CC Chemokine Receptor Research Articles

CCR5 Antagonist Maraviroc Inhibits Acute Exacerbation of Lung Inflammation Triggered by Influenza Virus in Cigarette Smoke-Exposed Mice.

Influenza A virus (IAV) infection is a common cause of acute exacerbations of chronic obstructive pulmonary disease (AECOPD). Since macrophage inflammatory protein 1 α, a chemokine that acts through CC-chemokine receptor (CCR)-5, appears elevated in COPD patients’ airways, we evaluated whether CCR5 antagonist Maraviroc could inhibit the exacerbated lung inflammatory response noted after IAV H1N1 infection in mice exposed to cigarette smoke (Cs). C57BL/6 mice, subjected or not to Cs inhalation for 11 days, were infected with H1N1 at day 7. Maraviroc (10 mg/kg) or dexamethasone (1 mg/kg) were given in a therapeutic schedule, followed by the analyses of lung function, survival rate, and inflammatory changes. As compared to mice subjected to Cs or H1N1 alone, the insult combination significantly worsened airway obstruction, neutrophil infiltration in the airways, and the survival rate. All changes were sensitive to Maraviroc but not dexamethasone. Maraviroc also reduced the accumulation of neutrophils and macrophages as well as CXCL1 production in the lung tissue, and serum levels of IL-6, whereas comparable viral titers in the lungs were noted in all infected groups. Collectively, these findings suggest that Maraviroc oral treatment could be an effective therapy for controlling acute exacerbations of respiratory diseases such as COPD.

HIF-2α Upregulates CCR1 to Promote Dissemination of Plasma Cells in Multiple Myeloma

Introduction: Multiple myeloma (MM) disease progression and relapse is dependent on the ability of the MM plasma cells (PC) to leave the bone marrow, re-enter the circulation and disseminate to other sites throughout the skeleton. Recent data suggests that increased bone marrow hypoxia is associated with increased recirculation of MM PC (Azab et. al. Blood 2012), suggesting that hypoxia may act as a stimulus for dissemination of MM PC. However, the mechanisms responsible for this phenomenon remain unclear. In this study, we identified a potential driver of hypoxia-mediated dissemination of MM PC.Methods and results: To identify which genes were regulated by hypoxia in MM PC, we utilised microarrays to assess the effects of HIF-2α overexpression on gene expression in the human MM cell line, LP-1. The most highly upregulated gene was the C-C chemokine receptor 1 (CCR1) (6.5-fold upregulated), the receptor for C-C chemokine ligand 3 (CCL3; also known as macrophage inflammatory protein-1α [MIP-1α]), a potent inducer of MM PC migration in vitro . In addition, overexpression of HIF-2α in the human MM cell line RPMI-8226 resulted in an almost three-fold increase in migration towards CCL3, indicating that upregulation of CCR1 in response to HIF-2α confers an increase in the migratory response to CCL3.Previous studies have indicated that over two-thirds of newly diagnosed MM patients have circulating MM PC, with increased PC numbers associated with a poorer prognosis (Nowakowski et. al. Blood 2005). CCR1 expression on peripheral blood MM PC, as assessed by flow cytometry, positively correlated with the number of circulating MM PC. Moreover, in silico analysis of CCR1 expression in publically-available microarray data from newly diagnosed MM patients found that above median CCR1 expression was associated with poor prognosis in these patients in an independent dataset (p=0.025; HR=2.3 [95% CI: 1.1-4.9]; n=142 patients; E-TABM-1138).To identify the potential role of CCR1 in driving MM PC dissemination, the migration and proliferation of MM PC expressing CCR1 was assessed in vitro and in vivo . The role of CCR1 in tumour growth and dissemination of MM PC was investigated using the C57BL/KalwRijHsd mouse model of MM. In this model, luciferase-expressing mouse 5TGM1 MM PC cells were injected into the tibia, where they establish in the bone marrow and initiate systemic MM disease. Using flow cytometry, our studies show that C57BL/KalwRijHsd mice bearing 5TGM1 CCR1 overexpressing cells (5TGM1-CCR1) have increased numbers of circulating tumour cells, when normalized to tumour burden, compared with mice bearing control 5TGM1 cells. These data suggest that expression of CCR1 drives the egress of MM PC from the bone marrow into the circulation in vivo .Conclusion: This study identified CCR1 as a novel driver for hypoxia-driven MM PC migration both in vitro and in vivo . Furthermore, this study highlights an association between increased CCR1 expression, poorer prognosis and an increase in circulating tumour cells in newly diagnosed MM patients. Importantly, these findings suggest that therapies targeting CCR1 may inhibit MM PC egress from the bone marrow and limit disease progression and relapse. DisclosuresNo relevant conflicts of interest to declare.

