CCL2 mRNA Levels Research Articles

Activation of an Innate Immune Receptor, Nod1, Accelerates Atherogenesis in Apoe−/− Mice

Atherosclerosis is essentially a vascular inflammatory process in the presence of an excess amount of lipid. We have recently reported that oral administration of a nucleotide-binding oligomerization domain (Nod)-1 ligand, FK565, induced vascular inflammation in vivo. No studies, however, have proven the association between Nod1 and atherosclerosis in vivo. To investigate a potential role of NOD1 in atherogenesis, we orally administered FK565 to apolipoprotein E knockout (Apoe(-/-)) mice for 4 wk intermittently and performed quantification of atherosclerotic lesions in aortic roots and aortas, immunohistochemical analyses, and microarray-based gene expression profiling of aortic roots. FK565 administration accelerated the development of atherosclerosis in Apoe(-/-) mice, and the effect was dependent on Nod1 in non-bone marrow origin cells by bone marrow transplantation experiments. Immunohistochemical studies revealed the increases in the accumulation of macrophages and CD3 T cells within the plaques in aortic roots. Gene expression analyses of aortic roots demonstrated a marked upregulation of the Ccl5 gene during early stage of atherogenesis, and the treatment with Ccl5 antagonist significantly inhibited the acceleration of atherosclerosis in FK565-administered Apoe(-/-) mice. Additionally, as compared with Apoe(-/-) mice, Apoe and Nod1 double-knockout mice showed reduced development of atherosclerotic lesions from the early stage as well as their delayed progression and a significant reduction in Ccl5 mRNA levels at 9 wk of age. Data in the present study show that the Nod1 signaling pathway in non-bone marrow-derived cells contributes to the development of atherosclerosis.

Propolis Ethanol Extract Stimulates Cytokine and Chemokine Production through NF-κB Activation in C2C12 Myoblasts.

Myoblast activation is a triggering event for muscle remodeling. We assessed the stimulatory effects of propolis, a beehive product, on myoblasts. After an 8 h treatment with 100 μg/mL of Brazilian propolis ethanol extract, expression of various chemokines, including CCL-2 and CCL-5, and cytokines, such as IL-6, increased. This propolis-induced cytokine production appears to depend on NF-κB activation, because the IKK inhibitor BMS-345541 repressed mRNA levels of CCL-2 by ~66%, CCL-5 by ~81%, and IL-6 by ~69% after propolis treatment. Supernatant from propolis-conditioned C2C12 cells upregulated RAW264 macrophage migration. The supernatant also stimulated RAW264 cells to produce angiogenic factors, including VEGF-A and MMP-12. Brazilian green propolis therefore causes myoblasts to secrete cytokines and chemokines, which might contribute to tissue remodeling of skeletal muscle.

Inhibitory effects of astaxanthin on azoxymethane-induced colonic preneoplastic lesions in C57/BL/KsJ-db/db mice.

BackgroundObesity and related metabolic abnormalities, including excess oxidative stress and chronic inflammation, are associated with colorectal carcinogenesis. Astaxanthin, a xanthophyll carotenoid found in aquatic animals, is known to possess antioxidant, anti-inflammatory, and antineoplastic properties. The present study examined the effects of astaxanthin on the development of azoxymethane (AOM)-induced colonic premalignant lesions in C57BL/KsJ-db/db (db/db) obese mice.MethodMale db/db mice were administered 4 weekly subcutaneous injections of AOM (15 mg/kg body weight) from 5 weeks of age and subsequently, from 1 week after the last injection of AOM, were fed a diet containing 200 ppm astaxanthin throughout the experiment (8 weeks).ResultThe development of colonic premalignant lesions, i.e., aberrant crypt foci and β-catenin accumulated crypts, was significantly inhibited in mice treated with astaxanthin than in mice fed the basal diet. Astaxanthin administration markedly reduced urinary levels of 8-OHdG and serum levels of d-ROMs, which are oxidative stress markers, while increasing the expression of mRNA for the antioxidant enzymes GPx1, SOD1, and CAT in the colonic mucosa of AOM-treated db/db mice. The expression levels of IL-1β, IL-6, F4/80, CCL2, and CXCL2 mRNA in the colonic mucosa of AOM-treated mice were significantly decreased by astaxanthin. Dietary feeding with astaxanthin also resulted in a reduction in the numbers of NF-κB- and PCNA-positive cells that were increased by AOM exposure, in the colonic epithelium.ConclusionThese findings suggest that astaxanthin inhibits the development of colonic premalignant lesions in an obesity-related colorectal carcinogenesis model by reducing oxidative stress, attenuating chronic inflammation, and inhibiting NF-κB activation and cell proliferation in the colonic mucosa. Astaxanthin, therefore, may be a potential candidate as a chemoprevention agent against colorectal carcinogenesis in obese individuals.

