Early Growth Response Gene 1 Benefits Autoimmune Disease by Promoting Regulatory T Cell Differentiation as a Regulator of Foxp3

Abstract

Foxp3⁺ regulatory T (T_reg) cells, as one of the subtypes of CD4⁺ T cells, are the crucial gatekeeper in the pathogenesis of self-antigen reactive diseases. In this context, we demonstrated that the selective ablation of early growth response gene 1 (Egr-1) in CD4⁺ T cells exacerbated experimental autoimmune encephalomyelitis (EAE) in murine models. The absence of Egr-1 in CD4⁺ T cells, obtained from EAE mice and naïve CD4⁺ T cells, impeded the differentiation and influence of T_reg. Importantly, in CD4⁺ T cells of multiple sclerosis patients, both Egr-1 and Foxp3 were found to decrease. Further studies showed that distinct from the classical Smad3 route, TGF-β could activate Egr-1 through the Raf-Erk signaling route to promote Foxp3 genetic modulation, thereby promoting T_reg cell differentiation and reducing EAE inflammation. A novel natural Egr-1 agonist, calycosin, was found to attenuate EAE progression by regulating the differentiation of T_reg. Together, the above results indicate the value of Egr-1, as a novel Foxp3 transactivator, for the differentiation of T_reg cells in the development of self-antigen reactive diseases.

Fig. 1.

Expression of Egr-1 in CD4⁺ T cells was inversely related to the severity of EAE in mice. (A) RNA sequencing screened genes differentially expressed in CD4⁺ T cells in the spleen of EAE mice with mild disease (neurofunctional score of 0.5) and severe disease (neurofunctional score of 4). (B) Comparison of mRNA manifestation of Egr-1 in CD4⁺ T cells of EAE mice with mild disease (neurofunctional score of 0.5) and severe disease (neural functional score of 4) by qPCR. n = 4. (C and D) Egr-1 manifestation in EAE mice with different scores and correlation analysis of Egr-1 manifestation in CD4⁺ T cells with EAE scores. n = 4. SP, spleen; LN, lymph nodes. (E to G) CD4⁺ T cells in PBMCs of treatment-naïve MS patients and healthy donors. Afterward, the cells were stimulated with anti-CD3 and anti-CD28 in vitro for 24 h, and the manifestation of Egr-1 and Foxp3 was analyzed. n = 5. (H) EAE was induced in CWT and CKO mice by immunizing with MOG_33–55 peptide and neurobehavioral defects. n = 11. (I and J) H&E and LFB staining pathological assessment of spinal cords from CWT and CKO EAE mice on day 21 post-immunization. n = 4. Data are expressed as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 2.

CD4⁺Egr-1 knockout regulated the subtypes of CD4⁺ T cell differentiation in EAE mice. (A) Proportions of T_reg/T_H17/T_H1 cell subsets in spleen, lymph nodes, and CNS from CWT (Egr-1^f/f) and CKO (Egr-1^f/f CD4-cre⁺) EAE mice. (B) Percentage of Foxp3-, IL-17-, and IFN-γ-positive cells. n = 4. (C and D) Representative flow cytometric chart and statistical bar graphs of the percentages of Foxp3- and IL-17-positive cells. n = 3. (E) Representative CFSE fluorescence flow cytometric chart of the inhibition of T_conv cells by T_reg polarized from naïve CD4⁺ T cells sorted from CWT and CKO mice and incubated for 3 d under T_reg polarization conditions. These cells were co-incubated with CFSE-labeled responder naïve CD4⁺ T cells. (F) Statistical analysis of the suppression rate of the T_reg from CWT and CKO mice on T_conv cells. Suppression % = (1 − CFSE fluorescence reduction percentage of T_conv cells) × 100%. n = 4. Data are expressed as mean ± SD. ns (not significant), P > 0.05; *P < 0.05; ***P < 0.001.

Fig. 3.

Adoptive transfer of CD4⁺ T cells of CKO EAE mice led to the aggravation of EAE in WT mice. (A) Experimental scheme of the adoptive transfer. CKO and CWT mice were immunized with MOG_35–55 for 15 d. Then, CD4⁺ T cells from lymph node cells were incubated with MOG_35–55 (20 μg/ml) and IL-12 (0.5 ng/ml) for 3 d. The treated cells (1 × 10⁷ cells/mouse) were then injected into WT C56BL/6 recipient mice through the tail vein. (B) Clinical score. n = 6. (C) H&E and LFB staining and inflammatory infiltration and demyelination scores in the spinal cord. (D) Proportion of CD4⁺ T cells in spleen, lymph nodes, and CNS of mice. n = 3 to 4. (E) Proportion of CD4⁺Foxp3⁺T_reg cells in CNS, spleen, and lymph nodes of mice. (F) Proportion of CD4⁺IL-17⁺T_H17 cells in CNS, spleen, and lymph nodes of mice. (G) Proportion of CD4⁺IFN-γ⁺T_H1 cells in CNS, spleen, and lymph nodes of mice. Data are expressed as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 by Student’s t test.

