Sp3 улучшила экспериментальный аутоиммунный энцефаломиэлит, спровоцировав Socs3 в клетках Th17.

Introduction

Background

EAE is the ideal mouse model for human MS with important significance in clinical neuroimmunology research. Both MS and EAE are believed to be T helper (Th) cell-mediated chronic inflammatory and degenerative diseases characterized by the infiltration of mononuclear cells, demyelination, and axonal breakdown degeneration in the central nervous system (CNS) [1] , [2] , [3] , [4] , [5] , [6] . Recent data have established the pivotal role of Th17 cells in the pathogenesis of EAE. The differentiation of Th17 cells must be carefully controlled to avoid inflammation-induced pathogenesis [7] , [8] , [9] . Numerous transcription factors negatively affect the development of Th17 cells. As negative regulators of Th17 cell development, for instance, forkhead box P3 (Foxp3), growth factor independent 1 (Gfi1), signal transducer and activator of transcription 5 (Stat5), and Ets1 have been found [10] , [11] . Recent reports have shown that Foxo1 [12] , [13] , [14] , [15] , [16] , PPAR, choline acetyltransferase [9] negatively regulate Th17 cell differentiation. Despite extensive research on Th17-specific regulators, the reasons behind aberrant Th17 production during MS pathogenesis are still unknown.

Grekova MC reported that the majority of individuals with MS exhibited a deficiency in the presence of SP3 in their peripheral blood mononuclear cells [17] . Subsequent research has indicated that typical levels of SP3 mRNA are present in normal CD4 + and CD8 + T cells, B cells, and macrophages. Conversely, natural killer cells from healthy individuals and SP3-negative MS patients do not exhibit any SP3 expression [18] . It is suggested that the transcription of the SP3 gene is hindered in immune cells of the majority of MS patients, thereby contributing to the onset of CNS inflammation in the condition [19] . Inadequate SP3 gene expression has been observed in Chinese individuals with MS, and it is proposed that SP3 expression may be linked to the clinical symptoms of MS and may have a role in its immunological pathogenesis [20] . It is possible that Sp3 gene transcription is suppressed in immune cells during EAE and MS progression. However, the role of Sp3 in autoimmune CNS inflammation is unknown, and whether Sp3 plays a role in neuroinflammation remains unclear.

EAE is a multifactorial disorder with prominent involvement of both cellular and epigenetic components. Among the epigenetic factors, miRNAs are currently intensively investigated in the context of MS [21] , [22] , [23] . Moreover, accumulating evidence has shown that miRNAs play a crucial role in regulating the differentiation and functionality of T helper cells. Specifically, miR-17-92, miR-21, miR-23b, miR-132, miR-155, miR-326, and miR-374c have been identified as key promoters of Th17 cell differentiation and exacerbation of EAE disease severity [24] , [25] , [26] , [27] , [28] , [29] . miR-142a-5p overexpression in activated lymphocytes shifts the T cell differentiation profile towards Th1 cells and contributes to the development of autoimmune neuroinflammation [30] . Furthermore, miRNA sequencing analysis has shown that miR-30a is significantly downregulated during the differentiation of Th17 cells in mice and in CD4 T cells isolated from the peripheral blood of individuals with MS and EAE [31] . Previously, we found that miR-181c facilitates the differentiation of Th17 cells and mediates EAE. Additionally, the non-coding RNA lincRNA-Cox2 was found to regulate the NLRP3 inflammasome and autophagy, which are involved in neuroinflammation [32] , [33] . We also demonstrated that miR-223 inhibits microglia autophagy and enhances CNS inflammation [34] . MiR-223 is ubiquitously present in various tissues such as the brain, thymus, muscle, and bone marrow, and its levels have been observed to fluctuate in reaction to stress. Clinical investigations have indicated a significant upregulation of miR-223 in individuals with MS, suggesting that the expression pattern of miR-223 holds promise as a diagnostic biomarker for MS [35] . Kasey C. Vickers has indicated that the overexpression of miR-223 leads to a significant repression of Sp3 mRNA levels, while inhibition of miR-223 results in a marked upregulation of Sp3 mRNA levels [36] . MiR-223 knockout mice exhibit a less severe form of EAE, characterized by elevated numbers of myeloid-derived suppressor cell (MDSC) [37] , as well as reduced infiltration of Th1 and Th17 cells in the spinal cord [38] . However, there have no reports concerning whether miR-223 directly affects CD4 + T cells during EAE or MS progression.

In this study, we discovered the deficient expression of Sp3 and the high expression of miR-223 in EAE mice. Through the use of TargetScan software and luciferase reporter system analysis, we determined that miR-223 regulates Sp3 by targeting its 3′UTR. Subsequent, overexpression of Sp3 or knockdown of its regulator miR-223 resulted in improved CNS inflammation, demyelination and EAE clinical signs. Additionally, our findings revealed that the regulation of Socs3 by Sp3 played a crucial role in modulating the Stat3, TGF-β-Smad2/3 and RORγt signaling pathways, which are involved in Th17 cell differentiation and CNS inflammation.

Results

Sp3 expression is deficient in MS patients and the spleen of EAE mouse

Grekova MC [17] and researchers from China [20] have demonstrated that there is a reduced Sp3 expression in MS patients. They proposed that Sp3 expression could be involved in the immunological pathogenesis of MS. The clinical single cell sequencing dataset GSE131282 was utilized, which encompassed the mRNA expression profiles through array analysis in the brain of 37 MS patients and 42 individuals. It was found the deficiency expression of SP3 in MS patients ( Fig. 1 a, p  < 0.05). Sp3 is found to be overexpressed in CD4 T cells, CD8 T cells, NK cells, and Lymph nodes in 92 normal tissues or compartments, as indicated by Genecards database analysis ( Fig. 1 b). We also detected decreased expression of Sp3 during the EAE peak phase ( Fig. 1 c and 1d).

Sp3 is high expressed in normal tissues and was blocked in the spleen, overexpression of Sp3 attenuates the clinical symptoms and inflammation in the CNS of EAE mice. a The expression of SP3 in brain from 42 health and 37 MS patients in GEO ( GSE131282 ). b The expression of SP3 from 92 normal tissues or compartments of human in genecards ( www.genecards.org ). c The expression of Sp3 was checked by real-time PCR in the spleen from normal wild type C57BL/6 and EAE mice. Changes in mRNA levels were quantified using the 2-ΔΔCT method with mGapdh mRNA as a control. Data were from three independent experiments (mean ± SD). * p  < 0.05; ** p  < 0.01, as determined using the Student’s t test. d The protein levels of Sp3 was tested by immunoblotting. β-actin was used as the loading control. EAE was induced by MOG 35-55 in female C57BL/6 mice (n = 6).The clinical scores of all the EAE mice were assessed daily according to the same criteria for 24 continuous days. Incidence ( e ), disease onset ( f ), daily clinical scores ( g ), cumulative disease scores ( h ), and peak disease scores ( i ) were monitored. e The incidence of EAE (mice with a clinical score ≥ 1 for 2 continuous days). The disease incidence is represented by the percentage of mice suffering from EAE. f The line graph depicts the survival rate of EAE between WT and LV-Sp3 mice. g The clinical scores of all the mice for 24 continuous days. h The mean disease cumulative score. i The mean maximum clinical score. j Hematoxylin and eosin (H&E) staining of representative spinal cord sections from LV-Sp3 mice and WT mice showing the infiltration of inflammatory cells in the white matter. k , m Showing the infiltration of inflammatory cells or demyelination in the white matter. l Luxol fast blue staining of intact myelin (blue) and demyelination (pink). The data are representative of at least two experiments with similar results. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Overexpression of Sp3 ameliorated clinical symptoms and inflammation in the CNS of EAE mice

