Atg5 deficiency in macrophages protects against kidney fibrosis via the CCR6-CCL20 axis.

BACKGROUND: Autophagy is a lysosome-dependent degradation pathway that regulates macrophage activation, differentiation, and polarization. Autophagy related 5 (Atg5) is a key protein involved in phagocytic membrane elongation in autophagic vesicles that forms a complex with Atg12 and Atg16L1. Alterations in Atg5 are related to both acute and chronic kidney diseases in experimental models. However, the role of macrophage-expressed Atg5 in acute kidney injury remains unclear. METHODS: Using a myeloid cell-specific Atg5 knockout (MΦ atg5-/-) mouse, we established renal ischemia/reperfusion and unilateral ureteral obstruction models to evaluate the role of macrophage Atg5 in renal macrophage migration and fibrosis. RESULTS: Based on changes in the serum urea nitrogen and creatinine levels, Atg5 deletion had a minimal effect on renal function in the early stages after mild injury; however, MΦ atg5-/- mice had reduced renal fibrosis and reduced macrophage recruitment after 4 weeks of ischemia/reperfusion injury and 2 weeks of unilateral ureteral obstruction injury. Atg5 deficiency impaired the CCL20-CCR6 axis after severe ischemic kidneys. Chemotactic responses of bone marrow-derived monocytes (BMDMs) from MΦ atg5-/- mice to CCL20 were significantly attenuated compared with those of wild-type BMDMs, and this might be caused by the inhibition of PI3K, AKT, and ERK1/2 activation. CONCLUSIONS: Our data indicate that Atg5 deficiency decreased macrophage migration by impairing the CCL20-CCR6 axis and inhibited M2 polarization, thereby improving kidney fibrosis.

HOXD3 promotes the migration and angiogenesis of hepatocellular carcinoma via modifying hepatocellular carcinoma cells exosome-delivered CCR6 and regulating chromatin conformation of CCL20.

Angiogenesis plays an essential role in the microenvironment of hepatocellular carcinoma (HCC). HOXD3 is involved in the metastasis and invasion of HCC cells; Whereas the underlying molecular mechanisms in the microenvironment of HCC remain unknown. Wound healing, transwell invasion, tube formation and spheroid sprouting assays were carried out to identify the effects of HCC-HOXD3-exosomes and genes on the migration of HCC cells. ChIP-PCR was applied to test the binding region of HOXD3 on CCR6, Med15, and CREBBP promoter. Exosome isolation and mRNA-seq were applied to examine the morphological characteristics of exosomes and the contained mRNA in exosomes. Co-IP and Immunofluorescence assays were used to demonstrate the role of CREBBP in the chromatin conformation of CCL20. The nude mice were used to identify the function of genes in regulating migration of HCC in vivo. In this study, integrated cellular and bioinformatic analyses revealed that HOXD3 targeted the promoter region of CCR6 and induced its transcription. CCR6 was delivered by exosomes to endothelial cells and promoted tumour migration. Overexpression of CCR6 promoted metastasis, invasion in HCCs and angiogenesis in endothelial cells (ECs), whereas its downregulation suppressed these functions. The role of HOXD3 in the metastasis and invasion of HCC cells was reversed after the suppression of CCR6. Furthermore, CCL20 was demonstrated as the ligand of CCR6, and its high expression was found in HCC tissues and cells, which was clinically associated with the poor prognosis of HCC. Mechanistically, HOXD3 targets the promoter regions of CREBBP and Med15, which affect CCL20 chromatin conformation by regulating histone acetylation and expression of Pol II to enhance the migration of HCCs. This study demonstrated the function of the HOXD3-CREBBP/Med15-CCL20-CCR6 axis in regulating invasion and migration in HCC, thus providing new therapeutic targets for HCC.

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.

