CCL2 chemokine inhibition primes the tumor vasculature for improved nanomedicine delivery and efficacy.

Blood vessel functionality is crucial for efficient tumor-targeted drug delivery. Heterogeneous distribution and perfusion of angiogenic blood vessels contribute to suboptimal accumulation of (nano-) therapeutics in tumors and metastases. To attenuate pathological angiogenesis, an L-RNA aptamer inhibiting the CC motif chemokine ligand 2 (CCL2) was administered to mice bearing orthotopic 4T1 triple-negative breast cancer tumors. The effect of CCL2 inhibition on tumor blood vessel functionality and tumor-targeted drug delivery was evaluated via multimodal and multiscale optical imaging, employing fluorophore-labeled polymeric (10 nm) and liposomal (100 nm) nanocarriers. Anti-CCL2 treatment induced a dose-dependent anti-angiogenic effect, reflected by a decreased relative blood volume, increased blood vessel maturity and functionality, and reduced macrophage infiltration, accompanied by a shift in the polarization of tumor-associated macrophages (TAM) towards a less M2-like and more M1-like phenotype. In line with this, CCL2 inhibitor treatment improved the delivery of polymers and liposomes to tumors, and enhanced the antitumor efficacy of free and liposomal doxorubicin. Together, these findings demonstrate that blocking the CCL2-CCR2 axis modulates TAM infiltration and polarization, resulting in vascular normalization and improved tumor-targeted drug delivery.

CCL2 Knockdown Attenuates Inflammatory Response After Spinal Cord Injury Through the PI3K/Akt Signaling Pathway: Bioinformatics Analysis and Experimental Validation.

Spinal cord injury (SCI) is a common clinical problem in orthopedics with a lack of effective treatments and drug targets. In the present study, we performed bioinformatic analysis of SCI datasets GSE464 and GSE45006 in the Gene Expression Omnibus (GEO) public database and experimentally validated CCL2 expression in an animal model of SCI. This was followed by stimulation of PC-12 cells using hydrogen peroxide to construct a cellular model of SCI. CCL2 expression was knocked down using small interfering RNA (si-CCL2), and PI3K signaling pathway inhibitors and activators were used to validate and observe the changes in downstream inflammation. Through data mining, we found that the inflammatory chemokine CCL2 and PI3K/Akt signaling pathways after SCI expression were significantly increased, and after peroxide stimulation of PC-12 cells with CCL2 knockdown, their downstream cellular inflammatory factor levels were decreased. The PI3K/Akt signaling pathway was blocked by PI3K inhibitors, and the downstream inflammatory response was suppressed. In contrast, when PI3K activators were used, the inflammatory response was enhanced, indicating that the CCL2-PI3K/Akt signaling pathway plays a key role in the regulation of the inflammatory response. This study revealed that the inflammatory chemokine CCL2 can regulate the inflammatory response of PC-12 cells through the PI3K/Akt signaling pathway, and blocking the expression of the inflammatory chemokine CCL2 may be a promising strategy for the treatment of secondary injury after SCI.

IDO Regulates Macrophage Functions by Inhibiting the CCL2/CCR2 Signaling Pathway in Fungal Keratitis.

PURPOSE: The aim of this study was to investigate the effects of indoleamine 2,3-dioxygenase (IDO) on macrophage polarization, phagocytosis, and killing through regulation of the CCL2/CCR2 signaling pathway in Aspergillus fumigatus keratitis. METHODS: In vivo and in vitro experiments were conducted in mice and mouse peritoneal macrophages after infection with A. fumigatus . Clinical scoring, reverse transcription-polymerase chain reaction, and immunofluorescence staining were used to evaluate the fungal keratitis lesions, macrophage-related cytokines, and macrophage recruitment. The expression of CCL2 and CCR2 was detected by reverse transcription-polymerase chain reaction and western blot after pretreatment with or without an IDO inhibitor (1-MT). After pretreatment with 1-MT, a CCR2 antagonist, a CCL2 neutralizing antibody, an IDO agonist (IFNG), and recombinant CCL2 protein (CCL2), the flow cytometry and colony-forming unit counts were used to detect the polarization, phagocytosis, and killing function. RESULTS: Compared with the control group, the infected eyes showed increased clinical scores, macrophage-related cytokine expression, and macrophage recruitment. 1-MT pretreatment increased the expression of CCL2 and CCR2 and the proportion of CD206+/CD86+ macrophages; macrophages polarized toward the M2 type, with enhanced killing function. CCR2 antagonists and CCL2 neutralizing antibodies reversed the effects of 1-MT. Compared with the infected group, IFNG pretreatment decreased the proportion of CD206+/CD86+ macrophages, and macrophages polarized toward the M1 type, with decreased phagocytosis and impaired killing function. CCL2 reversed the effect of IFNG. CONCLUSIONS: IDO can promote the polarization of macrophages to the M1 type by blocking the CCL2/CCR2 signaling pathway, inhibiting the phagocytosis and killing function of macrophages, and mediating the protective immune role of A. fumigatus .

