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*
Children’s Research Institute, Children’s Hospital, Columbus, OH 43205;
Division of Pediatric Pathology, Department of Pediatrics, College of Medicine and Public Health, Ohio State University, Columbus, OH 43210;
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232; and
Vaccine Research Center, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-/--infected mice. In
cytokine analyses of infected lung tissue, IFN- was induced in both
STAT1-/- and wild-type mice, with preferential IL-4,
IL-5, and IL-13 induction only in the STAT1-/- animals.
Eotaxin was detected in the lungs of both wild-type and
STAT1-/- mice following infection, with a 1.7-fold
increase over wild-type in the STAT1-/- mice. Using a
peptide epitope newly identified in the RSV fusion protein, we were
able to demonstrate that wild-type memory CD4+ T cells
stimulated by this peptide produce primarily IFN-, while
STAT1-/-CD4+ cells produce primarily IL-13.
These findings suggest that STAT1 activation by both type I (
)
and type II () IFNs plays an important role in establishing a
protective, Th1 Ag-specific immune response to RSV
infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Concerns about vaccine safety and efficacy have led to the study of
mechanisms underlying vaccine-enhanced illness and their relationship
to disease pathogenesis during primary RSV infection. Mouse models of
enhanced RSV pathology have been achieved by priming BALB/c animals
with FI-RSV, recombinant vaccinia virus expressing the RSV G protein,
or purified RSV glycoproteins, followed by intranasal (i.n.) virus
challenge (6, 7, 8, 9, 10, 11). In this study we report enhanced
disease and lung eosinophilia in animals homozygous for a targeted
disruption of the STAT1 gene following primary RSV
challenge. In STAT1-/- mice, signal
transduction in response to either type I () or type II () IFN
is ablated (12, 13).
Many laboratories have demonstrated that the eosinophilic lung disease
observed following FI-RSV or G protein priming and challenge is a
Th2-driven process (11, 14, 15), although the cytokine
determinants of eosinophilia are somewhat different (16).
Hussell et al. (17) have observed that the development of
lung eosinophilia is mitigated by CTL IFN- production and enhanced
by anti-IFN- or anti-CD8 Abs. However, work by
Srikiatkhachorn et al. (18) shows similar lung pathology
following RSV infection of wild-type and IFN--deficient animals.
In this study we have observed that in the absence of both
IFN- and IFN- signaling, or STAT1, mice show exacerbated
eosinophilic lung disease and a Th2-biased cytokine response. We
propose that the proinflammatory, Th2-biased immune response to primary
RSV infection in these animals is due to the lack of IFN- effects
that are normally induced by virus infection as well as IFN-
produced by activated NK cells and T lymphocytes. Our data suggest that
in addition to their role in limiting virus replication, activation of
STAT1 by type I () IFNs may also play a role in directing the
development of an appropriate, Th1-biased cell-mediated response to
virus infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pathogen-free wild-type BALB/c and CD1 mice were purchased from
Charles River Breeding Laboratories (Wilmington, MA) and Taconic Farms
(Germantown, NY). IFN--/- mice
(19) on a BALB/c background were purchased from The
Jackson Laboratory (Bar Harbor, ME). STAT1-/-
mice (12) on a CD1 background were bred from stocks and
maintained in a specific pathogen-free environment.
STAT1-/- mice on a BALB/c background were
obtained by back-cross; mice from the ninth back-cross generation were
used in these experiments. Mice on the 129SvEv background, IFN-R
(IFNAR)-/- (20), IFN-R
(IFNGR)-/- (21), and
IFN-R double knockout mice
(IFNARGR-/-) (22) were provided by
M. Aguet (Swiss Institute for Experimental Cancer Research, Lausanne,
Switzerland) and R. Schreiber (Washington University, St. Louis, MO).
STAT1-/- mice on the 129SvEv background were
achieved by back-cross. Stocks of the human A2 strain of RSV
(originally provided by R. Chanock, National Institutes of Health,
Bethesda, MD) were prepared as previously described (6).
RSV was grown and assayed for infectivity in HEp2 cells. Six- to
8-wk-old animals were lightly anesthetized and inoculated i.n.
with RSV.
Plaque assays
Lung tissue was removed, weighed, and quick-frozen in Eagle’s MEM supplemented with 10% FBS. Tissues were individually ground at 4°C in PBS using a PowerGen125 homogenizer (Fisher Scientific, Pittsburgh, PA). Dilutions of clarified lung supernatants were inoculated onto subconfluent HEp2 cell monolayers. After 1 h, plates were covered with 0.5% methylcellulose in Eagle’s MEM and incubated for 4 days at 37°C. Monolayers were fixed with 2% buffered formalin and stained with crystal violet.
Pathology
Lungs were harvested from animals 8 days postinoculation then inflated and fixed in 10% buffered formalin. Paraffin sections were stained with H&E and evaluated for the presence of inflammatory infiltrates. Eosinophils were identified by their size, bilobed nuclei, and eosinophilic cytoplasmic granules.
ELISA
RSV-specific Ig isotypes were quantitated by incubating dilutions of sera from convalescent animals with soluble RSV fusion protein (F protein) fixed to Immulon II 96-well plates (Nunc, Roskilde, Denmark). Plates were washed and incubated a second time with goat anti-murine IgG1, IgG2a, or total Ig conjugated to HRP (Zymed Laboratories, San Francisco, CA) at a dilution of 1/5000. After washing, TMB substrate was added, and the reaction was stopped after a 10-min incubation by the addition of 2.5 M H2SO4. Wells were considered positive if the OD at 450 nm was >0.2 after subtraction of background. Total IgE and cytokine levels were determined using an ELISA kit purchased from BD PharMingen (San Diego, CA).
