Targeting Interleukin-1ß Protects from Aortic Aneurysms Induced by Disrupted Transforming Growth Factor ß Signaling.

Aortic aneurysms are life-threatening conditions with effective treatments mainly limited to emergency surgery or trans-arterial endovascular stent grafts, thus calling for the identification of specific molecular targets. Genetic studies have highlighted controversial roles of transforming growth factor ß (TGF-ß) signaling in aneurysm development. Here, we report on aneurysms developing in adult mice after smooth muscle cell (SMC)-specific inactivation of Smad4, an intracellular transducer of TGF-ß. The results revealed that Smad4 inhibition activated interleukin-1ß (IL-1ß) in SMCs. This danger signal later recruited innate immunity in the adventitia through chemokine (C-C motif) ligand 2 (CCL2) and modified the mechanical properties of the aortic wall, thus favoring vessel dilation. SMC-specific Smad4 deletion in Il1r1- or Ccr2-null mice resulted in milder aortic pathology. A chronic treatment with anti-IL-1ß antibody effectively hampered aneurysm development. These findings identify a mechanistic target for controlling the progression of aneurysms with compromised TGF-ß signaling, such as those driven by SMAD4 mutations.

Cenicriviroc, a CCR2/CCR5 antagonist, promotes the generation of type 1 regulatory T cells.

Cenicriviroc, a dual CCR2/CCR5 antagonist, initially developed as an anti-HIV drug, has shown promising results in nonalcoholic steatohepatitis phase 2 clinical trials. It inhibits the infiltration and activation of CCR2+/CCR5+ monocytes and macrophages to the site of liver injury, preventing liver fibrosis. However, the role of Cenicriviroc in the modulation of helper T cell differentiation and functions remains to be explored. In inflamed colons of Crohn's disease patients, CCR2+ and CCR5+ CD4+ T cells are enriched. Considering the role of CCR2+ and CCR5+ T cells in IBD pathogenesis, we investigated the potential role of Cenicriviroc in colitis. Our in vitro studies revealed that Cenicriviroc inhibits Th1-, Th2-, and Th17-cell differentiation while promoting the generation of type 1 regulatory T cells (Tr1), known for preventing inflammation through induction of IL-10. This study is the first to report that Cenicriviroc promotes Tr1 cell generation by up-regulating the signature of Tr1 cell transcription factors such as c-Maf, Prdm1, Irf-1, Batf, and EGR-2. Cenicriviroc displayed a protective effect in experimental colitis models by preventing body weight loss and intestinal inflammation and preserving epithelial barrier integrity. We show that Cenicriviroc induced IL-10 and inhibited the generation of pro-inflammatory cytokines IFN-γ, IL-17, IL-6, and IL-1ß during colitis. Based on our data, we propose Cenicriviroc as a potential therapeutic in controlling tissue inflammation by inhibiting the generation and functions of effector T cells and promoting the induction of anti-inflammatory Tr1 cells.

CCR2 inhibitor strengthens the adiponectin effects against myocardial injury after infarction.

Little evidence demonstrated the effects of nitric oxide (NO) hydrogel with adipocytes in vivo. We aimed to investigate the effects of adiponectin (ADPN) and CCR2 antagonist on cardiac functions and macrophage phenotypes after myocardial infarction (MI) using chitosan caged nitric oxide donor (CSNO) patch with adipocytes. 3T3-L1 cell line was induced to adipocytes and ADPN expression was knocked down. CSNO was synthesized and patch was constructed. MI model was constructed and patch was placed on the infarcted area. ADPN knockdown adipocytes or control was incubated with CSNO patch, and CCR2 antagonist was also used to investigate the ADPN effects on myocardial injury after infarction. On day 7 after operation, cardiac functions of the mice using CSNO with adipocytes or ADPN knockdown adipocytes improved more than in mice only using CSNO for treatment. Lymphangiogenesis increased much more in the MI mice using CSNO with adipocytes. After treating with CCR2 antagonist, Connexin43+ CD206+ cells and ZO-1+ CD206+ cells increased, suggesting that CCR2 antagonist promoted M2 polarization after MI. Besides, CCR2 antagonist promoted ADPN expression in adipocytes and cardiomyocytes. ELISA was also used and CKMB expression was much lower than other groups at 3 days after operation. On day 7 after operation, the VEGF and TGFß expressions were high in the adipocytes CSNO group, illustrating that higher ADPN led to better treatment. In all, CCR2 antagonist enhanced the ADPN effects on macrophage M2 polarization and cardiac functions. The combination used in border zone and infarcted areas may help improve patients' prognosis in surgery, such as CABG.

Discovery of a CCR2-targeting pepducin therapy for chronic pain.