Reduced Levels of microRNAs miR-124a and miR-150 Are Associated with Increased Proinflammatory Mediator Expression in Krüppel-like Factor 2 (KLF2)-deficient Macrophages

Previous studies have shown that the myeloid-specific deficiency of the transcription factor Krüppel-like factor 2 (KLF2) accelerates atherosclerosis in hypercholesterolemic Ldlr(-/-) mice due to the enhanced adhesion of myeloid cells to activated endothelial cells in the vessel wall. This study revealed elevated basal inflammation with elevated plasma levels of Ccl2, Ccl4, Ccl5, and Ccl11 in the myeloid-specific KLF2 knock-out (myeKlf2(-/-)) mice. Peritoneal macrophages isolated from myeKlf2(-/-) mice showed increased mRNA levels of several inflammatory mediators, including Ccl2, Ccl5, Ccl7, Cox-2, Cxcl1, and IL-6. In contrast, the levels of two microRNAs, miR-124a and miR-150, were lower in Klf2(-/-) macrophages compared with Klf2(+/+) macrophages. Additional studies showed a direct inverse relationship between miR-124a levels with Ccl2 expression, with anti-miR-124a increasing Ccl2 mRNA levels in Klf2(+/+) macrophages, whereas the restoration of miR-124a levels in Klf2(-/-) macrophages significantly reduced Ccl2 mRNA expression. Likewise, the inverse relationship was observed between miR-150 levels and Cxcl1 expression in Klf2(+/+) and Klf2(-/-) mice. Moreover, miR150 likely regulates the miR124a expression and thus augments expression of inflammatory mediators in myeKlf2(-/-) macrophages. This study documented that the transcription factor KLF2 modulates inflammatory chemokine production via regulation of microRNA expression levels in immune cells.

Effect of repeated alcohol exposure during the third trimester-equivalent on messenger RNA levels for interleukin-1β, chemokine (C–C motif) ligand 2, and interleukin 10 in the developing rat brain after injection of lipopolysaccharide

Microglia undergo maturation during the third trimester of human development (equivalent to the first 1–2 weeks of postnatal life in rodents), during which these cells may be particularly sensitive to insult. Alcohol exposure during this period can activate the neuroimmune system, an effect that may contribute to the pathophysiology of fetal alcohol spectrum disorders. Here, we investigated whether repeated alcohol exposure during the third trimester-equivalent in rats has a priming effect on the neuroimmune response to injection of bacterial lipopolysaccharide (LPS). Pups were exposed to alcohol in vapor chambers for 4 h daily from postnatal day (PD)2 to PD16 (peak blood alcohol concentrations ∼150 mg/dL). On PD17, rats were injected with either saline or LPS (50 μg/kg) and the frontal cortex, cerebellar vermis, and dentate gyrus were collected 2 h later. Messenger RNA (mRNA) levels for the pro-inflammatory agents interleukin 1β (IL-1β) and chemokine (C–C) motif ligand 2 (CCL2), as well as levels of the anti-inflammatory cytokine interleukin 10 (IL-10), were measured using reverse transcriptase polymerase chain reaction. LPS consistently increased IL-1β and CCL2 mRNA levels in the dentate gyrus, frontal cortex, and cerebellum of both male and female rats. Furthermore, the LPS-induced increase of IL-1β mRNA levels was significantly blunted in the frontal cortex of alcohol-exposed female rats. Conversely, LPS only minimally affected IL-10 mRNA expression and there were no significant differences between air- and alcohol-exposed rats. Taken together with the literature regarding the effect of third-trimester alcohol exposure on the neuroimmune system, our findings suggest that chronic exposure to lower levels is less disruptive to the neuroimmune system than binge-like exposure to high doses of alcohol.