Key role of the CCR2-CCL2 axis in disease modification in a mouse model of tauopathy

BackgroundFor decades, dementia has been characterized by accumulation of waste in the brain and low-grade inflammation. Over the years, emerging studies highlighted the involvement of the immune system in neurodegenerative disease emergence and severity. Numerous studies in animal models of amyloidosis demonstrated the beneficial role of monocyte-derived macrophages in mitigating the disease, though less is known regarding tauopathy. Boosting the immune system in animal models of both amyloidosis and tauopathy, resulted in improved cognitive performance and in a reduction of pathological manifestations. However, a full understanding of the chain of events that is involved, starting from the activation of the immune system, and leading to disease mitigation, remained elusive. Here, we hypothesized that the brain-immune communication pathway that is needed to be activated to combat tauopathy involves monocyte mobilization via the C-C chemokine receptor 2 (CCR2)/CCL2 axis, and additional immune cells, such as CD4+ T cells, including FOXP3+ regulatory CD4+ T cells.MethodsWe used DM-hTAU transgenic mice, a mouse model of tauopathy, and applied an approach that boosts the immune system, via blocking the inhibitory Programmed cell death protein-1 (PD-1)/PD-L1 pathway, a manipulation previously shown to alleviate disease symptoms and pathology. An anti-CCR2 monoclonal antibody (αCCR2), was used to block the CCR2 axis in a protocol that partially eliminates monocytes from the circulation at the time of anti-PD-L1 antibody (αPD-L1) injection, and for the critical period of their recruitment into the brain following treatment.ResultsPerformance of DM-hTAU mice in short-term and working memory tasks, revealed that the beneficial effect of αPD-L1, assessed 1 month after a single injection, was abrogated following blockade of CCR2. This was accompanied by the loss of the beneficial effect on disease pathology, assessed by measurement of cortical aggregated human tau load using Homogeneous Time Resolved Fluorescence-based immunoassay, and by evaluation of hippocampal neuronal survival. Using both multiparametric flow cytometry, and Cytometry by Time Of Flight, we further demonstrated the accumulation of FOXP3+ regulatory CD4+ T cells in the brain, 12 days following the treatment, which was absent subsequent to CCR2 blockade. In addition, measurement of hippocampal levels of the T-cell chemoattractant, C-X-C motif chemokine ligand 12 (Cxcl12), and of inflammatory cytokines, revealed that αPD-L1 treatment reduced their expression, while blocking CCR2 reversed this effect.ConclusionsThe CCR2/CCL2 axis is required to modify pathology using PD-L1 blockade in a mouse model of tauopathy. This modification involves, in addition to monocytes, the accumulation of FOXP3+ regulatory CD4+ T cells in the brain, and the T-cell chemoattractant, Cxcl12.

Mogamulizumab Monotherapy in the Treatment of Relapsed/Refractory Peripheral T-Cell Lymphoma or Cutaneous T-Cell Lymphoma Patients in Single-Institution Experience

Objectives; T-cell lymphomas generally have a poor outcome with shorter long-term survival compared with B-cell lymphomas. CC chemokine receptor 4 (CCR4) is highly expressed by aggressive peripheral T-cell lymphoma, not otherwise specified (PTCLs), particularly adult T-cell leukemia/lymphoma (ATL) and cutaneous T-cell lymphomas (CTCLs). Mogamulizumab is a humanized immunoglobulin G1 monoclonal antibody that targets CCR4. In phase II study, Mogamulizumab has been shown to high clinical efficacy and low toxicity. However, its efficacy has not been well evaluated in the clinical setting. We retrospectively analyzed the efficacy and safety of Mogamulizumab of against relapsed/refractory non- ATL T-cell lymphomas.Methods; Fourteen patients with relapsed/ refractory CCR4 positive PTCLs or CTCL were treated with weekly Mogamulizumab (1.0 mg/kg) alone at Iwate Prefectural Central Hospital between January 2015 and June 2017. CCR4 expression was determined by immunohistochemistry by a central laboratory center (SRL, Tokyo, Japan). In the first line, most patients receive commonly anthracycline-containing induction chemotherapy regimens such as CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone). The patients had received a median of one previous chemotherapy regime (range 1-3).We retrospectively evaluated (1) the best overall response, (2) progression-free survival (PFS), (3) overall survival (OS), (4) the best response according to disease site(peripheral blood; PB, lymph nodes; LN and skin) and (5) safety. Response assessments were performed after eighth mogamulizumab infusions according to the 1999 IWG criteria guidelines for response assessments for NHL (IWG-NHL). Treatment efficacy in patients with CTCL was evaluated by using a Global Response Score. Treatment was discontinued if progressive disease (PD) was evident.For safety evaluations, adverse events (AEs) were graded according to the National Cancer Institute Common Terminology Criteria for AEs, version 4.0.Results; The median age was 69 years old (range 48-85). Eight patients are male. The diagnosis comprised PTCLs (n=12) and CTCL (n=2). The median follow up periods and median number of Mogamulizumab infusion were 293 days (range 49- 681) and 8 courses (range 2- 9). (1) Complete remission (CR) was achieved in two patients (16.7%) and partial remission in one patients (8.3%), stable disease (SD) in two patients (16.7%), respectively. And (2) median PFS was 88 days and (3) median OS was 475 days. Peripheral blood, lymph node and skin lesion were affected in two, ten, and six patients, respectively. (4) In PB, complete response (CR) was obtained in all two patients (100%) and two patients were achieved CR in skin lesion (33.3%). In LN, only one of ten patient manifested CR(10.0%). This patient's tumor burden is low. (5) With regard to safety issues, hematological AEs were frequently observed, and lymphocytopenia occurred in 62.5% of patients. However, the AEs were controllable and tolerated. Non-hematological AEs, including fever, infusion reactions and skin trouble , were frequently observed (37.5%, 25.0%, and 37.5%, respectively). Grade 3/4 non-hematological AEs were scarcely observed.Conclusions; Although Mogamulizumab monotherapy is effective with highly safety, the patients who had swollen lymph node may need combination chemotherapy with mogamulizumab. The number of patients was small in this retrospective analysis, further investigation are required. DisclosuresNo relevant conflicts of interest to declare.

