Interleukin-17-producing innate lymphoid cells and the NLRP3 inflammasome facilitate obesity-associated airway hyperreactivity

Abstract

Obesity is associated with the development of asthma, which is often difficult to control. To understand the immunological pathways that lead to obesity-associated asthma, we fed mice a high-fat diet for 12 weeks, which resulted in obesity and the development of airway hyperreactivity (AHR), a cardinal feature of asthma. This AHR was independent of adaptive immunity, as it occurred in obese Rag1(-/-) mice, which lack B and T cells, and was dependent on interleukin-17A (IL-17A) and the NLRP3 inflammasome, as it did not develop in obese Il17a(-/-) or Nlrp3(-/-) mice. AHR was also associated with the expansion of CCR6(+) type 3 innate lymphoid cells (ILCs) producing IL-17A (ILC3 cells) in the lung, which could by themselves mediate AHR when adoptively transferred into Rag2(-/-); Il2rg(-/-) mice treated with recombinant IL-1β. Macrophage-derived IL-1β production was induced by HFD and expanded the number of lung ILC3 cells. Blockade of IL-1β with an IL-1 receptor antagonist abolished obesity-induced AHR and reduced the number of ILC3 cells. As we found ILC3-like cells in the bronchoalveolar lavage fluid of individuals with asthma, we suggest that obesity-associated asthma is facilitated by inflammation mediated by NLRP3, IL-1β and ILC3 cells.

Figures

Figure 1. High fat diet feeding induces AHR in wildtype mice

(a–c). Wild type mice fed normal chow or 60% HFD, starting at weaning for 12–14 weeks. a. Comparison of bodyweight gain in wild-type C57BL/6 mice on normal chow or the HFD. ***p<0.001, HFD group compared to chow fed group. (Student’s t-test) b. Representative picture of mice and organs after chow or HFD feeding. Scale bar indicates 5mm c. Wild-type mice develop AHR, as assessed by the response to increasing doses of methacholine. Results represent the changes in lung resistance (RL) as a measure of AHR. **p<0.01, and ***p<0.001, compared to chow fed group. (Two-way ANOVA). Data are representative of at least three independent experiments. Error bars represent SEM. d. 7 wks old ob/ob mice fed on normal chow diet develop AHR. Results represent the changes in lung resistance (RL) as a measure of AHR. *p<0.05, and **p<0.01, compared to wild type mice. (Two-way ANOVA).

Figure 2. HFD induced AHR requires the presence of IL-17A

a. Cells were isolated from lungs of mice fed on normal chow or on the HFD, and supernatants were analyzed for IL-17A by ELISA. b. Comparison of bodyweight gain between WT and Il17−/− mice on either chow or HFD. NS, WT mice on HFD vsIl17−/− mice on HFD (Student’s t-test). c. AHR was measured using mice represented in Fig. 2b. Graph represents the changes in lung resistance (RL). *p<0.05, and **p<0.01, Il17−/− mice on HFD compared to WT mice on HFD (Two-way ANOVA). d. Total lung cells from mice fed normal chow or HFD were sorted for CD4+ or CD4− cell fraction and analyzed for Il17A mRNA expression by qRT-PCR. ***p<0.001 (chow CD4− vs HFD CD4− fraction), and *p<0.05 (chow CD4+ vs HFD CD4+ fraction), (Student’s t-test). e. ILC3 cells (CD45+Lineage−IL-17A+cells) from HFD fed WT mice (upper panel) and normal chow fed ob/ob mice (lower panel). Graph (right) represents total number of ILC3 cells in the lungs (upper panel; wild type chow vs HFD, ***p<0.001, lower panel; chow fed wild type vsob/ob, **p<0.01). f. ILC3 cells in Figure 2e were further characterized. Grey histograms represent isolype control staining. g. The changes in lung resistance (RL) as a measure of AHR in Rag1−/− mice fed a high fat diet. *p<0.05, and **p<0.01, comparing normal chow fed and high diet fed groups (Two-way ANOVA). h. ILC3 cells in the lungs of Rag1−/− mice. Graph (right) represents total number of ILC3 cells (chow vs HFD groups, ***p<0.001).

Figure 3. IL-1β production and M1 macrophages are increased in the lungs of obese mice

(a–f). Total RNA was extracted from the lung and adipose tissue for qRT-PCR. Fold induction of Il1b (a,d), Il6 (b,e), and Il23 (c,f) were calculated based on GAPDH expression. *p<0.05, and **p<0.01, mRNA expression from HFD group was compared to chow group. g. Expression of M1/M2 macrophage markers, CD206 (M2) and CD11c (M1), were analyzed after gating on of CD45+F4/80+ cells. Graph represents the total numbers of macrophages in the lung (left, *p<0.05 and ***p<0001) and adipose tissue (right, ***p<0.001). (Student’s t-test) h. IL-1β production from M1/M2 macrophages. Cells were taken from lung or adipose tissue and IL-1β producing M1 (CD11c+, left panel) or M2 (CD206+, right panel) macrophages were assessed by flow cytometry.

