Antagonism of the Platelet-Activating Factor Pathway Mitigates Inflammatory Adverse Events Driven by Anti-erythrocyte Antibody Therapy in Mice.

Immune thrombocytopenia (ITP) is an autoimmune disease characterized by low platelet counts primarily due to antiplatelet autoantibodies. Anti-D is a donor-derived polyclonal Ab against the rhesus D Ag on erythrocytes used to treat ITP. Unfortunately, adverse inflammatory/hypersensitivity reactions and a Food and Drug Administration-issued black box warning have limited its clinical use. This underscores the imperative to understand the inflammatory pathway associated with anti-erythrocyte Ab-based therapies. TER119 is an erythrocyte-specific Ab with anti-D-like therapeutic activity in murine ITP, while also exhibiting a distinct inflammatory signature involving production of CCL2, CCL5, and CXCL9 but not IFN-γ. Therefore, TER119 has been used to elucidate the potential mechanism underlying the adverse inflammatory activity associated with anti-erythrocyte Ab therapy in murine ITP. Prior work has demonstrated that TER119 administration is associated with a dramatic decrease in body temperature and inflammatory cytokine/chemokine production. The work presented in the current study demonstrates that inhibiting the highly inflammatory platelet-activating factor (PAF) pathway with PAF receptor antagonists prevents TER119-driven changes in body temperature and inhibits the production of the CCL2, CCL5, and CXCL9 inflammatory cytokines in CD-1 mice. Phagocytic cells and a functional TER119 Fc region were found to be necessary for TER119-induced body temperature changes and increases in CXCL9 and CCL2. Taken together, this work reveals the novel requirement of the PAF pathway in causing adverse inflammatory activity associated with anti-erythrocyte Ab therapy in a murine model and provides a strategy of mitigating these potential reactions without altering therapeutic activity.

SHIV-C109p5 NHP induces rapid disease progression in elderly macaques with extensive GI viral replication.

CCR5-tropic simian/human immunodeficiency viruses (SHIV) with clade C transmitted/founder envelopes represent a critical tool for the investigation of HIV experimental vaccines and microbicides in nonhuman primates, although many such isolates lead to spontaneous viral control post infection. Here, we generated a high-titer stock of pathogenic SHIV-C109p5 by serial passage in two rhesus macaques (RM) and tested its virulence in aged monkeys. The co-receptor usage was confirmed before infecting five geriatric rhesus macaques (four female and one male). Plasma viral loads were monitored by reverse transcriptase-quantitative PCR (RT-qPCR), cytokines by multiplex analysis, and biomarkers of gastrointestinal damage by enzyme-linked immunosorbent assay. Antibodies and cell-mediated responses were also measured. Viral dissemination into tissues was determined by RNAscope. Intravenous SHIV-C109p5 infection of aged RMs leads to high plasma viremia and rapid disease progression; rapid decrease in CD4+ T cells, CD4+CD8+ T cells, and plasmacytoid dendritic cells; and wasting necessitating euthanasia between 3 and 12 weeks post infection. Virus-specific cellular immune responses were detected only in the two monkeys that survived 4 weeks post infection. These were Gag-specific TNFα+CD8+, MIP1ß+CD4+, Env-specific IFN-γ+CD4+, and CD107a+ T cell responses. Four out of five monkeys had elevated intestinal fatty acid binding protein levels at the viral peak, while regenerating islet-derived protein 3α showed marked increases at later time points in the three animals surviving the longest, suggesting gut antimicrobial peptide production in response to microbial translocation post infection. Plasma levels of monocyte chemoattractant protein-1, interleukin-15, and interleukin-12/23 were also elevated. Viral replication in gut and secondary lymphoid tissues was extensive.IMPORTANCESimian/human immunodeficiency viruses (SHIV) are important reagents to study prevention of virus acquisition in nonhuman primate models of HIV infection, especially those representing transmitted/founder (T/F) viruses. However, many R5-tropic SHIV have limited fitness in vivo leading to many monkeys spontaneously controlling the virus post acute infection. Here, we report the generation of a pathogenic SHIV clade C T/F stock by in vivo passage leading to sustained viral load set points, a necessity to study pathogenicity. Unexpectedly, administration of this SHIV to elderly rhesus macaques led to extensive viral replication and fast disease progression, despite maintenance of a strict R5 tropism. Such age-dependent rapid disease progression had previously been reported for simian immunodeficiency virus but not for R5-tropic SHIV infections.

