Chrysin Inhibits NF-κB-Dependent CCL5 Transcription by Targeting IκB Kinase in the Atopic Dermatitis-Like Inflammatory Microenvironment.

Chrysin (5,7-dihydroxyflavone) is a natural polyphenolic compound that induces an anti-inflammatory response. In this study, we investigated the molecular mechanism underlying the chrysin-induced suppression of C-C motif chemokine ligand 5 (CCL5) gene expression in atopic dermatitis (AD)-like inflammatory microenvironment. We showed that chrysin inhibited CCL5 expression at the transcriptional level through the suppression of nuclear factor kappa B (NF-κB) in the inflammatory environment. Chrysin could bind to the ATP-binding pocket of the inhibitor of κB (IκB) kinase (IKK) and, subsequently, prevent IκB degradation and NF-κB activation. The clinical efficacy of chrysin in targeting IKK was evaluated in 2,4-dinitrochlorobenzene-induced skin lesions in BALB/c mice. Our results suggested that chrysin prevented CCL5 expression by targeting IKK to reduce the infiltration of mast cells to the inflammatory sites and at least partially attenuate the inflammatory responses. These findings suggested that chrysin might be useful as a platform for the design and synthesis of small-molecule IKK-targeting drugs for the treatment of chronic inflammatory diseases, such as AD.

PD-L1 is a direct target of cancer-FOXP3 in pancreatic ductal adenocarcinoma (PDAC), and combined immunotherapy with antibodies against PD-L1 and CCL5 is effective in the treatment of PDAC.

High expression of PD-L1 marks the poor prognosis of pancreatic ductal adenocarcinomas (PDAC). However, the regulatory mechanism of PD-L1 remains elusive. We recently reported that cancer Forkhead box protein 3 (Cancer-FOXP3 or C-FOXP3) promoted immune evasion of PDAC by recruiting Treg cells into PDAC via upregulation of CCL5. In this study, we confirmed that PD-L1 was overexpressed in PDAC samples from two independent cohorts of patients with radical resection. Moreover, C-FOXP3 was colocalized and correlated with the expression of PD-L1 in tumor cells at the mRNA and protein levels, and this finding was confirmed by the The Cancer Genome Atlas (TCGA) database. Chromatin immunoprecipitation (ChIP) revealed that C-FOXP3 directly bound to the promoter region of PD-L1 in pancreatic cancer cells. Furthermore, overexpression of C-FOXP3 activated the luciferase reporter gene under the control of the PD-L1 promoter. However, mutation of the binding motif-a completely reversed the luciferase activity. In addition, C-FOXP3-induced upregulation of PD-L1 effectively inhibited the activity of CD8+ T cells. Based on our recent finding that the CCL-5 antibody achieved a better response to PDAC models with high C-FOXP3 levels, we further demonstrated that the PD-L1 antibody strengthened the antitumor effect of CCL-5 blockade in xenograft and orthotopic mouse models with high C-FOXP3 levels. In conclusion, C-FOXP3 directly activates PD-L1 and represents a core transcription factor that mediates the immune escape of PDAC. Combined blockade of PD-L1 and CCL-5 may provide an effective therapy for patients with PDAC that have high C-FOXP3 levels.

Effects of CCL5 on the biological behavior of breast cancer and the mechanisms of its interaction with tumor­associated macrophages.

The recurrence and metastasis of breast cancer limit the effectiveness of clinical treatments, making them important issues for clinicians to address. Tumor­associated macrophages (TAMs) contribute to regulating the immune system. C­C motif chemokine ligand 5 (CCL5) is an inflammatory chemokine that promotes chemotaxis on cells involved in the immune/inflammatory response. Breast cancer cells that secrete CCL5 act on THP­1 cells, influencing the invasion and metastasis of tumors. However, knowledge remains limited regarding the mechanism underlying the effects of CCL5 on breast cancer cells and TAMs, as well as the mechanisms promoting the migration and invasion of breast cancer. The present study demonstrated that the positive expression of CCL5 was associated with lymph node status and tumor­node­metastasis stage. Treatment with ≥20 ng/ml CCL5 significantly promoted the migration and invasion of MCF­7 and MDA­MB­231 cells. CCL5­small interfering RNA intervention significantly decreased the migration and invasion of the two cell types. In vitro, THP­1 cells were successfully induced to become TAMs, which were then recruited via the chemotactic effects of CCL5. This process was achieved through the co­stimulation of phorbol­12­myristate­13­â€‹acetate, interleukin­4 (IL­4) and IL­13. The nuclear factor­κB (NF­κB) signaling pathway was activated to regulate EMT, as well as the migration and invasion process of MCF­7 cells, when co­cultured with TAMs. We also reported that blocking the expression of CCL5 in vivo may significantly inhibit the growth of human breast cancer xenografts. Therefore, targeting CCL5 may be considered as a novel therapeutic strategy for suppressing the invasion and metastasis of breast cancer.