Identification of the CCL2 PI3K/Akt axis involved in autophagy and apoptosis after spinal cord injury.

Spinal cord injury (SCI) is a devastating neurological disease with no cure that usually results in irreversible loss of sensory and voluntary motor functions below the injury site. We conducted an in-depth bioinformatics analysis combining the gene expression omnibus spinal cord injury database and the autophagy database and found that the expression of the autophagy gene CCL2 was significantly upregulated and the PI3K/Akt/mTOR signaling pathway was activated after SCI. The results of the bioinformatics analysis were verified by constructing animal and cellular models of SCI. We then used small interfering RNA to inhibit the expression of CCL2 and PI3K to inhibit and activate the PI3K/Akt/mTOR signaling pathway; western blot, immunofluorescence, monodansylcadaverine, and cell flow techniques were used to detect the expression of key proteins involved in downstream autophagy and apoptosis. We found that when PI3K inhibitors were activated, apoptosis decreased, the levels of autophagy-positive proteins LC3-I/LC3-II and Bcl-1 increased, the levels of autophagy-negative protein P62 decreased, the levels of pro-apoptotic proteins Bax and caspase-3 decreased, the levels of the apoptosis-inhibiting protein Bcl-2 increased. In contrast, when a PI3K activator was used, autophagy was inhibited, and apoptosis was increased. This study revealed the effect of CCL2 on autophagy and apoptosis after SCI through the PI3K/Akt/mTOR signaling pathway. By blocking the expression of the autophagy-related gene CCL2, the autophagic protective response can be activated, and apoptosis can be inhibited, which may be a promising strategy for the treatment of SCI.

CD40×HER2 bispecific antibody overcomes the CCL2-induced trastuzumab resistance in HER2-positive gastric cancer.

BACKGROUND: There was much hard work to study the trastuzumab resistance in HER2-positive gastric cancer (GC), but the information which would reveal this abstruse mechanism is little. In this study, we aimed to investigate the roles of tumor cell-derived CCL2 on trastuzumab resistance and overcome the resistance by treatment with the anti-CD40-scFv-linked anti-HER2 (CD40 ×HER2) bispecific antibody (bsAb). METHODS: We measured the levels of CCL2 expression in HER2-positive GC tissues, and revealed biological functions of tumor cell-derived CCL2 on tumor-associated macrophages (TAMs) and the trastuzumab resistance. Then, we developed CD40 ×HER2 bsAb, and examined the targeting roles on HER2 and CD40, to overcome the trastuzumab resistance without systemic toxicity. RESULTS: We found the level of CCL2 expression in HER2-postive GC was correlated with infiltration of TAMs, polarization status of infiltrated TAMs, trastuzumab resistance and survival outcomes of GC patients. On exposure to CCL2, TAMs decreased the M1-like phenotype, thereby eliciting the trastuzumab resistance. CCL2 activated the transcription of ZC3H12A, which increased K63-linked deubiquitination and K48-linked auto-ubiquitination of TRAF6/3 to inactivate NF-κB signaling in TAMs. CD40 ×HER2 bsAb, which targeted the CD40 to restore the ubiquitination level of TRAF6/3, increased the M1-like phenotypic transformation of TAMs, and overcame trastuzumab resistance without immune-related adversary effects (irAEs). CONCLUSIONS: We revealed a novel mechanism of trastuzumab resistance in HER2-positive GC via the CCL2-ZC3H12A-TRAF6/3 signaling axis, and presented a CD40 ×HER2 bsAb which showed great antitumor efficacy with few irAEs.

