lncRNA-GM targets Foxo1 to promote T cell-mediated autoimmunity

Abstract

RNA-RBP interaction is important in immune regulation and implicated in various immune disorders. The differentiation of proinflammatory T cell subset T_H17 and its balance with regulatory T cell (T_reg) generation is closely related to autoimmune pathogenesis. The roles of RNA-RBP interaction in regulation of T_H17/T_reg differentiation and autoinflammation remain in need of further investigation. Here we report that lncRNA-GM polarizes T_H17 differentiation but inhibits iT_reg differentiation by reducing activity of Foxo1, a transcriptional factor that is important in inhibiting T_H17 differentiation but promoting T_reg generation. lncRNA-GM-deficient mice were protected from experimental autoimmune encephalomyelitis. Mechanistically, lncRNA-GM directly binds to cytoplasmic Foxo1, thus inhibiting its activity through blocking dephosphorylation of Foxo1 by phosphatase PP2A to promote Il23r transcription. The human homolog of lncRNA-GM (AK026392.1) also polarizes human T_H17 differentiation. Our study provides mechanistic insight into the interaction of lncRNA and transcriptional factor in determining T cell subset differentiation during T cell-mediated autoimmune diseases.

Fig. 1.. lncRNA-GM is selectively up-regulated in T_H17 cells.

(A) Quantitative polymerase chain reaction (qPCR) analysis of lncRNA-GM mRNA expression in T_H1, T_H2, nonpathogenic T_H17 (IL-6 + TGF-β), pathogenic T_H17 (IL-6 + IL-1β + IL-23), optimal pathogenic T_H17 (IL-6 + TGF-β + IL-1β + IL-23) cells, and iT_reg cells from mice spleen (n = 3 to 4). (B) qPCR of nT_reg cells (CD4⁺ CD25⁺) in thymocytes and iT_reg cells in splenocytes for lncRNA-GM mRNA expression (n = 3 to 4). (C) qPCR analysis of human lncRNA-GM mRNA expression in T_H1, T_H2, T_H17, and iT_reg cells from human peripheral blood mononuclear cells (n = 3). (D) Copy number analysis of lncRNA-GM determined by qPCR in T_H1 and T_H17 (optimal pathogenic induction) cells (n = 3). (E) Chromatin immunoprecipitation (ChIP)–qPCR analysis of the enrichment of H3K9ac, H3K27ac, and H4K4me3 in lncRNA-GM promoter region in naïve T, T_H1, T_H17, and iT_reg cells (n = 3 to 6). (F) qPCR analysis of lncRNA-GM mRNA expression in T_H17 cells treated with 20 μM CPI-637 (CPI; n = 4). Results are presented as means ± SD (A to F). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. NS, not significant; DMSO, dimethyl sulfoxide.

Fig. 2.. lncRNA-GM promotes T_H17 cell differentiation but inhibits T_reg cell differentiation in vivo and in vitro.

(A) Flow cytometric analysis of CD44⁺ and CD62L⁺ in splenic CD4⁺ T cells from WT and lncRNA-GM^−/− mice (n = 4). (B and C) Flow cytometric analysis IFN-γ⁺, IL-4⁺, IL-17A⁺, and Foxp3⁺ cells in splenic CD4⁺ T cells (B) and differentiated T_H1, T_H2, nonpathogenic T_H17 (IL-6 + TGF-β), pathogenic T_H17 (IL-6 + IL-1β + IL-23), optimal pathogenic T_H17 (IL-6 + TGF-β + IL-1β + IL-23), and iT_reg cells for 3 days (C) (n = 4 to 5). (D) Quantification of IFN-γ⁺, IL-4⁺, IL-17A⁺, and Foxp3⁺ T cells in differentiated T_H1, T_H2, nonpathogenic T_H17, pathogenic T_H17, optimal pathogenic T_H17, and iT_reg cells (n = 3 to 5). (E) Flow cytometric analysis and quantification of IL-17A⁺ cells transfected with control lentivirus and lncRNA-GM overexpression lentivirus in T_H17 cells (n = 4). Results are presented as means ± SD (A, B, D, and E). One of three representative experiments is shown. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3.. lncRNA-GM promotes T_H17 cell differentiation by activating mTORC1 signaling.

