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The Journal of Immunology, 2002, 168: 2944-2952.
Copyright © 2002 by The American Association of Immunologists

The Role of IFN in Respiratory Syncytial Virus Pathogenesis

Joan E. Durbin2,*,{dagger}, Teresa R. Johnson§, Russell K. Durbin*,{dagger}, Sara E. Mertz*,{dagger}, Rafaella A. Morotti*,{dagger}, R. Stokes Peebles{ddagger} and Barney S. Graham§

* Children’s Research Institute, Children’s Hospital, Columbus, OH 43205; {dagger} Division of Pediatric Pathology, Department of Pediatrics, College of Medicine and Public Health, Ohio State University, Columbus, OH 43210; {ddagger} Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232; and § Vaccine Research Center, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Formalin-inactivated respiratory syncytial virus (RSV) vaccine preparations have been shown to cause enhanced disease in naive hosts following natural infection. In this study we demonstrate a similar pattern of enhanced disease severity following primary RSV infection of IFN-nonresponsive STAT1-/- mice. STAT1-/- mice showed markedly increased illness compared with wild-type BALB/c animals following RSV inoculation despite similar lung virus titers and rates of virus clearance. Histologically, STAT1-/- animals had eosinophilic and neutrophilic pulmonary infiltrates not present in wild-type or IFN-{gamma}-/--infected mice. In cytokine analyses of infected lung tissue, IFN-{gamma} 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-{gamma}, while STAT1-/-CD4+ cells produce primarily IL-13. These findings suggest that STAT1 activation by both type I ({alpha}{beta}) and type II ({gamma}) IFNs plays an important role in establishing a protective, Th1 Ag-specific immune response to RSV infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Respiratory syncytial virus (RSV)3 is the leading cause of bronchiolitis and viral pneumonia in infants (1). Primary RSV infection occurs within the first 2 years of life, with the most severe disease affecting infants between 2 and 4 mo. RSV vaccine development is a high priority, but the failure of vaccine trials in the 1960s has slowed progress. In the failed trials, vaccinees receiving a formalin-inactivated whole virus vaccine (FI-RSV) were not protected from infection. The vaccinated children experienced greater morbidity and mortality than did controls (2, 3, 4, 5).

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 ({alpha}{beta}) or type II ({gamma}) 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-{gamma} production and enhanced by anti-IFN-{gamma} or anti-CD8 Abs. However, work by Srikiatkhachorn et al. (18) shows similar lung pathology following RSV infection of wild-type and IFN-{gamma}-deficient animals.

In this study we have observed that in the absence of both IFN-{alpha}{beta} and IFN-{gamma} 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-{alpha}{beta} effects that are normally induced by virus infection as well as IFN-{gamma} 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 ({alpha}{beta}) IFNs may also play a role in directing the development of an appropriate, Th1-biased cell-mediated response to virus infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and virus

Pathogen-free wild-type BALB/c and CD1 mice were purchased from Charles River Breeding Laboratories (Wilmington, MA) and Taconic Farms (Germantown, NY). IFN-{gamma}-/- 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-{alpha}{beta}R (IFNAR)-/- (20), IFN-{gamma}R (IFNGR)-/- (21), and IFN-{alpha}{beta}{gamma}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-{gamma} 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-{gamma}-containing spots were visualized by sequential incubations with a biotinylated polyclonal anti-IFN-{gamma} Ab, an anti-biotin tertiary Ab, and the detection reagent.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Peak RSV titers are equivalent in wild-type, IFN-{gamma}-/-, 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-{alpha}{beta} 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 IGo. Viral lung titers from wild-type and IFN-{gamma}-/- 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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  		Table I. RSV lung titers in wild-type and mutant BALB/c mice1

 
To control for possible kinetic differences in virus growth in the absence of IFN signaling, lung titers from wild-type and STAT1-/- mice were compared at days 2, 4, and 6 postinfection with 107 PFU RSV. Virus production did peak earlier in the STAT1-/- cohort, giving a 1-log increase over wild-type levels, which were maximal at day 4 (Table IIGo). Clearance was slightly delayed in the STAT1-/- mice but was complete by day 8.


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  		Table II. Early virus peak in STAT1-/- BALB/c mice1

 
Increased RSV titers were also found in an experiment comparing wild-type CD1 mice to STAT1-/- animals on the CD1 background (Table IIIGo). 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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  		Table III. Virus load in lungs and nose of wild-type and STAT1-/- CD1 mice1

 
STAT1-/- mice have exacerbated disease compared with wild-type controls following primary RSV infection

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. 1Go). 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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  		FIGURE 1. Wild-type and STAT1-/- BALB/c mice were weighed daily following i.n. infection with 106 PFU of RSV. Mean daily weight loss for each strain is plotted; error bars reflect the SEM. Statistically significant differences were observed between the two groups on days 5 (p < 0.05), 6 (p = 0.005), 7 (p = 0.009), and 8 (p < 0.05). n = 11 for wild-type; n = 10 for the STAT1-/- groups.

