A lymphocyte chemoaffinity axis for lung, non-intestinal mucosae and CNS.

Tissue-selective chemoattractants direct lymphocytes to epithelial surfaces to establish local immune environments, regulate immune responses to food antigens and commensal organisms, and protect from pathogens. Homeostatic chemoattractants for small intestines, colon and skin are known1,2, but chemotropic mechanisms selective for respiratory tract and other non-intestinal mucosal tissues remain poorly understood. Here we leveraged diverse omics datasets to identify GPR25 as a lymphocyte receptor for CXCL17, a chemoattractant cytokine whose expression by epithelial cells of airways, upper gastrointestinal and squamous mucosae unifies the non-intestinal mucosal tissues and distinguishes them from intestinal mucosae. Single-cell transcriptomic analyses show that GPR25 is induced on innate lymphocytes before emigration to the periphery, and is imprinted in secondary lymphoid tissues on activated B and T cells responding to immune challenge. GPR25 characterizes B and T tissue resident memory cells and regulatory T lymphocytes in non-intestinal mucosal tissues and lungs in humans and mediates lymphocyte homing to barrier epithelia of the airways, oral cavity, stomach, and biliary and genitourinary tracts in mouse models. GPR25 is also expressed by T cells in cerebrospinal fluid and CXCL17 by neurons, suggesting a role in central nervous system (CNS) immune regulation. We reveal widespread imprinting of GPR25 on regulatory T cells, suggesting a mechanistic link to population genetics evidence that GPR25 is protective in autoimmunity3,4. Our results define a GPR25-CXCL17 chemoaffinity axis with the potential to integrate immunity and tolerance at non-intestinal mucosae and the CNS.

Unique characteristics of lung-resident neutrophils are maintained by PGE2/PKA/Tgm2-mediated signaling.

Lung-resident neutrophils need to be tightly regulated to avoid degranulation- and cytokine-associated damage to fragile alveolar structures that can lead to fatal outcomes. Here we show that lung neutrophils (LNs) express distinct surface proteins and genes that distinguish LNs from bone marrow and blood neutrophils. Functionally, LNs show impaired migratory activity toward chemoattractants and produce high levels of interleukin-6 (IL-6) at steady state and low levels of tumor necrosis factor-α in response to lipopolysaccharide (LPS) challenge. Treating bone marrow neutrophils with bronchoalveolar lavage fluid or prostaglandin E2 induces LN-associated characteristics, including the expression of transglutaminase 2 (Tgm2) and reduced production of inflammatory cytokines upon LPS challenge. Neutrophils from Tgm2-/- mice release high levels of inflammatory cytokines in response to LPS. Lung damage is significantly exacerbated in Tgm2-/- mice in an LPS-induced acute respiratory distress syndrome model. Collectively, we demonstrate that prostaglandin E2 is a key factor for the generation of LNs with unique immune suppressive characteristics, acting through protein kinase A and Tgm2, and LNs play essential roles in protection of the lungs against pathogenic inflammation.

Chemerin triggers migration of a CD8 T cell subset with natural killer cell functions.

The recruitment of cells with effector functions into the tumor microenvironment holds potential for delaying cancer progression. We show that subsets of human CD28-effector CD8 T cells, CCR7- CD45RO+ effector memory, and CCR7- CD45RO- effector memory RA phenotypes, express the chemerin receptor CMKLR1 and bind chemerin via the receptor. CMKLR1-expressing human CD8 effector memory T cells present gene, protein, and cytotoxic features of NK cells. Active chemerin promotes chemotaxis of CMKLR1-expressing CD8 effector memory cells and triggers activation of the α4ß1 integrin. In an experimental prostate tumor mouse model, chemerin expression is downregulated in the tumor microenvironment, which is associated with few tumor-infiltrating CD8+ T cells, while forced overexpression of chemerin by mouse prostate cancer cells leads to an accumulation of intra-tumor CD8+ T cells. Furthermore, α4 integrin blockade abrogated the chemerin-dependent recruitment of CD8+ T effector memory cells into implanted prostate tumors in vivo. The results identify a role for chemerin:CMKLR1 in defining a specialized NK-like CD8 T cell, and suggest the use of chemerin-dependent modalities to target effector CMKLR1-expressing T cells to the tumor microenvironment for immunotherapeutic purposes.

