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IL-33 markedly activates murine eosinophils by an NF-?B-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-?B, 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-4R? 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-?B-dependent mechanism. However, the induction of most IL-33-regulated transcripts (e.g., Il6 and Il13) was IL-4 independent and blocked by NF-?B 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.

SUBMITTER: Bouffi C

PROVIDER: S-EPMC3807853 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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

Bouffi Carine C Rochman Mark M Zust Christopher B CB Stucke Emily M EM Kartashov Andrey A Fulkerson Patricia C PC Barski Artem A Rothenberg Marc E ME

Journal of immunology (Baltimore, Md. : 1950) 20130916 8

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 an ...[more]

PMID: 24043894

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Project description:ObjectiveEosinophils are hallmarks in allergic type 2 inflammation and are known to release cytotoxic granule proteins that contribute to inflammation. Eosinophils develop in the bone marrow from hematopoietic stem cells and once mature, have a limited lifespan in culture, making them difficult to study ex vivo. IL-33 has increasingly been shown as a key regulator of type 2 inflammation via signaling through its receptor, ST2. The present study was conducted to detail a method of eosinophil differentiation from hematopoietic stem cells and determine the response to IL-33.MethodsCD34+ and CD14+ cells were isolated from donor apheresis cones and differentiated into eosinophils or macrophage controls, respectively. Morphologic, transcriptional and protein analyses were performed to validate this method of eosinophil differentiation. The effect of IL-33 on differentiated eosinophils was assessed using qPCR, immunofluorescence, and multiplex cytokine array.ResultsCD34 differentiated eosinophils appear morphologically similar by H&E and express eosinophil peroxidase (EPX) protein as well as the conventional eosinophil transcripts EPX, CLC, and MBP. In addition, differentiated eosinophils expressed both isoforms of the IL-33 receptor, ST2L and sST2 throughout the differentiation process. Transcript levels of both IL-33 receptors were up-regulated by treatment with IL-33 at earlier timepoints in the differentiation. These cells also expressed IL-4 and IL-13 mRNA which were up-regulated by IL-33 as well. Notably, IL-13 expression was significantly higher with IL-33 treatment compared to media control at every timepoint measured. IL-33 significantly increased cellular secretion of IL-13 protein at most timepoints throughout differentiation. IL-8, LIF, CCL1, CCL5, CCL7, and CCL8 were also significantly secreted after IL-33 stimulation.ConclusionsOur findings suggest that CD34 differentiated eosinophils are morphologically and phenotypically similar to peripheral eosinophils. The release of specific cytokines in direct response to IL-33 may contribute to the pathogenesis of type 2 inflammation and facilitates new avenues for studying eosinophils as effector cells in vitro.

Project description:In many types of cancer, presence of eosinophils in tumors correlate with an improved disease outcome. In line with this, activated eosinophils have been shown to reduce tumor growth in colorectal cancer (CRC). Interleukin (IL)-33 has recently emerged as a cytokine that is able to inhibit the development of tumors through eosinophils and other cells of the tumor microenvironment thereby positively influencing disease progress. Here, we asked whether eosinophils are involved in the effects of IL-33 on tumor growth in CRC.In models of CT26 cell engraftment and colitis-associated CRC, tumor growth was reduced after IL-33 treatment. The growth reduction was absent in eosinophil-deficient ΔdblGATA-1 mice but was restored by adoptive transfer of ex vivo-activated eosinophils indicating that the antitumor effect of IL-33 depends on the presence of eosinophils. In vitro, IL-33 increased the expression of markers of activation and homing in eosinophils, such as CD11b and Siglec-F, and the degranulation markers CD63 and CD107a. Increased expression of Siglec-F, CD11b and CD107a was also seen in vivo in eosinophils after IL-33 treatment. Viability and cytotoxic potential of eosinophils and their migration properties toward CCL24 were enhanced indicating direct effects of IL-33 on eosinophils. IL-33 treatment led to increased levels of IL-5 and CCL24 in tumors.Our data show that the presence of eosinophils is mandatory for IL-33-induced tumor reduction in models of CRC and that the mechanisms include eosinophil recruitment, activation and degranulation. Our findings also emphasize the potential use of IL-33 as an adjuvants in CRC immunotherapy.AbbreviationsAOM: azoxymethane; bmRPMI: bone marrow RPMI; CRC: colorectal cancer; CFSE: carboxyfluorescein succinimidyl ester; DSS: dextran sulfate sodium; EPX: eosinophil peroxidase; INF-γ: interferon gamma; ILC: innate lymphoid cell; IL-33: interleukin-33; IL-5: interleukin-5; MDSC: myeloid derived suppressor cells; NK cells: natural killer cells; P/S: penicillin/streptomycin; rm: recombinant mouse; T regs: regulatory T cells; TATE: tumor associated tissue eosinophilia; TNF-α: tumor necrosis factor alpha.

Dataset Information

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

Publications

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

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