Correlation of HMGB1, PON-1, MCP-1, and Periodontal P. gingivalis with Amniotic Fluid Fecal Dye.

BACKGROUND: This paper aims to investigate the correlation between high mobility group protein-1 (HMG-b1), antioxidant enzyme-1 (paraoxon-1, PON-1), monocyte chemoattractant protein-1 (monocyte chemoattractant protein-1, MCP-1), P. gingivalis, and MSAF. MATERIALS AND METHODS: The total sample size comprised of 73 cases in both groups. These patients were further subdivided into 2 groups: the MSAF group and the control group. 38 women were in the MSAF group and 35 women with term amniotic fluid serum were in the control group. The MSAF group was selected as a full-term singleton amniotic fluid fecal infection group. Clinical data were collected, and specimens were collected. Fecal staining of amniotic fluid and full-term amniotic fluid removes the placenta and umbilical cord blood. The expression of HMGB1 in the placenta was observed by immune-histochemical staining of MSAF and control groups. The content of PON-1 in cord blood was determined by ELISA. RESULTS: Correlation between maternal and neonatal clinical data and MSAF was done; MSAF group mean gestational age was 41.38 ± 1.40 weeks; control group mean gestational age was 39.20 ± 1.24 weeks. This study found no correlation between the birth weight, maternal age, sex, first/transmaternal, hyperthyroidism, hypothyroidism, and anemia between the MSAF and control group with nonsignificant P value (P > 0.05). However, the fatal age, gestational diabetes, gestational hypertension, umbilical cord abnormalities, placental abnormalities, and neonatal asphyxia factors were statistically different with a significant P value of <0.05 between both groups. HMGB1 and Periodontal P. gingivalis are mostly expressed in placental trophoblast, vascular endothelial cells, and amniotic epithelial and interstitial cells. After HE staining of 72 placentas by HE in MSAF and control, 6 had acute chorioamnionitis (5.1 control), 32 had chronic (23.9), 35 had abnormal placentas, and three in MSAF had chorionic columnar metaplasia. In immune-histochemistry experiments, the HMGB1 expression intensity of placental tissue was higher in the MSAF group (P < 0.05); however, the level of PON-1 was lower in the MSAF group as compared to the controls (P < 0.05). CONCLUSIONS: Gestational age and placental abnormalities are clinical high-risk factors for MSAF. HMGB1, PON-1, MCP-1, and Periodontal P. gingivalis may be involved in the development of MSAF, suggesting an oxidative/antioxidant imbalance with inflammation, and may be one of the mechanisms for MSAF development.

Quercetin for COVID-19 and DENGUE co-infection: a potential therapeutic strategy of targeting critical host signal pathways triggered by SARS-CoV-2 and DENV.

BACKGROUND: The clinical consequences of SARS-CoV-2 and DENGUE virus co-infection are not promising. However, their treatment options are currently unavailable. Current studies have shown that quercetin is both resistant to COVID-19 and DENGUE; this study aimed to evaluate the possible functional roles and underlying mechanisms of action of quercetin as a potential molecular candidate against COVID-19 and DENGUE co-infection. METHODS: We used a series of bioinformatics analyses to understand and characterize the biological functions, pharmacological targets and therapeutic mechanisms of quercetin in COVID-19 and DENGUE co-infection. RESULTS: We revealed the clinical characteristics of COVID-19 and DENGUE, including pathological mechanisms, key inflammatory pathways and possible methods of intervention, 60 overlapping targets related to the co-infection and the drug were identified, the protein-protein interaction (PPI) was constructed and TNFα, CCL-2 and CXCL8 could become potential drug targets. Furthermore, we disclosed the signaling pathways, biological functions and upstream pathway activity of quercetin in COVID-19 and DENGUE. The analysis indicated that quercetin could inhibit cytokines release, alleviate excessive immune responses and eliminate inflammation, through NF-κB, IL-17 and Toll-like receptor signaling pathway. CONCLUSIONS: This study is the first to reveal quercetin as a pharmacological drug for COVID-19 and DENGUE co-infection. COVID-19 and DENGUE co-infection remain a potential threat to the world's public health system. Therefore, we need innovative thinking to provide admissible evidence for quercetin as a potential molecule drug for the treatment of COVID-19 and DENGUE, but the findings have not been verified in actual patients, so further clinical drug trials are needed.

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.

Dissecting the differential structural and dynamics features of CCL2 chemokine orthologs.

