Women have increased prevalence of Th17-mediated autoimmune diseases, including lupus and multiple sclerosis, and severe asthma. While estradiol and progesterone increased IL-17A production in Th17 cells by inhibiting Let7f miRNA expression and increasing IL-23 receptor (IL-23R) expression, it remained unclear how estrogen signaling through the canonical nuclear receptors, estrogen receptor α (ERα) and/or ERβ, regulated this pathway. We hypothesized that estrogen signaling through ERα increased IL-23R expression and IL-17A production from Th17 cells. To test this hypothesis, naïve T cells from WT female, WT male, Esr1−/− and Esr2−/− female mice were differentiated into Th17 cells. IL-17A production and IL-23R expression were significantly increased in Th17 cells from WT female mice compared to Th17 cells from WT male mice. Deletion of ERα (Esr1−/−), but not ERβ (Esr2−/−), significantly decreased IL-17A production and IL-23R expression in Th17 cells by limiting IL-23R expression in a Let-7f dependent manner. ERα deficiency also decreased Th17 cell proliferation as well as decreased T cell metabolism as measured by ATP-linked oxygen consumption rate and proton leakage. Further, we found that Cox20 expression, a protein involved in mitochondrial respiration through assembly of cytochrome c oxidase in the electron transport chain, was increased in Th17 cells from WT female mice compared to Th17 cells from WT male and Esr1−/− female mice. Inhibition of Cox20 decreased IL-17 production in Th17 cells from WT female mice. Combined these studies showed that ERα signaling increased IL-17A production in Th17 cells by upregulating IL-23R expression and promoting mitochondrial respiration and proliferation.

Background: Group 2 innate lymphoid cells (ILC2) are stimulated by IL-33 to increase IL-5 and IL-13 production and airway inflammation. While sex hormones regulate airway inflammation, it remained unclear whether estrogen signaling through estrogen receptor-α (ER-α, Esr1) or ER-β (Esr2) increased ILC2-mediated airway inflammation. We hypothesize that estrogen signaling increases allergen-induced IL-33 release, ILC2 cytokine production, and airway inflammation. Methods: Female Esr1-/-, Esr2-/-, wild-type (WT), and IL33 fl/fl eGFP mice were challenged with Alternaria extract (Alt Ext) or vehicle for 4 days. In select experiments, mice were administered tamoxifen or vehicle pellets for 21 days prior to challenge. Lung ILC2, IL-5 and IL-13 production, and BAL inflammatory cells were measured on day 5 of Alt Ext challenge model. Bone marrow from WT and Esr1-/female mice was transferred (1:1 ratio) into WT female recipients for 6 weeks followed by Alt Ext challenge. hBE33 cells and normal human bronchial epithelial cells (NHBE) were pretreated with 17β-estradiol (E2), propyl-pyrazole-triol (PPT, ER-α agonist), or diarylpropionitrile (DPN, ER-β agonist) before allergen challenge to determine IL-33 gene expression and release, extracellular ATP release, DUOX-1 production, and necrosis. Results: Alt Ext challenged Esr1-/-, but not Esr2-/-, mice had decreased IL-5 and IL-13 production, BAL eosinophils, and IL-33 release compared to WT mice. Tamoxifen decreased IL-5 and IL-13 production and BAL eosinophils. IL-33eGFP + epithelial cells were decreased in Alt Ext challenged Esr1-/mice compared to WT mice. 17β-E2 or PPT, but not DPN, increased IL-33 gene expression, release, and DUOX-1 production in hBE33 or NHBE cells. Conclusion: Estrogen receptor-α signaling increased IL-33 release and ILC2-mediated airway inflammation.

RATIONALE: The purpose of this study was to determine whether conventionally used ILC2 markers, ST2 and CD127, are sufficient to include all Th2-cytokine producing ILCs. We hypothesized that ST2-and CD127-negative ILC2 populations exist in mouse lung and that these nontraditional populations might be activated by the clinically relevant fungal allergen Alternaria, a known potent inducer of IL-33. METHODS: Mice were challenged intranasally with Alternaria alternata for three consecutive days, and 24 hours after the last challenge brochoalveolar lavage fluid and lungs were harvested for flow cytometric analysis, ELISA, and lung section staining. ILCs were identified by flow cytometry as CD45+Lineage-Thy1.2+ lymphocyte-sized cells and were separated into four subsets based on ST2 and CD127 expression. Intracellular cytokine and transcription factor staining were performed on the ILC populations. RESULTS: Th2 cytokine-producing ILCs were identified in all four subpopulations, regardless of ST2 and CD127 expression, as evidenced by high GATA-3, IL-5, IL-13 and ILC2 surface marker expression. Further, ILC2 cytokines and surface markers were upregulated following Alternaria challenge, irrespective of ST2 and CD127 expression. The four ILC subpopulations were also found in RAG2 knockout mice, which is supportive of their presence being T-cell independent. Some of the unconventional ILC2s also produced IFNg and IL-17A suggesting the existence of ILC2 plasticity in these populations. CONCLUSIONS: Unconventional ST2-and CD127-negative ILC2 populations exist in mouse lung and are further induced by Alternaria. Thus, future mouse ILC2 investigations should be aware of ILC2 populations that do not express ST2 and CD127.

No abstract

RATIONALE: Rapid diagnosis of drug-induced allergy (DIA) is required in clinical practice to give the patient appropriate treatment. Analyzing cell kinetics as an index against chemotactic factors in the sample is a promising approach to diagnose DIA. We assessed this novel highsensitive allergy test (HiSAT) using patients' serum and investigated the optimal cells. METHODS: The serum of patients with or without the cryptogenic allergy-like symptoms were obtained in Fukuoka University hospital with the informed consent. Cytokine concentration was determined by using Cytokine Bead Array kit. Cell migration was sequentially recorded by EZ-TAXIScanä and analyzed by ImageJ software. The granulocytes and HL-60 cells, human promyelocytic leukemia cell line, were assessed as a chemotactic cell in HiSAT. The inhibitory effect of the specific antibody against the chemokine receptors was examined using the patients' serum. RESULTS: Cytokine/chemokine concentration in the serum of patients with the allergy-like symptoms differed largely by individuals. Fifty-nine percent of patients with the symptoms were considered to be an allergy as a result of HiSAT. After excluding non-allergy case according to the clinical record, the positive rate became 89%. The specific antibody against IL-8R or LTBR4 was significantly inhibited the cell migration against serum in HiSAT using granulocytes. Although the reactivity of HL-60 cells was lower than the granulocytes, the same results were obtained in HiSAT using patients' serum. CONCLUSIONS: HiSAT might be one of the promising approaches to diagnose DIA rapidly in clinical practice. The HL-60 cells are the candidate as an optimal chemotactic cell for HiSAT.