The Anti-Viral Compound Cosalane Attenuates Acute Graft-Versus-Host Disease in Murine Hematopoietic Stem Cell Transplant Models

Introduction : Acute graft-versus-host disease (aGVHD) remains a major barrier to the success of allogeneic hematopoietic stem cell transplantation (HSCT). Mechanistically, aGVHD depends on the trafficking of donor conventional T cells (Tcons) into host secondary lymphoid tissues (SLT) where they are activated by host antigens. Previously our group demonstrated that donor Tcons deficient in the lymphoid trafficking receptor CC-Chemokine Receptor 7 (CCR7) generated attenuated aGVHD responses but retained their graft-versus-leukemia (GVL) potential (Coghill JM et al. Blood. 2010; 115(23): 4914-22). This suggested that the receptor might be targeted therapeutically. Since there were no drugs affecting the CCR7 axis, we undertook a high throughput screen (HTS) of over 200,000 compounds to identify small molecule antagonists of the receptor. This effort demonstrated that cosalane, a compound originally developed as a novel HIV therapeutic but with additional activity against numerous other enveloped viruses including cytomegalovirus and herpes simplex virus, also possessed an intrinsic ability to block human and murine CCR7 signaling in response to both of its ligands. As a result, we examined the ability of cosalane to modulate aGVHD in vivo in mouse HSCT models.Results : Cosalane did not substantially alter aGVHD outcomes when dosed intravenously post-transplant to murine recipients in a standard C57BL/6 “B6” (H-2b) into C57BL/6-DBA2 F1 “B6D2” (H-2bxd) haplotype matched HSCT model. However, when donor Tcons were incubated with cosalane in vitro for one hour prior to transplant, aGVHD was significantly attenuated and survival was prolonged compared to cells incubated with DMSO vehicle (Figure 1A, *P=0.004 for curve comparison between BM with Tcons and BM with Tcons/cosalane groups by log rank test). Cosalane exposure did not appear to result in any cytotoxicity, and did not affect Tcon activation and expansion in vitro in response to CD3 stimulation in the presence of supplemental IL-2. Furthermore, when the entire stem cell product (bone marrow cells plus Tcons) was incubated with cosalane prior to transplant, myeloid engraftment was unaffected and survival times were once again improved with no evidence of bone marrow aplasia (Figure 1B, **P=0.001 for curve comparison by log rank test).While cosalane was originally identified as an inhibitor of CCR7, its effects on donor Tcon trafficking into host lymphoid sites post-transplant were surprisingly modest. No major differences in donor Tcon accumulation within the host inguinal lymph nodes, mesenteric lymph nodes, or spleen were noted on either transplant day +7 or day +14. A strong trend for reduced Tcon numbers was noted, however, within the host colon and liver by the second week post-transplant, implying an additional mechanism of action beyond its activity against CCR7.Cosalane's effects did not appear to be strain dependent, as survival times were also improved in a completely MHC mismatched B6 into BALB/c (H-2d) model system, although to a lesser extent. In addition, cosalane pretreatment of human peripheral blood mononuclear cells resulted in prolonged survival times in a mouse xenogeneic aGVHD transplant model. We went on to examine cosalane's effects on beneficial GVL effects using a well described luciferase transduced p815 (H-2d) murine mastocytoma model. Here survival times were prolonged in B6D2 mice receiving cosalane treated B6 Tcons/tumor versus those receiving DMSO treated B6 Tcons/tumor (who all succumbed to aGVHD) and those receiving bone marrow alone/tumor (who all died from malignancy). We have found cosalane's effects on aGVHD to be dose dependent in a B6 into B6D2 system (treatment range 13uM-39uM), and additional work is ongoing to optimize its administration so as to maximize anti-inflammatory effects while sparing anti-tumor immunity.Conclusions : The anti-viral compound cosalane is able to attenuate aGVHD in multiple allogeneic and xenogeneic model systems after a brief incubation with the donor Tcon and/or bone marrow product in vitro prior to transplant. Although identified in our HTS as a CCR7 antagonist, its effects on lymphoid trafficking after HSCT have thus far been limited, and additional mechanistic studies are currently ongoing. Given its known anti-viral effects against numerous pathogens common to the HSCT setting, we are also exploring its anti-infective properties in murine model systems. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Cutaneous T-cell-attracting chemokine as a novel biomarker for predicting prognosis of idiopathic pulmonary fibrosis: a prospective observational study

