While the CD4 T cells accounted for approximately 50C60% of the IL-13 and IL-17A produced in the lung, IL-13+ILC2 and IL-17A+ T cells were also increased in HDM-challenged sham-operated female mice and HDM-challenged gonadectomized male mice compared to HDM-challenged gonadectomized female and HDM-challenged sham-operated male mice (Figure 4D, 4G)
While the CD4 T cells accounted for approximately 50C60% of the IL-13 and IL-17A produced in the lung, IL-13+ILC2 and IL-17A+ T cells were also increased in HDM-challenged sham-operated female mice and HDM-challenged gonadectomized male mice compared to HDM-challenged gonadectomized female and HDM-challenged sham-operated male mice (Figure 4D, 4G). 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 WT male and female mice and ARtfm male mice that are unable to transmission through the AR, we decided 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. Th2 and Th17 differentiation experiments showed AR signaling experienced no direct effect on Th2 cell differentiation, but decreased IL-17A protein expression and IL-23 receptor mRNA relative expression from Th17 cells. Combined, these findings show AR signaling attenuated type 2 and IL-17A inflammation through IkappaBalpha different mechanisms and provide a potential explanation for the increased prevalence of asthma in women compared to men. extract and house dust mite (HDM) induced, ILC2-mediated airway inflammation (24, 25). Our group has also showed that Pectolinarigenin ovarian hormones increased Th17 cell differentiation and IL-17A production through an IL-23 receptor (R) dependent mechanism in mice and humans as well as increasing neutrophilic airway inflammation in mice (26). However, how testosterone regulates dual type 2 and IL-17A mediated airway inflammation remains unknown. We hypothesized that testosterone signaling through AR attenuates type 2 and IL-17A mediated airway inflammation. Our results showed that testosterone decreased HDM-induced total numbers of IL-13+ Th2 and IL-17A+ Th17 cells in the lung and that AR signaling directly attenuated total numbers of IL-17A+ Th17 cells in the lung and indirectly attenuated total numbers of IL-13+ Th2 cells by decreasing HDM-induced IL-4 production in the lung. Materials and Methods Mice Wild-type (WT) BALB/cJ 6C8 week aged female and male were purchased from Charles River Laboratories (Wilmington, MA). WT female, WT male, and AR testicular feminized (ARtfm) male C57BL/6J mice were purchased from Jackson Laboratory (Bar Harbor, Pectolinarigenin ME), and breeding colonies were established at Vanderbilt University or college Medical Center. CD90.1/CD90.2 mice were bred in house using homozygous CD90.1 and CD90.2 C57BL/6J mice from Jackson Laboratory. Gonadectomy or sham surgeries were conducted at 3 to 4 4 weeks of age by Charles River laboratories veterinary staff, and experiments were started when gonadectomized or sham-operated mice were 6C8 weeks aged. All animal experiments were conducted in adherence to the rules and regulations of the Association for Assessment and Accreditation of Laboratory Animal Care and were approved by the International Animal Care and Use Committee (IACUC) at Vanderbilt University or college Medical Center. House dust mite challenge protocol Mice were intranasally administered 40 g of HDM from Greer laboratories (Lenoir, NC) or PBS as vehicle control in a 100 l total volume for 4 occasions a week for 3 weeks as shown in Physique 1A. Open in a separate window Physique 1: HDM-induced BAL eosinophils and neutrophils were decreased in male compared Pectolinarigenin to female mice.(A) Experimental design for HDM-induced airway inflammation. (B-C) IL-13 and IL-17A protein expression in the lung homogenates as measured by ELISA. (D-H) Total BAL cells and total BAL eosinophils, neutrophils, macrophages, and lymphocytes. * p 0.05, 1-way ANOVA with Tukey post-hoc analysis, n= 6C9 mice/group. Data pooled from 2 impartial experiments. BAL and analysis of inflammatory cell infiltration in the airway BAL was performed by instilling 800 l of saline answer through a tracheostomy tube and then withdrawing the fluid with gentle suction through a syringe, as previously explained (27). The total cell count in the BAL fluid was counted using a hemocytometer and 0.04% trypan blue exclusion dye (Sigma Aldrich). The cells from your BAL were then fixed to a slide and stained using the Three Step Stain system (Richard-Allan Scientific, Thermo Scientific, Waltham, MA). Two hundred BAL cells were classified as eosinophils, neutrophils, lymphocytes, or macrophages using standard morphologic criteria and percentages of these inflammatory cells were decided. Total numbers of inflammatory cells were determined by multiplying the percentage of the inflammatory cells by the total numbers of viable cells in the BAL fluid. Cytokine and IgE measurements Cytokine levels were measured from BAL fluid, lung homogenates and/or Th0, Th2 and Th17 cell culture supernatants by ELISA using Quantikine and Duoset packages (R & D systems). Total serum IgE levels were decided using an ELISA (Biolegend). All experiments were performed according to the manufacturers instructions. Any OD450 value less than the lower limit of detection was assigned half the value of the lowest detectable standard. AHR measurements Mice were anesthetized with pentobarbital sodium (85 mg/kg) and an 18 gauge tracheostomy tube was placed in the trachea of the mice. Mice were then mechanically ventilated.