IL-33-mediated IL-13 secretion by ST2+ Tregs controls inflammation after lung injury

Abstract

Acute respiratory distress syndrome is an often fatal disease that develops after acute lung injury and trauma. How released tissue damage signals, or alarmins, orchestrate early inflammatory events is poorly understood. Herein we reveal that IL-33, an alarmin sequestered in the lung epithelium, is required to limit inflammation after injury due to an unappreciated capacity to mediate Foxp3+ Treg control of local cytokines and myeloid populations. Specifically, Il33-/- mice are more susceptible to lung damage-associated morbidity and mortality that is typified by augmented levels of the proinflammatory cytokines and Ly6Chi monocytes in the bronchoalveolar lavage fluid. Local delivery of IL-33 at the time of injury is protective but requires the presence of Treg cells. IL-33 stimulates both mouse and human Tregs to secrete IL-13. Using Foxp3Cre × Il4/Il13fl/fl mice, we show that Treg expression of IL-13 is required to prevent mortality after acute lung injury by controlling local levels of G-CSF, IL-6, and MCP-1 and inhibiting accumulation of Ly6Chi monocytes. Our study identifies a regulatory mechanism involving IL-33 and Treg secretion of IL-13 in response to tissue damage that is instrumental in limiting local inflammatory responses and may shape the myeloid compartment after lung injury.

Keywords: Cytokines; Immunology; Macrophages; Pulmonology; T cells.

Figure 1. IL-33 deficiency increases mortality after chemically induced acute lung injury (ALI).

(A) Weight loss and (B) mortality rate of WT and Il33^–/– B6 mice injected intratracheally (i.t.) with bleomycin (1.5 IU/kg). Data depicted are representative of 2 independent experiments (n = 6–10 mice per group). (C) Representative hematoxylin and eosin (H&E) staining of lung sections from WT and Il33^–/– mice at day 7 after 1.5 IU/kg bleomycin i.t. delivery. (D) Scoring of H&E–stained lung sections collected at day 7 after bleomycin administration for perivascular and peribronchiolar inflammation, fibrosis, intra-alveolar macrophages and hemorrhage, and consolidated parenchyma. Data depicted are from 2 pooled experiments and representative of 3 experiments completed (n = 6 mice per group). Data are the mean ± SD. P values were determined by 2-tailed Student’s t test (A and D), log-rank test (B), or Mann-Whitney test (D, “percent consolidation”). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 2. Local delivery of IL-33 protects Il33^–/– mice from mortality after ALI and reduces the frequency of proinflammatory cytokines and myeloid cells in alveoli.

(A) Survival of WT or IL33^–/– B6 mice injected i.t. with 1.0 IU/kg bleomycin alone or with 1 μg rIL-33. Data are from 2 pooled experiments. (B) At days 7–8 after bleomycin delivery, bronchoalveolar lavage fluid (BALF) from Il33^–/– mice had increased IL-6, G-CSF, LIF, IL-1α, and MCP-1 concentrations, which were significantly reduced by rIL-33 restoration. Data are from 1 experiment representative of 2, and n = 5–6 mice per group. (C and D) Flow cytometric assessment of BALF cells from WT and Il33^–/– treated as in A. (C) Frequencies of alveolar macrophages (CD45⁺CD11b^loSiglec-F⁺) and neutrophils (CD45⁺CD11b^hiSiglec-F^–Ly6G^hi). (D) Frequencies of alveolar Ly6C^hi inflammatory monocytes (CD45⁺CD11b^hiCD11c^loLy6C^hi) and Ly6C^lo immunosuppressive and reparative monocytes (CD45⁺CD11b^hiCD11c^loLy6C^lo). Data are representative of 2 independent experiments, with 5–6 mice per group in each experiment. Data are the mean ± SD. P values were determined by log-rank test (A), or 1-way ANOVA followed by Tukey’s multiple comparisons test (B–D). *P < 0.05; **P < 0.01.

Figure 3. IL-33 increases Tregs that are required for protective functions of IL-33 after ALI.

(A and B) Flow cytometric assessment of CD45⁺CD3⁺CD4⁺CD25⁺ cells isolated from enzymatically digested WT B6 and B6 Il33^–/– mouse lung tissue at day 8 after bleomycin revealed that delivery of IL-33 increased ST2⁺ Tregs in damaged Il33^–/– lung tissue. Data depicted in B are the mean ± SD and are representative of 2 independent experiments, with 4–5 mice per group in each experiment. (C) Survival of B6 Foxp3^DTR mice injected i.t. with bleomycin alone or with rIL-33 (1 μg i.t.) in the presence of Tregs or following their deletion with diphtheria toxin (15 μg/kg on day –3, –2, and –1, and every other day starting from day 1). Depicted data represent 1 experiment with 6–7 mice per group. P values were determined by 1-way ANOVA followed by Tukey’s multiple comparisons test (B) or log-rank test (C). *P < 0.05; **P < 0.01; ***P < 0.001. NS, not significant.