To assess the influence of Sp3 on MS, we analyzed the neurobehavioral impairments in mice treated with LV-Sp3 and a control substance after inducing EAE with MOG35–55. First, we assessed the lentivirus effect of LV-Ctrl, LV-anti-Sp3, and LV-Sp3 through immunoblot assays ( Fig. S1 a). The EAE mouse model exhibited a chronic progressive-relapsing disease course, as illustrated in Fig. 1 . Overexpressing Sp3 reduced neurobehavioral impairments, cumulative scores, and maximum neurological disability in EAE mice ( Fig. 1 e-1i, p  < 0.05). To assess the advancement of the disease in terms of CNS damage, a histological examination was performed on day 18. Histologically, a noticeable decrease in the infiltration of mononuclear cells, which are crucial components of the immune system, and a marked reduction in the process of demyelination, characterized by the loss of the protective myelin sheath surrounding nerve fibers, were observed during the acute phase of EAE ( Fig. 1 j-1m, p  < 0.05), suggesting a profound alteration in the immune response and its impact on the CNS during this critical period of the disease. Thus, the overexpression of Sp3 demonstrated an ameliorative effect on both the symptoms and pathology of EAE.

We also examined the effect of overexpression of the Sp3-2 isoform during EAE progression. However, no improvements of the clinical symptoms of EAE were observed ( Fig. S1 b-1f). It is possible that the Sp3-2 isoform did not contribute on EAE progression, even if it is the target of miR-223. Simultaneously, knockdown of Sp3 (Sp3 +/- or LV-anti-Sp3) could convert the Sp3 effect on EAE progression, and led to more serious or similar clinical features as in LV-Ctrl mice ( Fig. S1 b-1j). These findings indicated that Sp3 contributed to the advancement of EAE.

Overexpression of Sp3 suppressed Th1 and Th17 cells both in vivo and in vitro

Next, we investigated the impact of Sp3 on the differentiation of CD4 + T cell in various tissues of mice with EAE. The findings revealed that the overexpression of Sp3 led to a notable reduction in the polarization of CD4 + IL-17A + and CD4 + IFN-γ + cells in the spleen, lymph node, and CNS ( Fig. 2 a-2c). We tested the relationships and key cytokine gene expression of IL-17A and IFN-γ by real-time PCR in spleen and spinal cord, demonstrating a significant decrease in their expression upon Sp3 overexpression ( Fig. 2 d and 2e, p  < 0.05).

Overexpression of Sp3 suppressed Th1 and Th17cells in vivo and in vitro . Flow cytometry analysis of activated signature markers of Th1 and Th17 (IFN-γ and IL-17A) cells on the range of CD4 + cells isolated from the spleen, lymph nodes and CNS of LV-Ctrl and LV-Sp3-treated EAE mice (n = 6 mice per group) detected on the day 18 after the induction of EAE. Data are shown in a representative plot of the Th1 and Th17 cell subpopulations in three types of tissues ( a - c ). The percentage of respective subpopulations in CD4 + cells summarized by histograms. Treatment of cells isolated from the spleen, lymph nodes and CNS of LV-Ctrl and LV-Sp3-treated EAE mice was performed according to the description in the “Intracellular cytokine staining” section. Cells from the spleen ( d ) and lymph nodes ( e ) of LV-Ctrl and LV-Sp3-treated EAE mice were collected, and total RNA was extracted based on the manufacturer’s instructions using TRIzol reagent. The quantity of mRNA was recorded using real-time PCR. Gene expression was normalized based on the mGapdh content. f Naïve CD4 + T cells were separated from the spleen of female C57BL/6 mice, and then Th1 and Th17 cells were induced under various conditions according to the “Naïve CD4 + T cell preparation and differentiation” section. The cells were evaluated per the description in the “Intracellular cytokine staining” section. g , h ELISA was used to assess the expression of IFN-γ and IL-17A in the supernatants of Th1 and Th17 cells. Data were from three independent experiments (mean ± SEM). * p  < 0.05; ** p  < 0.01, as determined using the Student’s t test.

Consistent results were obtained in vitro , where similar outcomes were observed ( Fig. 2 f). Furthermore, ELISA analysis of the supernatant from Th17 and Th1 cell cultures indicated a significant decrease in the secretion of cytokines IL-17A and IFN-γ in cells transfected with LV-Sp3 ( Fig. 2 g and 2h, p  < 0.05).

MiR-223 target Sp3 and is elevated in MS and other autoimmune diseases patient

Sp3 expression is deficient in individuals with MS and was found to be inhibited in immune cells. Furthermore, investigations using Southern blot and polymerase chain reaction techniques on genomic DNA have not revealed any discernible variations between PBMCs from individuals with MS and those from control subjects [18] . However, the underlying mechanism and how the deficiency of Sp3 occurs in MS patients remain unclear. We hypothesize that Sp3 may be regulated in mRNA level and miRNAs have some unknown effects on MS. miRNAs serve as effective regulators of gene expression through their ability to target the 3′UTR of genes. We search the miRNAs in 3′UTR of Sp3 by TargetScan in mouse and human ( Fig. 3 a). It was found that 11 miRNAs could target in 3′UTR of Sp3 in both mouse and human. Research indicates that miRNAs exhibit notable stability in human blood and are recognized as potential clinical biomarkers for specific diseases. In order to investigate the changes in miRNA expression among individuals with MS, an examination was conducted on the Gene Expression Omnibus database to retrieve and evaluate raw data pertaining to miRNA expression profiles. Specifically, the clinical dataset GSE31568 was utilized, which encompassed the miRNA expression profiles of 863 miRNAs through array analysis in the peripheral blood cells of 23 MS patients and 22 individuals without the condition [39] . In our analysis, it was observed that miR-223 exhibited a notable upregulated in MS patients within the clinical datasets ( Fig. 3 b). Specifically, the clinical dataset GSE17846 contained the expression profiles of 866 miRNAs through array analysis conducted on peripheral blood cells obtained from 20 individuals diagnosed with relapsing-remitting MS, alongside 21 healthy control subjects [40] . Notably, our findings indicated a significant upregulation of miR-223 in MS patients within these clinical datasets ( Fig. 3 b).

