Steroid-resistant asthma comprises an important source of morbidity in patient populations. TH17 cells represent a distinct population of CD4+ Th cells that mediate neutrophilic inflammation and are characterized by the production of IL-17, IL-22, and IL-6. To investigate the function of TH17 cells in the context of Ag-induced airway inflammation, we polarized naive CD4+ T cells from DO11.10 OVA-specific TCR-transgenic mice to a TH2 or TH17 phenotype by culturing in conditioned medium. In addition, we also tested the steroid responsiveness of TH2 and TH17 cells. In vitro, TH17 cytokine responses were not sensitive to dexamethasone (DEX) treatment despite immunocytochemistry confirming glucocorticoid receptor translocation to the nucleus following treatment. Transfer of TH2 cells to mice challenged with OVA protein resulted in lymphocyte and eosinophil emigration into the lung that was markedly reduced by DEX treatment, whereas TH17 transfer resulted in increased CXC chemokine secretion and neutrophil influx that was not attenuated by DEX. Transfer of TH17 or TH2 cells was sufficient to induce airway hyperresponsiveness (AHR) to methacholine. Interestingly, AHR was not attenuated by DEX in the TH17 group. These data demonstrate that polarized Ag-specific T cells result in specific lung pathologies. Both TH2 and TH17 cells are able to induce AHR, whereas TH17 cell-mediated airway inflammation and AHR are steroid resistant, indicating a potential role for TH17 cells in steroid-resistant asthma.

Interleukin-5 (IL-5), which is produced by CD4؉ T helper 2 (Th2) cells, but not by Th1 cells, plays a key role in the development of eosinophilia in asthma. Despite increasing evidence that the outcome of many diseases is determined by the ratio of the two subsets of CD4 ؉ T helper cells, Th1 and Th2, the molecular basis for Th1-and Th2-specific gene expression remains to be elucidated. We previously established a critical role for the transcription factor GATA-3 in IL-5 promoter activation in EL-4 cells, which express both Th1-and Th2-type cytokines. Our studies reported here demonstrate that GATA-3 is critical for expression of the IL-5 gene in bona fide

Glucocorticoids, which are widely used as anffinflammatory agents, downregulate the expression of the interleukin 6 gene and of additional cytokine genes involved in inflammatory responses. Conversely, the transcription factor NF-ucB, a member ofthe Rel family oftranscription factors, has been implicated in the induction of multiple genes involved in the early processes of immune and inflammatory responses. This prompted us to investigate whether one of the mechanisms by which glucocorticoids exert their aninflammatory activities is through inhibition ofgene activation mediated by NF-KB. We report that, in intact cells, activation of the interieukin 6promoter by a combination of the factor NF-EL6 and the p65 subunit of NF-ucB is inhibited by dexamethasone (ligand)-activated glucocorticoid receptor. Conversely, activation of the mouse mammary tumor virus promoter by a combination of dexamethasone and glucocorticoid receptor is inhibited by overexpression of p65. Furthermore, we provide evidence for physical association between glucocorticoid receptor and p65 in protein crosslinking and coimmunoprecipitation experiments, using either in vitro translated proteins or those present in cell extracts. These studies suggest that direct interactions between NF-cB and glucocorticoid receptor may partly account for the antiinflammatory properties of glucocorticoids in vivo.

The transcription factor GATA-3 is expressed in T helper 2 (TH2) but not TH1 cells and plays a critical role in TH2 differentiation and allergic airway inflammation in vivo. Mice that lack the p50 subunit of nuclear factor kappa B (NF-kappa B) are unable to mount airway eosinophilic inflammation. We show here that this is not due to defects in TH2 cell recruitment but due to the inability of the p50-/- mice to produce interleukin 4 (IL-4), IL-5 and IL-13: cytokines that play distinct roles in asthma pathogenesis. CD4+ T cells from p50-/- mice failed to induce Gata3 expression under TH2-differentiating conditions but showed unimpaired T-bet expression and interferon gamma (IFN-gamma) production under TH1-differentiating conditions. Inhibition of NF-kappa B activity prevented GATA-3 expression and TH2 cytokine production in developing, but not committed, TH2 cells. Our studies provide a molecular basis for the need for both T cell receptor and cytokine signaling for GATA-3 expression and, in turn, TH2 differentiation.

Results SA subjects harbor more IFN-γ + CD4+ T cells in their airways compared with MMA subjects. A total of 66 subjects, 33 classified with MMA and 33 classified with SA, were included in this study; details of patient characteristics are included in Table 1. Of note, biological samples, such as cells in BAL fluid used for differential cell counts and cytokine expression, were analyzed from a subset of these subjects based on availability, as described in each figure legend. Since the recovery of BAL Severe asthma (SA) is a challenge to control, as patients are not responsive to high doses of systemic corticosteroids (CS). In contrast, mild-moderate asthma (MMA) is responsive to low doses of inhaled CS, indicating that Th2 cells, which are dominant in MMA, do not solely orchestrate SA development. Here, we analyzed broncholalveolar lavage cells isolated from MMA and SA patients and determined that IFN-γ (Th1) immune responses are exacerbated in the airways of individuals with SA, with reduced Th2 and IL-17 responses. We developed a protocol that recapitulates the complex immune response of human SA, including the poor response to CS, in a murine model. Compared with WT animals, Ifng -/-mice subjected to this SA model failed to mount airway hyperresponsiveness (AHR) without appreciable effect on airway inflammation. Conversely, AHR was not reduced in Il17ra -/-mice, although airway inflammation was lower. Computer-assisted pathway analysis tools linked IFN-γ to secretory leukocyte protease inhibitor (SLPI), which is expressed by airway epithelial cells, and IFN-γ inversely correlated with SLPI expression in SA patients and the mouse model. In mice subjected to our SA model, forced SLPI expression decreased AHR in the absence of CS, and it was further reduced when SLPI was combined with CS. Our study identifies a distinct immune response in SA characterized by a dysregulated IFN-γ/SLPI axis that affects lung function.