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Chemokines are important protein-signaling molecules that regulate various immune responses by activating chemokine receptors which belong to the G protein-coupled receptor (GPCR) superfamily. Despite the substantial progression of our structural understanding of GPCR activation by small molecule and peptide agonists, the molecular mechanism of GPCR activation by protein agonists remains unclear. Here, we present a 3.3-Å cryo-electron microscopy structure of the human chemokine receptor CCR6 bound to its endogenous ligand CCL20 and an engineered Go. CCL20 binds in a shallow extracellular pocket, making limited contact with the core 7-transmembrane (TM) bundle. The structure suggests that this mode of binding induces allosterically a rearrangement of a noncanonical toggle switch and the opening of the intracellular crevice for G protein coupling. Our results demonstrate that GPCR activation by a protein agonist does not always require substantial interactions between ligand and the 7TM core region. Chemokine receptors are GPCRs involved in immune responses and regulated by small protein ligands known as chemokines. A structural study of the human CCR6/CCL20–Go complex reveals that CCL20 binds in a shallow extracellular pocket, and suggests that activation of CCR6 by CCL20 binding involves an allosteric effect on a noncanonical toggle switch.

Estrogen deficiency causes a gut microbiome-dependent expansion of BM Th17 cells and TNF-α-producing T cells. The resulting increased BM levels of IL-17a (IL-17) and TNF stimulate RANKL expression and activity, causing bone loss. However, the origin of BM Th17 cells and TNF+ T cells is unknown. Here, we show that ovariectomy (ovx) expanded intestinal Th17 cells and TNF+ T cells, increased their S1P receptor 1-mediated (S1PR1-mediated) egress from the intestine, and enhanced their subsequent influx into the BM through CXCR3- and CCL20-mediated mechanisms. Demonstrating the functional relevance of T cell trafficking, blockade of Th17 cell and TNF+ T cell egress from the gut or their influx into the BM prevented ovx-induced bone loss. Therefore, intestinal T cells are a proximal target of sex steroid deficiency relevant for bone loss. Blockade of intestinal T cell migration may represent a therapeutic strategy for the treatment of postmenopausal bone loss.

ObjectiveA signature that unifies the colorectal cancer (CRC) microbiota across multiple studies has not been identified. In addition to methodological variance, heterogeneity may be caused by both microbial and host response differences, which was addressed in this study.DesignWe prospectively studied the colonic microbiota and the expression of specific host response genes using faecal and mucosal samples (‘ON’ and ‘OFF’ the tumour, proximal and distal) from 59 patients undergoing surgery for CRC, 21 individuals with polyps and 56 healthy controls. Microbiota composition was determined by 16S rRNA amplicon sequencing; expression of host genes involved in CRC progression and immune response was quantified by real-time quantitative PCR.ResultsThe microbiota of patients with CRC differed from that of controls, but alterations were not restricted to the cancerous tissue. Differences between distal and proximal cancers were detected and faecal microbiota only partially reflected mucosal microbiota in CRC. Patients with CRC can be stratified based on higher level structures of mucosal-associated bacterial co-abundance groups (CAGs) that resemble the previously formulated concept of enterotypes. Of these, Bacteroidetes Cluster 1 and Firmicutes Cluster 1 were in decreased abundance in CRC mucosa, whereas Bacteroidetes Cluster 2, Firmicutes Cluster 2, Pathogen Cluster and Prevotella Cluster showed increased abundance in CRC mucosa. CRC-associated CAGs were differentially correlated with the expression of host immunoinflammatory response genes.ConclusionsCRC-associated microbiota profiles differ from those in healthy subjects and are linked with distinct mucosal gene-expression profiles. Compositional alterations in the microbiota are not restricted to cancerous tissue and differ between distal and proximal cancers.

Th9 cells are a subset of CD4+ Th cells that produce the pleiotropic cytokine IL-9. IL-9/Th9 can function as both positive and negative regulators of immune response, but the role of IL-9/Th9 in tumor immunity is unknown. We examined the role of IL-9/Th9 in a model of pulmonary melanoma in mice. Lack of IL-9 enhanced tumor growth, while tumor-specific Th9 cell treatment promoted stronger antitumor responses in both prophylactic and therapeutic models. Th9 cells also elicited strong host antitumor CD8+ CTL responses by promoting Ccl20/Ccr6-dependent recruitment of DCs to the tumor tissues. Subsequent tumor antigen delivery to the draining LN resulted in CD8+ T cell priming. In agreement with this model, Ccr6 deficiency abrogated the Th9 cell-mediated antitumor response. Our data suggest a distinct role for tumor-specific Th9 cells in provoking CD8+ CTL-mediated antitumor immunity and indicate that Th9 cell-based cancer immunotherapy may be a promising therapeutic approach.

