls. CD8?T cells, rather than eosinophils, continue to dom-
inate the process. In the absence of both IFNRs, disease is mark-
edly eosinophilic and without the usual expansion of the lung lym-
phocyte compartment (44, 45). This is consistent with a report
from Hussell et al. (46) showing a correlation between the mag-
nitude of the CD8?T cell response and the propensity of a mouse
strain to develop eosinophilia. Therefore, the elevated eotaxin lev-
els, the absence of IP-10 induction, and the lack of CTLs in IFN-
???R?/?animals work together to create differences between two
strains of animals that both generate Th2 responses. Fig. 10 depicts
a model showing how the summation of these influences will de-
termine the composition of the inflammatory infiltrate.
We propose that innate IFN-?? and -? affect local chemokine
production by increasing levels of the Th1 and Tc1 chemoattrac-
tants (I-TAC, IP-10, and MIG) as well as stimulating CD8?T cell
proliferation and activation. An important role for IFN-?? in CTL
survival and IFN-? production has also been shown in other sys-
tems (20, 21, 47). If virus-specific Th1 cells and CTLs are present,
they will be drawn to the site of infection and augment local IFN-?
production. If activated Th2 lymphocytes are present, they will
preferentially accumulate in response to eotaxin, increasing local
IL-13 and eotaxin production and thereby amplifying an allergic-
type inflammatory response. In animals lacking the IFN-?R?/?, it
appears that the presence of type I IFNs does not block eosinophil
accumulation, but can still inhibit a primarily allergic response by
promoting CD8?T cell accumulation. It has been suggested that in
addition to their roles as CXCR3 agonists, I-TAC, IP-10, and MIG
may also act to block Th2 cell and eosinophil accumulation by
binding the CCR3 expressed by those cell types and acting as
antagonists (48). If this is a biologically significant process, this
antagonism could also play a role in shaping the inflammatory
response. Overall, our findings in the mouse model suggest that
although the link between RSV and asthma (6) is probably due to
the ability of this virus to promote Th2 lymphocyte differentiation,
it may be exacerbated by the limited type I IFN induction by RSV
infection in vivo (49, 50).
We thank Michel Aguet and Bob Schreiber for providing IFN-??R?/?,
IFN-??/?, and IFN-???R?/?mice; Tom Hamilton for anti-IP-10 anti-
serum; and Philip R. Johnson for RSV F protein peptides. We also thank
Chris Walker, Naglaa Shoukry, Andrew Cawthon, and Christine Biron for
helpful discussions, and Karen Watkins for manuscript preparation.
The authors have no financial conflict of interest.
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