Sphingosylphosphorylcholine Promotes Th9 Cell Differentiation Through Regulation of Smad3, STAT5, and β-Catenin Pathways

Abstract

Sphingosylphosphorylcholine (SPC) is one of sphingomyelin-derived sphingolipids. SPC levels are increased in ascitic fluids of ovarian cancer patients and stratum corneum of atopic dermatitis (AD) patients. SPC has antitumor activity against several cancer cells by reducing proliferation and migration and increasing apoptosis in vitro. SPC can also cause scratching, potentially exacerbating symptoms of AD. However, the role of SPC in modulating immune responses, particularly in the differentiation of Th9 cells, which carry the most powerful antitumor activity among CD4⁺ T cells, has yet to be investigated. In this study, we found that SPC is another inducer of Th9 cell differentiation by replicating TGF-β. SPC upregulated Smad3, STAT5, and β-catenin signaling pathways. Increased Smad3 and STAT5 signaling pathways by SPC promoted the differentiation of Th9 cells and increased β-catenin signaling pathway resulted in a less-exhausted, memory-like phenotype of Th9 cells. Increased Smad3, STAT5 and β-catenin signaling pathways by SPC were mediated by increased mitochondrial ROS. These results suggest that SPC is an important endogenous inducer of Th9 cell differentiation and may be one of the targets for treating Th9-related diseases, and that enhancing Th9 differentiation by SPC may be helpful in adoptive T cell therapy for cancer treatment.

Keywords: Beta-catenin; Interleukin-9; Reactive oxygen species; STAT5 transcription factor; Smad3 protein; Sphingosylphosphorylcholine.

Figure 1. SPC enhances Th9 cell differentiation. (A-E) Mouse naïve CD4⁺ T cells were cultured under Th9 polarizing condition with various concentration of SPC for 3 days. (A) Flow cytometry analysis of IL-9⁺CD4⁺ T cells. (B) IL-9 production was measured by ELISA in the culture medium. (C) Expression levels of Il9 and (D) Irf4 were measured by real-time quantitative PCR. (E) Flow cytometry analysis of IRF4. (F) Mouse naïve CD4⁺ T cells were cultured under Th9 polarizing condition with or without SPC for 5 days. Expression level of Il9 was measured by quantitative PCR. (G) Human naïve CD4⁺ T cells were cultured under Th9 polarizing condition with or without SPC for 5 days. Flow cytometry analysis of IL-9⁺ CD4⁺ T cells. The results shown are representative of 3 independent experiments (A left and G left). Data are expressed as mean ± SD (n=3 for A right, B, E and G right, n=4 for C, D and F).

^*p<0.05, ^***p<0.001, and ^****p<0.0001 (Student’s t-test).

Figure 2. SPC enhances Smad3 signaling in a TGF-β independent manner. (A, B) Mouse naïve CD4⁺ T cells were cultured under Th9 polarizing condition with or without SPC. (A) GSEA of TGF-β signaling in SPC-treated Th9 cells compared with vehicle-treated Th9 cells. (B) Smad2/3 phosphorylation was measured by flowcytometry at 48 h. (C, D) Mouse naïve CD4⁺ T cells were cultured under Th2 polarizing condition with or without SPC. (C) Smad2/3 phosphorylation was measured by flowcytometry at 120 h. (D) Production of TGF-β was measured by ELISA in the cell culture medium. (E) Mouse naïve CD4⁺ T cells were cultured under Th2 polarizing condition with or without SPC in the presence of vehicle (D.W) or anti-TGF-β (10 µg/ml) for 5 days. IL-9 expression was detected by flow cytometry. (F-H) Mouse naïve CD4⁺ T cells were cultured under Th2 polarizing condition with or without SPC in the presence of vehicle (0.05% DMSO), SB431542 (10 µM) or SIS3 (3 µM) for 5 days. (F) Flow cytometry analysis of IL-9⁺ CD4⁺ T cells. (G) IL-9 production was measured by ELISA in the supernatant. (H) Expression level of Il9 was determined by quantitative PCR. The results shown are representative of 3 independent experiments (E and F left). Data are expressed as mean ± SD (n=3 for B, C, D, F right and G, n=4 for H).

D.W, distilled water. ^*p<0.05, ^***p<0.001, and ^****p<0.0001 (Student’s t-test).

