Chemokine Receptor CCR5 Research Articles

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.

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 Chemokine Receptor CCR1 Mediates Microglia Stimulated Glioma Invasion

Glioblastoma multiforme (GBM) is the most aggressive form of adult brain tumor which is highly resistant to conventional treatment and therapy. Glioma cells are highly motile resulting in infiltrative tumors with poorly defined borders. Another hallmark of GBM is a high degree of tumor macrophage/microglia infiltration. The level of these tumor-associated macrophages/microglia (TAMs) correlates with higher malignancy and poorer prognosis. We previously demonstrated that inhibition of TAM infiltration into glioma tumors with the CSF-1R antagonist pexidartinib (PLX3397) can inhibit glioma cell invasion in-vitro and in-vivo. In this study, we demonstrate an important role for the chemokine receptor CCR1 in mediating microglia/TAM stimulated glioma invasion. Using two structurally distinct CCR1 antagonists, including a novel inhibitor "MG-1-5", we were able to block microglial activated GL261 glioma cell invasion in a dose dependent manner. Interestingly, treatment of a murine microglia cell line with glioma conditioned media resulted in a strong induction of CCR1 gene and protein expression. This induction was attenuated by inhibition of CSF-1R. In addition, glioma conditioned media treatment of microglia resulted in a rapid upregulation of gene expression of several CCR1 ligands including CCL3, CCL5, CCL6 and CCL9. These data support the existence of tumor stimulated autocrine loop within TAMs which ultimately mediates tumor cell invasion.

Efficient Redirection of NK Cells by Genetic Modification with Chemokine Receptors CCR4 and CCR2B

Natural killer (NK) cells are a subset of lymphocytes that offer great potential for cancer immunotherapy due to their natural anti-tumor activity and the possibility to safely transplant cells from healthy donors to patients in a clinical setting. However, the efficacy of cell-based immunotherapies using both T and NK cells is often limited by a poor infiltration of immune cells into solid tumors. Importantly, regulatory immune cell subsets are frequently recruited to tumor sites. In this study, we overexpressed two chemokine receptors, CCR4 and CCR2B, that are naturally found on T regulatory cells and tumor-resident monocytes, respectively, on NK cells. Using the NK cell line NK-92 as well as primary NK cells from peripheral blood, we show that genetically engineered NK cells can be efficiently redirected using chemokine receptors from different immune cell lineages and migrate towards chemokines such as CCL22 or CCL2, without impairing the natural effector functions. This approach has the potential to enhance the therapeutic effect of immunotherapies in solid tumors by directing genetically engineered donor NK cells to tumor sites. As a future therapeutic option, the natural anti-tumor activity of NK cells at the tumor sites can be increased by co-expression of chemokine receptors with chimeric antigen receptors (CAR) or T cell receptors (TCR) on NK cells can be performed in the future.

Molecular determinants of antagonist interactions with chemokine receptors CCR2 and CCR5.

CC chemokine receptors 2 and 5 (CCR2 and CCR5) are GPCRs that guide monocytes towards regions of inflammation, thus shaping the inflammatory response. CCR2 & CCR5 have been extensively studied as potential targets in Nonalcoholic Fatty Liver Disease (NAFLD) and Nonalcoholic Steatohepatitis (NASH), where antagonizing CCR2 reduces Kupffer cell activation, lowers monocyte infiltration, and prevents liver fibrosis. In the case of pancreatic ductal adenocarcinoma (PDAC), a lethal cancer, CCR2-mediated macrophage infiltration of the tumor microenvironment leads to poor patient survival and increased resistance to chemo-, radiation, and immuno-therapies, thus motivating studies of CCR2 antagonists in combination with these therapies. However, no CCR2 antagonist has yet reached the clinic. Redundancies inherent to the chemokine system make targeting this receptor difficult. Many inhibitors of CCR2 have comparable affinity for CCR5; while dual-selective CCR2/CCR5 antagonists may be indicated for NASH, dual-selectivity hinders their use for PDAC. To guide the design of better CCR2- and CCR5-targeting small-molecule therapeutics, it is necessary to understand the structural basis of antagonist interaction with these highly homologous receptors.

Maximizing biological discovery through global computational analyses of unbiased apex biotinylation data on chemokine receptors.