Effects of l-Carnitine, Erythritol and Betaine on Pro-inflammatory Markers in Primary Human Corneal Epithelial Cells Exposed to Hyperosmotic Stress

Purpose: To explore the effects of osmoprotectants on pro-inflammatory mediator production in primary human corneal epithelial cells (HCECs) exposed to hyperosmotic stress.Methods: HCECs cultured in iso-osmolar medium (312 mOsM) were switched to hyperosmotic media with or without prior incubation with 2–20 mM of l-carnitine, erythritol or betaine for different time periods. The mRNA expression and protein production of pro-inflammatory markers in HCECs were evaluated by RT-qPCR and ELISA.Results: Hyperosmolar media significantly stimulated the mRNA and protein expression of pro-inflammatory cytokines, TNF-α, IL-1β and IL-6, and chemokines, IL-8, CCL2 and CCL20 in HCECs in an osmolarity dependent manner. The stimulated expression of these pro-inflammatory mediators was significantly but differentially suppressed by l-carnitine, erythritol or betaine. l-Carnitine displayed the greatest inhibitory effects and down-regulated 54–77% of the stimulated mRNA levels of TNF-α (down from 12.3–5.7 fold), IL-1β (2.2–0.9 fold), IL-6 (7.3–2.9 fold), IL-8 (4.6–2.0 fold), CCL2 (15.3–3.5 fold) and CCL20 (4.1–1.5 fold) in HCECs exposed to 450 mOsM. The stimulated protein production of TNF-α, IL-1β, IL-6 and IL-8 was also significantly suppressed by l-carnitine, erythritol and betaine. l-carnitine suppressed 49–79% of the stimulated protein levels of TNF-α (down from 81.3 to 17.4 pg/ml), IL-1β (56.9–29.2 pg/ml), IL-6 (12.8–4.6 ng/ml) and IL-8 (21.2–10.9 ng/ml) by HCECs exposed to 450 mOsM. Interestingly, hyperosmolarity stimulated increase in mRNA and protein levels of TNF-α, IL-1β and IL-6 were significantly suppressed by a transient receptor potential vanilloid channel type 1 (TRPV1) activation inhibitor capsazepine.Conclusions: l-carnitine, erythritol and betaine function as osmoprotectants to suppress inflammatory responses via TRPV1 pathway in HCECs exposed to hyperosmotic stress. Osmoprotectants may have efficacy in reducing innate inflammation in dry eye disease.

Abstract 4725: Elevated co-expression of CCL2 and CX3CL1 is associated with apoptosis and good prognosis in soft tissue sarcoma patients

Abstract Background: Chemokines represent a group of chemotactic cytokines, which are involved in both negative and positive regulation of inflammatory processes, angiogenesis, cancer cell proliferation and chemoattraciton of tumor cells to metastatic sites. Material and methods: mRNA expression of CCL2 and CX3CL1 was determined in 126 patients with soft-tissue sarcoma using quantitative real-time PCR. Patient groups displaying low or high expression in their tumors were separated by the median expression of the markers. Kaplan-Meier analysis and multivariate Cox's regression analysis were performed to assess the correlation between chemokine expression levels and disease-specific survival in STS. To gain insight into the cell physiological consequences of inhibition of CCL2 and CX3CL1, siRNA-mediated knockdown was used. The effect on proliferation was analyzed by a BrdU Elisa. The effect on inhibition of apoptosis was analyzed by adding etoposide after transfection with the respective siRNA, followed by FACS analysis. Results: The mRNA levels of CCL2, CCL7, and CX3CL1 were analyzed in tumor tissues from 126 STS patients using qPCR. Low mRNA expression of CCL2 and CX3CL1 was significantly correlated with a worse prognosis (RR = 1.98; p = 0.019 and RR = 2.10; p = 0.014; multivariate Cox's regression analysis). A combined low expression of CCL2 and CX3CL1 was associated with a significantly increased risk of tumor-related death compared to patients with high expression levels of both chemokines (RR = 3.08; p = 0.003). A gender-specific multivariate analysis revealed that female STS patients with low CX3CL1 mRNA expression had a 3.46-fold increased risk of death (p = 0.004). Low expression of both CCL2 and CX3CL1 mRNAs resulted in an additive 5.37-fold increased risk of tumor-related death (p = 0.003) compared to those with high expression of both parameters in female patients. Treatment with siRNA against CX3CL1 revealed a significantly increased cell proliferation after 24h but this effect disappeared after 48h and 72 h, respectively. To test the effect of siRNA treatment against CCL2 or CX3CL1 on induction of apoptosis, after transfection with the respective siRNA, etoposide was added to induce apoptosis. A significant decrease in apoptosis induction could be detected after combined treatment with etoposide and siRNA against CCL2 (p = 0.0047) or against CX3CL1 (p = 0.0043) compared with the etoposide and non-targeting siRNAs treated control cells. Conclusion: Our findings demonstrate that mRNA expression of CCL2 and CX3CL1 was significantly correlated with prognosis. Interestingly, there was a gender-specific impact of CCL2 on disease-specific survival. A significant decrease in apoptosis induction could be detected when cells were transfected with siRNA targeting CCL2 or CX3CL1 compared to the cells transfected with non-targeting siRNA. Citation Format: Elke Nolte, Astrid Kehlen, Thomas Greither, Sven Wach, Matthias Kappler, Matthias Bache, Hans-Jürgen Holzhausen, Christine Lautenschläger, Steffen Göbel, Peter Würl, Uta-Dorothee Immel, Abbas Agaimy, Bernd Wullich, Helge Taubert. Elevated co-expression of CCL2 and CX3CL1 is associated with apoptosis and good prognosis in soft tissue sarcoma patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4725. doi:10.1158/1538-7445.AM2014-4725

