ROS-induced PADI2 downregulation accelerates cellular senescence via the stimulation of SASP production and NFκB activation.

Cellular senescence is closely related to tissue aging including bone. Bone homeostasis is maintained by the tight balance between bone-forming osteoblasts and bone-resorbing osteoclasts, but it undergoes deregulation with age, causing age-associated osteoporosis, a main cause of which is osteoblast dysfunction. Oxidative stress caused by the accumulation of reactive oxygen species (ROS) in bone tissues with aging can accelerate osteoblast senescence and dysfunction. However, the regulatory mechanism that controls the ROS-induced senescence of osteoblasts is poorly understood. Here, we identified Peptidyl arginine deiminase 2 (PADI2), a post-translational modifying enzyme, as a regulator of ROS-accelerated senescence of osteoblasts via RNA-sequencing and further functional validations. PADI2 downregulation by treatment with H2O2 or its siRNA promoted cellular senescence and suppressed osteoblast differentiation. CCL2, 5, and 7 known as the elements of the senescence-associated secretory phenotype (SASP) which is a secretome including proinflammatory cytokines and chemokines emitted by senescent cells and a representative feature of senescence, were upregulated by H2O2 treatment or Padi2 knockdown. Furthermore, blocking these SASP factors with neutralizing antibodies or siRNAs alleviated the senescence and dysfunction of osteoblasts induced by H2O2 treatment or Padi2 knockdown. The elevated production of these SASP factors was mediated by the activation of NFκB signaling pathway. The inhibition of NFκB using the pharmacological inhibitor or siRNA effectively relieved H2O2 treatment- or Padi2 knockdown-induced senescence and osteoblast dysfunction. Together, our study for the first time uncover the role of PADI2 in ROS-accelerated cellular senescence of osteoblasts and provide new mechanistic and therapeutic insights into excessive ROS-promoted cellular senescence and aging-related bone diseases.

Intratumoral delivery of CCL25 enhances immunotherapy against triple-negative breast cancer by recruiting CCR9+ T cells.

CCR9+ T cells have an increased potential to be activated and therefore may mediate strong antitumor responses. Here, we found, however, that CCL25, the only chemokine for CCR9+ cells, is not expressed in human or murine triple-negative breast cancers (TNBCs), raising a hypothesis that intratumoral delivery of CCL25 may enhance antitumor immunotherapy in TNBCs. We first determined whether this approach can enhance CD47-targeted immunotherapy using a tumor acidity-responsive nanoparticle delivery system (NP-siCD47/CCL25) to sequentially release CCL25 protein and CD47 small interfering RNA in tumor. NP-siCD47/CCL25 significantly increased infiltration of CCR9+CD8+ T cells and down-regulated CD47 expression in tumor, resulting in inhibition of tumor growth and metastasis through a T cell-dependent immunity. Furthermore, the antitumor effect of NP-siCD47/CCL25 was synergistically enhanced when used in combination with programmed cell death protein-1/programmed death ligand-1 blockades. This study offers a strategy to enhance immunotherapy by promoting CCR9+CD8+ T cell tumor infiltration.

Discovery of CCL18 antagonist blocking breast cancer metastasis.

Our previous studies have proved that CCL18 is the most secreted chemokine in breast cancer microenvironment by tumor associated macrophages (TAMs). CCL18 promotes breast cancer invasiveness by binding to its cognate receptor PITPNM3 and activating the downstream signaling pathways. The high level of CCL18 in serum or tumor stroma is associated with tumor metastasis and poor patients overall survival. In this study, we identify an effective small molecular compound (SMC) to antagonize the effect of CCL18. We screen more than 1000 SMCs from Sun Yat-sen University SMC library and select 15 top scored SMCs by using computer-aided virtual screening based on the structure of CCL18. Then in vitro cell migration assay narrows down the selected 15 SMCs to the most effective SMC-21598. We find 10 µM SMC-21598 significantly inhibits CCL18-induced breast cancer cells adherence, invasiveness, and migration. Our further surface plasmon resonance (SPR), fluorescence spectroscopy and isothermal titration calorimetry (ITC) assays reveal that SMC-21598 binds tightly to CCL18, which blocks the binding of CCL18 with its receptor PITPNM3. The in vivo animal experiments show that SMC-21598 doesn't significantly affect xenografts growth, but inhibits lung metastasis. Our study provides a potential lead compound to antagonize CCL18 function. It would be of great significance to develop SMC drugs to ameliorate breast cancer metastasis and prolong patients' survival.