C-C motif chemokine ligand 20 (CCL20) participates in multiple oncogenic processes, but its role in lung adenocarcinoma (LUAD) is unclear. Herein, we explored the mechanism by which CCL20 works in LUAD progression. We performed bioinformatical analyses based on the complete transcriptome sequencing data from 1544 LUAD cases in 4 independent cohorts to evaluate signaling pathways regulated by CCL20. We established A549 and H358 cell lines with CCL20 knockdown to explore how CCL20 promotes tumor progression and experiments. Using another independent cohort of 348 urothelial carcinoma patients treated with the anti-PD-L1 agent (atezolizumab), we explored the synergistic effect of CCL20 and TGF-β on immunotherapy efficacy. High CCL20 expression is a poor prognostic marker for LUAD patients, and is associated with enhanced epithelial-mesenchymal transition (EMT), inflammatory response, and activated TNF pathway in LUAD. CCL20 knockdown restrained the EMT process and cell proliferation of LUAD cells and . Low CCL20 expression blocked the detrimental effects of high TGF-β on survival and effectively improved patients' response to anti-PD-L1 therapy. Collectively, we revealed the underlying mechanisms by which CCL20 promotes LUAD progression based on the largest sample size. The synergistic inhibitory effect of CCL20 and TGF-β on immune-checkpoint blockade therapy efficacy provides new views of immunotherapy resistance.

The suppression of androgen receptor (AR) expression exacerbates the migration potential of prostate cancer. This study identified a previously unrecognized regulation of the AR‐controlled pathway that promotes migration potential in prostate cancer cells. Prostate cancer cells that pass through a transwell membrane (mig cells) have a higher migration potential with a decreased AR expression than parental cells. In this study, we aimed to elucidate the mechanism of migration enhancement associated with the suppression of AR signaling. Expression of C–C motif ligand 20 (CCL20) is upregulated in mig cells, unlike in the parental cells. Knockdown of AR with small interfering RNA (siAR) in LNCaP and C4‐2B cells increased CCL20 secretion and enhanced the migration of cancer cells. Mig cells, CCL20‐treated cells, and siAR cells promoted cell migration with an enhancement of AKT phosphorylation and Snail expression, while the addition of a C–C chemokine receptor 6 (CCR6, the specific receptor of CCL20) inhibitor, anti‐CCL20 antibody, and AKT inhibitor suppressed the activation of AKT and Snail. With 59 samples of prostate cancer tissue, CCL20 secretion was profuse in metastatic cases despite low AR expression levels. Snail expression was associated with the expression of CCL20 and CCR6. A xenograft study showed that the anti‐CCL20 antibody significantly inhibited Snail expression, thereby suggesting a new therapeutic approach for castration‐resistant prostate cancer with the inhibition of the axis between CCL20 and CCR6. This study is the first to show that AR suppression enhances CCL20 expression and that the CCL20‐CCR6 axis contributes to the migration potential of prostate cancer cells. The CCL20‐CCR6 axis‐AKT‐snail pathway, activated by AR suppression in prostate cancer, may be a novel therapeutic target for prostate cancer.

Background Radioresistance of HNSCCs remains a major challenge for effective tumor control. Combined radiotherapy (RT) and immunotherapy (IT) treatment improved survival for a subset of patients with inflamed tumors or tumors susceptible to RT-induced inflammation. To overcome radioresistance and improve treatment outcomes, an understanding of factors that suppress anti-tumor immunity is necessary. In this regard, regulatory T cells (Tregs) are critical mediators of immune suppression in HNSCCs. In this study, we investigated how radiation modulates Treg infiltration in tumors through the chemokine CCL20. We hypothesized that radiation induces CCL20 secretion resulting in Treg infiltration and suppression of anti-tumor immunity.Methods Human and mouse HNSCC cell lines with different immune phenotypes were irradiated at doses of 2 or 10 Gy. Conditioned media, RNA and protein were collected for assessment of CCL20. qPCR was used to determine CCL20 gene expression. In vivo, MOC2 cells were implanted into the buccal cavity of mice and the effect of neutralizing CCL20 antibody was determined alone and in combination with RT. Blood samples were collected before and after RT for analysis of CCL20. Tumor samples were analyzed by flow cytometry to determine immune infiltrates, including CD8 T cells and Tregs. Mass-spectrometry was performed to analyze proteomic changes in the tumor microenvironment after anti-CCL20 treatment.Results Cal27 and MOC2 HNSCCs had a gene signature associated with Treg infiltration, whereas SCC9 and MOC1 tumors displayed a gene signature associated with an inflamed TME. In vitro, tumor irradiation at 10 Gy significantly induced CCL20 in Cal27 and MOC2 cells relative to control. The increase in CCL20 was associated with increased Treg migration. Neutralization of CCL20 reversed radiation-induced migration of Treg cells in vitro and decreased intratumoral Tregs in vivo. Furthermore, inhibition of CCL20 resulted in a significant decrease in tumor growth compared to control in MOC2 tumors. This effect was further enhanced after combination with RT compared to either treatment alone.Conclusion Our results suggest that radiation promotes CCL20 secretion by tumor cells which is responsible for the attraction of Tregs. Inhibition of the CCR6-CCL20 axis prevents infiltration of Tregs in tumors and suppresses tumor growth resulting in improved response to radiation.