BackgroundIdiopathic pulmonary fibrosis (IPF) is a chronic, progressive fibrotic lung disease that leads to respiratory failure and death. Although there is a greater understanding of the etiology of this disease, accurately predicting the disease course in individual patients is still not possible. This study aimed to evaluate serum cytokines/chemokines as potential biomarkers that can predict outcomes in IPF patients.MethodsA multi-institutional prospective two-stage discovery and validation design using two independent cohorts was adopted. For the discovery analysis, serum samples from 100 IPF patients and 32 healthy controls were examined using an unbiased, multiplex immunoassay of 48 cytokines/chemokines. The serum cytokine/chemokine values were compared between IPF patients and controls; the association between multiplex measurements and survival time was evaluated in IPF patients. In the validation analysis, the cytokines/chemokines identified in the discovery analysis were examined in serum samples from another 81 IPF patients to verify the ability of these cytokines/chemokines to predict survival. Immunohistochemical assessment of IPF-derived lung samples was also performed to determine where this novel biomarker is expressed.ResultsIn the discovery cohort, 18 cytokines/chemokines were significantly elevated in sera from IPF patients compared with those from controls. Interleukin-1 receptor alpha (IL-1Rα), interleukin-8 (IL-8), macrophage inflammatory protein 1 alpha (MIP-1α), and cutaneous T-cell-attracting chemokine (CTACK) were associated with survival: IL-1Rα, hazard ratio (HR) = 1.04 per 10 units, 95% confidence interval (95% CI) 1.01–1.07; IL-8, HR = 1.04, 95% CI 1.01–1.08; MIP-1α, HR = 1.19, 95% CI 1.00–1.36; and CTACK, HR = 1.12 per 100 units, 95% CI 1.02–1.21. A replication analysis was performed only for CTACK because others were previously reported to be potential biomarkers of interstitial lung diseases. In the validation cohort, CTACK was associated with survival: HR = 1.14 per 100 units, 95% CI 1.01–1.28. Immunohistochemistry revealed the expression of CTACK and CC chemokine receptor 10 (a ligand of CTACK) in airway and type II alveolar epithelial cells of IPF patients but not in those of controls.ConclusionsCTACK is a novel prognostic biomarker of IPF.Trial registration None (because of no healthcare intervention)

Dual role for CXCR3 and CCR5 in asthmatic type 1 inflammation

Many patients with severe asthma (SA) fail to respond to type 2 inflammation-targeted therapies. We previously identified a cohort of subjects with SA expressing type 1 inflammation manifesting with IFN-γ expression and variable type 2 responses. We investigated the role of the chemotactic receptors C-X-C chemokine receptor 3 (CXCR3) and C-C chemokine receptor 5 (CCR5) in establishing type 1 inflammation in SA. Bronchoalveolar lavage microarray data from the Severe Asthma Research Program I/II were analyzed for pathway expression and paired with clinical parameters. Wild-type, Cxcr3-/-, and Ccr5-/- mice were exposed to a type 1-high SA model with analysis of whole lung gene expression and histology. Wild-type and Cxcr3-/- mice were treated with a US Food and Drug Administration-approved CCR5 inhibitor (maraviroc) with assessment of airway resistance, inflammatory cell recruitment by flow cytometry, whole lung gene expression, and histology. A cohort of subjects with increased IFN-γ expression showed higher asthma severity. IFN-γ expression was correlated with CXCR3 and CCR5 expression, but in Cxcr3-/- and Ccr5-/- mice type 1 inflammation was preserved in a murine SA model, most likely owing to compensation by the other pathway. Incorporation of maraviroc into the experimental model blunted airway hyperreactivity despite only mild effects on lung inflammation. IFNG expression in asthmatic airways was strongly correlated with expression of both the chemokine receptors CXCR3 and CCR5. Although these pathways provide redundancy for establishing type 1 lung inflammation, inhibition of the CCL5/CCR5 pathway with maraviroc provided unique benefits in reducing airway hyperreactivity. Targeting this pathway may be a novel approach for improving lung function in individuals with type 1-high asthma.

Structural basis of the activation of the CC chemokine receptor 5 by a chemokine agonist.

The human CC chemokine receptor 5 (CCR5) is a G protein-coupled receptor (GPCR) that plays a major role in inflammation and is involved in cancer, HIV, and COVID-19. Despite its importance as a drug target, the molecular activation mechanism of CCR5, i.e., how chemokine agonists transduce the activation signal through the receptor, is yet unknown. Here, we report the cryo-EM structure of wild-type CCR5 in an active conformation bound to the chemokine super-agonist [6P4]CCL5 and the heterotrimeric Gi protein. The structure provides the rationale for the sequence-activity relation of agonist and antagonist chemokines. The N terminus of agonist chemokines pushes onto specific structural motifs at the bottom of the orthosteric pocket that activate the canonical GPCR microswitch network. This activation mechanism differs substantially from other CC chemokine receptors that bind chemokines with shorter N termini in a shallow binding mode involving unique sequence signatures and a specialized activation mechanism.