Figure 4. IL-33 stimulates the secretion of IL-13 by murine ST2⁺ Tregs.

(A) Volcano plot depicting RNA-Seq analysis comparing ST2⁺ Tregs stimulated for 6 hours in the presence (condition B) or absence of IL-33 (20 ng/ml) (condition A) revealed increased transcripts for Il10 and Il13 after IL-33 stimulation. (B) qRT-PCR for Il1rl1, Il10, and Il13 expressed by sorted CD4⁺Foxp3⁺ (RFP⁺) ST2^– or ST2⁺ cells from PBS- or IL-33–treated Foxp3-IRES-mRFP reporter mice confirmed RNA-Seq findings. Each column represents 3 mice. Data are from 1 experiment representative of 3 completed. (C–E) Representative flow cytometry plot (C) depicting the sorting strategy used for assessment of cytokine secretion by indicated CD4⁺ T cell populations after culture with or without IL-33 (20 ng/ml). Supernatants were harvested on day 3 of culture and assessed by Cytometric Bead Array (CBA) for (D) IL-10 and (E) IL-13. Statistical analysis with ANOVA was used to establish significance between values indicated. *P < 0.05; **P < 0.01; ****P < 0.0001.

Figure 5. IL-33 expands suppressive human Tregs that secrete IL-13.

(A–D) Human Tregs (CD4⁺CD25^hiCD127^lo) sorted from peripheral blood mononuclear cells (PBMCs) were cultured with allogeneic monocyte-derived dendritic cells (Mono-DCs) and recombinant human IL-2 (300 U/ml) alone or with recombinant human IL-33 (50 ng/ml) added. (A) Cells were counted on the seventh day of culture and mean ± SD fold increase in Tregs depicted. Data represent 8 individual experiments with 18 different Treg and Mono-DC combinations. (B) Ex vivo–expanded Tregs from each culture condition were tested for their ability to suppress Teff proliferative responses to CD40L-activated allogeneic B cells. Treg suppressive function is expressed as percentage suppression of T cell proliferation and the mean ± SD from 5 cultures of expanded Tregs are depicted. (C and D) Supernatants were harvested on day 7 of culture and assessed by Cytometric Bead Array (CBA) for (C) IL-10 and (D) IL-13. Data depicted are the mean ± SD. (E) Mean frequency of IL-13⁺CD4⁺Foxp3^hi cells in cultures with and without IL-33 was determined by intracellular IL-13 and Foxp3 staining of one donor’s Tregs following 7 days of culture with Mono-DCs generated from 2 different donors. Data depict Tregs from 3 different donors and are the mean ± SEM. Statistical significance was determined between each condition by unpaired, 2-tailed Student’s t test. *P < 0.01, **P < 0.01.

Figure 6. Treg expression of IL-13 reduces inflammatory cytokines and myeloid cell infiltration to protect against mortality after ALI.

(A) Survival of B6 Il33^–/– mice instilled i.t. with bleomycin (1.0 IU/kg) and also treated with i.t. PBS (n = 10) or 1 μg recombinant (r) mouse IL-13 (n = 8). (B) Survival of Treg-depleted mice i.t. instilled with bleomycin (1.0 IU/kg) and 1 μg rIL-33 with or without rIL-13 (1 μg). (C) Weight changes of BALB/c Foxp3^Cre mice (n = 7) injected i.t. with bleomycin (6.0 IU/kg) compared with bleomycin-treated Foxp3^Cre × Il4/Il13^fl/fl (n = 8) or PBS-treated Foxp3^Cre mice (n = 3). Data were pooled from 2 separate experiments. (D) Survival of Foxp3^Cre (n = 12) and Foxp3^Cre × Il4/Il13^fl/fl (n = 11) injected i.t. with bleomycin. (E) At day 7 after bleomycin delivery, BALF from Foxp3^Cre × Il4/Il13^fl/fl had increased IL-6, G-CSF, and MCP-1 concentrations. (F) Flow cytometric analysis of BALF cells at day 7 after bleomycin delivery for alveolar macrophages (CD45⁺CD11b^loSiglec-F⁺), neutrophils (CD45⁺CD11b^hiSiglec-F^–Ly6G^hi), and inflammatory monocytes (CD45⁺CD11b^hiCD24^loMHC-II^loLy6C^hi). In E and F, data were pooled from 2 independent experiments (n = 5–6 mice per group total). (G) qRT-PCR data of Arg1 expression in cultures of F4/80-purified BALB/c splenic macrophages (Il1rl1^+/+ or Il1rl1^–/–) incubated for 18 hours under the conditions indicated. Data are from 1 experiment representative of 3 completed. Animal survival was compared by Kaplan-Meier analysis and the log-rank test. Data are the mean ± SD and P values were determined by 2-tailed Student’s t test or 1-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.