Tissue-Specific Dependence of Th1 Cells on the Amino Acid Transporter SLC38A1 in Inflammation.

Amino acid (AA) uptake is essential for T cell metabolism and function, but how tissue sites and inflammation affect CD4+ T cell subset requirements for specific AA remains uncertain. Here we tested CD4+ T cell AA demands with in vitro and multiple in vivo CRISPR screens and identify subset- and tissue-specific dependencies on the AA transporter SLC38A1 (SNAT1). While dispensable for T cell persistence and expansion over time in vitro and in vivo lung inflammation, SLC38A1 was critical for Th1 but not Th17 cell-driven Experimental Autoimmune Encephalomyelitis (EAE) and contributed to Th1 cell-driven inflammatory bowel disease. SLC38A1 deficiency reduced mTORC1 signaling and glycolytic activity in Th1 cells, in part by reducing intracellular glutamine and disrupting hexosamine biosynthesis and redox regulation. Similarly, pharmacological inhibition of SLC38 transporters delayed EAE but did not affect lung inflammation. Subset- and tissue-specific dependencies of CD4+ T cells on AA transporters may guide selective immunotherapies.

Cystic Fibrosis Reprograms Airway Epithelial IL-33 Release and Licenses IL-33-Dependent Inflammation.

Rationale: Type 2 inflammation has been described in people with cystic fibrosis (CF). Whether loss of CFTR (cystic fibrosis transmembrane conductance regulator) function contributes directly to a type 2 inflammatory response has not been fully defined. Objectives: The potent alarmin IL-33 has emerged as a critical regulator of type 2 inflammation. We tested the hypothesis that CFTR deficiency increases IL-33 expression and/or release and deletion of IL-33 reduces allergen-induced inflammation in the CF lung. Methods: Human airway epithelial cells (AECs) grown from non-CF and CF cell lines and Cftr+/+ and Cftr-/- mice were used in this study. Pulmonary inflammation in Cftr+/+ and Cftr-/- mice with and without IL-33 or ST2 (IL-1 receptor-like 1) germline deletion was determined by histological analysis, BAL, and cytokine analysis. Measurements and Main Results: After allergen challenge, both CF human AECs and Cftr-/- mice had increased IL-33 expression compared with control AECs and Cftr+/+ mice, respectively. DUOX1 (dual oxidase 1) expression was increased in CF human AECs and Cftr-/- mouse lungs compared with control AECs and lungs from Cftr+/+ mice and was necessary for the increased IL-33 release in Cftr-/- mice compared with Cftr+/+ mice. IL-33 stimulation of Cftr-/- CD4+ T cells resulted in increased type 2 cytokine production compared with Cftr+/+ CD4+ T cells. Deletion of IL-33 or ST2 decreased both type 2 inflammation and neutrophil recruitment in Cftr-/- mice compared with Cftr+/+ mice. Conclusions: Absence of CFTR reprograms airway epithelial IL-33 release and licenses IL-33-dependent inflammation. Modulation of the IL-33/ST2 axis represents a novel therapeutic target in CF type 2-high and neutrophilic inflammation.

Androgen receptor signaling promotes Treg suppressive function during allergic airway inflammation.

Women have higher prevalence of asthma compared with men. In asthma, allergic airway inflammation is initiated by IL-33 signaling through ST2, leading to increased IL-4, IL-5, and IL-13 production and eosinophil infiltration. Foxp3+ Tregs suppress and ST2+ Tregs promote allergic airway inflammation. Clinical studies showed that the androgen dehydroepiandrosterone (DHEA) reduced asthma symptoms in patients, and mouse studies showed that androgen receptor (AR) signaling decreased allergic airway inflammation. Yet the impact of AR signaling on lung Tregs remains unclear. Using AR-deficient and Foxp3 fate-mapping mice, we determined that AR signaling increased Treg suppression during Alternaria extract (Alt Ext; allergen) challenge by stabilizing Foxp3+ Tregs and limiting the number of ST2+ ex-Tregs and IL-13+ Th2 cells and ex-Tregs. AR signaling also decreased Alt Ext-induced ST2+ Tregs in mice by limiting expression of Gata2, a transcription factor for ST2, and by decreasing Alt Ext-induced IL-33 production from murine airway epithelial cells. We confirmed our findings in human cells where 5α-dihydrotestosterone (DHT), an androgen, decreased IL-33-induced ST2 expression in lung Tregs and decreased Alt Ext-induced IL-33 secretion in human bronchial epithelial cells. Our findings showed that AR signaling stabilized Treg suppressive function, providing a mechanism for the sex difference in asthma.