miR-223 is high expressed in MS patients, EAE, and Th17 cells. a Heatmaps indicate the expression of miR-223 from samples of human MS patient blood data sets. b The network indicates miR-223 expression upregulation in human diseases by red nodes and downregulation by blue nodes. The expression of miR-223 was tested by real-time PCR. Changes in mRNA levels were quantified using the 2-ΔΔCT method with mU6 mRNA as a control. Data were from three independent experiments (mean ± SD). * p  < 0.05; ** p  < 0.01, as determined using the Student’s t test. c, d The expression of miR-223 was checked by real-time PCR in the spleen and spinal cord from normal wild type C57BL/6 and EAE mice. e Naïve CD4 + T cells were separated from the spleen of female C57BL/6 mice and then differentiated to Th1, Th2, Th17 and iTreg cells under various conditions for 4 days. The cells were harvested, and RNA was extracted to evaluate mRNA expression. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

In order to investigate the potential associations of the notably dysregulated miR-223 with various human diseases, an analysis was conducted utilizing the ‘‘Human microRNA Disease Database (HMDD)’’, which encompasses data on dysregulated miRNAs across over 100 human diseases. A bipartite graph was constructed, with red nodes denoting elevated expression levels and blue nodes representing reduced miR-223 expression. The network, which is shown in Fig. 3 c, contained 42 kinds of diseases involving significantly abnormal expression of miR-223. HMDD has collected disease information up to 2014. Thus, we added new diseases to the network that have been described since 2014. MiR-223 was downregulated in 17 and upregulated in 26 kinds of disease. It is noteworthy that the expression of miR-223 was elevated in various autoimmune conditions such as MS, systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA). This observation led to the hypothesis that miR-223 could potentially have significant implications in the modulation of autoimmune disorders.

High expression of miR-223 in the spleen and spinal cord of EAE mice and the Th17 cell population in vitro

To probe the roles of miR-223 in expanding EAE, CD4 + T cells were extracted from the spleen and spinal cord of both EAE and healthy control mice. Subsequently, miRNA levels were assessed using real-time PCR. Our analysis revealed an increased expression of miR-223 in both the spleen and spinal cord as EAE progressed ( Fig. 3 d and 3e, p  < 0.05). Furthermore, in an in vitro examination, heightened levels of miR-223 were observed in Th17 cells during their differentiation from CD4 + T cells extracted from the spleen ( Fig. 3 f).

Knockdown of miR-223 ameliorated clinical symptoms and inflammation in the CNS of EAE mice

Previous research has indicated the significant involvement of specific miRNAs in the regulation of MS and EAE. For instance, a study highlighted the role of miR-223 in regulating EAE through MDSCs [41] . However, the direct impact of miR-223 on Th17 cells, which are crucial mediators of EAE, has not been fully elucidated. In this study, it was observed that miR-223 was elevated during Th17 cell differentiation. The investigation aimed to assess whether endogenous miR-223 levels in Th17 cells could influence the clinical manifestations of EAE and the underlying mechanism within Th17 cells. To evaluate this hypothesis, the effect of miR-223 knockdown by LV-anti-miR-223 lentivirus was examined in EL4 cells and mice during EAE. The lentiviral knockdown effectively suppressed the gene expression of miR-223 ( Fig. S2 a and 2b, p  < 0.05). Interestingly, the inhibition of miR-223 (LV-anti-miR-223) resulted in a significant improvement in the clinical symptoms of EAE, as evidenced by lower EAE clinical scores and reduced disease incidence ( Fig. S2 c-2e, p  < 0.05). Moreover, the severity of the disease, as measured by maximal and cumulative clinical score, was notably diminished in mice treated with LV-anti-miR-223 ( Fig. S2 f and 2 g, p  < 0.05). These findings suggest that endogenous levels of miR-223 have an impact on the clinical outcomes of EAE.

To further characterize the progression of the disease in terms of CNS injury, a histological analysis of the lumbar spinal cord was conducted. In the lumbar spinal cords of LV-anti-miR-223 treated mice during EAE's acute phase, hematoxylin and eosin (H&E) and Luxolfast blue (LFB) staining showed a decrease in mononuclear cell infiltration and demyelination ( Fig. S2 h and 2i). Thus, the knockdown of miR-223 was found to enhance both the pathological and clinical manifestations of EAE.

Knockdown of miR-223 suppressed Th17 cell differentiation in vivo and in vitro

Th17 cell disorders have the potential to serve as strong inducers of EAE and MS. Our investigation focused on the impact of miR-223 on the differentiation of Th17 cell differentiation in the spleen, lymph nodes, and CNS of EAE-afflicted mice. Notably, splenic cells and lymph nodes harvested from mice with reduced miR-223 expression exhibited significantly lower levels of the Th17 cytokine IL-17A and the Th1 cytokine IFN-γ ( Fig. 4 a and 4b). Examination of CNS-infiltrating CD4 T cells revealed a decrease in CD4 + IL-17A + and CD4 + IFN-γ + cells in EAE mice treated with LV-anti-miR-223 ( Fig. 4 c). Overactive CD4 T cells can result in severe neurological impairments. Our findings indicate that LV-anti-miR-223 reduced Th17 and Th1 cells in both peripheral immune organs and the CNS during the progression of EAE. To study the relationship and key cytokine gene expression of IL-17A and IFN-γ, we isolated cells from the spleen and spinal cord and analyzed their expression levels through real-time PCR. In the spleen, miR-223 knockdown significantly decreased the expression of IL-17A and IFN-γ, while in the spinal cord, only IL-17A expression was decreased ( Fig. 4 d and 4e, p  < 0.05).

Knockdown of miR-223 suppressed Th1 and Th17 cells in vivo and in vitro . Flow cytometry analysis of activated signature markers of Th1 and Th17 (IFN-γ and IL-17A) on the range of CD4 + cells isolated from the spleen, lymph nodes and CNS of LV-Ctrl and LV-anti-miR-223-treated EAE mice (n = 6 mice per group) at day 18 after the induction of EAE. Data are shown in a representative plot of the Th1 and Th17 cell subpopulations in three types of tissues ( a-c ). The percentage of respective subpopulations in CD4 + cells summarized by histograms. Cells from spleen ( d ) and lymph nodes ( e ) of LV-Ctrl and LV-anti-miR-223-treated EAE mice were collected, and total RNA was extracted. The quantity of mRNA was recorded using real-time PCR. Gene expression was normalized based on the mGapdh content. f Naïve CD4 + T cells were separated from the spleen of female C57BL/6 mice and then induced to differentiate intoTh1 and Th17 cells. g,h ELISA was used to test the expression of IFN-γ and IL-17A in the supernatants of Th1 and Th17 cells. Data were from three independent experiments (mean ± SEM). * p  < 0.05; ** p  < 0.01, as determined using the Student’s t test.

Additionally, in order to investigate the impact of miR-223 on the differentiation of CD4 + T cells in vitro , we separated naive CD4 + T cells from the spleen to induce Th17 and Th1 cells under various Th-polarizing conditions. Subsequently, we assessed the differentiation of Th cells by FACS ( Fig. 4 f, p  < 0.05). Furthermore, we tested the culture medium supernatant of Th17 and Th1 cells by ELISA. The secretion of IL-17A and IFN-γ notably decreased upon miR-223 knockdown ( Fig. 4 g and 4 h, p  < 0.05).