CCL18 enhances migration, invasion and EMT by binding CCR8 in bladder cancer cells.

Increased expression of CCL18 has been observed in various malignancies and in the urine samples of patients with bladder cancer (BC). However, the roles of CCL18 in the development, progression and metastasis of BC remain unclear. The present study demonstrated that CCL18 expression was significantly associated with advanced clinical stages of BC. Furthermore, exogenous CCL18 promoted cell invasion and migration, and induced cell epithelial­mesenchymal transition (EMT) in BC cells. Western blotting demonstrated that E­cadherin, an epithelial marker, was decreased, whereas matrix metalloproteinase (MMP)­2 and vascular endothelial growth factor (VEGF)­C were increased in CCL18­treated cells. Blocking CCR8 via a small molecule inhibitor or short hairpin (sh)RNA mitigated the decrease in E­cadherin, and increase in MMP­2 and VEGF­C, caused by human recombinant (r)CCL18. CCR8 knockdown by shRNA reversed rCCL18­induced cancer cell invasion, migration and EMT. In conclusion, these data suggested that CCL18 may promote migration, invasion and EMT by binding CCR8 in BC cells. Inhibition of CCL18 activity by blocking CCR8 could be a potential therapeutic strategy for preventing the progression of BC.

Leukocyte-Independent Effects of CC-Chemokines on Vascular Remodeling.

BACKGROUND: Vascular remodeling is an alteration in the structure of vessels in response to injury or hemodynamic changes. Disturbance of the structural and functional integrity of the endothelial cell layer can be observed in vascular remodeling associated with inflammation. Chemokines have been implicated in a wide range of diseases with prominent inflammatory components, and also in vascular remodeling. Among them, CC-chemokines are of great interest. They act through conventional CC-chemokine receptors (CCRs), widely expressed by leucocytes which are attracted to sites of chronic inflammation. However, many experimental data show that CCRs are expressed by vascular cells, suggesting a direct, leukocyte-independent effect on vascular remodeling. OBJECTIVE: Here, we discuss the role of CC-chemokines in atherosclerosis, angiogenesis, restenosis and renal dysfunction through direct activation of endothelial cells, endothelial progenitors, vascular smooth muscle cells, platelets, erythrocytes, mesangial cells and fibroblasts. RESULTS: The pathophysiological role of CC-chemokines has become more interesting since the discovery of the atypical chemokine receptor (ACKR) subfamily, that does not couple with G proteins and fails to transmit conventional intracellular signals. It has been demonstrated to be a chemokine scavenger or decoy receptor with a role in the regulation of acute inflammatory responses. CONCLUSION: At the vascular level, ACKRs are expressed by endothelial cells and endothelial lymphatic cells that seem to regulate angio- and lymph-angiogenesis. Pleiotropic effects of CC-chemokines on vascular wall cells and leukocytes increase their importance in vascular remodeling and suggest new drugs to counteract vascular dysfunction.

CCL28 promotes breast cancer growth and metastasis through MAPK-mediated cellular anti-apoptosis and pro-metastasis.

Breast cancer is one of the most commonly diagnosed cancers worldwide and the second leading cause of cancer-related deaths among females. CCL28 (mucosa-associated epithelial chemokine, MEC), a CC subfamily chemokine, has been well studied in the process of inflammation, and recently increasing evidence indicates that CCL28 is related to tumor progression. However, little is known concerning its function in breast cancer. In the present study, we generated a CCL28-overexpressing breast cancer cell line MDA-MB-231HM/CCL28 from parental MDA-MB­231HM cells. We found that overexpression of CCL28 promoted cell proliferation and tumor formation, and also enhanced migration, invasion and metastasis both in vitro and in vivo. Mechanistic studies revealed that CCL28 mediated intracellular activation of the mitogen-activated protein kinase (MAPK) signaling pathway to promote breast cancer cell proliferation and metastasis by upregulating anti-apoptotic protein Bcl-2 and suppressing cell adhesion protein ß-catenin. However, overexpression of CCL28 did not influence the expression of metastasis­related protein matrix metalloproteinase MMP2 and MMP9 and VEGF. Tissue sample analysis from animal models also indicated that overexpression of CCL28 was associated with enhanced pERK expression and reduced ß-catenin expression in breast carcinomas. Thus, our results show for the first time that CCL28 contributes to breast cancer progression through the ERK/MAPK­mediated anti-apoptotic and metastatic signaling pathway. Antagonists of CCL28 and the MAPK signaling pathway may be used synergistically to treat breast cancer patients.

