Rhinovirus infection of allergen-sensitized and -challenged mice induces eotaxin release from functionally polarized macrophages

Abstract

Human rhinovirus is responsible for the majority of virus-induced asthma exacerbations. To determine the immunologic mechanisms underlying rhinovirus (RV)-induced asthma exacerbations, we combined mouse models of allergic airways disease and human rhinovirus infection. We inoculated OVA-sensitized and challenged BALB/c mice with rhinovirus serotype 1B, a minor group strain capable of infecting mouse cells. Compared with sham-infected, OVA-treated mice, virus-infected mice showed increased lung infiltration with neutrophils, eosinophils and macrophages, airway cholinergic hyperresponsiveness, and increased lung expression of cytokines including eotaxin-1/CCL11, IL-4, IL-13, and IFN-gamma. Administration of anti-eotaxin-1 attenuated rhinovirus-induced airway eosinophilia and responsiveness. Immunohistochemical analysis showed eotaxin-1 in the lung macrophages of virus-infected, OVA-treated mice, and confocal fluorescence microscopy revealed colocalization of rhinovirus, eotaxin-1, and IL-4 in CD68-positive cells. RV inoculation of lung macrophages from OVA-treated, but not PBS-treated, mice induced expression of eotaxin-1, IL-4, and IL-13 ex vivo. Macrophages from OVA-treated mice showed increased expression of arginase-1, Ym-1, Mgl-2, and IL-10, indicating a shift in macrophage activation status. Depletion of macrophages from OVA-sensitized and -challenged mice reduced eosinophilic inflammation and airways responsiveness following RV infection. We conclude that augmented airway eosinophilic inflammation and hyperresponsiveness in RV-infected mice with allergic airways disease is directed in part by eotaxin-1. Airway macrophages from mice with allergic airways disease demonstrate a change in activation state characterized in part by altered eotaxin and IL-4 production in response to RV infection. These data provide a new paradigm to explain RV-induced asthma exacerbations.

Figures

Figure 1. OVA/RV treated mice show increased tissue eosinophils and macrophages in response to RV infection

Wild type BALB/c mice were sensitized intraperitoneally with endotoxin-free OVA and alum on days 1 and 7, and challenged intranasally on days 14, 15, and 16 with OVA. Controls were treated with PBS. Mice were inoculated with RV1B or sham (HeLa cell supernatant) on day 16. Mouse lungs were harvested 1, 2 and 4 after infection. Lungs were digested for 1 h in Type IV collagenase in serum free RPMI. Strained cells were treated with RBC lysis buffer, spun and enriched for leukocytes with 40% Percoll. Resulting pellets were resuspended in PBS and total cell count determined. Cytospins of leukocytes were stained with Diff-Quik and differential cell count determined for 200 cells. Time courses for tissue neutrophils (A), lymphocytes (B), eosinophils (C) and macrophages (D) are shown. (N=4-5 mice per group, bars represent mean±SEM, *different from respective sham group, †different from respective PBS group, P<0.05, one-way ANOVA.)

Figure 2. RV infection of OVA-sensitized and -challenged mice increases cytokine production

Twenty-four h after sham or RV infection, lung BAL fluid was centrifuged at 1500g and the resulting supernatant subjected to multiplex immunoassay. Results are shown for eotaxin-1/CCL-11 (A), IL-4 (B), IL-13 (C) and IL-5 (D). (N=5 mice per group, bars represent mean ± SEM, *different from respective sham group, p<0.05; †different from respective PBS group, P<0.05 one-way ANOVA.)

Figure 3. RV infection of OVA-sensitized and -challenged mice increases cytokine production four days after infection

Lung BAL fluid was centrifuged at 1500g and the resulting supernatant subjected to multiplex immunoassay. Results are shown for eotaxin-1/CCL11 (A and D), IL-4 (B) and IL-13 (C). cDNA for eotaxin-1/CCL-11 was synthesized using reverse transcriptase and subjected to quantitative real time PCR employing a Taqman probe. (N=5 mice per group, bars represent mean ± SEM, *different from respective sham group, p<0.05; †different from respective PBS group, P<0.05 one-way ANOVA.)

Figure 4. RV infection of OVA-sensitized and -challenged mice induces eotaxin-1-mediated airways cholinergic responsiveness

A. Mice were anesthetized and endotracheally intubated, and changes in respiratory system resistance to nebulized methacholine measured using the FlexiVent system (Scireq, Montreal, CA). Four days after infection, RV-infected OVA mice demonstrated significantly higher airways responses than all other groups at methacholine doses of 10 and 20 mg/ml. B. Measurement of viral copy number from lungs of PBS/RV and OVA/RV treated mice 1 day post infection. OVA/RV treatment significantly reduced viral copy number by 1 log. (N= 5 mice per group, bars represent mean ± SEM, *different from respective sham group, p<0.05; †different from respective PBS group, P<0.05 one-way ANOVA.) C. Selected RV-infected, OVA-sensitized and -challenged mice were given two systemic injections of rabbit anti-mouse eotaxin-1. Additional mice were treated with the isotype control antibody. Mice given anti-eotaxin displayed reduced tissue eosinophils 4 days after infection. D. Neutralizing antibody and isotype control-treated mice were administered increasing doses of aerosolized methacholine. Treatment with anti-eotaxin-1 significantly reduced airway cholinergic responsiveness compared to the IgG-treated group. (Bars represent mean ± SEM, *different from respective sham group, †different from IgG group, P<0.05, one-way ANOVA.)