Chemokine receptors CCR2 and CCR5 shape inflammatory responses by driving monocyte recruitment to sites of injury. Unfortunately, through the same mechanism they contribute to the formation of onco-protective tumor microenvironments in cancer and tissue fibrosis in chronic inflammatory conditions. These associations motivated targeting CCR2 and CCR5 in nonalcoholic fatty liver disease and pancreatic cancer, among others.

“Glyco-sulfo barcodes” regulate chemokine receptor function

Chemokine ligands and receptors regulate the directional migration of leukocytes. Post-translational modifications of chemokine receptors including O-glycosylation and tyrosine sulfation have been reported to regulate ligand binding and resulting signaling. Through in silico analyses, we determined potential conserved O-glycosylation and sulfation sites on human and murine CC chemokine receptors. Glyco-engineered CHO cell lines were used to measure the impact of O-glycosylation on CC chemokine receptor CCR5, while mutation of tyrosine residues and treatment with sodium chlorate were performed to determine the effect of tyrosine sulfation. Changing the glycosylation or tyrosine sulfation on CCR5 reduced the receptor signaling by the more positively charged CCL5 and CCL8 more profoundly compared to the less charged CCL3. The loss of negatively charged sialic acids resulted only in a minor effect on CCL3-induced signal transduction. The enzymes GalNAc-T1 and GalNAc-T11 were shown to be involved in the process of chemokine receptor O-glycosylation. These results indicate that O-glycosylation and tyrosine sulfation are involved in the fine-tuning and recognition of chemokine interactions with CCR5 and the resulting signaling.

Chemokine Receptor CCR8 and CCR8-Bearing Cells Are Involved in Regulation of Type 2 Immunity in Response to Airborne Allergens

Chemokines play diverse roles in immunity and immune homeostasis. Increased frequency of CCR8+CD3+ T cells are observed in asthma patients, suggesting an association between CCR8 and asthma pathogenesis. We hypothesized that CCR8 pathway has a regulatory role and suppressive effects on allergic airway inflammation. The goal of this project is to investigate the CCR8-mediated mechanisms involved in the regulation of type 2 immunity.

T follicular helper 17 (Tfh17) cells are superior for immunological memory maintenance.

A defining feature of successful vaccination is the ability to induce long-lived antigen-specific memory cells. T follicular helper (Tfh) cells specialize in providing help to B cells in mounting protective humoral immunity in infection and after vaccination. Memory Tfh cells that retain the CXCR5 expression can confer protection through enhancing humoral response upon antigen re-exposure but how they are maintained is poorly understood. CXCR5+ memory Tfh cells in human blood are divided into Tfh1, Tfh2, and Tfh17 cells by the expression of chemokine receptors CXCR3 and CCR6 associated with Th1 and Th17, respectively. Here, we developed a new method to induce Tfh1, Tfh2, and Tfh17-like (iTfh1, iTfh2, and iTfh17) mouse cells in vitro. Although all three iTfh subsets efficiently support antibody responses in recipient mice with immediate immunization, iTfh17 cells are superior to iTfh1 and iTfh2 cells in supporting antibody response to a later immunization after extended resting in vivo to mimic memory maintenance. Notably, the counterpart human Tfh17 cells are selectively enriched in CCR7+ central memory Tfh cells with survival and proliferative advantages. Furthermore, the analysis of multiple human cohorts that received different vaccines for HBV, influenza virus, tetanus toxin or measles revealed that vaccine-specific Tfh17 cells outcompete Tfh1 or Tfh2 cells for the persistence in memory phase. Therefore, the complementary mouse and human results showing the advantage of Tfh17 cells in maintenance and memory function supports the notion that Tfh17-induced immunization might be preferable in vaccine development to confer long-term protection.

Gram-Positive Bacteria Cell Wall Peptidoglycan Polymers Activate Human Dendritic Cells to Produce IL-23 and IL-1β and Promote TH17 Cell Differentiation.

Gram-positive bacterial infections are a major cause of organ failure and mortality in sepsis. Cell wall peptidoglycan (PGN) is shed during bacterial replication, and Bacillus anthracis PGN promotes a sepsis-like pathology in baboons. Herein, we determined the ability of polymeric Bacillus anthracis PGN free from TLR ligands to shape human dendritic cell (DC) responses that are important for the initiation of T cell immunity. Monocyte-derived DCs from healthy donors were incubated with PGN polymers isolated from Bacillus anthracis and Staphylococcus aureus. PGN activated the human DCs, as judged by the increased expression of surface HLA-DR, CD83, the T cell costimulatory molecules CD40 and CD86, and the chemokine receptor CCR7. PGN elicited the DC production of IL-23, IL-6, and IL-1β but not IL-12p70. The PGN-stimulated DCs induced the differentiation of naïve allogeneic CD4+ T cells into T helper (TH) cells producing IL-17 and IL-21. Notably, the DCs from a subset of donors did not produce significant levels of IL-23 and IL-1β upon PGN stimulation, suggesting that common polymorphisms in immune response genes regulate the PGN response. In sum, purified PGN is a highly stimulatory cell wall component that activates human DCs to secrete proinflammatory cytokines and promote the differentiation of TH17 cells that are important for neutrophil recruitment in extracellular bacterial infections.