POL7085 or anti-CCL28 treatment inhibits development of post-paramyxoviral airway disease.

INTRODUCTION: Asthma is major health burden throughout the world, and there are no therapies that have been shown to be able to prevent the development of disease. A severe respiratory paramyxoviral infection early in life has been demonstrated to greatly increase the risk of developing asthma. We have a mouse model of a severe respiratory paramyxoviral infection (Sendai virus, SeV) that mimics human disease, and requires early expression of the cytokine CCL28 to drive the development of post-viral airway disease. The known receptors for CCL28 are CCR3 and CCR10. However, it is not known if blockade of these receptors will prevent the development of post-viral airway disease. The objective of this study was to determine if treatment with a protein epitope mimetic antagonist of CCR10, POL7085, will provide sufficient protection against the development of post-viral airway disease. METHODS: C57BL6 mice were inoculated with SeV or UV inactivated SeV. From day 3-19 post inoculation (PI), mice were subcutaneously administered daily POL7085 or saline, or every other day anti-CCL28 mAb. On days 8, 10, and 12 PI bronchoalveolar cytokines, serum IgE, and lung cellular constituents were measured. At day 21 PI airway hyper-reactivity to methacholine and mucous cell metaplasia was measured. RESULTS: Treatment with either anti-CCL28 or POL7085 significantly reduced development of airway hyper-reactivity and mucous cell metaplasia following SeV infection. The prevention of post-viral airway disease was associated with early reductions in innate immune cells, but did not appear to be due to a reduction in IL-13 or IgE. CONCLUSIONS: Blockade of CCL28 or CCR10 during an acute severe respiratory paramyxoviral infection is sufficient to prevent the development of post-viral airway disease. However, the mechanism of action is unclear and requires further exploration.

CC-chemokine class inhibition attenuates pathological angiogenesis while preserving physiological angiogenesis.

Increasing evidence shows that CC-chemokines promote inflammatory-driven angiogenesis, with little to no effect on hypoxia-mediated angiogenesis. Inhibition of the CC-chemokine class may therefore affect angiogenesis differently depending on the pathophysiological context. We compared the effect of CC-chemokine inhibition in inflammatory and physiological conditions. In vitro, the broad-spectrum CC-chemokine inhibitor "35K" inhibited inflammatory-induced endothelial cell proliferation, migration, and tubulogenesis, with more modest effects in hypoxia. In vivo, adenoviruses were used to overexpress 35K (Ad35K) and GFP (AdGFP, control virus). Plasma chemokine activity was suppressed by Ad35K in both models. In the periarterial femoral cuff model of inflammatory-driven angiogenesis, overexpression of 35K inhibited adventitial neovessel formation compared with control AdGFP-infused mice. In contrast, 35K preserved neovascularization in the hindlimb ischemia model and had no effect on physiological neovascularization in the chick chorioallantoic membrane assay. Mechanistically, 2 key angiogenic proteins (VEGF and hypoxia-inducible factor-1α) were conditionally regulated by 35K, such that expression was inhibited in inflammation but was unchanged in hypoxia. In conclusion, CC-chemokine inhibition by 35K suppresses inflammatory-driven angiogenesis while preserving physiological ischemia-mediated angiogenesis via conditional regulation of VEGF and hypoxia-inducible factor-1α. CC-chemokine inhibition may be an alternative therapeutic strategy for suppressing diseases associated with inflammatory angiogenesis without inducing the side effects caused by global inhibition.- Ridiandries, A., Tan, J. T. M., Ravindran, D., Williams, H., Medbury, H. J., Lindsay, L., Hawkins, C., Prosser, H. C. G., Bursill, C. A. CC-chemokine class inhibition attenuates pathological angiogenesis while preserving physiological angiogenesis.

