IL-33 markedly activates murine eosinophils by an NF-κB-dependent mechanism differentially dependent upon an IL-4-driven autoinflammatory loop

Abstract

Eosinophils are major effector cells in type 2 inflammatory responses and become activated in response to IL-4 and IL-33, yet the molecular mechanisms and cooperative interaction between these cytokines remain unclear. Our objective was to investigate the molecular mechanism and cooperation of IL-4 and IL-33 in eosinophil activation. Eosinophils derived from bone marrow or isolated from Il5-transgenic mice were activated in the presence of IL-4 or IL-33 for 1 or 4 h, and the transcriptome was analyzed by RNA sequencing. The candidate genes were validated by quantitative PCR and ELISA. We demonstrated that murine-cultured eosinophils respond to IL-4 and IL-33 by phosphorylation of STAT-6 and NF-κB, respectively. RNA sequence analysis of murine-cultured eosinophils indicated that IL-33 induced 519 genes, whereas IL-4 induced only 28 genes, including 19 IL-33-regulated genes. Interestingly, IL-33 induced eosinophil activation via two distinct mechanisms, IL-4 independent and IL-4 secretion/autostimulation dependent. Anti-IL-4 or anti-IL-4Rα Ab-treated cultured and mature eosinophils, as well as Il4- or Stat6-deficient cultured eosinophils, had attenuated protein secretion of a subset of IL-33-induced genes, including Retnla and Ccl17. Additionally, IL-33 induced the rapid release of preformed IL-4 protein from eosinophils by a NF-κB-dependent mechanism. However, the induction of most IL-33-regulated transcripts (e.g., Il6 and Il13) was IL-4 independent and blocked by NF-κB inhibition. In conclusion, we have identified a novel activation pathway in murine eosinophils that is induced by IL-33 and differentially dependent upon an IL-4 auto-amplification loop.

Figure 1. Bone marrow-derived eosinophils respond to IL-33 and IL-4 but not IL-13

Hematoxylin/eosin staining of bone marrow-derived eosinophils at Day 14 of culture (magnification x400) (A). Characterization of eosinophils by the co-expression of Siglec-F and CCR3, using flow cytometry analysis (B). Western blot of p-NFκB and NFκB from eosinophils (4x10⁶/ml) treated with 100 ng/ml of IL-33 for 5 or 30 minutes (C). Flow cytometry of intracellular staining of p-p38 or p-STAT-6 from eosinophils (4×10⁶/ml) treated with 100 ng/ml of IL-33 for 5 minutes (D) or 100ng/ml of IL-4 or IL-13 for 30 minutes (E). The results shown are representative of 3 independent experiments.

Figure 2. Transcriptome analysis in eosinophils activated by IL-33 or IL-4

Eosinophils were treated for 1 or 4 hours with IL-4 or IL-33 at 10 ng/ml. RNA was sequenced by RNA-sequencing technology. Heatmap represents the genes differentially regulated by IL-4 and IL-33 after 1 and 4 hours of exposure (A). Venn diagram represents the number of genes (with a fold change ≥ 2 in either direction) differentially regulated by IL-4 (red), IL-33 (green), or both (grey) (B). Validation by qRT-PCR analysis of genes identified by RNA sequencing as upregulated in eosinophils activated by IL-4 or IL-33 at 1 or 4 hours (C). Bars represent the mean of 2 wells and the error bars represent the SEM values. Data are representative of 3 independent experiments.

Figure 3. Effect of IL-33 and IL-4 on cytokine/chemokine expression by eosinophils

Eosinophils (4×10⁶/ml) were activated for 24 hours in the presence of the indicated concentrations of IL-33 or IL-4 (IL-4 at 100 ng/ml for A and B). Supernatants were collected and IL-6 and IL-13 (A), CCL17(B), IL-4 (C), and RELM-α (E) were measured by ELISA. qRT-PCR analysis represents the kinetics of Il6, Ccl17, and Il4 induction by IL-33 in eosinophils (D). Bars (A-C, E) or symbols (D) represent the mean of 2 wells, and error bars represent the SEM values. Flow cytometry on eosinophils after overnight incubation with the different cytokines at 100 ng/ml to evaluate SiglecF+/RELM-α+ cells (F). The percentage of cells in each quadrant is indicated (F). Data are representative of 3 independent experiments. ND, not detected. **p < 0.01, ***p < 0.001.

