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R Gene-Deleted Mice1
* Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892;
Western College of Veterinary Medicine, Saskatoon, Saskatchewan, Canada;
State University of New York, Upstate Medical University, Syracuse, NY 13210; and
Department of Biological Sciences, University of Warwick, Coventry, United Kingdom
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Abstract |
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R gene-deleted (IFN-R–/–) mice. However, we demonstrate that PVM infection results in enhanced expression of eotaxin-2 (CCL24), thymus and activation-regulated chemokine (CCL17), and the proinflammatory RNase mouse eosinophil-associated RNase (mEar) 11, and decreased expression of monocyte chemotactic protein-5, IFN-
-inducible protein-10, and TLR-3 in lung tissue of IFN-R–/– mice when compared with wild type. No differential expression of chemokines MIP-1 or MIP-2 or Th2 cytokines IL-4 or IL-5 was observed. Differential expression of proinflammatory mediators was associated with distinct patterns of lung pathology. The widespread granulocytic infiltration and intra-alveolar edema observed in PVM-infected, wild-type mice are replaced with patchy, dense inflammatory foci localized to the periphery of the larger blood vessels. Bronchoalveolar lavage fluid from IFN-R–/– mice yielded 7- to 8-fold fewer leukocytes overall, with increased percentages of eosinophils, monocytes, and CD4+ T cells, and decreased percentage of CD8+ T cells. Differential pathology is associated with prolonged survival of the IFN-R–/– mice (50% survival at 10.8 ± 0.6 days vs the wild type at 9.0 ± 0.3 days; p < 0.02) despite increased virus titers. Overall, our findings serve to identify novel transcripts that are differentially expressed in the presence or absence of IFN-R-mediated signaling, further elucidating interactions between the IFN and antiviral inflammatory responses in vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Of particular recent interest are the ways in which IFNs and IFN-mediated signaling mechanisms interact with proinflammatory pathways and modulate the production of chemoattractant cytokines. Among the responses that have been studied thus far, the transcriptional activator, IFN-response factor (IRF)-3 is an absolute requirement for the production of the CC chemokine, RANTES, in response to infection with the paramyxoviruses Sendai (11, 12) or hRSV (13) in tissue culture. Interestingly, RANTES was not detected in a gene microarray study designed to identify gene transcription in vitro in response to overexpression of IRF-3 alone in the absence of virus infection (14); neither were any proinflammatory chemokine transcripts detected in a microarray study designed to evaluate responses to IRF-5 and IRF-7 (15). At the same time, intriguing relationships between inflammatory responses to virus infection and interactions with IFN- have been described (16, 17, 18).
Pneumonia virus of mice (PVM) is a virus of the family Paramyxoviridae, subfamily Pneumovirinae, and is the only pathogen of this subfamily that includes mice as its natural host. Intranasal inoculation with as few as 10–30 PFU of the mouse-passaged J3666 strain of PVM results in severe respiratory virus infection with robust replication to 108 PFU/g lung, accompanied by an inflammatory response mediated at least in part by the actions of the chemokine MIP-1 and its signaling through its major receptor CCR1 on granulocytes. Unless mice are treated with both antiviral (ribavirin) and immunomodulatory therapies (anti-MIP-1 or anti-CCR1) (19, 20), virus replication and the ensuing granulocytic inflammatory response lead to the significant morbidity and mortality. PVM has also been shown to be an important model for the study of postvirus Th2 responses (21).
In this work, we study the pathogenesis of PVM infection in mice devoid of the receptor for type I IFNs (IFN-R gene-deleted (IFN-R–/–) mice; Ref.21), and we identify a group of proinflammatory mediators whose expression is modulated (directly or indirectly) by IFN receptor-mediated signaling. The differential expression of these mediators is accompanied by differential pathology and cellular inflammation in response to this respiratory virus infection.
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Materials and Methods |
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R–/– gene-deleted mice
IFN-R–/– mice (22) (C57BL/6 background) were generated from heterozygotes and identified by standard PCR of genomic DNA isolated from tailsnips (primer sequences: sense UM4, 5'-AAGATGTGCTGTTCCCTTCCTCTGCTCTGA-3'; antisense UM5, 5'-AAGATGTGCTGTTCCCTTCCTCTGCTCTGA-3' (150-bp band from wild-type allele only); Neo sense, 5'-TCAGCGCAGGGGCGCCCGGTTCTTT-3'; and Neo antisense, 5'-TCAGCGCAGGGGCGCCCGGTTCTTT-3' (340-bp band from Neo cassette only).