Crosstalk between ß-Catenin and CCL2 Drives Migration of Monocytes towards Glioblastoma Cells.

Isocitrate dehydrogenase (IDH)-wildtype glioblastoma (GBM) is a fast growing and highly heterogeneous tumor, often characterized by the presence of glioblastoma stem cells (GSCs). The plasticity of GSCs results in therapy resistance and impairs anti-tumor immune response by influencing immune cells in the tumor microenvironment (TME). Previously, ß-catenin was associated with stemness in GBM as well as with immune escape mechanisms. Here, we investigated the effect of ß-catenin on attracting monocytes towards GBM cells. In addition, we evaluated whether CCL2 is involved in ß-catenin crosstalk between monocytes and tumor cells. Our analysis revealed that shRNA targeting ß-catenin in GBMs reduces monocytes attraction and impacts CCL2 secretion. The addition of recombinant CCL2 restores peripheral blood mononuclear cells (PBMC) migration towards medium (TCM) conditioned by shß-catenin GBM cells. CCL2 knockdown in GBM cells shows similar effects and reduces monocyte migration to a similar extent as ß-catenin knockdown. When investigating the effect of CCL2 on ß-catenin activity, we found that CCL2 modulates components of the Wnt/ß-catenin pathway and alters the clonogenicity of GBM cells. In addition, the pharmacological ß-catenin inhibitor MSAB reduces active ß-catenin, downregulates the expression of associated genes and alters CCL2 secretion. Taken together, we showed that ß-catenin plays an important role in attracting monocytes towards GBM cells in vitro. We hypothesize that the interactions between ß-catenin and CCL2 contribute to maintenance of GSCs via modulating immune cell interaction and promoting GBM growth and recurrence.

The anti-inflammatory peptide Catestatin blocks chemotaxis.

Increased levels of the anti-inflammatory peptide Catestatin (CST), a cleavage product of the pro-hormone chromogranin A, correlate with less severe outcomes in hypertension, colitis, and diabetes. However, it is unknown how CST reduces the infiltration of monocytes and macrophages (Mϕs) in inflamed tissues. Here, it is reported that CST blocks leukocyte migration toward inflammatory chemokines. By in vitro and in vivo migration assays, it is shown that although CST itself is chemotactic, it blocks migration of monocytes and neutrophils to inflammatory attracting factor CC-chemokine ligand 2 (CCL2) and C-X-C motif chemokine ligand 2 (CXCL2). Moreover, it directs CX3 CR1+ Mϕs away from pancreatic islets. These findings suggest that the anti-inflammatory actions of CST are partly caused by its regulation of chemotaxis.

Chemokine (C-C motif) ligand 2-enhanced adipogenesis and angiogenesis of human adipose-derived stem cell and human umbilical vein endothelial cell co-culture system in adipose tissue engineering.

Human adipose-derived stem cells (hADSCs) and human umbilical vein endothelial cells (HUVECs) co-cultured in vitro are widely used in adipose tissue engineering but exhibit various limitations. Chemokine (C-C motif) ligand 2 (CCL2) has been proved essential during adipogenesis and angiogenesis in vivo. We examined whether adipogenesis and angiogenesis could also be directly promoted by CCL2 in vitro. Cells were cultured with 0, 10, 50, and 100 ng/ml CCL2. The effects of CCL2 on adipogenesis of hADSCs, and lipid accumulation in the positive control group (hADSCs), blank control group (hADSCs + HUVECs), and experimental group (hADSCs + HUVECs + CCL2) in the hADSC and HUVEC direct co-culture system were evaluated by Oil Red O staining. Angiogenesis in the presence of CCL2 was evaluated by Matrigel tube formation assay. Angiogenic- and adipogenic-associated gene and protein expression in the co-culture system were measured by Quantitative Real-time Polymerase Chain Reaction and western blotting, respectively. All concentrations of CCL2 promoted hADSC adipogenic differentiation and HUVEC tube formation (P < 0.05). Following direct co-culture, the experimental group accumulated more lipid droplets than the positive control (P < 0.0001), whereas the latter showed better adipogenesis than the blank control group. 50 ng/ml CCL2 exhibited stronger adipogenic and angiogenic potential than other concentrations. After 72 h of direct co-culture, the mRNA expression of adipogenic differentiation (peroxisome proliferators-activated receptorsγ, CCAAT/enhancer binding protein-α, Leptin, and lipoprotein lipase) and angiogenic genes (vascular endothelial growth factor-A, vascular endothelial growth factor receptor 2, matrix metalloprotein (MMP) 9, and 14) in the experimental group was much higher than in the control (P < 0.05). The addition of 50 ng/ml CCL2 in the system resulted in elevated phosphorylated Protein kinase B/AKT expression. In summary, CCL2 directly promoted adipogenesis of hADSCs and angiogenesis of HUVECs under both mono-culture and co-culture condition in vitro possibly by enhancing AKT phosphorylation. An optimal concentration of 50 ng/ml CCL2 could improve the adipogenesis and angiogenesis of hADSC and HUVEC co-culture system.

