7e), the number of eosinophils in BAL (Fig

7e), the number of eosinophils in BAL (Fig. were also found in the lungs of asthmatic patients11. Th9 cells as a source of IL-9 contribute to allergic inflammation4,5. In humans, allergic donors have substantially more circulating Th9 cells than non-allergic donors12. Th9 cells are generated in vitro by culturing na?ve CD4+ T cells with IL-4 and TGF2,3,13. This pair of cytokines forms the basic environment for driving Th9 cells, the polarizing of which can be enhanced by the presence of IL-1 (ref. 14, 15), IL-2 (ref. 13), and IL-25 (ref. 16). We report here that nitric oxide (NO), MBX-2982 a free radical, is a potent enhancer of Th9 polarization and maintenance. NO is a crucial mediator of a range of biological functions, including vascular relaxation, platelet aggregation, neurotransmission, tumoricidal and microbicidal activities, and immune regulation (reviewed in ref. 17-20). NO is also associated with some of the most important immune pathologies, including rheumatoid arthritis, diabetes, systemic lupus erythematosus and septic shock. NO is derived from the guanidino nitrogen atom(s) and molecular oxygen in a reaction catalyzed by three forms of nitric oxide synthase (NOS). The neuronal form (nNOS or NOS1) and endothelial form (eNOS or NOS3) produce physiological level of NO at steady state. The cytokine-inducible form (iNOS or NOS2) is activated by a number of immunological stimuli, including IFN, TNF and LPS generated during infection, and catalyzes high output of NO, which can be cytotoxic and kill intracellular pathogens. We have recently shown21 that NO can suppress the proliferation and function of polarized murine and human Th17 cells via the down regulation of the expression of aryl hydrocarbon receptor which participates in the induction of Th17. We now show that, in contrast to its effect on Th17, NO markedly enhances the polarization and function of Th9 cells. NO does so by elevating the expression of p53, which in turn increases the production of IL-2 and activates the down stream MBX-2982 events including phosphorylation of STAT5 and the Itgb7 expression of IRF4. administration of an NO donor increases airway inflammation whereas message was clearly decreased but and mRNA were markedly up regulated (Fig. 1a). In this report, we elected to MBX-2982 focus on IL-9. The enhancement of expression by NO was confirmed by quantitative PCR (qPCR) assay (Fig. 1b), and by ELISA of the culture supernatants (Fig. 1c). FACS analysis demonstrated that decrease in the percentage of IL-17+ T cells was accompanied by an increase in the percentage of IL-9+ T cells and IL-17/IL-9 double positive T cells (Fig. 1d, e). We then determined whether NO can influence the differentiation of Th17 and Th9 cells under the mixed polarization conditions for Th17 and Th9. CD4+ T cells were cultured for 4 days with plate-bounded anti-CD3 and soluble anti-CD28 + IL-4, TGF, IL-6, IL-1, IL-23, and anti-IFN (without APC) in the presence of NOC-18. These culture conditions resulted in a modest percentage of Th17 cells MBX-2982 and a distinct population of Th9 cells. NO decreased MBX-2982 the percentage of IL-17+ cells and enhanced the percentage of IL-9+ T cells, without producing significant number of IL-17/IL-9 double positive T cells (Fig. 1f). The differential effect of NO on Th17 and Th9 cell polarization was also reflected in the concentrations of IL-9 and IL-17 in the supernatants of these cultures (Fig. 1g). These results therefore demonstrate that NO inhibits Th17 development but enhances the differentiation of Th9 cells under Th17 and Th17/9 polarizing conditions. Open in a separate window Figure 1 NO inhibits Th17 but enhances Th9 development. Purified BALB/c CD4+ T cells were cultured for 3 d under Th17 polarizing conditions (round-bottom 96-well plate with APC, CD3, IL-6, TGF-, IL-23, IL-1, IFN and IL-4). NO donor (NOC-18) was added at the beginning of culture. (a) Microarray analysis of differential mRNA expression between cells cultured with or without NO. Full microarray information is provided in Supplementary Fig. 1 and deposited in MIAME (E-MEXP-3959). (b) mRNA was further quantified by qPCR. (c) IL-9 concentration in the supernatant determined by ELISA. (d) IL-17 and IL-9 producing cells were analyzed by FACS. Numbers in quadrants indicate percentage of cells and are summarized in (e). (f) CD4+ T cells were cultured under Th17/Th9 conditions (CD3 + IL-4, TGF, IL-6, IL-1, IL-23, anti-IFN) NO. Percentage of cells producing IL-9 and IL-17 determined by.