Pro-Fibrotic Effects of CCL18 on Human Lung Fibroblasts Are Mediated via CCR6.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease of unknown origin, with a median patient survival time of ~3 years after diagnosis without anti-fibrotic therapy. It is characterized by progressive fibrosis indicated by increased collagen deposition and high numbers of fibroblasts in the lung. It has been demonstrated that CCL18 induces collagen and αSMA synthesis in fibroblasts. We aimed to identify the CCL18 receptor responsible for its pro-fibrotic activities. METHODS: We used a random phage display library to screen for potential CCL18-binding peptides, demonstrated its expression in human lungs and fibroblast lines by PCR and immunostaining and verified its function in cell lines. RESULTS: We identified CCR6 (CD196) as a CCL18 receptor and found its expression in fibrotic lung tissue and lung fibroblast lines derived from fibrotic lungs, but it was almost absent in control lines and tissue. CCL18 induced receptor internalization in a CCR6-overexpressing cell line. CCR6 blockade in primary human lung fibroblasts reduced CCL18-induced FGF2 release as well as collagen-1 and αSMA expression. Knockdown of CCR6 in a mouse fibroblast cell line abolished the induction of collagen and α-smooth muscle actin expression. CONCLUSION: Our data indicate that CCL18 triggers pro-fibrotic processes via CCR6, highlighting its role in fibrogenesis.

Role of the CCL20/CCR6 axis in tubular epithelial cell injury: Kidney-specific translational insights from acute kidney injury to chronic kidney disease.

This study investigated the role of the axis involving chemokine receptor 6 (CCR6) and its ligand chemokine (C-C motif) ligand 20 (CCL20) in acute kidney disease (AKD) using an ischemia-reperfusion injury (IRI) model. The model was established by clamping the unilateral renal artery pedicle of C57BL/6 mice for 30 min, followed by evaluation of CCL20/CCR6 expression at 4 weeks post-IRI. In vitro studies were conducted to examine the effects of hypoxia and H2 O2 -induced oxidative stress on CCL20/CCR6 expression in kidney tissues of patients with AKD and chronic kidney disease (CKD). Tubular epithelial cell apoptosis was more severe in C57BL/6 mice than in CCL20 antibody-treated mice, and CCR6, NGAL mRNA, and IL-8 levels were higher under hypoxic conditions. CCL20 blockade ameliorated apoptotic damage in a dose-dependent manner under hypoxia and reactive oxygen species injury. CCR6 expression in IRI mice indicated that the disease severity was similar to that in patients with the AKD phenotype. Morphometry of CCL20/CCR6 expression revealed a higher likelihood of CCR6+ cell presence in CKD stage 3 patients than in stage 1-2 patients. Kidney tissues of patients with CKD frequently contained CCL20+ cells, which were positively correlated with interstitial inflammation. CCL20/CCR6 levels were increased in fibrotic kidneys at 4 and 8 weeks after 5/6 nephrectomy. These findings suggest that modulating the CCL20/CCR6 pathway is a potential therapeutic strategy for managing the progression of AKD to CKD.

Increased gene expression of CCR6 and RORγt in peripheral blood cells of rheumatoid arthritis patients and their correlation with anti-cyclic citrullinated peptide and disease activity.

OBJECTIVES: The significance of T helper 17 (Th17) cells in the pathogenesis of rheumatoid arthritis (RA) has recently been demonstrated in many studies. Retinoic acid receptor-related orphan receptor γt (RORγt) is a transcription factor that is specifically involved in the generation of Th17 cells. Besides, the chemokine receptor CCR6, the receptor for CCL20, is characteristically expressed by these cells. Considering the pivotal roles of Th17 cells in RA pathogenesis, in this study, we assessed the gene expression of CCR6 and RORγt in the peripheral blood leukocytes of new case RA patients. Also, we evaluated their association with anticyclic citrullinated peptide (anti-CCP) antibodies and disease activity. METHODS: Forty-five new case RA patients and 45 healthy persons have been recruited in this investigation. The gene expression of CCR6 and RORγt was evaluated by quantitative real-time PCR (qRT-PCR), and anti-CCP antibodies plasma levels were measured using the enzyme-linked immunosorbent assay (ELISA) technique. Disease activity was measured according to the disease activity score-28 (DAS-28) formula. RESULTS: The gene expression of CCR6 and RORγt increased remarkably in new case RA patients compared to healthy controls (p < .05 and p < .01, respectively). Moreover, there was a positive correlation between RORγt gene expression and parameters, including gene expression of CCR6 (p = .001, r = .461), plasma levels of CCL20 (p = .0009, r = .477), ESR (p = .004, r = .419), DAS-28 (p = .006, r = .402), anti-CCP (p = .019, r = .346), and RF (p = .001, r = .451). Also, CCR6 gene expression was positively associated with the DAS-28 (p = .037, r = .310), plasma levels of anti-CCP (p = .037, r = .312), and ESR (p = .029, r = .327). CONCLUSION: Increased gene expression of CCR6 and RORγt in peripheral blood leukocytes of new case RA patients may contribute to the exacerbation and pathogenesis of RA.