Inoculation with PVM
Virus stocks (PVM strain J3666 at 106 PFU/ml) were prepared from mouse lung homogenates, and titers were determined as described previously (23). Mice were anesthetized briefly via inhalation of 20% halothane solution and, unless otherwise indicated, inoculated intranasally with 60 PFU PVM (strain J3666) in a 50- to 80-µl volume with IMDM as diluent. Mice were sacrificed at time points indicated by gentle cervical dislocation. All of the procedures were reviewed and approved by the National Institute of Allergy and Infectious Diseases Animal Care and Use Committee per animal study protocol LAD-8E.
Microscopic histology
Lungs for histology were inflated transtracheally with 0.2–0.3 ml of ice-cold phosphate-buffered 10% formalin before excision and fixation in same. Paraffin blocks, slide preparation, and H&E staining were done commercially (American Histolabs). Three slides each from three individual mice of each genotype (wild-type IFN-R+/+ and gene-deleted IFN-R–/–) at three distinct time points (total 54 slides) were coded by one investigator and assessed in that form by the veterinary pathologist (J. A. Ellis) to assure an unbiased reading.
Bronchoalveolar lavage fluid and cell differentials
Lungs were inflated transtracheally with cold 0.8 ml of PBS + 0.1% BSA + 1 mM EDTA, repeated once for a recovered volume of 1.5 ml. Cytospin preparations (100 µl per slide; Thermo Shandon) were prepared and stained with DiffQuik (Fisher Scientific) for leukocyte differential counts. One hundred to 200 hundred cells were scored per slide; n = 3–4 mice per point.
Preparation of mouse lung tissue homogenates and RNA
For analysis of protein, lung tissue was immersed in 1 ml of cold IMDM and subjected to blade homogenization. Tissue debris was removed by centrifugation at 4°C, and clarified supernatant was frozen on dry ice-ethanol and stored at –80°C before analysis. For analysis of RNA, mouse lung was immersed in 3–4 ml of RNAlater (Ambion), stored overnight at 4°C, then at –80°C until use. For preparation of RNA, samples were defrosted at room temperature, and lung tissue was removed from RNAlater, immersed in 5 ml of RNAzol, and processed as described previously (24).
Western blots
Clarified homogenates of mouse lung tissue (6 mg protein/ml) were diluted 1/1 in reducing sample buffer, heated to 65°C for 5 min and subjected to electrophoresis on 14% Tris-glycine SDS acrylamide gels and transfer to nitrocellulose membranes by standard methods. Membranes were probed with either 1) 1/500 dilution of rabbit polyclonal anti-PVM N protein antiserum or 2) 1/300 dilution of rabbit polyclonal anti-mouse eosinophil-associated RNase (mEars) antiserum, followed by a 1/1000 dilution of alkaline-phosphatase-linked goat anti-rabbit IgG and standard developing reagents (25).
ELISAs
ELISAs (R&D Systems) were performed to detect IFN-, MIP-1, MIP-2, thymus and activation-regulated chemokine (TARC), macrophage-derived chemokine (MDC), IL-4, IL-5, monocyte chemotactic protein-5, IFN-induced protein (IP)-10, and IFN-, per manufacturer’s instructions. Protein concentrations were determined by BCA assay (Pierce).
RNase assay
The RNase assay is essentially as described (25). Twenty microliters of mouse lung homogenate were added to 0.8 ml of 40 mM sodium phosphate (pH 7.4), all maintained at room temperature. The reaction was initiated with the addition of 10 µl of a stock of 20 mg/ml tRNA (Sigma-Aldrich), and reaction stopped at t = 20 min by the addition of ice-cold 20 mM lanthanum nitrate and 3% perchloric acid. A t = 0 spectrophotometric blank was prepared by adding stop solution to the buffer and RNase mix before addition of tRNA. Acid insoluble substrate (polymeric tRNA) was separated from acid soluble product (ribonucleotides) by centrifugation. Data obtained were corrected for total protein concentration as determined by BCA assay.
