Chemokines and lymphocyte homing in Sjögren's syndrome.

Sjögren's syndrome (SS) is a chronic systemic autoimmune disease that typically presents with lymphocyte, dendritic cell, and macrophage infiltration of exocrine gland ducts and the formation of ectopic germinal centers. The interactions of lymphocyte homing receptors and addressins and chemokines and their receptors, such as α4ß7/MAdCAM-1, LFA-1/ICAM-1, CXCL13/CXCR5, CCL25/CCR9, CX3CL1/CX3CR1, play important roles in the migration of inflammatory cells to the focal glands and the promotion of ectopic germinal center formation in SS. A variety of molecules have been shown to be involved in lymphocyte homing, including tumor necrosis factor-α, interferon (IFN)-α, IFN-ß, and B cell activating factor. This process mainly involves the Janus kinase-signal transducer and activator of transcription signaling pathway, lymphotoxin-ß receptor pathway, and nuclear factor-κB signaling pathway. These findings have led to the development of antibodies to cell adhesion molecules, antagonists of chemokines and their receptors, compounds interfering with chemokine receptor signaling, and gene therapies targeting chemokines and their receptors, providing new targets for the treatment of SS in humans. The aim of this study was to explore the relationship between lymphocyte homing and the pathogenesis of SS, and to provide a review of recent studies addressing lymphocyte homing in targeted therapy for SS.

The chemerin/CMKLR1 axis regulates intestinal graft-versus-host disease.

Gastrointestinal graft-versus-host disease (GvHD) is a major cause of mortality and morbidity following allogeneic bone marrow transplantation (allo-BMT). Chemerin is a chemotactic protein that recruits leukocytes to inflamed tissues by interacting with ChemR23/CMKLR1, a chemotactic receptor expressed by leukocytes, including macrophages. During acute GvHD, chemerin plasma levels were strongly increased in allo-BM-transplanted mice. The role of the chemerin/CMKLR1 axis in GvHD was investigated using Cmklr1-KO mice. WT mice transplanted with an allogeneic graft from Cmklr1-KO donors (t-KO) had worse survival and more severe GvHD. Histological analysis demonstrated that the gastrointestinal tract was the organ mostly affected by GvHD in t-KO mice. The severe colitis of t-KO mice was characterized by massive neutrophil infiltration and tissue damage associated with bacterial translocation and exacerbated inflammation. Similarly, Cmklr1-KO recipient mice showed increased intestinal pathology in both allogeneic transplant and dextran sulfate sodium-induced colitis. Notably, the adoptive transfer of WT monocytes into t-KO mice mitigated GvHD manifestations by decreasing gut inflammation and T cell activation. In patients, higher chemerin serum levels were predictive of GvHD development. Overall, these results suggest that CMKLR1/chemerin may be a protective pathway for the control of intestinal inflammation and tissue damage in GvHD.

IL-17D affects the chemokines and chemokine receptors of intestinal epithelial cells under hyperoxia.

IL-17D is a new member of the IL-17 family. Currently, it is believed that IL-17D can directly act on immune cells or may indirectly modulate immune responses by regulating cytokine expression. Herein, we hypothesized that IL-17D regulates the expression of chemokines in intestinal epithelial cells, in turn modulating the immune response within intestinal mucosa under hyperoxia. To explore this notion, newborn rats were divided into a hyperoxia group (85 % O2) and control group (21 % O2). Small intestinal tissues were obtained from neonatal rats at 3, 7, 10, and 14 days. Similarly, intestinal epithelial cells were treated by hyperoxia (85 % O2) as the hyperoxia group or were incubated under normal oxygen (21 % O2) as the control group. Finally, intestinal epithelial cells subjected to hyperoxia were treated with recombinant IL-17D and IL-17D antibodies for 24, 48, and 72 h. Immunohistochemistry, western blot, and reverse transcription-quantitative polymerase chain reaction were used to detect the expression levels of chemokines and chemokine receptors in intestinal tissues of newborn rats and intestinal epithelial cells. We found that hyperoxia affected chemokine expression both in vivo and in vitro. Under hyperoxia, IL-17D promoted the expression of CCL2, CCL25, CCL28, and CCR9 in intestinal epithelial cells while downregulating CCR2, CCR5, CCL5, and CCL20. Our findings provide a basis for further study on the effects of hyperoxia-induced intestinal inflammation and intestinal injury.