JAK-STAT6 Pathway Inhibitors Block Eotaxin-3 Secretion by Epithelial Cells and Fibroblasts from Esophageal Eosinophilia Patients: Promising Agents to Improve Inflammation and Prevent Fibrosis in EoE.

BACKGROUND: Although most studies on treatments for eosinophilic esophagitis (EoE) have focused on effects in the epithelium, EoE is a transmural disease. Eosinophils that infiltrate the subepithelial layers of the esophagus lead to fibrosis and the serious complications of EoE, and current therapies have shown minimal effects on this fibrosis. We aimed to elucidate T helper (Th)2 cytokine effects on esophageal fibroblasts and to explore potential fibroblast-targeted therapies for EoE. METHODS: We established telomerase-immortalized fibroblasts from human esophageal biopsies. We stimulated these esophageal fibroblasts with Th2 cytokines, and examined effects of omeprazole and inhibitors of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT6) pathway (AS1517499, leflunomide, and ruxolitinib) on STAT6 phosphorylation, STAT6 nuclear translocation, and eotaxin-3 expression. We also measured the effects of these inhibitors in esophageal epithelial cells stimulated with Th2 cytokines. RESULTS: As in esophageal epithelial cells, Th2 cytokines increased STAT6 phosphorylation, STAT6 nuclear translocation, eotaxin-3 transcription and protein secretion in esophageal fibroblasts. Unlike in epithelial cells, however, omeprazole did not inhibit cytokine-stimulated eotaxin-3 expression in fibroblasts. In contrast, JAK-STAT6 pathway inhibitors decreased cytokine-stimulated eotaxin-3 expression in both fibroblasts and epithelial cells. CONCLUSIONS: Omeprazole does not inhibit Th2 cytokine-stimulated eotaxin-3 expression by esophageal fibroblasts, suggesting that PPIs will have limited impact on subepithelial EoE processes such as fibrosis. JAK-STAT6 pathway inhibitors block Th2 cytokine-stimulated eotaxin-3 expression both in fibroblasts and in epithelial cells, suggesting a potential role for JAK-STAT inhibitors in treating both epithelial inflammation and subepithelial fibrosis in EoE.

Inhibition of Epithelial CC-Family Chemokine Synthesis by the Synthetic Chalcone DMPF-1 via Disruption of NF-κB Nuclear Translocation and Suppression of Experimental Asthma in Mice.

Asthma is associated with increased pulmonary inflammation and airway hyperresponsiveness. The interaction between airway epithelium and inflammatory mediators plays a key role in the pathogenesis of asthma. In vitro studies evaluated the inhibitory effects of 3-(2,5-dimethoxyphenyl)-1-(5-methylfuran-2-yl)prop-2-en-1-one (DMPF-1), a synthetic chalcone analogue, upon inflammation in the A549 lung epithelial cell line. DMPF-1 selectively inhibited TNF-α-stimulated CC chemokine secretion (RANTES, eotaxin-1, and MCP-1) without any effect upon CXC chemokine (GRO-α and IL-8) secretion. Western blot analysis further demonstrated that the inhibitory activity resulted from disruption of p65NF-κB nuclear translocation without any effects on the mitogen-activated protein kinase (MAPK) pathway. Treatment of ovalbumin-sensitized and ovalbumin-challenged BALB/c mice with DMPF-1 (0.2-100 mg/kg) demonstrated significant reduction in the secretion and gene expression of CC chemokines (RANTES, eotaxin-1, and MCP-1) and Th2 cytokines (IL-4, IL-5, and IL-13). Furthermore, DMPF-1 treatment inhibited eosinophilia, goblet cell hyperplasia, peripheral blood total IgE, and airway hyperresponsiveness in ovalbumin-sensitized and ovalbumin-challenged mice. In conclusion, these findings demonstrate the potential of DMPF-1, a nonsteroidal compound, as an antiasthmatic agent for further pharmacological evaluation.

Puerarin attenuates airway inflammation by regulation of eotaxin-3.