Gene microarray
Gene microarray was performed on lung RNA from PVM-infected and sham (diluent control)-inoculated mice sacrificed on day 6 postinoculation. All of the microarray procedures were conducted at the Microarray Core Facility (Rochester, NY) as described in our previous publication (24). This study used the M430 mouse gene microarray chip (Affymetrix) and was analyzed using GeneSpring 6.1 Software (Silicon Genetics).
Quantitative RT-PCR (Q-RT-PCR)
RNA samples (5 µg each) from three to five mice were pooled and treated with RNase-free DNase I (Invitrogen Life Technologies) before reverse transcription (1st strand synthesis kit; Roche Diagnostics) to cDNA. All of the reactions include reverse-transcriptase negative controls. Reactions for detection of the GAPDH housekeeping gene or the PVM virus SH gene include the ABI TaqMan reagent (x1) (Applied Biosystems), 100 nM primers (see below), 200 nM probe, 2 µl of cDNA template generated by reverse transcription step above, and distilled water to 25 µl. Primers and probes were as follows: rodent GAPDH set, Vic-labeled probe, (ABI catalog no. 4308313); PVM SH set, probe 5'-6FAM-CGCTGATAATGGCCTGCAGCA TAMRA-3', primer 1, 5'-GCCTGCATCAACACAGTGTGT-3'; primer 2, 5'-GCCTGATGTGGCAGTGCTT-3'. Reactions to detect all other genes described included x20 concentrated primer probe sets designed by ABI Assay by Design, which were diluted in the reaction mixture per manufacturer’s instructions. Primers and probes from ABI as follows: mEar11, Mm00519056_s1; Infb, Mm00439546_s1; CCL24, Mm00111701_m1; Oas1b, Mm00449297_m1; Oas3, Mm00460944_m1; Mx1, Mm00487796_m1; TLR3, Mm00446577_g1; Oas1g, Mm00726868_s1; and Oas2, Mm00460961_m1. Reactions were performed in an ABI 7700 Sequence detector,50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Data were normalized to GAPDH expression, and statistical significance was determined using the t test assuming equal variance.
Flow cytometric analysis
Total cells from BAL fluid from wild-type (IFN-R+/+) and IFN-R–/– mice on day 6 postinoculation were stained with the following mAb-fluorochrome conjugates: CD3 FITC, CD4 PE, CD8 allophycocyanin, and I-Ab PE/Cy5 (all from BD Pharmingen) in PBS with 0.1% BSA for 30 min. After staining, cells were washed twice in PBS with 0.1% BSA, stained with propidium iodide, and analyzed by flow cytometry. Control samples were stained with isotype-matched mAbs replacing CD4 and CD8. Each mouse BAL sample was analyzed individually. Data were acquired with a 2-laser, 4-parameter FACSCalibur flow cytometer (BD Biosciences) and analyzed on CellQuest software (BD Biosciences). Viable T cells were identified by first gating on cells of typical lymphocyte forward and side scatter, followed by gating on CD3+, I-Ab-negative, propidium iodide-negative cells. CD4 vs CD8 dot plots were generated, and the frequency of each subpopulation was determined. Quadrant statistical markers were placed on the basis of the isotype-matched controls. Typically, 50,000–200,000 total events were acquired to obtain 1,500–4,500 T cells per sample.
Statistical analysis
Statistical analysis was performed using Student’s t test or Mann-Whitney U test as appropriate.
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Results |
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R-deficient mice
We performed gene microarray analysis comparing transcripts expressed in PVM-infected wild-type (IFN-R+/+) mice to those expressed in PVM-infected gene-deleted (IFN-R–/–) mice at day 6 postinoculation. The initial half of the microarray analysis, which lists transcripts preferentially expressed in the PVM-infected wild-type mice, is shown in Table I. Most prominent among these preferentially expressed transcripts are the classic IFN response genes: the IFN-induced proteins with tetratricopeptide repeats, the IFN-activated genes 203 and 204, IFN regulator factor 7, Stat 2, and the antiviral IFN response genes, including the 2'-5' oligoadenylate synthetases, RNA-dependent protein kinase, and myxovirus resistance 1 gene, the latter set confirmed by Q-RT-PCR and shown in Fig. 1. TLR-3 (Fig. 1), the dsRNA receptor, previously identified as an IFN response gene by Heinz et al. (26), is also preferentially expressed in the wild-type mice, as is ubiquitin-specific protease 18 (also known as UBP43), which specifically removes the IFN-stimulated protein, ISG15, from intracellular protein conjugates (27). Two other transcripts that were preferentially expressed in the wild-type mice, those encoding ubiquitin-specific protease 1 (+36-fold) and RNA binding motif 11 (+17-fold), have no previously recorded relationship to IFN receptor-mediated signal transduction, and may bear some unique relationship to this experimental circumstance.
