7alpha-Hydroxycholesterol Elicits TLR6-Mediated Expression of IL-23 in Monocytic Cells

We investigated the question of whether 7-oxygenated cholesterol derivatives could affect inflammatory and/or immune responses in atherosclerosis by examining their effects on expression of IL-23 in monocytic cells. 7alpha-Hydroxycholesterol (7alphaOHChol) induced transcription of the TLR6 gene and elevated the level of cell surface TLR6 protein in THP-1 monocytic cells. Addition of an agonist of TLR6, FSL-1, to TLR6-expressing cells by treatment with 7alphaOHChol resulted in enhanced production of IL-23 and transcription of genes encoding the IL-23 subunit alpha (p19) and the IL-12 subunit beta (p40). However, treatment with 7-ketocholesterol (7K) and 7beta-hydroxycholesterol (7betaOHChol) did not affect TLR6 expression, and addition of FSL-1 to cells treated with either 7K or 7betaOHChol did not influence transcription of the genes. Pharmacological inhibition of ERK, Akt, or PI3K resulted in attenuated transcription of TLR6 induced by 7alphaOHChol as well as secretion of IL-23 enhanced by 7alphaOHChol plus FSL-1. Inhibition of p38 MAPK or JNK resulted in attenuated secretion of IL-23. These results indicate that a certain type of 7-oxygenated cholesterol like 7alphaOHChol can elicit TLR6-mediated expression of IL-23 by monocytic cells via PI3K/Akt and MAPKs pathways.

Establishment and application of TLR2 receptor-based cell screening model / 药学学报

TLR2 activity plays an important role in the pathogenesis of autoimmune diseases, tumor carcinogenesis and cardio-cerebrovascular diseases. To establish a TLR2 receptor-based cell screening model, NF-kappaB promoter-driven luciferase reporter plasmids were transfected into human embryonic kidney cells (HEK293) stably expressing human TLR2 and co-receptors CD14, TLR1 and TLR6. Single clones were then isolated and characterized. Using this screening system, a human TLR2-binding peptide C8 was obtained from the Ph.D.-7 Phage Display Peptide Library through biopanning and rapid analysis of selective interactive ligands (BRASIL). The binding characteristic of C8 with human TLR2 was evaluated by ELISA, flow cytometry and immunofluorescence. The NF-kappaB luciferase activity assay showed that C8 could activate the TLR2/TLR1 signaling pathway and induce the production of cytokines TNF-alpha and IL-6. In conclusion, the TLR2 receptor-based cell screening system is successfully established and a new TLR2-binding peptide is identified by using this system.

Association Between Toll-Like Receptors/CD14 Gene Polymorphisms and Inflammatory Bowel Disease in Korean Population

The innate immune response in patients who develop inflammatory bowel disease (IBD) may be abnormal. However, the exact role of Toll-like receptors (TLRs) / CD14 gene in the pathogenesis of IBD has not been fully elucidated. We aimed to investigate the association between polymorphisms of TLR1, 2, 4, 6, and CD14 gene and susceptibility to IBD in Korean population. A total 144 patients of IBD (99 patients with ulcerative colitis, 45 patients with Crohn's disease) and 178 healthy controls were enrolled. Using a PCR-RFLP, we evaluated mutations of TLR1 (Arg80Thr), TLR2 (Arg753Gln and Arg677Trp), TLR4 (Asp299Gly and Thr399Ile), TLR6 (Ser249Pro) genes and the -159 C/T promoter polymorphism of CD14 gene. No TLR polymorphisms were detected in Korean subjects. T allele and TT genotype frequencies of CD14 gene were significantly higher in IBD patients than in healthy controls. In subgroup analysis, T allelic frequency was higher in pancolitis phenotype of ulcerative colitis. In Korean population, the promoter polymorphism at -159 C/T of the CD14 gene is positively associated with IBD, both ulcerative colitis and Crohn's disease.

Prophylactic effect of TLR5 agonist flagellin on acute graft versus host disease after allogeneic hematopoietic stem cell transplantation and its mechanism / 中国实验血液学杂志

This study was aimed to investigate the prophylactic effect of Toll like receptor (TLR)5 agonist flagellin on acute graft versus host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and its possible mechanism. The animal model with allo-HSCT aGVHD was established by using purebred mice (male mouse C57BL/6 as donor, female mouse BALB/c as recipient) with complete-unidentical major histocompatibility antigen. The recipient mice were randomly divided into 3 groups: group 1 in which mice were injected with high purity (95%) flagellin before and after allo-HSCT respectively, group 2 in which mice received allo-HSCT without injection of flagellin, group 3 in which mice were radiated alone. The aGVHD features of mice in group 1 and 2 were observed and compared. The results showed that the typical symptoms of aGVHD appeared in transplanted mice. The death peak of mice in group 2 appeared at day 4-5 after transplantation. The aGVHD symptoms were obviously alleviated and the mean survival time was prolonged significantly in mice group 1 as compared with mice in group 2 (P < 0.05). The comparison of WBC count in peripheral blood of mice in 3 groups before transplantation showed no significant difference (P > 0.05), while WBC count of mice in group 1 and 2 showed the significant difference at days 14 and 21 after transplantation (P < 0.05). The pathological appearances of aGVHD in mice of group 1 were obviously reduced as compared with mice in group 2. The flow cytometric detection of Treg cell/CD4(+) T cell levels at different time before and after transplantation demonstrated that the Treg cell level in mice of group 1 at weeks 2-4 after transplantation significantly increased as compared with mice in group 2 (P < 0.05). It is concluded that flagellin can effectively prevent the aGVHD occurrence after allo-HSCT, reduce the symptoms and pathological changes of aGVHD, obviously prolong mean survival time of mice in group 1. The mechanism of flagellin effect may be associated to increase of Treg cell level in mice after allo-HSCT.

Multiple Signaling Molecules are Involved in Expression of CCL2 and IL-1beta in Response to FSL-1, a Toll-Like Receptor 6 Agonist, in Macrophages

TLR6 forms a heterodimer with TLR2 and TLR4. While proinflammatory roles of TLR2 and TLR4 are well documented, the role of TLR6 in inflammation is poorly understood. In order to understand mechanisms of action of TLR6 in inflammatory responses, we investigated the effects of FSL-1, the TLR6 ligand, on expression of chemokine CCL2 and cytokine IL-1beta and determined cellular factors involved in FSL-1-mediated expression of CCL2 and IL-1beta in mononuclear cells. Exposure of human monocytic leukemia THP-1 cells to FSL-1 resulted not only in enhanced secretion of CCL2 and IL-1beta, but also profound induction of their gene transcripts. Expression of CCL2 was abrogated by treatment with OxPAPC, a TLR-2/4 inhibitor, while treatment with OxPAPC resulted in partially inhibited expression of IL-1beta. Treatment with FSL-1 resulted in enhanced phosphorylation of Akt and mitogen-activated protein kinases and activation of protein kinase C. Treatment with pharmacological inhibitors, including SB202190, SP6001250, U0126, Akt inhibitor IV, LY294002, GF109203X, and RO318220 resulted in significantly attenuated FSL-1-mediated upregulation of CCL2 and IL-1beta. Our results indicate that activation of TLR6 will trigger inflammatory responses by upregulating expression of CCL2 and IL-1beta via TLR-2/4, protein kinase C, PI3K-Akt, and mitogen-activated protein kinases.