Early-life heterologous rhinovirus infections induce an exaggerated asthma-like phenotype

Abstract

Background: Early-life wheezing-associated respiratory tract infection by rhinovirus (RV) is a risk factor for asthma development. Infants are infected with many different RV strains per year.

Objective: We previously showed that RV infection of 6-day-old BALB/c mice induces a mucous metaplasia phenotype that is dependent on type 2 innate lymphoid cells (ILC2s). We hypothesized that early-life RV infection alters the response to subsequent heterologous infection, inducing an exaggerated asthma-like phenotype.

Methods: Wild-type BALB/c mice and Rora^fl/flIl7r^cre mice lacking ILC2s were treated as follows: (1) sham on day 6 of life plus sham on day 13 of life, (2) RV-A1B on day 6 plus sham on day 13, (3) sham on day 6 plus RV-A2 on day 13, and (4) RV-A1B on day 6 plus RV-A2 on day 13.

Results: Mice infected with RV-A1B at day 6 and sham at day 13 showed an increased number of bronchoalveolar lavage eosinophils and increased expression of IL-13 mRNA but not expression of IFN-γ mRNA (which is indicative of a type 2 immune response), whereas mice infected with sham on day 6 and RV-A2 on day 13 of life demonstrated increased IFN-γ expression (which is a mature antiviral response). In contrast, mice infected with RV-A1B on day 6 before RV-A2 infection on day 13 showed increased expression of IL-13, IL-5, Gob5, Muc5b, and Muc5ac mRNA; increased numbers of eosinophils and IL-13-producing ILC2s; and exaggerated mucus metaplasia and airway hyperresponsiveness. Compared with Rora^fl/fl mice, Rora^fl/flIl7r^cre mice showed complete suppression of bronchoalveolar lavage eosinophils and mucous metaplasia.

Conclusion: Early-life RV infection alters the response to subsequent heterologous infection, inducing an intensified asthma-like phenotype that is dependent on ILC2s.

Keywords: Asthma; IL-13; ILC2; RV-A1B; RV-A2; childhood; early-life; rhinovirus; trained immunity; type 2 innate lymphoid cell.

Figure 1.. Heterologous RV infection induces exaggerated airway responses.

A) Baby mice were inoculated with sham or RV-A1B or on day 6 of life and sham or RV-A2 on day 13 of life. B) Lungs from sham + sham, sham + RV-A1B, sham + RV-A2, and RV-A1B + RV-A2-infected mice were harvested on day 20 of life. Shown is lung mRNA expression of IL-5, IL-13, IL-5, IFN-γ, Gob5 (Clca1), Muc5b, Muc5ac, TNF-ɑ and IL-12b. C) BAL inflammatory cell counts for sham + sham, sham + RV-A1B, sham + RV-A2, and RV-A1B + RV-A2-infected mice. Data shown are mean ± SEM; n=3-9/group from two different experiments; *different from sham + sham, †different from RV-A1B + sham, P < 0.05 by one-way ANOVA and Tukey multiple comparison test. D) RV positive-strand RNA was measured on days 14, 15 or 20 of age (1, 2 or 7 days post RV-A2 infection), and presented as viral copy number in total lung. Data shown are mean±SEM, N =4-9, *different from day-6-sham + day-13-RV-A2, p<0.05, one-way ANOVA and Tukey multiple comparison test. E) mRNA expression in male and female mice.

Figure 2.. Heterologous RV infection induces exaggerated mucus metaplasia.

Baby mice were inoculated with sham or RV-A1B on day 6 and sham or RV-A2 on day 13 of life. Lungs were harvested on day 20 and processed for histology. Lungs sections were stained for PAS and Muc5ac and quantified using NIH ImageJ. A) PAS staining in sham, RV-A1B, RV-A2, RV-A1B + RV-A2-infected mice. The black bar is 50 microns (μ). B) Muc5ac staining in RV-A1B, RV-A2, RV1B + RV-A2-infected mice. The white bar is 50 μ. C) Quantification of Muc5ac staining in the airways. Data are represented are Muc5ac-positive cells per micron of basement membrane length. Images were taken at ×200 magnification. Data shown are mean ± SEM; n=3-4 airways/mouse, 4 mice per group from two different experiments; *different from sham + sham, †different from RV-A1B + sham, P < 0.05 by one-way ANOVA and Tukey multiple comparison test.

Figure 3.. RV infection induces ILC2 expansion.