Metabolic Reprogramming of Nasal Airway Epithelial Cells Following Infant Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) is a seasonal mucosal pathogen that infects the ciliated respiratory epithelium and results in the most severe morbidity in the first six months of life. RSV is a common cause of acute respiratory infection during infancy and is an important early-life risk factor strongly associated with asthma development. While this association has been repeatedly demonstrated, limited progress has been made on the mechanistic understanding in humans of the contribution of infant RSV infection to airway epithelial dysfunction. An active infection of epithelial cells with RSV in vitro results in heightened central metabolism and overall hypermetabolic state; however, little is known about whether natural infection with RSV in vivo results in lasting metabolic reprogramming of the airway epithelium in infancy. To address this gap, we performed functional metabolomics, 13C glucose metabolic flux analysis, and RNA-seq gene expression analysis of nasal airway epithelial cells (NAECs) sampled from infants between 2-3 years of age, with RSV infection or not during the first year of life. We found that RSV infection in infancy was associated with lasting epithelial metabolic reprogramming, which was characterized by (1) significant increase in glucose uptake and differential utilization of glucose by epithelium; (2) altered preferences for metabolism of several carbon and energy sources; and (3) significant sexual dimorphism in metabolic parameters, with RSV-induced metabolic changes most pronounced in male epithelium. In summary, our study supports the proposed phenomenon of metabolic reprogramming of epithelial cells associated with RSV infection in infancy and opens exciting new venues for pursuing mechanisms of RSV-induced epithelial barrier dysfunction in early life.

Prostaglandin I2 signaling licenses Treg suppressive function and prevents pathogenic reprogramming.

Tregs restrain both the innate and adaptive immune systems to maintain homeostasis. Allergic airway inflammation, characterized by a Th2 response that results from a breakdown of tolerance to innocuous environmental antigens, is negatively regulated by Tregs. We previously reported that prostaglandin I2 (PGI2) promoted immune tolerance in models of allergic inflammation; however, the effect of PGI2 on Treg function was not investigated. Tregs from mice deficient in the PGI2 receptor IP (IP KO) had impaired suppressive capabilities during allergic airway inflammatory responses compared with mice in which PGI2 signaling was intact. IP KO Tregs had significantly enhanced expression of immunoglobulin-like transcript 3 (ILT3) compared with WT Tregs, which may contribute to the impairment of the IP KO Treg's ability to suppress Th2 responses. Using fate-mapping mice, we reported that PGI2 signaling prevents Treg reprogramming toward a pathogenic phenotype. PGI2 analogs promoted the differentiation of naive T cells to Tregs in both mice and humans via repression of ß-catenin signaling. Finally, a missense variant in IP in humans was strongly associated with chronic obstructive asthma. Together, these data support that PGI2 signaling licenses Treg suppressive function and that PGI2 is a therapeutic target for enhancing Treg function.

Estrogen receptor-α signaling increases allergen-induced IL-33 release and airway inflammation.

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-ß (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 IL33fl/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 17ß-estradiol (E2), propyl-pyrazole-triol (PPT, ER-α agonist), or diarylpropionitrile (DPN, ER-ß 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. 17ß-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.

ERα Signaling Increased IL-17A Production in Th17 Cells by Upregulating IL-23R Expression, Mitochondrial Respiration, and Proliferation.

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 ERß, 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 ERß (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.

Testosterone Decreases House Dust Mite-Induced Type 2 and IL-17A-Mediated Airway Inflammation.