Simulating CXCR5 Dynamics in Complex Tissue Microenvironments.

To effectively navigate complex tissue microenvironments, immune cells sense molecular concentration gradients using G-protein coupled receptors. However, due to the complexity of receptor activity, and the multimodal nature of chemokine gradients in vivo, chemokine receptor activity in situ is poorly understood. To address this issue, we apply a modelling and simulation approach that permits analysis of the spatiotemporal dynamics of CXCR5 expression within an in silico B-follicle with single-cell resolution. Using this approach, we show that that in silico B-cell scanning is robust to changes in receptor numbers and changes in individual kinetic rates of receptor activity, but sensitive to global perturbations where multiple parameters are altered simultaneously. Through multi-objective optimization analysis we find that the rapid modulation of CXCR5 activity through receptor binding, desensitization and recycling is required for optimal antigen scanning rates. From these analyses we predict that chemokine receptor signaling dynamics regulate migration in complex tissue microenvironments to a greater extent than the total numbers of receptors on the cell surface.

Mechanism of interaction between virus and host is inferred from the changes of gene expression in macrophages infected with African swine fever virus CN/GS/2018 strain.

BACKGROUND: African swine fever virus (ASFV) is a highly lethal virus that can infect porcine alveolar macrophages (PAMs). Since ASFV, China has dealt with a heavy blow to the pig industry. However, the effect of infection of ASFV strains isolated from China on PAM transcription level is not yet clarified. METHODS: In this study, RNA sequencing (RNA-seq) was used to detect the differential expression of genes in PAMs at different time points after ASFV-CN/GS/2018 infection. The fluorescent quantitative polymerase chain reaction (qPCR) method was used to confirm the altered expression of related genes in PAMs infected with ASFV. RESULTS: A total of 1154 differentially expressed genes were identified after ASFV-CN/GS/2018 infection, of which 816 were upregulated, and 338 were downregulated. GO and KEGG analysis showed that these genes were dynamically enriched in various biological processes, including innate immune response, inflammatory response, chemokines, and apoptosis. Furthermore, qPCR verified that the DEAD box polypeptide 58 (DDX58), Interferon-induced helicase C domain-containing protein 1 (IFIH1), Toll-like receptor 3 (TLR3), and TLR7 of PAMs were upregulated after ASFV infection, while TLR4 and TLR6 had a significant downward trend during ASFV infection. The expression of some factors related to antiviral and inflammation was altered significantly after ASFV infection, among which interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), IFIT2, Interleukin-6 (IL-6) were upregulated, and Ewing's tumor-associated antigen 1 homolog (ETAA1) and Prosaposin receptor GPR37 (GPR37) were downregulated. In addition, we discovered that ASFV infection is involved in the regulation of chemokine expression in PAMs, and the chemokines, such as C-X-C motif chemokine 8 (CXCL8) and CXCL10, were upregulated after infection. However, the expression of chemokine receptor C-X-C chemokine receptor type 2 (CXCR2) is downregulated. Also, that the transcriptional levels of pro-apoptotic and anti-apoptotic factors changed after infection. CONCLUSIONS: After ASFV-CN/GS/2018 infection, the expression of some antiviral and inflammatory factors in PAMs changed significantly. The ASFV infection may activates the RLR and TLR signaling pathways. In addition, ASFV infection is involved in regulating of chemokine expression in PAMs and host cell apoptosis.

PD-L1+ and XCR1+ dendritic cells are region-specific regulators of gut homeostasis.