The immunosuppressive tumor microenvironment (TME) drives radioresistance, but the role of γδ T cells in regulating radiosensitivity remains incompletely understood. In this study, we found that γδ T cell infiltration in the TME substantially increased after radiotherapy and contributed to radioresistance. Depletion of γδ T cells enhanced radiosensitivity. Single-cell RNA-seq revealed that γδ T cells in the postradiotherapy TME were characterized by the expression of Zbtb16, Il23r, and Il17a, and served as the primary source of IL-17A. These γδ T cells promoted radioresistance by recruiting myeloid-derived suppressor cells and suppressing T cell activation. Mechanistically, radiotherapy-induced tumor cell-derived microparticles containing dsDNA activated the cGAS-STING/NF-κB signaling pathway in macrophages, upregulating the expression of the chemokine CCL20, which was critical for γδ T cell recruitment. Targeting γδ T cells and IL-17A enhanced radiosensitivity and improved the efficacy of radiotherapy combined with anti-PD-1 immunotherapy, providing potential therapeutic strategies to overcome radioresistance.

Intestinal epithelial overexpression of the Th17 cell chemoattractant CCL20 is implicated in inflammatory bowel disease and influenced by NOD2 mutations in Crohn’s disease. Vitamin D metabolites have been shown to ameliorate inflammatory bowel disease. Considering NOD2 mutations in Crohn’s disease, we investigated whether Vitamin D deficiency (serum 25-hydroxyvitamin D concentration < 20 ng/mL) increases circulating CCL20 levels in inflammatory bowel disease patients and healthy controls and whether active 1,25-dihydroxyvitamin D (calcitriol) downregulates systemic and intestinal CCL20 expression. In a cross-sectional study, serum concentrations of CCL20, 25-hydroxyvitamin D, and calcitriol were measured in 170 NOD2 -genotyped Crohn’s disease patients, 80 ulcerative colitis patients, and 60 healthy controls. Additionally, the effect of calcitriol on experimentally induced CCL20 expression was examined using human intestinal epithelial HT-29 cells. Multivariable linear regression analyses revealed that both the diagnosis of inflammatory bowel disease and vitamin D deficiency were independently associated with elevated CCL20 levels. Compared to healthy controls, Crohn’s disease patients and ulcerative colitis patients exhibited significantly higher circulating CCL20 levels. Unlike in Crohn’s disease patients, vitamin D deficiency was associated with higher CCL20 levels in healthy controls and ulcerative colitis patients, whereas the calcitriol/25-hydroxyvitamin D activation ratios were negatively correlated with serum CCL20 levels in healthy controls and ulcerative colitis patients with sufficient serum 25-hydroxyvitamin D status. Furthermore, calcitriol markedly inhibited intestinal epithelial induction of CCL20. In Crohn’s disease patients, cholecalciferol supplementation was associated with lower serum CCL20 levels, which were unaffected by NOD2 mutations. These findings suggest that although vitamin D metabolites may downregulate CCL20 expression in healthy controls and ulcerative colitis patients, this regulatory effect appears to be impaired in Crohn’s disease patients.

T helper (Th) 17 cells have recently been implicated in psoriasis pathogenesis, but mechanisms of how these cells traffic into inflamed skin are unknown. By immunostaining for interleukin (IL)-17A and IL-22, we show numerous cells present in psoriasis lesions that produce these cytokines. We next found that Th17 cytokines (IL-17A, IL-22, and tumor necrosis factor (TNF)-α) markedly increased the expression of CC chemokine ligand (CCL) 20, a CC chemokine receptor (CCR)6 ligand, in human keratinocyte monolayer and raft cultures in a dose- and time-dependent manner. Lastly, we showed in mice that subcutaneous injection with recombinant IL-17A, IL-22, or TNF-α led to the upregulation of both CCL20 and CCR6 expression in skin as well as cutaneous T-cell infiltration. Taken together, these data show that Th17 cytokines stimulate CCL20 production in vitro and in vivo, and thus provide a potential explanation of how CCR6-positive Th17 cells maintain their continual presence in psoriasis through a positive chemotactic feedback loop.