DGAT1 inhibits retinol-dependent regulatory T cell formation and mediates autoimmune encephalomyelitis.

The balance of effector versus regulatory T cells (Tregs) controls inflammation in numerous settings, including multiple sclerosis (MS). Here we show that memory phenotype CD4+ T cells infiltrating the central nervous system during experimental autoimmune encephalomyelitis (EAE), a widely studied animal model of MS, expressed high levels of mRNA for Dgat1 encoding diacylglycerol-O-acyltransferase-1 (DGAT1), an enzyme that catalyzes triglyceride synthesis and retinyl ester formation. DGAT1 inhibition or deficiency attenuated EAE, with associated enhanced Treg frequency; and encephalitogenic, DGAT1-/- in vitro-polarized Th17 cells were poor inducers of EAE in adoptive recipients. DGAT1 acyltransferase activity sequesters retinol in ester form, preventing synthesis of retinoic acid, a cofactor for Treg generation. In cultures with T cell-depleted lymphoid tissues, retinol enhanced Treg induction from DGAT1-/- but not from WT T cells. The WT Treg induction defect was reversed by DGAT1 inhibition. These results demonstrate that DGAT1 suppresses retinol-dependent Treg formation and suggest its potential as a therapeutic target for autoimmune inflammation.

The antimicrobial activity of chemerin-derived peptide p4 requires oxidative conditions.

Chemerin is a leukocyte attractant, adipokine, and antimicrobial protein abundantly produced in the skin epidermis. Despite the fact that most of the bactericidal activity present in human skin exudates is chemerin-dependent, just how chemerin shapes skin defenses remains obscure. Here we demonstrate that p4, a potent antimicrobial human chemerin peptide derivative, displays killing activity against pathogenic methicillin-resistant Staphylococcus aureus strains and suppresses microbial growth in a topical skin infection model. Mechanistically, we show that p4 homodimerization is required for maximal bactericidal activity and that an oxidative environment, such as at the skin surface, facilitates p4 disulfide bridge formation, required for the dimerization. p4 led to rapid damage of the bacterial internal membrane and inhibited the interaction between the membranous cytochrome bc1 complex and its redox partner, cytochrome c These results suggest that a chemerin p4-based defense strategy combats bacterial challenges at the skin surface.

A Chimeric Antibody against ACKR3/CXCR7 in Combination with TMZ Activates Immune Responses and Extends Survival in Mouse GBM Models.

Glioblastoma (GBM) is the least treatable type of brain tumor, afflicting over 15,000 people per year in the United States. Patients have a median survival of 16 months, and over 95% die within 5 years. The chemokine receptor ACKR3 is selectively expressed on both GBM cells and tumor-associated blood vessels. High tumor expression of ACKR3 correlates with poor prognosis and potential treatment resistance, making it an attractive therapeutic target. We engineered a single chain FV-human FC-immunoglobulin G1 (IgG1) antibody, X7Ab, to target ACKR3 in human and mouse GBM cells. We used hydrodynamic gene transfer to overexpress the antibody, with efficacy in vivo. X7Ab kills GBM tumor cells and ACKR3-expressing vascular endothelial cells by engaging the cytotoxic activity of natural killer (NK) cells and complement and the phagocytic activity of macrophages. Combining X7Ab with TMZ allows the TMZ dosage to be lowered, without compromising therapeutic efficacy. Mice treated with X7Ab and in combination with TMZ showed significant tumor reduction by MRI and longer survival overall. Brain-tumor-infiltrating leukocyte analysis revealed that X7Ab enhances the activation of M1 macrophages to support anti-tumor immune response in vivo. Targeting ACKR3 with immunotherapeutic monoclonal antibodies (mAbs) in combination with standard of care therapies may prove effective in treating GBM.