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R+/+) miceMonocyte chemotactic protein-5 (CCL-12) is produced in macrophages, smooth muscle cells, and mast cells and is an agonist for the receptor CCR2. The gene microarray results indicate a 3.3-fold increase in transcript encoding monocyte chemotactic protein-5 in PVM-infected wild-type lung tissue as compared with wild type on day 6 postinoculation. This finding is confirmed at the level of immuoreactive protein by ELISA (Table II).
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R-deficient mice (Table II). Reduction in the synthesis of IP-10 may be related to the reduced expression of TLR3 observed among IFN-R-deficient mice; Rudd et al. (28) demonstrated that targeted disruption of TLR3 expression by small interfering RNA resulted in reduced expression of IP-10 in response to hRSV in the MRC-5 fibroblast cell line. Targeted disruption of TLR3 also resulted in reduced expression of RANTES (CCL5) in response to hRSV infection in the MRC-5 cell line (28), and we observed a 2.36-fold greater expression of RANTES transcript in the wild-type mice. However, no differential expression of immunoreactive protein was observed in lung tissue, potentially due to the high levels of RANTES present at baseline in uninfected mouse lung tissue (data not shown).
Expression of type I IFNs in wild-type and IFN-R–/– mice
We evaluated the production in both wild-type and IFN-R–/– mice in response to PVM infection (Fig. 2). Immunoreactive IFN- was detected by ELISA in uninfected wild-type and IFN-R–/– mice, and PVM infection resulted in a significant (3.6-fold) increase to 99.1 ± 24 pg/ml/mg protein in wild-type mice. In contrast, PVM infection resulted in a statistically significant but comparatively minimal increase in IFN- production (1.6-fold, to 37.8 ± 6.9 pg/ml/mg protein) in the IFN-R–/– gene-deleted mice. The majority of the differential expression appears to be from the Ifna1 gene locus (Table I), although it would be necessary to sequence a statistically significant number of gene transcripts to confirm this. The differential expression of type I IFNs in response to infection is similar to that described by Malmgaard et al. (29) in their study of these mice infected with lymphocytic choriomeningitis virus in IFN-R–/– mice.
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in wild-type mice, as compared with a 1.5 ± 0.3-fold increase among IFN-R–/– mice. However, this degree of differential expression was not apparent in the gene array or on immunoassay of lung homogenate or BAL fluid, both of which contained minimal levels of immunoreactive IFN- (<50 pg/ml throughout). Although this is not inconsistent with our quantitative PCR results (Fig. 2), because this method can detect very small amounts of differentially expressed mRNA, one must conclude that IFN- is probably not contributing as much as IFN- to the responses observed.
Identification of transcripts preferentially expressed in PVM-infected, IFN-R-deficient mice vs wild type
In the second half of the gene microarray analysis, we focus on the transcripts preferentially expressed in the IFN-R–/– mice (Table III; N.B.: this table is organized as wild-type vs IFN-R–/–, and values are shown as negative numbers so as to distinguish them clearly from those presented in Table I). Transcripts that are preferentially expressed in PVM-infected IFN-R–/– mice are in effect suppressed by one or more aspects of IFN-R-mediated signaling. Most prominent among these IFN-suppressed transcripts are those encoding the CC chemokines eotaxin-2 and TARC (confirmed by Q-RT-PCR and ELISA, respectively; Table IV) and mEar 11 (confirmed by Q-RT-PCR; Fig. 3A). Despite its name, mEar 11 is also produced by lung macrophages (30) and belongs to a large cluster of homologous ribonucleases that are produced in response to inflammation (30, 31, 32). The 20 ± 3-fold increase in mEar 11 transcript (day 6 postinoculation, infected IFN-R–/– vs infected wild type) correlates with a 
5-fold increase in mEar protein (Fig. 3B) and 3-fold increase in RNase activity (Fig. 3C). Although TARC is known as a chemoattractant for Th2 CD4+ cells, and mEar 11 is produced in lung macrophages in response to Th2 stimulus, no elevations in Th2 cytokines IL-4 or IL-5 were detected either on gene microarray or by ELISA (Table IV). This can be contrasted to the findings of Durbin et al. (33) who reported induction of Th2 cytokines in response to respiratory syncytial virus in Stat 1–/– mice and concomitant eosinophilic inflammation in lung tissue.