Figure 4. IL-33 requires the IL-4 autocrine loop to induce RELM-αand CCL17

Wild-type (WT) or Il4^−/− eosinophils (4×10⁶/ml) treated for 24 hours with IL-4 (10 ng/ml, A) or IL-33 (10 ng/ml, A–D). RELM-α, CCL17, IL-6, and IL-13 levels in the supernatants were determined by ELISA (A–C). qRT-PCR from wild-type (WT) or Il4^−/− eosinophils (4×10⁶/ml) incubated for 4 hours in the presence of IL-33 (10 ng/ml); graph represents the fold change of gene expression in IL-33-treated eosinophils compare to untreated (D). Eosinophils (4×10⁶/ml) were activated for 4 hours (F) or 24 hours (E, G and H) by IL-33 (10 ng/ml) in the absence or presence of IgG1 (isotype control) or anti-IL-4 antibody (10 μg/ml). Gene expression of Cxcl2, Cxcl10, Clec4e, Il6, Il13, Adora2b, Ccl17, Tfec, and Retnla was analyzed by qRT-PCR (F). The levels of IL-4 (E), RELM-αand CCL17 (G), and IL-6 and IL-13 (H) in the supernatants were measured by ELISA. Bars represent the mean of 2 wells and the error bars represent the SEM values. Data are representative of 3 independent experiments. ND, not detected; ns, not significant. *p < 0.05, **p < 0.01, ***p < 0.001, § p=0.055 and # p=0.083.

Figure 5. Mature eosinophils respond to IL-33 and require IL-4 to induce RELM-αand CCL17

Eosinophils from Il5-transgenic mice were activated 24 hours with IL-33 in the presence of anti-IL-4 antibody or isotype control (IgG1). IL-4, RELM-α, CCL17, IL-6 and IL-13 released in the supernatants were measured by ELISA. *p < 0.05, **p<0.001, ns, not significant.

Figure 6. IL-4Rα, but not IL-13Rα1, is involved in IL-33-induced eosinophil activation

Eosinophils (4×10⁶/ml) activated for 4 hours (A) or 24 hours (B) by IL-33 (10 ng/ml) in the absence or presence of IgG2a (isotype control) or anti-IL-4Rαantibody, at 10 μg/ml. Gene expressions of Cxcl2, Cxcl10, Clec4e, Il6, Il13, Adora2b, Ccl17, Tfec, and Retnla were analyzed by qRT-PCR (A). The levels of RELM-α, CCL17, IL-4, IL-6, and IL-13 (B) in the supernatants were measured by ELISA. Wild-type (WT) or Il13ra1^−/− eosinophils (4×10⁶/ml) were treated for 24 hours in the presence of IL-33 (10ng/ml), and the levels of RELM-α, CCL17, IL-13, and IL-4 in the supernatants were measured by ELISA (C). Bars represent the mean of 2 wells and the error bars represent the SEM values. Data are representative of 3 independent experiments. ND, not detected; ns, not significant. *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 7. Role of STAT-6 in eosinophil activation

Wild-type (WT) and Stat6-deficient eosinophils were activated with IL-33 (100 ng/ml) or IL-4 (100 ng/ml). RELM-α (A), CCL17 (B), IL-4, IL-6, and IL-13 (C) released in the supernatants were measured by ELISA. Bars represent the mean of 2 wells and the error bars represent the SEM values. Data are representative of 3 independent experiments. ND, not detected. **p < 0.01, ***p < 0.001.

Figure 8. Effect of NFκB inhibitor in the regulation in IL-33-induced gene expression

Eosinophils (4×10⁶/ml) were pretreated for 1 hour with NFκB inhibitor (BAY11-7082 [BAY]) at 5 μM and then cultured for 4 hours in the presence of IL-33 (AB) or IL-4 (A) at 10 ng/ml. Gene expression of Cxcl2, Cxcl10, Clec4e, Il6, Il13, Adora2b, Ccl17, Tfec, and Retnla were analyzed by qRT-PCR (A), and IL-4 in the supernatant was measured by ELISA (B). Bars represent the mean of 2 wells and the error bars represent the SEM values. Data are representative of 3 independent experiments. **p < 0.01, ***p < 0.001, ****p < 0.0001 (A–B).

Figure 9. Proposed model summarizing our results

After binding to its receptor, IL-33 induces eosinophil activation, as measured by gene transcription and release of several mediators, through the phosphorylation of NFκB. Il6, Il13, Cxcl2, Cxcl10, and Clec4e are directly induced by IL-33 (IL-4-independent pathway). Although IL-4 transcription is not increased after IL-33 treatment, the protein release of preformed IL-4 is dependent on the IL-33/ST2 signaling pathway. Retnla, Ccl17 and Tfec induction by IL-33 is indirect and requires the activation of the IL-4/IL4Rα pathway (IL-4-dependent pathway) via the phosphorylation of STAT-6. This figure was produced using Servier Medical Art (www.servier.com).