Derivation of RSV-F peptide epitopes
RSV-F (RSV B strain) protein peptides recognized by lymphocytes derived from RSV-immune wild-type and STAT1-/- BALB/c mice were identified by screening a set of overlapping peptides synthesized by Chiron Mimotopes (Clayton, Australia). Peptides were 20 aa in length, with a 10-aa overlap. They were screened for their ability to stimulate immune CD4+ cells to proliferate. By this method only peptide no. 6 (F6), corresponding to amino acids 51–70 of the RSV B F protein sequence (GWYTSVITIELSNIKETKCN), gave a positive result. Splenocytes from two mice of each strain were used for each trial. The OVA-derived SIINFEKL peptide was regularly included as a negative control.
In vitro stimulation of memory splenocytes
Splenocytes were harvested from wild-type or STAT1-/- animals (two mice of each strain per experiment) inoculated i.n. 28 days before with 106 PFU of RSV. These were cultured in RPMI 1640 with 5% FCS and 5% rat T-STIM (Collaborative Biomedical Products, Bedford, MA) added day 3, in the presence of peptide (10 µg/ml) for 1 wk. On day 7, wild-type and STAT1-/- cells were restimulated with peptide for 6 h in the presence of gamma-irradiated feeder cells (splenocytes harvested from naive wild-type mice) and then lysed in TRIzol (Life Technologies, Rockville, MD).
RNase protection assay
Cytokine transcripts from infected lung homogenates or in vitro stimulated lymphocytes were assayed by RNase protection using the RiboQuant mck1 multiprobes (BD PharMingen) according to the manufacturer’s instructions. RNA was isolated from lung tissue harvested 4 days following i.n. RSV inoculation and homogenized in TRIzol (Life Technologies). Twenty-five micrograms of lung RNA or 10–20 µg of splenocyte RNA was used per sample. Each lung sample consisted of RNA derived from a single mouse.
Proliferation assay
Splenocytes (0.5 x 105) taken from RSV-immune animals were plated after 7 days of peptide stimulation, with 1.5 x 105 naive, gamma-irradiated BALB/c splenocytes and 10 µg/ml peptide in wells of a round-bottom 96-well plate. Each sample was plated in triplicate wells. At 2 days after peptide restimulation, 1 µCi of [3H]thymidine was added to each well. Incorporation of [3H]thymidine was determined after a 12-h incubation at 37°C.
ELISPOT assay
The ELISPOT assay was conducted using a kit purchased from
U-Cytech (Utrecht, The Netherlands). Splenocytes harvested from
RSV-immune BALB/c (wild-type and STAT1-/-) mice
were plated at a concentration of 2.5 x 105
cells per well on 96-well plates coated with a monoclonal
anti-IFN- Ab. The cells were cultured in RPMI 1640 containing
5% heat-inactivated autologous mouse serum. Peptide F6 (described
above) and the control peptide (SIINFEKL) were included at a
concentration of 10 µg/ml. After 36 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-/-, and STAT1-/- mice
Despite the well-established role of type I IFN in innate
antiviral responses, the susceptibility of
STAT1-/- animals to virus infection appears to
be somewhat pathogen specific (12, 13, 23). To determine
the role of IFN- in protection against RSV, viral titers were
determined at day 4 and again at day 8 following i.n. inoculation with
107 PFU of RSV A2. Four to six animals of each
strain were used at each time point in this and subsequent experiments.
Lungs were harvested from animals at the specified times, and viral
titers were determined by plaque assay of lung homogenates. Results
from three separate experiments comparing wild-type and mutant BALB/c
animals are shown in Table I
. Viral lung
titers from wild-type and IFN--/- mice were
not significantly different. STAT1-/- mice
showed a 0.5- to 1-log increase in virus load at day 4 postinoculation
in some experiments; rates of virus clearance were equivalent.
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). Clearance was slightly
delayed in the STAT1-/- mice but was complete
by day 8.
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). As before,
107 PFU were administered i.n. In CD1 animals,
both nose and lung titers in STAT1-/- mice were
increased 1–2 logs over wild-type levels at day 4. More residual virus
was present in STAT1-/- animals at day 8
postinoculation, and the rate of virus clearance was delayed.
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Given the somewhat surprising finding that in BALB/c mice virus
titers and clearance appear to be comparable in the presence or absence
of IFN responsiveness, we wished to determine whether illness was also
comparable. Mice are relatively nonpermissive for RSV and the widely
used BALB/c animal model employs the highest practical i.n. dose for
inoculation, which is 107 PFU per animal. At
lower doses, wild-type mice show little if any evidence of illness
(Fig. 1). The most striking indication
that illness is more severe in the STAT1-deficient BALB/c animals is
the LD50. Although
STAT1-/- animals survive a dose of
106 PFU, 6 of 10 animals died—or were found
moribund and were euthanized—in the second week following i.n.
instillation of 107 PFU RSV.
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charts the average weight change for
STAT1-/- BALB/c animals compared with age- and
strain-matched controls using 10 animals of each strain. The weights of
wild-type BALB/c animals remained steady or increased.
