Th2 LCR is essential for regulation of Th2 cytokine genes and for pathogenesis of allergic asthma

Abstract

Previous studies have shown that Th2 cytokine genes on mouse chromosome 11 are coordinately regulated by the Th2 locus control region (LCR). To examine the in vivo function of Th2 LCR, we generated CD4-specific Th2 LCR-deficient (cLCR KO) mice using Cre-LoxP recombination. The number of CD4 T cells in the cLCR KO mouse was comparable to that in wild-type mice. The expression of Th2 cytokines was dramatically reduced in in vitro-stimulated naïve CD4 T cells. Deletion of the LCR led to a loss of general histone H3 acetylation and histone H3-K4 methylation, and demethylation of DNA in the Th2 cytokine locus. Upon ovalbumin challenge in the mouse model of allergic asthma, cLCR KO mice exhibited marked reduction in the recruitment of eosinophils and lymphocytes in the bronchoalveolar lavage fluid, serum IgE level, lung airway inflammation, mucus production in the airway walls, and airway hyperresponsiveness. These results directly demonstrate that the Th2 LCR is critically important in the regulation of Th2 cytokine genes, in chromatin remodeling of the Th2 cytokine locus, and in the pathogenesis of allergic asthma.

Fig. 1.

Expression of cytokines in CD4-conditional LCR(^−/−) mice. Naïve CD4 T cells from wild-type and conditional LCR KO mice were stimulated with Th0, Th1, or Th2 conditions for 5 days. Cells were restimulated with a plate-bound anti-CD3 antibody for 16 h. Cytokines produced in supernatants were measured by ELISA (A) or transcripts of cytokines were measured by quantitative RT-PCR using total RNA isolated from the cells.

Fig. 2.

Histone H3 acetylation and histone H3-K4 methylation in the Th2 cytokine locus. Naïve CD4 T cells from wild-type or cLCR KO mice were in vitro-stimulated into either Th1 (A) or Th2 (B) cells, and histone H3 acetylation and histone H3-K4 methylation at several regulatory regions in the Th2 cytokine locus in the cells were examined by chromatin immunoprecipitation using anti-acetyl-H3 and anti-methyl-H3-K4 antibodies. Relative amount of precipitated DNA compared to input DNA was measured by quantitative RT-PCR.

Fig. 3.

DNA methylation in the il4 promoter and CNS-1 regions. Naïve CD4 T cells from wild-type or cLCR KO mice were stimulated in vitro into either Th1 (A) or Th2 (B) cells, and DNA methylation at the il4 promoter (A) and CNS-1 (B) regions of the Th2 cytokine locus was examined by bisulfate modification and DNA sequencing (n = 20–30).

Fig. 4.

Differential cell count (A), airway inflammation (B and C), and mucus secretion (D and E). Wild-type or cLCR KO mice were sensitized and challenged with OVA or PBS as described in Materials and Methods. (A) Differential cell counting of the BAL fluids was done by staining with Diff-Quik solution. (B and C) Paraffin-embedded lung sections were stained with hematoxylin and eosin. (B) Wild-type mice challenged with OVA. (C) The cLCR KO mice were challenged with OVA. (D and E) Paraffin-embedded lung sections were stained with PAS for mucus staining. (D) Wild-type mice were challenged with OVA. (E) The cLCR KO mice were challenged with OVA.

Fig. 5.

Airway resistance (A) and serum IgE (B). Wild-type or cLCR KO mice were sensitized and challenged with OVA or PBS as described in Materials and Methods. (A) Airway hyperresponsiveness was measure as average lung resistance (R_L) in anesthetized ventilated mice. Data are mean ± standard deviation of six mice. **, P < 0.01 compared with wild-type mice. (B) Serum IgE amount was measured by ELISA.