Elevated plasma RANTES in fibrodysplasia ossificans progressiva - A novel therapeutic target?

Fibrodysplasia ossificans progressiva (FOP) is a rare hereditary disease caused by a mutation in the intracellular domain of the activin A receptor type I and is characterized by episodes (flare-ups) of progressive heterotopic endochondral ossification (HO) in the soft tissues. The mutation alone is not sufficient for the occurrence of HO since flare-ups are triggered by inflammation and activation of the innate immune system. A number of cellular and humoral mediators have been implicated in animal and in vitro models. Observations in humans support the inflammatory nature of the condition, but data on the involved mediators are variable. We hypothesize that for induction of flare-ups in patients with FOP increase in at least one of the pro-inflammatory cytokines is both essential and sufficient to trigger the entire process of the inflammatory cells influx resulting in the novel ectopic bone formation and we suggest that C-C motif ligand 5 (CCL5), a pro-inflammatory chemokine also known as Regulated on activation, normal T-cell expressed and secreted (RANTES), might be the key candidate. CCL5 is a chemoattractant for all cellular types implicated in HO and is produced by the cells of the tissue microenvironment at the sites of HO as well as by the pro-inflammatory cellular mediators. CCL5 induces ossification in cultured human pluripotent mesenchymal cells (hMSCs) and in the primary culture of monocytes from FOP patients (but not from their healthy relatives), stimulation with lipopolysaccharide induces CCL5 expression. Finally, in a pilot study we used a panel of 23 cytokines and chemokines to screen the plasma samples of three subjects: a female patient with FOP during a flare-up; a female patient with hyperostosis corticalis generalisata (van Buchem disease), another rare disease characterized by excessive bone formation at the sites where it regularly occurs that does not include inflammatory events; and a healthy woman without bone disorders. There appeared a rather clear-cut signal of a 2-fold higher level of CCL5 in the FOP patient vs. the healthy subject and the van Buchem patient. Evaluation of the hypothesis would require an international prospective study, with main motivation being the lack of a conclusive treatment as the major unmet need in FOP. A treatment targeting CCL5 receptor already exists and is used in HIV-infected patients.

Breast Phyllodes Tumors Recruit and Repolarize Tumor-Associated Macrophages via Secreting CCL5 to Promote Malignant Progression, Which Can Be Inhibited by CCR5 Inhibition Therapy.

PURPOSE: Malignant phyllodes tumor (PT) is a fast-progression neoplasm derived from periductal stromal cells of the breast, which currently still lack effective treatment strategies. Our previous studies showed that the high density of tumor-associated macrophages (TAM) plays an important role in the malignant progression of PTs. TAMs secreted large amount of CCL18 to promote myofibroblast differentiation and invasion via binding to its receptor PIPTNM3 on myofibroblasts. Herein, we investigate the mechanism of how TAMs are recruited and repolarized by PTs to drive the malignant progression. EXPERIMENTAL DESIGN: The cytokines secreted by PTs were identified by the cytokine array. The clinical and pathologic correlations of the cytokine with PTs were estimated with IHC. The mechanisms of the cytokine that recruited and polarized the macrophage were explored with a coculture model of primary PT cells and macrophages in vitro and in vivo. The patient-derived xenografts (PDX) of malignant PTs were used to evaluate the therapeutic effect of CCR5 inhibitor. RESULTS: A high level of malignant PT-secreted CCL5 correlated with poor outcome of PTs and could be an independent prognostic factor of PTs. CCL5 bound to its receptor, CCR5, on macrophages thus activated AKT signaling to recruit and repolarize TAMs. Subsequently, the TAMs released CCL18 to further promote the aggressive phenotype of malignant PTs by enhancing and maintaining the myofibroblast differentiation and invasion in vitro and in vivo. In a murine PDX model of human malignant PTs, the CCL5-CCR5 axis blocked by maraviroc, an FDA-proved CCR5 inhibitor, prevented recruitment of monocytes to the tumor and dramatically suppressed tumor growth. CONCLUSIONS: Our findings indicate that malignant PTs recruit and repolarize TAMs through a CCL5-CCR5-driven signaling cascade. Thus, a positive feedback loop of CCL5-CCR5 and CCL18-PIPTNM3 between myofibroblast and TAMs is constituted to drive the malignant progression of PTs. Furthermore, targeting CCR5 with maraviroc represents a potential clinically available strategy to treat malignant PTs.