Endothelial expression of CXCR7 and the regulation of systemic CXCL12 levels.

The concentration of CXCL12/SDF-1 in the bloodstream is tightly regulated, given its central role in leucocyte and stem/progenitor cell egress from bone marrow and recruitment to sites of inflammation or injury. The mechanism responsible for this regulation is unknown. Here we show that both genetic deletion and pharmacological inhibition of CXCR7, a high-affinity CXCL12 receptor, caused pronounced increases in plasma CXCL12 levels. The rise in plasma CXCL12 levels was associated with an impairment in the ability of leucocytes to migrate to a local source of CXCL12. Using a set of complementary and highly sensitive techniques, we found that CXCR7 protein is expressed at low levels in multiple organs in both humans and mice. In humans, CXCR7 was detected primarily on venule endothelium and arteriole smooth muscle cells. CXCR7 expression on venule endothelium was also documented in immunodeficient mice and CXCR7(+/lacZ) mice. The vascular expression of CXCR7 therefore gives it immediate access to circulating CXCL12. These studies suggest that endothelial CXCR7 regulates circulating CXCL12 levels and that CXCR7 inhibitors might be used to block CXCL12-mediated cell migration for therapeutic purposes.

Expression, regulation, and function of atypical chemerin receptor CCRL2 on endothelial cells.

Chemokine (CC motif) receptor-like 2 (CCRL2) binds leukocyte chemoattractant chemerin and can regulate local levels of the attractant, but does not itself support cell migration. In this study, we show that CCRL2 and VCAM-1 are upregulated on cultured human and mouse vascular endothelial cells (EC) and cell lines by proinflammatory stimuli. CCRL2 induction is dependent on NF-κB and JAK/STAT signaling pathways, and activated endothelial cells specifically bind chemerin. In vivo, CCRL2 is constitutively expressed at high levels by lung endothelial cells and at lower levels by liver endothelium; and liver but not lung EC respond to systemic LPS injection by further upregulation of the receptor. Plasma levels of total chemerin are elevated in CCRL2(-/-) mice and are significantly enhanced after systemic LPS treatment in CCRL2(-/-) mice compared with wild-type mice. Following acute LPS-induced pulmonary inflammation in vivo, chemokine-like receptor 1 (CMKLR1)(+) NK cell recruitment to the airways is significantly impaired in CCRL2(-/-) mice compared with wild-type mice. In vitro, chemerin binding to CCRL2 on endothelial cells triggers robust adhesion of CMKLR1(+) lymphoid cells through an α(4)ß(1) integrin/VCAM-1-dependent mechanism. In conclusion, CCRL2 is expressed by EC in a tissue- and activation-dependent fashion, regulates circulating chemerin levels and its bioactivity, and enhances chemerin- and CMKLR1-dependent lymphocyte/EC adhesion in vitro and recruitment to inflamed airways in vivo. Its expression and/or induction on EC by proinflammatory stimuli provide a novel and specific mechanism for the local enrichment of chemerin at inflammatory sites, regulating the recruitment of CMKLR1(+) cells.

Phospholipase C activator m-3M3FBS protects against morbidity and mortality associated with sepsis.

Although phospholipase C (PLC) is a crucial enzyme required for effective signal transduction and leukocyte activation, the role of PLC in polymicrobial sepsis remains unclear. In this study, we show that the direct PLC activator m-3M3FBS treatment significantly attenuates vital organ inflammation, widespread immune cell apoptosis, and mortality in a mouse sepsis model induced by lethal cecal ligation and puncture challenge. Mechanistically, m-3M3FBS-dependent protection was largely abolished by pretreatment of mice with the PLC-selective inhibitor U-73122, thus confirming PLC agonism by m-3M3FBS in vivo. PLC activation enhanced the bactericidal activity and hydrogen peroxide production of mouse neutrophils, and it also enhanced the production of IFN-γ and IL-12 while inhibiting proseptic TNF-α and IL-1ß production in cecal ligation and puncture mice. In a second model of sepsis, PLC activation also inhibited the production of TNF-α and IL-1ß following systemic LPS challenge. In conclusion, we show that agonizing the central signal transducing enzyme PLC by m-3M3FBS can reverse the progression of toxic shock by triggering multiple protective downstream signaling pathways to maintain organ function, leukocyte survival, and to enhance microbial killing.