Targeting the CCL2/CCR2 chemokine axis has been shown to be effective at relieving pain in rodent models of inflammatory and neuropathic pain, therefore representing a promising avenue for the development of non-opioid analgesics. However, clinical trials targeting this receptor for inflammatory conditions and painful neuropathies have failed to meet expectations and have all been discontinued due to lack of efficacy. To overcome the poor selectivity of CCR2 chemokine receptor antagonists, we generated and characterized the function of intracellular cell-penetrating allosteric modulators targeting CCR2, namely pepducins. In vivo, chronic intrathecal administration of the CCR2-selective pepducin PP101 was effective in alleviating neuropathic and bone cancer pain. In the setting of bone metastases, we found that T cells infiltrate dorsal root ganglia (DRG) and induce long-lasting pain hypersensitivity. By acting on CCR2-expressing DRG neurons, PP101 attenuated the altered phenotype of sensory neurons as well as the neuroinflammatory milieu of DRGs, and reduced bone cancer pain by blocking CD4+ and CD8+ T cell infiltration. Notably, PP101 demonstrated its efficacy in targeting the neuropathic component of bone cancer pain, as evidenced by its anti-nociceptive effects in a model of chronic constriction injury of the sciatic nerve. Importantly, PP101-induced reduction of CCR2 signaling in DRGs did not result in deleterious tumor progression or adverse behavioral effects. Thus, targeting neuroimmune crosstalk through allosteric inhibition of CCR2 could represent an effective and safe avenue for the management of chronic pain.

CCR2/CCR5 antagonist cenicriviroc reduces colonic inflammation and fibrosis in experimental colitis.

BACKGROUND AND AIM: Cenicriviroc (CVC) is a CCR2/CCR5 antagonist that has been shown to be effective in the treatment of inflammatory and fibrotic diseases. Our study evaluated its efficacy in colitis. METHODS: Mouse models of DSS-induced acute and chronic colitis were established. The efficacy of CVC in colitis was assessed by disease activity index (DAI) scores, histological assessment of inflammation and fibrosis, and expression assays of key molecules. In in vitro experiments, HT29 cell line was exposed to TNFα to study inflammatory signaling in intestinal epithelial cells. CCD-18Co colonic myofibroblasts and human primary colonic fibroblasts were activated by TGFß1 to mimic fibroblast activation. RESULTS: In HT29 cells, CVC significantly reduced mRNA expression of CCL5 (P < 0.01) but had no effect on CCL2. Furthermore, CVC reduced downstream CX3CL1 (P < 0.01) and TNFα (P < 0.05) expression, thereby inhibiting inflammatory progression. In acute colitis mice, CVC significantly reduced DAI scores and serum TNFα levels (P < 0.05) and attenuated colonic inflammation as shown by HE staining. Meanwhile, CVC had no adverse effects on the liver, heart, and kidney of mice. On the other hand, in cellular models of chronic colitis, CVC decreased the expression of fibrosis markers, including FN, CTGF, α-SMA, and MMP9, and inhibited TGFß1-induced fibrotic activation (P < 0.01). In addition, CVC attenuated colonic fibrosis in chronic colitis mice. Moreover, CVC significantly promoted autophagy, which contributed to its regulation of inflammation. CONCLUSIONS: CVC significantly inhibited inflammation through CCL5/CCR5 signaling without damaging vital organs and suppressed fibrotic activation in chronic colitis, suggesting its great potential to relieve colonic inflammation and fibrosis.

Macrophages promote the transition from myocardial ischemia reperfusion injury to cardiac fibrosis in mice through GMCSF/CCL2/CCR2 and phenotype switching.