Essential role of the CCL2-CCR2 axis in Mayaro virus-induced disease.

Mayaro virus (MAYV) is an emerging arbovirus member of the Togaviridae family and Alphavirus genus. MAYV infection causes an acute febrile illness accompanied by persistent polyarthralgia and myalgia. Understanding the mechanisms involved in arthritis caused by alphaviruses is necessary to develop specific therapies. In this work, we investigated the role of the CCL2/CCR2 axis in the pathogenesis of MAYV-induced disease. For this, wild-type (WT) C57BL/6J and CCR2-/- mice were infected with MAYV subcutaneously and evaluated for disease development. MAYV infection induced an acute inflammatory disease in WT mice. The immune response profile was characterized by an increase in the production of inflammatory mediators, such as IL-6, TNF, and CCL2. Higher levels of CCL2 at the local and systemic levels were followed by the significant recruitment of CCR2+ macrophages and a cellular response orchestrated by these cells. CCR2-/- mice showed an increase in CXCL-1 levels, followed by a replacement of the macrophage inflammatory infiltrate by neutrophils. Additionally, the absence of the CCR2 receptor protected mice from bone loss induced by MAYV. Accordingly, the silencing of CCL2 chemokine expression in vivo and the pharmacological blockade of CCR2 promoted a partial improvement in disease. Cell culture data support the mechanism underlying the bone pathology of MAYV, in which MAYV infection promotes a pro-osteoclastogenic microenvironment mediated by CCL2, IL-6, and TNF, which induces the migration and differentiation of osteoclast precursor cells. Overall, these data contribute to the understanding of the pathophysiology of MAYV infection and the identification future of specific therapeutic targets in MAYV-induced disease.IMPORTANCEThis work demonstrates the role of the CCL2/CCR2 axis in MAYV-induced disease. The infection of wild-type (WT) C57BL/6J and CCR2-/- mice was associated with high levels of CCL2, an important chemoattractant involved in the recruitment of macrophages, the main precursor of osteoclasts. In the absence of the CCR2 receptor, there is a mitigation of macrophage migration to the target organs of infection and protection of these mice against bone loss induced by MAYV infection. Much evidence has shown that host immune response factors contribute significantly to the tissue damage associated with alphavirus infections. Thus, this work highlights molecular and cellular targets involved in the pathogenesis of arthritis triggered by MAYV and identifies novel therapeutic possibilities directed to the host inflammatory response unleashed by MAYV.

The primary macrophage chemokine, CCL2, is not necessary after a peripheral nerve injury for macrophage recruitment and activation or for conditioning lesion enhanced peripheral regeneration.

BACKGROUND: Peripheral nerve injuries stimulate the regenerative capacity of injured neurons through a neuroimmune phenomenon termed the conditioning lesion (CL) response. This response depends on macrophage accumulation in affected dorsal root ganglia (DRGs) and peripheral nerves. The macrophage chemokine CCL2 is upregulated after injury and is allegedly required for stimulating macrophage recruitment and pro-regenerative signaling through its receptor, CCR2. In these tissues, CCL2 is putatively produced by neurons in the DRG and Schwann cells in the distal nerve. METHODS: Ccl2fl/fl mice were crossed with Advillin-Cre, P0-Cre, or both to create conditional Ccl2 knockouts (CKOs) in sensory neurons, Schwann cells, or both to hypothetically remove CCL2 and macrophages from DRGs, nerves or both. CCL2 was localized using Ccl2-RFPfl/fl mice. CCL2-CCR2 signaling was further examined using global Ccl2 KOs and Ccr2gfp knock-in/knock-outs. Unilateral sciatic nerve transection was used as the injury model, and at various timepoints, chemokine expression, macrophage accumulation and function, and in vivo regeneration were examined using qPCR, immunohistochemistry, and luxol fast blue staining. RESULTS: Surprisingly, in all CKOs, DRG Ccl2 gene expression was decreased, while nerve Ccl2 was not. CCL2-RFP reporter mice revealed CCL2 expression in several cell types beyond the expected neurons and Schwann cells. Furthermore, macrophage accumulation, myelin clearance, and in vivo regeneration were unaffected in all CKOs, suggesting CCL2 may not be necessary for the CL response. Indeed, Ccl2 global knockout mice showed normal macrophage accumulation, myelin clearance, and in vivo regeneration, indicating these responses do not require CCL2. CCR2 ligands, Ccl7 and Ccl12, were upregulated after nerve injury and perhaps could compensate for the absence of Ccl2. Finally, Ccr2gfp knock-in/knock-out animals were used to differentiate resident and recruited macrophages in the injured tissues. Ccr2gfp/gfp KOs showed a 50% decrease in macrophages in the distal nerve compared to controls with a relative increase in resident macrophages. In the DRG there was a small but insignificant decrease in macrophages. CONCLUSIONS: CCL2 is not necessary for macrophage accumulation, myelin clearance, and axon regeneration in the peripheral nervous system. Without CCL2, other CCR2 chemokines, resident macrophage proliferation, and CCR2-independent monocyte recruitment can compensate and allow for normal macrophage accumulation.

