IMPDH2 facilitates CD4+ T cell activation through AKT/mTOR pathway by upregulating SRPK1 in myasthenia gravis

Abstract

Background

Myasthenia gravis (MG) is a T cell-mediated autoimmune disease characterized by abnormal immune responses, particularly the hyperactivation of CD4+ T cells, which may disrupt signal transmission at the neuromuscular junction. Inosine-5′-monophosphate dehydrogenase-2 (IMPDH2) has been reported to participate in immune activation and is likely associated with T cells, but its role in the pathogenesis of MG remains unclear. Therefore, the present study aimed to elucidate the mechanism through which IMPDH2 regulates CD4+ T cells in MG.

Methods

In this study, IMPDH2 expression was measured by qRT-PCR in peripheral blood mononuclear cells (PBMCs) collected from 60 MG patients and 60 healthy controls. Western blotting was additionally performed to detect IMPDH2 protein expression in six MG patients (three ocular and three generalized), compared with six healthy controls matched by age, gender, and sample collection time. CD4+ T cells were then isolated from PBMCs of MG patients and healthy controls by immunomagnetic bead sorting, and IMPDH2 expression was further analyzed by qRT-PCR. Subsequently, correlations between IMPDH2 expression levels and clinical indices (neutrophil and lymphocyte counts) as well as disease severity (Myasthenia Gravis Activities of Daily Living scores and Quantitative Myasthenia Gravis scores) were assessed. Additionally, flow cytometry, EdU assays, and CCK-8 assays were employed to evaluate the effects of IMPDH2 knockdown or overexpression on CD4+ T cell apoptosis and proliferation. The expression of apoptosis-related proteins was detected by western blotting. Mass spectrometry (MS), co-immunoprecipitation (Co-IP), and kinase inhibitor-based Co-IP validation assays were used to screen and verify proteins potentially interacting with IMPDH2 in CD4+ T cells. The colocalization of IMPDH2 and its binding proteins in CD4+ T cells was confirmed by confocal fluorescence microscopy and quantitative analysis. Furthermore, western blotting was performed to assess regulatory interactions between IMPDH2 and its binding proteins upon knockdown of either molecule. Western blotting was also used to detect protein levels within MG-related signaling pathways following IMPDH2 knockdown or overexpression.

Results

IMPDH2 expression was significantly elevated in PBMCs and CD4+ T cells from MG patients compared with healthy controls. Clinical data analysis demonstrated a positive correlation between IMPDH2 expression and both lymphocyte and neutrophil counts in MG patients. Additionally, IMPDH2 expression positively correlated with MG disease severity. Functionally, upregulation or downregulation of IMPDH2 correspondingly promoted or suppressed CD4+ T cell proliferation and apoptosis. Mechanistically, direct interactions between IMPDH2 and SRPK1 were confirmed in vitro, and IMPDH2 was found to regulate SRPK1 expression, subsequently affecting CD4+ T cell proliferation and apoptosis in MG. Furthermore, IMPDH2 was shown to activate the AKT/mTOR signaling pathway by modulating SRPK1 expression.

Conclusions

This study revealed that IMPDH2 is highly expressed in PBMCs and CD4+ T cells from MG patients, implicating its role in aberrant T cell activation during MG pathogenesis. IMPDH2 potentiates the AKT/mTOR signaling pathway in CD4+ T cells through its interaction with and upregulation of SRPK1 expression, thereby inhibiting CD4+ T cell apoptosis and promoting their proliferation in MG. These findings provide novel insights and potential therapeutic targets for modulating autoimmune responses in MG.