Increased regulated on activation, normal T-cell expressed and secreted levels and cysteine–cysteine chemokine receptor 5 upregulation in omental adipose tissue and peripheral blood mononuclear cells are associated with testosterone level and insulin resistance in polycystic ovary syndrome

To study the relationship between circulating chemokine cysteine-cysteine motif ligand (CCL) 5 levels and cysteine-cysteine chemokine receptor type 5 (CCR5) expression in peripheral blood mononuclear cells (PBMCs) and adipose tissue with hyperandrogenism and insulin resistance in patients with polycystic ovary syndrome (PCOS). Case-control study. University teaching hospital. Fifteen women with PCOS and 15 controls matched for body mass index and age were enrolled in this study. None. Plasma levels of CCL3, CCL4, and CCL5 were determined using enzyme-linked immunosorbent assay kits, and omental adipose tissue and PBMCs were analyzed using real-time polymerase chain reaction to determine the expression level of CCR5 in participants. Levels of CCL5 were significantly higher in women with PCOS. Expression of CCR5 in adipose tissue and PBMCs was significantly higher in women with PCOS compared with that in women in the control group. Cysteine-cysteine chemokine receptor type 5 expression also was upregulated in THP-1 cells after chronic exposure to testosterone. Levels of CCL5 had a significant positive correlation with testosterone levels in women with PCOS. Moreover, CCR5 showed a positive correlation with fasting glucose levels, homeostasis model insulin resistance index, and C-reactive protein. Increased levels of CCL5 and overexpression of CCR5 in PBMCs and adipose tissue are associated with hyperandrogenism and insulin resistance in women with PCOS. Additionally, CCR5 and CCL5 may be used as biomarkers in the pathogenesis of PCOS.

Combined tumor-directed recruitment and protection from immune suppression enable CAR T cell efficacy in solid tumors.

CAR T cell therapy remains ineffective in solid tumors, due largely to poor infiltration and T cell suppression at the tumor site. T regulatory (Treg) cells suppress the immune response via inhibitory factors such as transforming growth factor-β (TGF-β). Treg cells expressing the C-C chemokine receptor 8 (CCR8) have been associated with poor prognosis in solid tumors. We postulated that CCR8 could be exploited to redirect effector T cells to the tumor site while a dominant-negative TGF-β receptor 2 (DNR) can simultaneously shield them from TGF-β. We identified that CCL1 from activated T cells potentiates a feedback loop for CCR8+ T cell recruitment to the tumor site. This sustained and improved infiltration of engineered T cells synergized with TGF-β shielding for improved therapeutic efficacy. Our results demonstrate that addition of CCR8 and DNR into CAR T cells can render them effective in solid tumors.

CCL17-CCR4 axis contributes to the onset of vitiligo in mice.

BackgroundDestruction of melanocytes mediated by autoimmunity is currently believed as the main cause of vitiligo. This article aims to identify the role of CC chemokine ligand 17 (CCL17)–CC chemokine receptor 4 (CCR4) axis in vitiligo and provide new possibilities for the clinical treatment of vitiligo.MethodsA total of 30 patients with vitiligo from Affiliated Huaian No. 1 People's Hospital of Nanjing Medical University were recruited based on the inclusion and exclusion criteria. Trephine was used to obtain skin samples from the lesion area and its surrounding normal areas, and the expression levels of CCL17, CCR4, Tbx21, Eomes, and Blimp1 were determined by quantitative reverse transcription polymerase chain reaction. Vitiligo mouse model was established by adoptively transferring CFP‐PMEL CD8+ T cells into sublethally irradiated Krt14‐Kitl* mice. Recipient mice received intraperitoneal injection of 1 × 106 plaque‐forming units of rVV‐hPMEL on the same day of transfer. The degree of depigmentation was scored blindly by one observer 5 weeks after vitiligo induction. CFP‐PMEL CD8+ T cells migration to skin, draining lymph nodes, spleen, and blood were detected by flow cytometry. CCR4 blockade was performed by intraperitoneal injection of neutralizing antibody.ResultsThe expression levels of CCL17, CCR4, Tbx21, Eomes, and Blimp1 in skin lesions were significantly increased compared with that in surrounding normal areas. CCL17−/− and CCR4−/− mice exhibited significantly lower disease scores than WT mice. The CFP‐PMEL CD8+ T cells accumulation was significantly decreased in the skin of CCL17−/− and CCR4−/− mice, but was not changed in draining lymph nodes, spleen, and blood. Administration of CCR4 neutralizing antibody decreased the degree of depigmentation and the recruitment of CFP‐PMEL CD8+ T cells to the skin, while keeping the number of T cells in draining lymph nodes unchanged.ConclusionTargeting CCL17‐CCR4 axis might inhibit T cell migrating to skin and alleviate vitiligo progression.

