IL-13Ralpha2 and IL-10 coordinately suppress airway inflammation, airway-hyperreactivity, and fibrosis in mice

Abstract

Development of persistent Th2 responses in asthma and chronic helminth infections are a major health concern. IL-10 has been identified as a critical regulator of Th2 immunity, but mechanisms for controlling Th2 effector function remain unclear. IL-10 also has paradoxical effects on Th2-associated pathology, with IL-10 deficiency resulting in increased Th2-driven inflammation but also reduced airway hyperreactivity (AHR), mucus hypersecretion, and fibrosis. We demonstrate that increased IL-13 receptor alpha 2 (IL-13Ralpha2) expression is responsible for the reduced AHR, mucus production, and fibrosis in BALB/c IL-10(-/-) mice. Using models of allergic asthma and chronic helminth infection, we demonstrate that IL-10 and IL-13Ralpha2 coordinately suppress Th2-mediated inflammation and pathology, respectively. Although IL-10 was identified as the dominant antiinflammatory mediator, studies with double IL-10/IL-13Ralpha2-deficient mice illustrate an indispensable role for IL-13Ralpha2 in the suppression of AHR, mucus production, and fibrosis. Thus, IL-10 and IL-13Ralpha2 are both required to control chronic Th2-driven pathological responses.

Figure 1. Th2-polarized SEA-induced airway inflammation.

Animals were sensitized with 10 μg SEA on days 1 and 14, followed by 2 intratracheal airway challenges with 10 μg SEA on days 28 and 31. Animals were euthanized 24 hours after the final airway challenge. *P < 0.05, Mann-Whitney U test. (A) Total airway infiltrates and airway eosinophilia recovered in BAL. (B) BAL fluid eotaxin levels measured by ELISA. (C) BAL fluid IL-5 levels measured by ELISA. (D) Lung tissue mRNA, IFNγ (Th1) and IL-4 (Th2). (E) BAL and lung cells stimulated with PMA and ionomycin with brefeldin A for 3 hours and then stained with anti-CD4 (APC) and anti-IL-13 (PE) before acquisition and analysis by FACS. (F) Five-micrometer sections were cut from paraffin-embedded lung tissue and stained with H&E (lower panels) to show cellular infiltration or alcian blue–PAS (upper panels) showing mucin⁺ goblet cells lining the airways. The mucin response to SEA was 7+, while the inflammatory response to PBS was 2+ and to SEA, 4+. Five lungs from each group were scored in a blinded fashion; original magnification, ×100. (G) Lung cells and BAL recoveries from C57BL/6 IL-10–GFP mice were stained with anti-CD4 (APC) and anti-CD25 (PE) for lung cells, with GFP expression observed in the FL1 channel. (H) RNA was extracted from lung tissue, with IL-13Rα2 mRNA quantified by quantitative reverse transcription PCR (qRT-PCR). (I) Soluble IL-13Rα2 was measured by ELISA, in serum collected from mice bled 24 hours after the last airway challenge.

Figure 2. Airway inflammation, but not AHR, is regulated by IL-10.

Animals were sensitized and challenged as in Figure 1. Animals were euthanized 24 hours after the final airway challenge. *P < 0.05, Mann-Whitney U test. (A) Airway eosinophils (left), neutrophils (right), and macrophages (center) recovered from BAL. (B) BAL fluid IL-5 levels measured by ELISA. (C) BAL fluid eotaxin levels measured by ELISA. (D) AHR. Penh measurements 24 hours after the final airway challenge using a Buxco system with mice exposed to increasing doses of methacholine. n, number of mice in each group. (E) RNA was extracted from lung tissue with Muc5ac (top) and IL-13 (bottom) mRNA quantified by qRT-PCR. (F) RNA was extracted from lung tissue, with IL-13Rα2 mRNA quantified by qRT-PCR. (G) Soluble IL-13Rα2 was measured by ELISA in serum collected from mice bled 24 hours after the last airway challenge.

Figure 3. Exacerbated airway inflammation and airway pathology in IL-10^–/–IL-13Rα2^–/– dKO mice following SEA challenge.