(A) Heatmap showing the representative up-regulated or down-regulated genes from WT and lncRNA-GM^−/− T_H17 cells (fold change > 1.5, P < 0.05). (B) qPCR analysis of Il17, Il17f, and Il23r mRNA expression in naïve T and T_H17 cells (n = 3). (C) qPCR analysis of Foxp3, Il10rb, and Gpr83 mRNA expression in naïve T and iT_reg cells (n = 3). (D) Cytometric bead array analysis of IL-17A and IL-17F in T_H17 cell supernatants and IL-10 in iT_reg cell supernatants from WT and lncRNA-GM^−/− mice (n = 3 to 4). (E) Gene set enrichment analysis of T_H17 cells from WT and lncRNA-GM^−/− mice. (F) Heatmap showing down-regulated mTOR pathway–related genes in lncRNA-GM^−/− T_H17 cells (P < 0.05). (G) Immunoblot analysis of mTORC1 and downstream signaling pathway in WT and lncRNA-GM^−/− naïve T, T_H0 (α-CD3/CD28), and T_H17 (α-CD3/CD28 plus IL-6 + TGF-β + IL-1β + IL-23) cells for 3 days. (H) Phosphorylation of mTOR, p70S6K, and 4EBP1 was measured by flow cytometry from WT and lncRNA-GM^−/− T_H17 cells (n = 3 to 4). (I and J) Flow cytometric analysis (I) and quantification (J) of CD4⁺ IL-17A⁺ cells in WT and lncRNA-GM^−/− T_H17 cells after treatment with DMSO and 50 nM rapamycin (Rapa; n = 4). Naïve CD4⁺ T cells were cultured in vitro under optimal pathogenic T_H17 cell polarizing conditions for 3 days. Results are presented as means ± SD (B, C, D, H, and J). One of three representative experiments is shown. *P < 0.05; **P < 0.01; ***P < 0.001. FDR, false discovery rate. NES, normalized enrichment scores; MFI, mean fluorescence intensity.

Fig. 4.. lncRNA-GM directly interacts with Foxo1 and attenuates Foxo1 dephosphorylation by PP2A.

(A and B) RIP-qPCR analysis of lncRNA-GM and lncLrrc55-AS immunoprecipitated by Flag antibody in HEK293T (A) and lncRNA-GM and lnc-Dpf3 immunoprecipitated by Foxo1 or IgG in T cells (B) (n = 3 to 4). (C) RNA FISH analysis of lncRNA-GM (red) and Foxo1 (green) in T_H17 cells. DNA (blue) was stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar, 2.5 μm. (D) Immunoblot analysis of Foxo1 levels in T_H17 and iT_reg cells. (E and F) ChIP-qPCR analysis of the recruitment of Foxo1 to Il23r and Il17 promoter regions (E) and RORγt to Il23r and Il17 promoter regions (F) in T_H17 cells and the recruitment of Foxo1 to Foxp3 promoter regions in WT iT_reg cells (n = 3 to 4). (G) ChIP-qPCR analysis of the recruitment of Foxo1 to Foxp3 promoter regions in iT_reg cells (n = 3). (H) Immunoblot analysis of Foxo1 and p-Foxo1 (Ser²⁵⁶) levels in cytoplasm and nuclear extracts from T_H17 cells. (I) Immunofluorescence analysis of Foxo1 (green) or IgG (green) in naïve T or T_H17 cells. DNA (blue) was stained with DAPI. Scale bar, 2.5 μm. (J) Immunoblot analysis of PP2A in T cells after IP with anti-Foxo1 antibody. (K and L) Flow cytometric analysis of IL-17A⁺ cells in T_H17 (K) after treatment with DMSO or 50 nM AS1842856 (AS) and Foxp3⁺ cells in iT_reg (L) after treatment with 200 nM AS1842856 (n = 4 to 5). Results are presented as means ± SD (A, B, E, F, G, K, and L). One of three representative experiments is shown. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 5.. Deficiency of lncRNA-GM attenuates pathogenesis of active EAE.

(A and B) Mean clinical score (A) and peak clinical score (B) of WT and lncRNA-GM^−/− mice after induction of EAE with MOG_35–55 and pertussis toxin (n = 6). (C to H) At day 28 after EAE induction, WT and lncRNA-GM^−/− mice were analyzed for spleen size and weight (n = 5) (C), quantification of CD4⁺ T cells in the brain and mesenteric lymph node (mLN; n = 3 to 6) (D), quantification of IL-17A⁺, IFN-γ⁺, and Foxp3⁺ T cells in the brain, spleen, and spinal cord (SC; n = 4 to 6) (E and F), and histology of hematoxylin and eosin–stained brain (G) and spinal cord (H). Results are presented as means ± SD (A, B, C, D, E, and F). One of three representative experiments is shown. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 6.. Deficiency of lncRNA-GM reduces severity of EAE by T cell adoptive transfer.

(A and B) Mean clinical score (A) and peak clinical score (B) of Rag2^−/− recipients of WT and lncRNA-GM^−/− CD4⁺ T cells after induction of EAE (n = 7). (C and D) At day 28 after EAE induction, Rag2^−/− recipient mice were analyzed for quantification of CD4⁺ and CD8⁺ T cells in the spleen (n = 4) (C) and quantification of CD4⁺ IL-17A⁺, IFN-γ⁺, and Foxp3⁺ T cells in the spleen, brain, and spinal cord (n = 3 to 6) (D). Results are presented as means ± SD (A to D). One of three representative experiments is shown. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.