The intestinal mucosa constitutes an environment of closely regulated immune cells. Dendritic cells (DC) interact with the gut microbiome and antigens and are important in maintaining gut homeostasis. Here, we investigate DC transcriptome, phenotype and function in five anatomical locations of the gut lamina propria (LP) which constitute different antigenic environments. We show that DC from distinct gut LP compartments induce distinct T cell differentiation and cytokine secretion. We also find that PD-L1+ DC in the duodenal LP and XCR1+ DC in the colonic LP comprise distinct tolerogenic DC subsets that are crucial for gut homeostasis. Mice lacking PD-L1+ and XCR1+ DC have a proinflammatory gut milieu associated with an increase in Th1/Th17 cells and a decrease in Treg cells and have exacerbated disease in the models of 5-FU-induced mucositis and DSS-induced colitis. Our findings identify PD-L1+ and XCR1+ DC as region-specific physiologic regulators of intestinal homeostasis.

Selective targeting of ligand-dependent and -independent signaling by GPCR conformation-specific anti-US28 intrabodies.

While various GPCRs, including US28, display constitutive, ligand-independent activity, it remains to be established whether ligand-dependent and -independent active conformations differ and can be selectively modulated. Previously, the agonist-bound conformation of US28 was stabilized and its structure was solved using the anti-US28 nanobody Nb7. Here we report the recognition of the constitutively active, apo-conformation of US28 by another nanobody VUN103. While the Nb7 intrabody selectively inhibits ligand-induced signaling, the VUN103 intrabody blocks constitutive signaling, indicating the existence of distinct US28 conformational states. By displacing Gαq protein, VUN103 prevents US28 signaling and reduces tumor spheroids growth. Overall, nanobodies specific for distinct GPCR conformational states, i.e. apo- and agonist-bound, can selectively target and discern functional consequences of ligand-dependent versus independent signaling.

Fractalkine Elicits Chemotactic, Phenotypic, and Functional Effects on CX3CR1+CD27- NK Cells in Obesity-Associated Cancer.

Esophagogastric adenocarcinomas (EAC) are obesity-associated malignancies underpinned by severe immune dysregulation and inflammation. Our previous work indicates that NK cells migrate to EAC omentum, where they undergo phenotypic and functional alterations and apoptosis. In this study, we investigate whether such erroneous chemotaxis to omentum is paralleled by compromised NK cell infiltration of EAC patient tumor and examine the role of the inflammatory chemokine fractalkine in shaping the NK cell-mediated response. Our data show diminished NK cell frequencies in EAC tumor compared with those in the circulation and reveal that intratumoral NK cell frequencies decline as visceral obesity increases in EAC patients. Our in vitro findings demonstrate that antagonism of fractalkine receptor CX3CR1 significantly reduces NK cell migration to EAC patient-derived, omental adipose tissue-conditioned media, but not toward tumor-conditioned media. These data suggest fractalkine is a key driver of NK cell chemotaxis to omentum but has a lesser role in NK cell homing to tumor in EAC. We propose that this may offer a novel therapeutic strategy to limit NK cell depletion in the omentum of obese EAC patients, and our data suggest the optimal timing for CX3CR1 antagonism is after neoadjuvant chemoradiotherapy. Our functional studies demonstrate that fractalkine induces the conversion from CX3CR1+CD27- to CX3CR1-CD27+ NK cells and increases their IFN-γ and TNF-α production, indicative of its role in shaping the dominant NK cell phenotype in EAC omentum. This study uncovers crucial and potentially druggable pathways underpinning NK cell dysfunction in obesity-associated cancer and provides compelling insights into fractalkine's diverse biological functions.

Comprehensive analysis of a chemokine- and chemokine receptor family-based signature for patients with lung adenocarcinoma.