Chemokines are a sub-group of cytokines that regulate the leukocyte migration. Monocyte chemoattractant protein-1 (MCP/CCL2) is one of the essential CC chemokine that regulates the migration of monocytes into inflamed tissues. It has been observed that the primary sequences of CCL2 orthologs among rodents and primates vary significantly at the C-terminal region. However, no structural details are available for the rodentia family CCL2 proteins. The current study unravelled the structural, dynamics and in-silico functional characteristics of murine CCL2 chemokine using a comprehensive set of NMR spectroscopy techniques and evolutionary approaches. The study unravelled that the N-terminal portion of the murine CCL2 forms a canonical CC chemokine dimer similar to that of human CCL2. However, unlike human CCL2, the murine ortholog exhibits extensive dynamics in the µs-ms timescales. The presence of C-terminal region of the murine CCL2 protein/rodentia family is highly glycosylated, completely disordered, and inhibits the folding of the structured CCL2 regions. Further, it has been observed that the glycosaminoglycan binding surfaces of these orthologs proteins are greatly differed. In a nut shell, this comparative study provided the role of molecular evolution in generating orthologous proteins with differential structural and dynamics characteristics to engage them in specific molecular interactions.

A self-assembled peptide hydrogel for cytokine sequestration.

Cytokine-directed monocyte infiltration is involved in multiple pathological processes. Immuno-isolating matrices that can sequester cell-released chemokines in a microenvironment may prolong the viability and functionality of implanted materials. We describe a self-assembling peptide-based hydrogel that can capture the cytokine CCL2 released in the extracellular space by immune cells and stromal cells. The shear-responsive matrix can absorb and retain this signaling molecule needed for the chemotaxis of the infiltrating monocytes and their differentiation into phagocytic macrophages. Such cytokine-sequestering biomaterials may be useful as adjunctive materials with the delivery of exogenous implants or cell suspensions for tissue regeneration, without the administration of systemic immunosuppressants. Our work highlights the versatility of nanofibrous peptide hydrogels for modulating the biological response in tissue niches.

Elevated Synovial Fluid Concentration of Monocyte Chemoattractant Protein-1 and Interleukin-8 in Dogs with Osteoarthritis of the Stifle.

Chemokines such as monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) have been shown to cause monocyte and natural killer cell chemotaxis and polymorphonuclear cell chemotaxis, respectively. Additionally, MCP-1 signalling has been implicated in modulating pain. Elevated synovial fluid concentrations of MCP-1 and IL-8 have been demonstrated in humans with osteoarthritis, but currently there are no studies evaluating synovial MCP-1 or IL-8 concentrations in dogs. Additionally, there are no canine studies evaluating the correlation between these chemokines and caregiver perceived pain and mobility, as measured by the clinical metrology instrument, Liverpool Osteoarthritis in Dogs. This study documented elevated synovial fluid concentrations of IL-8 and MCP-1 in the stifle of dogs with secondary osteoarthritis compared with normal stifles. However, this study found no correlation between MCP-1 or IL-8 and Liverpool Osteoarthritis in Dogs or radiographic severity of osteoarthritis.

A sensitive sandwich-type immunosensor for the detection of MCP-1 based on a rGO-TEPA-Thi-Au nanocomposite and novel RuPdPt trimetallic nanoalloy particles.

A novel electrochemical immunosensor was proposed for the detection of monocyte chemoattractant protein-1 (MCP-1), a biomarker of cardiovascular disease. Due to thionine (Thi) possessing electroactive redox properties, a one-step approach was utilized to synthesize a reduced graphene oxide-tetraethylene-thionine-Au (rGO-TEPA-Thi-Au) nanocomposite at room temperature using the synergistic effect of Thi and rGO-TEPA towards HAuCl4. We obtained the excellent matrix material, which immobilized more primary antibody MCP-1-Ab1 on rGO-TEPA on a modified glassy carbon electrode (GCE). To further enhance the sensitivity of the sensor, a novel signal generation and amplification strategy was developed for detection. RuPdPt trimetallic nanoalloy particles (RuPdPt TNPs), a novel nanomaterial, were synthesized by a one-pot method, displayed a uniform morphology as well as good electrochemical activity and bound with the secondary antibodies against MCP-1 via the Pt-NH2 bond. Most importantly, RuPdPt TNPs have a significant ability to catalyze H2O2 to produce an electron. The electrochemical signal was highly amplified because the electrochemical signal was primarily derived from the synergistic catalysis of H2O2 by RuPdPt TNPs and recorded by chronoamperometry. Under the optimal conditions, this newly designed biosensor exhibited sensitive detection of MCP-1 in the range from 20 fg mL-1 to 1000 pg mL-1, with a detection limit of 8.9 fg mL-1 (based on a S/N = 3). Additionally, the designed immunosensor showed acceptable selectivity, reproducibility and stability. This immunosensor is a promising strategy for analyzing clinical serum samples in the future.