PPARδ dysregulation of CCL20/CCR6 axis promotes gastric adenocarcinoma carcinogenesis by remodeling gastric tumor microenvironment.

BACKGROUND: Peroxisome proliferator-activated receptor delta (PPARδ) promotes inflammation and carcinogenesis in many organs, but the underlying mechanisms remains elusive. In stomachs, PPARδ significantly increases chemokine Ccl20 expression in gastric epithelial cells while inducing gastric adenocarcinoma (GAC). CCR6 is the sole receptor of CCL20. Here, we examine the role of PPARδ-mediated Ccl20/Ccr6 signaling in GAC carcinogenesis and investigate the underlying mechanisms. METHODS: The effects of PPARδ inhibition by its specific antagonist GSK3787 on GAC were examined in the mice with villin-promoter-driven PPARδ overexpression (PpardTG). RNAscope Duplex Assays were used to measure Ccl20 and Ccr6 levels in stomachs and spleens. Subsets of stomach-infiltrating immune cells were measured via flow cytometry or immunostaining in PpardTG mice fed GSK3787 or control diet. A panel of 13 optimized proinflammatory chemokines in mouse sera were quantified by an enzyme-linked immunosorbent assay. RESULTS: GSK3787 significantly suppressed GAC carcinogenesis in PpardTG mice. PPARδ increased Ccl20 level to chemoattract Ccr6+ immunosuppressive cells, including tumor-associated macrophages, myeloid-derived suppressor cells and T regulatory cells, but decreased CD8+ T cells in gastric tissues. GSK3787 suppressed PPARδ-induced gastric immunosuppression by inhibiting Ccl20/Ccr6 axis. Furthermore, Ccl20 protein levels increased in sera of PpardTG mice starting at the age preceding gastric tumor development and further increased with GAC progression as the mice aged. GSK3787 decreased the PPARδ-upregulated Ccl20 levels in sera of the mice. CONCLUSIONS: PPARδ dysregulation of Ccl20/Ccr6 axis promotes GAC carcinogenesis by remodeling gastric tumor microenvironment. CCL20 might be a potential biomarker for the early detection and progression of GAC.

Neutrophils for Revascularization Require Activation of CCR6 and CCL20 by TNFα.

BACKGROUND: Activation of immune-inflammatory pathways involving TNFα (tumor necrosis factor alpha) signaling is critical for revascularization and peripheral muscle tissue repair after ischemic injury. However, mechanisms of TNFα-driven inflammatory cascades directing recruitment of proangiogenic immune cells to sites of ischemia are unknown. METHODS: Muscle tissue revascularization after permanent femoral artery ligation was monitored in mutant mice by laser Doppler imaging and light sheet fluorescence microscopy. TNFα-mediated signaling and the role of the CCL20 (C-C motif chemokine ligand 20)-CCR6 (C-C chemokine receptor 6) axis for formation of new vessels was studied in vitro and in vivo using bone marrow transplantation, flow cytometry, as well as biochemical and molecular biological techniques. RESULTS: TNFα-mediated activation of TNFR (tumor necrosis factor receptor) 1 but not TNFR2 was found to be required for postischemic muscle tissue revascularization. Bone marrow-derived CCR6+ neutrophil granulocytes were identified as a previously undescribed TNFα-induced population of proangiogenic neutrophils, characterized by increased expression of VEGFA (vascular endothelial growth factor A). Mechanistically, postischemic activation of TNFR1 induced expression of the CCL20 in vascular cells and promoted translocation of the CCL20 receptor CCR6 to the cell surface of neutrophils, essentially conditioning VEGFA-expressing proangiogenic neutrophils for CCL20-dependent recruitment to sites of ischemia. Moreover, impaired revascularization of ischemic peripheral muscle tissue in diabetic mice was associated with reduced numbers of proangiogenic neutrophils and diminished CCL20 expression. Administration of recombinant CCL20 enhanced recruitment of proangiogenic neutrophils and improved revascularization of diabetic ischemic skeletal muscles, which was sustained by sequential treatment with fluvastatin. CONCLUSIONS: We demonstrate that site-specific activation of the CCL20-CCR6 axis via TNFα recruits proangiogenic VEGFA-expressing neutrophils to sites of ischemic injury for initiation of muscle tissue revascularization. The findings provide an attractive option for tissue revascularization, particularly under diabetic conditions.