Kaempferol reduces obesity, prevents intestinal inflammation, and modulates gut microbiota in high-fat diet mice.

Kaempferol, a flavonoid identified in a wide variety of dietary sources, has been reported to possess anti-obesity properties; however, its underlying mechanism was poorly understood. Chronic, low-grade gut inflammation and dysbacteria are proposed as underlying factors as well as novel treatment approaches for obesity-associated pathologies. This present study aims to investigate the benefits of experimental treatment with kaempferol on intestinal inflammation and gut microbial balance in animal model of obesity. High fat diet (HFD) was applied to C57BL/6J mice for 16 weeks, during which the supplement of kaempferol served as a variable. Clearly, HFD induced obesity, fat accumulation, glucose intolerance and adipose inflammation, the metabolic syndrome of which was the main finding. All these metabolic disorders can be alleviated through kaempferol supplementation. In addition, increased intestinal permeability, infiltration of immunocytes (macrophage, dendritic cells and neutrophils) and overexpression of inflammatory cytokines (tumor necrosis factor-alpha, interleukin-1beta, interleukin-6, monocyte chemoattractant protein-1) were also found in the HFD-induced mice. Kaempferol supplementation improved intestinal barrier integrity and inhibited gut inflammation, by reducing the activation of TLR4/NF-κB pathway. Furthermore, the characterization of the cecal microbiota by sequencing showed that kaempferol supplementation was able to counteract the dysbiosis associated to obesity. Our study delineated the multiple mechanism of action underlying the anti-obesity effect of kaempferol, and provide scientific evidence to support the development of kaempferol as a dietary supplement for obesity treatment.

Targeting the CCL2/CCR2 Axis in Cancer Immunotherapy: One Stone, Three Birds?

CCR2 is predominantly expressed by monocytes/macrophages with strong proinflammatory functions, prompting the development of CCR2 antagonists to dampen unwanted immune responses in inflammatory and autoimmune diseases. Paradoxically, CCR2-expressing monocytes/macrophages, particularly in tumor microenvironments, can be strongly immunosuppressive. Thus, targeting the recruitment of immunosuppressive monocytes/macrophages to tumors by CCR2 antagonism has recently been investigated as a strategy to modify the tumor microenvironment and enhance anti-tumor immunity. We present here that beneficial effects of CCR2 antagonism in the tumor setting extend beyond blocking chemotaxis of suppressive myeloid cells. Signaling within the CCL2/CCR2 axis shows underappreciated effects on myeloid cell survival and function polarization. Apart from myeloid cells, T cells are also known to express CCR2. Nevertheless, tissue homing of Treg cells among T cell populations is preferentially affected by CCR2 deficiency. Further, CCR2 signaling also directly enhances Treg functional potency. Thus, although Tregs are not the sole type of T cells expressing CCR2, the net outcome of CCR2 antagonism in T cells favors the anti-tumor arm of immune responses. Finally, the CCL2/CCR2 axis directly contributes to survival/growth and invasion/metastasis of many types of tumors bearing CCR2. Together, CCR2 links to two main types of suppressive immune cells by multiple mechanisms. Such a CCR2-assoicated immunosuppressive network is further entangled with paracrine and autocrine CCR2 signaling of tumor cells. Strategies to target CCL2/CCR2 axis as cancer therapy in the view of three types of CCR2-expessing cells in tumor microenvironment are discussed.