Abstract 3585: Metastasis of 4T1 murine breast cancer cells to the lung is dependent on the chemokine CCL2/MCP-1 produced by stromal cells in the primary tumor

Abstract The chemokine CCL2, also known as MCP-1, plays an important role in the initiation and progression of cancer. Since tumor cells produce CCL2, they are considered to be the main source of this chemokine. Here, we examined the capacity of CCL2 produced by non-tumor cells to affect the growth and lung metastasis of 4T1 mouse breast cancer cells. Primary tumors formed in the mammary pad of WT or CCL2-/- mice grew at a similar rate; however, lung metastases were markedly reduced in CCL2-/- mice, with significantly longer mouse survival. High levels of CCL2 mRNA were detected in tumors growing in WT, but not CCL2-/- mice. Serum CCL2 levels were increased in tumor-bearing WT, but not CCL2-/- mice. Transplantation of CCL2-/- bone marrow cells into WT mice did not alter the incidence of lung metastasis, but transplantation of WT bone marrow cells into CCL2-/- mice increased lung metastasis. The primary tumors of CCL2-/- mice consistently developed necrosis earlier than those of WT mice and showed decreased infiltration by macrophages and reduced angiogenesis. Interestingly, 4T1 cells that metastasized to the lung constitutively expressed elevated levels of CCL2. Intravenous injection of 4T1 cells producing a high level of CCL2 resulted in increased tumor foci in the lung of CCL2-/- mice. Finally, the culture supernatants of 4T1 cells markedly induced CCL2 production in mouse macrophages. Thus, CCL2 produced by tumor cell-activated stromal cells in the primary tumors promotes lung metastasis of 4T1 cells, while tumor cell-derived CCL2 also contributes to the metastasis once tumor cells enter the circulation. A greater understanding of the source and role of CCL2 during tumor progression and the mechanisms by which stromal cells produce this chemokine may lead to novel strategies for cancer treatment. Citation Format: Teizo Yoshimura, O.M. Zack Howard, Toshihiro Ito, Masaki Kuwabara, Akihiro Matsukawa, Keqiang Chen, Ying Liu, Mingyong Liu, Ji Ming Wang. Metastasis of 4T1 murine breast cancer cells to the lung is dependent on the chemokine CCL2/MCP-1 produced by stromal cells in the primary tumor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3585. doi:10.1158/1538-7445.AM2014-3585

Identification of a novel neutrophil population: proangiogenic granulocytes in second-trimester human decidua.

The maternal leukocytes of the first-trimester decidua play a fundamental role in implantation and early development of the fetus and placenta, yet little is known regarding the second-trimester decidual environment. Our multicolor flow cytometric analyses of human decidual leukocytes detected an elevation in tissue resident neutrophils in the second trimester. These cells in both human and murine samples were spatially restricted to decidua basalis. In comparison with peripheral blood neutrophils (PMNs), the decidual neutrophils expressed high levels of neutrophil activation markers and the angiogenesis-related proteins: vascular endothelial growth factor-A, Arginase-1, and CCL2, similarly shown in tumor-associated neutrophils. Functional in vitro assays showed that second-trimester human decidua conditioned medium stimulated transendothelial PMN invasion, upregulated VEGFA, ARG1, CCL2, and ICAM1 mRNA levels, and increased PMN-driven in vitro angiogenesis in a CXCL8-dependent manner. This study identified a novel neutrophil population with a physiological, angiogenic role in human decidua.

Molecular and cellular neuroinflammatory status of mouse brain after systemic lipopolysaccharide challenge: importance of CCR2/CCL2 signaling.