Sp3 gene is a direct target of miR-223

To evaluate the mechanism of miR-223 on the polarization of Th17 cells, we conducted a search for genes associated with immune response and Th17 cell differentiation that harbor potential miR-223 binding sites in their 3′UTRs. This search was performed utilizing bioinformatics tools such as Miranda, TargetRank and TargetScan. Previous studies have identified hspa1a (gene encoding Hsp72, GenBank accession number: NM_010479.2 , 445-451) [42] as a target of miR-223, playing a crucial role in the regulation of immune responses and T cell differentiation. In our luciferase reporter assay [43] , [44] , [45] , Hsp72 was selected as a control. Additionally, through the utilization of three bioinformatics tools, Sp3 (GenBank accession number: NM_001018042.3 , 436–443) was identified as a target of miR-223 ( Fig. 5 a). The predicted interaction between miR-223 and the 3′UTR of Sp3 and Hsp72 is illustrated in Fig. 5 b. The Sp3 gene is highly conserved and has two isoforms ( NM_001018042.3 , NM_001098425.1 ). Both 3′UTRs are targeted by miR-223. The Sp3 gene exhibits conserved regions with the 3′UTR across 80 different mammalian species, as identified through miR-223 targeting analysis. In fact, knockdown of miR-223 could significantly increase the gene expression of Hsp72 ( Fig. 5 c, p  < 0.05). Sp3 gene expression also increased while there were no changes in the Sp1 gene in EL4 cells transfected with LV-anti-miR-223. We also measured the gene expression of Sp1 in the spleen during EAE progression ( Fig. S3 a). The gene expression of Sp1 was not affected by miR-223 in EL4 cells and the spleen of mice. Sp3 is a kind of transcription factor that is present in all organs. To validate the predictions made through bioinformatics analysis, we conducted immunoblot assays on EL4 cell extracts that were control, miR-223 knockdown-transfected, or miR-223 overexpression-transfected. A Sp3-specific antibody was utilized for this purpose. Our findings revealed elevated levels of Sp3 translation in the nucleus of EL4 cells transfected with LV-anti-miR-223, irrespective of PMA/I stimulation. Conversely, the overexpression of miR-223 led to a notable suppression in Sp3 translation ( Fig. 5 d). Similarly, the same results were obtained for Hsp72, with higher translation in the nucleus of EL4 cells transfected with LV-anti-miR-223, aligning with previous studies in osteosarcoma [42] .

Sp3 as a direct target of miR-223. a Potential target genes of miR-223 as predicted by miRanda, TargetRank and TargetScan. b The miR-223 target sequence in the 3′UTR of Sp3 and Hsp72 in mouse. c EL4 cells were transfected with LV-Ctrl or LV-anti-miR-223. Quantitative PCR (real-time PCR) analysis of Hsp72, Sp3 and Sp1 mRNA levels. d The protein level of Sp3 and Hsp72 was tested by immunoblotting. H3 and β-actin were used as the nuclear and cytoplasm loading controls. e A schematic representing the luciferase constructs with the wild-type (CN) or mutant 3′UTR miR-223 MRE sequences of Sp3 and Hsp72. Mutations are marked in lowercase letters and underlined. f Normalized luciferase activity in lysates from 293 T cells co-transfected with wild-type or mutant Sp3-luciferase constructs and miR-223 or control mimics (mean ± SEM, n = 3, * p  < 0.05, ** p  < 0.01). g - i EL4 cells were transfected with LV-Ctrl or LV-anti-miR-223 and stimulated with PMA/I for 0 min, 10 min, 30 min, and 60 min. Quantitative PCR analysis of IFN-γ, IL-17A and IL-17F mRNA levels (g). h , i The protein levels of Smad2/3, phospho-Smad2/3, and Stat3 were tested by immunoblotting. β-actin was used as the loading control. The data are expressed as the mean ± SEM; experiments were performed in triplicate (* p  < 0.05, ** p  < 0.01).

In order to confirm the impact of miR-223 on Sp3 and Hsp72, a segment within the 3′UTR of the Sp3 and Hsp72 mRNAs containing a potential miR-223 response element was employed. Additionally, a modified version of the construct was generated with alterations to key binding sites, particularly within the miR-223 seed sequence-binding area ( Fig. 5 e). 293 T cells exhibited reduced luciferase activity upon co-transfection of the miR-223 overexpression vector ( Fig. 5 f, target). Conversely, miR-223 did not significantly affect luciferase activity from the modified construct, where luciferase activity remained comparable to control levels ( Fig. 5 f, mutant). Consistent with previous findings, Hsp72 yielded similar outcomes [42] indicating that miR-223 regulates Sp3 and Hsp72 levels by directly targeting the aforementioned miRNA response element in the 3′UTR region of the Sp3 and Hsp72 genes.

Another Sp3 isoform ( NM_001098425.1 , denoted Sp3-2) 3′UTR region was also tested with miR-223 using the luciferase report system ( Fig. S4 a, p  < 0.05). MiR-223 also directly targeted the Sp3-2 isoform.

Upon PMA/I stimulation, the levels of IFN-γ, IL-17A and IL-17F in EL4 cells transfected with LV-anti-miR-223 were significantly decreased based on real-time PCR analysis ( Fig. 5 g). Additionally, the expression of IL-17A was downregulated in human Jurkat cells transfected with LV-anti-miR-223 ( Fig. S3 b, p  < 0.05). Classical theory suggests that the IL-6/Stat3 and TGF-β/Smad2 signaling pathways play a role in the control of Th17 cell polarization. The phosphorylation levels of Smad2 protein were suppressed in miR-223 knockdown EL4 cells under PMA/I conditions ( Fig. 5 h and 5i).

Sp3 regulates the Smad2 and Stat3 signaling pathway during Th17 cell differentiation via Socs3

IL-6/JAK2/Stat3 and TGF-β/Smad2/3 are the key cytokine signal transduction pathways controlling Th17 cell differentiation. Stat3 and Smad2/3 phosphorylation play crucial roles in the regulation of T cell differentiation [46] . JAK2, as a Janus kinase family member, contributes to signaling through the gp130 receptor family (e.g., IL-6R). The JAK2/Stat3 signaling pathway is closely associated with cell differentiation and proliferation, making it a significant focus for research on information transfer and regulation of Th17 cell differentiation. In the spleen of LV-Sp3 mice during EAE progression, there was a notable inhibition of JAK2 protein. ( Fig. S4 b). Additionally, the level of phospho-Stat3 protein were reduced in both the spleen and CNS of LV-Sp3 mice during EAE progression ( Fig. 6 a and 6b). In contrast, knockdown of Sp3 increased the protein level of phospho-Stat3 in comparison to overexpression of Sp3 in CNS ( Fig. 6 b, p  < 0.05).