Glioma-derived CCL2 and CCL7 mediate migration of immune suppressive CCR2+/CX3CR1+ M-MDSCs into the tumor microenvironment in a redundant manner.

Glioblastoma (GBM) is the most common and malignant primary brain tumor, resulting in poor survival despite aggressive therapies. GBM is characterized in part by a highly heterogeneous and immunosuppressive tumor microenvironment (TME) made up predominantly of infiltrating peripheral immune cells. One significant immune cell type that contributes to glioma immune evasion is a population of immunosuppressive, hematopoietic cells, termed myeloid-derived suppressor cells (MDSCs). Previous studies suggest that a potent subset of myeloid cells, expressing monocytic (M)-MDSC markers, distinguished by dual expression of chemokine receptors CCR2 and CX3CR1, utilize CCR2 to infiltrate into the TME. This study evaluated the T cell suppressive function and migratory properties of CCR2+/CX3CR1+ MDSCs. Bone marrow-derived CCR2+/CX3CR1+ cells adopt an immune suppressive cell phenotype when cultured with glioma-derived factors. Recombinant and glioma-derived CCL2 and CCL7 induce the migration of CCR2+/CX3CR1+ MDSCs with similar efficacy. KR158B-CCL2 and -CCL7 knockdown murine gliomas contain equivalent percentages of CCR2+/CX3CR1+ MDSCs compared to KR158B gliomas. Combined neutralization of CCL2 and CCL7 completely blocks CCR2-expressing cell migration to KR158B cell conditioned media. CCR2+/CX3CR1+ cells are also reduced within KR158B gliomas upon combination targeting of CCL2 and CCL7. High levels of CCL2 and CCL7 are also associated with negative prognostic outcomes in GBM patients. These data provide a more comprehensive understanding of the function of CCR2+/CX3CR1+ MDSCs and the role of CCL2 and CCL7 in the recruitment of these immune suppressive cells and further support the significance of targeting this chemokine axis in GBM.

Chemokine Receptors CCR1, CCR3, CCR7 and Chemokines CX3CL1 and CCL5 are Significantly Up-Regulated and Very Reliable for Acute Rejection Diagnosis of Kidney Transplants

Chemokine Receptors CCR1, CCR3, CCR7 and Chemokines CX3CL1 and CCL5 are Significantly Up-Regulated and Very Reliable for Acute Rejection Diagnosis of Kidney Transplants

Disulfiram Ophthalmic Solution Inhibited Macrophage Infiltration by Suppressing Macrophage Pseudopodia Formation in a Rat Corneal Alkali Burn Model

FROUNT is an intracellular protein that promotes pseudopodia formation by binding to the chemokine receptors CCR2 and CCR5 on macrophages. Recently, disulfiram (DSF), a drug treatment for alcoholism, was found to have FROUNT inhibitory activity. In this study, we investigated the effect of DSF eye drops in a rat corneal alkali burn model. After alkali burn, 0.5% DSF eye drops (DSF group) and vehicle eye drops (Vehicle group) were administered twice daily. Immunohistochemical observations and real-time reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed at 6 h and 1, 4, and 7 days after alkali burn. Results showed a significant decrease in macrophage accumulation in the cornea in the DSF group, but no difference in neutrophils. RT-PCR showed decreased expression of macrophage-associated cytokines in the DSF group. Corneal scarring and neovascularization were also suppressed in the DSF group. Low-vacuum scanning electron microscopy imaging showed that macrophage length was significantly shorter in the DSF group, reflecting the reduced extension of pseudopodia. These results suggest that DSF inhibited macrophage infiltration by suppressing macrophage pseudopodia formation.