Following acute myocardial ischemia reperfusion (MIR), macrophages infiltrate damaged cardiac tissue and alter their polarization phenotype to respond to acute inflammation and chronic fibrotic remodeling. In this study we investigated the role of macrophages in post-ischemic myocardial fibrosis and explored therapeutic targets for myocardial fibrosis. Male mice were subjected to ligation of the left coronary artery for 30 min. We first detected the levels of chemokines in heart tissue that recruited immune cells infiltrating into the heart, and found that granulocyte-macrophage colony-stimulating factor (GMCSF) released by mouse cardiac microvascular endothelial cells (MCMECs) peaked at 6 h after reperfusion, and c-c motif chemokine ligand 2 (CCL2) released by GMCSF-induced macrophages peaked at 24 h after reperfusion. In co-culture of BMDMs with MCMECs, we demonstrated that GMCSF derived from MCMECs stimulated the release of CCL2 by BMDMs and effectively promoted the migration of BMDMs. We also confirmed that GMCSF promoted M1 polarization of macrophages in vitro, while GMCSF neutralizing antibodies (NTABs) blocked CCL2/CCR2 signaling. In MIR mouse heart, we showed that GMCSF activated CCL2/CCR2 signaling to promote NLRP3/caspase-1/IL-1ß-mediated and amplified inflammatory damage. Knockdown of CC chemokine receptor 2 gene (CCR2-/-), or administration of specific CCR2 inhibitor RS102895 (5 mg/kg per 12 h, i.p., one day before MIR and continuously until the end of the experiment) effectively reduced the area of myocardial infarction, and down-regulated inflammatory mediators and NLRP3/Caspase-1/IL-1ß signaling. Mass cytometry confirmed that M2 macrophages played an important role during fibrosis, while macrophage-depleted mice exhibited significantly reduced transforming growth factor-ß (Tgf-ß) levels in heart tissue after MIR. In co-culture of macrophages with fibroblasts, treatment with recombinant mouse CCL2 stimulated macrophages to release a large amount of Tgf-ß, and promoted the release of Col1α1 by fibroblasts. This effect was diminished in BMDMs from CCR2-/- mice. After knocking out or inhibiting CCR2-gene, the levels of Tgf-ß were significantly reduced, as was the level of myocardial fibrosis, and cardiac function was protected. This study confirms that the acute injury to chronic fibrosis transition after MIR in mice is mediated by GMCSF/CCL2/CCR2 signaling in macrophages through NLRP3 inflammatory cascade and the phenotype switching.

A New Application for Cenicriviroc, a Dual CCR2/CCR5 Antagonist, in the Treatment of Painful Diabetic Neuropathy in a Mouse Model.

The ligands of chemokine receptors 2 and 5 (CCR2 and CCR5, respectively) are associated with the pathomechanism of neuropathic pain development, but their role in painful diabetic neuropathy remains unclear. Therefore, the aim of our study was to examine the function of these factors in the hypersensitivity accompanying diabetes. Additionally, we analyzed the analgesic effect of cenicriviroc (CVC), a dual CCR2/CCR5 antagonist, and its influence on the effectiveness of morphine. An increasing number of experimental studies have shown that targeting more than one molecular target is advantageous compared with the coadministration of individual pharmacophores in terms of their analgesic effect. The advantage of using bifunctional compounds is that they gain simultaneous access to two receptors at the same dose, positively affecting their pharmacokinetics and pharmacodynamics and consequently leading to improved analgesia. Experiments were performed on male and female Swiss albino mice with a streptozotocin (STZ, 200 mg/kg, i.p.) model of diabetic neuropathy. We found that the blood glucose level increased, and the mechanical and thermal hypersensitivity developed on the 7th day after STZ administration. In male mice, we observed increased mRNA levels of Ccl2, Ccl5, and Ccl7, while in female mice, we observed additional increases in Ccl8 and Ccl12 levels. We have demonstrated for the first time that a single administration of cenicriviroc relieves pain to a similar extent in male and female mice. Moreover, repeated coadministration of cenicriviroc with morphine delays the development of opioid tolerance, while the best and longest-lasting analgesic effect is achieved by repeated administration of cenicriviroc alone, which reduces pain hypersensitivity in STZ-exposed mice, and unlike morphine, no tolerance to the analgesic effects of CVC is observed until Day 15 of treatment. Based on these results, we suggest that targeting CCR2 and CCR5 with CVC is a potent therapeutic option for novel pain treatments in diabetic neuropathy patients.

Inhibition of CCR2 attenuates neuroinflammation and neuronal apoptosis after subarachnoid hemorrhage through the PI3K/Akt pathway.

BACKGROUND: Neuroinflammation and neuronal apoptosis are closely associated with a poor prognosis in patients with subarachnoid hemorrhage (SAH). We investigated the role of C-C motif chemokine receptor 2 (CCR2) in SAH. METHODS: Pre-processed RNA-seq transcriptome datasets GSE167110 and GSE79416 from the Gene Expression Omnibus (GEO) database were screened for genes differentially expressed between mice with SAH and control mice, using bioinformatics analysis. The endovascular perforation model was performed to establish SAH. RS504393 (a CCR2 antagonist) and LY294002 (PI3K inhibitor) were administered to explore the mechanism of neuroinflammation after SAH. SAH grading, neurological scoring, brain water content and blood-brain barrier (BBB) permeability determination, enzyme-linked immunosorbent assay (ELISA), western blotting, and immunofluorescence were performed. An in vitro model of SAH was induced in H22 cells by hemin treatment. The protective mechanism of CCR2 inhibition was studied by adding RS504393 and LY294002. Clinical cerebrospinal fluid (CST) samples were detected by ELISA. RESULTS: Expression of CCR2 was upregulated in both datasets and was identified as a hub gene. CCR2 expression was significantly upregulated in the cytoplasm of neurons after SAH, both in vitro and in vivo. RS significantly reduced the brain water content and blood-brain barrier permeability, alleviated neuroinflammation, and reduced neuronal apoptosis after SAH. Additionally, the protective effects of CCR2 inhibition were abolished by LY treatment. Finally, the levels of CCR2, inflammatory factors, and apoptotic factors were elevated in the CSF of patients with SAH. CCR2 levels were associated with patient outcomes at the 6-month follow-up. CONCLUSION: CCR2 expression was upregulated in both in vitro and in vivo SAH models. Additionally, inhibition of CCR2, at least partly through the PI3K/AKT pathway, alleviated neuroinflammation and neuronal apoptosis in vivo and in vitro. CCR2 levels in the CSF have a moderate diagnostic value for 6-month outcome prediction in patients with SAH.

