[Effect of semaphorin-3A on the cellular stability of CD4+CD25+ regulatory T cells induced by lipopolysaccharide]

Abstract

Objective: To investigate the effect and mechanism of semaphorin-3A (Sema3A) in maintaining the cellular stability of CD4⁺CD25⁺ regulatory T cells (Tregs) induced by lipopolysaccharide (LPS).

Methods: In vitro, using immunomagnetic beads, splenic CD4⁺CD25⁺ Tregs of C57BL/6J mice were isolated and cultured. According to the random number table, the isolated cells were divided into control group (treated with anti-CD3e and anti-CD28 for polyclonal activation), LPS group (on the basis of control group, treated with LPS at the dose of 100 μg/L), LPS + nuclear factor kappa B (NF-κB) inhibitor pyrrolidine dithiocarbamate (PDTC) group (treated with LPS at the dose of 100 μg/L and PDTC at the dose of 25 mg/L), LPS + phosphate buffer solution (PBS) group (treated with LPS at the dose of 100 μg/L and PBS at the volume of 10 μL), LPS + PDTC + recombinant Sema3A (rSema3A) group (treated with LPS at the dose of 100 μg/L, PDTC at the dose of 25 mg/L and rSema3A at the dose of 300 μg/L), and LPS + PBS + rSema3A group (treated with LPS at the dose of 100 μg/L, PBS at the volume of 10 μL and rSema3A at the dose of 300 μg/L). mRNA and protein expressions of the specific markers of CD4⁺CD25⁺ Tregs, including forkhead box protein P-3 (Foxp-3), cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and membrane-associated transforming growth factor-β1 (TGF-β1^m+) were detected by reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence methods after 24 hours. The supernatant interleukin-10 (IL-10) and secretory type TGF-β1 (sTGF-β1) were detected by enzyme-linked immunosorbent assay (ELISA). The apoptotic level was detected by immunofluorescence. The demethylation of Foxp3-Tregs-specific demethylated region (Foxp-3-TSDR) was detected by methylation specific PCR (MSP) in order to reflect the cellular stability of CD4⁺CD25⁺ Tregs. DNA binding activity of NF-κB signaling pathway was determined by electrophoretic mobility shift assay (EMSA), and activity of NF-κB signaling pathway was determined by Western blotting.

Results: Compared with control group, LPS could increase the cellular stability, including an increase in the mRNA and protein expressions of Foxp-3, CTLA-4 and TGF-β1^m+ and secretion of IL-10 and sTGF-β1, decrease in the level of apoptosis and increase in the methylation of Foxp-3-TSDR. At the same time, LPS increased DNA binding activity of NF-κB signaling pathway and phosphorylation levels of the major molecules of NF-κB, including inhibitory protein kinaseβ (IKKβ) and p65, suggesting that the mechanism of enhancing cellular stability by LPS was related to the NF-κB signaling pathway. Compared with LPS group, PBS had no effect on cellular stability and NF-κB signaling pathway. However, administration of rSema3A further promoted cellular stability and activated NF-κB signaling pathway. Compared with LPS + PBS + rSema3A group, PDTC inhibited the function of rSema3A to increase cellular stability, as the mRNA and protein expressions of Foxp-3, CTLA-4 and TGF-β1^m+ were significantly down-regulated in the LPS + PDTC + rSema3A group [Foxp-3 mRNA (2^-ΔΔCt): 8.092±1.117 vs. 18.509±1.068, Foxp-3 protein (relative fluorescence intensity): 1.224±0.033 vs. 1.826±0.181; CTLA-4 mRNA (2^-ΔΔCt): 3.254±0.760 vs. 11.840±0.827, CTLA-4 protein (relative fluorescence intensity): 1.305±0.058 vs. 1.842±0.111; TGF-β1^m+ mRNA (2^-ΔΔCt): 3.589±1.180 vs. 8.509±0.472, TGF-β1^m+ protein (relative fluorescence intensity): 1.319±0.033 vs. 1.822±0.063, all P < 0.01], the secretion of IL-10 and sTGF-β1 were significantly decreased [IL-10 (ng/L): 445.33±54.08 vs. 992.67±83.10, sTGF-β1 (ng/L): 1 116.67±65.25 vs. 1 494.67±94.45, both P < 0.01], the apoptosis was significantly increased (fluorescence intensity: 0.398±0.031 vs. 0.268±0.046, P < 0.01), the methylation of Foxp-3-TSDR was significantly decreased (grey value: 0.467±0.048 vs. 1.780±0.119, P < 0.01), the DNA binding activity of NF-κB signaling pathway was significantly inhibited (grey value: 1.23±0.02 vs. 3.95±0.06, P < 0.01), and the phosphorylation levels of IKKβ and p65 were significantly decreased [p-IKKβ (p-IKKβ/IKKβ): 0.97±0.07 vs. 1.97±0.04, p-p65 (p-p65/p65): 0.95±0.08 vs. 1.93±0.06, both P < 0.01].

Conclusions: LPS increases the cellular stability of CD4⁺CD25⁺ Tregs through the NF-κB signaling pathway, and Sema3A further increases the cellular stability of CD4⁺CD25⁺ Tregs, and is related to the NF-κB signaling pathway.