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Examples of characteristic lung pathology of wild-type (IFN-R+/+) and gene-deleted (IFN-R–/–) mice on day 6 postinoculation are shown in Fig. 4. We observe multifocal acute alveolitis with intra-alveolar edema in the parenchymal lung tissue of wild-type mice (Fig. 4, A and C), with occasional hemorrhage and moderate granulocytic infiltrates throughout. This is accompanied by bronchiolar cell hyperplasia and occasional necrosis of epithelial lining cells. In contrast, the IFN-R–/–-infected mice display a more pronounced multifocal bronchiolitis with infiltration by a mixed population of inflammatory cells (Fig. 4, B and D). This is also associated with perivascular infiltration with margination of leukocytes frequently appreciated. Edema was not as prominent as in the wild-type infected mice.
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R–/– mice (Table V). Among the recovered leukocytes, we observed an increased proportion of eosinophils (from 3 to 9%) and mononuclear leukocytes (from 4 to 24%) when compared with samples obtained from the wild-type mice.
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R–/–, 2.0 ± 0.41%; n = 5; no significant difference). However, the proportions of CD4+ and CD8+ T cells differed significantly (Fig. 5). Among the wild-type mice, 39.1 ± 3.5% of the CD3+ T cells were CD4+, and 40.5 ± 1.7% were CD8+; among the IFN-R–/– mice, 67.8 ± 3.1% of the CD3+ T cells were CD4+, and 10.3 ± 2.0% were CD8+ (p < 0.01; median values presented). These results are consistent with 1) a limited degree of preferential recruitment of CD4+ T cells in response to TARC and eotaxin-2 (given that there were fewer leukocytes overall (Table V)) and/or 2) a defective primary CD8+ T cell response in the IFN-R–/– mice (34).
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and MIP-2
Given the differential recruitment of leukocytes in response to PVM, we considered the possible contribution of the chemokines MIP-1 and/or MIP-2, despite the absence of evidence of differential expression on gene microarray (Table I). In our previous work, we have demonstrated local production of the CC chemokine MIP-1 and the CXC chemokine MIP-2 in response to PVM infection (23, 35). Although MIP-1-mediated inflammatory response blocks virus replication, and CCR1 gene-deleted mice tend to survive somewhat longer in response to moderate virus inocula (23), in the end, the negative sequelae of the MIP-1-mediated inflammatory response are clearly in evidence. Furthermore, we have shown that genetic and/or biochemical blockade of MIP-1 or its receptor, CCR1, eliminates the granulocytic inflammatory response characteristic of this infection (19, 20). As shown in Tables I and III, we observed no differential expression of transcripts encoding MIP-1 or of MIP-2 on gene microarray. Furthermore, both chemokines were detected in mouse lung homogenates, but no differential expression was observed (Table VI). Thus, neither MIP-1 nor MIP-2 expression can explain the differential pathology observed between the wild-type and IFN-R–/– gene-deleted strains of mice in response to PVM infection.
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R–/– mice
Virus replication was determined qualitatively by Western blotting of clarified lung homogenates probed with rabbit polyclonal anti-PVM N nucleoprotein (Fig. 6A) and quantitatively by Q-RT-PCR of lung RNA from PVM-infected wild-type (IFN-R+/+) and gene-deleted (IFN-R–/–) mice (Fig. 6B). The Western blot demonstrates a time-dependent increase in intensity of the band representing PVM N protein with increased intensity on days 3–5 postinoculation among the IFN-R–/– mice. Q-RT-PCR was performed to determine a more precise copy number, using the SH transcript as a target. No virus RNA was detected in mice before inoculation (Fig. 5B) or directly after inoculation with 60 PFU (data not shown). Virus recovery on day 6 was 3.0 ± 1.4 x 104 copies/µg lung RNA from wild-type mice, and 9.5 ± 1.2 x 104 copies/µg lung RNA for the IFN-R–/– mice (*, p < 0.05).