BackgroundGenetic and environmental factors are critical elements influencing the etiology of major depression. It is now accepted that neuroinflammatory processes play a major role in neuropsychological disorders. Neuroinflammation results from the dysregulation of the synthesis and/or release of pro- and anti-inflammatory cytokines with central or peripheral origin after various insults. Systemic bacterial lipopolysaccharide (LPS) challenge is commonly used to study inflammation-induced depressive-like behaviors in rodents. In the present study, we investigated immune-to-brain communication in mice by examining the effects of peripheral LPS injection on neuroinflammation encompassing cytokine and chemokine production, microglia and central nervous system (CNS)-associated phagocyte activation, immune cell infiltration and serotonergic neuronal function.MethodsLPS was administered to C57BL/6 J mice by intraperitoneal injection; brains were collected and pro-inflammatory cytokine mRNA and proteins were measured. To examine the relative contribution of the different populations of brain immune cells to the occurrence of neuroinflammation after acute systemic inflammation, we precisely characterized them by flow cytometry, studied changes in their proportions and level of activation, and measured the amount of cytokines they released by Cytometric Bead Array™ after cell sorting and ex vivo culture. Because of the central role that the chemokine CCL2 seems to play in our paradigm, we studied the effect of CCL2 on the activity of serotonergic neurons of the raphe nucleus using electrophysiological recordings.ResultsWe report that systemic LPS administration in mice caused a marked increase in pro-inflammatory IL-1β, IL-6, TNFα and CCL2 (monocyte chemoattractant protein-1) mRNA and protein levels in the brain. Moreover, we found that LPS caused microglia and CNS-associated phagocyte activation characterized by upregulation of CCR2, TLR4/CD14, CD80 and IL-4Rα, associated with overproduction of pro-inflammatory cytokines and chemokines, especially CCL2. LPS also induced a marked and selective increase of CCR2+ inflammatory monocytes within the brain. Finally, we showed that CCL2 hyperpolarized serotonergic raphe neurons in mouse midbrain slices, thus probably reducing the serotonin tone in projection areas.ConclusionTogether, we provide a detailed characterization of the molecular and cellular players involved in the establishment of neuroinflammation after systemic injection of LPS. This highlights the importance of the CCL2/CCR2 signaling and suggests a possible link with depressive disorders.

Silencing of CCR2 in myocarditis.

Myocarditis is characterized by inflammatory cell infiltration of the heart and subsequent deterioration of cardiac function. Monocytes are the most prominent population of accumulating leucocytes. We investigated whether in vivo administration of nanoparticle-encapsulated siRNA targeting chemokine (C-C motif) receptor 2 (CCR2)-a chemokine receptor crucial for leucocyte migration in humans and mice--reduces inflammation in autoimmune myocarditis. In myocardium of patients with myocarditis, CCL2 mRNA levels and CCR2(+) cells increased (P < 0.05), motivating us to pursue CCR2 silencing. Flow cytometric analysis showed that siRNA silencing of CCR2 (siCCR2) reduced the number of Ly6C(high) monocytes in hearts of mice with acute autoimmune myocarditis by 69% (P < 0.05), corroborated by histological assessment. The nanoparticle-delivered siRNA was not only active in monocytes but also in bone marrow haematopoietic progenitor cells. Treatment with siCCR2 reduced the migration of bone marrow granulocyte macrophage progenitors into the blood. Cellular magnetic resonance imaging (MRI) after injection of macrophage-avid magnetic nanoparticles detected myocarditis and therapeutic effects of RNAi non-invasively. Mice with acute myocarditis showed enhanced macrophage MRI contrast, which was prevented by siCCR2 (P < 0.05). Follow-up MRI volumetry revealed that siCCR2 treatment improved ejection fraction (P < 0.05 vs. control siRNA-treated mice). This study highlights the importance of CCR2 in the pathogenesis of myocarditis. In addition, we show that siCCR2 affects leucocyte progenitor trafficking. The data also point to a novel therapeutic strategy for the treatment of myocarditis.

Diabetes mellitus potentiates diffuse large B‑cell lymphoma via high levels of CCL5.

There is much evidence suggesting that CCL5 is one of the chemoattractant cytokines involved in diabetes mellitus (DM) with diffuse large B‑cell lymphoma (DLBCL). However, the pathological impact is unclear. In the current study, in order to improve understanding regarding the role of CCL5 in DM with DLBCL, the expression levels of CCL5 mRNA were examined in normal B cells, human DLBCL cell lines (Ly1, Ly8 and Ly10) and a mouse DLBCL cell line (A20), as well as those in cells cultured with either 5 or 30 mmol/l glucose. A20‑CCL5+ (CCL5 overexpression) and A20‑CCL5‑ (CCL5 knockdown) subclones were obtained through cell transduction with a lentiviral vector, and were subcutaneously injected into BALB/c DM mice and normal mice. Tumor growth was observed by calculating the tumor volume. The results demonstrated that CCL5 mRNA levels in DLBCL cells were significantly higher than those in the normal cells (P<0.05); and levels in DLBCL cells in 30 mmol/l Glu were significantly higher than in those of DLBCL cells in 5 mmol/l Glu (P<0.05). A20‑CCL5+ cells led to tumor formation in DM mice compared with A20 and A20‑CCL5‑ cells. These results indicate that high levels of CCL5 expression may accelerate DLBCL formation in DM.

