Lung CD103+ dendritic cells restrain allergic airway inflammation through IL-12 production

Abstract

DCs are necessary and sufficient for induction of allergic airway inflammation. CD11b+ DCs direct the underlying Th2 immunity, but debate surrounds the function of CD103+ DCs in lung immunity and asthma after an allergic challenge. We challenged Batf3-/- mice, which lacked lung CD103+ DCs, with the relevant allergen house dust mite (HDM) as a model to ascertain their role in asthma. We show that acute and chronic HDM exposure leads to defective Th1 immunity in Batf3-deficient mice. In addition, chronic HDM challenge in Batf3-/- mice results in increased Th2 and Th17 immune responses and exacerbated airway inflammation. Mechanistically, Batf3 absence does not affect induction of Treg or IL-10 production by lung CD4+ T cells following acute HDM challenge. Batf3-dependent CD103+ migratory DCs are the main source of IL-12p40 in the mediastinal lymph node DC compartment in the steady state. Moreover, CD103+ DCs selectively increase their IL-12p40 production upon HDM administration. In vivo IL-12 treatment reverts exacerbated allergic airway inflammation upon chronic HDM challenge in Batf3-/- mice, restraining Th2 and Th17 responses without triggering Th1 immunity. These results suggest a protective role for lung CD103+ DCs to HDM allergic airway inflammation through the production of IL-12.

Keywords: Immunology; Inflammation.

Figure 1. Batf3^–/– mice lack CD103⁺ cDC1s in the lung and mLN and show a reduced CD8α⁺ cDC1 compartment in mLN.

(A) HDM sensitization and challenge regime (acute protocol). (B) Gating strategy for lung Batf3-dependent DCs. (C) Staining of lung CD103⁺ and CD11b⁺ DCs in mice treated as indicated in A. Left: representative plots. Right: percentage of CD103⁺ cDC1s in the CD11c⁺MHCII⁺ compartment in the lung. (D) HDM administration protocol to evaluate early DC response following allergen challenge. (E) Gating strategy to identify migratory (mig) and resident (res) DCs in mLNs. (F and G) Res-DCs (F) and mig-DCs (G) in mLNs from mice i.n. challenged with 100 μg HDM. Left: representative plots. Right: percentage of Batf3-dependent DCs in the CD11c⁺MHCII⁺ compartment in the mLN. (C, F, and G) Individual data (n = 3–4 PBS, n = 6–7 HDM) and mean ± SEM of 1 representative experiment of 3 performed. *P < 0.05, ***P < 0.001, 1-way ANOVA followed by Bonferroni’s post-test. HDM, house dust mite; mLN, mediastinal lymph nodes.

Figure 2. Batf3 deficiency does not impact on airway inflammation following acute HDM exposure.

(A) Inflammatory infiltrates in BAL of HDM-sensitized and -challenged Batf3^–/– and WT mice (n = 3 PBS, n = 7 HDM). (B) Top: representative H&E and PAS staining in formalin-fixed lung sections. Scale bars: 200 μm (PAS) or 100 μm (H&E). Bottom: inflammation and mucus score (n = 4 PBS, n = 6 HDM). (C and D) Serum HDM-specific IgG1 (C) and IgG2a (D) determined by ELISA (n = 5 PBS, n = 10 HDM). (A–D) Individual data and mean ± SEM of 1 representative experiment of at least 3 performed. ***P < 0.001, Mann Whitney U test. HDM, house dust mite; PAS, periodic acid Schiff.

Figure 3. Batf3^–/– mice show impaired local and systemic IFN-γ production upon acute HDM challenge.

mLNs (A–D) and splenocytes (E–H) from HDM-sensitized and HDM-challenged mice were restimulated with HDM, and released cytokines were measured by ELISA. Individual data and mean ± SEM from a representative experiment of at least 3 performed (n = 3 PBS, n = 7 HDM); *P < 0.05, ***P < 0.001, Mann Whitney U test. HDM, house dust mite; mLN, mediastinal lymph node.

Figure 4. Batf3^–/– mice develop exacerbated HDM-induced chronic airway inflammation.

(A) Schematic regime of chronic HDM exposure. (B) Inflammatory infiltrates in BAL from the indicated treatments and genotypes (n = 3 PBS, n = 7 HDM). (C) Representative histological staining of mice of the indicated genotype chronically exposed to HDM. Top: H&E staining (arrowheads point at perivascular and peribronchial infiltration) and PAS staining (insets depict mucus-producing goblet cells). Bottom: staining for collagen (Sirius red). Scale bars: 200 μm (PAS-PBS or H&E) or 100 μm (PAS-HDM or Sirius red). Right: inflammation score (n = 5 PBS, 9 HDM). (D–F) Total serum IgE (D) and HDM-specific IgG2a (E) and IgG1 (F) (n = 5 PBS, n = 10 HDM). (B–F) Individual data and mean ± SEM from 1 experiment representative of at least 3 independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001, Mann Whitney U test. HDM, house dust mite; PAS, periodic acid Schiff.

