Hedgehog signaling promotes TH2 differentiation in naive human CD4 T cells.

To the Editor:

Hedgehog (Hh) proteins are intercellular signaling molecules that control development and tissue homeostasis. They also regulate thymocyte development and peripheral T-cell activation in mice and human subjects and have recently been shown to promote TH2 differentiation and function in mice.1, 2, 3, 4 Sonic Hedgehog homologue (SHH) is involved in homeostasis of many epithelial tissues, and because these tissues are the sites of allergic disease, it is important to understand how Hh signaling influences human CD4 TH differentiation. Here we show that Hh signaling promotes human TH2 differentiation by using materials and methods described in Furmanski et al and Yanez et al and in the Methods section in this article's Online Repository at www.jacionline.org.

We used quantitative RT-PCR to evaluate gene expression of components of the Hh signaling pathway in naive human CD4 T cells stimulated for 48 hours in TH0-, TH1-, or TH2-polarizing conditions. Expression levels of the Hh-responsive transcription factors glioma-associated oncogene 1 (GLI1) and GLI2 and the Hh cell-surface receptor patched 1 (PTCH1) were greater in CD4 T cells cultured under TH2-skewing conditions compared with those cultured under TH0 or TH1 conditions (Fig 1, A), suggesting that Hh signaling is involved in human TH differentiation or function. Because GLI1 and PTCH1 are Hh target genes, their greater expression in TH2-differentiated cells indicates that this population has overall greater Hh-mediated transcription.

Fig 1

Shh treatment increases TH2 differentiation in vitro. Naive CD4 T cells (n = 12 donors) stimulated under TH-skewing conditions with or without rShh (B-I) analyzed at 48 hours (A), at day 4 (B-E), and at day 7 plus restimulation (F-I) are shown. Plots indicate means ± SEMs; each point represents an individual donor. Fig 1, A, Gene expression (quantitative RT-PCR; n = 3). FACS histograms show intracellular expression (gated on CD4+ cells) of GATA-3 (Fig 1, B) and T-bet (Fig 1, C). Gray overlays show control stain. Scatterplots show percentages of positive cells. Fig 1, D and E, Cytokine concentration (ELISA) in supernatants. Fig 1, F and G, FACS plots show CD4 and intracellular cytokine expression. Scatterplots show cytokine-positive percentages. Fig 1, H and I, Gene expression (quantitative RT-PCR; n = 3). *P < .05 and **P < .01, paired 2-tailed t test. ns, Not significant.

To test the influence of SHH signaling on TH differentiation, we stimulated purified naive human CD4 T cells from 12 independent, randomly selected anonymous donors for 4 days under skewing conditions with or without a single dose of recombinant Shh (rShh). Treatment with rShh significantly enhanced expression of the TH2 transcription factor GATA-3 in cells stimulated under TH2 conditions, whereas GATA-3 expression under TH0 conditions and T-bet expression under TH0 or TH1 conditions were not affected (Fig 1, B and C). Treatment of TH2-skewing cultures with rShh also increased the concentration of IL-4 in supernatants after 4 days of culture compared with control TH2-skewing cultures (Fig 1, D). Interestingly, the concentration of IFN-γ was lower when rShh was added compared with control TH1 cultures (Fig 1, E). After 7 days of culture and anti-CD3/CD28 restimulation, the proportion of CD4 T cells that expressed IL-4 was significantly increased in the presence of rShh under TH2 conditions (Fig 1, F). In contrast, the percentage of cells that expressed IFN-γ was reduced in TH1 plus rShh cultures compared with TH1 cells (Fig 1, G). Shh treatment increased GATA3 and IL4 expression in TH2 cultures (Fig 1, H), whereas rShh treatment decreased IFNG and TBX21 (T-bet) expression in TH1 cultures (Fig 1, I). These data indicate that Hh signaling promotes TH2 differentiation in human CD4 T cells, with simultaneous repression of IFN-γ and T-bet.

