Toll-Like Receptor 4 Signaling in High Mobility Group Box-1 Protein 1 Mediated the Suppression of Regulatory T-Cells

Abstract

BACKGROUND Treg cells play a central role in the suppression of immune response, and their suppressive capacity can be modulated by toll-like receptor (TLR) ligands. However, the detailed pathway of TLR ligand modulation is still unknown. The present study aimed to evaluate the effect of the high mobility group box-1 protein 1 (HMGB1) and lipopolysaccharide (LPS) on Treg cells through TLR4 signaling. MATERIAL AND METHODS Treg cells were purified from healthy human peripheral blood mononuclear cells (PBMCs) by magnetic-bead activity cell sorting (MACS), blocked by anti-TLR4 monoclonal antibody, and then incubated with different concentration of LPS or HMGB1. The level of gene expression of IL-1β, IL-10, IFN-γ, and TGF-β were detected using quantitative real-time polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA), and the proliferation of Treg cells after treating by LPS and HMGB1 was analyzed by flow cytometry. The NF-κB expression in Treg cells was examined by Western blotting. RESULTS LPS treated CD4 CD25 Treg cells directly increased the expression of IL-1b and IL-10 and decreased the expression of IFN-γ and TGF-β. However, HMGB1 treatment resulted in a marked decreased expression of IL-1β, IL-10, IFN-γ, and TGF-β. The proliferation of CD4+ T cells was significantly inhibited by Treg cells in the LPS treatment group, but weaken in the HMGB1 treatment group. These data suggest that HMGB1 and LPS stimulation could downregulate the expression NF-κB p65 in cytoplasmic proteins and increase the expression in nuclear proteins, thus leading to modulation of IL-1β, IL-10, IFN-γ, and TGF-β expression; moreover, the suppressive function of Treg cells could be regulated by TLR4. CONCLUSIONS TLR4 signaling in HMGB1 mediated the suppressive function of Treg cells through the activation of the NF-κB pathway.

Figure 1

(A) The purity of negatively sorted CD4⁺ T cells was (97.01±2.65%). The purity of positively sorted CD4⁺ CD25⁺ Treg cells was (84.52±2.10%). (B) Purified CD4⁺ CD25⁺ Treg cells were treated with 1 μg/mL HMGB1 for different times (8, 16, 24, 32, 48, or 72 hours); cytokines in the supernatant were detected by ELISA. (C) Purified CD4⁺ CD25⁺ Treg cells were treated with 10 μg/mL LPS for different times (8, 16, 24, 32, 48, or 72 hours); cytokines in the supernatant were detected by ELISA.

Figure 2

Quantization of cytokines produced after HMGB1 (0.1 μg/mL and 1 μg/mL for 16 hours) treatment in the non-anti-TLR4 group and the anti-TLR4 group. Cells cultured without HMGB1 were used as controls. (A) Cytokines in the supernatant were detected by ELISA, and (B) their expression was also detected by quantitative PCR.

Figure 3

Quantization of cytokines produced after LPS (0.1 μg/mL, 1 μg/mL, and 10 μg/mL for 24 hours) treatment in the non-anti-TLR4 group and the anti-TLR4 group. Cells cultured without HMGB1 were used as controls. (A) Cytokines in the supernatant were detected by ELISA, and (B) their expression was also detected by quantitative PCR.

Figure 4

Proliferation of CD4⁺ T cells in different culture systems detected by flow cytometry.

Figure 5

CD4⁺ CD25⁺ Treg cells treated with HMGB1 (A) and LPS (B) could inhibit the proliferation of CD4⁺ T cells. Data were shown as the mean ±SD.

Figure 6

NF-κB p65 expression of CD4⁺ CD25⁺ Treg cells treated with HMGB1 and LPS.