Figure 4. HFD increases NLRP3, which is required for AHR

a. Total RNA was extracted from the lung, liver and adipose tissue and assessed for Nlrp3. Fold induction of Nlrp3 mRNA was calculated based on GAPDH expression. **p<0.01 and ***p<0.001, mRNA level from mice fed on HFD compared to chow group. (Student’s t-test) b. Graph represent grams of weight gain in WT or Nlrp3−/− mice. **p<0.01, WT mice on HFD compared to Nlrp3−/− mice on HFD group. (Student’s t-test) c. WT and Nlrp3−/− mice 3 months after HFD (left). Representative pictures of lung, liver and epididymal adipose tissue from Nlrp3−/− mice fed on chow or HFD (right). Scale bar indicates 5mm d. Development of obesity induced AHR was compared in WT and Nlrp3−/− mice. Graph represents the changes in lung resistance (RL). *p<0.05, Nlrp3−/− mice on HFD were compared to WT mice on HFD group (Two-way ANOVA). e. IL-1β levels in the culture supernatant of lung cells from WT and Nlrp3−/− mice. The data shown are means ± SEM. **p<0.01, supernatants from HFD group were compared to chow group. (Student’s t-test) f. Lungs were taken from chow or HFD fed mice and stimulated with PMA/ionomycin for 5 hr. −IL-17A+ ILC3 cells in WT mice (upper panel) or Nlrp3−/− mice (lower panel) were assessed by FACS. Graph represents the total number of ILC3 cells in the lung. ***p<0.001, ILC3 cells in WT mice were compared to Nlrp3−/− mice. (Student’s t-test).

Figure 5. IL-17 producing innate lymphoid cells are required for the development of AHR

Graphs represent the changes in lung resistance (RL) (upper panels) and BAL fluid cell counts (lower panels) after rIL-1β (a) or rIL-1α (b) treatment. For AHR, **p<0.05, and ***p<0.001, rIL-1β or rIL-1α treated mice were compared to saline treated mice (Two-way ANOVA). For BAL fluid, **p<0.01, and ***p<0.001, rIL-1β vs saline (a), **p<0.01, rIL-1α vs saline (b) (Student’s t-test). c. Changes in lung resistance (RL) (upper panel) and BAL fluid cell counts (lower panel) after depletion of ILCs. For AHR, **p<0.01, rIL-1β treated mice were compared to anti-Thy1.2 mAb treated mice (Two way ANOVA). For BAL fluid, **p<0.01, and ***p<0.001, rIL-1β vs anti-Thy1.2 mAb+rIL-1β (Student’s t-test). d. ILC3 cells in the lung (left panel), and total number of ILC3 cells in each group (right panel). **p<0.01, rIL-1β treated mice were compared to anti-Thy1.2 mAb treated mice (Student’s t-test). e. Changes in lung resistance (RL) in Rag2−/−Il2rγ−/− mice after adoptive transfer of ILC3 cells (Lin−CCR6+). ***p<0.001, comparing Rag2−/−Il2rγ−/− mice treated with rIL-1β receiving or not receiving ILC3 cells (Two-way ANOVA). Cells in BAL fluid (right panel). *p<0.05, and ***p<0.001, ILC3 cells +rIL-1β vs rIL-1β group (Student’s t-test). f. IL-17A and IL-13 production from adoptively transferred ILC3 cells. g. Changes in lung resistance (RL) in Il4/Il13−/− and Il17−/− mice after rIL-1β treatment. *p<0.05, and **p<0.01, rIL-1β treated Il4/Il13−/− mice were compared to rIL-1β treated Il17−/− mice (Two way- ANOVA). Cells in BAL fluid (right panel). ** p<0.01, and ***p<0.001, rIL-1β treated Il4/Il13−/− mice were compared to rIL-1β treated Il17−/− mice (Student’s t-test). h. IL-1R expression on ILC3 cells. Lin−IL-17+ cells (red) express higher level of IL-1R compared to Lin−IL-17− cells (blue). Shaded histogram: isotype control. i. Cells were taken from rIL-1β treated Rag2−/− mice then cultured in vitro to induce ILC3 cells.

Figure 6. Treatment of obese mice with anti-IL-1, prevents AHR

a. Schedule of feeding and anti-IL1R antagonist treatment. To block IL-1β (and IL-1α), obese mice were treated with IL1R antagonist (50 mg/kg) subcutaneously for 7 days before measuring AHR. b. AHR was measured 24hr after last IL1R antagonist treatment. Graph represents the changes in lung resistance (RL). ***p<0.001, IL1R antagonist treated obese mice were compared to HFD fed obese mice. (Two-way ANOVA). c. ILC3 cells were analyzed after PMA/Ionomycin stimulation (left panel). Graph represents the percentage of IL-17A producing ILC3 cells of the total lung lymphocytes in each group (right panel). d. Cells from BAL fluid were cultured in vitro in the presence of rIL-2, rIL-7 and rIL-1β for 72hr. To measure IL-17 production from Lin− population, cells were re-stimulated with PMA and ionomycin for 5hr. Graph represents the percentage of IL-17 producing CD45+Lin− lymphocytes in each group (bottom), *p<0.05 (Student’s T-test). e. Possible mechanisms of obesity induced AHR. High fat diet results in the activation of the NLRP3 inflammasome, through fatty acids or cholesterol crystals in macrophages in adipose tissue and in the lungs. This results in IL-1β production, which drives the development of ILC3 cells in the lungs. Treatment with IL-1R antagonist blocks the effects of IL-1β (and IL-1α), and blocks the development of pulmonary ILC3 cells.