TH17 cells mediate steroid-resistant airway inflammation and airway hyperresponsiveness in mice

Abstract

Steroid-resistant asthma comprises an important source of morbidity in patient populations. T(H)17 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 T(H)17 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 T(H)2 or T(H)17 phenotype by culturing in conditioned medium. In addition, we also tested the steroid responsiveness of T(H)2 and T(H)17 cells. In vitro, T(H)17 cytokine responses were not sensitive to dexamethasone (DEX) treatment despite immunocytochemistry confirming glucocorticoid receptor translocation to the nucleus following treatment. Transfer of T(H)2 cells to mice challenged with OVA protein resulted in lymphocyte and eosinophil emigration into the lung that was markedly reduced by DEX treatment, whereas T(H)17 transfer resulted in increased CXC chemokine secretion and neutrophil influx that was not attenuated by DEX. Transfer of T(H)17 or T(H)2 cells was sufficient to induce airway hyperresponsiveness (AHR) to methacholine. Interestingly, AHR was not attenuated by DEX in the T(H)17 group. These data demonstrate that polarized Ag-specific T cells result in specific lung pathologies. Both T(H)2 and T(H)17 cells are able to induce AHR, whereas T(H)17 cell-mediated airway inflammation and AHR are steroid resistant, indicating a potential role for T(H)17 cells in steroid-resistant asthma.

FIGURE 1

T_H17 cells are not sensitive to DEX treatment in vitro. CD4⁺CD62L⁺CD25⁻ naive T cells isolated from DO11.10 OVA TCR-transgenic mice were cultured with WT BALB/c splenocytes that had been pulsed with OVA_323–339 under polarizing conditions. On day 6, cells were collected and stimulated with PMA/ionomycin for precursor frequency by ELISPOT or pretreated for 2 h with the indicated doses of DEX before stimulation with CD3/CD28 beads + IL-2. A, Spot frequencies of IL-17-producing T cells. B, Spot frequencies of IL-4-producing T cells. Cells were plated under the following conditions: T cells indicate un-stimulated T cells cultured without bead stimulation; 1 μM DEX indicates T cells pretreated with 1 μM DEX but not stimulated with CD3/CD28 microbeads; beads indicates untreated T cells stimulated with CD3/CD28 microbeads; μM DEX + bead indicates T cells pretreated with the indicated dose of DEX and stimulated with CD3/CD28 microbeads. Cell culture supernatant was collected at 36 h after stimulation and the levels of T_H2 and T_H17 cytokines were determined by multiplex suspension array assay. C, IL-5 levels from T_H2 cells. D, IL-13 levels from T_H2 cells. E, IL-17 levels from T_H17 cells. F, IL-22 levels from T_H17 cells. All data are graphed as mean ± SEM for n = 5–8;*, p < 0.01.

FIGURE 2

In vivo cytokine and chemokine profiles induced by T_H17 cell transfer and allergen provocation are not attenuated by DEX. A, SCID mice on a BALB/c background were challenged with OVA protein (50 μg/mouse) i.t. on day −1. On day 0, mice were pretreated with DEX (2.5 mg/kg, i.p.) or PBS control 2 h before cell transfer (1 × 10⁶ in vitro-polarized T_H cells). Mice were subsequently challenged with 50 μg/mouse OVA for 3 consecutive days. Animals were pretreated with DEX on day 2, 2 h before challenge. Mice were sacrificed on day 4, 24 h after the last airway challenge. Cytokine and chemokine levels in the BAL fluid (data not shown, unless indicated) and LH (B–E) were determined by multiplex suspension cytokine array. Data are graphed as mean ± SEM for n = 4 – 8;*, p < 0.05 T_H2_PBS vs T_H2_DEX.

FIGURE 3

T_H2- and T_H17-mediated airway inflammation and AHR. BAL fluid differential was determined by counting at least 100 cells from cytospins prepared of 100,000 cells. Data for A, lymphocytes; B, eosinophils; C, neutrophils; and D, macrophages are graphed as mean ± SEM percentage of total cells for n = 4 – 6; *, p < 0.05 PBS vs DEX in same transfer group; #, p < 0.05 T_H2_PBS vs T_H17_PBS.

FIGURE 4

The inflammatory response associated with T_H17 cell transfer is mediated by IL-17. T_H17 cells were transferred to IL-17R KO mice and BALB/c controls that had been challenged with OVA 1 day before transfer. Mice were challenged with 50 μg of OVA per mouse for 3 consecutive days after transfer. BAL fluid differential and LH levels of cytokines and chemokines 24 h after the last airway challenge were determined. Levels of KC (A), G-CSF (B), and IL-6 (C) in the LH were determined by multiplex suspension cytokine array. BAL fluid neutrophils (D), and eosinophils (E) are expressed as cells/ml × 10⁶. Data are graphed as mean ± SEM for n = 4 – 6; *, p < 0.05 compared with WT control.

FIGURE 5

IL-17 is sufficient to induce mucus secretion and AHR. A, gob5 expression was determined in whole lung by quantitative real-time PCR and normalized to the housekeeping gene 18S. Data are graphed as mean ± SEM for n = 4; *, p < 0.05 vs T_H2_PBS. B, Expression data were confirmed by PAS staining of lung sections from T_H2, and T_H17, transferred animals. C, An adenovirus overexpressing IL-4, IL-17, or luciferase control was given i.t. to WT BALB/c (10⁸ PFU/mouse) and airway responsiveness to methacholine was determine on day 3. *, p < 0.05 AdIL-4 vs Adluc; τ, p < 0.5 AdIL-17 vs Adluc. D, Methacholine dose-response curves in control, T_H2 vehicle-treated, or T_H2 DEX-treated mice. Control mice received all OVA challenges but PBS i.v. at time of cell transfer to experimental groups. Data are graphed as mean ± SEM for n = 6 – 8; *, p < 0.05 for T_H2 vs T_H2 + DEX. E, Methacholine dose-response curves in control, T_H17 vehicle- treated, or T_H17 + DEX-treated mice. Control mice received all OVA challenges but PBS i.v. at time of cell transfer to experimental groups. Data are graphed as mean ± SEM for n = 6 – 8.

FIGURE 6

Mechanism of T_H17 cell resistance to DEX treatment. T_H17 cells are not deficient in their ability to translocate GRα. A, GR staining by immunofluorescent microscopy in DEX-treated (1 μM) and nontreated T_H2 and T_H17 cells. B, Cytoplasmic protein fractions of DEX-treated and nontreated T_H2 and T_H17 cells were immunoblotted for GRα and β-actin. Samples were loaded in the following order: T_H2_PBS, T_H2_DEX, T_H17_PBS, and T_H17_DEX. C, Florescent intensity of nuclear GRα staining in T_H2 and T_H17 cells with and without DEX treatment. Data are graphed as mean ± SEM; *, p < 0.05 for DEX compared with no DEX conditions. D and E, T_H2 (C) or T_H17 (D) cells were transfected with NFAT-luciferase before stimulation with anti-CD3 and anti-CD28 microbeads (6 h) with or without pretreatment (2 h before beads) with DEX; *, p < 0.05 control (CNTRL) vs CD3/CD28; #, p < 0.05 CD3/CD28 vs CD3/CD28 + DEX.