CD200Rhigh neutrophils with dysfunctional autophagy establish systemic immunosuppression by increasing regulatory T cells.

Distinct neutrophil populations arise during certain pathological conditions. The generation of dysfunctional neutrophils during sepsis and their contribution to septicemia-related systemic immune suppression remain unclear. In this study, using an experimental sepsis model that features immunosuppression, we identified a novel population of pathogenic CD200Rhigh neutrophils that are generated during the initial stages of sepsis and contribute to systemic immune suppression by enhancing regulatory T (Treg) cells. Compared to their CD200Rlow counterparts, sepsis-generated CD200Rhigh neutrophils exhibit impaired autophagy and dysfunction, with reduced chemotactic migration, superoxide anion production, and TNF-α production. Increased soluble CD200 blocks autophagy and neutrophil maturation in the bone marrow during experimental sepsis, and recombinant CD200 treatment in vitro can induce neutrophil dysfunction similar to that observed in CD200Rhigh neutrophils. The administration of an α-CD200R antibody effectively reversed neutrophil dysfunction by enhancing autophagy and protecting against a secondary infection challenge, leading to increased survival. Transcriptome analysis revealed that CD200Rhigh neutrophils expressed high levels of Igf1, which elicits the generation of Treg cells, while the administration of an α-CD200R antibody inhibited Treg cell generation in a secondary infection model. Taken together, our findings revealed a novel CD200Rhigh neutrophil population that mediates the pathogenesis of sepsis-induced systemic immunosuppression by generating Treg cells.

Serum amyloid A stimulates macrophage foam cell formation via lectin-like oxidized low-density lipoprotein receptor 1 upregulation.

Elevated levels of serum amyloid A (SAA) is a risk factor for cardiovascular diseases, however, the role of SAA in the pathophysiology of atherosclerosis remains unclear. Here we show that SAA induced macrophage foam cell formation. SAA-stimulated foam cell formation was mediated by c-jun N-terminal kinase (JNK) signaling. Moreover, both SAA and SAA-conjugated high density lipoprotein stimulated the expression of the important scavenger receptor lectin-like oxidized low-density lipoprotein receptor 1 (LOX1) via nuclear factor-κB (NF-κB). A LOX1 antagonist carrageenan significantly blocked SAA-induced foam cell formation, indicating that SAA promotes foam cell formation via LOX1 expression. Our findings therefore suggest that SAA stimulates foam cell formation via LOX1 induction, and thus likely contributes to atherogenesis.

Regulation of chemerin chemoattractant and antibacterial activity by human cysteine cathepsins.

Chemerin, a ligand for the G-protein coupled receptor chemokine-like receptor 1, requires C-terminal proteolytic processing to unleash its chemoattractant activity. Proteolytically processed chemerin selectively attracts specific subsets of immunoregulatory APCs, including chemokine-like receptor 1-positive immature plasmacytoid dendritic cells (pDC). Chemerin is predicted to belong to the structural cathelicidin/cystatin family of proteins composed of antibacterial polypeptide cathelicidins and inhibitors of cysteine proteinases (cystatins). We therefore hypothesized that chemerin may interact directly with cysteine proteases, and that it might also function as an antibacterial agent. In this article, we show that chemerin does not inhibit human cysteine proteases, but rather is a new substrate for cathepsin (cat) K and L. cat K- and L-cleaved chemerin triggered robust migration of human blood-derived pDC ex vivo. Furthermore, cat K- and L-truncated chemerin also displayed antibacterial activity against Enterobacteriaceae. Cathepsins may therefore contribute to host defense by activating chemerin to directly inhibit bacterial growth and to recruit pDC to sites of infection.

Development of a rapid image-based high-content imaging screening assay to evaluate therapeutic antibodies against the monkeypox virus.

