Pam3Csk4 regulates glycolysis in PMN-MDSCs and attenuates their immunosuppressive effect on T cells

To investigate the effects of Pam3Csk4 treatment on glycolysis, proliferation-related features, and immunosuppressive functions of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), as well as the subsequent impact of PMN-MDSCs on T cell proliferation. Because MRSA infection is frequently associated with antibiotic resistance and host immunosuppression, this study aimed to explore Pam3Csk4 as a potential non-antibiotic immunomodulatory strategy. An immunosuppression mouse model was established via daily LPS injections. Bone marrow MDSCs were magnetically sorted, and PMN-MDSCs versus M-MDSCs were identified by flow cytometry. PMN-MDSCs derived from both in vitro induction and in vivo models were treated with Pam3Csk4. The expression of glycolytic markers (HK2/3, LDHA, PKM2) was analyzed using qPCR and Western blot. ARG1 levels were measured by ELISA, while proliferation-related proteins were detected via WB. Additionally, CD3⁺ T cells isolated from heat-inactivated MRSA-immunized mice were co-cultured with Pam3Csk4-pretreated PMN-MDSCs for 72 h to evaluate CD4⁺ and CD8⁺ T cell distribution. The LPS-induced model was used to generate an immunosuppressive MDSC-enriched condition, whereas heat-inactivated MRSA immunization was used to assess antigen-associated T-cell responses under controlled experimental conditions. LPS-treated mice showed an MDSC-enriched immunosuppressive condition characterized by increased MDSC-related myeloid populations in bone marrow. Pam3Csk4 treatment of PMN-MDSCs modulated the expression of glycolysis-related markers, including HK2/3, LDHA, and PKM2, in a time-dependent manner and significantly increased lactate production. Functionally, Pam3Csk4 treatment modestly reduced M-MDSC proportions and markedly decreased ARG1 levels in supernatants, while modulating proteins associated with PMN-MDSC proliferation/differentiation and immunosuppression. Notably, combined treatment with Pam3Csk4 and MRSA enhanced the expression of selected glycolysis-related markers in a time- and marker-dependent manner. In co-culture experiments, although heat-inactivated MRSA immunization increased splenic CD3⁺ T cells, Pam3Csk4 pretreatment significantly attenuated the inhibitory effect of PMN-MDSCs specifically on CD3⁺ CD4⁺ T cell proliferation and altered CD4⁺/CD8⁺ T-cell subset distribution. Pam3Csk4 treatment activates the glycolytic pathway in PMN-MDSCs and modulates proliferation/differentiation-related markers and immunosuppressive function, thereby weakening the ability of PMN-MDSCs to suppress CD3⁺ CD4⁺ T cell proliferation. These findings suggest that metabolic regulation of PMN-MDSCs by Pam3Csk4 may represent a potential immunomodulatory approach for improving host immune responses during MRSA-associated immunosuppression or immune dysregulation.