Structure of CC chemokine receptor 2 with orthosteric and allosteric antagonists.

CC chemokine receptor 2 (CCR2) is one of 19 members of the chemokine receptor subfamily of human class A G-protein-coupled receptors. CCR2 is expressed on monocytes, immature dendritic cells, and T-cell subpopulations, and mediates their migration towards endogenous CC chemokine ligands such as CCL2 (ref. 1). CCR2 and its ligands are implicated in numerous inflammatory and neurodegenerative diseases including atherosclerosis, multiple sclerosis, asthma, neuropathic pain, and diabetic nephropathy, as well as cancer. These disease associations have motivated numerous preclinical studies and clinical trials (see http://www.clinicaltrials.gov) in search of therapies that target the CCR2-chemokine axis. To aid drug discovery efforts, here we solve a structure of CCR2 in a ternary complex with an orthosteric (BMS-681 (ref. 6)) and allosteric (CCR2-RA-[R]) antagonist. BMS-681 inhibits chemokine binding by occupying the orthosteric pocket of the receptor in a previously unseen binding mode. CCR2-RA-[R] binds in a novel, highly druggable pocket that is the most intracellular allosteric site observed in class A G-protein-coupled receptors so far; this site spatially overlaps the G-protein-binding site in homologous receptors. CCR2-RA-[R] inhibits CCR2 non-competitively by blocking activation-associated conformational changes and formation of the G-protein-binding interface. The conformational signature of the conserved microswitch residues observed in double-antagonist-bound CCR2 resembles the most inactive G-protein-coupled receptor structures solved so far. Like other protein-protein interactions, receptor-chemokine complexes are considered challenging therapeutic targets for small molecules, and the present structure suggests diverse pocket epitopes that can be exploited to overcome obstacles in drug design.

Structural basis for ligand modulation of the CCR2 conformational landscape.

CC chemokine receptor 2 (CCR2) is a part of the chemokine receptor family, an important class of therapeutic targets. These class A G-protein coupled receptors (GPCRs) are involved in mammalian signaling pathways and control cell migration toward endogenous CC chemokine ligands, named for the adjacent cysteine motif on their N terminus. Chemokine receptors and their associated ligands are involved in a wide range of diseases and thus have become important drug targets. CCR2, in particular, promotes the metastasis of cancer cells and is also implicated in autoimmunity-driven type-1 diabetes, diabetic nephropathy, multiple sclerosis, asthma, atherosclerosis, neuropathic pain, and rheumatoid arthritis. Although promising, CCR2 antagonists have been largely unsuccessful to date. Here, we investigate the effect of an orthosteric and an allosteric antagonist on CCR2 dynamics by coupling long-timescale molecular dynamics simulations with Markov-state model theory. We find that the antagonists shift CCR2 into several stable inactive conformations that are distinct from the crystal structure conformation and disrupt a continuous internal water and sodium ion pathway, preventing transitions to an active-like state. Several metastable conformations present a cryptic drug-binding pocket near the allosteric site that may be amenable to targeting with small molecules. Without antagonists, the apo dynamics reveal intermediate conformations along the activation pathway that provide insight into the basal dynamics of CCR2 and may also be useful for future drug design.

The molecular structure and role of CCL2 (MCP-1) and C-C chemokine receptor CCR2 in skeletal biology and diseases.