Antibody-based therapy is emerging as a critical therapeutic countermeasure to treat acute viral infections by offering rapid protection against clinical disease. The advancements in structural biology made it feasible to rationalize monoclonal antibodies (mAbs) by identifying key and, possibly, neutralizing epitopes of viral proteins for therapeutic purposes. A critical component in assessing mAbs during pandemics requires the development of rapid but detailed methods to detect and quantitate the neutralization activity. In this study, we developed and optimized two high-content image (HCI)-based assays: one to detect viral proteins by staining and the second to quantify cytopathic viral effects by a label-free phenotypic assay. These assays were employed to screen for therapeutic antibodies against the monkeypox virus (MPXV) using surrogate poxviruses such as vaccinia virus (VACV). Plaque-based neutralization results confirmed the HCI data. The phenotypic assay found pox virus-induced syncytia formation in various cells, and we were able to quantitate and use this phenotype to screen mAbs. The HCI identified several potent VACV-neutralizing antibodies that showed in vitro efficacy against both clades of MPXV. In addition, a combination study of ST-246/tecovirimat/TPOXX a single neutralizing antibody Ab-40, showed synergistic activity against VACV in an in-vitro neutralization assay. This rapid high-content method utilizing state-of-the-art technologies enabled the evaluation of hundreds of mAbs quickly to identify several potent anti-MPXV neutralizing mAbs for further development.

Endothelial CCRL2 induced by disturbed flow promotes atherosclerosis via chemerin-dependent ß2 integrin activation in monocytes.

AIMS: Chemoattractants and their cognate receptors are essential for leucocyte recruitment during atherogenesis, and atherosclerotic plaques preferentially occur at predilection sites of the arterial wall with disturbed flow (d-flow). In profiling the endothelial expression of atypical chemoattractant receptors (ACKRs), we found that Ackr5 (CCRL2) was up-regulated in an endothelial subpopulation by atherosclerotic stimulation. We therefore investigated the role of CCRL2 and its ligand chemerin in atherosclerosis and the underlying mechanism. METHODS AND RESULTS: By analysing scRNA-seq data of the left carotid artery under d-flow and scRNA-seq datasets GSE131776 of ApoE-/- mice from the Gene Expression Omnibus database, we found that CCRL2 was up-regulated in one subpopulation of endothelial cells in response to d-flow stimulation and atherosclerosis. Using CCRL2-/-ApoE-/- mice, we showed that CCRL2 deficiency protected against plaque formation primarily in the d-flow areas of the aortic arch in ApoE-/- mice fed high-fat diet. Disturbed flow induced the expression of vascular endothelial CCRL2, recruiting chemerin, which caused leucocyte adhesion to the endothelium. Surprisingly, instead of binding to monocytic CMKLR1, chemerin was found to activate ß2 integrin, enhancing ERK1/2 phosphorylation and monocyte adhesion. Moreover, chemerin was found to have protein disulfide isomerase-like enzymatic activity, which was responsible for the interaction of chemerin with ß2 integrin, as identified by a Di-E-GSSG assay and a proximity ligation assay. For clinical relevance, relatively high serum levels of chemerin were found in patients with acute atherothrombotic stroke compared to healthy individuals. CONCLUSIONS: Our findings indicate that d-flow-induced CCRL2 promotes atherosclerotic plaque formation via a novel CCRL2-chemerin-ß2 integrin axis, providing potential targets for the prevention or therapeutic intervention of atherosclerosis.

Chemokine-like receptor 1 regulates skeletal muscle cell myogenesis.