Overexpression of the Chemokine Receptor CCR7 in Patients with Hepatocellular Carcinoma: Correlation with Disseminated Circulating Tumor Cells

Overexpression of the Chemokine Receptor CCR7 in Patients with Hepatocellular Carcinoma: Correlation with Disseminated Circulating Tumor Cells

Discovery and characterization of a functional scFv for CCR2 inhibition via an extracellular loop

The chemokine receptor CCR2 plays a key role in cellular migration and inflammatory processes. While tremendous progress has been made in elucidating CCR2 function and inhibition, the majority of approaches target its N-terminal domain and less is known about the function of the remaining extracellular loops and their potential as targets. Here, we used phage display to identify an antibody-derived scFv (single chain variable fragment) clone that specifically targets the second extracellular epitope of CCR2 (ECL2) for inhibition. Using in silico molecular docking, we identified six potential primary binding conformations of the novel scFv to the specified CCR2 epitope. In silico molecular dynamic analysis was used to determine conformational stability and identify protein–protein interactions. Umbrella sampling of a range of configurations with incrementally increasing separation of scFv and target generated by force pulling simulations was used to calculate binding energies. Downstream characterization by ELISA showed high binding affinity of the ECL2-scFv to CCR2. Furthermore, we showed that blocking the second extracellular loop inhibits macrophage migration and polarized macrophages towards M1 inflammatory cytokine production as potently as lipopolysaccharide (LPS). These studies highlight the applicability of epitope-specific targeting, emphasize the importance of in silico predictive modeling, and warrant further investigation into the role of the remaining epitopes of CCR2.

Higher Frequencies of T-Cells Expressing NK-Cell Markers and Chemokine Receptors in Parkinson’s Disease

Immune cells are thought to be involved in a destructive cycle of sterile cerebral inflammatory responses in neurodegenerative diseases such as Parkinson’s Disease (PD). Despite their peripheral origin, immune cells may enter the CNS due to impaired blood–brain barrier function and may potentially contribute to neuronal damage. Hence, specific characteristics of peripherally activated immune cells could help in understanding neurodegeneration in PD and could potentially serve as accessible disease markers. To investigate immune cell activation status, the expression of receptors for cell surface molecules CD161, NKG2A, NKG2C and NKG2D as well as chemokine receptors CCR6, CXCR2, CXCR3 and CCR5 associated with neurodegenerative diseases was investigated. The frequencies of peripheral CD8+ T-cells expressing the inhibitory and activating receptors NKG2A and NKG2C, and the activating receptor NKG2D were higher in PD patients than in healthy matched controls. The frequencies of NKG2C+CD8− cells were also higher, whereas the frequencies of CD161+ cells were not significantly different. Of the chemokine receptor-expressing cells, only the proportion of CD4−CD56+CCR5+ T-cells was higher in PD patients than in the controls. These observations support the hypothesis that an imbalance in the activation state of T-cells plays a role in the pathological processes of PD and suggest that peripheral blood immune cell phenotypes could be specific early markers for inflammation in PD.

MAIT cells in chronic HBV infection‐related liver fibrosis: Friends or foes?

MAIT cells in chronic HBV infection‐related liver fibrosis: Friends or foes?

Inflammasome is a central player in B cell development and homing.

Whereas the role of the nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing protein (NLRP) 3 pathway in innate immunity has been extensively studied, little attention has been paid to its contribution to adaptive immunity. Studies in animal models and human subjects have shown the contribution of NLRP3 to the T cell compartment, and its role in B lymphocyte functions has been proposed. Here, we report that ablation of nlrp3 in mice led to altered B cell development in the bone marrow, and distorted expression of B cell subsets that play innate-like functions, that is, marginal zone B cells in the spleen and B-1a cells in the peritoneal cavity. Mechanistically, in the absence of NLRP3 expression, the transcription factor IRF4, previously found to interact with NLRP3 in the nucleus of lymphocytes, was up-regulated. NLRP3 ablation reduced the expression of the chemokine receptors CXCR4 and CCR7 in an IRF4-dependent manner, indicating that the presence of NLRP3 is critical for optimal expression of chemokine receptors on B cells. We conclude that activation of the NLRP3 inflammasome plays a role in B cell development, homing, and retention in lymphoid organs.