Establishment of a Novel Anti-Human CCR6 Monoclonal Antibody C6Mab-19 with the High Binding Affinity in Flow Cytometry.

CC chemokine receptor 6 (CCR6) is a member of the G-protein-coupled receptor family that is highly expressed in B lymphocytes, effector and memory T cells, regulatory T cells, and immature dendritic cells. CCR6 has been revealed to have important functions in many pathological conditions, such as cancer, intestinal bowel disease, psoriasis, and autoimmune diseases. The only CCR6 chemokine ligand, CC motif chemokine ligand 20 (CCL20), is also involved in pathogenesis by interacting with CCR6. The CCL20/CCR6 axis is drawing attention as an attractive therapeutic target for various diseases. In this study, we developed novel monoclonal antibodies (mAbs) against human CCR6 (hCCR6) using the peptide immunization method, which are applicable to flow cytometry and immunohistochemistry. The established anti-hCCR6 mAb, clone C6Mab-19 (mouse IgG1, kappa), reacted with hCCR6-overexpressed Chinese hamster ovary-K1 (CHO/hCCR6), human liver carcinoma (HepG2), and human differentiated hepatoma (HuH-7) cells in flow cytometry. The dissociation constant (KD) of C6Mab-19 was determined as 3.0 × 10-10 M for CHO/hCCR6, 6.9 × 10-10 M for HepG2, and 1.8 × 10-10 M for HuH-7. Thus, C6Mab-19 could bind to exogenously and endogenously expressed hCCR6 with extremely high affinity. Furthermore, C6Mab-19 could stain formalin-fixed paraffin-embedded lymph node tissues from a patient with non-Hodgkin lymphoma by immunohistochemistry. Therefore, C6Mab-19 is suitable for detecting hCCR6-expressing cells and tissues and could be useful for pathological analysis and diagnosis.

Panel of miR-150 and linc00673, regulators of CCR6/CCL20 may serve as non-invasive diagnostic marker of non-small cell lung cancer.

The C-C motif ligand 20 (CCL20) is a chemokine that specifically binds to the chemokine receptor 6 (CCR6) and the CCL20/CCR6 axis has been implicated in the non-small lung cancer (NSCLC) development and progression. Its expression is regulated by mutual interactions of non-coding RNAs (ncRNAs). This goals of presented study was to evaluate the expression level of CCR6/CCL20 mRNA in NSCLC tissue comparative to selected ncRNAs: miR-150, linc00673. The expression level of the studied ncRNAs was also assessed in serum extracellular vesicles (EVs). Thirty patients (n = 30) were enrolled as the study cohort. Total RNA was isolated from tumor tissue, adjacent macroscopically unchanged tissue and serum EVs. The expression level of studied genes and ncRNAs were estimated based on the qPCR method. Higher expression level of CCL20 mRNA but lower expression level of CCR6 mRNA were observed in tumor in comparison to control tissue. Relative to the smoking status, higher CCL20 (p < 0.05) and CCR6 mRNA (p > 0.05) expression levels were observed in current smokers than in never smokers. In serum EVs the expression level of miR-150 has a negative correlation with AJCC tumor staging, whereas the expression level of linc00673 positively correlated (p > 0.05). The lower expression level of miR-150 and higher expression level of linc00673 in serum EVs were observed in NSCLC patients with lymph nodes metastases (p > 0.05). Regarding the histopathological type, significantly lower expression level of miR-150 and higher expression level of linc00673 were observed in the serum EVs of patients with AC compared to patient with SCC. Our findings revealed that smoking significantly changed the expression level of CCL20 mRNA in NSCLC tissue. Changes in expression levels of miR-150 and linc00673 in the serum EVs of NSCLC patients in relation to presence of lymph node metastases and the stage of cancer development may serve as a non-invasive molecular biomarkers of tumor progression. Furthermore, expression levels of miR-150 and linc00673 may serve as non-intrusive diagnostic biomarkers differentiating adenocarcinoma from squamous cell carcinoma.

CCL20 inhibition for treating inflammation in ankylosing spondylitis.