STAT1-/- mice all demonstrated a marked weight
loss between 6 and 8 days postinfection (averaging 4.04 ±
0.63 g), accounting for 12–25% of their day 0 weight. By 12
days, all mice had returned to their preillness baseline.
Because the increased and prolonged weight loss following RSV infection
of STAT1-/- mice suggested exacerbated disease,
we next looked at lung pathology 8 days after inoculation using four to
six animals of each strain (Fig. 2).
Lungs from wild-type animals showed typical peribronchiolar,
perivascular, and interstitial lymphocytic infiltrates
(24). Inflammation in STAT1-/-
mice infected with the same inoculum was quantitatively more intense,
diffusely involving the alveolar septae in addition to peribronchiolar
and perivascular infiltrates, and was made up of neutrophils,
macrophages, and eosinophils as well as lymphocytes. The picture of
enhanced eosinophilic pulmonary disease in the
STAT1-/- mice resembles that described in
wild-type BALB/c mice challenged with RSV following FI-RSV priming
(6, 8, 25). Microscopically, lung inflammation in
IFN--/- BALB/c mice was similar to that seen
in wild-type animals (data not shown).
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Serum samples from convalescent mice of each genotype were assayed
for the presence of RSV-F protein-specific Abs 28 days after i.n.
inoculation. Total RSV-specific Ig titers were equivalent among the
three strains, although differences in isotype were evident with
increased IgG1/IgG2a ratios in both IFN--/-
and STAT1-/- animals (Fig. 3). Preferential IgG1 production is
characteristic of B lymphocytes maturing under the influence of
Th2-type cytokines (26). Upon rechallenge with RSV,
IFN--/-, STAT1-/-,
and wild-type mice on both the CD1 and BALB/c strain backgrounds were
protected and showed no signs of illness (data not shown).
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The exacerbated illness, eosinophilic lung infiltrates, and IgG1
Ab predominance in the STAT1-/- animals led us
to ask whether their RSV-specific immune response was Th2 in nature. To
examine this possibility we sacrificed either wild-type and
IFN--/- or wild-type and
STAT1-/- BALB/c animals 4 days after i.n.
inoculation with 107 PFU RSV. Five to six animals
of each genotype were used for each experiment. One lung from each
mouse was homogenized in PBS for the measurement of the cytokines IL-4,
IL-5, IL-13, IFN-, and eotaxin by ELISA. In comparing cytokine
induction between wild-type and IFN--/-
mice, we found statistically significant differences only in IFN-
production (Fig. 4A). When
wild-type and STAT1-/- mice were compared,
strong induction of IL-13, as well as weak but detectable up-regulation
of IL-4 and IL-5, were seen only in the
STAT1-/- animals (Fig. 4B). These
values were statistically significant only for IL-13
(p = 0.001). Although eotaxin was detectable in
lung homogenates from both wild-type and
STAT1-/- BALB/c animals, eotaxin concentrations
were elevated nearly 2-fold in the lungs of the
STAT1-/- mice (p =
0.001). These lung cytokine profiles, taken together with the
exacerbated, eosinophilic lung disease, confirm a Th2 bias in the
response to primary RSV infection by STAT1-/-
animals.
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. Very little cytokine production is
evident on day 2, and day 4 results are comparable to those described
above, although in this experiment there is significantly increased
production of IL-4, IL-5, and IL-13 by the
STAT1-/- animals. Day 6 profiles are remarkable
for increased IFN- production by mice of both genotypes, increased
nearly 5-fold in the STAT1-/- animals relative
to wild type.
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We wished to determine the source of cytokines measured in infected lung tissue by RNase protection and ELISA. To do this we required a RSV epitope recognized by RSV immune CD4+ lymphocytes of BALB/c mice. Peptides derived from the RSV-F protein were screened for their ability to stimulate immune CD4+ cells to proliferate. In this screen only peptide F6, GWYTSVITIELSNIKETKCN, gave a stimulation index of 10-fold over background when splenocytes from RSV-immune STAT1-/- mice were used. Peptide F6 corresponds to amino acids 51–70 of the RSV B F protein sequence. This sequence is conserved between the RSV A2 and B strains with a single amino acid difference at residue 67, a position occupied by asparagine in RSV A2 and by threonine in the RSV B strain. When the proliferation assay was conducted in parallel with immune splenocytes harvested from wild-type BALB/c controls, only a 1.6-fold stimulation over background was seen (data not shown). This result was consistent over multiple experiments. Splenocytes from two immune mice of each strain were used for each trial.
Reasoning that peptide F6 was recognized by wild-type mice but at a
lower precursor frequency, we performed an IFN- ELISPOT assay to
quantitate the F6 response. Splenocytes from RSV-immune mice of either
genotype were plated at a concentration of 2.5 x
105 cells/well. Peptide was added at a
concentration of 10 µg/ml with the MHC class I-restricted OVA-derived
SIINFEKL peptide included as a negative control. Although T cells from
wild-type mice showed a modest stimulation over background when
presented with peptide F6, no IFN- synthesis over background was
detected in wells containing STAT1-/- cells
(Fig. 5). CD4+ and
CD8+ cell depletion controls showed that only
CD4+ cells responded to peptide F6 (data not
shown). Thus, despite the fact that STAT1-/-
cells proliferate more vigorously when stimulated with the RSV-F
protein CD4+ epitope, they do not respond by
secreting IFN-.
