Targeting Neuropilin-1 Suppresses the Stability of CD4+ CD25+ Regulatory T Cells via the NF-κB Signaling Pathway in Sepsis

Abstract

Neuropilin-1 (Nrp-1) contributes to maintaining the stability of CD4⁺ CD25⁺ regulatory T cells (T_regs). We investigated the impact of Nrp-1 on the stability of CD4⁺ CD25⁺ T_regs, and the underlying signaling pathways, in a model of sepsis. Splenic CD4⁺ CD25⁺ T_regs were either treated with anti-Nrp-1, transfected to silence Nrp-1 and inhibitor of NF-κB kinase subunit beta (IKKβ), or administered ammonium pyrrolidine dithiocarbamate (PDTC), followed by recombinant semaphorin 3A (rSema3A), in a simulation of sepsis. After the creation of a sepsis model in mice, anti-Nrp-1 was administered. The expression of the gene encoding forkhead box protein P-3 foxp3-T_reg-specific demethylated region (foxp3-TSDR), the apoptosis rate, the expression of Foxp-3, cytotoxic T-lymphocyte-associated protein-4 (CTLA-4), and transforming growth factor β1 (TGF-β1), interleukin 10 (IL-10) and TGF-β1 secretion, and the NF-κB signaling activity of CD4⁺ CD25⁺ T_regs were determined. Sepsis simulation with or without rSema3A increased the stability of CD4⁺ CD25⁺ T_regs, including an increase in the expression of Foxp-3, CTLA-4, and TGF-β1, decreases in apoptosis and the methylation of foxp3-TSDR, increases in the secretion of TGF-β1 and IL-10, and an increase in the immunosuppressive effect on CD4⁺ T lymphocytes. Silencing of Nrp-1 or anti-Nrp-1 treatment abrogated lipopolysaccharide (LPS) stimulation with or without an rSema3A-mediated effect. Sepsis simulation increased the DNA-binding activity of NF-κB, as well as the ratios of phosphorylated IKKβ (p-IKKβ) to IKKβ and p-P65 to P65 in vitro and vivo Silencing of IKKβ expression or PDTC treatment suppressed the stability of CD4⁺ CD25⁺ T_regs in LPS-induced sepsis. Weakening Nrp-1 reduced the stability of CD4⁺ CD25⁺ T_regs by regulating the NF-κB signaling pathway; thus, Nrp-1 could be a new target for immunoregulation in sepsis.

Keywords: NF-κB; immunoregulation; immunosuppression; neuropilin-1; regulatory T cells; sepsis.