The chemokine-like receptor-1 (CMKLR1) is a G protein-coupled receptor that is activated by chemerin, a secreted plasma leukocyte attractant and adipokine. Previous studies identified that CMKLR1 is expressed in skeletal muscle in a stage-specific fashion during embryogenesis and in adult mice; however, its function in skeletal muscle remains unclear. Based on the established function of CMKLR1 in cell migration and differentiation, we investigated the hypothesis that CMKLR1 regulates the differentiation of myoblasts into myotubes. In C(2)C(12) mouse myoblasts, CMKLR1 expression increased threefold with differentiation into multinucleated myotubes. Decreasing CMKLR1 expression by adenoviral-delivered small-hairpin RNA (shRNA) impaired the differentiation of C(2)C(12) myoblasts into mature myotubes and reduced the mRNA expression of myogenic regulatory factors myogenin and MyoD while increasing Myf5 and Mrf4. At embryonic day 12.5 (E12.5), CMKLR1 knockout (CMKLR1(-/-)) mice appeared developmentally delayed and displayed significantly lower wet weights and a considerably diminished myotomal component of somites as revealed by immunolocalization of myosin heavy chain protein compared with wild-type (CMKLR1(+/+)) mouse embryos. These changes were associated with increased Myf5 and decreased MyoD protein expression in the somites of E12.5 CMKLR1(-/-) mouse embryos. Adult male CMKLR1(-/-) mice had significantly reduced bone-free lean mass and weighed less than the CMKLR1(+/+) mice. We conclude that CMKLR1 is essential for myogenic differentiation of C(2)C(12) cells in vitro, and the CMKLR1 null mice have a subtle skeletal muscle deficit beginning from embryonic life that persists during postnatal life.

CXCR7 protein is not expressed on human or mouse leukocytes.

Since the discovery that CXCR7 binds to CXCL12/SDF-1α, the role of CXCR7 in CXCL12-mediated biological processes has been under intensive scrutiny. However, there is no consensus in the literature on the expression of CXCR7 protein by peripheral blood cells. In this study we analyzed human and mouse leukocytes and erythrocytes for CXCR7 protein expression, using a competitive CXCL12 binding assay as well as by flow cytometry and immunohistochemistry using multiple CXCR7 Abs. CXCR7(-/-) mice were used as negative controls. Together, these methods indicate that CXCR7 protein is not expressed by human peripheral blood T cells, B cells, NK cells, or monocytes, or by mouse peripheral blood leukocytes. CXCR7 protein is, however, expressed on mouse primitive erythroid cells, which supply oxygen to the embryo during early stages of development. These studies therefore suggest that, whereas CXCR7 protein is expressed by primitive RBCs during murine embryonic development, in adult mammals CXCR7 protein is not expressed by normal peripheral blood cells.

Activation of CXCR2 by extracellular matrix degradation product acetylated Pro-Gly-Pro has therapeutic effects against sepsis.

RATIONALE: Acetylated Pro-Gly-Pro (Ac-PGP) is an endogenous degradation product of extracellular collagen that binds to leukocyte-expressed chemoattractant receptor CXCR2. Although certain agents that block CXCR2-mediated signaling protect against experimental sepsis, the roles of Ac-PGP and CXCR2 in sepsis are unclear. OBJECTIVES: To investigate the role of Ac-PGP and its receptor, CXCR2, in murine models of cecal ligation and puncture (CLP)-induced polymicrobial sepsis and organ injury. METHODS: The impact of in vivo Ac-PGP treatment on animal survival after induction of experimental sepsis was assessed. Vital organ inflammation and immune cell apoptosis were evaluated by histology, and the modulation of proinflammatory cytokine production and bactericidal activity by Ac-PGP in mouse and human blood leukocytes was measured. MEASUREMENTS AND MAIN RESULTS: The activation of CXCR2 by tripeptide agonist Ac-PGP dramatically improved survival in three experimental sepsis models. Ac-PGP elicited bactericidal activity via the generation of hydrogen peroxide, inhibited lung inflammation, and reduced immune cell apoptosis. Fluorescein isothiocyanate-labeled PGP bound directly to CXCR2, and the protective effect of Ac-PGP in sepsis was abolished in CXCR2-deficient mice. Ac-PGP treatment enhanced the production of type 1 cytokines (IFN-γ and IL-12) but inhibited the production of proinflammatory cytokines (tumor necrosis factor [TNF]-α, IL-1ß, and IL-6) in vivo. In vitro, Ac-PGP directly increased IFN-γ production and decreased the LPS-stimulated production of TNF-α by mouse splenocytes and human leukocytes. Furthermore, direct treatment of LPS-stimulated splenocytes with IFN-γ resulted in diminished secretion of TNF-α and IL-6. CONCLUSIONS: CXCR2 and Ac-PGP are thus novel target and starting molecules, respectively, for the development of therapeutic agents against sepsis.