Monocyte chemoattractant protein-1, also called chemokine (C-C motif) ligand 2 (CCL2) or small inducible cytokine A2, is an inflammatory mediator capable of recruiting monocytes, memory T cells, and dendritic cells. CCL2 is a member of the CC chemokine superfamily, which binds to its receptor, C-C motif chemokine receptor-2 (CCR2), for the induction of chemotactic activity and an increase of calcium influx. It exerts multiple effects on a variety of cells, including monocytes, macrophages, osteoclasts, basophils, and endothelial cells, and is involved in a diverse range of diseases. This review discusses the molecular structure and role of CCL2 and CCR2 in skeletal biology and disease. Molecular structure analyses reveal that CCL2 shares a conserved C-C motif; however, it has only limited sequence homology with other CCL family members. Likewise, CCR2, as a member of the G-protein-coupled seven-transmembrane receptor superfamily, shares conserved cysteine residues, but exhibits very limited sequence homology with other CCR family members. In the skeletal system, the expression of CCL2 is regulated by a variety of factors, such as parathyroid hormone/parathyroid hormone-related peptide, interleukin 1b, tumor necrosis factor-α and transforming growth factor-beta, RANKL, and mechanical forces. The interaction of CCL2 and CCR2 activates several signaling cascades, including PI3K/Akt/ERK/NF-κB, PI3K/MAPKs, and JAK/STAT-1/STAT-3. Understanding the role of CCL2 and CCR2 will facilitate the development of novel therapies for skeletal disorders, including rheumatoid arthritis, osteolysis and other inflammatory diseases related to abnormal chemotaxis.

Effects of a CCR2 antagonist on macrophages and Toll-like receptor 9 expression in a mouse model of diabetic nephropathy.

The pathogenesis of diabetic nephropathy (DN) is related to macrophage (Mφ) recruitment to the kidneys, tumor necrosis factor-α (TNF-α) production, and oxidative stress. Toll-like receptor 9 (TLR9) activation is reportedly involved in systemic inflammation, and it exacerbates this condition in metabolic syndrome. Therefore, we hypothesized that TLR9 plays a role in the pathogenesis of DN. Two subsets of kidney Mφs in DN model (db/db) mice were analyzed using flow cytometry to evaluate their distribution and TLR9 expression and function. Mice were administered the CCR2 antagonist INCB3344 for 8 wk; changes in Mφ distribution and function and its therapeutic effects on DN pathology were examined. Bone marrow-derived CD11bhigh (BM-Mφ) and tissue-resident CD11blow Mφs (Res-Mφ) were identified in the mouse kidneys. As DN progressed, the BM-Mφ number, TLR9 expression, and TNF-α production increased significantly. In Res-Mφs, reactive oxygen species (ROS) production and phagocytic activity were enhanced. INCB3344 decreased albuminuria, serum creatinine level, BM-Mφ abundance, TLR9 expression, and TNF-α production by BM-Mφs and ROS production by Res-Mφs. Both increased activation of BM-Mφ via TLR9 and TNF-α production and increased ROS production by Res-Mφs were involved in DN progression. Thus, inactivating Mφs and their TLR9 expression by INCB3344 is a potential therapeutic strategy for DN.NEW & NOTEWORTHY We classified kidney macrophages (Mφs) into bone marrow-derived Mφs (BM-Mφs) expressing high CD11b and tissue-specific resident Mφ (Res-Mφs) expressing low CD11b. In diabetic nephropathy (DN) model mice, Toll-like receptor 9 (TLR9) expression and TNF-α production via TLR9 activation in BM-Mφs and ROS production in Res-Mφs were enhanced. Furthermore, CCR2 antagonist suppressed the kidney infiltration of BM-Mφs and their function and the ROS production by Res-Mφs, with concomitant TLR9 suppression. Our study presents a new therapeutic strategy for DN.

Mechanistic insights into the role of the chemokine CCL2/CCR2 axis in dorsal root ganglia to peripheral inflammation and pain hypersensitivity.

