An IL-9-pulmonary macrophage axis defines the allergic lung inflammatory environment

Abstract

Despite IL-9 functioning as a pleiotropic cytokine in mucosal environments, the IL-9-responsive cell repertoire is still not well defined. Here, we found that IL-9 mediates proallergic activities in the lungs by targeting lung macrophages. IL-9 inhibits alveolar macrophage expansion and promotes recruitment of monocytes that develop into CD11c⁺ and CD11c^- interstitial macrophage populations. Interstitial macrophages were required for IL-9-dependent allergic responses. Mechanistically, IL-9 affected the function of lung macrophages by inducing Arg1 activity. Compared with Arg1-deficient lung macrophages, Arg1-expressing macrophages expressed greater amounts of CCL5. Adoptive transfer of Arg1⁺ lung macrophages but not Arg1^- lung macrophages promoted allergic inflammation that Il9r^-/- mice were protected against. In parallel, the elevated expression of IL-9, IL-9R, Arg1, and CCL5 was correlated with disease in patients with asthma. Thus, our study uncovers an IL-9/macrophage/Arg1 axis as a potential therapeutic target for allergic airway inflammation.

Fig. 1.. Deficiency of IL-9R inhibits allergic airway inflammation.

(A), Schematic of HDM extract–induced airway inflammation model. All measurements, unless indicated, are at the end of week 6. (B), Airway resistance was measured in response to methacholine challenge one day after the last HDM treatment (n = 3–4 per group). (C), Lung section with H&E staining. (D–E), BALF total cell number and BALF eosinophil number were analyzed (n = 4–5 per group). (F), Lung eosinophils were analyzed by flow cytometry (n = 3–5 per group). (G), Serum HDM specific IgE level was analyzed by ELISA (n = 4 per group). (H), Lung section with Periodic Acid-Schiff Stain (PAS) staining, arrows showed mucus production. (I) Lung Clca1 mRNA expression was analyzed (n = 3–4 per group). (J), Trachea section with toluidine blue staining, arrows show mast cells. (K), Lung mast cells were analyzed by flow cytometry (n = 3–4 per group). (L), Serum MCPT1 expression was analyzed by ELISA (n = 3–4 per group) (M), IL-9⁺ cells were analyzed in HDM treated Il9 reporter (infer) mice (n=3–8). (N-S), WT or Il9r^−/− bone marrow cells were transferred to lethally irradiated WT or Il9r^−/− mice. After reconstitution, the recipient mice were challenged with HDM as shown in (A). BALF total cell number (O), BALF eosinophils (P), Serum HDM specific IgE (Q), Lung mast cells (R) and serum MCPT1 level was analyzed (n = 4–5). Data are presented as mean ± SEM from two independent experiments. Unpaired two-tailed Student t-test was used for comparison to generate p values in D–G, I, K–L. One-way ANOVA with Tukey’s multiple comparisons was used to generate p values in M, O-S. Two-way ANOVA with Sidak’s multiple comparisons was used to generate p values in B. ns p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ND: Undetected. See also Fig. S1.

Fig. 2.. Lung macrophages are key IL-9 responders in allergic response.

Mice were intranasally treated with HDM three times a week for six weeks. (A and B), Flow cytometry analysis of lung macrophages (n = 3 for WT-PBS and Il9r^−/−-PBS group, n = 6 for WT-HDM group, n = 3 for Il9r^−/−-HDM group). (C), WT (CD45.1) and Il9r^−/− mice (CD45.2) bone marrow cells were mixed in 1:1 ratio and transferred to lethally irradiated recipient mice (CD45.1⁺CD45.2⁺ mice). After reconstitution, chimeric mice were treated with HDM, lung macrophages from donor cells were analyzed by flow cytometry (n = 9). (D–F), Lung cells from PBS- or HDM-treated mice were stained with IL-9R and other surface markers for analysis by flow cytometry. (D), Pie chart analysis of IL-9R⁺ cells. (E), Fluoresence intensity of IL-9R among various populations were analyzed, ΔgMFI is the gMFI of each population minus the gMFI of isotype controls in that population; there were too few IMs for analysis in PBS-treated mice (n = 4–7). (F), Histogram of IL-9R in lung macrophage populations. (G and H), Mice were intranasally treated with IL-9 for three days (G). (H), Lung macrophages were analyzed by flow cytometry (n = 5 per group). (I-K), Mice were treated with HDM for six weeks, anti-IL-9, anti-IL-13 or isotype-matched control antibodies were intravenously injected every other day during the last two weeks (I). (J and K), Lung macrophages were analyzed by flow cytometry (n = 3–5). Data are presented as mean ± SEM from three independent experiments. Unpaired two-tailed Student t-test was used for comparison to generate p values in B, E, H, and J. Paired two-tailed t-tests were used to generate p values in C. *p<0.05, **p<0.01, ****p<0.0001. See also Fig. S2.

