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. 2014 Nov:468-470:140-149.
doi: 10.1016/j.virol.2014.07.039. Epub 2014 Aug 28.

Sustained inflammation and differential expression of interferons type I and III in PVM-infected interferon-gamma (IFNγ) gene-deleted mice

Stephanie F Glineur  1 , Aaron B Bowen  1 , Caroline M Percopo  1 , Katia E Garcia-Crespo  1 , Kimberly D Dyer  1 , Sergei I Ochkur  2 , Nancy A Lee  2 , James J Lee  2 , Joseph B Domachowske  3 , Helene F Rosenberg  1
Affiliations

Sustained inflammation and differential expression of interferons type I and III in PVM-infected interferon-gamma (IFNγ) gene-deleted mice

Stephanie F Glineur et al. Virology. 2014 Nov.

Abstract

Interferon gamma (IFNγ) has complex immunomodulatory and antiviral properties. While IFNγ is detected in the airways in response to infection with the pneumovirus pathogen, pneumonia virus of mice (PVM; Family Paramyxoviridae), its role in promoting disease has not been fully explored. Here, we evaluate PVM infection in IFNγ(-/-) mice. Although the IFNγ gene-deletion has no impact on weight loss, survival or virus kinetics, expression of IFNβ, IFNλ2/3 and IFN-stimulated 2-5' oligoadenylate synthetases was significantly diminished compared to wild-type counterparts. Furthermore, PVM infection in IFNγ(-/-) mice promoted prominent inflammation, including eosinophil and neutrophil infiltration into the airways and lung parenchyma, observed several days after peak virus titer. Potential mechanisms include over-production of chemoattractant and eosinophil-active cytokines (CXCL1, CCL11, CCL3 and IL5) in PVM-infected IFNγ(-/-) mice; likewise, IFNγ actively antagonized IL5-dependent eosinophil survival ex vivo. Our results may have clinical implications for pneumovirus infection in individuals with IFNγ signaling defects.

Keywords: Eosinophils; Inflammation; Interferon; Pneumovirus.