Resistin Enhances Monocyte Chemoattractant Protein-1 Production in Human Synovial Fibroblasts and Facilitates Monocyte Migration.

BACKGROUND/AIMS: The adipocyte-secreting adipokine, resistin, may play a critical role in the modulation of inflammatory diseases. Migration and infiltration of mononuclear cells into inflammatory sites are critical events during the development of osteoarthritis (OA). Monocyte chemoattractant protein-1 (MCP-1), also known as chemokine ligand 2 (CCL2), plays a critical role in the regulation of monocyte migration and infiltration. In this study, we show how resistin promotes MCP-1 expression in OA synovial fibroblasts and monocyte migration. METHODS: We used qPCR to detect MCP-1 and miRNA expression. THP-1 migration was investigated by Transwell assay. The Western blotting was used to examine the resistinmediated signaling pathways. RESULTS: Resistin activated the phosphatidylinositol-3-kinase (PI3K), Akt and mammalian target of rapamycin (mTOR) signaling pathways, while PI3K, Akt and mTOR inhibitors or small interfering RNAs diminished resistin-induced MCP-1 expression and monocyte migration. We also demonstrate that resistin stimulates MCP-1mediated monocyte migration by suppressing microRNA (miR)-33a and miR-33b via the PI3K, Akt and mTOR signaling pathways. CONCLUSION: These results provide new insights into the mechanisms of resistin action that may have therapeutic implications for patients with OA.

A novel CC chemokine ligand 2 like gene from ayu Plecoglossus altivelis is involved in the innate immune response against to Vibrio anguillarum.

Chemokine (CC motif) ligand 2 (CCL2), also known as monocyte chemoattractant protein 1 (MCP-1), is one of the key chemokines that regulate migration and infiltration of monocytes/macrophages (MO/MФ) in mammals. However, the functional repertoire of fish CCL2 remains unclear. Here, we identified a cDNA sequence encoding a novel CCL2-like protein (PaCCL2L) in ayu, Plecoglossus altivelis. Sequence analysis revealed that PaCCL2L grouped with CCL2 homologs, and is most closely related to Mexican tetra (Astyanax mexicanus) and zebrafish (Danio rerio) homologs. PaCCL2 transcripts were expressed in all tested tissues from healthy ayu, with the highest level in the spleen. Upon Vibrio anguillarum infection, PaCCL2L transcripts increased significantly in tested tissues, including the liver, spleen, and head kidney. We then produced the recombinant PaCCL2L mature peptide (rPaCCL2L) by prokaryotic expression and generated the corresponding antibodies (anti-PaCCL2L). A significant increase in PaCCL2L protein and mRNA expression was observed in ayu MO/MФ following V. anguillarum challenge. Intraperitoneal injection of rPaCCL2L resulted in significantly improved survival and reduced tissue bacterial load in V. anguillarum-infected ayu. rPaCCL2L had a positive effect on the chemotaxis of MO/MΦ and neutrophils both in vitro and in vivo. Meanwhile, rPaCCL2L exhibited a positive effect on the chemotaxis of LPS-stimulated MO/MΦ (M1 type) in vitro, whereas it exhibited no chemotaxis effect on cAMP-stimulated MO/MΦ (M2 type). In addition, rPaCCL2L treatment exhibited an enhanced effect on MO/MΦ phagocytosis, bacterial killing, respiratory burst, and mRNA expression of proinflammatory cytokines, whereas anti-PaCCL2L treatment had an inhibitory effect. Our study demonstrates that PaCCL2L might play a role in the immune response of ayu against V. anguillarum infection through chemotactic recruitment and activation of MO/MΦ.

Molecular dynamics simulations of the chemokine CCL2 in complex with pull down-derived heparan sulfate hexasaccharides.