Figure 5. Enhanced Th2 and Th17 immunity upon chronic HDM challenge in Batf3^–/– mice.

mLNs (A–D) and splenocytes (E–H) from mice challenged over 6 weeks with HDM (3 × 10 μg doses per week) were restimulated with HDM, and released cytokines were measured by ELISA. Individual data and mean ± SEM from a representative independent experiment of at least 2 performed (n = 3 PBS, n = 7 HDM); *P < 0.05, **P < 0.01, ***P < 0.001, Mann Whitney U test. HDM, house dust mite; mLN, mediastinal lymph node.

Figure 6. Batf3-dependent DCs are neither required for lung Treg expansion nor necessary for IL-10 production by lung CD4⁺ T cells, following HDM airway exposure.

Lungs from WT and Batf3 ^–/– mice sensitized and challenged with HDM were collected at day 14. (A) Representative staining for FoxP3⁺CD25⁺ Tregs, either depicted as plots (left) or scatter plot graphs (right), representing frequency and number of cells expressing FoxP3 and CD25 (n = 3–4 PBS, n = 6–7 HDM). (B and C) Frequencies of Tregs expressing Helios and ICOS (n = 3–4 PBS, n = 7 HDM). (D) Lung cells were restimulated with anti-CD3 and anti-CD28 for 6 hours in the presence of Brefeldin A for the last 4 hours, and CD4⁺CD44⁺ T cells were analyzed for intracellular IL-10; representative plots (left) and percentages and numbers (right) are shown (n = 4 PBS, n = 6 HDM). (A–D) Individual data and mean ± SEM from a representative independent experiment of 3 performed. HDM, house dust mite.

Figure 7. CD103⁺ mig-DCs are the main DC source of IL-12 in mLNs after HDM exposure.

Mice were challenged with 100 μg HDM i.n., and mLNs were collected 3 days later. (A) IL-12p40 and (B) IL-6 mRNA expression was analyzed in purified CD11c⁺ cells, and mRNA was normalized against β-actin. Data shown (mean ± SEM) is a pool of 3 independent experiments; each symbol represents 1 experiment (5–10 mice pooled per experiment); **P < 0.01, 2-tailed Student’s t test. (C–E) mLN cells were stained for CD11c, MHC class II, CD103, CD11b, and intracellular IL-12p40. (C) Numbers of IL-12p40–producing cells in the mLN CD11c⁺MHCII^hi fraction. (D) Representative plots showing IL-12p40 staining in CD103⁺ and CD11b⁺ mig-DCs. (E) Frequencies (left) and numbers (right) of IL-12p40–producing mig-DC subsets. (C and E) Individual data and mean ± SEM from a representative independent experiment of 3 performed (n = 4 PBS, n = 7 HDM); **P < 0.01, ***P < 0.001, 1-way ANOVA followed by Bonferroni’s post-test (C) and Mann Whitney U test (A and E). HDM, house dust mite; mLN, mediastinal lymph node.

Figure 8. Intranasal administration of IL-12 dampens allergic airway inflammation in Batf3^–/– mice chronically exposed to HDM.

WT and Batf3^–/– mice were challenged for 6 weeks with HDM, as depicted in Figure 4A. Batf3^–/– mice subjected to this chronic HDM challenge were treated or not with IL-12p70 (i.n.), as indicated in Methods. (A) Inflammatory infiltrates in BAL (n = 4 PBS, n = 7 HDM). (B) Total serum IgE was determined in serum by ELISA (n = 7 PBS, n = 13 HDM). mLNs (C–E) and splenocytes (F–H) were restimulated with HDM, and the indicated released cytokines were measured by ELISA (n = 6 PBS, n = 13 HDM). One representative experiment (A) or a pool of 2 independent experiments (B–H) of 3 performed is shown. Symbols denote individual mice, and lines represent mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, Mann Whitney U test. HDM, house dust mite; mLN, mediastinal lymph node.

Figure 9. Proposed function of pulmonary CD103⁺ DCs in HDM-induced asthma.

Upon HDM exposure, CD103⁺ cDC1s migrate from the lung to the mLNs, where they produce IL-12 that restrains Th2 and Th17 immune responses and contributes to Th1 differentiation. Batf3-dependent DCs might, thus, play a protective role in HDM-induced airway inflammation, dampening the classical features of asthma, including eosinophilia, mucus secretion, and IgE production.