The clinical significance and comprehensive features of chemokines and their receptors in lung adenocarcinoma (LUAD) have not been clarified. We aimed to characterize the expression profiles of chemokine and chemokine receptor family members and construct a chemokine- and chemokine receptor-based prognosis signature. A total of 1511 patients with LUAD from seven independent cohorts were included in the study. The training set collected from The Cancer Genome Atlas (TCGA) database containing 468 cases. The validation was performed on the basis of six different cohorts downloaded from Gene Expression Omnibus (GEO) database. A five-chemokine- and chemokine receptor-(CXCL2, CXCL13, CCL26, CCL20, CX3CR1) based prognosis signature was constructed with TCGA dataset using LASSO Cox regression and Cox proportional hazards regression analysis. A multivariate analysis verified that this signature was an independent prognostic factor. The predictive value of this signature was further verified by other six independent cohorts and multiple clinical subtypes. We performed immune cell infiltration analysis and biological pathway analysis which provided more insight into this signature-related immune and inflammatory landscape and clarified the intrinsic molecular mechanism by which this signature could be used to predict clinical prognosis. Furthermore, we explored the close relationship between this signature and tumor mutation burden (TMB), neoantigen burden, PD-1, PD-L1, CTLA4, TIDE score, T cell-inflamed score. This signature provided a robust prognostic biomarker for LUAD and could serve as a predictor for immunotherapy response, which may be used as an important supplement to immunotherapy to achieve individualized tumor treatment by optimizing the prognostic management and immunotherapy for patients with LUAD.

XCR1+ type 1 conventional dendritic cells drive liver pathology in non-alcoholic steatohepatitis.

Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are prevalent liver conditions that underlie the development of life-threatening cirrhosis, liver failure and liver cancer. Chronic necro-inflammation is a critical factor in development of NASH, yet the cellular and molecular mechanisms of immune dysregulation in this disease are poorly understood. Here, using single-cell transcriptomic analysis, we comprehensively profiled the immune composition of the mouse liver during NASH. We identified a significant pathology-associated increase in hepatic conventional dendritic cells (cDCs) and further defined their source as NASH-induced boost in cycling of cDC progenitors in the bone marrow. Analysis of blood and liver from patients on the NAFLD/NASH spectrum showed that type 1 cDCs (cDC1) were more abundant and activated in disease. Sequencing of physically interacting cDC-T cell pairs from liver-draining lymph nodes revealed that cDCs in NASH promote inflammatory T cell reprogramming, previously associated with NASH worsening. Finally, depletion of cDC1 in XCR1DTA mice or using anti-XCL1-blocking antibody attenuated liver pathology in NASH mouse models. Overall, our study provides a comprehensive characterization of cDC biology in NASH and identifies XCR1+ cDC1 as an important driver of liver pathology.

Role of Myeloid-Derived Suppressor Cells in High-Dose-Irradiated TRAMP-C1 Tumors: A Therapeutic Target and an Index for Assessing Tumor Microenvironment.

PURPOSE: To investigate the temporal and spatial infiltration of TRAMP-C1 tumors by myeloid-derived suppressor cells (MDSCs) after high-dose radiation therapy (RT), and to explore their effect on tumor growth. METHODS AND MATERIALS: TRAMP-C1 intramuscularly tumors were irradiated with a single dose of 8 Gy or 25 Gy. The dynamics of infiltrated MDSCs and their intratumoral spatial distribution were assessed by immunohistochemistry and flow cytometry. Cytokine levels in the blood and tumor were analyzed by multiplex immunoassay. Mice were injected with anti-Gr-1 antibody to determine whether MDSCs affect tumor growth after RT. RESULTS: CD11b+Gr-1+ MDSCs infiltrated TRAMP-C1 tumors irradiated with 25 Gy, but not 8 Gy, within 4 hours and recruitment persisted for at least 2 weeks. Both CD11b+Ly6G+Ly6C+ polymorphonuclear-MDSCs (PMN-MDSCs) and CD11b+Ly6G-Ly6Chi monocytic-MDSCs (M-MDSCs) were involved. Tumor RT also increased the representation of both MDSC subpopulations in the spleen and peripheral blood. Levels of multiple cytokines were increased in the tumors at 2 weeks, including GM-CSF, G-CSF, CCL-3, CCL-5, CXCL-5, IL-6, IL-17α, and VEGF-a; while G-CSF, IL-6, and TNF-α levels increased in the blood. PMN-MDSCs aggregated in the central necrotic region of the irradiated tumors over time, where they were associated with avascular hypoxia (CD31-PIMO+). MDSCs expressed the proangiogenic factor, matrix metalloproteinase-9, and, within the necrotic area, high levels of arginase-1 and indoleamine 2,3-dioxygenase. Depletion of PMN-MDSCs by Gr-1 antibody increased the efficacy of high-dose RT. CONCLUSIONS: PMN-MDSCs infiltrate TRAMP-C1 tumors after high-dose RT. Their spatial distribution suggests they are involved in the evolution of an intratumoral state of necrosis associated with avascular hypoxia, and their phenotype is consistent with them being immunosuppressive. They appear to promote tumor growth after RT, making them a prime therapeutic target for therapeutic intervention. Assessment of MDSCs and cytokine levels in blood could be an index of the need for such an intervention.