CCL20/CCR6 chemokine signaling is not essential for pathogenesis in an experimental autoimmune encephalomyelitis mouse model of multiple sclerosis

Multiple sclerosis is an autoimmune disease in which the immune system attacks the nerve myelin sheath. The balance between pathogenic Th17 cells and regulatory Treg cells, both of which express the chemokine receptor CCR6 is critical for determining disease activity. It has been postulated that CCL20, the cognate ligand of CCR6, produced by the blood-brain barrier attracts these immune cells to the central nervous system (CNS). However, the pathological phenotypes of the experimental model of multiple sclerosis in CCR6-knockout (KO) mice are inconclusive, while this has not been addressed in CCL20-KO mice.To address this, we generated CCL20-KO and CCR6-KO mice using the CRISPR/Cas9 system. Clinical phenotypes of experimental autoimmune encephalomyelitis (EAE) in the chronic phase were slightly exacerbated in both mutant mice relative to those in wild-type (WT) mice. Inflammatory cell infiltration and demyelination in the CNS were similar in the KO and WT mice. CNS CD4+ T cell counts were the same for mutant and WT mice. The mutant and WT mice did not differ significantly in the proportions of Th17 and Treg cells in the CNS, or in IL-17 and TGF-β mRNA expression in the CNS.These findings suggest that CCL20/CCR6-mediated cell migration is not necessarily required for the onset of EAE, and may be compensated for by other chemokine signals.

Multifaceted Defects in Monocytes in Different Phases of Chronic Hepatitis B Virus Infection: Lack of Restoration after Antiviral Therapy.

Monocytes play an important role in the control of microbial infection, but monocyte biology during chronic hepatitis B virus (HBV) infection (CHI) remains inadequately studied. We investigated the frequency, phenotype, and functions of monocyte subsets in different phases of CHI, namely, immune tolerance (IT), hepatitis B early antigen (HBeAg)-positive/HBeAg-negative chronic hepatitis B (EP-/EN-CHB, respectively), and inactive carrier (IC), identified factors responsible for their functional alterations, and determined the impact of antiviral therapy on these cells. Flow cytometric analysis indicated that HLA-DR+ CD14++ CD16- classical monocytes were significantly reduced while HLA-DR+ CD14++ CD16+ intermediate and HLA-DR+ CD14+ CD16++ nonclassical monocytes were expanded in IT and EP-/EN-CHB compared with those in IC and healthy controls (HC). In comparison to IC/HC, monocytes in IT and CHB exhibited diminished expression of Toll-like receptor 2 (TLR-2)/TLR-4/TLR-9 and cytokines interleukin-12 (IL-12)/tumor necrosis factor alpha (TNF-α)/IL-6 but produced higher levels of IL-10/transforming growth factor β (TGF-β). Further, monocytes in CHB/IT showed impaired phagocytosis and oxidative response relative to those in IC/HC. In vitro assays indicated that high titers of hepatitis B surface antigen (HBsAg) present in IT/CHB and of IL-4 in CHB triggered the functional defects in monocytes via induction of β-catenin. Additionally, monocyte-derived M1 macrophages of CHB/IT produced fewer proinflammatory and more anti-inflammatory cytokines than those of IC/HC, while in CHB/IT, the monocytes skewed the differentiation of CD4+ T cells more toward regulatory T cells and a Th2 phenotype. Moreover, monocytes in CHB and IT overexpressed chemokine receptor CCR2, which coincided with increased intrahepatic accumulation of β-catenin+ CD14+ cells. One year of tenofovir therapy failed to normalize monocyte functions or reduce serum HBsAg/IL-4 levels. Taken together, monocytes are functionally perturbed mostly in IT and EP-/EN-CHB phases. Targeting intramonocytic β-catenin or reducing HBsAg/IL-4 levels might restore monocyte function and facilitate viral clearance. IMPORTANCE Chronic HBV infection (CHI) is a major cause of end-stage liver disease for which pharmacological treatments currently available are inadequate. Chronically HBV-infected patients fail to mount an efficient immune response to the virus, impeding viral clearance and recovery from hepatitis. Monocytes represent a central part of innate immunity, but a comprehensive understanding on monocyte involvement in CHI is still lacking. We here report a multitude of defects in monocytes in chronically HBV-infected patients that include alteration in subset distribution, Toll-like receptor expression, cytokine production, phagocytic activity, oxidative response, migratory ability, polarization of monocyte-derived macrophages, and monocyte-T-cell interaction. We demonstrated that high levels of hepatitis B virus surface antigen and IL-4 potentiate these defects in monocytes via β-catenin induction while therapy with the nucleotide analog tenofovir fails to restore monocyte function. Our findings add to the continuing effort to devise new immunotherapeutic strategies that could reverse the immune defects in CHI.