Fig. 3.. Heterogeneity of pulmonary macrophages and blood monocytes in allergic inflammation.

(A-G), CITE-seq combined with cell hashing analysis for blood monocytes and lung macrophages from HDM-challenged mice, 15 mice were pooled per group before FACS sorting (A). (B), UMAP showing clusters based on gene expression. (C), UMAP based on cell hashing antibodies. (D), UMAP based on ADT antibodies. (E), Annotation of the lung macrophages within the UMAP based on both transcriptome and surface proteome. (F), Pseudotime analysis using CCR2⁺ blood monocytes as the root cell type. (G), Lung macrophages were sorted from HDM-treated mice and morphology were analyzed by cytospin. (H), Dot plot showing the gene expression in different clusters. The number on the y-axis are the cluster numbers shown in (B). (I), UMAP showing Nos2 expression. (J), Heatmap showing differential expressed genes in different clusters. The number on the top are the cluster numbers shown in (B). (K-L), UMAP showing gene expression of the indicated genes. See also Fig. S3.

Fig. 4.. Lung macrophages amplify IL-9 mediated allergic lung inflammation.

(A), Mice were intranasally treated with HDM three times a week for six weeks. Clodronate was intranasally or intravenously injected to the mice every other day for the last two weeks of the HDM model. (B-F), Mice were treated as described in A. Lung macrophages (B-C) and eosinophils (D) were analyzed by flow cytometry from lavaged lung. Dot plots indicate the percentage of total lung macrophage populations. Bar graphs quantify live lung macrophage populations. (E), Airway resistance was measured in response to methacholine challenge one day after the last dose of HDM treatment. (F), H&E staining of lung sections (n = 3–4 per group). (G-L), Boy/J and Il9r^−/− mice were treated with HDM three times a week for 4 weeks. Macrophage populations were sorted from Boy/J mice one day after the last HDM treatment and intranasally transferred to Il9r^−/− mice followed by HDM challenge every other day for another two weeks (G). (H), Airway resistance was measured in response to methacholine challenge one day after the last HDM treatment at the end of week 6. Statistical differences are indicated between groups in the panel key for clarity. Lung eosinophils (I) and lung donor cells (J) were analyzed by flow. MLN size (K) and cell numbers (L) were analyzed (n = 3–5 per group). Data are presented as mean ± SEM from two independent experiments. One-way ANOVA with Tukey’s correction for multiple comparisons was used to generate p values in I, J and L. Two-way ANOVA with Sidak’s multiple comparisons was used to generate p values in C and D. Two-way ANOVA with Tukey’s multiple comparisons was used to generate p values in H to compare group effect by using mean of every group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See also Fig. S4.

Fig. 5.. IL-9 enhances allergic inflammation via recruiting monocytes.

(A-D), Mice were intranasally treated with HDM three times a week for 6 weeks. Blood (A) and bone marrow (B) monocytes were analyzed (n = 3–5 per group). (C and D), CCR2^lo and CCR2^hi monocytes were gated and IL-9R expression was further analyzed (n = 4–7 per group). (E), Bone marrow monocytes were isolated and plated on the top chamber of a transwell, IL-9 or CCL2 was added to the lower chamber, and cells were allowed to migrate for 3 hours before counting (n = 4 per group). (F-H), Monocytes were isolated from human PBMCs, IL-9R (F) and CCR2 (G) expression was analyzed by flow, migration assay was performed as described in (E), migrated cells were analyzed (H) (n = 4 per group). (I), IL-9R expression were analyzed in non-asthma or asthma donors’ blood CD14⁺ monocytes (n = 3 for non-asthma group, n = 4 for asthma group). (J-M), Mice were treated with HDM three times a week for 5 weeks. CCR2 inhibitor was intravenously injected to the mice twice a week for 4 weeks (J). Bone marrow monocytes (K), lung monocytes (L) and lung macrophages (M) were analyzed by flow cytometry (n = 5 for control group, n = 4 for CCR2 inhibitor group). (N), Fluorescent bead-labeled monocytes were transferred to Il9r^−/− mice, recipient mice were treated with IL-9 and HDM, lung macrophages and eosinophils were analyzed on day 7, dot plots were gated on MerTK⁺ CD64⁺ SiglecF⁻ live cells (n=4 per group). Data are presented as mean ± SEM from two independent experiments. Unpaired two-tailed Student t-test was used for comparison to generate p values in A-B, E, G-I, K-L and N. Two-way ANOVA with Sidak’s multiple comparisons was used to generate p values in C-D and M. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See also Fig. S5.