 
To compare disease severity, wild-type and STAT1-/- animals were inoculated i.n. with 106 PFU RSV and illness was determined by daily weight measurements. Fig. 1Go 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. 2Go). 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-{gamma}-/- BALB/c mice was similar to that seen in wild-type animals (data not shown).



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  		FIGURE 2. Lungs from wild-type (BALB/c) and STAT1-/- mice (on a BALB/c background) were harvested 8 days after i.n. RSV infection. Photomicrographs of representative sections were taken at x100, x200, and x1000 magnification. Lungs from wild-type animals (A, x100; B, x200; C, x1000) and STAT1-/- mice (D, x100; E, x200; F, x1000) are pictured.

 
In the absence of IFN signaling, virus-specific Ab titers are normal

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-{gamma}-/- and STAT1-/- animals (Fig. 3Go). Preferential IgG1 production is characteristic of B lymphocytes maturing under the influence of Th2-type cytokines (26). Upon rechallenge with RSV, IFN-{gamma}-/-, 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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  		FIGURE 3. Serum samples were collected from four to six mice of each genotype (wild-type, STAT1-/-, and IFN-{gamma} -/- BALB/c mice) 28 days after primary infection with RSV and assayed for the presence of RSV F protein-specific Ab. Virus-specific IgG/A/M, IgG1, and IgG2a levels were measured using isotype-specific HRP-conjugated secondary Abs. The highest dilution giving a positive signal is plotted. Error bars represent the SEM. Only the IgG2a levels in IFN-{gamma} -/- mice differed significantly (p < 0.001) from wild-type.

 
Wild-type BALB/c mice have a Th1 response to primary RSV infection while STAT1-/- BALB/c mice display a Th2-biased cytokine profile

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-{gamma}-/- 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-{gamma}, and eotaxin by ELISA. In comparing cytokine induction between wild-type and IFN-{gamma}-/- mice, we found statistically significant differences only in IFN-{gamma} production (Fig. 4GoA). 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. 4GoB). 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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  		FIGURE 4. Cytokine levels were measured by ELISA in lung homogenates obtained from wild-type, IFN-{gamma}-/-, and STAT1-/- BALB/c mice 4 days following i.n. inoculation with RSV. Comparisons between wild-type and IFN-{gamma}-/- (A) or STAT1-/- (B) mice are shown. A, In the absence of IFN-{gamma}, cytokine profiles differ significantly only for IFN-{gamma}. B, In the absence of STAT1, IL-4, IL-5, IL-13, IFN-{gamma}, and eotaxin all appear elevated relative to the control, but these differences are statistically significant only for IL-13 (p < 0.001) and eotaxin (p < 0.001).

 
To determine whether the 4-day time point was providing a complete picture of cytokine secretion following RSV infection, ELISA were repeated on lung homogenates from wild-type and STAT1-/- mice 2, 4, and 6 days after inoculation. These results are tabulated in Table IVGo. 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-{gamma} production by mice of both genotypes, increased nearly 5-fold in the STAT1-/- animals relative to wild type.


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  		Table IV. Lung cytokine profiles1

 
Genotype determines cytokine secretion patterns by memory CD4+ T cells responsive to the same RSV epitope

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-{gamma} 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-{gamma} synthesis over background was detected in wells containing STAT1-/- cells (Fig. 5Go). 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-{gamma}.



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  		FIGURE 5. An IFN-{gamma} ELISPOT was performed to compare the responses of RSV-immune wild-type and STAT1-/- splenocytes to the RSV F protein-derived peptide F6, the control peptide (SIINFEKL), and medium alone.

 
To determine which cytokines were produced by STAT1-/-CD4+ T cells recognizing peptide F6, splenocytes from two BALB/c wild-type and two STAT1-/- RSV immune mice were cultured for 1 wk in the presence of peptide, and then restimulated with peptide F6 for an additional 6 h. RNA from the stimulated cells was extracted with TRIzol and analyzed by RNase protection assay. These results are shown in Fig. 6Go. We see that when wild-type CD4+ cells are stimulated by peptide F6 (Fig. 6Go, lane 1) they produce IFN-{gamma} transcripts in addition to low levels of the IL-13 transcript. In the STAT1-/- mice (Fig. 6Go, lane 2) the situation is reversed, with very little IFN-{gamma} 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-{gamma} 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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  		FIGURE 6. Splenocytes were harvested from RSV-immune wild-type or STAT1-/- animals (two mice of each strain per experiment). After a 1-wk expansion in the presence of peptide F6, cells were restimulated with peptide for 6 h and then lysed in TRIzol. RNA from each sample (20 µg) was then analyzed by RNase protection assay for the presence of cytokine transcripts. Lane 1, RNA from the restimulated wild-type cells; lane 2, RNA from the STAT1-/- cells.