OBJECTIVES: Th17 cells are known to play a significant role in AS. C-C motif chemokine ligand 20 (CCL20) binds to C-C chemokine receptor 6 (CCR6) on Th17 cells, promoting their migration to inflammation sites. The aim of this research is to examine the effectiveness of CCL20 inhibition in treating inflammation in AS. METHODS: Mononuclear cells from peripheral blood (PBMC) and SF (SFMC) were collected from healthy individuals and AS. Flow cytometry was used to analyse cells producing inflammatory cytokines. CCL20 levels were determined using ELISA. The impact of CCL20 on Th17 cell migration was verified using a Trans-well migration assay. The in vivo efficacy of CCL20 inhibition was evaluated using an SKG mouse model. RESULTS: The presence of Th17 cells and CCL20 expressing cells was higher in SFMCs from AS patients compared with their PBMCs. The CCL20 level in AS SF was significantly higher than in OA patients. The percentage of Th17 cells in PBMCs from AS patients increased when exposed to CCL20, whereas the percentage of Th17 cells in SFMCs from AS patients decreased when treated with CCL20 inhibitor. The migration of Th17 cells was found to be influenced by CCL20, and this effect was counteracted by the CCL20 inhibitor. In the SKG mouse model, the use of CCL20 inhibitor significantly reduced joint inflammation. CONCLUSION: This research validates the critical role of CCL20 in AS and suggests that targeting CCL20 inhibition could serve as a novel therapeutic approach for AS treatment.

Intestinal endothelial cells increase HIV infection and latency in resting and activated CD4 + T cells, particularly affecting CCR6 + CD4 + T cells.

BACKGROUND: With suppressive antiretroviral therapy, HIV infection is well-managed in most patients. However, eradication and cure are still beyond reach due to latent viral reservoirs in CD4 + T cells, particularly in lymphoid tissue environments including the gut associated lymphatic tissues. In HIV patients, there is extensive depletion of T helper cells, particularly T helper 17 cells from the intestinal mucosal area, and the gut is one of the largest viral reservoir sites. Endothelial cells line lymphatic and blood vessels and were found to promote HIV infection and latency in previous studies. In this study, we examined endothelial cells specific to the gut mucosal area-intestinal endothelial cells-for their impact on HIV infection and latency in T helper cells. RESULTS: We found that intestinal endothelial cells dramatically increased productive and latent HIV infection in resting CD4 + T helper cells. In activated CD4 + T cells, endothelial cells enabled the formation of latent infection in addition to the increase of productive infection. Endothelial-cell-mediated HIV infection was more prominent in memory T cells than naïve T cells, and it involved the cytokine IL-6 but did not involve the co-stimulatory molecule CD2. The CCR6 + T helper 17 subpopulation was particularly susceptible to such endothelial-cell-promoted infection. CONCLUSION: Endothelial cells, which are widely present in lymphoid tissues including the intestinal mucosal area and interact regularly with T cells physiologically, significantly increase HIV infection and latent reservoir formation in CD4 + T cells, particularly in CCR6 + T helper 17 cells. Our study highlighted the importance of endothelial cells and the lymphoid tissue environment in HIV pathology and persistence.

A Novel C-C Chemoattractant Cytokine (Chemokine) Receptor 6 (CCR6) Antagonist (PF-07054894) Distinguishes between Homologous Chemokine Receptors, Increases Basal Circulating CCR6+ T Cells, and Ameliorates Interleukin-23-Induced Skin Inflammation.

