Abstract
IL17-producing T cells have been recently described as a distinct T cell helper population (Th17 cells) characterized by expression of membrane CD4 and IL23R and intracellular expression of the orphan nuclear receptor RORgt. In Th17 cells the transcription factor RORgt induces the transcription of IL17 gene, whereas in Th1 cells the transcription factor Tbet is responsible for the transcription of IFNg gene. Th1 along with Th17 cells are thought to contribute to the pathogenesis of autoimmune diseases. In murine models Th17 cells are fully polarized. In humans a proportion of Th17 cells are also positive for interferon gamma (IFN-g); they are named Th1/17 cells and their function is yet unclear. In patients with colitis and seronegative arthritis Th17 cells are increased. The induction of Th17 and Th1/17 in patients with MDS has not been previously evaluated. To examine the expression of Th17 and Th1/17 cells in this disease, peripheral blood mononuclear cells (PBMC) from patients with MDS were cultured in vitro for 6 days in RPMI-1640, 15% FBS supplemented with PHA (0.1 μg/mL) and IL-2 (10 ng/mL). Percentages of CD4+IL23R+IL-17+ T cells (Th17) and CD4+IL23R+IL17+IFN-g+ T cells (Th1/17) in patients with MDS were determined by flow cytometry: Th17 cells were markedly increased in patients (n=30) compared to healthy controls (n=15), (17.5% ± 3.4 vs 2.5% ± 0.4, p=0.008). Th1/Th17 cells were also significantly increased in MDS patients compared to controls (15.17% ± 2.80 vs 2.56% ± 0.80, p=0.008). None of the patients had been on immunosuppressive treatment or transfused before sampling. In multi-transfused patients with no underlying hematologic disease examined (n=3) the Th17 and Th1/17 populations were comparable to those of healthy donors. In patients with MDS the majority of the Th17 cells expressed also IFNg (90.07% ± 2.87) whereas in healthy controls only 59.7% ± 5.5 of the Th17 cells were also positive for IFNg (p<0.0001). There were no differences between different subtypes of MDS (RA, RARS, and RAEB). Using confocal microscopy, purified CD4+ T cells from PBMC cultures from patients (n=5) showed increased Tbet and RORgt expression at the single-cell level compared to controls (n=3),(T-bet: 22.03 ± 1.20 vs 11.60 ± 0.35 arbitrary units respectively, p<0.0001 and RORãt: 28.90 ± 0.35 vs 21.03 ± 1.20 arbitrary units, p=0.0008. For each sample 100 cells were analyzed). We next asked whether kinases involved in the induction of Tbet are also involved in the induction of RORgt. We analyzed the effects of rottlerin, a PKC-theta inhibitor, SB203580, a p38 MAPK pathway inhibitor, and PD98059, an ERK pathway inhibitor, on Th17 and Th1/17 cell induction in patients (n=7) and controls (n=4). Rottlerin decreased the Th17 content in patients and controls by 45.0%, and the Th1/17 content by 64.8%. SB203580 showed a 17% and 18% decrease on Th17 and on Th1/17 content, respectively, in patients and controls. PD98059 showed no effect on Th17 and Th1/17 populations in patients and controls. By immunoblots, in normal CD4+T cells rottlerin decreased both T-bet and RORgt protein levels by 50% and 20%, respectively. SB203580, decreased RORgt levels by 25%, and PD98059 did not obviously decrease Tbet but decreased RORgt levels by 20%.
CD4+IL23R+IL-17+ T cells and CD4+IL23R+IL17+IFN-g+ T cells are increased in most patients with MDS. T cells have recently been implicated in MDS pathogenesis. Although more studies are needed in order to define the role of Th17 and Th1/17 cells in the pathogenesis of MDS, our in vitro data with the kinase inhibitors may suggest a probable therapeutic target for patients with MDS.
Cutting Edge: The Transcription Factor Kruppel-Like Factor 4 Regulates the Differentiation of Th17 Cells Independently of RORγt