High endogenous CCL2 expression promotes the aggressive phenotype of human inflammatory breast cancer.

Inflammatory Breast Cancer (IBC) is a highly aggressive malignancy with distinct clinical and histopathological features whose molecular basis is unresolved. Here we describe a human IBC cell line, A3250, that recapitulates key IBC features in a mouse xenograft model, including skin erythema, diffuse tumor growth, dermal lymphatic invasion, and extensive metastases. A3250 cells express very high levels of the CCL2 chemokine and induce tumors enriched in macrophages. CCL2 knockdown leads to a striking reduction in macrophage densities, tumor proliferation, skin erythema, and metastasis. These results establish IBC-derived CCL2 as a key factor driving macrophage expansion, and indirectly tumor growth, with transcriptomic analysis demonstrating the activation of multiple inflammatory pathways. Finally, primary human IBCs exhibit macrophage infiltration and an enriched macrophage RNA signature. Thus, this human IBC model provides insight into the distinctive biology of IBC, and highlights potential therapeutic approaches to this deadly disease.

Fetuin-A regulates adipose tissue macrophage content and activation in insulin resistant mice through MCP-1 and iNOS: involvement of IFNγ-JAK2-STAT1 pathway.

In the context of obesity-induced adipose tissue (AT) inflammation, migration of macrophages and their polarization from predominantly anti-inflammatory to proinflammatory subtype is considered a pivotal event in the loss of adipose insulin sensitivity. Two major chemoattractants, monocyte chemoattractant protein-1 (MCP-1) and Fetuin-A (FetA), have been reported to stimulate macrophage migration into inflamed AT instigating inflammation. Moreover, FetA could notably modulate macrophage polarization, yet the mechanism(s) is unknown. The present study was undertaken to elucidate the mechanistic pathway involved in the actions of FetA and MCP-1 in obese AT. We found that FetA knockdown in high fat diet (HFD) fed mice could significantly subdue the augmented MCP-1 expression and reduce adipose tissue macrophage (ATM) content thereby indicating that MCP-1 is being regulated by FetA. Additionally, knockdown of FetA in HFD mice impeded the expression of inducible nitric oxide synthase (iNOS) reverting macrophage activation from mostly proinflammatory to anti-inflammatory state. It was observed that the stimulating effect of FetA on MCP-1 and iNOS was mediated through interferon γ (IFNγ) induced activation of JAK2-STAT1-NOX4 pathway. Furthermore, we detected that the enhanced IFNγ expression was accounted by the stimulatory effect of FetA upon the activities of both cJun and JNK. Taken together, our findings revealed that obesity-induced FetA acts as a master upstream regulator of AT inflammation by regulating MCP-1 and iNOS expression through JNK-cJun-IFNγ-JAK2-STAT1 signaling pathway. This study opened a new horizon in understanding the regulation of ATM content and activation in conditions of obesity-induced insulin resistance.

HCV Core Protein Induces Chemokine CCL2 and CXCL10 Expression Through NF-κB Signaling Pathway in Macrophages.

HCV core protein is the first structural protein synthesized during hepatitis C virus (HCV) infection and replication. It is released from virus infected liver cells and mediates multiple functions to affect host cell response. The innate immune response is the first line of defense against viral infection. After HCV infection, Kupffer cells (KCs) which are liver macrophages play an important role in host innate immune response. Kupffer cells act as phagocytes and release different cytokines and chemokines to counter viral infection and regulate inflammation and fibrosis in liver. Earlier, we have demonstrated that HCV core protein interacts with gC1qR and activates MAPK, NF-κB and PI3K/AKT pathways in macrophages. In this study, we explored the effect of HCV core protein on CCL2 and CXCL10 expression in macrophages and the signaling pathways involved. Upon silencing of gC1qR, we observed a significant decrease expression of CCL2 and CXCL10 in macrophages in the presence of HCV core protein. Inhibiting NF-κB pathway, but not P38, JNK, ERK and AKT pathways greatly reduced the expression of CCL2 and CXCL10. Therefore, our results indicate that interaction of HCV core protein with gC1qR could induce CCL2 and CXCL10 secretion in macrophages via NF-κB signaling pathway. These findings may shed light on the understanding of how leukocytes migrate into the liver and exaggerate host-derived immune responses and may provide novel therapeutic targets in HCV chronic inflammation.