Blocking chemokine receptor C-C chemoattractant cytokine (chemokine) receptor (CCR) 6-dependent T cell migration has therapeutic promise in inflammatory diseases. PF-07054894 is a novel CCR6 antagonist that blocked only CCR6, CCR7, and C-X-C chemoattractant cytokine (chemokine) receptor (CXCR) 2 in a ß-arrestin assay panel of 168 G protein-coupled receptors. Inhibition of CCR6-mediated human T cell chemotaxis by (R)-4-((2-(((1,4-Dimethyl-1H-pyrazol-3-yl)(1-methylcyclopentyl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3-hydroxy-N,N-dimethylpicolinamide (PF-07054894) was insurmountable by CCR6 ligand, C-C motif ligand (CCL) 20. In contrast, blockade of CCR7-dependent chemotaxis in human T cells and CXCR2-dependent chemotaxis in human neutrophils by PF-07054894 were surmountable by CCL19 and C-X-C motif ligand 1, respectively. [3H]-PF-07054894 showed a slower dissociation rate for CCR6 than for CCR7 and CXCR2 suggesting that differences in chemotaxis patterns of inhibition could be attributable to offset kinetics. Consistent with this notion, an analog of PF-07054894 with fast dissociation rate showed surmountable inhibition of CCL20/CCR6 chemotaxis. Furthermore, pre-equilibration of T cells with PF-07054894 increased its inhibitory potency in CCL20/CCR6 chemotaxis by 10-fold. The functional selectivity of PF-07054894 for inhibition of CCR6 relative to CCR7 and CXCR2 is estimated to be at least 50- and 150-fold, respectively. When administered orally to naïve cynomolgus monkeys, PF-07054894 increased the frequency of CCR6+ peripheral blood T cells, suggesting that blockade of CCR6 inhibited homeostatic migration of T cells from blood to tissues. PF-07054894 inhibited interleukin-23-induced mouse skin ear swelling to a similar extent as genetic ablation of CCR6. PF-07054894 caused an increase in cell surface CCR6 in mouse and monkey B cells, which was recapitulated in mouse splenocytes in vitro. In conclusion, PF-07054894 is a potent and functionally selective CCR6 antagonist that blocks CCR6-mediated chemotaxis in vitro and in vivo. SIGNIFICANCE STATEMENT: The chemokine receptor, C-C chemoattractant cytokine (chemokine) receptor 6 (CCR6) plays a key role in the migration of pathogenic lymphocytes and dendritic cells into sites of inflammation. (R)-4-((2-(((1,4-Dimethyl-1H-pyrazol-3-yl)(1-methylcyclopentyl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3-hydroxy-N,N-dimethylpicolinamide (PF-07054894) is a novel CCR6 small molecule antagonist that illustrates the importance of binding kinetics in achieving pharmacological potency and selectivity. Orally administered PF-07054894 blocks homeostatic and pathogenic functions of CCR6, suggesting that it is a promising therapeutic agent for the treatment of a variety of autoimmune and inflammatory diseases.

Human CCR6+ Th Cells Show Both an Extended Stable Gradient of Th17 Activity and Imprinted Plasticity.

Th17 cells have been investigated in mice primarily for their contributions to autoimmune diseases. However, the pathways of differentiation of Th17 and related Th cells (type 17 cells) and the structure of the type 17 memory population in humans are not well understood; such understanding is critical for manipulating these cells in vivo. By exploiting differences in levels of surface CCR6, we found that human type 17 memory cells, including individual T cell clonotypes, form an elongated continuum of type 17 character along which cells can be driven by increasing RORγt. This continuum includes cells preserved within the memory pool with potentials that reflect the early preferential activation of multiple over single lineages. The phenotypes and epigenomes of CCR6+ cells are stable across cell divisions under noninflammatory conditions. Nonetheless, activation in polarizing and nonpolarizing conditions can yield additional functionalities, revealing, respectively, both environmentally induced and imprinted mechanisms that contribute differentially across the type 17 continuum to yield the unusual plasticity ascribed to type 17 cells.

Chemokine Receptors CCR6 and PD1 Blocking scFv E27 Enhances Anti-EGFR CAR-T Therapeutic Efficacy in a Preclinical Model of Human Non-Small Cell Lung Carcinoma.

Chimeric antigen receptor (CAR)-T cells, a therapeutic agent for solid tumors, are not completely effective due to a lack of infiltration of T cells into the tumor site and immunity caused by Programmed Death Receptor 1(PD1). Here, an epidermal growth factor receptor (EGFR) CAR-T cell was engineered to express the chemokine receptor CCR6 and secrete PD1 blocking Single-chain antibody fragment (scFv) E27 to enhance their anti-tumor effects. The findings showed that CCR6 enhanced the migration of EGFR CAR-E27-CCR6 T cells in vitro by the Transwell migration assay. When incubated with tumor cells, EGFR CAR-E27-CCR6 T cells specifically exerted potent cytotoxicity and produced high levels of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-2 (IL-2), and interferon-γ (IFN-γ). A non-small cell lung carcinoma (NSCLC) cell line-derived xenograft model was constructed by implanting modified A549 cell lines into immunodeficient NOD.PrkdcscidIl2rgem1/Smoc (NSG) mice. In comparison with traditional EGFR CAR-T cells, live imaging indicated that EGFR CAR-E27-CCR6 T cells displayed superior anti-tumor function. In addition, the histopathological examination of mouse organs showed no obvious organic damage. Our findings confirmed that PD1 blocking and CCR6 can enhance the anti-tumor function of EGFR CAR-T cells in an NSCLC xenograft model, providing an effective treatment strategy to improve the efficacy of CAR-T in NSCLC.

