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.

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.

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.

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.

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.

Crystal structure of a mirror-image L-RNA aptamer (Spiegelmer) in complex with the natural L-protein target CCL2.

We report the crystal structure of a 40 mer mirror-image RNA oligonucleotide completely built from nucleotides of the non-natural L-chirality in complex with the pro-inflammatory chemokine L-CLL2 (monocyte chemoattractant protein-1), a natural protein composed of regular L-amino acids. The L-oligonucleotide is an L-aptamer (a Spiegelmer) identified to bind L-CCL2 with high affinity, thereby neutralizing the chemokine's activity. CCL2 plays a key role in attracting and positioning monocytes; its overexpression in several inflammatory diseases makes CCL2 an interesting pharmacological target. The PEGylated form of the L-aptamer, NOX-E36 (emapticap pegol), already showed promising efficacy in clinical Phase II studies conducted in diabetic nephropathy patients. The structure of the L-oligonucleotide[Symbol: see text]L-protein complex was solved and refined to 2.05 Å. It unveils the L-aptamer's intramolecular contacts and permits a detailed analysis of its structure-function relationship. Furthermore, the analysis of the intermolecular drug-target interactions reveals insight into the selectivity of the L-aptamer for certain related chemokines.

Monocyte-targeting supramolecular micellar assemblies: a molecular diagnostic tool for atherosclerosis.

Atherosclerosis is a multifactorial inflammatory disease that can progress silently for decades and result in myocardial infarction, stroke, and death. Diagnostic imaging technologies have made great strides to define the degree of atherosclerotic plaque burden through the severity of arterial stenosis. However, current technologies cannot differentiate more lethal "vulnerable plaques," and are not sensitive enough for preventive medicine. Imaging early molecular markers and quantifying the extent of disease progression continues to be a major challenge in the field. To this end, monocyte-targeting, peptide amphiphile micelles (PAMs) are engineered through the incorporation of the chemokine receptor CCR2-binding motif of monocyte chemoattractant protein-1 (MCP-1) and MCP-1 PAMs are evaluated preclinically as diagnostic tools for atherosclerosis. Monocyte-targeting is desirable as the influx of monocytes is a marker of early lesions, accumulation of monocytes is linked to atherosclerosis progression, and rupture-prone plaques have higher numbers of monocytes. MCP-1 PAMs bind to monocytes in vitro, and MCP-1 PAMs detect and discriminate between early- and late-stage atherosclerotic aortas. Moreover, MCP-1 PAMs are found to be eliminated via renal clearance and the mononuclear phagocyte system (MPS) without adverse side effects. Thus, MCP-1 PAMs are a promising new class of diagnostic agents capable of monitoring the progression of atherosclerosis.

Breast cancer: coordinated regulation of CCL2 secretion by intracellular glycosaminoglycans and chemokine motifs.

The chemokine CCL2 (MCP-1) has been identified as a prominent tumor-promoting factor in breast cancer. The major source for CCL2 is in the tumor cells; thus, identifying the mechanisms regulating CCL2 release by these cells may enable the future design of modalities inhibiting CCL2 secretion and consequently reduce tumorigenicity. Using cells deficient in expression of glycosaminoglycans (GAGs) and short hairpin RNAs reducing heparan sulfate (HS) and chondroitin sulfate (CS) expression, we found that intracellular HS and CS (=GAGs) partly controlled the trafficking of CCL2 from the Golgi toward secretion. Next, we determined the secretion levels of GFP-CCL2-WT and GFP-CCL2-variants mutated in GAG-binding domains and/or in the 40s loop of CCL2 ((45)TIVA(48)). We have identified partial roles for R18+K19, H66, and the (45)TIVA(48) motif in regulating CCL2 secretion. We have also demonstrated that in the absence of R24 or R18+K19+(45)TIVA(48), the secretion of CCL2 by breast tumor cells was almost abolished. Analyses of the intracellular localization of GFP-CCL2-mutants in the Golgi or the endoplasmic reticulum revealed particular intracellular processes in which these CCL2 sequences controlled its intracellular trafficking and secretion. The R24, (45)TIVA(48) and R18+K19+(45)TIVA(48) domains controlled CCL2 secretion also in other cell types. We propose that targeting these chemokine regions may lead to reduced secretion of CCL2 by breast cancer cells (and potentially also by other malignant cells). Such a modality may limit tumor growth and metastasis, presumably without affecting general immune activities (as discussed below).