Homeobox Transcription Factor VentX Regulates Differentiation and Maturation of Human Dendritic Cells

Dendritic cells (DCs) are specialized antigen presentation cells that play critical roles in the initiation and regulation of immune responses. The molecular determinants of DC differentiation and maturation are target of extensive investigation. VentX is a human homeobox transcriptional factor that regulates proliferation and differentiation of hematopoietic cells. In the current study, we report that ablation of VentX expression in monocytes significantly impaired their differentiation into DCs. Conversely, overexpression of VentX in monocytic THP1 cells accelerated their differentiation toward DCs. We showed that VentX regulates DC differentiation, in part, through modulating IL6 expression. Clinically, we found that VentX expression was elevated in intestinal lamina propria DCs (LPDCs) of inflamed mucosa from inflammatory bowel disease patients. Knockdown experiments suggested that VentX is essential for the maturation of LPDCs. In addition, corticosteroid treatment markedly decreased VentX expression in LPDCs and enforced expression of VentX counteracted the effects of corticosteroid on DCs maturation. Our data suggest that VentX is a critical transcriptional regulator of DC differentiation and maturation, and a potential target of immune regulation and therapy.

High coexpression of CCL2 and CX3CL1 is gender‐specifically associated with good prognosis in soft tissue sarcoma patients

Chemokines are involved in both the negative and positive regulation of inflammatory processes, angiogenesis and cancer/cancer stem cell proliferation as well as the chemoattraction of tumor cells to metastatic sites. The aim of this study was to measure the mRNA expression levels of three chemokines, CCL2, CCL7 and CX3CL1, in soft tissue sarcomas (STSs) and to assess the correlations between these levels as well as their correlations with clinicopathological data and the disease-specific survival of STS patients. The mRNA levels of CCL2, CCL7 and CX3CL1 were analyzed in tumor tissues from 126 STS patients using qPCR. Low mRNA expression of CCL2 and CX3CL1 was significantly correlated with a worse prognosis (RR = 1.98; p = 0.019 and RR = 2.10; p = 0.014; multivariate Cox's regression analysis). A combined low expression of CCL2 and CX3CL1 was associated with a significantly increased risk of tumor-related death as compared to patients with high expression levels of both chemokines (RR = 3.08; p = 0.003). A gender-specific multivariate analysis revealed that female STS patients with low CX3CL1 mRNA expression had a 3.46-fold increased risk of death (p = 0.004). Low expression of both CCL2 and CX3CL1 mRNAs resulted in an additive 5.37-fold increased risk of tumor-related death (p = 0.003) as compared to those with high expression of both parameters in female patients. In conclusion, this is the first study to show a significant correlation between combined low expression of CCL2 and CX3CL1 and a poor prognosis for STS patients, particularly in female patients.

Low-Dose Rapamycin Unmasks the Protective Potential of Targeting Intragraft NF-κB for Islet Transplants

Islet grafts can contribute to their own destruction via the elaboration of proinflammatory genes, many of which are transcriptionally regulated by nuclear factor κ-light-chain-enhancer of activated B-cells (NF-κB). Thus, NF-κB constitutes an enticing gene therapy candidate to improve the success of islet transplantation. To test this hypothesis in vivo, we blocked NF-κB in BALB/c (H2(d)) to C57/BL6 (H2(b)) mouse islet allografts by genetically engineering islets to express the NF-κB superrepressor, IκBα. Here we show by microarray and RTqPCR that islets exhibit an intrinsic early immediate proinflammatory response, with the most highly upregulated proinflammatory genes comprising the chemokines Cxcl1, Cxcl2, Cxcl10, and Ccl2; the cytokines Tnf-α and Il-6; and the adhesion molecule Icam1. Overexpression of IκBα inhibited the expression of these genes by 50-95% in islets and MIN6 β-cells in vitro, by inhibiting NF-κB-dependent gene transcription. Histological and RTqPCR analysis at postoperative day (POD) 10 revealed that IκBα-transduced islet allografts exhibited improved islet architecture and strong insulin-labeling with decreased Ccl2 and Il-6 mRNA levels compared to the GFP-transduced control grafts. Despite these protective effects, NF-κB-blocked islet allografts were promptly rejected in our MHC-mismatched mouse model. However, IκBα-expressing grafts did harbor localized "pockets" of Foxp3(+) mononuclear cells not evident in the control grafts. This result suggested that the effect of the NF-κB blockade might synergize with regulatory T-cell-sparing rapamycin. Indeed, combining intragraft IκBα expression with low-dose rapamycin increased the mean survival time of islet allografts from 20 to 81 days, with 20% of the grafts surviving for greater than 100 days. In conclusion, rapamycin unmasks the protective potential of intragraft NF-κB blockade, which can, in some cases, permit permanent allograft survival without continuous systemic immunosuppression.