The CCR6/CCL20 axis expands RORγt+ Tregs to protect from glomerulonephritis.

Previous studies have identified a unique Treg population, which expresses the Th17 characteristic transcription factor RORγt. These RORγt+ Tregs possess enhanced immunosuppressive capacity, which endows them with great therapeutic potential. However, as a caveat, they are also capable of secreting pro-inflammatory IL-17A. Since the sum function of RORγt+ Tregs in glomerulonephritis (GN) remains unknown, we studied the effects of their absence. Purified CD4+ T cell populations, containing or lacking RORγt+ Tregs, were transferred into immunocompromised RAG1 knockout mice and the nephrotoxic nephritis model of GN was induced. Absence of RORγt+ Tregs significantly aggravated kidney injury, demonstrating overall kidney-protective properties. Analyses of immune responses showed that RORγt+ Tregs were broadly immunosuppressive with no preference for a particular type of T cell response. Further characterization revealed a distinct functional and transcriptional profile, including enhanced production of IL-10. Expression of the chemokine receptor CCR6 marked a particularly potent subset, whose absence significantly worsened GN. As an underlying mechanism, we found that chemokine CCL20 acting through receptor CCR6 signaling mediated expansion and activation of RORγt+ Tregs. Finally, we also detected an increase of CCR6+ Tregs in kidney biopsies, as well as enhanced secretion of chemokine CCL20 in 21 patients with anti-neutrophil cytoplasmic antibody associated GN compared to that of 31 healthy living donors, indicating clinical relevance. Thus, our data characterize RORγt+ Tregs as anti-inflammatory mediators of GN and identify them as promising target for Treg directed therapies.

Intestinal recruitment of CCR6-expressing Th17 cells by suppressing miR-681 alleviates endotoxemia-induced intestinal injury and reduces mortality.

INTRODUCTION: Sepsis or endotoxemia can induce intestinal dysfunction in the epithelial and immune barrier. Th17 cells, a distinct subset of CD4+ T-helper cells, act as "border patrol" in the intestine under pathological condition and in the previous studies, Th17 cells exhibited an ambiguous function in intestinal inflammation. Our study will explore a specific role of Th17 cells and its relevant mechanism in endotoxemia-induced intestinal injury. MATERIALS AND METHODS: Lipopolysaccharide was used to establish mouse model of endotoxemia. miR-681 was analyzed by RT-PCR and northern blot analysis and its regulation by HIF-1α was determined by chromatin immunoprecipitation and luciferase reporter assay. Intestinal Th17 cells isolated from endotoxemic mice were quantitatively evaluated by flow cytometry and its recruitment to the intestine controlled by miR-681/CCR6 pathway was assessed by using anti-miRNA treatment and CCR6 knockout mice. Intestinal histopathology, villus length, intestinal inflammation, intestinal permeability, bacterial translocation and survival were investigated, by histology and TUNEL analysis, ELISA, measurement of diamine oxidase, bacterial culture, with or without anti-miR-681 treatment in endotoxemic wild-type and (or) CCR6 knockout mice. RESULTS: In this study, we found that miR-681 was significantly promoted in intestinal Th17 cells during endotoxemia, which was dependent on hypoxia-inducible factor-1α (HIF-1α). Interestingly, miR-681 could directly suppress CCR6, which was a critical modulator for Th17 cell recruitment to the intestines. In vivo, anti-miR-681 enhanced survival, increased number of intestinal Th17 cells, reduced crypt and villi apoptosis, decreased intestinal inflammation and bacterial translocation, resulting in protection against endotoxemia-induced intestinal injury in mice. However, CCR6 deficiency could neutralize the beneficial effect of anti-miR-681 on the intestine during endotoxemia, suggesting that the increment of intestinal Th17 cells caused by anti-miR-681 relies on CCR6 expression. CONCLUSION: The results of the study indicate that control of intestinal Th17 cells by regulating novel miR-681/CCR6 signaling attenuates endotoxemia-induced intestinal injury.

Probing the effects of MR120 in preclinical chronic colitis: A first-in-class anti-IBD agent targeting the CCL20/CCR6 axis.