A general method for site specific fluorescent labeling of recombinant chemokines.

Chemokines control cell migration in many contexts including development, homeostasis, immune surveillance and inflammation. They are also involved in a wide range of pathological conditions ranging from inflammatory diseases and cancer, to HIV. Chemokines function by interacting with two types of receptors: G protein-coupled receptors on the responding cells, which transduce signaling pathways associated with cell migration and activation, and glycosaminoglycans on cell surfaces and the extracellular matrix which organize and present some chemokines on immobilized surface gradients. To probe these interactions, imaging methods and fluorescence-based assays are becoming increasingly desired. Herein, a method for site-specific fluorescence labeling of recombinant chemokines is described. It capitalizes on previously reported 11-12 amino acid tags and phosphopantetheinyl transferase enzymes to install a fluorophore of choice onto a specific serine within the tag through a coenzyme A-fluorophore conjugate. The generality of the method is suggested by our success in labeling several chemokines (CXCL12, CCL2, CCL21 and mutants thereof) and visualizing them bound to chemokine receptors and glycosaminoglycans. CXCL12 and CCL2 showed the expected co-localization on the surface of cells with their respective receptors CXCR4 and CCR2 at 4 °C, and co-internalization with their receptors at 37 °C. By contrast, CCL21 showed the presence of large discrete puncta that were dependent on the presence of both CCR7 and glycosaminoglycans as co-receptors. These data demonstrate the utility of this labeling approach for the detection of chemokine interactions with GAGs and receptors, which can vary in a chemokine-specific manner as shown here. For some applications, the small size of the fluorescent adduct may prove advantageous compared to other methods (e.g. antibody labeling, GFP fusion) by minimally perturbing native interactions. Other advantages of the method are the ease of bacterial expression, the versatility of labeling with any maleimide-fluorophore conjugate of interest, and the covalent nature of the fluorescent adduct.

Molecular basis of lateral force spectroscopy nano-diagnostics: computational unbinding of autism related chemokine MCP-1 from IgG antibody.

Monocyte-chemoattractant protein-1 (MCP-1), also known as CCL2, is a potent chemoattractant of T cells and monocytes, involved in inflammatory and angio-proliferative brain and retinal diseases. Higher expression of MCP-1 is observed in metastatic tumors. Unusual levels of MCP-1 in the brain may be correlated with autism. Immunochemistry where atomic force microscope (AFM) tips functionalized with appropriate antibodies against MCP-1 are used could in principle support medical diagnostics. Useful signals from single molecule experiments may be generated if interaction forces are large enough. The chemokine-antibody unbinding force depends on a relative motion of the interacting fragments of the complex. In this paper the stability of the medically important MCP-1- immunoglobulin G antibody Fab fragment complex has been studied using steered molecular dynamics (SMD) computer simulations with the aim to model possible arrangements of nano-diagnostics experiments. Using SMD we confirm that molecular recognition in MCP1-IgG is based mainly on six pairs of residues: Glu39A - Arg98H, Lys56A - Asp52H, Asp65A - Arg32L, Asp68A - Arg32L, Thr32A - Glu55L, Gln61A - Tyr33H. The minimum external force required for mechanical dissociation of the complex depends on a direction of the force. The pulling of the MCP-1 antigen in the directions parallel to the antigen-antibody contact plane requires forces about 20 %-40 % lower than in the perpendicular one. Fortunately, these values are large enough that the fast lateral force spectroscopy may be used for effective nano-diagnostics purposes. We show that molecular modeling is a useful tool in planning AFM force spectroscopy experiments.

Targeting monocyte chemotactic protein-1 synthesis with bindarit induces tumor regression in prostate and breast cancer animal models.