Immunosuppressive Property of MSCs Mediated by Cell Surface Receptors.

In the past decade, mesenchymal stem cells (MSCs) tend to exhibit inherent tropism for refractory inflammatory diseases and engineered MSCs have appeared on the market as therapeutic agents. Recently, engineered MSCs target to cell surface molecules on immune cells has been a new strategy to improve MSC applications. In this review, we discuss the roles of multiple receptors (ICAM-1, Gal-9, PD-L1, TIGIT, CD200, and CXCR4) in the process of MSCs' immunosuppressive properties. Furthermore, we discuss the principles and strategies for developing receptor-regulated MSCs and their mechanisms of action and the challenges of using MSCs as immunosuppressive therapies.

Recent advances in CAR-T cell engineering.

Chimeric antigen receptor T (CAR-T) cell therapy is regarded as an effective solution for relapsed or refractory tumors, particularly for hematological malignancies. Although the initially approved anti-CD19 CAR-T therapy has produced impressive outcomes, setbacks such as high relapse rates and resistance were experienced, driving the need to discover engineered CAR-T cells that are more effective for therapeutic use. Innovations in the structure and manufacturing of CAR-T cells have resulted in significant improvements in efficacy and persistence, particularly with the development of fourth-generation CAR-T cells. Paired with an immune modifier, the use of fourth-generation and next-generation CAR-T cells will not be limited because of cytotoxic effects and will be an efficient tool for overcoming the tumor microenvironment. In this review, we summarize the recent transformations in the ectodomain, transmembrane domain, and endodomain of the CAR structure, which, together with innovative manufacturing technology and improved cell sources, improve the prospects for the future development of CAR-T cell therapy.

Tissue-resident memory T cell reactivation by diverse antigen-presenting cells imparts distinct functional responses.

CD8+ tissue-resident memory T cells (TRM cells) are poised at the portals of infection and provide long-term protective immunity. Despite their critical roles, the precise mechanics governing TRM cell reactivation in situ are unknown. Using a TCR-transgenic Nur77-GFP reporter to distinguish "antigen-specific" from "bystander" reactivation, we demonstrate that lung CD8+ TRM cells are reactivated more quickly, yet less efficiently, than their counterparts in the draining LNs (TLN cells). Global profiling of reactivated memory T cells revealed tissue-defined and temporally regulated recall response programs. Unlike the reactivation of CD8+ TLN cells, which is strictly dependent on CD11c+XCR1+ APCs, numerous antigen-presenting partners, both hematopoietic and non-hematopoietic, were sufficient to reactivate lung CD8+ TRM cells, but the quality of TRM cell functional responses depended on the identity of the APCs. Together, this work uncovers fundamental differences in the activation kinetics, mechanics, and effector responses between CD8+ memory T cells in peripheral vs. lymphoid organs, revealing a novel tissue-specific paradigm for the reactivation of memory CD8+ T cells.

IL-12 from endogenous cDC1, and not vaccine DC, is required for Th1 induction.