Animals were sensitized and challenged as in Figure 1. Animals were euthanized 24 hours after the final airway challenge. *P < 0.05, 1-way ANOVA, except in Figure 3G, where a Kruskal-Wallis test was used. (A) Total airway infiltrates recovered in BAL. (B) Eosinophils recovered in BAL. (C) Eotaxin levels measured in BAL fluid by ELISA. (D) IL-5 levels measured by in BAL fluid by ELISA. (E) Lungs were excised, broken down into a single-cell suspension, and stimulated with PMA and ionomycin, with brefeldin A, for 3 hours and then stained with anti-CD4 (APC) and anti–IL-13 (PE) before cells were acquired and analyzed by FACS. (F) The procedure in F was identical to E apart from the final staining step using anti–IFN-γ and not anti–IL-13. (G) Perivascular/bronchial inflammation. H&E-stained sections from fixed and sectioned lungs were scored in a blinded fashion for cellular tissue infiltration. (H) Perivascular/bronchial edema. H&E-stained sections were scored in a blinded fashion for signs of edema. (I) RNA was extracted from lung tissue, with gob 5 mRNA quantified by qRT-PCR. (J) As in I, except Muc5ac mRNA was quantified.

Figure 4. Removing IL-13Rα2 in IL-10^–/– mice reveals a role for IL-13Rα2 in controlling goblet cell hyperplasia and mucus production.

Animals were sensitized and challenged as in Figure 1. Animals were euthanized 24 hours after the final airway challenge. Alcian blue–PAS staining of fixed lung sections taken from IL-10^–/– (A) and IL-10^–/–IL-13Rα2^–/– dKO (B) mice; original magnification, ×100. Mucin-containing cells were graded as 2+ in A and 4+ in B. The inflammatory response was 8+ in both photomicrographs.

Figure 5. Removing IL-13Rα2 in IL-10^–/– mice reveals a role for IL-13Rα2 in controlling AHR.

Animals were sensitized and challenged as in Figure 1. Animals were euthanized 24 hours after the final airway challenge. (A) AHR. Penh measurements 24 hours after the final airway challenge using a Buxco system with mice exposed to increasing doses of methacholine. Results from 1 of 3 representative experiments are shown. (B) AHR. R_L measurements 24 hours after the final airway challenge with mice exposed to increasing doses of methacholine. Results from 1 of 2 representative experiments are shown.

Figure 6. Both IL-12 and IL-13Rα2 regulate AHR and goblet cell hyperplasia in the absence of IL-10.

Animals were sensitized and challenged as in Figure 1. *P < 0.05, 1-way ANOVA. (A) Five-micrometer sections were cut from paraffin-embedded lung tissue and stained with alcian blue–PAS. Five lungs from each group were scored in a blinded fashion. (B) AHR. Penh measurements 24 hours after the final airway challenge using a Buxco system with mice exposed to increasing doses of methacholine. Results from 1 of 2 representative experiments are shown.

Figure 7. Recruitment of IL-10–GFP⁺CD4⁺CD25⁺ cells into the liver and increased serum IL-13Rα2 following infection with S. mansoni.

IL-10–GFP reporter (tiger) mice were infected with 30 S. mansoni cercariae and euthanized on indicated weeks after infection. (A) Livers were excised from infected IL-10–GFP reporter mice on the indicated weeks after infection. Tissues were prepared into a single-cell suspension and stained with fluorescently labeled antibody anti-CD4 (APC) and anti-CD25 (PE). (B and C) Serum IL-13Rα2 was measured by ELISA. (D) RNA was extracted from liver tissue, with IL-10 mRNA quantified by qRT-PCR.

Figure 8. Increased granuloma volume and fibrosis in IL-10^–/–IL-13Rα2^–/– dKO mice.

Mice were infected with 30 S. mansoni cercariae and euthanized at week 8 (early) and week 12 (late) during the chronic stages of egg-induced pathology. *P < 0.05, Mann-Whitney U test for single comparisons or 1-way ANOVA for multiple comparisons. Similar numbers of paired adult parasites and eggs were found in the tissues of all groups, so the observed pathological changes were not attributed to differences in parasite burden. (A) Liver tissue was excised, fixed, sectioned, and stained with Wright-Giemsa. Granuloma volume was calculated in a blinded fashion from Giemsa-stained liver sections at weeks 8 (left) and 12 (right) after infection. (B) Liver fibrosis (μmol of hydroxyproline per worm pair) was calculated from liver biopsies taken at weeks 8 (left) and 12 (right) after infection. (C) A survival study was conducted with mice infected with 30 S. mansoni cercariae.