Anti-psoriasis effect of 18ß-glycyrrhetinic acid by breaking CCL20/CCR6 axis through its vital active group targeting GUSB/ATF2 signaling.

BACKGROUND: Psoriasis is an immune-mediated chronic inflammatory skin disease. Current research suggests that the long-term persistence and recurrence of psoriasis are closely related to the feedback loop formed between keratinocytes and immune cells, especially in Th 17 or DC cells expressing CCR6. CCL20 is the ligand of CCR6. Therefore, drugs that block the expression of CCL20 or CCR6 may have a certain therapeutic effect on psoriasis. Glycyrrhetinic acid (GA) is the main active ingredient of the plant drug licorice and is often used to treat autoimmune diseases, including psoriasis. However, its mechanism of action is still unclear. METHODS: Psoriasis like skin lesion model was established by continuously applying imiquimod on the back skin of normal mice and CCR6-/- mice for 7 days. The therapeutic and preventive effects of glycyrrhetinic acid (GA) on the model were observed and compared. The severity of skin injury is estimated through clinical PASI scores and histopathological examination. qRT-PCR and multiple cytoline assay were explored to detect the expression levels of cytokines in animal dorsal skin lesions and keratinocyte line HaCaT cells, respectively. The dermis and epidermis of the mouse back were separated for the detection of CCL20 expression. Transcription factor assay was applied to screen, and luciferase activity assay to validate transcription factors regulated by GA. Technology of surface plasmon laser resonance with LC-MS (SPR-MS), molecular docking, and enzyme activity assay were used to identified the target proteins for GA. Finally, we synthesized different derivatives of 18beta-GA and compared their effects, as well as glycyrrhetinic acid (GL), on the skin lesion of imiquimod-induced mice to evaluate the active groups of 18beta-GA. RESULTS: 18ß-glycyrrhetinic acid (GA) improved IMQ-induced psoriatic lesions, and could specifically reduce the chemokine CCL20 level of the epidermis in lesion area, especially in therapeutic administration manner. The process was mainly regulated by transcription factor ATF2 in the keratinocytes. In addition, GUSB was identified as the primary target of 18ßGA. Our findings indicated that the subject on molecular target research of glycyrrhizin should be glycyrrhetinic acid (GA) instead of glycyrrhizic acid (GL), because GL showed little activity in vitro or in vivo. Apart from that, α, ß, -unsaturated carbonyl in C11/12 positions was crucial or unchangeable to its activity of 18ßGA, while proper modification of C3 or C30 position of 18ßGA may vastly increase its activity. CONCLUSION: Our research indicates that 18ßGA exerted its anti-psoriasis effect mainly by suppressing ATF2 and downstream molecule CCL20 predominately through α, ß, -unsaturated carbonyl at C11/12 position binding to GUSB in the keratinocytes, and then broke the feedback loop between keratinocytes and CCR6-expressing immune cells. GA has more advantages than GL in the external treatment of psoriasis. A highlight of this study is to investigate the influence of special active groups on the pharmacological action of a natural product, inspired by the molecular docking result.

Production of IL-17 by MAIT Cells Is Increased in Multiple Sclerosis and Is Associated with IL-7 Receptor Expression.

Multiple sclerosis (MS) is a T cell-driven inflammatory disease of the CNS. Research on T cell subsets involved in MS pathogenesis has mainly focused on classical CD4+ T cells, especially Th17 cells, as they produce the proinflammatory, MS-associated cytokine IL-17. However, the abundant unconventional mucosal-associated invariant T (MAIT) cells are also able to produce IL-17. MAIT cells are characterized by high CD161 expression and a semi-invariant Vα7.2 TCR, with which they recognize bacterial and yeast Ags derived from the riboflavin (vitamin B2) metabolism. In this study, we characterized MAIT cells from the peripheral blood of MS patients in comparison with healthy individuals with respect to their type-17 differentiation. We found a specific increase of IL-17+ MAIT cells as well as an increased expression of retinoic acid-related orphan receptor (ROR)γt and CCR6 in MAIT cells from MS patients, whereas the expression of T cell activation markers HLA-DR and CD38 was not different. IL-17 production by MAIT cells furthermore correlated with the surface expression level of the IL-7 receptor α-chain (CD127), which was significantly increased on MAIT cells from MS patients in comparison with healthy individuals. In summary, our findings indicate an augmented type-17 differentiation of MAIT cells in MS patients associated with their IL-7 receptor surface expression, implicating a proinflammatory role of these unconventional T cells in MS immunopathology.

Protein engineering of the chemokine CCL20 prevents psoriasiform dermatitis in an IL-23-dependent murine model.