Fig. 6.. IL-9 impacts lung macrophage function by regulating Arg1 expression.

(A), UMAP showing Arg1 expression from CITE-seq experiment described in Fig. 3. (B), Arg1⁺ cells from HDM-treated mice were analyzed by flow cytometry (n=4 per group). Dot plots were gated on lived IMs. (C), Mixed bone marrow chimeric mice were generated and treated as described in Fig. S2D, donor derived Arg1⁺ IMs were analyzed by flow cytometry (n = 8 per group). (D and E), Arginase activity was analyzed in lung macrophages and serum from HDM-treated mice (n=3–4 per group). (F), Naïve mice were treated with IL-9 for three days, Arg1 production from IMs were analyzed (n=5 for PBS group, n=4 for IL-9 treated group). (G), IMs were sorted from WT HDM-treated mice and stimulated with IL-9 for 24hs, Arg1 mRNA expression was analyzed (n=3). (H-M), YARG and Il9r^−/− mice were treated with HDM three times a week for 4 weeks. YFP^+/−macrophage populations were sorted from YARG mice one day after the last HDM treatment and intranasally transferred to Il9r^−/− mice followed by HDM challenge every other day for another two weeks (H). Lung Arg1⁺ macrophages (I) and YFP⁺ macrophages (J) were analyzed by flow cytometry. (K), Airway resistance was measured in response to methacholine challenge one day after the last HDM treatment at the end of week 6. Statistical differences are indicated between groups in the panel key for clarity. (L), Lung eosinophils were analyzed by flow. (M), MLN cell number was analyzed (n= 3–8 per group). Data are presented as mean ± SEM from two independent experiments. Paired two-tailed t-tests were used to generate p values in B and C. Unpaired two-tailed Student t-test was used for comparison in D–G and L–M. One-way ANOVA with Tukey’s multiple comparisons was used to generate p values in I. Two-way ANOVA with Tukey’s multiple comparisons was used to generate p values in K to comparing group effect by using mean of every group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.001. See also Fig. S6.

Fig. 7.. IL-9 promotes allergic inflammation by inducing CCL5 production from lung macrophages.

Arg1^fl/fl LysM-Cre^+/− mice were treated with HDM for 6 weeks. (A), Arg1 expression in IMs was analyzed. BALF total cell number (B), BALF eosinophils (C), Lung eosinophils (D) and MLN cell number were analyzed (n = 4–10). (F), UMAP showing Ccl5 expression from CITE-seq experiment described in Fig. 3. (G), CCL5 expression in IMs was analyzed by flow cytometry (n = 4–10). (H and I), Serum CCL5 level was analyzed by ELISA (n = 4–10). (J), FACS sorted Arg1^+/− macrophages were treated with IL-9 for 24 hours, CCL5 level was measured by ELISA (n=3). (K-L), Naïve mice were treated with IL-9 for three days, CCL5 and Arg1 expression from IMs were analyzed (n=4). (M), CCL5 expression was analyzed by gating on Arg1⁺ or Arg1⁻ IMs from WT HDM treated mice (n=11). (N), Human monocyte were cultured under M2 macrophage condition, CCL5 mRNA expression was analyzed (n = 3). (O), IL-9 production from PBMCs was analyzed from non-asthma donors and asthma patients (n=7–9). (P), Correlation between FEV1 corrected for height and IL-9 production from patient PBMCs. (Q-R), Arginase activity (Q) and CCL5 level (R) was analyzed from non-asthma donors and asthma patients (n=5–9). (S), Correlation of PBMC IL-9 and serum CCL5 concentration (n=11). Data are presented as mean ± SEM from two independent experiments. Unpaired two-tailed Student t-test was used for comparison in B–C, D–E, H–I, K–L, N–O and Q–R. A paired two-tailed t-test was used to generate the p value in M. Two-way ANOVA with Sidak’s multiple comparisons was used for comparisons in J. Spearman correlation was performed for P and S, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See also Fig. S6.