Sp3 regulated the Smad2/3 and Stat3 signaling pathways during Th17 cell differentiation through Socs3. a , b The protein levels of Smad2/3, phospho-Smad2, Stat3 and phospho-Stat3 were tested by immunoblotting in the spleen and CNS from LV-Ctrl, LV-Sp3 or LV-anti-Sp3-treated EAE mice. c RORγt, a key transcription factor in Th17 cells, was tested in CNS by immunoblotting. d EL4 cells were transfected with LV-Ctrl or LV-Sp3 and stimulated with PMA/I for 0 min, 10 min, 30 min, and 60 min. The protein levels of Smad2/3, phospho-Smad2/3, and Stat3 were assessed by immunoblotting. e Analysis SP3 binding genes by TRRUST, FIMO_JASPAR, GSE131282 database. f Analysis SOCS3 gene expression in MS patients in GEO ( GSE131282 ). g JASPAR database predicts that transcription factor SP3 binds to the upstream 1740–1750 bp position of the SOCS3 gene. h The protein level of Socs3 was tested by immunoblotting in EL4 cells. i The protein level of Sp3 was tested by immunoblotting in EL4 cells and 293 T cells. β-actin was used as the loading control. Densitometric ratios were quantified using ImageJ software. The data are expressed as the mean ± SEM (* p  < 0.05, ** p  < 0.01).

The differentiation of naïve CD4 + T cell into Th17 cells can be induced by the combined action of TGF-β and the inflammatory factor IL-6. By binding to TβRII, TGF-β further activated Smad2/3 through phosphorylation. During the progression of EAE, we noted a decrease in the phosphorylation of Smad2 in the spleen and CNS of LV-Sp3 mice ( Fig. 6 a and 6b, p  < 0.05). Knockdown of Sp3 increased the phosphorylation of Smad2 protein compared with overexpression of Sp3 in the CNS. Th17 cells are regulating by many transfer factors, among which RORγt is an important and key marker [47] . Concurrently, we found that overexpression of Sp3 could significantly reduce the expression of RORγt in the CNS ( Fig. 6 c). In EL4 cells transfected with LV-Sp3, the protein level of phospho-Smad2 was suppressed at 10 and 30 min ( Fig. 6 d).

As previously reported, as a negative feedback inhibitor, Socs3 can regulation Stat3 phosphorylation by inhibition of JAK2. We predicted human SP3 binding genes in the TRRUST and FIMO_JASPAR databases, respectively. 113 genes were identified in the TRRUST database and 12,973 genes were identified in the FIMO_JASPAR database. We also performed intersection processing with the downregulated genes from the GSE131282 data for MS gray matter lesions and healthy control groups. The resulting Venn plot shows 44 genes in the intersection elements of the three, including SOCS3 ( Fig. 6 e). In the GSE131282 single cell sequencing data, there were 37 cases of MS gray matter lesions, which showed a decrease in SOCS3 expression ( Fig. 6 f, p  < 0.05). It was predicts that transcription factor SP3 binds to the upstream 1740-1750 bp position of the SOCS3 gene (NC:000017.11: c78362925-78360826) promoter, and the correlation coefficient is 0.906211595 ( Fig. 6 g) by the JASPAR database. The expression of Socs3 protein was elevated in EL4 cells following transfection with LV-Sp3, whereas it was reduced in EL4 cells transfected with LV-anti-Sp3 ( Fig. 6 h). Research has shown that Sp3 is crucial for IL-6-induced activation of Socs3 transcription and serves as an essential enhancer, leading to increased levels of Socs3 mRNA and protein [48] . In the spleen, overexpression of Sp3 increased the expression of Socs3 protein during EAE progression ( Fig. S4 c). In our research, Socs3 played a role in the regulation of Th17 cell differentiation by influencing the IL-6/JAK2/Stat3 and TGF-β/Smad2/3 signaling pathways.

To date, there have been no reports on Socs3 feedback regulation of Sp3. We constructed a high expression Socs3 carrier with Flag tags and assessed it effects ( Fig. S5 a). We found that overexpression of Socs3 could significantly increase the expression of Sp3 in EL4 and 293 T cells upon TGF-β stimulation ( Fig. 6 i), demonstrating a feedback regulation relationship between Socs3 and Sp3.

Sp3 regulation of Smad2/3 independently of Smad7

As inhibitor of Smad2/3, Smad7 regulates Th17 cell differentiation during Echinococcus multilocularis infection [49] . In the present study, no changes in Smad7 expression were detected in the spleen and CNS of LV-Sp3 or LV-anti-Sp3 mice during EAE progression ( Fig. S4 d). It is possible that Smad7 was not involved in Sp3 regulation of phospho-Smad2/3 during EAE progression.

Sp3 regulates Smad2/3 signaling through Socs3 binding and degradation of TβRII

Many studies have described an inhibition by Socs3 of the JAK2/Stat3 pathway, while few studies have reported an interaction between Socs3 and Smad2/3. SPSB1, a protein containing a Spry domain within its Socs box, has been identified as a TGF-β target gene by microarray screening and PCR [50] , [51] , and it can degraded TβRII but not TβRI. In this study, it was observed that the upregulation of Socs3 gene expression was stimulated by TGF-β, whereas no alterations were noted in the expression of the Socs1 gene ( Fig. 7 a, p  < 0.05).

Socs3 targeted and degraded TβRII. a EL4 cells were stimulated with TGF-β. Real-time PCR analysis of the expression of Socs1 and Socs3. The data are representative of at least three experiments with similar results. b Immunoblot analysis of Socs3 and TβRII. c Immunoblot analysis of Smad2/3 and phospho-Smad2/3 in 293 T cells stimulated with TGFβ for 0 h, 0.5 h, 1 h, and 2 h. The 293 T cells were transfected with Ctrl or siSocs3. d Real-time PCR analysis of Socs3. e Immunoblot analysis of Socs3 and TβRII. f Immunoblot analysis of Smad2/3 and phospho-Smad2/3. g -i The 293 T cells were transfected or not with Ctrl, siSocs3 or LV-Socs3 upon TGF-β stimulation for 1 h. Mouse anti-Socs3 or rabbit anti-TβRII antibody and Alexa Fluor 594 donkey anti-mouse IgG and Alexa Fluor 488 donkey anti-rabbit IgG secondary antibody conjugates were used. Cells were inspected under 100 × magnification using a BX60 fluorescence microscope (Olympus, BX60).

The TGF-β signaling pathway necessitates the presence of two cell-surface serine/threonine kinase receptors, namely type II (TβRII) and type I (TβRI) TGF-β receptors. Typically, TGF-β binds to TβRII, which then recruits TβRI to establish a heteromeric complex of TβRI-TβRII. This complex formation triggers the phosphorylation of TβRI, followed by the phosphorylation of receptor-regulated Smads (R-Smads, i.e., Smad2, Smad3, etc.), which subsequently bind a co-Smad (Smad4). In order to assess TGF-β signaling activity, the study initially evaluated TβRII levels, which were characterized by the stable expression of an inducible Socs3 construct. As depicted in Fig. 7 b, the introduction of Socs3 led to a reduction in TGF-β-induced TβRII levels. Subsequently, an analysis of phospho-Smad2/3 levels was conducted to further explore TGF-β signaling activity. The upregulation of Socs3 was found to notably diminish the level of phospho-Smad2/3 with TGF-β ( Fig. 7 c). To confirm the interaction between Socs3 and TβRII, Socs3 expression was downregulated through siRNA transfection, resulting in the suppression of Socs3 at both the transcriptional and translational levels ( Fig. 7 d and 7e, p  < 0.05). The TβRII protein level was increased in 293 T cells transfected with siSocs3 regardless of TGF-β stimulation, while, the Smad2/3 phosphorylation level was concomitantly increased ( Fig. 7 e and 7f). This interaction pattern of Socs3 and TβRII, pospho-Smad2/3 was also evident ( Fig. S5 b-5e). Taken together, Socs3 interacted with TβRII.