Porcine Reproductive and Respiratory Syndrome Virus nsp11 Antagonizes Broad Antiviral Effects of MCPIP1 by Inducing Interleukin-17 Expression.

Monocyte chemotactic protein-induced protein 1 (MCPIP1) is an inflammatory regulator in immune response and has broad antiviral effects by targeting viral RNA. Porcine reproductive and respiratory syndrome virus (PRRSV), a major viral pathogen in pigs, causes immune suppression leading to coinfection of swine pathogens, but the mechanisms are not fully clarified. In this study, MCPIP1 expression was found to be significantly upregulated in lungs of PRRSV-infected piglets, as well as in Marc-145 and porcine pulmonary alveolar macrophage (PAM) cells upon PRRSV stimulation. MCPIP1 overexpression significantly inhibited PRRSV replication, while MCPIP1 knockdown increased the virus titer. Various mutations in RNase functional domains of MCPIP1 impaired the inhibitory activity against PRRSV, while those in deubiquitinase domains failed to do so. MCPIP1 expression started to decrease from 60 h after PRRSV infection in PAMs. Meanwhile, infection with higher dose of PRRSV further downregulated MCPIP1, indicating the antagonizing effects from PRRSV against MCPIP1. Moreover, it was confirmed that MCPIP1 expression was downregulated in 3D4 cells with either interleukin-17 (IL-17) or nsp11 overexpression, while IL-17 inhibitor abolished the decrease of MCPIP1 caused by nsp11, indicating nsp11 employs IL-17 induction to inhibit MCPIP1. Furthermore, the PRRSV nsp11 mutant with a deficiency in IL-17 induction showed the recovered expression of MCPIP1 in infected cells, inspiring a strategy for virus attenuation. This is the first report about the role of MCPIP1 against PRRSV and the function of PRRSV nsp11 against innate immunity to facilitate virus replication via IL-17. The study not only illuminates PRRSV infection machinery but also enlightens alternative antiviral strategies, such as vaccine candidates. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) suppresses the innate immunity and leads to coinfection of swine pathogens. Monocyte chemotactic protein-induced protein 1 (MCPIP1) is a broad-spectrum host antiviral protein. Therefore, to further clarify the mechanism of PRRSV against innate immunity, we explored the relationship between MCPIP1 and PRRSV infection. The results showed that MCPIP1 inhibited PRRSV infection in the early stage of virus infection. Importantly, PRRSV nsp11 subsequently employed IL-17 induction to suppress MCPIP1 expression and antagonized anti-PRRSV effects. Furthermore, PRRSV with mutation of nsp11 S74A failed to induce MCPIP1 reduction. These findings confirmed the function of MCPIP1 against PRRSV and revealed that PRRSV nsp11 plays an important role in virus against innate immunity. This study enlightens a new strategy to develop safer attenuated vaccines against PRRSV by nsp11 mutation.

Germacranolides from Carpesium divaricatum: Some New Data on Cytotoxic and Anti-Inflammatory Activity.