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A total of 30 age- (±3 days) and gender-matched wild-type (IFN-R+/+) and gene-deleted (IFN-R–/–) mice (15 per genotype) were compared for survival analysis (Fig. 6C). The first deaths occurred on day 7 postinoculation, which were two of the wild-type group; the wild-type group reached 100% mortality by day 12, with 50% survival calculated at 9.0 ± 0.3 days. In contrast, the first death among the gene-deleted (IFN-R–/–) mice did not occur until day 8, and the group included one long-term survivor (regained lost weight and maintained vigor through day 40, seroconversion documented). Using day 18 as the cutoff for the long-term survivor, 50% survival of the IFN-R–/– mice was calculated at 10.8 ± 0.6 days (p < 0.02). Thus, the IFN-R–/– mice have a clear survival advantage over their wild-type counterparts. Although seemingly paradoxical, we know from previous work that the inflammatory response to pneumovirus infection plays a crucial role in this infection (19, 20). It is likely that differential activation of the inflammatory response accounts for the paradoxical survival observed here.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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R+/+) and IFN-R-deficient (IFN-R–/–) mice, we demonstrate receptor-dependent transcriptional activation of many of the classic IFN response genes, including those of the antiviral cascade (multiple Oas genes, PRKR, Mx1). PVM replication in vivo was clearly diminished by the actions of the IFN response genes, demonstrating the fact that pneumoviruses are not resistant to the effects of endogenous type I IFNs in vivo. Molecular dissection of the bRSV and hRSV pathogens has suggested a role for the nonstructural NS-1 and NS-2 proteins (7, 8, 9, 10) in antagonizing the activities of the IFN-mediated antiviral proteins. However, Mohapatra and colleagues (36) have recently demonstrated that administration of small interfering RNA silencing respiratory syncytial virus NS1 expression results in reduced virus replication both in tissue culture and in vivo. Further RNA silencing and/or reverse genetics approaches might provide insight into the roles of the PVM nonstructural NS-1 and NS-2 proteins and their interactions with the IFN-mediated antiviral state.
We observe a small but significant survival differential, which, seemingly paradoxically, proves to be advantageous for the gene-deleted IFN-R–/– mice. This might be puzzling were it not for our earlier observations on the crucial contributions of the antiviral inflammatory responses associated with this disease (19, 20, 23). The microscopic pathology suggested distinct differences in the nature of the antiviral inflammatory response among the wild-type (IFN-R+/+) and gene-deleted (IFN-R–/–) mice, the former characterized by a more edematous pattern with neutrophils predominating, and the latter, one with an overall reduced inflammatory response, and preferential recruitment of eosinophils and mononuclear leukocytes. The chemokine MIP-1, which elicits primarily neutrophils, is not differentially expressed in wild-type vs IFN-R–/– mice, but we do observe reduced expression of the chemokines monocyte chemotactic protein-5 and IP-10 and preferential expression of eotaxin-2 (CCL-24), TARC (CCL-17), and the proinflammatory enzyme mEar 11 in IFN-R–/– gene-deleted mice. Eotaxin-2 is a chemoattractant for eosinophils (37), and TARC, for T cells, primarily memory effector CD4+, with a preference for Th2 cells (38). Mouse Ear11 is a member of the highly divergent mouse ribonuclease cluster that is orthologous to the eosinophil-derived neurotoxin/RNase 2 gene of the RNase A superfamily. The RNase A superfamily is a highly divergent, multilineage enzyme family that is unique to vertebrate species (39). Despite its name, mEar 11 is found not only in eosinophils, but in lung macrophages in response to allergic (Th2) stimuli (30) (although we observed no Th2 cytokines present in lung tissue in response to this infection model; see Table IV). Mouse Ear 11 is absent at homeostasis, but has been detected in response to allergic, biochemical, and now microbial provocation in lung tissue. Among its potential functions, the paralogous gene, mEar 2, is a chemoattractant for mouse dendritic cells in vitro and in vivo (40), and related RNase A ribonucleases have antiviral activity in vitro (41).