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. We see that
when wild-type CD4+ cells are
stimulated by peptide F6 (Fig. 6, lane 1) they produce
IFN- transcripts in addition to low levels of the IL-13 transcript.
In the STAT1-/- mice (Fig. 6, lane
2) the situation is reversed, with very little IFN- mRNA
production, large amounts of IL-13 mRNA, and a small but detectable
amount of transcript for the Th2 cytokine IL-5. In repeat experiments,
we see some variation in the amount of IL-13 transcript produced by
peptide-stimulated wild-type T cells, but the proportion of IL-13 and
IFN- produced by
STAT1-/-CD4+ cells is
invariant. This experiment is consistent with the ELISPOT data in
demonstrating that wild-type CD4+ T cells mount a
Th1 or Th0 response and
STAT1-/-CD4+ T cells
mount a Th2 response to the same RSV epitope.
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Our studies comparing wild-type,
IFN--/-, and
STAT1-/- BALB/c mice have established that the
loss of IFN- alone is not sufficient to promote exacerbated lung
disease and Th2 cytokine production in response to RSV infection. These
results implied that IFN- alone, in the absence of IFN-, could
protect against lung eosinophilia. For a direct determination of the
effects of type I and type II IFNs on the development of eosinophilic
disease, we repeated our RSV infection experiment using 129SvEv mice
with targeted disruptions of the IFNAR (20), IFNGR
(21), a double knockout mouse lacking both receptors
(IFNARGR) (22), and the STAT1-/-
mouse, all on the 129SvEv background. While there are inherent
difficulties in comparisons made between mouse strains, we took
advantage of these available mutants to examine lung histopathology
following primary RSV infection. A dose of 107
PFU was given i.n. Groups of five wild-type,
IFNAR-/-, IFNGR-/-,
IFNARGR-/-, and
STAT1-/- 129SvEv animals were used, and two
independent experiments were conducted.
Lungs for microscopic examination were harvested on day 8. Shown
in Fig. 7, A and B,
are representative microscopic sections that demonstrate the sparse
lymphocytic infiltrates found in 129 wild-type animals, very similar to
that seen in the BALB/c mice (Fig. 2). IFNAR-/-
and IFNGR-/- animals had histology similar to
wild-type and are not pictured. Fig. 7, C and D,
shows enhanced disease with eosinophilic infiltrates in lungs from
IFNARGR-/- mice. In these animals the
peribronchiolar, perivascular, and interstitial infiltrates consist
predominantly of eosinophils. Fig. 7, E and F,
represents the increasingly severe pathology of lungs from
STAT1-/- 129SvEv mice with diffuse
eosinophilic, neutrophilic, and macrophage infiltrates that are
peribronchial, perivascular, and interstitial, and involve the alveolar
spaces. This comparison across five genotypes confirms that STAT1
activation, by either IFN- or IFN-, is required to inhibit
RSV-mediated immunopathology.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-/-
mice to that seen in age-matched, wild-type controls. Although
IFN--/- mice were comparable to wild-type in
terms of illness, virus yield, and lung histopathology,
STAT1-/- animals developed markedly exacerbated
disease. Following primary RSV infection with a moderate dose of virus,
the STAT1-/- mice averaged a 25% weight loss
at a RSV dose that left wild-type mice unaffected (Fig. 1). Lung virus
titers in STAT1-/- animals were within 1 log of
those found in wild-type BALB/c controls (Table I) despite an earlier
peak in the absence of STAT1. This relative insensitivity of RSV to IFN
responsiveness demonstrated by our experiments is not surprising in
light of reports of poor IFN induction by RSV as well as poor IFN
effect on RSV replication in respiratory epithelium (27, 28). Unlike other paramyxoviridae, no specific disruption of
innate antiviral mechanisms by human RSV has yet been identified
(29), although bovine RSV mutants lacking nonstructural
proteins NS1 and NS2 lose their resistance to IFN in cultured cell
lines (30). It is likely that these proteins play a
similar role in HRSV infection in humans, but it is not known whether
HRSV NS1 and NS2 have any impact on murine type I IFNs. We have
observed that this lack of IFN effect is somewhat mouse strain
dependent and is most evident on the BALB/c background (Table I). In
experiments with CD1 STAT1-/- animals, a 1- to
2-log increase in peak nose and lung titers was seen compared with
RSV-infected wild-type mice (Table III), and the 129SvEv
STAT1-/- animals exhibited a similar
sensitivity (data not shown). It must be noted that the CD1
STAT1-/- animals were not extensively
back-crossed and therefore may not be entirely strain equivalent to the
control wild-type CD1 animals purchased from Charles River Breeding
Laboratories.
The exacerbated disease in STAT1-/-
mice was confirmed by microscopic examination of lung tissue from
infected BALB/c and 129SvEv animals. Histopathologic studies showed
large areas of consolidation in the lungs of
STAT1-/- mice with dense inflammatory
infiltrates (Fig. 2) which differed from wild-type in composition as
well as amount. Although RSV disease in wild-type mice consists
primarily of sparse perivascular and peribronchiolar lymphocytic
infiltrates, the responding cells in STAT1-/-
animals include large numbers of eosinophils, macrophages, and
neutrophils as well as lymphocytes.