Epigenetic upregulation of CCL2 and CCL3 via histone modifications in infiltrating macrophages after peripheral nerve injury

To gain insight into the epigenetic regulation of CC-chemokine ligand (CCL) 2 and CCL3, key players in the peripheral sensitization leading to neuropathic pain, we examined the relationship between histone H3 modification and the upregulation of these molecules using a mouse model of neuropathic pain after partial sciatic nerve ligation (PSL). We found that circuiting bone marrow (BM)-derived macrophages infiltrated into the injured sciatic nerve (SCN) using enhanced green fluorescent protein chimeric mice.The mRNA levels of CCL2, CCL3 and their receptors (CCR2 and CCR1/CCR5, respectively) were increased in the injured SCN. Chromatin immunoprecipitation assay revealed that levels of lysine 9-acetylated histone H3 (H3K9Ac) and lysine 4-trimethylated H3 (H3K4me3) in the promoter regions of the CCL2 and CCL3 genes were increased in the injured SCN after PSL, indicating the enhancement of gene expression. Immunoreactivity for H3K9Ac and H3K4me3 was localized in the nuclei of infiltrating BM-derived cells and CCL-expressing cells in the injured SCN. We observed H3K9Ac and H3K4me3 mainly in the nuclei of recruited macrophages on day 7 after PSL. Furthermore, upregulation of CCLs and CCRs were suppressed by histone acetyltransferase inhibitor, anacardic acid.Taken together, our findings demonstrate that CCL2 and CCL3 are upregulated in the injured peripheral nerve through epigenetic histone modification in infiltrating immune cells such as macrophages. These chemokine cascades may subsequently elicit chronic neuroinflammation following nerve injury.

Involvement of spinal chemokine CCL2 in the hyperalgesia evoked by bone cancer in mice: a role for astroglia and microglia.

The hypernociceptive role played by the chemokine CCL2, and its main receptor, CCR2, in pathological settings is being increasingly recognized. We aimed to characterize the involvement of spinal CCL2 in the hyperalgesia due to the intratibial inoculation of fibrosarcoma NCTC 2472 cells in mice. The intrathecal (i.t.) administration of the CCR2 antagonist RS 504393 (1–3 μg) or an anti-CCL2 antibody inhibited tumoral hyperalgesia. No change in the expression of spinal CCR2 was detected by western blot, whereas immunohistochemical experiments demonstrated increased CCL2 staining at the superficial laminae of the spinal cord ipsilateral to the tumor. This spinal CCL2 does not seem to be released from nociceptors since CCL2 mRNA and CCL2 levels in DRGs, as measured by RT-PCR and ELISA, remain unmodified in tumor-bearing mice. In contrast, immunohistochemical assays demonstrated the spinal up-regulations of GFAP and Iba-1, respective markers of astroglia and microglia, and the expression of CCL2 in both types of glial cells at the superficial laminae of the spinal cord of tumor-bearing mice. Finally, since CCL2 could induce astroglial or microglial activation, we studied whether the blockade of CCR2 could inhibit the increased spinal glial expression. GFAP, but not Iba-1, up-regulation was reduced in tumor-bearing mice treated for 3 days with i.t. RS 504393, indicating that spinal CCL2 acts as an astroglial activator in this setting. The participation at spinal level of CCL2/CCR2 in tumoral hypernociception, together with its previously described involvement at periphery, makes attractive the modulation of this system for the alleviation of neoplastic pain.