We then investigated whether pharmacologic inhibition of the Hh signaling pathway by treatment with an inhibitor of the nonredundant Hh signal transduction molecule smoothened (SMO; PF-04449913) would impair TH2 differentiation. The proportion of cells that expressed the TH1 lineage–specific transcription factor T-bet was not affected by SMO inhibitor treatment under skewing conditions (Fig 2, A). Likewise, no differences were found in expression of GATA-3 under neutral or TH1 conditions (Fig 2, B). However, SMO inhibitor treatment significantly reduced the proportion of CD4 T cells that expressed GATA-3 and Ki-67 (a marker of proliferation) when cultured under TH2-skewing conditions (Fig 2, B and C). SMO inhibition did not affect the percentage of cells that expressed IFN-γ under TH1 conditions, and as expected, IL-4 expression was low under TH1 conditions in both control and SMO inhibitor–treated cultures (Fig 2, D). However, when cultured under TH2 conditions, the percentage of cells that expressed IL-4 was significantly reduced by SMO inhibitor treatment (Fig 2, E). Analysis of cytokine concentrations in culture supernatants by means of ELISA showed that IFN-γ levels were similar in both groups under TH1 conditions (Fig 2, F), but under TH2 conditions, significantly lower concentrations of IL-4 were found in the SMO inhibitor group compared with the control group (Fig 2, G). Finally, we investigated transcript levels of IL4 and IFNG by using quantitative RT-PCR. In TH2-skewed cells IL4 expression was significantly lower in SMO inhibitor–treated cultures than control cultures (Fig 2, H), whereas IFNG transcript levels were not different between groups under TH1 conditions (Fig 2, I). Taken together, these analyses indicate that attenuation of Hh signal transduction by treatment with the SMO inhibitor reduced TH2 differentiation but did not affect TH1 fate.

Fig 2

SMO inhibition decreases TH2 differentiation in vitro. Naive CD4 T cells (n = 12 donors) stimulated under TH-skewing conditions with SMO inhibitor (gray squares) or DMSO (control; open bars/squares) on day 4 (A-C, F, and G) and day 7 plus restimulation (D, E, H, and I). Scatterplots show means ± SEMs; each point represents an individual donor. Fig 2, A-C, Percentage of CD4+ cells that were positive for intracellular staining against T-bet (Fig 2, A), GATA-3 (Fig 2, B), and Ki-67 (Fig 2, C). Fig 2, D and E, FACS plots show expression of CD4 and intracellular IFN-γ (upper plots) or intracellular IL-4 (lower plots) in cells cultured under TH1 (Fig 2, D) or TH2 (Fig 2, E) conditions. Scatterplots show percentages of CD4+ cells that stained positive with the stated cytokine. Fig 2, F and G, Cytokine concentration (ELISA) in supernatants from TH1 (Fig 2, F) and TH2 (Fig 2, G) cultures. Fig 2, H and I, Gene expression (quantitative RT-PCR) in cells from TH2 (Fig 2, H) and TH1 (Fig 2, I) cultures (3 random donors). *P < .05, **P < .01, and ***P < .001, paired 2-tailed t test. ns, Not significant.

Here we show that Hh signaling promotes TH2 differentiation in human CD4 T cells. We found that treatment of naive CD4 T cells with rShh under TH2-skewing conditions increased expression of the transcription factor GATA-3, a reliable indicator of TH2 transcriptional identity. In support of this, IL4 expression was enhanced and IL-4 cytokine production was increased in TH2 cultures on treatment with rShh. In contrast, rShh treatment antagonized TH1 differentiation in TH1 cultures, leading to lower IFNG and TBX21 expression and a lower proportion of cells expressing intracellular IFN-γ. Attenuation of Hh signal transduction by pharmacologic SMO inhibition reduced TH2 differentiation: both GATA3 expression and IL4 expression were significantly decreased.

In murine TH differentiation Hh signaling promotes TH2 differentiation, skewing the overall pattern of transcription to a TH2-like profile, and Il4 is a GLI2 target gene in murine T cells. Importantly, Hh pathway activation in T cells has physiologic relevance in a murine model of allergic asthma because by favoring TH2 polarization and cytokine production, it contributes to disease severity.3, 7

In human subjects a genome-wide association study linked components of the Hh signaling pathway to allergic asthma, and a recent study found that children with asthma presented with greater levels of SHH in airway epithelia than healthy control subjects.

Here we provide in vitro evidence that Hh signaling enhances TH2 differentiation in human CD4 T cells. One strength of our study is that our experiments were performed with cells isolated from 12 different unknown leukocyte cone donors, and we obtained consistent experimental results from all donors independent of their age or sex (of which we had no knowledge). A weakness of our study is that it was limited to in vitro experimentation. In the future, it will be interesting to assess the TH differentiation status of T-cell populations isolated from samples from patients with asthma to obtain further ex vivo evidence that Hh signaling is involved in human TH2 responses. This will be important to our understanding of human atopic diseases, such as asthma, in which TH2 T-cell responses drive disease.