The CC and CXC chemokine receptors in turbot (Scophthalmus maximus L.) and their response to Aeromonas salmonicida infection

Chemokines are crucial regulators of cell mobilization for development, homeostasis, and immunity. Chemokines signal through binding to chemokine receptors, a superfamily of seven-transmembrane domain G-coupled receptors. In the present study, eleven CC chemokine receptors (CCRs) and seven CXC chemokine receptors (CXCRs) were identified from turbot genome. Phylogenetic and syntenic analyses were performed to annotate these genes, indicating the closest relationship between the turbot chemokine receptors and their counterparts of Japanese flounders (Paralichthys olivaceus). Evolutionary analyses revealed that the tandem duplications of CCR8 and CXCR3, the whole genome duplications of CCR6, CCR9, CCR12, and CXCR4, and the teleost-specific CCR12 led to the expansion of turbot chemokine receptors. In addition, turbot chemokine receptors were ubiquitously expressed in nine examined healthy tissues, with high expression levels observed in spleen, gill, and head kidney. Moreover, most turbot chemokine receptors were significantly differentially expressed in spleen and gill after Aeromonas salmonicida infection, and exhibited general down-regulations at early time points and then gradually up-regulated. Finally, protein-protein interaction network (PPI) analyses indicated that chemokine receptors interacted with a few immune-related genes such as interleukins, Grk genes, CD genes, etc. These results should be valuable for comparative immunological studies and provide insights for further functional characterization of chemokine receptors in turbots.

Development of Anti-Human CC Chemokine Receptor 9 Monoclonal Antibodies for Flow Cytometry.

CC chemokine receptor 9 (CCR9) belongs to the beta chemokine receptor family and is mainly distributed on the surface of immature T lymphocytes and enterocytes. This receptor is highly expressed in rheumatoid arthritis, colitis, type 2 diabetes, and various tumors. Therefore, more sensitive monoclonal antibodies (mAbs) need to be developed to predict the prognosis of many high CCR9 expression diseases. Because CCR9 is a structurally unstable G protein-coupled receptor, it has been difficult to develop anti-CCR9 mAbs using the traditional method. This study developed anti-human CCR9 (hCCR9) mAbs for flow cytometry using a Cell-Based Immunization and Screening (CBIS) method. Two mice were immunized with hCCR9-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/hCCR9), and hybridomas showing strong signals from CHO/hCCR9 and no signals from CHO-K1 cells were selected by flow cytometry. We established an anti-hCCR9 mAb, C9Mab-1 (IgG1, kappa), which detected hCCR9 in MOLT-4 leukemia T lymphoblast cells and CHO/hCCR9 cells by flow cytometry. Our study showed that an anti-hCCR9 mAb was developed more rapidly by the CBIS method than the previous method.

CC Chemokine Ligand 7 Derived from Cancer-Stimulated Macrophages Promotes Ovarian Cancer Cell Invasion.

Simple SummaryOvarian cancer is the most lethal of gynecological malignancies. Although metastasis leads to a bleak prognosis and high mortality, the molecular and cellular processes of ovarian cancer metastasis remain largely unclear. Non-malignant cells including macrophages constitute a unique microenvironment that can completely change the nature of malignant cells at various levels. In this study, we found that the expression of chemokine (C-C motif) ligand 7 (CCL7), a soluble inflammatory factor, was enhanced in macrophages stimulated by human ovarian cancer cells as well as peritoneal macrophages from patients with ovarian cancer; CCL7 promoted the invasion of ovarian cancer cells. Our study elucidates the crosstalk between macrophages and ovarian cancer cells that contributes to ovarian cancer metastasis.In the tumor microenvironment, macrophages have been suggested to be stimulated by tumor cells, becoming tumor-associated macrophages that promote cancer development and progression. We examined the effect of these macrophages on human ovarian cancer cell invasion and found that conditioned medium of macrophages stimulated by ovarian cancer cells (OC-MQs) significantly increased cell invasion. CC chemokine ligand 7 (CCL7) expression and production were significantly higher in OC-MQs than in the control macrophages. Peritoneal macrophages from patients with ovarian cancer showed higher CCL7 expression levels than those from healthy controls. Inhibition of CCL7 using siRNA and neutralizing antibodies reduced the OC-MQ-CM-induced ovarian cancer cell invasion. CC chemokine receptor 3 (CCR3) was highly expressed in human ovarian cancer cells, and a specific inhibitor of this receptor reduced the OC-MQ-CM-induced invasion. Specific signaling and transcription factors were associated with enhanced CCL7 expression in OC-MQs. CCL7-induced invasion required the expression of matrix metalloproteinase 9 via activation of extracellular signal-related kinase signaling in human ovarian cancer cells. These data suggest that tumor-associated macrophages can affect human ovarian cancer metastasis via the CCL7/CCR3 axis.