Soluble mediators in the function of the epidermal-immune-neuro unit in the skin.

Skin is the largest, environmentally exposed (barrier) organ, capable of integrating various signals into effective defensive responses. The functional significance of interactions among the epidermis and the immune and nervous systems in regulating and maintaining skin barrier function is only now becoming recognized in relation to skin pathophysiology. This review focuses on newly described pathways that involve soluble mediator-mediated crosstalk between these compartments. Dysregulation of these connections can lead to chronic inflammatory diseases and/or pathologic conditions associated with chronic pain or itch.

Targeting PLD2 in adipocytes augments adaptive thermogenesis by improving mitochondrial quality and quantity in mice.

Phospholipase D (PLD)2 via its enzymatic activity regulates cell proliferation and migration and thus is implicated in cancer. However, the role of PLD2 in obesity and type 2 diabetes has not previously been investigated. Here, we show that during diet-induced thermogenesis and obesity, levels of PLD2 but not PLD1 in adipose tissue are inversely related with uncoupling protein 1, a key thermogenic protein. We demonstrate that the thermogenic program in adipose tissue is significantly augmented in mice with adipocyte-specific Pld2 deletion or treated with a PLD2-specific inhibitor and that these mice are resistant to high fat diet-induced obesity, glucose intolerance, and insulin resistance. Mechanistically, we show that Pld2 deletion in adipose tissue or PLD2 pharmacoinhibition acts via p62 to improve mitochondrial quality and quantity in adipocytes. Thus, PLD2 inhibition is an attractive therapeutic approach for obesity and type 2 diabetes by resolving defects in diet-induced thermogenesis.

The novel chemokine receptor CXCR7 regulates trans-endothelial migration of cancer cells.

BACKGROUND: Migration of metastatic tumor cells from the bloodstream into lymph nodes is thought to be facilitated by expression of the chemokine receptors CCR7, CXCR4 and, for B cell-derived tumors, CXCR5. Expression of their respective chemokine ligands (CCL19, CCL21, CXCL12 and CXCL13) by endothelial cells inside the lymph nodes facilitates the trans-endothelial migration (TEM) of these cells through high endothelial venules into the lymph node parenchyma. It is known that CXCR7, a second CXCL12 receptor, regulates TEM of CXCR4+CXCR7+ tumor cells towards a CXCL12 source. In this study, we set out to assess the potential stimulation by CXCL12 of tumor cell TEM towards other chemokines and whether CXCR7 might be able to regulate such effects. METHODS: The human Burkitt's lymphoma cell line NC-37, which expresses CXCR4, CXCR5, CXCR7 and CCR7, was selected as a model system. TEM of these cells through a human HUVEC endothelial cell monolayer was used as the main model system for these studies. Regulation of their TEM behavior by various concentrations of the various cognate chemokines for the above-mentioned receptors, placed in either the source or target wells of modified Boyden chamber migration plates, was assessed by quantifying the number of cells migrated under each experimental condition. RESULTS: Exposure of CXCR4⁺CXCR7⁺ cancer cells to CXCL12 greatly potentiated their TEM towards the chemokines CCL19 and CXCL13. This CXCL12-potentiated TEM was inhibited by the second CXCR7 chemokine ligand, CXCL11, as well as CXCR7-specific small molecule antagonists and antibodies. In contrast, the CXCR4 antagonist AMD3100 was less effective at inhibiting CXCL12-potentiated TEM. Thus, CXCR7 antagonists may be effective therapeutic agents for blocking CXCL12-mediated migration of CXCR4⁺CXCR7⁺ tumor cells into lymph nodes, regardless of whether the cancer cells follow a CXCL12 gradient or whether serum CXCL12 stimulates their migration towards CCR7 and CXCR5 chemokines in the lymph nodes.