TPN10475 Inhibits Effector T Lymphocyte Activation and Alleviates Experimental Autoimmune Encephalomyelitis by Promoting TGF-β Signaling

Introduction

Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) mediated by immune cells, with complex etiology and unclear pathogenesis. MS is characterized by neurological dysfunction in young people and is a non-traumatic disabling disease with increasing prevalence globally. Although existing drugs for treating MS have controlled the condition to some extent, their side effects and drug-related serious adverse events remain major medical challenges.

Experimental Autoimmune Encephalomyelitis (EAE) is commonly used as an animal model for MS to study disease mechanisms and potential treatments. The pathology and histology of EAE are highly similar to MS, mainly manifesting as abnormal inflammatory cell infiltration in the CNS, leading to demyelination of nerve axons.

In the pathogenesis of MS, CD4+ T cells are key participants, especially Th1 and Th17 cells, which damage the spinal cord and axons by producing inflammatory factors and cytokines, and attract more inflammatory cells to further exacerbate the inflammatory response. In addition, CD4+ T cells can also inhibit the function of effector CD4+ T cells through specific CD4+ T regulatory cells (Treg) and other regulatory feedback mechanisms, thereby downregulating inflammation.

Transforming Growth Factor Beta (TGF-β) is an important intercellular signal that controls cell proliferation, differentiation, and migration. It mediates signal transduction through cell surface receptor complexes and ultimately regulates the expression of target genes. Studies have shown that TGF-β plays an important role in immune regulation, including inhibiting the proliferation, differentiation, and activation of immune cells.

Artemisinin and its derivatives are not only famous for their antimalarial effects but have also shown potent immunosuppressive activity in recent years, demonstrating significant therapeutic effects in autoimmune diseases such as MS and rheumatoid arthritis. The artemisinin derivative TPN10466 can exert protective effects in mouse EAE disease by enhancing the proliferation and function of Treg cells.

This study focuses on a new artemisinin derivative, TPN10475, aiming to explore its alleviating effect on EAE and its possible mechanisms. The results show that TPN10475 can effectively resist the reduction of TGF-β signaling induced by TCR signal stimulation, inhibit the activation function of effector CD4+ T cells, and limit the differentiation of pathological Th1 and Th17 cells. Moreover, TPN10475 alleviated EAE by reducing the activation drive of peripheral autoreactive helper T lymphocytes and inhibiting the migration of inflammatory cells to the central nervous system.

Materials and Methods

Male C57BL/6 mice aged 6-8 weeks were used in the study, all purchased from Gempharmatech and raised in the Tongji University animal care facility. All animal experimental procedures were approved by the Animal Research Ethics Committee of Tongji University.

Isolation and in vitro polarization of CD4+ T cells

CD4+ T cells were purified from mouse splenocytes using magnetic cell sorting technology, then activated with anti-mouse CD3 and anti-mouse CD28 antibodies and polarized in RPMI 1640 medium containing different cytokines.

Cell proliferation and viability detection

Carboxyfluorescein diacetate succinimidyl ester (CFSE) dye was used to track lymphocyte cell division, and Cell Counting Kit-8 (CCK8) solution was used to assess the growth of HEK293T cells.

Flow cytometry analysis

Flow cytometry was used to analyze cell surface markers and cytokine expression in in vitro and in vivo experiments to detect cell activity and apoptosis.

Quantitative real-time PCR

Total RNA was extracted from CD4+ T cells, reverse transcribed to cDNA, and specific gene expression was detected by quantitative real-time PCR.

Enzyme-linked immunosorbent assay (ELISA)

Commercial ELISA kits were used to detect the concentrations of inflammatory cytokines and anti-inflammatory cytokines in cell culture supernatants and mouse serum.

EAE induction and assessment

EAE was induced by subcutaneous immunization of mice with MOG35-55 peptide. Changes in mouse body weight and clinical scores were assessed, and pathological analysis was used to evaluate spinal cord inflammatory cell infiltration and white matter demyelination.