Increase in chemokines CXCL10 and CCL2 in blood from pigs infected with high compared to low virulence African swine fever virus isolates

Modulation of the expression of chemokines and chemokine receptors in whole blood was compared following infection of pigs with high and low virulence isolates of African swine fever virus. Levels of mRNAs for CCL2, CCL3L1, CCL4, CXCL10, CCR1 and CCR5 were significantly increased in at least one time point following infection in two experiments and CCL5, CCR9 and CXCR4 mRNA were significantly increased in one of the experiments. The results showed that greatest fold increases in mRNAs for CXCL10 and CCL2 were observed following infection of pigs. CXCL10 mRNA was increased by up to 15 fold in infected compared to uninfected pigs. CXCL10 protein was also detected in serum from pigs infected with the high virulence Benin 97/1 isolate. Levels of CCL2 mRNA were increased in pigs infected with high virulence Benin 97/1 isolate compared to low virulence OURT88/3 isolate and this correlated with an increase of greater than 30 fold in levels of CCL2 protein detected in serum from pigs infected with this isolate. An increase in overall chemotaxis active compounds in defibrinated plasma samples from Benin 97/1 infected pigs was observed at 3 days post-infection (dpi) and a decrease by 7 dpi as measured by chemotaxis assay using normal pig leucocytes in vitro. Increased levels of CXCL10 may either contribute to the activation of lymphocyte priming toward the Th1 phenotype or induction of T lymphocyte apoptosis. Increased levels of CCL2, a chemoattractant for macrophages, may result in increased recruitment of monocytes from bone marrow thus increasing the pool of cells susceptible to infection.

Diet‐induced obesity alters immune cell infiltration and expression of inflammatory cytokine genes in mouse ovarian and peri‐ovarian adipose depot tissues

Dysregulation of immune cells and/or altered inflammatory signaling have been implicated with reproductive dysfunction. Physiological changes leading to perturbations in the profile of immune cells and/or pro-inflammatory cytokines in or around female reproductive tissue could potentially have profound effects on ovarian function. Obesity is associated with chronic low-grade inflammation due, in part, to increased immune cell infiltration and inflammation in visceral adipose depots. This study investigated the impact of diet-induced obesity on immune cell infiltration and inflammation in peri-ovarian adipose tissue and mRNA expression of key inflammatory markers and microRNAs (miRs) in ovarian tissue. Six-week-old female C57Bl/6J mice were fed a standard chow or high-fat diet (HFD; 60% kcal fat) for approximately 7 months, at which time peri-ovarian adipose tissue and ovarian tissues were collected. Histological analysis of peri-ovarian adipose tissue from obese mice revealed increased (P < 0.05) adipocyte size and the presence of crown-like structures, the morphological presentation of infiltrating immune cells in adipose tissue, along with increases (P < 0.05) in the mRNA levels of markers of T-cells, activated macrophages, inflammatory cytokines, and chemokines. Ovarian mRNA levels of Il1b, Il6, Tnfa, p55, p75, Ccl2, Ikbkb, and Rela were higher in obese tissue (P < 0.05), with a strong trend (P = 0.06) for an increase in Nos2 and RELA protein. Additionally, ovarian miR125b and miR143 levels were decreased (P = 0.1). These data demonstrate that diet-induced obesity elevates expression of inflammatory-mediator genes in both the ovary and surrounding adipose depot, potentially negatively affecting ovarian function.

65 : Production of CCL2 by astrocytes in the inflamed CNS is critical for the development of EAE through the recruitment of myeloid cells from the periphery

CCL2 (monocyte chemoattractant protein-1, MCP-1) is a potent chemoattractant for immune cells including monocytes and dendritic cells, and is believed to be one of the most important chemokines involved in the pathology of multiple sclerosis (MS). Perivascular astrocytes within the central nervous system (CNS) are the most common and prominent source of CCL2 in both MS and its animal model, experimental autoimmune encephalomyelitis (EAE). CCL2 production within the CNS activates CCR2, its predominant functional receptor, leading to the infiltration of CCR2+ monocytes into the CNS. We and others have shown that, CCL2 mRNA levels in the spinal cord of mice with EAE were at least 100 fold higher compared to control mice. Furthermore, mice deficient in CCL2 or its receptor, CCR2, show a significant reduction in CNS macrophage infiltrates, as well as an increased resistance to EAE. Using the Cre/loxP recombination system, where Cre is driven by an astrocyte specific promoter, we tested the central hypothesis that ablating CCL2 expression from astrocytes will reduce myeloid cell trafficking to the CNS during EAE, resulting in less severe disease. Our results show that conditional ablation of CCL2 in astrocytes led to a 0.5-fold reduction in CCL2 mRNA levels in the spinal cord during peak disease (Student’s t -test, P t -test, P P