To further confirm the interaction between Socs3 and TβRII, we performed co-immunofluorescence staining. Socs3 exhibited a dispersed distribution within the cytoplasm and redistributed to the cell membrane upon TGF-β stimulation ( Fig. 7 g). TβRII was located in the cell membrane and digested after stimulation by TGF-β ( Fig. 7 h). Knockdown of Socs3, TβRII resulted in no changes. In contrast, overexpression of Socs3 significantly decreased the level of TβRII ( Fig. 7 h). TβRII and Socs3 were extensively co-localized at the cell membrane, regardless of TGF-β presence ( Fig. 7 i).

Knock-in of Sp3 in CD4 T cells ameliorated clinical symptoms and suppressed Th17 cell differentiation in EAE mice

To assess the influence of overexpression of Sp3 in CD4 T cells (Sp3 +/+CD4Cre ) on the progression of MS course in vivo, we conducted an analysis of neurobehavioral impairments in Sp3 +/+ transgenic mice. Our findings, as illustrated in Fig. 8 , revealed a chronic progressive-relapsing pattern in the EAE mouse model. Notably, the heightened expression of Sp3 in CD4 T cells led to a notable reduction in the severity of neurobehavioral deficits, cumulative scores, and maximum neurological disability in EAE when compared to Sp3 fl/fl mice ( Fig. 8 a-8d, p  < 0.05). Consequently, the overexpression of Sp3 in CD4 T cells improved the pathological and clinical symptoms of EAE.

Sp3 in CD4 T cells attenuated or aggravated the clinical symptoms of Sp3 +/+CD4Cre or Sp3 -/-CD4Cre EAE mice. The clinical scores of all the EAE mice were assessed daily according to the same criteria for 24 continuous days. Incidence ( a ), Daily clinical scores ( b ), peak disease scores ( c ), and cumulative disease scores ( d ) were monitored Sp3 fl/fl and Sp3 +/+CD4Cre EAE mice. The data are representative of at least two experiments with similar results. Flow cytometry analysis of CD4 + cells isolated from the spleen and lymph node of Sp3 fl/fl and Sp3 +/+CD4Cre EAE mice (n = 6 mice per group) at day 18 after the induction of EAE. Data are shown in a representative plot of the CD4 + cell subpopulations in spleen and lymph node ( e, f, g ). Knockout of Sp3 in CD4 T cells of Sp3 -/-CD4Cre EAE mice. Incidence ( h ), Daily clinical scores ( i ), peak disease scores ( j ), and cumulative disease scores ( k ) were monitored. The data are representative of at least two experiments with similar results. Flow cytometry analysis of CD4 + cells isolated from the spleen of Sp3 fl/fl and Sp3 -/-CD4Cre EAE mice at day 15 after the induction of EAE. Data are shown in a representative plot of the CD4 + cell subpopulations in spleen ( l ). m Naïve CD4 + T cells were separated from the spleen of female Sp3 fl/fl and Sp3 -/-CD4Cre mice and then induced to differentiate into Th17 cells. Data were from three independent experiments (mean ± SEM). * p  < 0.05; ** p  < 0.01, as determined using the Student’s t test. n Schematic of the molecular mechanism by which Sp3 regulates Th17 cell differentiation. As the target of miR-223, Sp3 positively regulates Socs3, which regulates Sp3 via a feedback mechanism. Socs3 negatively regulates the IL-6/JAK2/Stat3 and TGF-β/Smad2/3 signal transduction pathways to control Th17 cell differentiation. Thus, RORγt contributes to Th17 cell differentiation and EAE induction.

Subsequently, an investigation was carried out to ascertain Sp3′s effect on CD4 + T cell in the spleen and lymph nodes of EAE mice. Overexpression of Sp3 in CD4 T cells significantly decreased the polarization of CD4 + IL-17A + cells in the spleen during EAE progression ( Fig. 8 e-8g, p  < 0.05).

Knockout of Sp3 in CD4 T cells exacerbated clinical symptoms in EAE mice and enhanced Th17 cell differentiation in vitro

Simultaneously, knockout of Sp3 in CD4 T cells (Sp3 -/-CD4Cre ) was found to alter the impact of Sp3 on the progression of EAE, resulting in exacerbated symptoms ( Fig. 8 h-8k, p  < 0.05). These findings indicate that Sp3 exerts its influence through CD4 T cells in the advancement of EAE.

Subsequently, an investigation was conducted to determine the effect of Sp3 on CD4 + T cell in the spleen and lymph nodes of EAE mice. Moreover, consistent outcomes were observed in vitro , demonstrating that Sp3 knockout enhanced the differentiation of spleen CD4 + T cell toward Th17 cells under various induced conditions ( Fig. 8 i-8m, p  < 0.05).

Discussion

MS has traditionally been understood as a condition primarily driven by CD4 T-cell activity, as evidenced by the strong genetic risk associated with major histocompatibility complex (MHC) class II [52] , [53] and myelin-specific T cells [54] , [55] . Recent research has indicated an upregulation of Th1/Th17 cells in EAE animal models [53] , [56] . Furthermore, microarray analysis has revealed an increased presence of IL-17-producing glial cells, CD4, and CD8 T cells in active MS lesions [57] , [58] , [59] . The activities of Th17 cells may contribute to disease progression and CNS dysfunction, as these cells have been shown to exhibit enhanced migration across BBB models and display neurotoxic effects in vitro
[60] . Studies have indicated a reduced presence of the bifunctional transcription factor Sp3 in various immune cells in the majority of MS [17] , with potential correlations between SP3 expression levels, clinical manifestations of MS, and its immunological pathogenesis [20] . This suggests that the transcription of the Sp3 gene is hindered in immune cells of most individuals with MS, thereby contributing to the onset of inflammation in CNS during the disease [19] . The Sp3 gene is found to be highly expressed in CD4 T cells, CD8 T cells, NK cells, and lymph nodes, but its expression is inhibited in immune cells of individuals with MS. In EAE mice, knock-in Sp3 in CD4 T cells (Sp3 +/+CD4Cre ), overexpression Sp3 led to a notable reduction of neurobehavioral impairments, cumulative scores, and maximum neurological disability. Knockdown (Sp3 +/- ) or knockout Sp3 in CD4 T cells (Sp3 -/-CD4Cre ) can exacerbate EAE symptoms and promote the differentiation of Th17 cells. These findings suggest that Sp3 moderates Th17 cell differentiation both in vivo and in vitro . However, the underlying mechanism and how the deficiency of Sp3 occurs in MS patients remain unclear.