Carpesium divaricatum Sieb. & Zucc., a traditional medicinal plant used as an inflammation-relieving remedy, is a rich source of terpenoids. At least 40 germacrane-type sesquiterpene lactones, representatives of four different structural groups, were isolated from the plant. Cytotoxicity against cancer cells in vitro is the most frequently described biological activity of the compounds. However, little is known about the selectivity of the cytotoxic effect. The anti-inflammatory activity of the germacranolides is also poorly documented. The objective of the present study was to assess the cytotoxic activity of selected C. divaricatum germacranolides-derivatives of 4,5,8,9-tetrahydroxy-3-oxo-germacran-6,12-olide towards cancer and normal cell lines (including cells of different p53 status). Moreover, to assess the anti-inflammatory effect of the compounds, the release of four proinflammatory cytokines/chemokines (IL-1ß, IL-8, TNF-α and CCL2) by lipopolysaccharide-stimulated human neutrophils was measured by ELISA. The investigated sesquiterpene lactones demonstrated nonselective activity towards prostate cancer (Du145 and PC3) and normal prostate epithelial cells (PNT2) as well as against melanoma cells (A375 and HTB140) and keratinocytes (HaCaT). Cytotoxic activity against osteosarcoma cells was independent of their p53 status. In sub-cytotoxic concentrations (0.5-2.5 µM) the studied compounds significantly decreased cytokine/chemokine release by lipopolysaccharide-stimulated human leukocytes.

Roles of CCL2-CCR2 Axis in the Tumor Microenvironment.

Chemokines are a small family of cytokines that were first discovered as chemotactic factors in leukocytes during inflammation, and reports on the relationship between chemokines and cancer progression have recently been increasing. The CCL2-CCR2 axis is one of the major chemokine signaling pathways, and has various functions in tumor progression, such as increasing tumor cell proliferation and invasiveness, and creating a tumor microenvironment through increased angiogenesis and recruitment of immunosuppressive cells. This review discusses the roles of the CCL2-CCR2 axis and the tumor microenvironment in cancer progression and their future roles in cancer therapy.

MCP-1: Function, regulation, and involvement in disease.

MCP-1 (Monocyte chemoattractant protein-1), also known as Chemokine (CC-motif) ligand 2 (CCL2), is from family of CC chemokines. It has a vital role in the process of inflammation, where it attracts or enhances the expression of other inflammatory factors/cells. It leads to the advancement of many disorders by this main mechanism of migration and infiltration of inflammatory cells like monocytes/macrophages and other cytokines at the site of inflammation. MCP-1 has been inculpated in the pathogenesis of numerous disease conditions either directly or indirectly like novel corona virus, cancers, neuroinflammatory diseases, rheumatoid arthritis, cardiovascular diseases. The elevated MCP-1 level has been observed in COVID-19 patients and proven to be a biomarker associated with the extremity of disease along with IP-10. This review will focus on involvement and role of MCP-1 in various pathological conditions.

Ex vivo mass cytometry analysis reveals a profound myeloid proinflammatory signature in psoriatic arthritis synovial fluid.

OBJECTIVES: A number of immune populations have been implicated in psoriatic arthritis (PsA) pathogenesis. This study used mass cytometry (CyTOF) combined with transcriptomic analysis to generate a high-dimensional dataset of matched PsA synovial fluid (SF) and blood leucocytes, with the aim of identifying cytokine production ex vivo in unstimulated lymphoid and myeloid cells. METHODS: Fresh SF and paired blood were either fixed or incubated with protein transport inhibitors for 6 hours. Samples were stained with two CyTOF panels: a phenotyping panel and an intracellular panel, including antibodies to both T cell and myeloid cell secreted proteins. Transcriptomic analysis by gene array of key expanded cell populations, single-cell RNA-seq, ELISA and LEGENDplex analysis of PsA SF were also performed. RESULTS: We observed marked changes in the myeloid compartment of PsA SF relative to blood, with expansion of intermediate monocytes, macrophages and dendritic cell populations. Classical monocytes, intermediate monocytes and macrophages spontaneously produced significant levels of the proinflammatory mediators osteopontin and CCL2 in the absence of any in vitro stimulation. By contrast minimal spontaneous cytokine production by T cells was detected. Gene expression analysis showed the genes for osteopontin and CCL2 to be among those most highly upregulated by PsA monocytes/macrophages in SF; and both proteins were elevated in PsA SF. CONCLUSIONS: Using multiomic analyses, we have generated a comprehensive cellular map of PsA SF and blood to reveal key expanded myeloid proinflammatory modules in PsA of potential pathogenic and therapeutic importance.

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

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

Potential Role of Macrophage Phenotypes and CCL2 in the Pathogenesis of Takayasu Arteritis.