Fig. 4.

Egr-1 induced the manifestation of Foxp3 by connection to its promoter site. (A) mRNA manifestation of Foxp3 and RORγt in CD4⁺ T cells sorted from the spleens of CWT and CKO mice, after stimulation with anti-CD3 (5 μg/ml) plus anti-CD28 (2 μg/ml) for 6 h. (B) mRNA manifestation of Foxp3 under T_reg polarization condition. (C) Protein manifestation of Egr-1 and Foxp3 in CD4⁺ T cells of CWT and CKO EAE mice. (D) Protein manifestation of Egr-1 and Foxp3 in Jurkat cells transfected with PCB6-Egr-1 and PCB6 empty plasmids. (E) Egr-1 protein manifestation in naïve CD4⁺ T cells sorted from normal mice and transfected with PCB6-Egr-1 plasmid and PCB6 control plasmid. (F) Expression of Foxp3 in naïve CD4⁺ T cells stimulated with TCR under neutral conditions and transfected with either PCB6-Egr-1 plasmid or PCB6 control plasmid in the presence of anti-TGF-β or control IgG. n = 4. (G) Schematic diagram depicting and the putative Egr-1 connection site (−131 bp to −124 bp). (H and I) ChIP analysis performed using a negative control immunoglobulin G (IgG) or anti-Egr-1 antibody in CD4⁺ T cells of EAE mice. (J) Schematic diagram depicting the sequences in the WT and mutant (MT) Foxp3 stimulator. (K) Luciferase activity analysis in EL4 T cells transfected with Foxp3 Promoter-WT plasmid and Foxp3 Promoter-MT plasmid after overexpressing Egr-1. n = 6. Data are expressed as mean ± SD. ns, P > 0.05; ***P < 0.001.

Fig. 5.

TGF-β regulated the manifestation of Egr-1 in CD4⁺ T cells through the Ras/Raf/Mek/Erk signaling route. (A) Representative flow cytometric chart of Egr-1 manifestation in naïve CD4⁺ T cells stimulated with TCR in the absence or presence of TGF-β and IL-2 for 24 h. (B) Statistical analysis of the percentage of Egr-1-positive cells. (C and D) Protein manifestation stimulated with TGF-β. (E and F) Protein manifestation after pretreatment with GW5074 (inhibitor of Raf, 10 μM). (G and H) Protein manifestation after pretreatment with U0126 (inhibitor of Erk and Mek, 10 μM). n = 4. (I and J) Expression of Raf/Mek/Erk/Egr-1 protein after adding SIS3 (1 μM) during T_reg differentiation. (K and L) Proportion of T_reg cell differentiation. Data are expressed as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 6.

Egr-1 agonist CAL and FN had curative impact on EAE mice. (A) Screening of Egr-1 agonists by using ERE luciferase reporter gene. n = 5 to 6. (B) Daily neurobehavioral assessment of EAE mice treated with CAL, FN, FTY-720, or vehicle. n = 8. (C and D) Daily neurobehavioral assessment of EAE mice treated with CAL (10, 20, and 40 mg/kg), FTY-720, or vehicle. n = 9. (E) Neurobehavioral assessment. n = 9. (F) Body weight of EAE mice. n = 9. (G) Cumulative score and peak score. n = 9. (H and I) H&E and LFB staining and inflammatory infiltration and demyelination scores in the spinal cord of EAE mice. n = 3. Data are expressed as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 7.

Egr-1 agonist CAL affected T_reg differentiation and influence through CD4⁺Egr-1. (A) Schematic diagram of T_reg differentiation. (B and C) Different concentrations of CAL were added during CWT/CKO T_reg differentiation, and the ratio of Foxp3⁺T_reg cells was detected. n = 3. (D) IL-10 secretion in T_reg cell incubation medium. n = 3. (E) Schematic diagram of T_reg immunosuppression. (F) Representative CFSE fluorescence flow cytometric chart of the inhibition of T_conv cells by T_reg polarized from naïve CD4⁺ T cells sorted from CWT and CKO mice and incubated with 40 μM CAL for 3 d under T_reg polarization conditions. These cells were co-incubated with CFSE-labeled responder naïve CD4⁺ T cells. (G) Statistical analysis of the suppression rate of the T_reg on T_conv cells. n = 3. Data are expressed as mean ± SD. ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 8.

Clinical drugs for MS such as FTY-720 and GA could activate Egr-1 manifestation. (A) After 11 d of EAE activation in ERE-EGFP mice, FTY-720 (1 mg/kg), GA (0.15 mg/mouse), IFN-β (1 × 10⁴ U/mouse), and DEX (0.07 mg/mouse) were administered. n = 5. (B) Daily neurobehavioral assessment and score statistics. n = 5. (C) Cumulative score and peak score of EAE mice. n = 5. (D and E) H&E and LFB staining and inflammatory infiltration and demyelination scores in the spinal cord. n = 4. (F and G) Proportion of CD4⁺Egr-1⁺ cells in lymph nodes of EAE mice. n = 3. Data are expressed as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001, versus EAE group.