Toll-like Receptor-4 Signaling Modulates Hepatocellular Carcinoma-infiltrating CD4⁺CD25^high FOXP3⁺Regulatory T Cells Function And Its Mechanism

Objectives To investigate the clinicopathological significance of systemic and local accumulation of Treg cells in hepatocellular carcinoma (HCC) patients, and further explore the theory of TLR4 participating the adaptive immune responses and analyze cellular interaction.Methods Blood and tissues samples were collected from a total of 60 HCC patients, who underwent hepatic resection in the center of Hepatobiliary Surgery, Union Hospital, Wuhan, China from March 2008 to October 2008. The proportion of CD4+CD25+Treg,CD4+CD25high Treg,CD4+CD25highFOXP3+Treg cells in peripheral blood and tumor tissues with 60 HCC patients was detected by flow cytomety, immunohistochemistry and Western blotting. TLR4 expression was examined in tumor tissues and cell lines. The correlation between FOXP3+Treg cells in peripheral blood and TLR4 expression of HCC tissues was analyzed. Murine hepatoma cell H22 pre-incubated with LPS for the activation of TLR4 was co-cultured with macrophage line RAW246.7, the synthesis of cytokines as CCL22, IL-10 by the two cell lines was detected by RT-PCR.Results The prevalence of CD4+CD25+Treg (16.765.53%, P＜0.01), CD4+CD25high Treg (8.57±6.31% , P=0.002) and FOXP3+Treg cells (67.51±20.59%, P＜0.05) in peripheral blood from HCC patients was significantly higher than in healthy donors (6.04±2.01% , 1.71±1.59% and 43.35±13.91% , respectively). The number of FOXP3 positive cells was higher in HCC group than in precancerous group. The proportion of CD4+CD25highFOXP3+ Treg cells was significantly higher in the patients with high serum AFP levels and multiple tumor foci (P<0.001, P=0.009). The proportion of CD4+CD25highFOXP3+Tregs did not correlate with other clinicopathologic characteristics of HCC patients Tumor and normal tissues. HepG2 and H22 cells both expressed TLR4 mRNA and protein. The TLR4 mRNA and protein expression in HCC tissues was stronger than in normal tissues. The expression level of TLR4 protein in HCC specimens was correlated with frequency of CD4+CD25highFOXP3+Tregs in peripheral blood. Cell cultivation experiments indicated that the mRNA levels of IL-10 and CCL22 were significantly up-regulated in RAW246.7 cell lines not H22 cell lines under the condition of co-cultivation of H22 pre-incubated with LPS, while the expression of TNF-a remained unchanged. Tumor cells without LPS pre-incubation induced negligible gene expression changes in the co-cultured RAW246.7 cells. H22 cells alone did not produce IL-10 and CCL22 under the condition of TLR4 activation by LPS stimulation or not.Conclusions Treg cells accumulate in HCC tumor and peripheral blood. The variation in the number of Treg cells correlated positively with that of more foci of tumor. It has been suggested that TLR4 on hepatoma cell lines may indirectly facilitate the recruitment of Tregs into tumor site and promote the intrahepatic metastasis under the interaction with macrophages by secreting IL-10 and CCL22. TLR4 signaling may enhance the suppressive function of Tregs via the interaction between cancerous cells and macrophages.