BACKGROUND: Pain is reported as the leading cause of disability in the common forms of inflammatory arthritis conditions. Acting as a key player in nociceptive processing, neuroinflammation, and neuron-glia communication, the chemokine CCL2/CCR2 axis holds great promise for controlling chronic painful arthritis. Here, we investigated how the CCL2/CCR2 system in the dorsal root ganglion (DRG) contributes to the peripheral inflammatory pain sensitization. METHODS: Repeated intrathecal (i.t.) administration of the CCR2 antagonist, INCB3344 was tested for its ability to reverse the nociceptive-related behaviors in the tonic formalin and complete Freund's adjuvant (CFA) inflammatory models. We further determined by qPCR the expression of CCL2/CCR2, SP and CGRP in DRG neurons from CFA-treated rats. Using DRG explants, acutely dissociated primary sensory neurons and calcium mobilization assay, we also assessed the release of CCL2 and sensitization of nociceptors. Finally, we examined by immunohistochemistry following nerve ligation the axonal transport of CCL2, SP, and CGRP from the sciatic nerve of CFA-treated rats. RESULTS: We first found that CFA-induced paw edema provoked an increase in CCL2/CCR2 and SP expression in ipsilateral DRGs, which was decreased after INCB3344 treatment. This upregulation in pronociceptive neuromodulators was accompanied by an enhanced nociceptive neuron excitability on days 3 and 10 post-CFA, as revealed by the CCR2-dependent increase in intracellular calcium mobilization following CCL2 stimulation. In DRG explants, we further demonstrated that the release of CCL2 was increased following peripheral inflammation. Finally, the excitation of nociceptors following peripheral inflammation stimulated the anterograde transport of SP at their peripheral nerve terminals. Importantly, blockade of CCR2 reduced sensory neuron excitability by limiting the calcium mobilization and subsequently decreased peripheral transport of SP towards the periphery. Finally, pharmacological inhibition of CCR2 reversed the pronociceptive action of CCL2 in rats receiving formalin injection and significantly reduced the neurogenic inflammation as well as the stimuli-evoked and movement-evoked nociceptive behaviors in CFA-treated rats. CONCLUSIONS: Our results provide significant mechanistic insights into the role of CCL2/CCR2 within the DRG in the development of peripheral inflammation, nociceptor sensitization, and pain hypersensitivity. We further unveil the therapeutic potential of targeting CCR2 for the treatment of painful inflammatory disorders.

Antifibrogenic effects of C-C chemokine receptor type 2 antagonist in a bleomycin-induced scleroderma model.

There have been several studies on the role of the monocyte chemotactic protein-1/C-C chemokine receptor type 2 (CCR2) signalling pathway in fibrotic diseases, which identified the blockade of this pathway as a potential therapeutic target for treating fibrosis. We examined the efficacy of CCR2 antagonist (RS-504393) in a mouse model of scleroderma induced by bleomycin. RS-504393 was administered via intradermal injection 6 hours prior to bleomycin injection, in the same sites. Histopathological examination showed that RS-504393 treatment suppressed dermal fibrosis and decreased dermal thickness. The numbers of mast cells and myofibroblasts in the skin of RS-504393-treated mice were significantly lower compared with those in PBS-treated mice. Moreover, the amount of collagen in the skin of RS-504393-treated mice was significantly lower compared with that in the PBS-treated mice. Additionally, mRNA levels of TGF-ß1 and collagen I alpha 1 in sclerotic skin were significantly decreased by RS-504393, and semiquantitative histopathological scoring of the lungs showed inhibition of fibrosis in RS-504393-treated mice. The amount of collagen in the lung of the RS-504393-treated mice was lower compared with that in the PBS-treated mice. These data suggest that CCR2 antagonist RS-504393 may be a therapeutic agent for human scleroderma.

CCR2-Mediated Uptake of Constitutively Produced CCL2: A Mechanism for Regulating Chemokine Levels in the Blood.

C-C chemokine receptor 2 (CCR2) is a key driver of monocyte/macrophage trafficking to sites of inflammation and has long been considered a target for intervention in autoimmune disease. However, systemic administration of CCR2 antagonists is associated with marked increases in CCL2, a CCR2 ligand, in the blood. This heretofore unexplained phenomenon complicates interpretation of in vivo responses to CCR2 antagonism. We report that CCL2 elevation after pharmacological CCR2 blockade is due to interruption in a balance between CCL2 secretion by a variety of cells and its uptake by constitutive internalization and recycling of CCR2. We observed this phenomenon in response to structurally diverse CCR2 antagonists in wild-type mice, and also found substantially higher CCL2 plasma levels in mice lacking the CCR2 gene. Our findings suggest that CCL2 is cleared from blood in a CCR2-dependent but G protein (Gαi, Gαs or Gαq/11)-independent manner. This constitutive internalization is rapid: on a given monocyte, the entire cell surface CCR2 population is turned over in <30 minutes. We also found that constitutive receptor internalization/recycling and ligand uptake are not universal across monocyte-expressed chemokine receptors. For example, CXCR4 does not internalize constitutively. In summary, we describe a mechanism that explains the numerous preclinical and clinical reports of increased CCL2 plasma levels following in vivo administration of CCR2 antagonists. These findings suggest that constitutive CCL2 secretion by monocytes and other cell types is counteracted by constant uptake and internalization by CCR2-expressing cells. The effectiveness of CCR2 antagonists in disease settings may be dependent upon this critical equilibrium.

PDX regulates inflammatory cell infiltration via resident macrophage in LPS-induced lung injury.