 
Eosinophilic lung disease is induced by RSV in mice lacking both IFNAR and IFNGR as well as in STAT1-/- mice

Our studies comparing wild-type, IFN-{gamma}-/-, and STAT1-/- BALB/c mice have established that the loss of IFN-{gamma} alone is not sufficient to promote exacerbated lung disease and Th2 cytokine production in response to RSV infection. These results implied that IFN-{alpha}{beta} alone, in the absence of IFN-{gamma}, 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. 7Go, 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. 2Go). IFNAR-/- and IFNGR-/- animals had histology similar to wild-type and are not pictured. Fig. 7Go, 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. 7Go, 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-{alpha}{beta} or IFN-{gamma}, is required to inhibit RSV-mediated immunopathology.



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  		FIGURE 7. Lungs from 129SvEv wild-type,IFNAR-/-, IFNGR-/-, IFNARGR-/-, and STAT1-/- mice were harvested 8 days after i.n. RSV infection. Photomicrographs of representative sections were taken at x200 and x1000 magnification. Lungs from wild-type (A, x200; B, x1000), IFNARGR-/- (C, x200; D, x1000), and STAT1-/- (E, x200; F, x1000) mice are pictured. Pathology in IFNAR-/- and IFNGR-/- animals was indistinguishable from wild-type.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In these studies, we compared primary RSV infection in STAT1-/- and IFN-{gamma}-/- mice to that seen in age-matched, wild-type controls. Although IFN-{gamma}-/- 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. 1Go). Lung virus titers in STAT1-/- animals were within 1 log of those found in wild-type BALB/c controls (Table IGo) 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 IGo). 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 IIIGo), 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. 2Go) 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 IGo 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-{gamma}, 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-{gamma} secretion by memory wild-type BALB/c splenocytes (Fig. 5Go), 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-{gamma}, while Th cells from STAT1-/- mice secreted primarily IL-13 (Fig. 6Go). In repeat experiments, the ratio of IL-13/IFN-{gamma} produced by peptide F6-stimulated wild-type CD4+ T cells varied, but IFN-{gamma} 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 ({alpha}{beta}) and type II ({gamma}) 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-{gamma} 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-{gamma}, 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-{gamma}-/- mice (Fig. 3Go). The lack of eosinophilic lung disease following primary RSV infection of IFN-{gamma}-deficient animals has also been reported by Srikiatkhachorn et al. (18), although in those studies IFN-{gamma}-/- 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-{gamma} 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-{alpha}{beta} acts in an autocrine or paracrine manner by binding to the specific IFN-{alpha}{beta} cell surface receptor (55). Binding of either IFN-{alpha}{beta} or IFN-{gamma} 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-{alpha}{beta} has an important role in dendritic cell maturation (57) as well as NK cell expansion and activation. Early IFN-{gamma} 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-{gamma} 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-{gamma} deficiency does not lead to eosinophilia (18), suggesting that IFN-{alpha}{beta} 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-{alpha}{beta} 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-{alpha}{beta} stimulation (64), STAT1-mediated processes are sufficient to promote Th1 differentiation even in the absence of IFN-{gamma}. We further showed that either IFN-{alpha}{beta} or -{gamma} 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-{alpha}{beta} and -{gamma} 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.


   Acknowledgments
 
We thank Frances Robinson, Rauf Kuli-Zade, and Tenicia Pitts for technical assistance.


   Footnotes
 
1 This work was supported by Grant RO1-AI-33933 (to B.S.G.) from the National Institutes of Health. J.E.D. is supported by Child Health Research Center Grants P30-HD-34615 and RO1-AI-47226, both from the National Institutes of Health. Back

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 Back

3 Abbreviations used in this paper: RSV, respiratory syncytial virus; i.n., intranasal; IFNAR, IFN-{alpha}{beta}R; IFNGR, IFN-{gamma}R; IFNARGR, IFN-{alpha}{beta}{gamma}R; FI-RSV, formalin-inactivated whole virus vaccine; F, fusion. Back

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. Back

Received for publication May 30, 2001. Accepted for publication January 8, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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