Viral induction of a chronic asthma phenotype and genetic segregation from the acute response

Abstract

Paramyxoviral infections cause most of the acute lower respiratory tract illness in infants and young children and predispose to the development of chronic wheezing, but the relationship between these short- and long-term viral effects are uncertain. Here we show that a single paramyxoviral infection of mice (C57BL6/J strain) not only produces acute bronchiolitis, but also triggers a chronic response with airway hyperreactivity and goblet cell hyperplasia lasting at least a year after complete viral clearance. During the acute response to virus, same-strain ICAM-1-null mice are protected from airway inflammation and hyperreactivity despite similar viral infection rates, but the chronic response proceeds despite ICAM-1 deficiency. Neither response is influenced by IFN-gamma deficiency, but the chronic response is at least partially prevented by glucocorticoid treatment. In contrast to viral infection, allergen challenge caused only short-term expression of asthma phenotypes. Thus, paramyxoviruses cause both acute airway inflammation/hyperreactivity and chronic airway remodeling/hyperreactivity phenotypes (the latter by a hit-and-run strategy, since viral effects persist after clearance). These two phenotypes can be segregated by their dependence on the ICAM-1 gene and so depend on distinct controls that appear critical for the development of lifelong airway diseases such as asthma.

Figures

Figure 1

ICAM-1 deficiency protects against virus-induced inflammation. Wild-type (+/+) and ICAM-1–null (–/–) mice were inoculated with SeV (5,000 EID50) and analyzed as follows. Lung sections were immunostained with anti–ICAM-1 (a) or anti-SeV Ab (b) and counterstained with hematoxylin on the indicated postinoculation days. Representative photomicrographs are shown for each genotype (four mice/genotype). Wild-type mice inoculated with PBS or SeV-UV revealed alveolar but not conducting airway epithelial staining for ICAM-1, and incubation of lung tissue with control nonimmune IgG resulted in no signal above background in either genotype (data not shown). Similarly, ICAM-1–null mice exhibited no detectable ICAM-1 staining above background (data not shown). Bar, 20 μm. (c) Bronchoalveolar lavage fluid was subjected to total and differential cell counts. Values represent mean ± SEM for four mice. For a–c, values obtained from +/+ and –/– cohorts inoculated with PBS or UV-inactivated SeV were no different from preinoculation values (data not shown). *Significant decrease compared with the wild-type cohort.

Figure 2

ICAM-1 deficiency protects against weight loss after viral infection without changing viral clearance. Wild-type and ICAM-1–null mice were inoculated with SeV (5,000 EID50) and analyzed as follows. (a) Body weights relative to initial values were determined as mean ± SEM of eight mice. *Significant increase compared with the wild-type cohort. (b) Lungs were subjected to Western blotting against anti-SeV Ab, and bands corresponding to SeV nucleocapsid protein (NP) and the Sp1 control were quantified by densitometry as mean ± SEM of three mice. (c and d) Lungs were also assayed for SeV plaque-forming units (c) and SeV copy number (d). Values for viral plaque-forming units and viral RNA copy number represent mean ± SEM for 1 g of lung tissue and 100 ng of total lung RNA, respectively (three mice/genotype). Viral RNA copy number was determined by real-time RT-PCR for SeV nucleocapsid protein and corrected for GAPDH control. For a–d, values obtained from +/+ and –/– cohorts inoculated with PBS or UV-inactivated SeV were no different from preinoculation values (data not shown).

Figure 3

ICAM-1 deficiency protects against acute but not chronic airway hyperreactivity induced by viral infection. Wild-type or ICAM-1–null mice were assessed for airway reactivity to inhaled methacholine by measurements of Penh at the indicated times before and after inoculation with SeV (5,000 EID50) or an equivalent amount of SeV-UV. Values are provided for baseline (B) and after exposure to vehicle (V) or methacholine (doubling concentrations, 5–160 mg/ml), and each value represents mean ± SEM of eight to nine mice. Values for Penh in cohorts that were inoculated with vehicle alone were no different from those for SeV-UV treatment (data not shown). *Significant increase from control mice that received SeV-UV. **Significant increase from control mice and from SeV-infected ICAM-1–/– mice.