This pattern of exacerbated disease in STAT1-/- mice following primary RSV infection is reminiscent of vaccine-enhanced RSV disease where an inappropriate Th2-like response to formalin-inactivated, alum-precipitated virus produced a debilitating inflammatory response rather than immunity (2, 3, 4, 5, 6, 8, 25, 31). The altered pathology following infection in STAT1-/- animals suggested a preexisting Th2 bias in the absence of IFN signaling. Further evidence of this bias in STAT1 knockout mice included the predominance of the IgG1 isotype of RSV-specific Ab and the production of elevated IL-4, IL-5, and IL-13 transcripts (data not shown) and protein in lung extracts from infected STAT1-/- animals.
Although the presence of Th2 cytokines has been found to correlate with
the eosinophilic, vaccine-enhanced lung pathology, it is not certain
that a Th2 response can entirely explain disease severity. In this
context, it was useful to examine T cell responses in the
STAT1-/- mouse where severe, eosinophilic lung
disease was present following primary infection. Table I demonstrates
comparable rates of viral clearance in wild-type and
STAT1-/- animals. As yet unpublished studies
conducted in our laboratory have demonstrated unimpaired anti-RSV
CTL activity by memory wild-type and
STAT1-/-CD8+ T
cells.4 In addition, virus
specific CD8+ T cells of either genotype have
been found to secrete IFN-, consistent with our influenza studies in
STAT1-/- animals (32). To
determine whether cytokine secretion profiles from RSV-specific
STAT1-/- Th cells were Th0 or Th2 in character,
we first identified a RSV-F peptide epitope (GWYTSVITIELSNIKETKCN),
designated F6, that was recognized by BALB/c mice. While peptide F6
induced IFN- secretion by memory wild-type BALB/c splenocytes (Fig. 5), none was detected from peptide-treated, immune,
STAT1-/-CD4+ cells even
though they demonstrated an enhanced proliferation response to peptide
F6. When cytokine transcript profiles were analyzed following peptide
F6 stimulation of wild-type or STAT1-/- T
cells, wild-type memory T cells were found to secrete primarily
IFN-, while Th cells from STAT1-/- mice
secreted primarily IL-13 (Fig. 6). In repeat experiments, the ratio of
IL-13/IFN- produced by peptide F6-stimulated wild-type
CD4+ T cells varied, but IFN- mRNA or protein
production was virtually absent in
STAT1-/-CD4+ T cells.
This is consistent with observations of Varga et al. (33)
and Srikiatkhachorn et al. (34) that a single
immunodominant epitope of the RSV-G protein is able to elicit both Th1
and Th2 responses. It seems clear from our studies that STAT1
activation is essential for establishing a Th1 response to RSV
infection; in its absence the response to the F6 viral protein epitope
is entirely Th2. This difference appears to correlate with disease
severity even though it cannot be concluded with certainty that Th2
cytokine profiles alone determine pathology. T cells with different
specificities may have different patterns because the cytokine pattern
in whole lungs is less polarized.
Nonetheless, it has been demonstrated that all three of the Th2 cytokines found in STAT1-/- lung homogenates, IL-4, IL-5, and IL-13, play important roles in eosinophil recruitment (35). IL-4 and IL-13 stimulate eotaxin synthesis by fibroblasts and epithelial cells (36) and up-regulate endothelial adhesion molecules (37). IL-5 acts systemically to mobilize eosinophils from bone marrow and acts as an eosinophil survival factor (38). Once the level of eotaxin rises in the lung, it acts to concentrate and activate eosinophils (39). Although eotaxin was detected in lung homogenates taken from both RSV-infected STAT1-/- mice and wild-type BALB/c controls, levels were nearly doubled (1.7-fold) in the mutant animals.
In addition to eosinophils, neutrophils are also prominent in the
atypical inflammatory infiltrate seen in
STAT1-/- mice following primary RSV infection.
The presence of neutrophils in the lung has also been noted in
wild-type BALB/c mice following FI-RSV or alum-precipitated RSV-F or -G
protein priming and challenge (10, 18, 40, 41). The
pathways mediating neutrophil accumulation are not as thoroughly
understood. The ELR+ (glutamic
acid-leucine-arginine motif-positive) subfamily of CXC chemokines is
known to be important for the recruitment and activation of neutrophils
in both man and mouse, particularly IL-8 in man. These ligands are
induced in response to a number of stressors, including RSV infection
(42, 43), and act through the CXCR1 (human only) and CXCR2
present on the granulocyte cell surface (44, 45, 46, 47). Reports
from a number of laboratories have suggested that expression of these
chemokines is negatively regulated by both type I () and type II
() IFNs in human peripheral blood monocytes and neutrophils as well
as in fibroblasts (48, 49, 50, 51, 52). These reports, coupled with
our previous observation of lung neutrophilia as well as eosinophilia
following influenza infection in the absence of
STAT1-/- (32), suggest that IFNs
may also inhibit neutrophil accumulation in the murine system.
The role of IFN- in promoting Th1 differentiation of naive Th
lymphocytes is well documented (26, 53), so the production
of Th2 cytokines by STAT1-/- animals was not
unanticipated. More surprising was the realization that, in mice
lacking IFN-, the outcome of RSV infection was essentially
equivalent to that seen in wild-type animals. The increased ratio of
RSV-F protein-specific IgG1/IgG2 Ab production was the only indication
of altered immune responsiveness following primary infection of
IFN--/- mice (Fig. 3). The lack of
eosinophilic lung disease following primary RSV infection of
IFN--deficient animals has also been reported by Srikiatkhachorn et
al. (18), although in those studies
IFN--/- animals did show increased lung
eosinophilia upon RSV challenge after VV-G priming. Similar results
have been described in a study of influenza infection in IFN-
knockout mice (54), whereas we have documented an
exacerbated Th2-like response to influenza infection in the absence of
STAT1 (32).