Success of DC vaccines relies on the quality of antigen presentation, costimulation, lymph node migration, and the release of IL-12, in case of Th1 priming. Here, we provide evidence for interaction between the injected vaccine DCs with endogenous lymph node-resident DCs for Th1 induction. While migration of the injected DCs was essential for antigen delivery to the lymph node, the injected DCs contributed only partially to Th0 priming and were unable to instruct Th1 generation. Instead, we provide evidence that the lymph node-resident XCR1+ DCs are activated by the injected DCs to present the cognate antigen and release IL-12 for Th1 polarization. The timing of interactions in the draining lymph nodes appeared step-wise as (a) injected DCs with cognate T cells, (b) injected DCs with bystander DCs, and (c) bystander DCs with T cells. The transcriptome of the bystander DCs showed a downregulation of Treg- and Th2/Th9-inducing genes and self-antigen presentation, as well as upregulation of MHC class II and genes required for Th1 instruction. Together, these data show that injected mature lymph node migratory DCs direct T cell priming and bystander DC activation, but not Th1 polarization, which is mediated by endogenous IL-12p70+XCR1+ resident bystander DCs. Our results are of importance for clinical DC-based vaccinations against tumors where endogenous DCs may be functionally impaired by chemotherapy.

CXCR1 and CXCR2 Chemokine Receptor Agonists Produced by Tumors Induce Neutrophil Extracellular Traps that Interfere with Immune Cytotoxicity.

Neutrophils are expanded and abundant in cancer-bearing hosts. Under the influence of CXCR1 and CXCR2 chemokine receptor agonists and other chemotactic factors produced by tumors, neutrophils, and granulocytic myeloid-derived suppressor cells (MDSCs) from cancer patients extrude their neutrophil extracellular traps (NETs). In our hands, CXCR1 and CXCR2 agonists proved to be the major mediators of cancer-promoted NETosis. NETs wrap and coat tumor cells and shield them from cytotoxicity, as mediated by CD8+ T cells and natural killer (NK) cells, by obstructing contact between immune cells and the surrounding target cells. Tumor cells protected from cytotoxicity by NETs underlie successful cancer metastases in mice and the immunotherapeutic synergy of protein arginine deiminase 4 (PAD4) inhibitors, which curtail NETosis with immune checkpoint inhibitors. Intravital microscopy provides evidence of neutrophil NETs interfering cytolytic cytotoxic T lymphocytes (CTLs) and NK cell contacts with tumor cells.

Chemokine Receptors and Phagocyte Biology in Zebrafish.

Phagocytes are highly motile immune cells that ingest and clear microbial invaders, harmful substances, and dying cells. Their function is critically dependent on the expression of chemokine receptors, a class of G-protein-coupled receptors (GPCRs). Chemokine receptors coordinate the recruitment of phagocytes and other immune cells to sites of infection and damage, modulate inflammatory and wound healing responses, and direct cell differentiation, proliferation, and polarization. Besides, a structurally diverse group of atypical chemokine receptors (ACKRs) are unable to signal in G-protein-dependent fashion themselves but can shape chemokine gradients by fine-tuning the activity of conventional chemokine receptors. The optically transparent zebrafish embryos and larvae provide a powerful in vivo system to visualize phagocytes during development and study them as key elements of the immune response in real-time. In this review, we discuss how the zebrafish model has furthered our understanding of the role of two main classes of chemokine receptors, the CC and CXC subtypes, in phagocyte biology. We address the roles of the receptors in the migratory properties of phagocytes in zebrafish models for cancer, infectious disease, and inflammation. We illustrate how studies in zebrafish enable visualizing the contribution of chemokine receptors and ACKRs in shaping self-generated chemokine gradients of migrating cells. Taking the functional antagonism between two paralogs of the CXCR3 family as an example, we discuss how the duplication of chemokine receptor genes in zebrafish poses challenges, but also provides opportunities to study sub-functionalization or loss-of-function events. We emphasize how the zebrafish model has been instrumental to prove that the major determinant for the functional outcome of a chemokine receptor-ligand interaction is the cell-type expressing the receptor. Finally, we highlight relevant homologies and analogies between mammalian and zebrafish phagocyte function and discuss the potential of zebrafish models to further advance our understanding of chemokine receptors in innate immunity and disease.

The Treponema pallidum outer membrane protein Tp92 activates endothelial cells via the chemerin/CMKLR1 pathway.