Puerarin is an isoflavonoid isolated from the root of the plant Pueraria lobata and has been used as a prescribed drug in China for the treatment of many diseases in the clinical practice. The present study aimed to determine the protective effects and the underlying mechanisms of puerarin on ovalbumin (OVA)-induced allergic inflammation in a mouse model of allergic asthma. Asthma mice model was established by ovalbumin. A total of 50 mice were randomly assigned to five experimental groups: control, model, dexamethasone (2 mg/kg), and puerarin (10 mg/kg, 20 mg/kg). Airway resistance (Raw) was measured by the forced oscillation technique, differential cell count in BAL fluid (BALF) was measured by Wright-Giemsa staining, histological assessment was measured by hematoxylin and eosin (HE) staining, BALF levels of Th1/Th2 cytokines were measured by enzyme-linked immunosorbent assay, eotaxin-3 was evaluated by western blotting. Our study demonstrated that, compared with model group, puerarin inhibited OVA-induced increases in Raw and eosinophil count; interleukin (IL)-4, IL-5, IL-13 levels were recovered in bronchoalveolar lavage fluid compared; increased IFN-γ level in bronchoalveolar lavage fluid; histological studies demonstrated that puerarin substantially inhibited OVA-induced eosinophilia in lung tissue compared with model group. Western blotting studies demonstrated that puerarin substantially inhibited eotaxin-3 compared with model group. Our findings support puerarin can prevent some signs of allergic asthma in the mouse model.

A positive feedback loop between mesenchymal-like cancer cells and macrophages is essential to breast cancer metastasis.

The close vicinity of cancer cells undergoing epithelial-mesenchymal transition (EMT) and tumor-associated macrophages (TAMs) at the invasive front of tumors suggests that these two cell type may mutually interact. We show that mesenchymal-like breast cancer cells activate macrophages to a TAM-like phenotype by GM-CSF. Reciprocally, CCL18 from TAMs induces cancer cell EMT, forming a positive feedback loop, in coculture systems and humanized mice. Inhibition of GM-CSF or CCL18 breaks this loop and reduces cancer metastasis. High GM-CSF expression in breast cancer samples is associated with more CCL18(+) macrophages, cancer cell EMT, enhanced metastasis, and reduced patient survival. These findings suggest that a positive feedback loop between GM-CSF and CCL18 is important in breast cancer metastasis.

Lentiviral small hairpin RNA knockdown of macrophage inflammatory protein-1γ ameliorates experimentally induced osteoarthritis in mice.

Immune cells are involved in the pathogenesis of osteoarthritis (OA). CD4(+) T cells were activated during the onset of OA and induced macrophage inflammatory protein (MIP)-1γ expression and subsequent osteoclast formation. We evaluated the effects of local knockdown of MIP-1γ in a mouse OA model induced by anterior cruciate ligament transection. The mouse macrophage cell lines and osteoclast-like cells generated from immature hematopoietic monocyte/macrophage progenitors of murine bone marrow were cocultured with either receptor activator of NFκB ligand (RANKL) or CD4(+) T cells. The levels of MIP-1γ and RANKL in cells and mice were examined by enzyme-linked immunosorbent assay (ELISA). The osteoclastogenesis was evaluated using tartrate-resistant acid phosphatase and cathepsin K staining. OA was induced in one hind-leg knee joint of B6 mice. Lentiviral vector encoding MIP-1γ small hairpin RNA (shRNA) and control vector were individually injected intra-articularly into the knee joints, which were histologically assessed for manifestations of OA. The expression of MIP-1γ and matrix metalloproteinase (MMP)-13 and the infiltration of CD4(+) T cells, macrophages, and osteoclastogenesis in tissues were examined using immunohistochemistry. CD4(+) T cells were involved in OA by inducing MIP-1γ expression in osteoclast progenitors and the subsequent osteoclast formation. Neutralizing MIP-1γ with a specific antibody abolishes RANKL-stimulated and CD4(+) T-cell-stimulated osteoclast formation. MIP-1γ levels were significantly higher in synovium and the chondro-osseous junction of joints 90 days postsurgery. The number of infiltrated CD4(+) T cells and macrophages and IL-1ß expression were reduced in the synovial tissues of mice treated with MIP-1γ shRNA. Histopathological examinations revealed that mice treated with MIP-1γ shRNA had less severe OA than control mice had, as well as decreased osteoclast formation and MMP-13 expression. Locally inhibiting MIP-1γ expression may ameliorate disease progression and provide a new OA therapy.

Anti-CCL25 antibody prolongs skin allograft survival by blocking CCR9 expression and impairing splenic T-cell function.