Adropin decreases endothelial monolayer permeability after cell-free hemoglobin exposure and reduces MCP-1-induced macrophage transmigration.

BACKGROUND: Cell-free heme-containing proteins mediate endothelial injury in a variety of disease states including subarachnoid hemorrhage and sepsis by increasing endothelial permeability. Inflammatory cells are also attracted to sites of vascular injury by monocyte chemotactic protein 1 (MCP-1) and other chemokines. We have identified a novel peptide hormone, adropin, that protects against hemoglobin-induced endothelial permeability and MCP-1-induced macrophage migration. METHODS: Human microvascular endothelial cells were exposed to cell-free hemoglobin (CFH) with and without adropin treatment before measuring monolayer permeability using a FITC-dextran tracer assay. mRNA and culture media were collected for molecular studies. We also assessed the effect of adropin on macrophage movement across the endothelial monolayer using an MCP-1-induced migration assay. RESULTS: CFH exposure decreases adropin expression and increases paracellular permeability of human endothelial cells. Treating cells with synthetic adropin protects against the increased permeability observed during the natural injury progression. Cell viability was similar in all groups and Hmox1 expression was not affected by adropin treatment. MCP-1 potently induced macrophage migration across the endothelial monolayer and adropin treatment effectively reduced this phenomenon. CONCLUSIONS: Endothelial injury is a hallmark of many disease states. Our results suggest that adropin treatment could be a valuable strategy in preventing heme-mediated endothelial injury and macrophage infiltration. Further investigation of adropin therapy in animal models and human tissue specimens is needed.

Central Nervous System (CNS) Viral Seeding by Mature Monocytes and Potential Therapies To Reduce CNS Viral Reservoirs in the cART Era.

The human immunodeficiency virus (HIV) enters the central nervous system (CNS) within a few days after primary infection, establishing viral reservoirs that persist even with combined antiretroviral therapy (cART). We show that monocytes from people living with HIV (PLWH) on suppressive cART harboring integrated HIV, viral mRNA, and/or viral proteins preferentially transmigrate across the blood-brain barrier (BBB) to CCL2 and are significantly enriched post-transmigration, and even more highly enriched posttransmigration than T cells with similar properties. Using HIV-infected ART-treated mature monocytes cultured in vitro, we recapitulate these findings and demonstrate that HIV+ CD14+ CD16+ ART-treated monocytes also preferentially transmigrate. Cenicriviroc and anti-JAM-A and anti-ALCAM antibodies significantly and preferentially reduce/block transmigration of HIV+ CD14+ CD16+ ART-treated monocytes. These findings highlight the importance of monocytes in CNS HIV reservoirs and suggest targets to eliminate their formation and reseeding.IMPORTANCE We characterized mechanisms of CNS viral reservoir establishment/replenishment using peripheral blood mononuclear cells (PBMC) of PLWH on cART and propose therapeutic targets to reduce/block selective entry of cells harboring HIV (HIV+) into the CNS. Using DNA/RNAscope, we show that CD14+ CD16+ monocytes with integrated HIV, transcriptionally active, and/or with active viral replication from PBMC of PLWH prescribed cART and virally suppressed, selectively transmigrate across a human BBB model. This is the first study to our knowledge demonstrating that monocytes from PLWH with HIV disease for approximately 22 years and with long-term documented suppression can still carry virus into the CNS that has potential to be reactivated and infectious. This selective entry into the CNS-and likely other tissues-indicates a mechanism of reservoir formation/reseeding in the cART era. Using blocking studies, we propose CCR2, JAM-A, and ALCAM as targets on HIV+ CD14+ CD16+ monocytes to reduce and/or prevent CNS reservoir replenishment and to treat HAND and other HIV-associated comorbidities.