Prostate and breast cancer are major causes of death worldwide, mainly due to patient relapse upon disease recurrence through formation of metastases. Chemokines are small proteins with crucial roles in the immune system, and their regulation is finely tuned in early inflammatory responses. They are key molecules during inflammatory processes, and many studies are focusing on their regulatory functions in tumor growth and angiogenesis during metastatic cell seeding and spreading. Bindarit is an anti-inflammatory indazolic derivative that can inhibit the synthesis of MCP-1/CCL2, with a potential inhibitory function in tumor progression and metastasis formation. We show here that in vitro, bindarit can modulate cancer-cell proliferation and migration, mainly through negative regulation of TGF-ß and AKT signaling, and it can impair the NF-κB signaling pathway through enhancing the expression of the NF-κB inhibitor IkB-α. In vivo administration of bindarit results in impaired metastatic disease in prostate cancer xenograft mice (PC-3M-Luc2 cells injected intra-cardially) and impairment of local tumorigenesis in syngeneic Balb/c mice injected under the mammary gland with murine breast cancer cells (4T1-Luc cells). In addition, bindarit treatment significantly decreases the infiltration of tumor-associated macrophages and myeloid-derived suppressor cells in 4T1-Luc primary tumors. Overall, our data indicate that bindarit is a good candidate for new therapies against prostate and breast tumorigenesis, with an action through impairment of inflammatory cell responses during formation of the tumor-stroma niche microenvironment.

Structural basis for high selectivity of anti-CCL2 neutralizing antibody CNTO 888.

Human CC chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein-1 (MCP-1), is a member of the ß chemokine family whose actions are mediated through the G-protein-coupled receptor CCR2. Binding of CCL2 to its receptor CCR2 triggers calcium mobilization and chemotaxis. CCL2 is implicated in the pathogenesis of certain inflammatory diseases and cancer. CNTO 888, a neutralizing human anti-CCL2 antibody, was derived by antibody phage display. The antibody binds human CCL2 with high affinity (K(D)=22 pM) and inhibits CCL2 binding to its receptor. The crystal structure of the CNTO 888 Fab alone and in complex with the monomeric form of CCL2 (P8A variant) was determined at 2.6 Å and 2.8 Å resolution, respectively. CNTO 888 recognizes a conformational epitope encompassing residues 18-24 and 45-51 that overlaps the mapped receptor binding site. The epitope of CNTO 888 does not overlap with the dimerization site of CCL2, and thus its inhibitory activity is not expected to result from interference with the oligomeric state of CCL2. Comparison of the X-ray-determined epitopes of CNTO 888 and another CCL2-neutralizing antibody, 11K2, provides insight into the molecular basis of antibody selectivity and functional inhibition.

Properties of 7ND-CCL2 are modulated upon fusion to Fc.

7ND, a truncated version of the chemokine MCP-1/CCL2 lacking amino acids 2-8, is a potent antagonist of CCR2. In contrast to CCL2, 7ND is an obligate monomer. Similar to other chemokines, the in vivo half-life of 7ND is very short and its use as an antagonist in disease models is thus limited. We therefore constructed a 7ND-Fc fusion protein to extend the half-life of 7ND and overcome its limitations as a potential therapeutic antagonist. When we tested the properties of the fusion molecule in vitro, we found to our surprise that 7ND-Fc, in contrast to 7ND, produced a distinct, albeit small, chemotactic response in THP-1 cells, and a robust chemotactic response in L1.2 cells stably transfected with CCR2. To test whether this unexpected observation might be due to the bivalency of 7ND-Fc stemming from the dimeric nature of Fc fusions, we produced a heterodimeric Fc fusion which displays only one 7ND moiety, using a technology called strand exchange of engineered CH3 domains (SEED). The monovalent construct had properties equivalent to the parent 7ND. Furthermore, partial agonist activity appears to depend on receptor density as well as the signaling pathway examined. However, we were able to show that 7ND-Fc, but not 7ND alone, has antagonistic activity in experimental autoimmune encephalomyelitis, a murine model of multiple sclerosis.

An in vitro analysis system using a fluorescence protein reporter for evaluating anti-inflammatory effects in macrophages.