Chemokines, by virtue of their ability to recruit immune cells into allografts, play critical roles in acute transplantation rejection. CCR9 and its ligand, CCL25, is one of the key regulators of thymocyte migration and maturation in normal and inflammatory conditions. Moreover, several studies have revealed that high expression of CCR9 and CCL25 participated in many kinds of diseases. However, the role of CCR9 in allograft rejection is still unclear. In this study, we established a murine skin transplantation model of acute rejection. Our findings showed that the proportion of CCR9-expressing T cells was significantly increased in the spleen of allotransplanted mice compared with syngeneic transplantation. Furthermore, expression of CCL25 in allograft was similarly increased. Neutralization of CCL25 by intravenous injection of anti-CCL25 monoclonal antibody significantly prolonged skin allograft survival, decreased the number of infiltrating cells, and simultaneously suppressed the chemotactic ability and the proliferation of the splenic T cells in response to allogeneic antigens. Finally, blockade of CCL25 also diminished the secretion of IFN-γ by splenic T cells. These studies indicated that CCR9/CCL25 was involved in acute transplantation rejection and anti-CCL25 strategies might be useful in preventing acute rejection.

Chemokine binding protein vCCI attenuates vaccinia virus without affecting the cellular response elicited by immunization with a recombinant vaccinia vector carrying the HPV16 E7 gene.

Viral CC chemokine inhibitor (vCCI) of the clone P13 vaccinia virus (VACV) strain PRAHA lacks eight amino acids in the signal peptide sequence. To study the influence of vCCI on virus biology, a virus with the vCCI gene coding for a prolonged signal sequence was prepared. We found that secreted vCCI attenuated the virus in vivo, and that it correlated with decreased levels of RANTES, eotaxin, TARC, and MDC in the blood in comparison with the parental virus. We determined the influence of vCCI on the CTL response against VACV E3((140-148)) (VGPSNSPTF) and HPV16 E7((49-57)) (RAHYNIVTF) H-2D(b)-restricted epitopes. The examination of the specific CTL response elicited by immunization with the recombinant VACV-expressing tumor-associated HPV16 E7 antigen by IFN-γ ELISPOT showed that the immunogenicity of the recombinant VACV-producing secretory vCCI was similar to that of the parent virus or deletion mutant in the C23L/B29R locus. Immunization with the secretory vCCI-producing recombinant virus has a lower therapeutic anti-tumor effect against TC-1 tumors. Viral CCI downregulated the E7-specific response induced by gene gun immunization with the DNA vaccines pBSC-SigE7 LAMP and pBSC-vCCI. We also observed that the immune response against vCCI elicited by the DNA vaccine did not affect the multiplication of VACV in vivo.

In silico design of multi-target inhibitors for C-C chemokine receptors using substructural descriptors.

Rational design of entry inhibitors is an active area for the discovery of new and effective anti-HIV agents. C-C Chemokine receptors represent key targets for the HIV entry process. Several of these proteins with features to be HIV co-receptors have not been sufficiently studied or used for the design of novel entry inhibitors. With the purpose to overcome this problem, we develop here a fragment-based approach for the design of multi-target inhibitors against four C-C chemokine receptors. This approach was focused on the construction of a multi-target QSAR discriminant model using a large and heterogeneous database of compounds and substructural descriptors for the classification and prediction of inhibitors for C-C chemokine receptors. The model correctly classified more than 89% of active and inactive compounds in both: training and prediction series. As principal advantage, this model permitted the automatic and fast extraction of fragments responsible for the inhibitory activity against the different C-C chemokine receptors under study and new molecular entities were suggested as possible versatile inhibitors for these proteins.

IκBα glutathionylation and reduced histone H3 phosphorylation inhibit eotaxin and RANTES.