Effect of rose extract treatment on soluble CCR5 and CXCR4 secretion by the endothelial cells in vitro

Background: CC-Chemokine Receptor 5 (CCR5) and Chemokine C-X-C-Motif Receptor 4 (CXCR4) are expressed in various tissues, and they are potential molecules involved in multiple pathways. CCR5 and CXCR4 targets are associated with immune regulation in patients in multiple tissues and numerous clinical conditions. The study was performed searching for a novel therapy for immune regulation on these CCR5 and CXCR4 receptors with rose extract.
 Methods: The crushed red rose extract was prepared, and it was processed for analysis. The HUVEC cells were obtained for seeding in the cell culture. The cells were tested in normal physiological conditions and varying degrees of hypoxia. The cells were treated with extract for 72 hours, and the resultant secreted supernatants were analyzed for expression of CCR5 and CXCR4 by the Elisa technique.
 Results: The CCR5 levels were significantly elevated at normoxia compared to untreated controls. The surge of CCR5 was persistent in 12% hypoxia, and at higher degrees of hypoxia, the levels were mildly lower than the untreated levels. The CXCR4 levels were not changed in normoxia, and even with significant hypoxia, the levels were similar or mildly reduced compared to untreated values.
 Conclusion: The rose extract has the potentials to induce the secretion of soluble CCR5 from the HUVEC cells, and it can prevent the reduction of soluble CXCR4 levels during the hypoxic challenge of the endothelial cells. This in-turn can modulate the receptor levels on the endothelial cells, which has clinical applications.

MO253A RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED DOSE-RANGING STUDY (LUMINA-1 STUDY) TO EVALUATE THE SAFETY AND EFFICACY OF CCX140 IN SUBJECTS WITH FOCAL SEGMENTAL GLOMERULOSCLEROISS (FSGS)*

Abstract Background and Aims Primary Focal Segmental Glomerulosclerosis (FSGS) is the most common primary glomerular disease in patients with end stage renal disease in the United States. Current treatment regimens target reduction in proteinuria, but may have limited response or exhibit disease recurrence. CCX140 is an orally-administered selective small molecule inhibitor of the C-C chemokine receptor 2 (CCR2) under investigation for the treatment reduction of proteinuria in patients with FSGS. The primary objectives of this study were to evaluate the safety and efficacy of CCX140 in patients with FSGS and a urine protein to creatinine ratio (UPCR) of ≥1 g/g. Efficacy was assessed by the change in UPCR. Method Forty-six adult patients with primary or genetic FSGS were randomized into a double-blind, placebo-controlled Phase 2 dose-ranging study designed to evaluate the safety and efficacy of CCX140. Changes of urinary protein excretion estimation (UPCR) from baseline to Week 12 were measured in four blinded treatment groups (three active CCX140 doses of 5 mg once daily, 10 mg and 15 mg twice-daily (BID) vs placebo). Starting at Week 12, all subjects including those in the placebo group received the CCX140, 15 mg BID for an additional 12 weeks, and UPCR changes from Week 12 to Week 24 were assessed. There was a 4-week follow-up period from Week 24 to Week 28 where no CCX140 was administered. Results In the intent to treat (ITT) analysis of UPCR changes at Week 12 relative to baseline, the 15 mg BID CCX140 group exhibited the greatest reduction of UPCR (median reduction from baseline 0.9 g/g or approximately 30%, and approximately 25% reduction from baseline for the geometric mean), but that did not differ significantly from the placebo group (median reduction from baseline 0.45 g/g; or approximately 22%, and approximately 23% reduction from baseline for the geometric mean). Also, after crossover of the blinded portion of the trial to 15 mg BID active dosing, the previous placebo group did not appear to exhibit an additional reduction of UPCR. CCX140 at all doses was well-tolerated, with no serious adverse events (SAEs) during the blinded trial and a numerically lower rate of treatment-emergent adverse events in the CCX140 treatment groups. Conclusion In the study, CCX140 did not demonstrate a therapeutically meaningful reduction in proteinuria relative to the control group after 12 weeks of blinded treatment. The study provides insights into the natural disease progression of patients with primary or genetic FSGS as part of a clinical trial setting.

Polymorphisms and haplotypes of IL2RA, IL10, IFNG, IRF5, and CCR2 are associated with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis in children.