Figure 4

Persistence of goblet cell hyperplasia after viral infection in wild-type and ICAM-1–null mice. (a) Wild-type and ICAM-1–null mice were inoculated with SeV (5,000 EID50) or SeV-UV, and lung sections were stained with PAS and immunostained for MUC5AC mucin. Representative photomicrographs are shown for each genotype (n = 5) at postinoculation day 21. Immunostaining with nonimmune IgG gave no signal above background (data not shown). Bar, 20 μm. (b) Quantification of results shown in a using values for MUC5AC+ cells per mm basement membrane (bm) and for MUC5AC+ staining as percentage of total epithelial area. Values represent mean ± SEM (n = 10 airways from three to five mice). Similar results were found by analysis of staining intensity (data not shown). (c) Wild-type and ICAM-1–null mice were inoculated with SeV or SeV-UV and subjected to analysis on postinoculation day 77 as described in a and b. Representative photomicrographs and corresponding quantification are shown for MUC5AC immunostaining for each genotype (n = 5). Bar, 50 μm. Results for PAS staining were similar to results at postinoculation day 21 (data not shown). (d) Quantification of results shown in c. Values represent mean ± SEM. No significant difference was detected for postinfection values for wild-type versus ICAM-1–null or for postinfection day 21 versus day 77. *Significant increase from control SeV-UV.

Figure 5

IFN-γ–null and wild-type mice exhibit the same acute and chronic responses to viral infection. Wild-type (+/+) and IFN-γ–null (–/–) mice were inoculated with SeV (5,000 EID50) and analyzed as follows. (a) Body weights relative to initial values were determined as mean ± SEM of eight to ten mice per group. No significant difference was found for wild-type versus IFN-γ–null cohorts. (b) Lung sections from IFN-γ–null mice were immunostained with anti-SeV Ab and counterstained with hematoxylin on the indicated postinoculation days. Representative photomicrographs are shown for each genotype (five mice/genotype). Bar, 20 μm. (c) Airway reactivity to inhaled methacholine was assessed at the indicated times before and after inoculation with SeV (5,000 EID50) or SeV-UV as described in Figure 3 legend. Values are shown for the final concentration of methacholine (160 mg/ml) and represent mean ± SEM of nine mice. The same pattern was observed at lower concentrations of methacholine, and the values for Penh in wild-type or IFN-γ–/– cohorts that received no virus were no different from those for SeV-UV (data not shown). *Significant increase compared with control mice that received SeV–UV.

Figure 6

Long-term persistence of goblet cell hyperplasia after viral infection in wild-type and IFN-γ–null mice. (a) Wild-type and IFN-γ–null mice were inoculated with SeV (5,000 EID50) or SeV-UV, and lung sections were stained for PAS and MUC5AC mucin as described in Figure 4 legend. Representative photomicrographs are shown for each genotype and condition (n = 5) at postinoculation day 365. Bar, 20 μm. (b) Quantification of results shown in a. Values represent mean ± SEM. The response of IFN-γ–null mice was no different from that of wild-type mice. *Significant change from control SeV-UV treatment.

Figure 7

Reversibility of airway hyperreactivity and goblet cell hyperplasia after allergen challenge. (a) On the indicated study days, wild-type mice were sensitized to Ova, then challenged with intranasal PBS or Ova either twice on day 0 (Ova ×2) or twice on day 0 and again on days 1 and 2 (Ova ×4), and finally were phenotyped for airway hyperreactivity and goblet cell hyperplasia. (b) For each experimental condition, airway reactivity was assessed as described in legends for Figure 3 and Figure 5. (c) For the same conditions, lung sections were immunostained for MUC5AC mucin, and immunopositive cells were quantified as described in Figure 4 legend. For b and c, all values represent mean ± SEM for groups of eight to ten mice. *Significant change from the corresponding PBS-challenged cohort.

Figure 8

Glucocorticoid suppression of airway hyperreactivity and goblet cell hyperplasia after viral infection in mice. (a) Wild-type mice were inoculated with SeV (5,000 EID50) or SeV-UV and then treated with saline or dexamethasone (0.5 mg/kg per day, given subcutaneously) on postinoculation days 13–21. At the end of this period, levels of airway reactivity were determined as described in Figure 3 legend. (b) For the same conditions described in a, MUC5AC+ cells were determined as described in Figure 4 legend. For a and b, all values represent mean ± SEM. n = 10 mice. *Significant decrease compared with saline-treated cohort. GC, glucocorticoid.