RNA sequencing analysis

Total RNA was extracted from CD4+ T cells for RNA-seq analysis to screen for differentially expressed genes, and heatmap, volcano plot, KEGG pathway analysis, and Gene Ontology (GO) analysis were performed.

Statistical analysis

Experimental data are presented as mean ± standard error. Unpaired two-tailed Student’s t-test or Mann-Whitney U test was used for statistical analysis, with statistical significance set at P < 0.05.

Results

TPN10475 inhibits CD4+ T cell proliferation and effector function

In vitro experiments demonstrated that TPN10475 significantly inhibited the generation of effector memory CD4+ T cells induced by anti-CD3/CD28 activation. Further analysis showed that it also had a significant inhibitory effect on IFN-γ and TNF-α secreted by effector memory CD4+ T cells. This indicates that TPN10475 has an inhibitory effect on the function of effector memory CD4+ T cells.

TPN10475 limits the development of Th1 and Th17 cells

Further studies found that TPN10475 could dose-dependently inhibit the generation of Th1 and Th17 cells, while slightly promoting Treg cells. TPN10475 also inhibited the ability of Th1 cells to secrete IFN-γ and TNF-α, as well as the ability of Th17 cells to secrete IL-17A and GM-CSF.

TPN10475 alleviates clinical symptoms in EAE mice

By orally administering different doses of TPN10475 to EAE mice, the results showed that TPN10475 not only alleviated the clinical symptoms of EAE mice but also delayed the onset time of the disease. Pathological analysis showed that TPN10475 significantly reduced inflammatory cell infiltration and white matter demyelination area in the spinal cord.

TPN10475 inhibits inflammatory responses in the CNS

Flow cytometry analysis showed that the proportion of infiltrating CD4+ T cells in the CNS of mice in the TPN10475 treatment group was significantly reduced, especially the proportion of Th17 and Th1-like Th17 cell subsets. This indicates that TPN10475 can reduce inflammatory cell infiltration in the CNS.

TPN10475 inhibits early inflammatory activation in EAE

The study found that the secretion of inflammatory cytokines in peripheral serum of mice in the treatment group was significantly reduced in the early stage of the disease, indicating that TPN10475 can produce a significant anti-inflammatory effect in the early stage of EAE onset.

TPN10475 regulates peripheral tissue immune responses in the early stages of EAE

Flow cytometry analysis showed that TPN10475 significantly reduced the proportion of CD4+ T cells in the spleen and peripheral lymph nodes of EAE mice, while significantly reducing the proportion of Th1, Th17, and Th1-like Th17 cells, and promoting the generation of Treg cells. In addition, TPN10475 significantly reduced the levels of inflammatory cytokines secreted by peripheral immune cells, further verifying its immunomodulatory effect.

TPN10475 inhibits the activation of effector CD4+ T cells by enhancing TGF-β receptor signaling pathway

RNA sequencing analysis showed that TPN10475 significantly affected the TGF-β receptor signaling pathway and negative regulation of inflammatory responses, and increased the expression levels of genes related to the TGF-β receptor signaling pathway. Experimental results further confirmed that TPN10475 inhibited the activation of effector CD4+ T cells and limited the differentiation of Th1 and Th17 cells by promoting the TGF-β signaling pathway.

Inhibition of TGF-β signaling reverses the effect of TPN10475 on activated CD4+ T cells

By intervening CD4+ T cells with TGF-β receptor inhibitor LY2109761, it was found that blocking TGF-β signaling could reverse the inhibitory effect of TPN10475 on activated CD4+ T cells, which further verified the mechanism of TPN10475 inhibiting effector CD4+ T cell activation by promoting the TGF-β signaling pathway.

Conclusion

This study found that the artemisinin derivative TPN10475 significantly inhibited the proliferation and activation of effector CD4+ T cells by promoting TGF-β signaling, limited the differentiation of pathological Th1 and Th17 cells, and alleviated EAE by reducing the activation drive of peripheral autoreactive helper T lymphocytes and the migration of inflammatory cells to the central nervous system. This research provides valuable reference for further study of the treatment of autoimmune diseases and provides a potential basis for regulating the TGF-β signaling pathway to alleviate EAE.