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Figures

Figure 1
Figure 1. IFNγ gene-deletion has no impact on survival or virus replication and clearance in response to acute PVM infection
(A) Survival of IFNγ−/− and wild-type BALB/c mice inoculated on day 0 with 0.033 (diamonds; n = 8 – 9 mice per group), 0.33 (squares; n = 15 – 17 mice per group) or 3.3 TCID50 units (circles; n = 5 mice per group) of pneumonia virus of mice (PVM strain J3666). (B) Body weight (g ± sd, n = 5 mice per group until day 9) after inoculation with 0.33 TCID50 units PVM per mouse on day 0. (C) IFNγ and (D) CXCL9/MIG are detected in lung tissue homogenates of wild-type, but not IFNγ-gene deleted mice inoculated with 0.033 TCID50 units PVM strain J3666 at day 0. (E) Virus replication and clearance evaluated by dual-standard curve qRT-PCR [Gabryszewski et al., 2011] in whole lung tissue of IFNγ−/− and wild-type mice inoculated with 0.033 TCID50 units PVM strain J3666 at day 0; “a.” indicates peak virus titer at day 7, and “b.” indicates that virus can no longer be detected at day 21 post-inoculation; n = 6–10 mice per time point.
Figure 2
Figure 2. Differential expression of type I and type III interferons in response to PVM infection in wild-type and IFNγ−/− mice
(A) Immunoreactive IFNα is detected in both PVM-infected wild-type and IFNγ−/− mice. (B) Differential expression of transcripts encoding IFNβ and (C) IFNλ2/3 in PVM-infected wild-type and IFNγ−/− mice. Mice were inoculated with 0.033 TCID50 units PVM strain J3666; n= 6–10 mice per time point. Shown are the means ± SEM, ***p < 0.001, two-way ANOVA with Bonferroni’s multiple comparisons test. (D) Differential expression of transcripts encoding IFNβ and (E) IFNλ2/3 in PVM-infected wild-type and IFNαβR−/− mice; n = 5 mice per point.
Figure 3
Figure 3. Differential expression of interferon-stimulated genes (ISGs) in response to PVM infection in wild-type and IFNγ−/− mice
Differential expression of transcripts encoding (A) Oas 1g (B) Oas2 (C) Oas3 and (D) Mx1 in PVM-infected wild-type and IFNγ−/− mice. Mice were inoculated with 0.033 TCID50 units PVM strain J3666; n = 4 – 8 mice per time point. Shown are the means ± SEM, **p < 0.01, ***p < 0.001, two-way ANOVA with Bonferroni’s multiple comparisons test.
Figure 4
Figure 4. IFNγ gene-deletion augments histopathology observed in response to non-lethal PVM infection
(A) Lung histopathology was scored as described by Hardy and colleagues [2009] as described in the Methods; shown are the means ± SEM, ***p < 0.001, two-way ANOVA with Bonferroni’s multiple comparisons test. (B) Lung tissue from IFNγ−/− mice on day 10 post-inoculation, in which lesions are significantly more profound and diffuse than those seen in (C) lung tissue from wild-type mice (original magnifications, 5x); (D) likewise, more profound cellular infiltration is observed in lung tissue from PVM-infected IFNγ−/− mice when compared to a corresponding section from (E) PVM-infected wild-type mice (H&E staining, original magnifications, 40x), all mice inoculated with 0.033 TCID50 units PVM strain J3666.
Figure 5
Figure 5. Granulocytes are recruited to the airways and lung parenchyma of IFNγ gene-deleted mice
(A) Percent eosinophils and (B) percent neutrophils detected in the bronchoalveolar lavage (BAL) fluid of PVM-infected IFNγ−/− and wild-type mice; n= 6 – 10 mice per time point; in (A), examples of eosinophils in BAL fluid (modified Giemsa stain), as indicated by arrows in the inset. (C) Percent eosinophils (SiglecF+CD11c−) and (D) percent neutrophils (Gr1+) recruited in the lung tissues of PVM-infected IFNγ gene-deleted and wild-type mice; cells were evaluated by flow cytometry, n = 7 mice pooled for each time point, data from 2 independent experiments. (E) Eosinophil degranulation evaluated by detection of immunoreactive eosinophil peroxidase (EPX) in BAL fluid, measured as eosinophil equivalents per µL [Ochkur et al. 2012]. EPX detected in BAL fluid in wild-type and IFNγ−/− mice are compared to results obtained from mice sensitized and challenged with Aspergillus fumigatus alone (Af), and mice sensitized and challenged and subsequently infected with PVM (Af + PVM), the latter set of conditions shown to promote eosinophil degranulation [Percopo et al., 2014]. Mice were inoculated with 0.033 TCID50 units PVM strain J3666. Shown are the means ± SEM, ***p < 0.001, two-way ANOVA with Bonferroni’s multiple comparisons test.
Figure 6
Figure 6. Chemoattractant and eosinophil-active cytokines detected preferentially in lung tissue of PVM-infected IFNγ gene-deleted mice
(A) CCL11/eotaxin-1; (B) CXCL1 / KC; (C) CCL3/MIP-1a; (C) IL-5; n = 6 – 10 mice per time point. Shown are the means ± SEM, ***p < 0.001, **p < 0.01, two-way ANOVA with Bonferroni’s multiple comparisons test.
Figure 7
Figure 7. IFNγ antagonizes IL-5-mediated survival of mouse eosinophils
(A) Eosinophils (t = 0 are day 12 cultures, 95–100% eosinophils) generated from bone marrow of (A) wild-type mice (bmEos) and (B) IFNγR gene-deleted mice (IFNγR−/− bmEos) with IL-5 alone (10 ng/mL), with IFNγ alone (100 ng/mL), or with IL-5 (10 ng/mL) and IFNγ (100 ng/mL) together; *p < 0.05. Shown are representatives of n = 3 experiments, each run in triplicate.
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