Objectives: To investigate vascular macrophage phenotype as well as vascular and peripheral chemokine (C-C motif) ligand 2 (CCL2) expression during different stages of disease progression in patients with Takayasu Arteritis (TA). Methods: In this study, 74 patients with TA and 50 controls were recruited. TA disease activity was evaluated with Kerr scores. Macrophage phenotype and CCL2 expression were examined by immunohistochemistry in vascular specimens from 8 untreated and 7 treated TA patients, along with 4 healthy controls. Serum CCL2 were quantified by enzyme-linked immune-absorbent assay from TA patients at baseline (n=59), at 6-months (n=38), and from 46 healthy volunteers. Vascular macrophage phenotype, vascular CCL2 expression and serum CCL2 levels during different stages, as well as the relationship between serum CCL2 and disease activity or other inflammatory parameters (erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and interleukin 6 (IL-6)) were investigated. Results: In untreated patients, vascular M1 macrophages and CCL2 showed increased expression, mainly in the adventitia. In contrast, in treated patients, vascular adventitial M1 and CCL2 expression were decreased, while vascular medial M2 macrophages and CCL2 levels were increased. Distribution of macrophages and CCL2 was consistent within the TA vascular lesions regardless of the disease stage. Furthermore, peripheral CCL2 was elevated in patients with TA (TA: 160.30 ± 120.05 vs. Control: 65.58 ± 54.56 pg/ml, P < 0.001). CCL2 levels were found to correlate with ESR, CRP, and IL-6 (all R values between 0.55 and 0.6, all P < 0.001). Receiver operating curve analysis demonstrated that CCL2 (at the cut-off value of 100.36 pg/ml) was able to predict disease activity (area under the curve = 0.74, P = 0.03). Decrease in CCL2 level was observed in patients with clinical remission (CR), but not in patients without CR, after 6 months of treatment (CR patients: baseline 220.18 ± 222.69 vs. post-treatment 88.71 ± 55.89 pg/ml, P = 0.04; non-CR patients: baseline 142.45 ± 104.76 vs. post-treatment 279.49 ± 229.46 pg/ml, P = 0.02). Conclusions: Macrophages contribute to vascular pathological changes in TA by undergoing phenotype transformation. CCL2 is an important factor for recruiting macrophages and a potential biomarker for disease activity.

Schistosoma mansoni infection is associated with decreased risk of respiratory allergy symptoms and low production of CCL2.

OBJECTIVES: We measured the production of cytokines, chemokines and antibodies involved in allergic responses and sCD23 levels during Schistosoma mansoni infection. METHODS: Individuals (n = 164) were selected using the ISAAC questionnaire and parasitological exams. The subjects were divided as follows: those infected individuals with allergy-related symptoms (A-I), those with allergy-related symptoms only (A-NI); those only infected (NA-I); and those non-infected individuals without allergy-related symptoms (NA-NI). We used supernatants from cell culture (mitogenic stimulation) to measure cytokine and chemokine levels using cytometric bead arrays. Serum levels of anti-Ascaris lumbricoides (Asc) and anti-Blomia tropicalis IgE were measured using ImmunoCAP, and sCD23 was measured using ELISA. RESULTS: Schistosoma mansoni infection was associated with a lower risk of allergy-related symptoms. In A-I, there were higher levels of TNF-α, IL-10, IL-6, IFN-γ and CXCL8 than in NA-NI group, with TNF-α and IL-6 also at higher levels compared to A-NI group. Levels of IL-6, CXCL8, total and anti-Asc IgE, as well as the numbers of eosinophils, were higher in NA-I than in NA-NI, and the antibodies were also lower in A-NI than in NA-I group. In AI and NA-I, there was less production of CCL2 than in NA-NI. There were no differences in the levels of IL-2, IL-4, IL-17, CCL5, sCD23 and anti-Blomia IgE. CONCLUSIONS: Patients with allergy-related symptoms and infected (simultaneously) had higher levels of IL-10; due to the infection, there was increased production of IL-6 and CXCL8 and less CCL2. These data may characterize deviation to Th1 or attenuation of the Th2 response in allergy sufferers in areas endemic for schistosomiasis.

The multifaceted roles of the chemokines CCL2 and CXCL12 in osteophilic metastatic cancers.