Transcriptome Profiling of Human Monocyte-Derived Macrophages Upon CCL2 Neutralization Reveals an Association Between Activation of Innate Immune Pathways and Restriction of HIV-1 Gene Expression.

Macrophages are key targets of human immunodeficiency virus type 1 (HIV-1) infection and main producers of the proinflammatory chemokine CC chemokine ligand 2 (CCL2), whose expression is induced by HIV-1 both in vitro and in vivo. We previously found that CCL2 neutralization in monocyte-derived macrophages (MDMs) strongly inhibited HIV-1 replication affecting post-entry steps of the viral life cycle. Here, we used RNA-sequencing to deeply characterize the cellular factors and pathways modulated by CCL2 blocking in MDMs and involved in HIV-1 replication restriction. We report that exposure to CCL2 neutralizing antibody profoundly affected the MDM transcriptome. Functional annotation clustering of up-regulated genes identified two clusters enriched for antiviral defense and immune response pathways, comprising several interferon-stimulated, and restriction factor coding genes. Transcripts in the clusters were enriched for RELA and NFKB1 targets, suggesting the activation of the canonical nuclear factor κB pathway as part of a regulatory network involving miR-155 up-regulation. Furthermore, while HIV-1 infection caused small changes to the MDM transcriptome, with no evidence of host defense gene expression and type I interferon signature, CCL2 blocking enabled the activation of a strong host innate response in infected macrophage cultures, and potently inhibited viral genes expression. Notably, an inverse correlation was found between levels of viral transcripts and of the restriction factors APOBEC3A (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 A), ISG15, and MX1. These findings highlight an association between activation of innate immune pathways and HIV-1 restriction upon CCL2 blocking and identify this chemokine as an endogenous factor contributing to the defective macrophage response to HIV-1. Therapeutic targeting of CCL2 may thus strengthen host innate immunity and restrict HIV-1 replication.

Effects of adipokine administration to the hypothalamic preoptic area on body temperature in rats.

The effects of adipokine administration to the hypothalamic preoptic area (POA), which is one of the body temperature (BT) regulation centers in the central nervous system, on BT were investigated in male Wistar rats. BT was measured in conscious rats using telemetry. Insulin-like growth factor-1 (IGF-1), interleukin-1ß (IL-1ß), monocyte chemoattractant protein-1 and lipocalin-2 produced hyperthermia, and the effects induced by IL-1ß (25 ng) and IGF-1 (5 µg) were sustainable and remarkable. IL-6 did not show any significant effect. The IGF-1-induced effect was inhibited by pretreatment with IGF binding protein 3 (IGFBP3) or NVP-AEW541 (NVP, a selective inhibitor of type 1 IGF receptor tyrosine kinase, IGF1R TK). NVP-induced inhibition was observed only in the early phase of IGF-1-induced hyperthermia. In addition, IGF-1 increased the IL-1ß concentration in the microdialysate of POA perfusion, but did not increase the IL-1ß concentration in the plasma or the PGE2 concentration in the microdialysate. These findings suggested that IGF-1 produced hyperthermia, which was mediated, at least a part, through an increased IL-1ß concentration after activation of IGF1R TK in the POA, and the IGF-IGFBP system possibly participates in BT homeostasis in the POA.

7ND protein exerts inhibitory effects on both osteoclast differentiation in vitro and lipopolysaccharide­induced bone erosion in vivo.

Excessive numbers of osteoclasts are responsible for inflammation­induced osteolysis. Identification of osteoclast­targeting agents may facilitate the development of a novel therapeutic approach for the treatment of pathological bone loss. Seven­amino acid truncated (7ND) protein, a mutant form of monocyte chemoattractant protein­1 (MCP­1), functions as a competitive inhibitor of MCP­1. However, the effects of 7ND protein on osteoclast differentiation remain unknown. Therefore, in the present study, the effects of 7ND protein on osteoclast differentiation induced by tumour necrosis factor superfamily member 11 were investigated. In the present study, 7ND protein inhibited the osteoclast differentiation of peripheral blood mononuclear cells without influencing cell proliferation. Furthermore, to evaluate the effects of 7ND protein in vivo, a lipopolysaccharide (LPS)­induced calvarial bone erosion animal model was established. The 7ND protein remarkably attenuated LPS­induced bone resorption, as assessed by micro­computed tomography and histological analysis. Taken together, the present results suggested the feasibility of local delivery of 7ND protein to mitigate osteoclast differentiation and LPS­induced osteolysis, which may represent a potential approach to treat inflammatory bone destruction.