Our study elucidates the role of miR-223 in targeting Sp3 to modulate the differentiation of Th17 cells, which in turn contributes to the alleviation of neurobehavioral deficits, cumulative scores, and the overall severity of neurological disability in EAE. Southern blot and polymerase chain reaction of genomic DNA have not revealed any discernible differences between MS and control PBMCs [18] . We hypothesize that miRNAs have some unknown effects on MS. Previous research by Kasey C. Vickers has indicated that the overexpression of miR-223 leads to a significant repression of Sp3 mRNA levels, while inhibition of miR-223 results in a marked upregulation of Sp3 mRNA levels [36] . Through gene reporter (luciferase) assays, it was demonstrated that miR-223 directly targets Sp3 [36] . We found that 11 miRNAs could target the 3′UTR of Sp3 by TargetScan in mice and humans, and found that a notable upregulation of miR-223 in MS patients within the clinical datasets GSE17846 and GSE31568 . This study further illustrated that knockdown of miR-223 increased Sp3 protein and mRNA levels, and facilitated Sp3 protein degradation in the presence of miR-223 overexpression. This effect was confirmed by the observation that knockdown of miR-223 improved clinical symptoms and neurobehavioral deficits during EAE progression, suggesting that miR-223 played a role in EAE progression. In vivo and in vitro, Th1 and Th17 cell populations were decreased by LV-anti-miR-223 treatment. Consequently, the results of this study establish Sp3 as a crucial target of miR-223 in the regulation of Th17 cell polarization during EAE progression.

Our research proposes a novel mechanism that encompasses the miR-223/Sp3/Socs3/TGF-β signaling pathway, which may serve as a promising therapeutic approach for the modulation of Th17 cells in the context of immunotherapy. Recent studies have indicated that circular plant peptide kalata B1, cathepsin B, the CCR1 chemokine receptor, specific histamine H4 receptor antagonists, phenols, and alkaloids may serve as potential therapeutic agents for the treatment of MS by modulating CD4 T cell activity [61] , [62] , [63] , [64] , [65] . The classical theory indicates that the differentiation of Th17 cell population is prompted by IL-6 and TGF-β through the JAK-STAT3 and TGFβ-Smad2/3 signaling pathways. The JAK/STAT signaling pathway plays a crucial role in the initiation, control, and cessation of immune responses, and any malfunction in this pathway can have pathological consequences in autoimmune and neuroinflammatory conditions [66] , [67] . The SOCS family comprises intracellular proteins that serve as vital regulators of the immune system. Among these, SOCS3 is a significant member that can negatively modulate IL-6-JAK2-STAT3 signaling pathway, which is essential for Th17 cell differentiation [68] , [69] . T cells lacking SOCS3 exhibit over activation of STAT3 and notably heightened IL-17 secretion induced by IL-6 [70] . The Socs3 gene harbors a GC-rich segment in its 5′-upstream region. Sp3, a widely expressed transcription factor, binds to this GC-rich motif instead of Sp1, indicating the involvement of Sp3 in the regulation of SOCS3 expression. The findings suggest that Sp3 plays a crucial role in the IL-6-induced transcriptional activation of the Socs3 gene promoter, acting as an enhancer of both basal and induced transcriptional activities, thereby leading to increased Socs3 mRNA and protein expression [48] . AP-1, pSTAT, and SP1/SP3 binding sites were required for SOCS3 gene activation [71] . In our research, overexpression of Sp3 significantly increased Socs3 expression. This finding is consistent with the other study. For the first time, we show that the negative feedback regulation of Sp3 by Socs3, and these results showed that Sp3 expression increased upon TGF-β treatment in EL4 and 293 T cells transfected with LV-Socs3. The protein level of JAK2 and phospho-Stat3 was notably reduced in LV-Sp3 mice as EAE advanced. In contrast, knockdown of Sp3 increased the protein level of phospho-Stat3 in the CNS. Overall, Sp3 regulated Th17 cell differentiation, potentially via the Socs3-JAK2-Stat3 signaling pathway during EAE progression.

Regarding the TGF-β-Smad2/3 signaling pathway, Smad7 functions as a negative regulator of TGF-β superfamily signaling, modulating a wide range of diverse responses elicited by TGF-β ligands through inhibition of Smad2/3 activity during Th17 cell polarization [72] . Our findings indicate that alterations in Smad7 expression were not observed in the spleen and CNS of mice with EAE following either upregulation or downregulation of Sp3. These results suggested that Sp3 regulated Smad2/3 and Th17 cell differentiation that were not involved in Smad7. A study identified SPSB1 as a new inhibitory factor of the TGF-β signaling pathway by interacting with TβRII. SPSB1 was found to downregulate TβRII levels by promoting ubiquitination and degradation of TβRII via its Socs box domain [51] . We hypothesize that Socs3 can directly target and degrade TβRII and then regulate the Smad2/3 signaling pathway. Here we demonstrated that overexpression of Socs3 degraded TβRII and inhibited phospho-Smad2/3, and the small interfering RNA of Socs3 had the reverse results. Next, we found that Socs3 redistributed to the cell membrane with TβRII upon TGF-β stimulation. These results confirmed our hypothesis that Socs3 regulated TβRII expression. Collectively, these findings indicate that Socs3 plays a significant role in regulating the TGF-β-Smad2/3 signaling pathway.

Limitations of the study

EAE serves as a pivotal animal model for MS, contributing significantly to the elucidation of disease mechanisms and the evaluation of novel therapeutic interventions. However, the model is not without its considerable limitations. EAE typically manifests as a more straightforward pathological process, lacking the diversity and heterogeneity that characterize the human condition [73] , [74] , [75] , failing to accurately replicate the natural progression observed in MS, particularly regarding the dynamic alterations associated with chronic inflammation and neurodegeneration [76] . This disparity underscores the necessity for a cautious interpretation of EAE-derived research findings when developing new therapies for MS, advocating for a synthesis of these results with extensive human clinical data for a more comprehensive analysis. This research identifies a novel target for the treatment of MS; however, preclinical or clinical trials have yet to be undertaken. Sp3 is associated with systemic sclerosis and neuroinflammatory diseases, including Parkinson's [77] , [78] . Subsequent investigations should prioritize the development of therapeutic strategies aimed at Sp3 and its associated regulatory pathways, as well as the assessment of their efficacy and safety in clinical settings.

Conclusion

In conclusion, as the target of miR-223, Sp3 can positively regulate Socs3, which regulate Sp3 via a feedback mechanism. IL-6/JAK2/Stat3 and TGF-β/Smad2/3 are key cytokine signal transduction pathways controlling Th17 cell differentiation. SOCS3 negatively controls JAK2 to suppress phospho-Stat3. Moreover, Socs3 degrades TβRII and reduces the production of phospho-Smad2/3. Hence, the suppression of miR-223 has the capacity to control the differentiation of Th17 cells and restrict the migration of activated CD4 + T cells into the CNS in the context of EAE advancement via the Sp3-Socs3 signaling cascade. Consequently, alterations in Sp3 concentrations could potentially influence the Th17 cell cohort and impact the pathological development of EAE. The need for further studies is evident to explore Sp3′s impact on other autoimmune diseases governed by Th17 cells.