Figure 5. RV colocalizes with CD68+ macrophages, eotaxin-1, and IL-4 in OVA-sensitized and -challenged mice

A. Lungs were formaldehyde fixed overnight, paraffin embedded, sectioned at 5 μm and incubated with a 1:2500 dilution of donkey-anti-mouse eotaxin-1 (Santa Cruz Biotechnology, CA) or isotype control IgG. Eotaxin was identified by DAB staining. Following OVA/RV treatment, eotaxin-1 localization is noted in macrophages (arrows) and eosinophils (arrowheads) but not in the airway epithelium (line segment = 50 μm). B-E. OVA/RV lung sections were co-stained with antiserum against RV1B which was directly conjugated to AF-594 (red), while CD68 was conjugated to AF-633 (far red, shown in blue). Secondary antibody to eotaxin-1 was conjugated to AF-488 (green). Cells with colocalization (white) are designated by arrows and a high magnification view is shown in panel F. Original magnification, 600X. G. RV infection of CD68-positive cells in the airway lumen and epithelial layer. In this panel, RV anti-serum was directly conjugated to AF-594 (red), while CD68 was conjugated to AF488 (green). Colocalization is yellow. Colocalization in the epithelium suggests infiltration by a macrophage. H. Sections incubated with secondary antibodies alone showed no staining. I-M. RV co-localizes with IL-4 in CD68-positive cells. RV and CD68 were conjugated with AF-594 (red), and AF-633 (shown in blue) respectively. There is some blue background staining of elastin in the epithelial basement membrane. IL-4 was directly conjugated to AF-488 (green). M. Co-localization of RV, IL-4, and CD68 is white. Original magnification, 400X.

Figure 6

Macrophage-depleted OVA-treated mice show reduced airway eosinophils and hyperresponsiveness following RV infection. Clodronate or PBS-containing liposomes were instilled into the trachea 24 h after the last OVA challenge. Twenty-four h following macrophage depletion, mice were inoculated with sham or RV and harvested 24 h after infection. Lung digests were performed as described in Figure 1. Differential counts were determined. Cytospins of leukocytes were stained with Diff-Quik and differential cell count determined for 200 cells. Macrophages (A), eosinophils (B), neutrophils (C) and lymphocytes (D) are shown. E. Airway resistance for each group was measured following treatment with 0, 10, and 20 mg/ml methacholine. (N=4 mice per group, bars represent mean±SEM, *different from respective sham group, †different from OVA/RV/ PBS liposome group, P<0.05, one-way ANOVA.)

Figure 7

Clodronate-mediated depletion of macrophages reduces OVA/RV induced cytokine expression. Lung mRNA was extracted and corresponding cDNA subjected to quantitative real time PCR. (N=4 mice per group, bars represent mean±SEM, *different from sham group, †different from OVA/RV/ PBS liposome group, P<0.05, one-way ANOVA.)

Figure 8

Macrophages from OVA-sensitized and -challenged mice show increased cytokine mRNA expression after RV stimulation ex vivo. BAL fluid was extracted from PBS-treated and OVA-sensitized and -challenged mice and seeded in 12-well plates. Cells were allowed to adhere to plates for 90 minutes. Adherent cells were subsequently infected with RV1B, or sham or media (controls). A. Eotaxin-1 expression was observed in adherent BAL cells from OVA-treated but not PBS-treated mice. Eotaxin-1 significantly increased following RV stimulation. B. IL-13. C. TNF-α. D. IL-4. E. IL-10. F. IFN-γ. (N=3-4, bars represent mean±SEM. Because some treatment conditions yielded no detectable mRNA expression, data were normalized to the condition with the lowest detectable mRNA signal. *different from respective sham group, †different from respective UV RV group, §different from respective PBS group, P<0.05, one-way ANOVA.)

Figure 9

Effect of Th2 environment on macrophage polarization. A. OVA sensitization and challenge alters the mRNA expression of macrophage activation markers. Data are fold-increase compared to macrophages from PBS-treated mice (N=3-4, bars represent mean±SEM). B. Effect of IL-4/IL-13 incubation on the eotaxin response to RV infection in macrophages from PBS-treated naïve mice. (N=3, bars represent mean±SEM.of fold increase in mRNA expression compared to control cells, mean±SEM, *P<0.05, one-way ANOVA).