Cytokine storms are central to the development of Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH). Previous studies have shown that single-nucleotide polymorphisms (SNPs) of cytokine genes may be associated with the development of EBV-HLH in children. As such, we investigated the association between susceptibility to EBV-HLH in children and SNPs and haplotypes of genes encoding interleukin-2 receptor subunit alpha (IL2RA), interleukin-10 (IL10), interferon gamma (IFNG), interferon regulatory factor 5 (IRF5), and C-C chemokine receptor 2 (CCR2). Sixty-six children with EBV-HLH and 58 healthy EBV-seropositive controls were enrolled in this study. SNPs of IL2RA rs2104286, rs12722489, and rs11594656; IL10 rs1800896, rs1800871, and rs1800872; IFNG rs2430561, IRF5 rs2004640, and CCR2 rs1799864 were assayed and genotyped using the SNaPshot technique. Frequencies of the A allele of IL2RA rs2104286 and IL10 rs1800896, and C allele of IL-10 rs1800872 were significantly higher in the EBV-HLH group than in the control group. The AA genotype of IL2RA rs2104286 and IL10 rs1800896, and the CC genotype of IL10 rs1800872 might be associated with a significantly high risk of EBV-HLH. However, the frequencies of genotypes and alleles of IL2RA rs2104286, IL10 rs1800871, IFNG rs2430561, IRF5 rs2004640, and CCR2 rs1799864 were similar in both groups. Additionally, IL2RA AGT (rs2104286-rs12722489-rs11594656) and IL10 ACC (rs1800896-rs1800871-rs1800872) haplotypes were also associated with an increased risk of EBV-HLH. SNPs of IL2RA rs2104286, IL10 rs1800896 and rs1800872 and the haplotypes of IL2RA AGT and IL10 ACC were highly associated with susceptibility to EBV-HLH in children.

What does elevated TARC/CCL17 expression tell us about eosinophilic disorders?

Eosinophilic disorders encompass a large spectrum of heterogeneous diseases sharing the presence of elevated numbers of eosinophils in blood and/or tissues. Among these disorders, the role of eosinophils can vary widely, ranging from a modest participation in the disease process to the predominant perpetrator of tissue damage. In many cases, eosinophilic expansion is polyclonal, driven by enhanced production of interleukin-5, mainly by type 2 helper cells (Th2 cells) with a possible contribution of type 2 innate lymphoid cells (ILC2s). Among the key steps implicated in the establishment of type 2 immune responses, leukocyte recruitment toward inflamed tissues is particularly relevant. Herein, the contribution of the chemo-attractant molecule thymus and activation-regulated chemokine (TARC/CCL17) to type 2 immunity will be reviewed. The clinical relevance of this chemokine and its target, C-C chemokine receptor 4 (CCR4), will be illustrated in the setting of various eosinophilic disorders. Special emphasis will be put on the potential diagnostic, prognostic, and therapeutic implications related to activation of the TARC/CCL17-CCR4 axis.

Astragalus membranaceus polysaccharides potentiate the growth-inhibitory activity of immune checkpoint inhibitors against pulmonary metastatic melanoma in mice

Astragalus membranaceus (A. membranaceus) is commonly used in various herbal formulations to treat several human and animal diseases. Polysaccharides, which are the major bioactive components in the A. membranaceus, exhibit various bioactive properties. However, the ability of A. membranaceus polysaccharides (APS) to activate the mucosal immune response has not been examined. We examined the effect of intranasal administration of APS on mucosal immune cell activation and the growth-inhibitory activity against pulmonary metastatic melanoma in mice by combination treatment with immune checkpoint blockade. The intranasal treatment of APS increased the number of lineage−CD11c+ dendritic cell (DCs) in the mesenteric lymph nodes (mLN) through the upregulation of CC-chemokine receptor 7 expression. Moreover, intranasal treatment of APS activated DCs, which further stimulated natural killer (NK) and T cells in the mLN. The APS/anti-PD-L1 antibody combination inhibited the pulmonary infiltration of B16 melanoma cells. The depletion of NK cells and CD8 T cells in mice mitigated the anti-cancer effect of this combination, thereby highlighting the critical role of NK cells and CD8 T cells in mediating anti-cancer immunity. These findings demonstrated that APS could be used as a topical mucosal adjuvant to enhance the immune check point inhibitor anti-cancer effect.

B cells modulate the expression of MHC-II on cardiac CCR2− macrophages

The uninjured murine heart contains a heterogeneous population of macrophages with disparate ontogenies and functions. These macrophages are often associated with blood vessels and can be subclassified based on the expression of CC chemokine receptor 2 (CCR2) and major histocompatibility complex class II (MHC-II). The biological cues that modulate these macrophage pool subpopulations have not been completely identified. It has been recently shown that a sub-population of circulating naïve B cells adheres to the myocardial microvasculature. We hypothesized that B cells might modulate the phenotype of myocardial macrophages. To test this hypothesis, we analyzed both the relative location of B cells and macrophages in myocardial histological section and the prevalence of myocardial macrophage subsets in hearts from B cell–deficient mice (μMT) and mice depleted of B cells through administration of an anti-CD20 antibody. We found that B cells pause in the microvasculature in proximity of macrophages and modulate the number of myocardial CCR2−MHC-IIhigh cells. Through in vitro studies we found that this is likely the result of a paracrine effect of B cells on the expression of MHC-II in CCR2− cells. These results reveal an unexpected relationship between B cells and resident macrophages and, highlighting a direct intramyocardial effect of circulating B cells, challenge the currently held belief that naïve recirculating B lymphocytes merely shuttle between lymphoid stations.