Breast and prostate cancers have a great propensity to metastasize to long bones. The development of bone metastases is life-threatening, incurable, and drastically reduces patients' quality of life. The chemokines CCL2 and CXCL12 and their respective receptors, CCR2 and CXCR4, are central instigators involved in all stages leading to cancer cell dissemination and secondary tumor formation in distant target organs. They orchestrate tumor cell survival, growth and migration, tumor invasion and angiogenesis, and the formation of micrometastases in the bone marrow. The bone niche is of particular importance in metastasis formation, as it expresses high levels of CCL2 and CXCL12, which attract tumor cells and contribute to malignancy. The limited number of available effective treatment strategies highlights the need to better understand the pathophysiology of bone metastases and reduce the skeletal tumor burden in patients diagnosed with metastatic bone disease. This review focuses on the involvement of the CCL2/CCR2 and CXCL12/CXCR4 chemokine axes in the formation and development of bone metastases, as well as on therapeutic perspectives aimed at targeting these chemokine-receptor pairs.

IL-6 deficiency promotes colitis by recruiting Ly6Chi monocytes into inflamed colon tissues in a CCL2-CCR2-dependent manner.

Interleukin 6 (IL-6) is a pleiotropic cytokine that is elevated in inflammatory bowel disease. However, the role of IL-6 deficiency in colitis is not well-defined. Some IL-6 and IL-6 receptor antagonists are associated with severe gastrointestinal immune adverse effects, but the mechanisms of the effects are not clear. This study aimed to investigate the effect of IL-6 in ulcerative colitis in Il6-/- mice. Results indicated that physiological deficiency of IL-6 promoted the development of colitis. Moreover, IL-6 deficiency significantly increased the mRNA levels of monocytes chemokine Ccl2 and its receptor Ccr2 in colon tissues. Similarly, the percentage of Ly6Chigh monocytes and neutrophils were increased in the colon of Il6-/- mice. Intestinal crypts more strongly increased the migration of Il6-/- macrophages than wild-type ones. Moreover, Il6-/- macrophages promoted the migration of neutrophils. Most importantly, RS102895, an antagonist of CCR2, diminished chemotaxis of macrophages and inhibited colitis in Il6-/- mice. Collectively, these results indicate that Il6-/- macrophages migrate to inflamed colon tissues and recruit neutrophils, thereby promoting the effect of Il6-/- on colitis. This study expands our understanding on the effect of IL-6 deficiency in colitis and the development of gastrointestinal immune adverse effects.

Loss of Speckle-Type POZ Protein Promotes Prostate Cancer Cell Migration and Invasion Through Upregulation of MCP-1.

BACKGROUND The goal of this study is to verify that the loss of speckle-type POZ protein (SPOP) promotes the migration and invasion of prostate cancer cells, and that this process is brought about by an increase in MCP-1. MATERIAL AND METHODS SPOP knockout C4-2 cells (C4-2 SPOP-/-) were verified by western blotting. Transwell and wound-healing assays were applied to verify different migration and invasion abilities between the C4-2 SPOP-/- and control cells. We used an antibody array to find different soluble chemokine factors in the C4-2 SPOP-/- cells. ELISA and qRT-PCR were applied for confirmation. To test MCP-1 function in conditioned medium, a transwell assay was applied with or without anti-MCP-1 antibody. RESULTS The western blot showed that SPOP was knocked out in sgSPOP-1 and sgSPOP-2 (different clones of C4-2 SPOP-/-). The transwell and wound-healing assays indicated that, compared with control cells, sgSPOP-1 and sgSPOP-2 had stronger migration and invasion abilities. The antibody array found that the expression of MCP-1 was upregulated in sgSPOP-1 and sgSPOP-2 conditioned medium. This result was verified by ELISA and qRT-PCR. In the prostate cancer cells, migration and invasion activity was greatly increased in C4-2 SPOP-/- conditioned medium, while this activity was decreased after anti-MCP-1 antibody neutralization. CONCLUSIONS Our findings suggest that the loss of SPOP in C4-2 cells promotes increased cell migration and invasion abilities. This may be realized by upregulating the expression of MCP-1. The inhibition of MCP-1 expression may be an effective treatment for SPOP-mutant prostate cancer.