The enhancement of CCL2 and CCL5 by human bone marrow-derived mesenchymal stem/stromal cells might contribute to inflammatory suppression and axonal extension after spinal cord injury.

Human bone marrow-derived mesenchymal stem/stromal cells (hMSCs) have shown potential in facilitating recovery from spinal cord injury (SCI) through communicating with microglia/macrophages (MG/MΦ). We here focused on chemokines as a candidate for the communication. Selected MG/MΦ-related chemokines were determined gene expression after SCI and further focused CCL2/CCR2 and CCL5/CCR5 to estimate role of the chemokines by hMSCs. Male C57/BL6 mice were subjected to spinal cord transection. Gene expression was assayed in the spinal cords following SCI for selected MG/MΦ-related chemokines and their receptors. hMSCs (5×105 cells) were then transplanted into parenchyma of the spinal cord, and the expressions of the Ccl2/Ccr2 and Ccl5/Ccr5 axes, inflammation, MG/MΦ-polarization, and axonal regeneration were evaluated to measure the influence of the hMSCs. Finally, mouse CCL5 was injected into the spinal cords. Acute increases in gene expression after SCI were observed for most chemokines, including Ccl2; chronic increases were observed for Ccl5. CCL2+-cells merged with NeuN+-neurons. CCR2+ immunoreactivity was principally observed in Ly-6G+/iNOS+-granulocytes on postoperative day (pod) 1, and CCL5+ and CCR5+ immunoreactivity overlapped with NeuN+-neurons and F4/80+-MG/MΦ on pod 14. The hMSC transplantation enhanced Ccl2 and Ccl5 and improved locomotor activity. The hMSC implantation did not alter the number of Ly-6G+/CCR2+ but decreased Il1, Elane, and Mpo on pod 3. Conversely, hMSC transplantation increased expression of Zc3h12a (encodes MCP-1-induced protein) on pod 14. Moreover, hMSC increased the Aif1, and two alternatively activated macrophage (AAM)-related genes, Arg1 and Chil3 (Ym1), as well as axonal regenerative markers, Dpysl2 and Gap43. Gene expression indicative of AAM polarization and axonal regeneration were partially recovered by CCL5 injection. These results suggest that hMSC implantation increases Ccl2 and Ccl5, improves locomotor activity, enhances MG/MΦ polarization to AAM, and increases the gene expression of axonal regenerative markers. These functions of hMSCs might be partially mediated by the CCL2/CCR2 and CCL5/CCR5 axes.

Distinct release strategies are required to modulate macrophage phenotype in young versus aged animals.

The role of innate immunity and macrophages in the host response to biomaterials has received renewed attention. A context-dependent spectrum of macrophage phenotypes are shown to affect tissue integration and performance of implanted biomaterials and medical devices. Recent studies by our group demonstrated that the host response in aged animals was characterized by delayed macrophage recruitment, differences in marker expression and a shifted pro-inflammatory (M1) response, associated with an unresolved host response in the long-term. The present work sought to study the effects of single and sequential cytokine delivery regimens in aged mice to restore delayed recruitment of macrophages and shift the inflammatory host response towards an M2-like phenotype, using MCP-1 (macrophage chemotactic protein-1) and IL-4 (interleukin-4), respectively. Implantation of cytokine-eluting implants showed a preserved response to MCP-1 in both young and aged animals, restoring delayed macrophage recruitment in aged mice. However, the response elicited by IL-4, sequential delivery of MCP-1/IL-4 and coating components was distinct in young versus aged mice. While single delivery of IL-4 did not counteract the high inflammatory response observed in aged mice, the sequential delivery of MCP-1/IL-4 was capable of restoring both recruitment and shifting the macrophage response towards an M2-like phenotype, associated with decreased implant scarring in the long-term. In young mice, sequential delivery was not as effective as IL-4 alone at promoting an M2-like response, but did result in a reduction of M1 macrophages and capsule deposition downstream. These results demonstrate that a proper understanding of patient/context-dependent biological responses are needed to design biomaterial-based therapies with improved outcomes in the setting of aging.