In virtually all cell types, virus infection results in transcriptional
up-regulation, synthesis, and secretion of type I IFNs. Once induced,
IFN- acts in an autocrine or paracrine manner by binding to the
specific IFN- cell surface receptor (55). Binding of
either IFN- or IFN- to their distinct receptors stimulates the
Janus kinase-STAT signaling cascade leading to induction of the
IFN-stimulated genes that mediate the biological effects of IFN
(56). Our findings suggest that in addition to limiting
virus replication within the lung, IFN activation of STAT1 coordinates
antiviral defenses more broadly by promoting the Th1 Ag-specific
responses best suited to the elimination of virus-infected cells.
IFN- has an important role in dendritic cell maturation
(57) as well as NK cell expansion and activation. Early
IFN- production by activated NK cells will influence
CD4+ cell differentiation (58) and
has been found to correlate with CD8+ cell
recruitment in the setting of murine RSV infection
(59).
The RSV G glycoprotein has an immunodominant epitope that is
known to induce a Th2 immune response in selected genetic backgrounds
(9, 10, 11, 16, 60, 61, 62). The eosinophilia induced by RSV G is
modulated by early IFN- produced either by
CD8+ CTL (17, 18) or NK cells
(59). Interestingly, primary infection with RSV in BALB/c
mice even in the setting of IFN- deficiency does not lead to
eosinophilia (18), suggesting that IFN- also plays a
role in regulating that response. Although induction by RSV may be weak
when compared with other viral pathogens (27, 63), in the
setting of RSV it appears to be sufficient to promote Th1
differentiation in the majority of cases. In
STAT1-/- mice the Th2 response and eosinophilia
are not based on a response specific to the G glycoprotein, as
demonstrated by the Th2-biased response to the epitope derived from the
F glycoprotein. Therefore, the cytokine milieu established by the
initial host response to RSV can determine the pattern of T cell
differentiation and composition of the subsequent virus-specific immune
response irrespective of the influence of selected Ag
specificities.
In summary, the experiments described in this study suggest a new role
for IFN- in the orchestration of antiviral defenses. In addition
to the antiviral effects and induction of NK cell blastogenesis, our
work with RSV and influenza suggests a complementary function of type I
IFNs, and perhaps other unidentified stimuli, that can influence T cell
differentiation through STAT1 activation. Although mice cannot activate
STAT4 through IFN- stimulation (64), STAT1-mediated
processes are sufficient to promote Th1 differentiation even in the
absence of IFN-. We further showed that either IFN- or -
could protect against eosinophilia by evaluating the effects of primary
RSV infection in background-matched IFNAR1-/-
mice and IFNGR1-/- mice, lacking IFNAR or IFNGR
function, respectively. As predicted by our studies with the BALB/c
mice, lung disease in either of these 129SvEv strains resembled that
seen in wild-type controls, suggesting that STAT1 activation by either
pathway was sufficient to inhibit eosinophilia.
Several laboratories have demonstrated STAT1 functions that are
independent of IFNR activation, as well as IFN effects independent of
STAT1 (65, 66, 67, 68), prompting us to compare RSV-mediated lung
pathology in IFNARGR-/- mice lacking both the
IFN- and - pathways to those lacking STAT1. Both strains
developed exacerbated, eosinophilic inflammation, indicating that
protection from eosinophilia is dependent on IFN-mediated activation of
STAT1. However, increased inflammation and illness in
STAT1-/- animals, beyond that seen in the
absence of both IFNRs, suggests that STAT1 activation through other
pathways (69) can also modulate T cell responses to RSV.
Although eosinophilia corresponds to the production of Th2 cytokines,
it is not required for disease, and it is likely that other effector
mechanisms influenced by these cytokines also contribute to exacerbated
disease following RSV infection of naive
STAT1-/- mice or vaccinated wild-type mice. The
shift to Fas ligand-mediated CTL killing in the presence of increased
IL-4 (70, 71), as well as other direct effects of
CD4+ Th2 cells, may be contributing to the
pathology seen in these settings.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Joan E. Durbin, Children’s Research Institute, Children’s Hospital, Wexner Institute for Pediatric Research, Room 411, 700 Children’s Drive, Columbus, OH 43205. E-mail address: durbinj{at}pediatrics.ohio-state.edu
3 Abbreviations used in this paper: RSV, respiratory syncytial virus; i.n., intranasal; IFNAR, IFN-R; IFNGR, IFN-R; IFNARGR, IFN-R; FI-RSV, formalin-inactivated whole virus vaccine; F, fusion.
4 J. E. Durbin, C. Beall, S. E. Mertz, P. R. Johnson, and C. M. Walker. Identification and characterization of a respiratory syncytial virus F protein-specific cytotoxic T-lymphocyte epitope in wild type and STAT1-/- BALB/c mice. Submitted for publication.