Psoriasis is a chronic inflammatory skin disease characterized by the infiltration of T cell and other immune cells to the skin in response to injury or autoantigens. Conventional, as well as unconventional, γδ T cells are recruited to the dermis and epidermis by CCL20 and other chemokines. Together with its receptor CCR6, CCL20 plays a critical role in the development of psoriasiform dermatitis in mouse models. We screened a panel of CCL20 variants designed to form dimers stabilized by intermolecular disulfide bonds. A single-atom substitution yielded a CCL20 variant (CCL20 S64C) that acted as a partial agonist for the chemokine receptor CCR6. CCL20 S64C bound CCR6 and induced intracellular calcium release, consistent with G-protein activation, but exhibited minimal chemotactic activity. Instead, CCL20 S64C inhibited CCR6-mediated T cell migration with nominal impact on other chemokine receptor signaling. When given in an IL-23-dependent mouse model for psoriasis, CCL20 S64C prevented psoriatic inflammation and the up-regulation of IL-17A and IL-22. Our results validate CCR6 as a tractable therapeutic target for psoriasis and demonstrate the value of CCL20 S64C as a lead compound.

Ginger Extract Modulates the Expression of Chemokines CCL20 and CCL22 and Their Receptors (CCR6 and CCR4) in the Central Nervous System of Mice with Experimental Autoimmune Encephalomyelitis.

Background Chemokines facilitate the leukocytes infiltration into the central nervous system (CNS) which is an essential step in the pathogenesis of multiple sclerosis. Ginger has also a broad anti-inflammatory properties. The aim was to evaluate the effects of ginger extract on the expression of CCL20 and CCL22 and their receptors (CCR6 and CCR4, respectively) in experimental autoimmune encephalomyelitis (EAE). Material and Methods Female C57BL/6 mice used for EAE induction by immunization with myelin oligodendroglial glycoprotein. Then, the EAE mice were treated with PBS or ginger extract, from day +3 to +30. At day 31, mice were scarified and the expression of CCL20 and CCL22 and their receptors in the spinal cord measured using real time-PCR. Results The expression of CCL20, CCL22 and CCR4 in the spinal cord of PBS-administrated EAE mice was significantly higher than healthy group (P<0.04, P<0.05 and P<0.02, respectively). In 200- and 300 mg/kg ginger extract-treated EAE mice, the expression of CCL20, CCL22 and CCR4 were significantly reduced as compared with PBS-administrated EAE group (P<0.04, P<0.01 and P<0.002 for 200 mg/kg ginger extract and P<0.01, P<0.005 and P<0.004 for 300 mg/kg ginger extract, respectively). The CCR6 expression in EAE mice treated with 200- or 300 mg/kg ginger extracts was lower than PBS-administrated EAE mice (P<0.01 and P=0.07, respectively). Conclusion Treatment of EAE mice with ginger extract down-regulate the expression of CCL20 and CCL22 and their receptors in EAE mice. The possible therapeutic potential of ginger for treatment of MS can be considered in future investigations.

Effect of NB-UVB on levels of MCP-1 and CCR6 mRNA in patients with psoriasis vulgaris.

The aim of this study is to explore the effect of narrow band ultraviolet B (NB-UVB) on the chemokine receptor CCR6 mRNA levels in patients with psoriasis. Psoriasis area and severity index (PASI) values were recorded before and after the treatment with NB-UVB phototherapy of 30 psoriasis vulgaris patients. The reverse transcription-polymerase chain reaction method was used to detect the expression level of CCR6 mRNA in peripheral blood mononuclear cells, and compared with 30 healthy subjects. The PASI value of the 30 psoriasis vulgaris patients decreased significantly after 15 iterations of phototherapy treatment (P < 0.01). The expression level of CCR6 mRNA in psoriasis patients was significantly higher than in the healthy controls (P < 0.01), while the expression level of CCR6 mRNA decreased significantly after phototherapy (P < 0.01). Reduction of CCR6 level may be one of the mechanisms through which NB-UVB can treat psoriasis.

CrkL meditates CCL20/CCR6-induced EMT in gastric cancer.

BACKGROUND: In recent years, Crk-like adapter protein (CrkL) has been identified as a key regulator in the epithelial-to-mesenchymal transition (EMT). However, the molecular mechanisms underlying the CC chemokine receptor 6 (CCR6) and chemokine (C-C motif) ligand 20 (CCL20)-induced EMT in gastric cancer are still unclear. METHODS: We conducted the immunohistochemistry and immunoblotting to detect the expression of CCR6 and CrkL in 90 cases of gastric cancer tissues and five kinds of cell lines. And then, gastric cancer cells were subjected to small interfering RNA (siRNA) treatment and in vitro assay. RESULTS: Both CCR6 and CrkL were aberrantly expressed in gastric cancer specimens and closely correlated with differentiation of cell lines. The expression of CCR6 and CrkL was also significantly associated with metastasis, stage, and poor prognosis of gastric cancer. In addition, we validated CCL20 activated the expression of p-CrkL, p-Erk1/2, p-Akt, vimentin, N-cadherin and MMP2 in MGC803 cells in a dose-dependent manner. However, si-CrkL abrogated the CCL20-induced p-Erk1/2, vimentin, N-cadherin and MMP2 expression. Most importantly, the knockdown of CrkL decreased migration and invasion of MGC803 cells. CONCLUSIONS: CrkL mediates CCL20/CCR6-induced EMT via Akt pathway, instead of Erk1/2 pathway in development of gastric cancer, which indicated CCL20/CCR6-CrkL-Erk1/2-EMT pathway may be targeted to antagonize the progression of gastric cancer.