PI3K/Akt signaling pathway may be involved in MCP-1-induced P2X4R expression in cultured microglia and cancer-induced bone pain rats.

P2X4 receptor (P2X4R), a subtype of P2 purinergic receptors, is an ATP-gated receptor through which activity of spinal microglia instigates pain hypersensitivity in various pain conditions. Accumulating evidence indicates that monocyte chemoattractant protein-1 (MCP-1) plays an important role in chronic pain facilitation, and it could stimulate microglia activation and involve in regulating P2X4R expression. However, the mechanism of MCP-1 in regulating the expression of P2X4R in microglia is poorly understood, and whether MCP-1 can aggravate pain via up-regulating spinal P2X4R expression in Cancer-induced Bone Pain (CIBP) remains unclear. In this study, we observed that Iba-1 and P2X4R expression is increased in microglia treated with MCP-1, and blockade with a selective CCR2 antagonist RS-504393 suppressed microglia activation and reduced P2X4R expression in cultured microglia. In response to MCP-1, the expression level of p-Akt was also increased and RS-504393 inhibited the increase. Besides, PI3K inhibitor LY 294002 could attenuate MCP-1-induced P2X4R expression in cultured microglia. MCP-1 was found to be associated with P2X4R expression and mechanical allodynia induced by CIBP in vivo since the expression of MCP-1 was increased in CIBP and RS-504393 alleviated the P2X4R expression and mechanical allodynia in CIBP. Moreover, RS-504393 also reduced the increase of p-Akt induced by CIBP. Inhibition of PI3K/Akt pathway may partly reduce MCP-1/CCR2-induced expression of P2X4R and mechanical allodynia in CIBP rats.

CCL2 induces resistance to the antiproliferative effect of cabazitaxel in prostate cancer cells.

Understanding the mechanism of chemoresistance and disease progression in patients with prostate cancer is important for developing novel treatment strategies. In particular, developing resistance to cabazitaxel is a major challenge in patients with docetaxel-resistant and castration-resistant prostate cancer (CRPC) because cabazitaxel is often administered as a last resort. However, the mechanism by which cabazitaxel resistance develops is still unclear. C-C motif chemokine ligands (CCL) were shown to contribute to the castration resistance of prostate cancer cells via an autocrine mechanism. Therefore, we focused on CCL as key factors of chemoresistance in prostate cancer cells. We previously established a cabazitaxel-resistant cell line, DU145-TxR/CxR, from a previously established paclitaxel-resistant cell line, DU145-TxR. cDNA microarray analysis revealed that the expression of CCL2 was upregulated in both DU145-TxR and DU145-TxR/CxR cells compared with DU145 cells. The secreted CCL2 protein level in DU145-TxR and DU145-TxR/CxR cells was also higher than in parental DU145 cells. The stimulation of DU145 cells with CCL2 increased the proliferation rate under treatments with cabazitaxel, and a CCR2 (a specific receptor of CCL2) antagonist suppressed the proliferation of DU145-TxR and DU145-TxR/CxR cells under treatments of cabazitaxel. The CCL2-CCR2 axis decreased apoptosis through the inhibition of caspase-3 and poly(ADP-ribose) polymerase (PARP). CCL2 is apparently a key contributor to cabazitaxel resistance in prostate cancer cells. Inhibition of the CCL2-CCR2 axis may be a potential therapeutic strategy against chemoresistant CRPC in combination with cabazitaxel.

Co-delivery of RNAi and chemokine by polyarginine nanocapsules enables the modulation of myeloid-derived suppressor cells.

