TH9 cells are required for tissue mast cell accumulation during allergic inflammation

Abstract

Background: IL-9 is important for the growth and survival of mast cells. IL-9 is produced by T cells, natural killer T cells, mast cells, eosinophils, and innate lymphoid cells, although the cells required for mast cell accumulation during allergic inflammation remain undefined.

Objective: We sought to elucidate the role of TH9 cells in promoting mast cell accumulation in models of allergic lung inflammation.

Methods: Adoptive transfer of ovalbumin-specific TH2 and TH9 cells was used to assess the ability of each subset to mediate mast cell accumulation in tissues. Mast cell accumulation was assessed in wild-type mice and mice with PU.1-deficient T cells subjected to acute and chronic models of allergic inflammation.

Results: Adoptive transfer experiments demonstrated that recipients of TH9 cells had significantly higher mast cell accumulation and expression of mast cell proteases compared with control or TH2 recipients. Mast cell accumulation was dependent on IL-9, but not IL-13, a cytokine required for many aspects of allergic inflammation. In models of acute and chronic allergic inflammation, decreased IL-9 levels in mice with PU.1-deficient T cells corresponded to diminished tissue mast cell numbers and expression of mast cell proteases. Mice with PU.1-deficient T cells have defects in IL-9 production from CD4(+) T cells, but not natural killer T cells or innate lymphoid cells, suggesting a TH cell-dependent phenotype. Rag1(-/-) mice subjected to a chronic model of allergic inflammation displayed reduced mast cell infiltration comparable with accumulation in mice with PU.1-deficient T cells, emphasizing the importance of IL-9 produced by T cells in mast cell recruitment.

Conclusion: TH9 cells are a major source of IL-9 in models of allergic inflammation and play an important role in mast cell accumulation and activation.

Keywords: PU.1; T(H)2 cells; T(H)9 cells; allergic inflammation; mast cells.

Figure 1

Adoptive transfer of Th9 cells increases mast cell numbers. A, Naive CD4⁺ T cells from DO11.10 mice were differentiated under Th9 / Th2 conditions with OVA_323–339 peptide and mitomycin C treated antigen presenting cells. Before transfer, the percentage of IL-13⁺ and IL-9⁺ cells were measured in Th9 / Th2 cell cultures by intracellular cytokine staining. B, Total number of inflammatory cells were determined in BAL fluid and numbers of eosinophils determined by flow cytometry. C and D, Mucus production was assessed in the lung tissues by PAS staining and expression of mucus genes Muc5ac and Clca3 quantified by qPCR. E, PBS, Th2 or Th9 DO11.10 cells were intravenously transferred to BALB/c mice that were subsequently challenged with OVA+TSLP for 5 days. Mast cells were counted after staining tracheal sections with toluidine blue and expression of mast cell proteases was measured in lung tissues by quantitative PCR. F and G, Th9 cell recipients received 10 μg of anti-IL-9, anti-IL-13, or IgG2b control mAb on days 1, 3 and 5 before challenge with OVA+TSLP. Mice were sacrificed 24 h after the last challenge and mast cell numbers were quantitated in tracheal tissue sections by toluidine blue staining and expression of mast cell proteases was determined by quantitative PCR. Boxes on low magnification micrographs indicate the region for the higher magnification panel. Arrow: mast cells. Data are the mean ± SEM of 3–5 mice per group and representative of 2 independent experiments with similar results. *p < 0.05.

Figure 2

Th9-dependent accumulation of mast cells in an acute model of allergic inflammation. A, Wild-type mice were sensitized and challenged using the OVA/alum protocol. Mice received control immunoglobulin (control Ab) or anti-IL-9, 30 min before the first, third and fifth intranasal challenge. Mice were sacrificed 48 h after the last challenge and tracheal tissues were evaluated for the presence of mast cells. B and C, Wild-type and Sfpi1^lck−/− mice were sensitized and challenged with the OVA/Alum protocol and mast cells were counted in toluidine blue stained tracheal tissue sections and quantitative PCR was performed for mast cell protease gene expression in lung tissues. Data shown represent the mean ± SEM of 5 to 6 mice per group from 2 independent experiments with similar results. *p < 0.05.

Figure 3

Th9-dependent accumulation of mast cells in an HDM-induced chronic model of allergic inflammation. A–F, Wild-type mice were immunized intranasally with HDM for 5 weeks and A, Cellular infiltration in the lungs of wild-type and Sfpi1^lck−/− mice was evaluated by hematoxylin and eosin (H&E) staining. Magnification is indicated. B, Inflammatory cells in the BAL fluid (T cells, B cells, Eosinophils (Eos), Neutrophils (Neu), Dendritic cells (DC) and Macrophages (Mac) were evaluated by flow cytometry. C, BAL CD4⁺ T cells were stimulated with PMA and ionomycin for 5 hours to assess cytokine production by intracellular staining. D, Cells from mediastinal lymph nodes were stimulated with HDM for 5 days. Cell-free supernatant was used to assess cytokine production using ELISA. E, Expression of mast cell proteases was determined by quantitative PCR analysis of lung RNA. F, Mast cell numbers were evaluated in tracheal sections. Data shown represent mean ± SEM of 5 to 7 mice per group from 2 independent experiments with similar results. *P < 0.05. G, Immunohistochemical staining of T cells (CD3) and mast cells (c-kit, CD117) with Vulcan Red and DAB in paraffin embedded tracheal tissue sections. Green arrowhead indicates T cells and black arrow shows mast cells. Boxes on low magnification micrographs indicate the region for the higher magnification panel.

Figure 4

T cell-dependent mast cell accumulation in HDM-induced chronic allergic airway inflammation. A, Numbers of inflammatory cells in the BAL fluid of wild-type, Sfpi1^lck−/−, Rag1^−/−, and unchallenged wild-type mice (UC) were determined. B and C, Mast cell infiltration in tracheal toluidine blue stained sections. Original magnification is indicated. Mast cell numbers were determined by counting in at least 5 hpfs. Boxes on low magnification micrographs indicate the region for the higher magnification panel. D, Serum mMCP-1 levels were determined by ELISA. Data are mean ± SEM of 5–7 mice per group and representative of 2 independent experiments with similar results. *P <0.05.