Airway smooth muscle cells (ASMCs) secrete eotaxin and RANTES (regulated on activation, normal T-cell expressed and secreted) in response to tumour necrosis factor (TNF)-α, which is inhibited by the nuclear factor (NF)-κB inhibitor dimethylfumarate (DMF). NF-κB/IκB (inhibitor of NF-κB) glutathionylation and changes in chromatin remodelling can inhibit NF-κB activity. In this study, we determined whether NF-κB/IκB glutathionylation and reduced histone H3 phosphorylation might underlie the inhibitory effect of DMF on NF-κB activity, and eotaxin and RANTES secretion. Primary human ASMCs were treated with DMF, diamide and/or glutathione (GSH) ethylester (OEt) prior to TNF-α stimulation and were subsequently analysed by ELISA, electrophoretic mobility shift assay, immunofluorescence, co-immunoprecipitation or immunoblotting. DMF reduced intracellular GSH and induced IκBα glutathionylation (IκBα-SSG), which inhibited IκBα degradation, NF-κB p65 nuclear entry and NF-κB/DNA binding. In addition, DMF inhibited the phosphorylation of histone H3, which was possibly mediated by the inhibitory effect of DMF on mitogen- and stress-activated protein kinase (MSK)-1. However, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase MAPK and MAPK phosphatase-1, upstream of MSK-1, were not inhibited by DMF. Importantly, DMF-mediated effects on NF-κB, histone H3, eotaxin and RANTES were reversed by addition of GSH-OEt. Our data suggest that DMF inhibits NF-κB-dependent eotaxin and RANTES secretion by reduction of GSH with subsequent induction of IκBα-SSG and inhibition of histone H3 phosphorylation. Our findings offer new potential drug targets to reduce airway inflammation in asthma.

Site-directed mutagenesis of the CC chemokine binding protein 35K-Fc reveals residues essential for activity and mutations that increase the potency of CC chemokine blockade.

Chemokines of the CC class are key mediators of monocyte recruitment and macrophage differentiation and have a well documented role in many inflammatory diseases. Blockade of chemokine activity is therefore an attractive target for anti-inflammatory therapy. 35K (vCCI) is a high-affinity chemokine binding protein expressed by poxviruses, which binds all human and murine CC chemokines, preventing their interaction with chemokine receptors. We developed an Fc-fusion protein of 35K with a modified human IgG1 Fc domain and expressed this construct in human embryonic kidney 293T cells. Purified 35K-Fc is capable of inhibiting CC chemokine-induced calcium flux, chemotaxis, and ß-arrestin recruitment in primary macrophages and transfected cells. To elucidate the residues involved in chemokine neutralization, we performed site-directed mutagenesis of six key amino acids in 35K and expressed the mutant Fc-fusion proteins in vitro. We screened the mutants for their ability to block chemokine-induced ß-arrestin recruitment in transfected cells and to inhibit primary macrophage signaling in an electric cell substrate impedance sensing assay. Using a sterile model of acute inflammation, zymosan-induced peritonitis, we confirmed that wild-type 35K-Fc can reduce monocyte recruitment, whereas one mutant (R89A) showed a more pronounced blockade of monocyte influx and another mutant (E143K) showed total loss of function. We believe that 35K-Fc will be a useful tool for exploring the role of CC chemokines in chronic inflammatory pathologies, and we have identified a higher potency form of the molecule that may have potential therapeutic applications in chronic inflammatory disease.

Pan-CC chemokine neutralization restricts splenocyte egress and reduces inflammation in a model of arthritis.

Chemokines are key regulators of leukocyte trafficking and play a crucial role under homeostatic and inflammatory conditions. Because chemokines are involved in multiple pathologies, they represent an attractive class of therapeutic targets. However, because of the redundancy of this system, neutralizing a single chemokine may be insufficient to achieve therapeutic benefit. Our strategy was to use a Fc-fusion recombinant protein form of the poxvirus-derived viral CC chemokine inhibitor protein (vCCI-Fc) that has the ability to specifically bind to multiple CC chemokines and neutralize their activity. In this study, we demonstrate first that, in vivo, vCCI-Fc prevents CC chemokine-dependent migration of macrophages into inflamed tissue of carageenan-challenged mice. We next studied this effect of inhibiting CC chemokine activity in a model more relevant to human disease, collagen-induced arthritis. Mice receiving vCCI-Fc revealed a striking retention of splenocytes, including activated and IFN-gamma-secreting CD4(+) and CD8(+) T cells, that was associated with a concomitant decrease of cells in the draining lymph nodes. These phenomena resulted in a significant decrease in the incidence of disease and a reduction in clinical score, joint inflammation, and cartilage destruction as compared with mice receiving isotype control. Taken together, these results define a role for CC chemokines in the control of disease, as interfering with their function leads to a previously unappreciated role of controlling inflammatory cell trafficking in and out of secondary lymphoid organs.