A) Figure showing flow cytometry analysis of live lineage-negative, CD25+ CD127+ ILC2s. B) Graph showing group mean data for ILC2s. Data shown are mean ± SEM; n=7 per group from three different experiments; *different from sham + sham, †different from RV-A1B + sham, P < 0.05 by one-way ANOVA and Tukey multiple comparison test. C) Flow cytometry analysis of lineage markers and IL-13 in sham, RV-A1B, RV-A2 and RV-A1B + RV-A2 groups. D) Lineage-negative, IL-13+ cells (left panel) and lineage-positive, IL-13+ cells (right panel) for the four groups. Data are mean ± SEM; n=3 per group from one experiment; *different from sham + sham, †different from RV-A1B + sham, P < 0.05 by one-way ANOVA and Tukey multiple comparison test. E) IL-13 production from sorted lineage-negative, CD25+, CD127+ ILC2s. ILC2s were stimulated with either PMA and ionomycin (left panel) or IL-33 (right panel). Data shown are mean ± SEM; n = 9-10 per group from three experiments for PMA and ionomycin stimulation; n=3 per group from one experiment for IL-33 stimulation; *different from sham + sham, †different from RV-A1B + sham, P < 0.05 by one-way ANOVA and Tukey multiple comparison test

Figure 4.. Airway immunofluorescence staining for IL-13, GATA3 and CD3.

Baby mice were inoculated with sham or RV-A1B on day 6 of life and sham or RV-A2 on day 13 of life. On day 20, lungs were harvested and fixed with paraformaldehyde. A) Lung sections were stained with anti-IL-13 (shown in red) anti-GATA3 (green), anti-CD3 (blue) and DAPI (black). Areas of IL-13 and GATA3 colocalization appear yellow; areas of GATA3 and CD3 colocalization appear cyan (if IL-13-negative) or white (if IL-13-positive). The white bar is 50 μ. B) Color breakdown of areas outlined in panel A. Solid box outlines IL-13+ GATA3+ CD3-negative cells; dashed box outlines IL-13+ GATA3+ CD3+ cells. The white bar is 10 μ. C) Group mean data showing IL-13+ GATA3+ CD3-negative (open bars) and CD3+ (shaded bars). Each point represents the average of four airways from one mouse. Data shown are mean ± SEM; *different from sham + sham, †different from RV-A1B + sham, P < 0.05 by one-way ANOVA and Tukey multiple comparison test.

Figure 5.. Effect of prior RV-A1B infection on the response to heterologous infection with RV-A2.

Baby mice were inoculated with sham or RV-A1B on day 6 of life and sham or RV-A2 on day 13 of life. On days 14 and 15, i.e., 24 and 48 h after RV-A2 infection, lungs were harvested for mRNA analysis by qPCR or lung immunofluorescence staining. A) mRNA expression of CXCL1, CXCL2, IL-13, IL-5, CCL11 and CCL24. Gene expression values were normalized to GAPDH and represented as fold increase over sham + sham inoculated mice. Data are mean ± SEM; n=6 per group from two different experiments; *different from sham + RV-A2, P < 0.05 by one-way ANOVA and Tukey multiple comparison test. B) Lung sections were stained with anti-CCL11 (shown in green; the white bar is 50 μ).

Figure 6.. Rora^fl/flIl7r^cre ILC2-deficient mice are protected from heterologous infection induced exaggerated type 2 inflammation and mucous metaplasia.

A) Flow cytometry analysis of lineage-, CD25+, CD127+ ILC2s. N=2-4 per group B) BAL inflammatory cell counts and C) mRNA expression in sham + sham, RV-A1B + sham, sham + RV-A2 and RV-A1B + RV-A2-infected Rora^fl/fl and Rora^fl/flIl7r^cre ILC2-deficient mice. For panels B and C, N=3-6 per group. For panels A-C, data are mean ± SEM; *different from sham + sham group of like mouse strain, †different from RV-A1B + sham of like mouse strain, ‡different from corresponding group in Rora^fl/fl mouse strain, P < 0.05 by one-way ANOVA and Tukey multiple comparison test. D). Viral copy number in RV-A1B + RV-A2-infected Rora^fl/fl and Rora^fl/flIl7r^cre ILC2-deficient mice. E. PAS-stained representative airways from RV-A1B + RV-A2-infected Rora^fl/fl mice (left) and Rora^fl/flIl7r^cre ILC2-deficient mice (right). The black bar is 50 μ. F) Changes in total respiratory system resistance in response to inhaled methacholine in anesthetized, tracheotomized Rora^fl/fl and Rora^fl/flIl7r^cre mice. Resistance data were normalized to baseline airways resistance. Data are mean ± SEM for 3-10 per group measured in 3 different experiments, *different from Rora^fl/fl mice, P < 0.05 by two-way ANOVA and Tukey multiple comparison test.