Curcumin relieves TPA-induced Th1 inflammation in K14-VEGF transgenic mice.

Curcumin has been confirmed to have anti-inflammatory properties in addition to the ability to decrease the expression of pro-inflammatory cytokines in keratinocytes. It was suggested that the interleukin-23 (IL-23)/IL-17A cytokine axis played a critical role in the pathogenesis of 12-O-tetradecanoyl phorbol 12-myristate 13-acetate (TPA)-induced K14-VEGF transgenic psoriasis-like mice model. Here, we report that topical use of a curcumin gel formulation inhibited TPA-induced Th1 inflammation in K14-VEGF transgenic mice ears but not Th17 inflammation as expected. Real-time PCR showed that mRNA levels of IL-23, IL-17A, IL-22, IL-6 and TNFα cytokines failed to increase after TPA-induction in K14-VEGF transgenic mice ear skin; but the mRNA level of IFNγ increased significantly at the same time. Furthermore, TPA-induction up-regulated the TCRγδ protein but failed to impact the CCR6 protein, which means that the proliferation of γδ T cells is incapable of IL-17A production. We find that curcumin is capable of relieving TPA-induced inflammation by directly down-regulating IFNγ production. In conclusion, curcumin inhibits TPA-induced Th1 inflammation in K14-VEGF transgenic mice which has not been previously described.

Functional characterization of ferret CCL20 and CCR6 and identification of chemotactic inhibitors.

CCL20 is currently the only known chemokine ligand for the receptor CCR6, and is a mucosal chemokine involved in normal and pathological immune responses. Although nucleotide sequence data are available for ccl20 and ccr6 sequences from multiple species, the ferret ccl20 and ccr6 sequences have not been determined. To increase our understanding of immune function in ferret models of infection and vaccination, we have used RT-PCR to obtain the ferret ccl20 and ccr6 cDNA sequences and functionally characterize the encoded proteins. The open reading frames of both genes were highly conserved across species and mostly closely related to canine sequences. For functional analyses, single cell clones expressing ferret CCR6 were generated, a ferret CCL20/mouse IgG(2a) fusion protein (fCCL20-mIgG(2a)) was produced, and fCCL20 was chemically synthesized. Cell clones expressing ferret CCR6 responded chemotactically to fCCL20-mIgG2a fusion protein and synthetic ferret CCL20. Chemotaxis inhibition studies identified the polyphenol epigallocatechin-3-gallate and the murine γ-herpesvirus 68 M3 protein as inhibitors of fCCL20. Surface plasmon resonance studies revealed that EGCG bound directly to fCCL20. These results provide molecular characterization of previously unreported ferret immune gene sequences and for the first time identify a broad-spectrum small molecule inhibitor of CCL20 and reveal CCL20 as a target for the herpesviral M3 protein.

[Expression and function analysis of chemokine receptor CCR6 in HEK293 cells].

AIM: To construct a cell line which stably expresses human chemokine receptor 6 (CCR6). METHODS: The human CCR6 cDNA and plasmid G were co-transfected into HEK 293 cells and the clones stably expressing CCR6 were picked out. The expression of CCR6 in HEK293 cells was detected by RT-PCR, Western blotting, immunofluorescence test, calcium mobilization and in vitro chemotaxis assay. RESULTS: The transfected HEK293 cells could stably express functional human CCR6. CONCLUSION: Successfully establish a cell line which stably express human CCR6 and lays the foundation for its biological function's study and specific antagonist screening.

TH1 and TH2 cell polarization increases with aging and is modulated by zinc supplementation.

Elderly subjects suffer from increased levels of activated T cells and a TH1/TH2 imbalance. Zinc deficiency of the aged is correlated with decreased cell-mediated immune responses. The association of age and zinc adjustment with the amounts of TH1 (CCR5+) and TH2 (CCR4+) cell populations in healthy aged old donors enrolled in the European ZINCAGE project was examined. Old and nonagenarian individuals revealed increased TH1, TH2 cell numbers and a decreased TH2/TH1 ratio in comparison to young individuals. The differences between TH2/TH1 ratios of young and old/nonagenarians arose from young females. Adjusted zinc status led to enhanced TH2 and TH1 amounts in fresh whole blood and thawed cells of aged donors whereas increased HLA-DR+ expression and a generally lower CCR5 expression was observed on thawed PBMC. In conclusion, aging is associated with an increase in T helper cell polarization, and changes in TH2/TH1 subsets are more obvious in women than in men. Advanced healthy aging is accompanied by TH cell polarization, too. Moderate zinc supplementation in vivo alters TH proportions. Longer zinc treatment will give more insight into the beneficial effect of zinc on T helper cell modulation.