BACKGROUND: Binding of chemokines to glycosaminoglycans (GAGs) is a crucial step in leukocyte recruitment to inflamed tissues. METHODS: A disaccharide compositional analysis of the HS dp6 fraction in combination with MS analysis of the CCL2-depleted dp6 fraction was the basis for target GAG ligand structure suggestions. Four experimentally-derived heparan sulfate hexasaccharides, two potentially chemokine-specific and two unspecific, have been docked to CCL2. Subsequent 300 ns molecular dynamics simulations were used to improve the docked complexes. RESULTS: Hexasaccharides with four sulfations and no acetylations are suggested for selective and high affinity chemokine binding. Using the Antithromin-III/heparin complex as positive control for docking, we were able to recover the correct complex structure only if the previously liganded ATIII structure was used as input. Since the liganded structure is not known for a CCL2-GAG complex, we investigated if molecular dynamics simulations could improve initial docking results. We found that all four GAG oligosaccharides ended up in close contact with the known binding residues after about 100 ns simulation time. CONCLUSIONS: A discrimination of specific vs. unspecific CCL2 GAG ligands is not possible by this approach. Long-time molecular dynamics simulations are, however, well suited to capture the delicate enthalpy/entropy balance of GAG binding and improve results obtained from docking. GENERAL SIGNIFICANCE: With the comparison of two methods, MS-based ligand identification and molecular modelling, we have shown the current limitations of our molecular understanding of complex ligand binding which is could be due to the numerical inaccessibility of ligand-induced protein conformational changes.

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.

Computational Signaling Protein Dynamics and Geometric Mass Relations in Biomolecular Diffusion.

We present an atomistic level computational investigation of the dynamics of a signaling protein, monocyte chemoattractant protein-1 (MCP-1), that explores how simulation geometry and solution ionic strength affect the calculated diffusion coefficient. Using a simple extension of noncubic finite size diffusion correction expressions, it is possible to calculate experimentally comparable diffusion coefficients that are fully consistent with those determined from cubic box simulations. Additionally, increasing the concentration of salt in the solvent environment leads to changes in protein dynamics that are not explainable through changes in solvent viscosity alone. This work in accurate computational determination of protein diffusion coefficients led us to investigate molecular-weight-based predictors for biomolecular diffusion. By introducing protein volume- and protein surface-area-based extensions of traditional statistical relations connecting particle molecular weight to diffusion, we find that protein solvent-excluded surface area rather than volume works as a better geometric property for estimating biomolecule Stokes radii. This work highlights the considerations necessary for accurate computational determination of biomolecule diffusivity and presents insight into molecular weight relations for diffusion that could lead to new routes for estimating protein diffusion beyond the traditional approaches.

Globular C1q receptor (p33) binds and stabilizes pro-inflammatory MCP-1: a novel mechanism for regulation of MCP-1 production and function.

The protein gC1qR (globular C1q receptor), also named p33, was originally identified as a binding partner of the globular heads of C1q in the complement system. gC1qR/p33 is abundantly expressed in many cell types, but the functional importance of this protein is not completely understood. Here, we investigate the impact of gC1qR/p33 on the production and function of the pathophysiologically important chemokine monocyte chemoattractant protein-1 (MCP-1) and the underlying molecular mechanisms. Knockdown of gC1qR/p33 negatively regulated the production of MCP-1, but had no effect on the expression of transcript for MCP-1 in human periodontal ligament cells, suggesting a translational/post-translational mechanism of action. Laser scanning confocal microscopy showed considerable cytosolic co-localization of gC1qR/p33 and MCP-1, and co-immunoprecipitation disclosed direct physical interaction between gC1qR/p33 and MCP-1. Surface plasmon resonance analysis revealed a high-affinity binding (KD = 10.9 nM) between gC1qR/p33 and MCP-1. Using a transwell migration assay, we found that recombinant gC1qR/p33 enhances MCP-1-induced migration of human THP-1 monocytes, pointing to a functional importance of the interaction between gC1qR/p33 and MCP-1. An in vitro assay revealed a rapid turnover of the MCP-1 protein and that gC1qR/p33 stabilizes MCP-1, hence preventing its degradation. We propose that endogenous gC1qR/p33 physically interacts with MCP-1 causing stabilization of the MCP-1 protein and stimulation of its activity in human periodontal ligament cells, suggesting a novel gC1qR/p33-mediated pro-inflammatory mechanism of action.

C-C motif chemokine ligand 2 regulates lps-induced inflammation and ER stress to enhance proliferation of bovine endometrial epithelial cells.