Received for publication May 30, 2001. Accepted for publication January 8, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
genes. Science 259:1739. receptor. Science 259:1742./ and interferon receptors. J. Virol. 69:4792.[Abstract]
/ interferon-induced antiviral response. J. Virol. 74:8234. and interferon- down-regulate the production of interleukin-8 and ENA-78 in human monocytes. J. Leukocyte Biol. 57:929.[Abstract]
suppresses IL-8 expression in human monocytes. J. Immunol. 151:2725.[Abstract]
. Biochem. J. 310:751.
gene. J. Exp. Med. 178:1725./-producing cells link innate and adaptive immunity. J. Exp. Med. 192:219. and interferons, in innate and adaptive immune responses to viral infections. Semin. Immunol. 10:383.[Medline]
expression in natural killer cells precedes lung CD8 T cell recruitment during RSV infection. J. Gen. Virol. 79:2593.[Abstract]
production by local T cells in RSV-induced eosinophilic lung disease. J. Gen. Virol. 79:1751.[Abstract]
receptor 1-TRADD signaling complex to inhibit NF-
B activation. Mol. Cell. Biol. 20:4505./-mediated inhibition and promotion of interferon-: STAT1 resolves a paradox. Nat. Immunol. 1:70.[Medline]
through Stat1-dependent and independent pathways. EMBO J. 19:263.[Medline]
activate stats in T cells. J. Biol. Chem. 273:309.This article has been cited by other articles:
|
|
 |
|
  J. Kovacs-Nolan, J. W. Mapletoft, Z. Lawman, L. A. Babiuk, and S. van Drunen Littel-van den Hurk Formulation of bovine respiratory syncytial virus fusion protein with CpG oligodeoxynucleotide, cationic host defence peptide and polyphosphazene enhances humoral and cellular responses and induces a protective type 1 immune response in mice J. Gen. Virol., August 1, 2009; 90(8): 1892 - 1905. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. K. Pribul, J. Harker, B. Wang, H. Wang, J. S. Tregoning, J. Schwarze, and P. J. M. Openshaw Alveolar Macrophages Are a Major Determinant of Early Responses to Viral Lung Infection but Do Not Influence Subsequent Disease Development J. Virol., May 1, 2008; 82(9): 4441 - 4448. [Abstract] [Full Text] [PDF]
|
|
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P. L. Collins and B. S. Graham Viral and Host Factors in Human Respiratory Syncytial Virus Pathogenesis J. Virol., March 1, 2008; 82(5): 2040 - 2055. [Full Text] [PDF]
|
|
|
|
|
|
E. M. Castilow, M. R. Olson, D. K. Meyerholz, and S. M. Varga Differential Role of Gamma Interferon in Inhibiting Pulmonary Eosinophilia and Exacerbating Systemic Disease in Fusion Protein-Immunized Mice Undergoing Challenge Infection with Respiratory Syncytial Virus J. Virol., March 1, 2008; 82(5): 2196 - 2207. [Abstract] [Full Text] [PDF]
|
|
|
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 L. P. Shornick, A. G. Wells, Y. Zhang, A. C. Patel, G. Huang, K. Takami, M. Sosa, N. A. Shukla, E. Agapov, and M. J. Holtzman Airway Epithelial versus Immune Cell Stat1 Function for Innate Defense against Respiratory Viral Infection J. Immunol., March 1, 2008; 180(5): 3319 - 3328. [Abstract] [Full Text] [PDF]
|
|
|
|
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|
 Y.-M. Lee, N. Miyahara, K. Takeda, J. Prpich, A. Oh, A. Balhorn, A. Joetham, E. W. Gelfand, and A. Dakhama IFN-{gamma} Production during Initial Infection Determines the Outcome of Reinfection with Respiratory Syncytial Virus Am. J. Respir. Crit. Care Med., January 15, 2008; 177(2): 208 - 218. [Abstract] [Full Text] [PDF]
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|
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|
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|
L. M. Pletneva, O. Haller, D. D. Porter, G. A. Prince, and J. C. G. Blanco Induction of type I interferons and interferon-inducible Mx genes during respiratory syncytial virus infection and reinfection in cotton rats J. Gen. Virol., January 1, 2008; 89(1): 261 - 270. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Mok, S. Lee, T. J. Utley, B. E. Shepherd, V. V. Polosukhin, M. L. Collier, N. L. Davis, R. E. Johnston, and J. E. Crowe Jr. Venezuelan Equine Encephalitis Virus Replicon Particles Encoding Respiratory Syncytial Virus Surface Glycoproteins Induce Protective Mucosal Responses in Mice and Cotton Rats J. Virol., December 15, 2007; 81(24): 13710 - 13722. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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|
|
|
|
|
|
M. Boxus, M. Tignon, S. Roels, J.-F. Toussaint, K. Walravens, M.-A. Benoit, P. Coppe, J.-J. Letesson, C. Letellier, and P. Kerkhofs DNA Immunization with Plasmids Encoding Fusion and Nucleocapsid Proteins of Bovine Respiratory Syncytial Virus Induces a Strong Cell-Mediated Immunity and Protects Calves against Challenge J. Virol., July 1, 2007; 81(13): 6879 - 6889. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. M. Johnson and P. Scott STAT1 Expression in Dendritic Cells, but Not T Cells, Is Required for Immunity to Leishmania major J. Immunol., June 1, 2007; 178(11): 7259 - 7266. [Abstract] [Full Text] [PDF]
|
|
|
|
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|
