IL-33 induces IL-9 production in human CD4+ T cells and basophils

Abstract

IL-33, an IL-1 family member and ligand for the IL-1 receptor-related protein ST2, has been associated with induction of Th2 cytokines such as IL-4, IL-5, and IL-13. Here, we report that IL-33 can initiate IL-9 protein secretion in vitro in human CD4+ T cells and basophils isolated from peripheral blood. TGF-β has been described as a critical factor for IL-9 induction in Th2 cells; however, we found that TGF-β also induces co-production of IL-9 in purified, naïve (>99%) CD4(+)CD45RA(+)CD45RO(-)CD25(-) T cells differentiated towards a Th1 profile. Subsequently, it was demonstrated that TGF-β is important, although not an absolute requirement, for IL-9 production in CD4+ T cells. IL-9 production by purified (>95%) human basophils, cultured for 24 h with IL-3 or IL-33, was found, with a strong synergy between the two, likely to be explained by the IL-3 upregulated ST2 expression. Collectively, these data indicate that barrier functioning cells are important for the regulation of IL-9 production by immune cells in inflamed tissue.

Figure 1. TGF-β induces IL-9 secretion in Th1 and Th2 cells.

Naïve CD4+ T cells were activated with fibroblast-bound anti-CD3/CD28 under classical Th1 and Th2 conditions, with IL-12 and anti-IL-4 or IL-4, anti-IFN-γ, and anti-IL-12 respectively for 5 days, restimulated at day 5 and split in cultures with and without TGF-β or anti-TGF-β for 5 more days of stimulation. (A, B and C) Supernatant concentrations of IL-9, IL-5 and IFN-γ at day 10 after restimulation with PMA and ionomycin for 6 h in the presence of Bref A for the last 4 h. Each donor is represented by a specific symbol and connected with a line. Data are from four independent experiments, each with two donors. * p<0.05, **p<0.01, ***p<0.001.

Figure 2. TGF-β induces IL-9 expression in Th1 and Th2 cells.

Naïve CD4+ T cells were activated with fibroblast-bound anti-CD3/CD28 under classical Th1 and Th2 conditions, with IL-12 and anti-IL-4 or IL-4, anti-IFN-γ, and anti-IL-12 respectively for 5 days, restimulated at day 5 and split in cultures with and without TGF-β or anti-TGF-β for 5 more days of stimulation. (A, B, C, D, E and F) Percentage positive LIVE⁺CD4+ cells for, respectively IL-9, PU.1, IL-13, IFN-γ, GATA-3, and T-bet at day 10 after restimulation with PMA and ionomycin for 6 h in the presence of Bref A for the last 4 h. Each donor is represented by a symbol and connected with a line. Data are from four independent experiments, each with two donors.* p<0.05, **p<0.01, ***p<0.001.

Figure 3. TGF-β induces IL-9 mRNA expression in Th1 and Th2 cells.

Naïve CD4+ T cells were activated with fibroblast-bound anti-CD3/CD28 under classical Th1 and Th2 conditions, with IL-12 and anti-IL-4 or IL-4, anti-IFN-γ, and anti-IL-12 respectively for 5 days, restimulated at day 5 and split in cultures with and without TGF-β or anti-TGF-β for 5 more days of stimulation. (A, C and D) qRT-PCR gene expression analysis of the relative gene expression of IL-9, GATA3, and Tbet at day 10 relative to the Th0 culture. (B) CT value from qRT-PCR gene expression analysis of the gene PU.1. Each donor is represented by a specific symbol and connected with a line. Data are from four independent experiments, each with two donors. *p<0.05, **p<0.01, ***p<0.001.

Figure 4. Induction of the IL-33 receptor ST2 by IL-4 and IL-33.

Naïve CD4+ T cells were activated with fibroblast-bound anti-CD3/CD28 for 5 days in presence of either blocking antibodies against IFN-γ and IL-12 (Th0) or these antibodies plus IL-4 (Th2). At day 5, these cultures were restimulated and split in cultures with and without TGF-β and/or IL-33 for an additional 5 days of stimulation. qRT-PCR gene expression analysis of the relative ST2 expression at day 10 to the Th0 culture. Each donor is represented by a specific symbol and connected with a line. Data are from four independent experiments, each with two donors. Horizontal lines represent means. **p<0.01, ***p<0.001.

Figure 5. A and B. IL-33 and TGF-β induce IL-9 secretion in Th cells.

Naïve CD4+ T cells were activated with fibroblast-bound anti-CD3/CD28 for five 5 days in the presence of blocking antibodies against IFN-γ and IL-12 (Th0) or these antibodies plus IL-4 (Th2). At day 5, these cultures were restimulated and split in cultures with TGF-β or anti-TGF-βplus IL-33 for an additional 5 days of stimulation. (A) Supernatant multiplex analysis of IL-9, at day 10, after restimulation with PMA and ionomycin for 6 h in the presence of Bref A for the last 4 h. (B) qRT-PCR gene expression analysis of the relative expression of IL9 at day 10 related to the Th0 culture. Data are from four independent experiments, each with two donors. Vertical lines represent means (SEM). All tested cultures are significantly different p<0.05 or less compared to Th0 culture. *p<0.05, **p<0.01, ***p<0.001.

Figure 6. ST2 expression and IL-9 secretion from basophils stimulated with IL-3 and IL-33.

Analysis of ST2 expression with FACS. Black line shows surface expression of ST2 on freshly isolated basophils at 0 h; tinted red shows negative control culture without cytokines after 24 h; blue lines show samples. Example from one donor included in both FACS and multiplex analysis is presented. In total, two independent experiments were performed, using two donors for each experiment, with similar results. Multiplex analysis of IL-9 in supernatant from a control culture or cultures with IL-3 and/or IL-33. Data are from two independent experiments, each with two donors. Vertical lines represent means (SEM). **p<0.01.