Chemokines play an important role in regulating the complex immune system at the maternal-fetal interface during pregnancy. Among various chemokines, CC motif chemokine ligand 2 (CCL2) plays a role in the recruitment of immune regulatory cells to implantation sites within the endometrium. In cattle, CCL2 is abundantly expressed in the uterine endometrium. However, its intracellular signaling has not been identified. In this study, we examined the effects of CCL2 on bovine endometrial (BEND) cell proliferation. CCL2 stimulated BEND cell proliferation by abundant expression of PCNA, accumulation of cells in the G2/M phase, and activation of the PI3 K/AKT and MAPK signaling pathways. Moreover, CCL2 reduced endoplasmic reticulum stress and restored the inflammation-induced reduction in BEND cell proliferation by regulating the unfolded protein response genes and cytokines. Collectively, these results demonstrated that CCL2 plays a pivotal role in reproductive tissues and may support maternal-fetal interface to improve efficiency of pregnancy.

Insights into CC Chemokine Ligand 2/Chemokine Receptor 2 Molecular Recognition: A Step Forward toward Antichemotactic Agents.

Chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein 1 (MCP-1), is a chemokine that recruits immune cells to inflammatory sites by interacting with G protein-coupled receptor CCR2. The CCL2/CCR2 axis is also involved in pathological processes such as tumor growth and metastasis and hence is currently considered as an important drug target. CCL2 exists in a dynamic monomer-dimer equilibrium that is modulated by CCR2 binding. We used solution nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations to study the interactions between CCL2 and a sulfopeptide corresponding to the N-terminal sequence of CCR2 (CCR218-31). Peptide binding induced the dissociation of CCL2 into monomers, forming stable CCL2/CCR218-31 complexes. NMR relaxation measurements indicated that residues around the CCR218-31 binding site, which are located at the dimer interface, undergo a complex regime of motions. NMR data were used to construct a three-dimensional structural model of the CCL2/CCR218-31 complex, revealing that CCR218-31 occupies a binding site juxtaposed to the dimer interface, partially replacing monomer-monomer contacts, explaining why CCR218-31 binding weakens the dimer interface and induces dissociation. We found that the main interactions governing receptor binding are highly stable salt bridges with conserved chemokine residues as well as hydrophobic interactions. These data provide new insights into the structure-function relationship of the CCL2-CCR2 interaction and may be helpful for the design of novel antichemotactic agents.

Key determinants of selective binding and activation by the monocyte chemoattractant proteins at the chemokine receptor CCR2.

Chemokines and their receptors collectively orchestrate the trafficking of leukocytes in normal immune function and inflammatory diseases. Different chemokines can induce distinct responses at the same receptor. In comparison to monocyte chemoattractant protein-1 (MCP-1; also known as CCL2), the chemokines MCP-2 (CCL8) and MCP-3 (CCL7) are partial agonists of their shared receptor CCR2, a key regulator of the trafficking of monocytes and macrophages that contribute to the pathology of atherosclerosis, obesity, and type 2 diabetes. Through experiments with chimeras of MCP-1 and MCP-3, we identified the chemokine amino-terminal region as being the primary determinant of both the binding and signaling selectivity of these two chemokines at CCR2. Analysis of CCR2 mutants showed that the chemokine amino terminus interacts with the major subpocket in the transmembrane helical bundle of CCR2, which is distinct from the interactions of some other chemokines with the minor subpockets of their receptors. These results suggest the major subpocket as a target for the development of small-molecule inhibitors of CCR2.

CCL2 nitration is a negative regulator of chemokine-mediated inflammation.

Chemokines promote leukocyte recruitment during inflammation. The oxidative burst is an important effector mechanism, this leads to the generation of reactive nitrogen species (RNS), including peroxynitrite (ONOO). The current study was performed to determine the potential for nitration to alter the chemical and biological properties of the prototypical CC chemokine, CCL2. Immunofluorescence was performed to assess the presence of RNS in kidney biopsies. Co-localisation was observed between RNS-modified tyrosine residues and the chemokine CCL2 in diseased kidneys. Nitration reduced the potential of CCL2 to stimulate monocyte migration in diffusion gradient chemotaxis assays (p < 0.05). This was consistent with a trend towards reduced affinity of the nitrated chemokine for its cognate receptor CCR2b. The nitrated chemokine was unable to induce transendothelial monocyte migration in vitro and failed to promote leukocyte recruitment when added to murine air pouches (p < 0.05). This could potentially be attributed to reduced glycosaminoglycan binding ability, as surface plasmon resonance spectroscopy showed that nitration reduced heparan sulphate binding by CCL2. Importantly, intravenous administration of nitrated CCL2 also inhibited the normal recruitment of leukocytes to murine air pouches filled with unmodified CCL2. Together these data suggest that nitration of CCL2 during inflammation provides a mechanism to limit and resolve acute inflammation.