Inflammatory cell infiltration contributes to the pathogenesis of acute respiratory distress syndrome (ARDS). Protectin DX (PDX), an endogenous lipid mediator, shows anti-inflammatory and proresolution bioactions. In vivo, the mice were intraperitoneally injected with PDX (0.1 µg/mouse) after intratracheal (1 mg/kg) or intraperitoneal (10 mg/kg) LPS administration. Flow cytometry was used to measure inflammatory cell numbers. Clodronate liposomes were used to deplete resident macrophages. RT-PCR, and ELISA was used to measure MIP-2, MCP-1, TNF-α and MMP9 levels. In vitro, sorted neutrophils, resident and recruited macrophages (1 × 106 ) were cultured with 1 µg/mL LPS and/or 100 nmol/L PDX to assess the chemokine receptor expression. PDX attenuated LPS-induced lung injury via inhibiting recruited macrophage and neutrophil recruitment through repressing resident macrophage MCP-1, MIP-2 expression and release, respectively. Finally, PDX inhibition of neutrophil infiltration and transmembrane was associated with TNF-α/MIP-2/MMP9 signalling pathway. These data suggest that PDX attenuates LPS-stimulated lung injury via reduction of the inflammatory cell recruitment mediated via resident macrophages.

CCL2 mediates early renal leukocyte infiltration during salt-sensitive hypertension.

Studies examining mechanisms of Dahl salt-sensitive (SS) hypertension have implicated the infiltration of leukocytes in the kidneys, which contribute to renal disease and elevated blood pressure. However, the signaling pathways by which leukocytes traffic to the kidneys remain poorly understood. The present study nominated a signaling pathway by analyzing a kidney RNA sequencing data set from SS rats fed either a low-salt (0.4% NaCl) diet or a high-salt (4.0% NaCl) diet. From this analysis, chemokine (C-C motif) ligand 2 (CCL2) and chemokine (C-C motif) receptor 2 (CCR2) were nominated as a potential pathway modifying renal leukocyte infiltration and contributing to SS hypertension. The functional role of the CCL2/CCR2 pathway was tested by daily administration of CCR2 antagonist (RS-102895 at 5 mg·kg-1·day-1 in DMSO) or DMSO vehicle for 3 or 21 days by intraperitoneal injections during the high salt challenge. Blood pressure, renal leukocyte infiltration, and renal damage were evaluated. The results demonstrated that RS-102895 treatment ameliorated renal damage (urinary albumin excretion; 43.4 ± 5.1 vs. 114.7 ± 15.2 mg/day in vehicle, P < 0.001) and hypertension (144.3 ± 2.2 vs. 158.9 ± 4.8 mmHg in vehicle, P < 0.001) after 21 days of high-salt diet. It was determined that renal leukocyte infiltration was blunted by day 3 of the high-salt diet (1.4 ± 0.1 vs. 1.9 ± 0.2 in vehicle × 106 CD45+ cells/kidney, P = 0.034). An in vitro chemotaxis assay validated the effect of RS-102895 on leukocyte chemotaxis toward CCL2. The results suggest that increased CCL2 in SS kidneys is important in the early recruitment of leukocytes, and blockade of this recruitment by administering RS-102895 subsequently blunted the renal damage and hypertension.

Chronic alcohol-induced neuroinflammation involves CCR2/5-dependent peripheral macrophage infiltration and microglia alterations.

BACKGROUND: Chronic alcohol consumption is associated with neuroinflammation, neuronal damage, and behavioral alterations including addiction. Alcohol-induced neuroinflammation is characterized by increased expression of proinflammatory cytokines (including TNFα, IL-1ß, and CCL2) and microglial activation. We hypothesized chronic alcohol consumption results in peripheral immune cell infiltration to the CNS. Since chemotaxis through the CCL2-CCR2 signaling axis is critical for macrophage recruitment peripherally and centrally, we further hypothesized that blockade of CCL2 signaling using the dual CCR2/5 inhibitor cenicriviroc (CVC) would prevent alcohol-induced CNS infiltration of peripheral macrophages and alter the neuroinflammatory state in the brain after chronic alcohol consumption. METHODS: C57BL/6J female mice were fed an isocaloric or 5% (v/v) ethanol Lieber DeCarli diet for 6 weeks. Some mice received daily injections of CVC. Microglia and infiltrating macrophages were characterized and quantified by flow cytometry and visualized using CX3CR1eGFP/+ CCR2RFP/+ reporter mice. The effect of ethanol and CVC treatment on the expression of inflammatory genes was evaluated in various regions of the brain, using a Nanostring nCounter inflammation panel. Microglia activation was analyzed by immunofluorescence. CVC-treated and untreated mice were presented with the two-bottle choice test. RESULTS: Chronic alcohol consumption induced microglia activation and peripheral macrophage infiltration in the CNS, particularly in the hippocampus. Treatment with CVC abrogated ethanol-induced recruitment of peripheral macrophages and partially reversed microglia activation. Furthermore, the expression of proinflammatory markers was upregulated by chronic alcohol consumption in various regions of the brain, including the cortex, hippocampus, and cerebellum. Inhibition of CCR2/5 decreased alcohol-mediated expression of inflammatory markers. Finally, microglia function was impaired by chronic alcohol consumption and restored by CVC treatment. CVC treatment did not change the ethanol consumption or preference of mice in the two-bottle choice test. CONCLUSIONS: Together, our data establish that chronic alcohol consumption promotes the recruitment of peripheral macrophages into the CNS and microglia alterations through the CCR2/5 axis. Therefore, further exploration of the CCR2/5 axis as a modulator of neuroinflammation may offer a potential therapeutic approach for the treatment of alcohol-associated neuroinflammation.