Figure 3. SPC enhances STAT5 signaling by promoting IL-2 production. (A-C) Mouse naïve CD4⁺ T cells were cultured under Th9 polarizing condition with or without SPC for 3 days. (A) GSEA of IL-2-STAT5 signaling pathway in SPC-treated Th9 cells compared with vehicle-treated Th9 cells. (B) STAT5 phosphorylation was measured by flow cytometry at 48 h. (C) Flow cytometry analysis of IL-2⁺ CD4⁺ T cells. (D, E) Mouse naïve CD4⁺ T cells were cultured under Th9 polarizing condition with or without SPC for 5 days. (D) Expression level of Il2 was measured by quantitative PCR. (E) IL-2 production was measured by ELISA in the culture medium. (F, G) Mouse naïve CD4⁺ T cells were cultured under Th9 polarizing condition with or without SPC in the presence of vehicle (D.W) or anti-IL-2 (20 µg/ml) for 3 days. (F) Flow cytometry analysis of IL-9⁺ CD4⁺ T cells. (G) IL-9 production was measured by ELISA in the supernatant. The results shown are representative of 3 independent experiments (C left and F left). Data are expressed as mean ± SD (n=3 for B, C right, F right and G, n=4 for D and E).

D.W, distilled water. ^**p<0.01, ^***p<0.001, and ^****p<0.0001 (Student’s t-test).

Figure 4. SPC-treated Th9 cells have less-exhausted features by enhanced β-catenin signaling pathway and exhibit increased antitumor activity in vivo. (A-G) Mouse naïve CD4⁺ T cells were cultured under Th9 polarizing condition with or without SPC for 3 days. (A) GSEA of exhaustion associated gene sets in SPC-treated Th9 cells compared with vehicle-treated Th9 cells. (B) Heatmap of indicated gene expressions analyzed by RNA-seq. (C) Flow cytometry analysis of CTLA4 and LAG3. (D) GSEA of memory T cell associated gene sets in SPC-treated Th9 cells compared with vehicle-treated Th9 cells. (E) Expression level of Tcf7 was measured by quantitative PCR. (F) Flow cytometry analysis of TCF1. (G) Flow cytometry analysis of β-catenin at 48 h. (H) Mouse naïve CD4⁺ T cells were cultured under Th9 polarizing condition with or without SPC in the presence of vehicle (0.05% DMSO) or LF3 (20 µM) for 3 days. Flow cytometry analysis of LAG3. (I) C57BL/6 mice were injected intravenously with 3×10⁵ B16-OVA tumor cells. Mice were injected intravenously with PBS, OT-II Th9 cells or SPC-treated Th9 cells (2×10⁶) 4 days after tumor cell injection. On day 15, tumor foci were measured. Data are expressed as mean ± SD (n=3 for C and F to H, n=4 for E). Data are pooled from 3 independent experiments with 5–6 mice per group (I).

^*p<0.05, ^**p<0.01, ^***p<0.001, and ^****p<0.0001 (Student’s t-test for C, E-H, one-way ANOVA for I).

Figure 5. SPC-induced Th9 cell differentiation is mediated by mitochondrial ROS. (A) Mouse naïve CD4⁺ T cells were cultured under Th9 polarizing condition with or without SPC. Mitochondrial ROS was measured by flow cytometry at 1 h after vehicle (D.W) or SPC treatment. (B-F) Mouse naïve CD4⁺ T cells were cultured under Th9 polarizing condition with or without SPC in the presence of vehicle (D.W) or MitoTEMPO (200 µM) for 3 days. (B) Flow cytometry analysis of phosphor-Smad2/3, phosphor-STAT5 and β-catenin at 48–72 h. (C) Flow cytometry analysis of IRF4 and TCF1 (D) Flow cytometry analysis of IL-9⁺ CD4⁺ T cells. (E) IL-9 and IL-2 production were measured by ELISA in the culture medium. (F) Flow cytometry analysis of LAG3. Data are expressed as mean ± SD (n=3).

D.W, distilled water. ^*p<0.05, ^**p<0.01, ^***p<0.001, and ^****p<0.0001 (Student’s t-test).

Figure 6. SPC enhances Th9 cell differentiation and function by upregulating Smad3, STAT5, and β-catenin signaling pathways, mediated by increased mitochondrial ROS.