Myeloid-Derived Suppressor Cells (MDSCs), immunosuppressive cells that promote tumor growth, represent an attractive target in cancer immunotherapy. However, the clinical success of this strategy is limited by the lack of efficient drug delivery vehicles targeting this cell compartment. The objective of this work was to develop a delivery carrier, multilayer polymer nanocapsules, with the capacity to co-encapsulate two types of immunomodulatory drugs, a chemokine and an RNAi sequence, aimed at reverting MDSC-mediated immunosuppression. The chemokine CCL2, intended to attract monocyte-macrophage MDSCs, was encapsulated within the L2 inverse micellar aqueous domains of the lipid core of these nanocapsules. On the other hand, two different RNAi sequences that modulate the CCAAT/enhancer-binding protein beta (C/EBPß) pathway, shC/EBPß and miR 142-3p, were successfully associated to their polymer shell. These RNAi sequences were covered by subsequent layers of polyarginine and hyaluronic acid, thereby creating multi-layered assemblies that protected them and facilitated their targeted delivery. The in vitro studies performed in primary MDSCs cultures showed the capacity of miR 142-3p-loaded nanocapsules to reduce the highly immunosuppressive monocyte-macrophage subset. Additionally, the encapsulation of CCL2 within the nanocapsules induced a potent monocyte-macrophage chemoattraction that could be used to direct the therapy to these cell subsets. Finally, in vitro and in vivo studies showed the capacity of shC/EBPß-loaded nanocapsules to downregulate C/EBPß levels in MDSCs and to reduce monocyte differentiation into tumor-associated macrophages in an MCA-203 fibrosarcoma mice model. In conclusion, the multilayer polymer nanocapsules described here are efficient vehicles for the co-delivery of proteins and RNA, and are potential candidates as nanomedicines for the modulation of MDSCs.

Matrix metalloproteinases (MMPs) mediate leukocyte recruitment during the inflammatory phase of zebrafish heart regeneration.

In zebrafish, the role of matrix metalloproteinases (MMPs) in the inflammatory phase of heart regeneration following cryoinjury remains poorly understood. Here, we demonstrated an increase in MMP enzymatic activity and elevated expression of mmp9 and mmp13 in the injured area (IA) of hearts from as early as 1 day post-cryoinjury (dpc). Treatment with the broad-spectrum MMP inhibitor, GM6001, during the first week after cryoinjury resulted in impaired heart regeneration, as indicated by the larger scar and reduced numbers of proliferating cardiomyocytes. GM6001 also significantly reduced the number of leukocytes to the IA at 0.5 dpc to 4 dpc. Specific inhibition of both MMP-9 and MMP-13 also resulted in impaired regeneration and leukocyte recruitment. However, chemokine rescue with recombinant CXCL8 and CCL2 restored the recruitment of macrophages and the cardiac regenerative capability in GM6001-treated fish. MMP-9 and MMP-13 cleaved zebrafish CXCL8 at the same site, and the truncated form was more chemotactic than the intact form. In contrast, CCL2 did not have an MMP-9 or MMP-13 cleavage site. Together, these data suggest that MMPs might play a key role in the inflammatory phase of heart regeneration in zebrafish, by mediating leukocyte recruitment via the activation of chemokines.

MCP-1 Feedback Loop Between Adipocytes and Mesenchymal Stromal Cells Causes Fat Accumulation and Contributes to Hematopoietic Stem Cell Rarefaction in the Bone Marrow of Patients With Diabetes.

Fat accumulates in bone marrow (BM) of patients with diabetes. In this study, we investigated the mechanisms and consequences of this phenomenon. BM mesenchymal stromal cells (BM-MSCs) from patients with type 2 diabetes (T2D) constitutively express adipogenic markers and robustly differentiate into adipocytes (ADs) upon in vitro induction as compared with BM-MSCs from subjects without diabetes. Moreover, BM-ADs from subjects with T2D (T2D BM-ADs) paracrinally stimulate a transcriptional adipogenic program in BM-MSCs. Antagonism of MCP-1, a chemokine pivotally expressed in T2D BM-ADs, prevented the T2D BM-AD secretome from converting BM-MSCs into ADs. Mechanistic validation of human data was next performed in an obese T2D mouse model. Systemic antagonism of MCP-1 improved metabolic control, reduced BM fat, and increased osteocyte density. It also indirectly re-established the abundance of long-term versus short-term hematopoietic stem cells. We reveal a diabetic feedback loop in which 1) BM-MSCs are constitutively inclined to make ADs, and 2) mature BM-ADs, via secreted MCP-1, relentlessly fuel BM-MSC determination into new fat. Pharmacological inhibition of MCP-1 signaling can contrast this vicious cycle, restoring, at least in part, the balance between adipogenesis and hematopoiesis in BM from subjects with T2D.