Mutant CCL2 protein coating mitigates wear particle-induced bone loss in a murine continuous polyethylene infusion model.

Wear particle-induced osteolysis limits the long-term survivorship of total joint replacement (TJR). Monocyte/macrophages are the key cells of this adverse reaction. Monocyte Chemoattractant Protein-1 (MCP-1/CCL2) is the most important chemokine regulating trafficking of monocyte/macrophages in particle-induced inflammation. 7ND recombinant protein is a mutant of CCL2 that inhibits CCL2 signaling. We have recently developed a layer-by-layer (LBL) coating platform on implant surfaces that can release biologically active 7ND. In this study, we investigated the effect of 7ND on wear particle-induced bone loss using the murine continuous polyethylene (PE) particle infusion model with 7ND coating of a titanium rod as a local drug delivery device. PE particles were infused into hollow titanium rods with or without 7ND coating implanted in the distal femur for 4 weeks. Specific groups were also injected with RAW 264.7 as the reporter macrophages. Wear particle-induced bone loss and the effects of 7ND were evaluated by microCT, immunohistochemical staining, and bioluminescence imaging. Local delivery of 7ND using the LBL coating decreased systemic macrophage recruitment, the number of osteoclasts and wear particle-induced bone loss. The development of a novel orthopaedic implant coating with anti-CCL2 protein may be a promising strategy to mitigate peri-prosthetic osteolysis.

Synthesis of polymers and nanoparticles bearing polystyrene sulfonate brushes for chemokine binding.

The movement of leukocytes to the site of inflammation in response to injury or infection is orchestrated by chemokines binding and signaling through cognate receptors. The interaction between sulfated tyrosine residues on the flexible N-terminal tail of the receptor with positively charged regions of the chemokine is one of the key recognition features that facilitates binding. In this manuscript we describe the synthesis of polymers and silica nanoparticles bearing polystyrene sulfonate brushes to mimic the sulfated tyrosine residues. We show that both the polymers and nanoparticles possess high binding affinity for the chemokine monocyte chemoattractant protein-1 (MCP-1) in monomeric and dimeric form. We also demonstrate key differences in the relative affinity for the chemokine for the free polymer versus the polymer-derived nanoparticle system.

Designing a mutant CCL2-HSA chimera with high glycosaminoglycan-binding affinity and selectivity.

Chemokines like CCL2 mediate leukocyte migration to inflammatory sites by binding to G-protein coupled receptors on the target cell as well as to glycosaminoglycans (GAGs) on the endothelium of the inflamed tissue. We have recently shown that the dominant-negative Met-CCL2 mutant Y13A/S21K/Q23R with improved GAG binding affinity is highly bio-active in several animal models of inflammatory diseases. For chronic indications, we have performed here a fusion to human serum albumin (HSA) in order to extend the serum half-life of the chemokine mutant. To compensate a potential drop in GAG-binding affinity due to steric hindrance by HSA, a series of novel CCL2 mutants was generated with additional basic amino acids which were genetically introduced at sites oriented towards the GAG ligand. From this set of mutants, the Met-CCL2 variant Y13A/N17K/S21K/Q23K/S34K exhibited high GAG-binding affinity and a similar selectivity as wild type (wt) CCL2. From a set of different HSA-chemokine chimeric constructs, the linked HSA(C34A)(Gly)4Ser-Met-CCL2(Y13A/N17K/S21K/Q23K/S34K) fusion protein was found to show the best overall GAG-binding characteristics. Molecular modeling demonstrated an energetically beneficial fold of this novel protein chimera. This was experimentally supported by GdmCl-induced unfolding studies, in which the fusion construct exhibited a well-defined secondary structure and a transition point significantly higher than both the wt and the unfused CCL2 mutant protein. Unlike the wt chemokine, the quaternary structure of the HSA-fusion protein is monomeric according to size-exclusion chromatography experiments. In competition experiments, the HSA-fusion construct displaced only two of seven unrelated chemokines from heparan sulfate, whereas the unfused CCL2 mutant protein displaced five other chemokines. The most effective concentration of the HSA-fusion protein in inhibiting CCL2-mediated monocyte attachment to endothelial cells, as detected in the flow chamber, was 8.6 µg/ml. This novel HSA-fusion protein exhibits not only high affinity but also selective displacement of chemokines from GAGs binding. HSA is therefore proposed to be a highly promising scaffold candidate for therapeutic, GAG-targeting chemokine mutants.