Defective mitophagy of CD4+T cells in ITP via NAD+/SIRT1 reduction

Abstract Introduction Immune thrombocytopenia (ITP), an acquired autoimmune disease, is characterized by immune-mediated platelet destruction and suppressed platelet production, resulting in isolated thrombocytopenia. A growing body of research indicates that the immune dysregulation of CD4+T cells plays a pivotal role in the pathogenesis of ITP. Our previous research revealed that PINK1/Parkin-mediated mitophagy deficiency contributed to mitochondrial dysfunction and altered the metabolism of ITP CD4+T cells, which affected their differentiation and function by regulating the epigenetic state of immune response genes. However, the upstream mechanism responsible for the mitophagy deficiency of CD4+T cells in ITP remains unclear. NAD+ is one of the most important metabolic products in the human body. As a common substrate for various enzymes, including PARPs, sirtuins, D38/CD157 and SARM1, NAD+ influences processes such as genomic stability, gene expression, inflammation, and circadian rhythms. Defects in the NAD+/SIRT1-PGC-1α axis reportedly lead to a deficiency in PINK1-related mitophagy, thereby mediating mitochondrial dysfunction in xeroderma pigmentosum. However, whether the NAD+/SIRT1 axis plays a role in mitophagy deficiency in ITP CD4+ T cells remains unclear. This study aimed to explore the upstream mechanism of mitophagy deficiency in ITP CD4+ T cells and to analyze the correlation of the NAD+/SIRT1 axis in ITP CD4+ T cells with mitophagy deficiency. Methods BM CD4+ T cells were isolated from ITP patients and healthy donors. The content of NAD+ in CD4+ T cells was analyzed using an NAD+ detection kit. The protein expression of SIRT1 in CD4+ T cells was analyzed by Western blotting. Nicotinamide riboside (NR) and a SIRT1 agonist (SRT1720) were administered to ITP CD4+ T cells in vitro to reveal the role of the NAD+/SIRT1 axis in mitophagy deficiency. An active ITP mouse model was established for in vivo verification. Results RNA-seq revealed that the activity of the NAD+ nucleotidase pathway was increased, and metabolomics revealed that nicotinamide was significantly reduced in the CD4+T cells of ITP patients, which suggested abnormal NAD+ metabolism in ITP CD4+ T cells. The NAD+ content and the expression level of SIRT1 in ITP CD4+ T cells were significantly reduced, indicating a defect in the NAD+/SIRT1 axis of CD4+ T cells in ITP. After in vitro treatment with a SIRT1 agonist, the expression level of PINK1 and the colocalization of mitochondria and lysosomes increased, suggesting an improvement in mitophagy. Moreover, the content of mitochondria and mtROS and the membrane potential of mitochondria in ITP CD4+ T cells were restored after SIRT1 agonist intervention. After in vitro treatment with NR, the expression of SIRT1 and the level of mitophagy were significantly increased. In vivo, a SIRT1 agonist and NR accelerated platelet recovery in an active ITP mouse model. After treatment, the Th1/Th2 imbalance and the Treg number were restored, and the contents of mitochondria and mtSOX and the potential of mitochondria were corrected. Moreover, mitophagy in CD4+ T cells was corrected in the bone marrow of the ITP model mice. The mtROS content of ITP CD4+ T cells was increased, whereas the glutathione content was significantly decreased. GSEA revealed that pathways related to oxidative stress and the DNA damage response in ITP CD4+ T cells were significantly upregulated, suggesting that mtROS may cause functional impairment of ITP CD4+ T cells through oxidative stress and DNA damage. Moreover, mitophagy deficiency could mediate cellular mitochondrial damage, leading to mtDNA leakage. GSEA revealed that the expression of type I interferon in ITP CD4+ T cells and the pathways positively regulated by type 1 interferon were significantly upregulated, suggesting that mitophagy deficiency may promote the production of type I interferons by CD4+ T cells in ITP, thereby participating in the immune imbalance in ITP. Conclusion Defects in the NAD+/SIRT1 axis lead to mitophagy deficiency in ITP CD4+ T cells, resulting in an immune imbalance in ITP. Targeted correction of the NAD+/SIRT1 axis can regulate CD4+ T-cell differentiation through the correction of mitophagy in ITP CD4+ T cells, providing new therapeutic ideas and targets for the clinical treatment of ITP.