Concerning the growing interest in the role played by the CCL20/CCR6 axis in IBD pathogenesis and in the search for novel anti-IBD small molecules, we have recently discovered the first small-molecule (MR120) endowed with protective action against TNBS-induced colitis and zymosan-induced peritonitis. This protective action occurs through interference with the CCL20/CCR6 signaling. The aim of the present work is to expand the preclinical investigation of MR120, evaluating its beneficial anti-inflammatory effect on a model of chronic colitis obtained by cyclically exposing C57BL/6 mice to 3% DSS. Subcutaneous administration of MR120 at 1 mg/kg, the same dose effective against acute inflammation, helped attenuate several systemic and local inflammatory responses induced by DSS. Besides significantly improving murine health conditions, MR120 counteracted mucosal macroscopic injury, the increase of colonic edema and neutrophils oxidative activity, and mitigated spleen enlargement, while not significantly lowering intestinal IL-6 concentration. Overall, repeated daily treatment with MR120 for approximately 30 days was well tolerated and showed moderate protection in a relevant model of chronic colitis, in line with the beneficial effect previously observed in acute models of intestinal inflammation. Although more potent analogues of MR120 will be needed to more fully evaluate their clinical translatability, the present work provides a valuable example of in vivo efficacy of CCL20/CCR6 modulators in a chronic model of IBD.

Clinical Significance of the Pre-Transplant CXCR3 and CCR6 Expression on T Cells In Kidney Graft Recipients.

BACKGROUND: T cells play a fundamental role in the processes that mediate graft rejection, tolerance, and defense against infections. The CXCR3 and CCR6 receptors, highly expressed in Th1 (type 1 T helper cells)/Tc1 (T cytotoxic cells, type 1), Th1-Tc1, and Th17-Tc17 lymphocytes, respectively, participate in cell migration toward inflamed tissues. The altered expression level of CXCR3 and CCR6 has been associated with different clinical events after renal transplantation, such as acute rejection (AR) and chronic graft dysfunction, but data are still limited. In this study, we evaluated the expression of the receptor CXCR3 and CCR6 in peripheral blood T lymphocytes from kidney transplant recipients (KTR) and their association with viral infections, AR, and allograft function. METHODS: Through flow cytometry, the peripheral blood expression of CXCR3 and CCR6 in T cells was evaluated in a pretransplant collection of KTR. The levels of these T subpopulations and their association with the incidence of AR, kidney graft function, viral infections, cytomegalovirus, and BK virus were studied. Adverse clinical events and graft function were monitored during the first year post transplant. RESULTS: KTRs with low pretransplantation levels of Th17 (CD4+CXCR3-CCR6+) (tertile 1, Th17<16.4%) had a higher risk of suffering AR during the first year post transplantation (P = .033). KTRs with viral infections or reactivations during the first 3 months post transplantation had significantly lower levels of Tc17 (CD8+CXCR3-CCR6+) and higher levels of Th1 (CD4+CXCR3+CCR6-). In patients with cytomegalovirus reactivations, the viral peak correlates negatively with the pretransplant levels of Th1 (r = -0.606, P = .037). CONCLUSIONS: Pretransplantation assessment of Th1-Th17 and Tc1-Tc17 levels may help predict post-transplant clinical events such as AR and reactivation of viral infections.

Cell autonomous expression of BCL6 is required to maintain lineage identity of mouse CCR6+ ILC3s.

Innate lymphoid cells (ILC) are similar to T helper (Th) cells in expression of cytokines and transcription factors. For example, RORγt is the lineage-specific transcription factor for both ILC3 and Th17 cells. However, the ILC counterpart for BCL6-expressing T follicular helper (Tfh) cells has not been defined. Here, we report that in the ILC compartment, BCL6 is selectively co-expressed with not only CXCR5 but also RORγt and CCR6 in ILC3 from multiple tissues. BCL6-deficient ILC3 produces enhanced levels of IL-17A and IL-22. More importantly, phenotypic and single-cell ATAC-seq analysis show that absence of BCL6 in mature ILC3 increases the numbers of ILC1 and transitional cells co-expressing ILC3 and ILC1 marker genes. A lineage-tracing experiment further reveals BCL6+ ILC3 to ILC1 trans-differentiation under steady state. Finally, microbiota promote BCL6 expression in colonic CCR6+ ILC3 and thus reinforce their stability. Collectively, our data have demonstrated that CCR6+ ILC3 have both Th17 and Tfh programs and that BCL6 expression in these cells functions to maintain their lineage identity.