The apparent differences in cellular recruitment are particularly intriguing, given the complex nature of the inflammatory response to this infection. It would be interesting to elucidate the signals that elicit the cellular inflammatory responses in the IFN-R–/– mice, and to determine what the unique relationship there might be between IFN-R-mediated signaling and the differential control of cellular inflammation. Based on the chemoattractant properties, the differential expression of these mediators may be causally associated with the recruitment of eosinophils and mononuclear leukocytes, and/or the differential recruitment/responses of T cell subsets observed among IFN-R–/–-infected mice.
Although future work will define the relationships of these transcripts and their protein products to the inflammatory response observed in the IFN-R–/– mice, as a group, they represent a novel set of IFN-suppressed genes, or transcripts expressed in response to virus infection only in the absence of IFN-R-mediated signaling. There are other examples of interactions between type I IFN signaling and innate/acquired immunity–specifically, induction of MHC I (41, 42) and RANTES (11, 12, 13), but in direct response to IFN- and IRF-3, respectively. In the situation here, expression of atypical proinflammatory genes correlates with improved survival in the setting of an otherwise fatal pneumovirus infection in IFN-R–/– mice. Of particularly intriguing interest–how might expression genes that would otherwise be suppressed in the presence of an intact IFN-signaling mechanism, particularly proinflammatory mediators, alter the disease outcomes in infections caused by viruses that have acquired the ability to antagonize IFN production and/or signaling at the receptor? Our work suggests an unexplored interaction between IFN receptor-mediated signaling and the antiviral inflammatory response that can now be dissected at the molecular level.
Of perhaps even greater interest–how can we exploit these findings to develop novel anti-inflammatory therapies for severe respiratory infections? Using the PVM as a model for the more severe forms of hRSV, we have already shown that the use of antivirals alone is insufficient, and only when antivirals are coupled with anti-inflammatory (or, more specifically, carefully crafted immunomodulatory therapy directed against the chemokine MIP-1 and/or its receptor CCR1) can we alter the course of disease and effect significant reductions in morbidity and mortality (reviewed in Ref.35). From this work, we observe that an alteration of the inflammatory milieu–including overexpression of the cytokines TARC, eotaxin-2, and potentially mEar11, and/or reduced expression of monocyte chemotactic protein-5 and IP-10–may alter the course of an otherwise fatal pneumonia by altering the nature of the inflammatory response. This warrants further exploration from a therapeutic perspective.
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Acknowledgments |
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R+/– mice on the C57BL/6 background, Drs. James Lee and Nancy Lee (Mayo Clinic, Scottsdale, AZ) for the anti-mEars polyclonal Ab, Dr. Andrew Brooks (Functional Genomics Center, Rochester, NY) for executing the gene microarray experiments, and the staff of the Building 14BS animal facility (National Institute of Allergy and Infectious Diseases) for care of the mice used in these experiments. |
Disclosures |
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Footnotes |
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1 This work was supported by National Institute of Allergy and Infectious Diseases, Division of Intramural Research (to H.F.R.), and a Scientist Development grant from the American Heart Association (to J.B.D.). 
2 Address correspondence and reprint requests to Dr. Helene F. Rosenberg, Building 10, Room 11N104, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892. E-mail address: hrosenberg{at}niaid.nih.gov
3 Abbreviations used in this paper: hRSV, human respiratory syncytial virus; bRSV, bovine respiratory syncytial virus; IRF, IFN-response factor; PVM, Pneumonia virus of mice; IP, IFN-induced protein; TARC, thymus and activation-regulated chemokine; MDC, macrophage-derived chemokine; Q-RT-PCR, quantitative RT-PCR.
Received for publication April 6, 2005. Accepted for publication July 18, 2005.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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B. A. Austin, W. P. Halford, B. R. G. Williams, and D. J. J. Carr Oligoadenylate Synthetase/Protein Kinase R Pathways and {alpha}beta TCR+ T Cells Are Required for Adenovirus Vector: IFN-{gamma} Inhibition of Herpes Simplex Virus-1 in Cornea J. Immunol., April 15, 2007; 178(8): 5166 - 5172. [Abstract] [Full Text] [PDF]
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