Monitoring of inflammation in adipose tissues, which causes insulin resistance, is valuable in evaluating insulin resistance. We developed an in vitro analysis system using a fluorescence protein (FP) as a reporter gene driven by pro-inflammatory cytokine promoters such as monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNFα). In the reporter-transfected RAW264 cells, the protein expression levels of green fluorescence protein (GFP) were increased by inflammatory stimulations such as lipopolysaccharide (LPS), conditioned medium prepared using hypertrophied 3T3-L1 adipocytes, and a co-culture system. The changes in fluorescence intensity were equivalent to those of the mRNA and protein expression levels for each cytokine. Moreover, the effects of 15-deoxy-12,14Δ-prostaglandine J(2), a natural anti-inflammatory compound, were detectable in this system. These data indicate that the FP system developed here is an analysis system of low cost with simple procedures for evaluating inflammation, suggesting usability in the large-scale screening of anti-inflammatory compounds.

Identification of CCR2-binding features in Cytl1 by a CCL2-like chemokine model.

Chemokines are small secreted proteins that play an important role in immune responses and have also been shown to be involved in cartilage development and contributing to pathogenesis of a variety of diseases. They present a conserved 3D structure, so-called IL8-like chemokine fold, which is supported by conserved cysteines forming intra-molecular disulfide bonds. These cysteine sequence motifs have often been used to find new chemokine family members by sequence-based database searches. However, it has been shown that different patterns can provide disulfide bonds fitting into an IL8-like architecture, which has been the key to identify new remote homologues of the IL8-like chemokine family. We report a structural-functional characterization of cytokine-like protein 1 (Cytl1) by a combination of different computational structure-based techniques. Previous studies based on sequence analysis and secondary structure predictions reported that Cytl1 might adopt a 4-helical cytokine fold. However, our detailed molecular modeling studies and structure-based functional analysis strongly suggest that Cytl1 is more likely to adopt an IL8-like chemokine fold, in particular similar to CCL2 (monocyte chemoattractant protein 1, MCP-1). Moreover, we identify in a CCL2-like 3D model of Cytl1 the necessary reported features to signal through the chemokine receptor CCR2. Those discovered structural features of Cytl1 as CCL2-like chemokine, together with the fact that both, CCL2 and Cytl1, are known to be involved in cartilage development and pathogenesis of osteoarthritis and rheumatoid arthritis, make us hypothesize that Cytl1 could be a structurally and functionally related analog of CCL2 signaling through the chemokine receptor CCR2.

Rationally evolving MCP-1/CCL2 into a decoy protein with potent anti-inflammatory activity in vivo.

Leukocyte recruitment from the blood into injured tissues during inflammatory diseases is the result of sequential events involving chemokines binding to their GPC receptors as well as to their glycosaminoglycan (GAG) co-receptors. The induction and the crucial role of MCP-1/CCL2 in the course of diseases that feature monocyte-rich infiltrates have been validated in many animal models, and several MCP-1/CCL2 as well as CCR2 antagonists have since been generated. However, despite some of them being shown to be efficacious in a number of animal models, many failed in clinical trials, and therapeutically interfering with the activity of this chemokine is not yet possible. We have therefore generated novel MCP-1/CCL2 mutants with increased GAG binding affinity and knocked out CCR2 activity, which were designed to interrupt the MCP-1/CCL2-related signaling cascade. We provide evidence that our lead mutant MCP-1(Y13A/S21K/Q23R) exhibits a 4-fold higher affinity toward the natural MCP-1 GAG ligand heparan sulfate and that it shows a complete deficiency in activating CCR2 on THP-1 cells. Furthermore, a significantly longer residual time on GAG ligands was observed by surface plasmon resonance. Finally, we were able to show that MCP-1(Y13A/S21K/Q23R) had a mild ameliorating effect on experimental autoimmune uveitis and that a marginal effect on oral tolerance in the group co-fed with Met-MCP-1(Y13A/S21K/Q23R) plus immunogenic peptide PDSAg was observed. These results suggest that disrupting wild type chemokine-GAG interactions by a chemokine-based antagonist can result in anti-inflammatory activity that could have potential therapeutic implications.