As adults, women are twice as likely as men to have asthma; however, the mechanisms explaining this sexual dimorphism remain unclear. Increased type 2 cytokines and/or IL-17A, leading to increased airway eosinophils and neutrophils, respectively, are associated with asthma. Previous studies showed that testosterone, signaling through the androgen receptor (AR), decreased Th2-mediated allergic inflammation and type 2 innate immune responses during allergic inflammation. Therefore, we hypothesized that testosterone and AR signaling attenuate type 2 and IL-17A-mediated airway inflammation. To test our hypothesis, sham-operated and gonadectomized female and male mice were intranasally challenged with house dust mite (HDM) or vehicle (PBS) for 3 wk. Testosterone decreased and ovarian hormones increased HDM-induced eosinophilic and neutrophilic inflammation, IgE production, and airway hyperresponsiveness, as well as decreased the numbers of IL-13+ CD4 Th2 cells and IL-17A+ CD4 Th17 cells in the lung. Next, using wild-type male and female mice and ARtfm male mice that are unable to signal through the AR, we determined AR signaling intrinsically attenuated IL-17A+ Th17 cells but indirectly decreased IL-13+ CD4 Th2 cells in the lung by suppressing HDM-induced IL-4 production. In vitro Th2 and Th17 differentiation experiments showed AR signaling had no direct effect on Th2 cell differentiation but decreased IL-17A protein expression and IL-23R mRNA relative expression from Th17 cells. Combined, these findings show AR signaling attenuated type 2 and IL-17A inflammation through different mechanisms and provide a potential explanation for the increased prevalence of asthma in women compared with men.

Testosterone Attenuates Group 2 Innate Lymphoid Cell-Mediated Airway Inflammation.

Sex hormones regulate many autoimmune and inflammatory diseases, including asthma. As adults, asthma prevalence is 2-fold greater in women compared to men. The number of group 2 innate lymphoid cells (ILC2) is increased in patients with asthma, and we investigate how testosterone attenuates ILC2 function. In patients with moderate to severe asthma, we determine that women have an increased number of circulating ILC2 compared to men. ILC2 from adult female mice have increased IL-2-mediated ILC2 proliferation versus ILC2 from adult male mice, as well as pre-pubescent females and males. Further, 5α-dihydrotestosterone, a hormone downstream of testosterone, decreases lung ILC2 numbers and IL-5 and IL-13 expression from ILC2. In vivo, testosterone attenuated Alternaria-extract-induced IL-5+ and IL-13+ ILC2 numbers and lung eosinophils by intrinsically decreasing lung ILC2 numbers, as well as by decreasing expression of IL-33 and thymic stromal lymphopoietin (TSLP), ILC2-stimulating cytokines. Collectively, these findings provide a foundational understanding of sexual dimorphism in ILC2 function.

Estrogen and progesterone decrease let-7f microRNA expression and increase IL-23/IL-23 receptor signaling and IL-17A production in patients with severe asthma.

BACKGROUND: Women have an increased prevalence of severe asthma compared with men. IL-17A is associated with severe asthma and requires IL-23 receptor (IL-23R) signaling, which is negatively regulated by let-7f microRNA. OBJECTIVE: We sought to Determine the mechanism by which 17ß-estradiol (E2) and progesterone (P4) increase IL-17A production. METHODS: IL-17A production was determined by using flow cytometry in TH17 cells from women (n = 14) and men (n = 15) with severe asthma. Cytokine levels were measured by using ELISA, and IL-23R and let-7f expression was measured by using quantitative PCR in TH17-differentiated cells from healthy women (n = 13) and men (n = 14). In sham-operated or ovariectomized female mice, 17ß-E2, P4, 17ß-E2+P4, or vehicle pellets were administered for 3 weeks before ex vivo TH17 cell differentiation. Airway neutrophil infiltration and CXCL1 (KC) expression were also determined in ovalbumin (OVA)-challenged wild-type female recipient mice with an adoptive transfer of OVA-specific TH17 cells from female and male mice. RESULTS: In patients with severe asthma and healthy control subjects, IL-17A production was increased in TH17 cells from women compared with men. IL-23R expression was increased and let-7f expression was decreased in TH17-differentiated cells from women compared with men. In ovariectomized mice IL-17A and IL-23R expression was increased and Let-7f expression was decreased in TH17 cells from mice administered 17ß-E2+P4 compared with those administered vehicle. Furthermore, transfer of female OVA-specific TH17 cells increased acute neutrophil infiltration in the lungs of OVA-challenged recipient mice compared with transfer of male OVA-specific TH17 cells. CONCLUSIONS: 17ß-E2+P4 increased IL-17A production from TH17 cells, providing a potential mechanism for the increased prevalence of severe asthma in women compared with men.