Methods

Animal experiments

Female C57BL/6 mice of the wild type, aged between 7 and 8 weeks, were procured from the Academy of Military Medical Science (Beijing, China). Sp3 fl/fl , Sp3 +/- gene knockout mouse, Sp3 +/+CD4Cre knock in mouse and Sp3 -/-CD4Cre knockout mouse were purchased from the Cyagen Biosciences (Guangzhou, China). Each group consists of 6 mice, with a body weight of 18–20 g. Accommodated and provided sustenance in a controlled environment free of specific pathogens at either the animal facility of Guangdong Pharmaceutical University (Guangzhou, China) or the Experimental Animal Center of Tianjin Medical University (Tianjin, China). The experiments were conducted following animal care guidelines and received approval from either Guangdong Pharmaceutical University’s Animal Ethics Committee (Guangzhou, China) or Tianjin Medical University (Tianjin, China).

EAE induction was performed according to our previous published studies [29] , [30] , [31] .

Histopathology

The lumbar spinal cords from the EAE mice were stained with H&E to detect inflammatory cell infiltration and with luxol fast blue (AlfaAesar, 1328–51–4) to assess demyelination, which were performed according to our previously published studies [29] , [30] , [31] .

Intracellular cytokine staining

Single-cell suspensions were obtained according to our previous published studies [29] , [31] . The cells were examined by the BD FACS Celesta (BD Biosciences, USA), and the acquired data were analyzed using FlowJo software. Refer to Fig. S6 for a comprehensive overview of the flow analysis gate strategy.

Naïve CD4 + T cell preparation and differentiation

Naïve CD4 + T cells isolated from the spleen were separated through the utilization of magnetic beads (MiltenyiBiotec, 130-106-643). Th1 and Tha7 cells differentiation were performed according to our previous published studies [29] , [31] . Following a 4-day incubation period, the cells underwent a washing process and were subsequently re-exposed to a leukocyte activation cocktail (BD, 550583) for a duration of 4 h. Subsequently, the cells were harvested for FACS and RNA analysis.

Cell culture and treatment

The EL4 mouse T cell line was procured from the Cell Resource Center at the Institute of Biomedical Sciences, Chinese Academy of Medical Sciences/Peking Union Medical College (Beijing, China). The cells were grown in RPMI 1640 medium. The cells were maintained in a 5 % CO2 humidified incubator at 37°C and subjected to treatment with PMA/I (Enzo Life Sciences, USA), or TGFβ.

Target prediction for miRNA

miRNA targets were determined through the utilization of publicly accessible bioinformatics resources including Miranda, TargetRank, and TargetScan. These tools are available online at the following URLs: https://34.236.212.39/microrna/home.do , http://hollywood.mit.edu/targetrank/ , https://www.targetscan.org/vert_71/ .

MicroRNA, plasmid, lentivirus, and transfection

The vector primers designed for LV-CN and LV-anti-miR-223 were synthesized by GENEWIZ (Suzhou, China) ( supplement table 1 ). In preparation for the dual-luciferase assay, MRE located within the 3′UTR region of Sp3 and Hsp72 mRNA, along with its corresponding mutated variant, were inserted into a pmirGLO-control vector (Promega, E1741) downstream of the luciferase gene using specific primers ( supplement table 2 ).

To clone the miR-223 sponge, a sponge containing miR-223 mature sites, 5′- TGTCAGTTTGTCAAATACCCCA-3′ driven by an H1 promoter, was cloned into the BamHI /EcoRI site of the pmiRZip lentivirus vector (SBI, CA).

LV-Socs3 was synthesized by Vigene Biosciences (Shandong, China). Socs3 was synthesized and cloned into the AsisI/MluI site of the pLent-EF1a-FH-CMV-GP lentivirus vector (Vigene Biosciences, China).

polyethyleneimine (PEI; Polysciences Inc., 23966) was used for transient transfection of EL4 or 293 T cells, as per the manufacturer’s instructions. Briefly, EL4 or 293 T cells were taken and lysed at 48 h after transfection.

Immunoblot analyses and antibodies

Western blot analysis was performed according to our previous published studies [29] , [30] , [31] . Primary antibodies were applied in 5 % milk-TBST: anti-Sp3 (Santa Cruz, sc-644), anti-Hsp72 (Proteintech, 10995-1-AP), anti-Smad2/3 (Cell Signaling Technology, 8685 s), anti-p-Smad2 (Cell Signaling Technology, 8828), anti-Smad7 (Proteintech, 25840-1-AP), anti-Stat3 (Cell Signaling Technology, 12640s), anti-p-Stat3 (Cell Signaling Technology, 9145), anti-Socs3 (Abcam, ab16030), anti-JAK2 (ABclonal, A11497), anti-JAK2-p (Abcam, ab32101), anti-TβRII (ABclonal, A1415), anti-RORγt (Abcam, ab78007), anti-β-actin (Sungene, KM9001L), and anti-H3 (Sungene, KM9005). Secondary anti-mouse or anti-rabbit antibodies coupled to horseradish peroxidase (anti-mouse SIGNALWAY ANTIBODY Co., Ltd., L3032; anti-rabbit SIGNALWAY ANTIBODY Co., Ltd., L3012). Protein bands were revealed with chemiluminescence HRP substrate (Millipore, WBKLS0500). Protein loading volume and strip exposure time for every antibody were detected before the formal experiment.

RNA isolation and RT-PCR analyses

Total RNA extraction and RT-PCR analysis were performed according to our previous published studies [29] , [30] , [31] .

Real-time-PCR for mRNA quantification

Real-time-PCR was performed according to our previous published studies [29] , [30] , [31] . The primers are shown in supplement table 3 . 7500 Fast PCR System (AB, USA) were used for the reactions.

ELISA

The MultiSciences cytokine assay system was used to assess IL-17A (MULTI SCIENCES, EK2171/2) and IFN-γ (MULTI SCIENCES, EK2801/2) secreted from cells. ELISA was performed according to the manufacturer’s instructions.

Immunofluorescence staining and confocal microscopy

Forty-eight hours after cotransfection of LV-CN, LV-Socs3, siRNA, 293 T cells were incubated in DMEM/FBS containing 2 ng/ml TGFβ for 4 h. Mouse anti-Socs3 antibody (Santa Cruz, sc-51699) or rabbit anti-TβRII antibody and Alexa Fluor 594 donkey anti-mouse IgG (Proteintech, SA00006-7) and Alexa Fluor 488 donkey anti-rabbit IgG (Proteintech, SA00006-2) secondary antibody conjugates were used. Cells were inspected under 100 × magnification using a BX60 fluorescence microscope (Olympus, Germany).

Statistical analyses

Statistical analyses were performed according to our previous published studies [29] , [30] , [31] .

Funding

This work was supported by the National Natural Science Foundation of China [No.81971540, 82173087], Guangzhou Municipal Science and Technology Bureau [2025A03J4088], the Project in Key Areas of Guangdong Province’s General Colleges and Universities [No. 2024ZDZX2079], Department of Science and Technology of Guangdong Province [No. 2024A1515013068], Innovation and Enhancement Engineering Team Project of Guangdong Pharmaceutical University [No. 2024ZZ03].

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

Not applicable

Footnotes

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jare.2025.01.051 .

Contributor Information

Yan Li, Email: yanli@gdpu.edu.cn.

Rongxin Zhang, Email: rxzhang@gdpu.edu.cn.

Supplementary material

The following are the Supplementary data to this article:

Supplementary Data 1

Supplementary Data 2

References

Associated Data

Supplementary Materials

Supplementary Data 1

Supplementary Data 2