Endothelium damage caused by Treponema pallidum is the key step in the systemic dissemination and pathophysiology of syphilis, particularly cardiovascular syphilis and neurosyphilis. However, the molecular mechanisms supporting endothelium damage of syphilis are undefined. The outer membrane proteins were thought to be involved. Tp92 was first identified as an outer membrane protein of T. pallidum. Homologous proteins to Tp92 play important roles in cell attachment, inflammation, and tissue destruction in other bacterial species. In this study, we investigated the effect of Tp92 on endothelial cells activation. The data showed that Tp92 induced chemerin production in activated endothelial cells. Endothelial cell-derived chemerin upregulated the expression of TNF-α and ICAM-1 in endothelial cells via CMKLR1. In addition, endothelial cell-derived chemerin promoted THP-1-derived macrophage migration towards endothelial cells. These findings suggest that Tp92 may play an important role in mediating endothelial cell activation by inducing the secretion of chemerin.

Analysis of lung stromal expression of the atypical chemokine receptor ACKR2 reveals unanticipated expression in murine blood endothelial cells.

Analysis of chemokine receptor, and atypical chemokine receptor, expression is frequently hampered by the lack of availability of high-quality antibodies and the species specificity of those that are available. We have previously described methodology utilizing Alexa-Fluor-labeled chemokine ligands as versatile reagents to detect receptor expression. Previously this has been limited to hematopoietic cells and methodology for assessing expression of receptors on stromal cells has been lacking. Among chemokine receptors, the ones most frequently expressed on stromal cells belong to the atypical chemokine receptor subfamily. These receptors do not signal in the classic sense in response to ligand but scavenge their ligands and degrade them and thus sculpt in vivo chemokine gradients. Here, we demonstrate the ability to use either intratracheal or intravenous, Alexa-Fluor-labeled chemokine administration to detect stromal cell populations expressing the atypical chemokine receptor ACKR2. Using this methodology, we demonstrate, for the first time, expression of ACKR2 on blood endothelial cells. This observation sets the lung aside from other tissues in which ACKR2 is exclusively expressed on lymphatic endothelial cells and suggest unique roles for ACKR2 in the pulmonary environment.

CCL3/CCR1 mediates CD14+CD16- circulating monocyte recruitment in knee osteoarthritis progression.

OBJECTIVES: Monocyte-derived macrophages, as the predominant immune cell type that is increased in inflamed synovium, play a vital role during knee osteoarthritis (KOA) progression. However, the mechanisms underlying the recruitment of circulating monocytes to osteoarthritic knees remain uncertain. Based on previous data obtained from plasma, we investigated the contributions of CCL2, CCL3, CCL4 and their cognate receptors in circulating monocyte chemotaxis and KOA development. METHODS: Using flow cytometry staining, we characterized the expression patterns of the chemokine receptors in CD14+CD16- circulating monocytes from KOA patients and healthy volunteers. The expression of chemokines in synovial fluids, synovium and cartilage was investigated in KOA patients and in patients without KOA. The role of chemokines and their cognate receptors in the chemotaxis of CD14+CD16- circulating monocytes was assessed using chemokine neutralizing antibodies (NA) and receptor antagonists in vitro and in vivo. RESULTS: The majority of CD14+CD16- circulating monocytes were CCR1-and CCR2-positive. CCL2, CCL3 and CCL4 were elevated in synovial fluid of KOA patients compared with that of controls. The most likely source of these chemokines is inflamed synovium and cartilage in the osteoarthritic knee. The CCL3/CCR1 and CCL2/CCR2 axes showed substantial ability to recruit CD14+CD16- monocytes in transwell assays. Similar results were confirmed in a mouse model of collagenase-induced KOA (CIA) in which blocking either the CCL3/CCR1 axis or the CCL2/CCR2 axis reduced synovial hyperplasia and F4/80+ macrophage infiltration. CONCLUSIONS: Our findings suggested that, analogous to the CCL2/CCR2 axis, CCL3 produced in osteoarthritic knees can chemoattract circulating monocytes to the inflamed synovium through CCR1.