Pharmacological Inhibition of CCR2/5 Signaling Prevents and Reverses Alcohol-Induced Liver Damage, Steatosis, and Inflammation in Mice.

Kupffer cell and macrophage (MØ) activation contributes to steatosis, inflammation, and fibrosis in alcoholic liver disease (ALD). We found increased frequency of MØ, T cells, and expression of C-C chemokine receptor type 2 (Ccr2) and C-C chemokine receptor type 5 (Ccr5) in the livers of patients with ALD, and increased circulating chemokines, C-C chemokine ligand types 2 (CCL2), and C-C chemokine ligand types 5 (CCL5) in patients with alcoholic hepatitis. We hypothesized that inhibition of CCL2 signaling with the dual CCR2/5 inhibitor, cenicriviroc (CVC), would attenuate ALD. In a mouse model of ALD, liver injury (alanine aminotransferase [ALT]) and steatosis were prevented by CVC whether administered as "prevention" throughout the alcohol feeding or as "treatment" started after the development of ALD. Alcohol-induced increases in early liver fibrosis markers (sirius red, hydroxyproline, and collagen-1) were normalized by both modes of CVC administration. We found that prevention and treatment with CVC reversed alcohol-related increases in liver mRNA and protein expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, and CCL2. CVC administration regimens prevented the increase in infiltrating MØ (F4/80lo CD11bhi ) and reduced proinflammatory Ly6Chi MØ in livers of alcohol-fed mice. CVC increased liver T-cell numbers and attenuated Il-2 expression without an effect on CD69+ or CD25+ T-cell expression. In vitro, CVC inhibited CCL2-induced increases in hepatocyte fatty acid synthase (Fasn) and adipose differentiation-related protein (Adrp), whereas it augmented acyl-coenzyme A oxidase 1 (Acox-1), proliferator-activated receptor gamma co-activator alpha (Pgc1α) and uncoupling protein 2 expression, suggesting mechanisms for attenuated hepatocyte steatosis. We found that CCL2 and CCL5 sensitized hepatocytes to lipopolysaccharide-induced liver injury (TNF-α, ALT, and lactate dehydrogenase release). Alcohol feeding induced apoptosis (poly ADP-ribose polymerase [PARP] and caspase-3 [CASP-3] cleavage) and pyroptosis (gasdermin D [GSDMD] cleavage) in livers, and CVC prevented both of these forms of cell death. Conclusion: Together, our data demonstrate preclinical evidence for CCR2/CCR5 inhibition with CVC as a potent intervention to ameliorate alcohol-induced steatohepatitis and liver damage.

Mesenchymal stem/stromal cells stably transduced with an inhibitor of CC chemokine ligand 2 ameliorate bronchopulmonary dysplasia and pulmonary hypertension.

Perinatal bronchopulmonary dysplasia (BPD) is defined as lung injury in preterm infants caused by various factors, resulting in serious respiratory dysfunction and high mortality. The administration of mesenchymal stem/stromal cells (MSCs) to treat/prevent BPD has proven to have certain therapeutic effects. However, MSCs can only weakly regulate macrophage function, which is strongly involved in the development of BPD. 7ND-MSCs are MSCs transfected with 7ND, a truncated version of CC chemokine ligand 2 (CCL2) that promotes macrophage activation, using a lentiviral vector. In the present study, we show in a BPD rat model that 7ND-MSC administration, but not MSCs alone, ameliorated the impaired alveolarization evaluated by volume density and surface area in the lung tissue, as well as pulmonary artery remodeling and pulmonary hypertension induced by BPD. In addition, 7ND-MSCs, but not MSCs alone, reduced M1 macrophages and the messenger RNA expressions of interleukin-6 and CCL2 in the lung tissue. Thus, the present study showed the treatment effect of 7ND-MSCs in a BPD rat model, which was more effective than that of MSCs alone.