 N. W. Lukacs, M. L. Moore, B. D. Rudd, A. A. Berlin, R. D. Collins, S. J. Olson, S. B. Ho, and R. S. Peebles Jr Differential Immune Responses and Pulmonary Pathophysiology Are Induced by Two Different Strains of Respiratory Syncytial Virus Am. J. Pathol., September 1, 2006; 169(3): 977 - 986. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Kotelkin, I. M. Belyakov, L. Yang, J. A. Berzofsky, P. L. Collins, and A. Bukreyev The NS2 Protein of Human Respiratory Syncytial Virus Suppresses the Cytotoxic T-Cell Response as a Consequence of Suppressing the Type I Interferon Response. J. Virol., June 1, 2006; 80(12): 5958 - 5967. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. L. Miller, C. Gerard, M. Schaller, A. D. Gruber, A. A. Humbles, and N. W. Lukacs Deletion of CCR1 Attenuates Pathophysiologic Responses during Respiratory Syncytial Virus Infection J. Immunol., February 15, 2006; 176(4): 2562 - 2567. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. L. Garvey, K. D. Dyer, J. A. Ellis, C. A. Bonville, B. Foster, C. Prussin, A. J. Easton, J. B. Domachowske, and H. F. Rosenberg Inflammatory Responses to Pneumovirus Infection in IFN-{alpha}{beta}R Gene-Deleted Mice J. Immunol., October 1, 2005; 175(7): 4735 - 4744. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Guerrero-Plata, S. Baron, J. S. Poast, P. A. Adegboyega, A. Casola, and R. P. Garofalo Activity and Regulation of Alpha Interferon in Respiratory Syncytial Virus and Human Metapneumovirus Experimental Infections J. Virol., August 15, 2005; 79(16): 10190 - 10199. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Holtzman, J. W. Tyner, E. Y. Kim, M. S. Lo, A. C. Patel, L. P. Shornick, E. Agapov, and Y. Zhang Acute and Chronic Airway Responses to Viral Infection: Implications for Asthma and Chronic Obstructive Pulmonary Disease Proceedings of the ATS, August 1, 2005; 2(2): 132 - 140. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. A. Tripp, C. Oshansky, and R. Alvarez Cytokines and Respiratory Syncytial Virus Infection Proceedings of the ATS, August 1, 2005; 2(2): 147 - 149. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M.-E. Hamelin, K. Yim, K. H. Kuhn, R. P. Cragin, M. Boukhvalova, J. C. G. Blanco, G. A. Prince, and G. Boivin Pathogenesis of Human Metapneumovirus Lung Infection in BALB/c Mice and Cotton Rats J. Virol., July 15, 2005; 79(14): 8894 - 8903. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. R. Johnson, S. E. Mertz, N. Gitiban, S. Hammond, R. LeGallo, R. K. Durbin, and J. E. Durbin Role for Innate IFNs in Determining Respiratory Syncytial Virus Immunopathology J. Immunol., June 1, 2005; 174(11): 7234 - 7241. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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|
|
|
|
|
|
B. D. Rudd, E. Burstein, C. S. Duckett, X. Li, and N. W. Lukacs Differential Role for TLR3 in Respiratory Syncytial Virus-Induced Chemokine Expression J. Virol., March 15, 2005; 79(6): 3350 - 3357. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Dakhama, J.-W. Park, C. Taube, K. Chayama, A. Balhorn, A. Joetham, X.-D. Wei, R.-H. Fan, C. Swasey, N. Miyahara, et al. The Role of Virus-specific Immunoglobulin E in Airway Hyperresponsiveness Am. J. Respir. Crit. Care Med., November 1, 2004; 170(9): 952 - 959. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-Z. Wang, Y.-X. Bao, C. L. Rosenberger, Y. Tesfaigzi, J. M. Stark, and K. S. Harrod IL-12p40 and IL-18 Modulate Inflammatory and Immune Responses to Respiratory Syncytial Virus Infection J. Immunol., September 15, 2004; 173(6): 4040 - 4049. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Rutigliano and B. S. Graham Prolonged Production of TNF-{alpha} Exacerbates Illness during Respiratory Syncytial Virus Infection J. Immunol., September 1, 2004; 173(5): 3408 - 3417. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. M. Spann, K.-C. Tran, B. Chi, R. L. Rabin, and P. L. Collins Suppression of the Induction of Alpha, Beta, and Gamma Interferons by the NS1 and NS2 Proteins of Human Respiratory Syncytial Virus in Human Epithelial Cells and Macrophages J. Virol., April 15, 2004; 78(8): 4363 - 4369. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. J. Easton, J. B. Domachowske, and H. F. Rosenberg Animal Pneumoviruses: Molecular Genetics and Pathogenesis Clin. Microbiol. Rev., April 1, 2004; 17(2): 390 - 412. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. M. Haynes, L. P. Jones, A. Barskey, L. J. Anderson, and R. A. Tripp Enhanced Disease and Pulmonary Eosinophilia Associated with Formalin-Inactivated Respiratory Syncytial Virus Vaccination Are Linked to G Glycoprotein CX3C-CX3CR1 Interaction and Expression of Substance P J. Virol., September 15, 2003; 77(18): 9831 - 9844. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. S. Harrod, R. J. Jaramillo, C. L. Rosenberger, S.-Z. Wang, J. A. Berger, J. D. McDonald, and M. D. Reed Increased Susceptibility to RSV Infection by Exposure to Inhaled Diesel Engine Emissions Am. J. Respir. Cell Mol. Biol., April 1, 2003; 28(4): 451 - 463. [Abstract] [Full Text] [PDF]
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