Lipingshu capsule improves atherosclerosis associated with lipid regulation and inflammation inhibition in apolipoprotein E-deficient mice.

BACKGROUND: Atherosclerosis (AS) is mainly responsible for cardiovascular diseases. The present study investigated whether Lipingshu capsule (LPS), whose ingredients are present in health food stores, has beneficial effect on AS. METHODS: C57BL/6 J mice were given a low fat rodent diet and assigned as control group (CON). ApoE-/- mice were placed on high fat rodent diet and randomly separated into high fat diet (HFD) group and HFD + LPS group whose animals were given 0.9 g/kg.BW LPS daily for 10 weeks. Atherosclerotic lesions in aorta and aortic root were evaluated. Serum lipids and multiple cytokine were measured. RESULTS: ApoE-/- mice fed with high fat diet had serious aortic lesions, whereas LPS markedly decreased plaque area of the total aorta and of the aortic root. LPS recovered the serum lipid profiles by substantially reducing TC, LDL-C, TG and Ox-LDL contents. Multi-cytokine analysis revealed greater serum levels of IL-1α, IL-1ß, IL-6, IFN-γ, GMCSF, RANTES and TNF-α induced by high fat diet slumped with LPS treatment. CONCLUSION: LPS reduces atherosclerotic lesions and thus alleviates AS by lipid profile modulation and inflammation inhibition.

Simultaneous blockade of IL-6 and CCL5 signaling for synergistic inhibition of triple-negative breast cancer growth and metastasis.

BACKGROUND: Metastatic triple-negative breast cancer (TNBC) is a heterogeneous and incurable disease. Numerous studies have been conducted to seek molecular targets to treat TNBC effectively, but chemotherapy is still the main choice for patients with TNBC. We have previously presented evidence of the important roles of interleukin-6 (IL-6) and chemokine (C-C motif) ligand 5 (CCL5) in TNBC tumor growth and metastasis. These experiments highlighted the importance of the crosstalk between cancer cells and stromal lymphatic endothelial cells (LECs) in tumor growth and metastasis. METHODS: We examined the viability and migration of MDA-MB-231-LN, SUM149, and SUM159 cells co-cultured with LECs when treated with maraviroc (CCR5 inhibitor) and tocilizumab (anti-IL-6 receptor antibody). To assess the anti-tumor effects of the combination of these two drugs in an athymic nude mouse model, MDA-MB-231-LN cells were implanted in the mammary fat pad and maraviroc (8 mg/kg, orally daily) and cMR16-1 (murine surrogate of the anti-IL-6R antibody, 10 mg/kg, IP, 3 days a week) were administrated for 5 weeks and effects on tumor growth and thoracic metastasis were measured. RESULTS: In this study, we used maraviroc and tocilizumab to confirm that IL-6 and CCL5 signaling are key pathways promoting TNBC cell proliferation and migration. Further, in a xenograft mouse model, we showed that tumor growth was dramatically inhibited by cMR16-1, the mouse version of the anti-IL6R antibody. The combination of maraviroc and cMR16-1 caused significant reduction of TNBC tumor growth compared to the single agents. Significantly, the combination of maraviroc and cMR16-1 abrogated thoracic metastasis. CONCLUSION: Taken together, these findings show that IL-6 and CCL5 signaling, which promote crosstalk between TNBC and lymphatic vessels, are key enhancers of TNBC tumor growth and metastasis. Furthermore, these results demonstrate that a drug combination inhibiting these pathways may be a promising therapy for TNBC patients.

Emodin inhibits epithelial­mesenchymal transition and metastasis of triple negative breast cancer via antagonism of CC­chemokine ligand 5 secreted from adipocytes.

Triple negative breast cancer (TNBC) has the lowest survival rate of the breast cancer subtypes owing to its aggressive and metastatic behavior. It has been reported that peritumoral adipose tissue contributes to the cell invasiveness and dissemination of TNBC. Emodin is an active anthraquinone derivative isolated from Rheum palmatum, with anticancer properties that have been reported to inhibit lung metastasis in a nude mouse xenograft model. In the present study, the effects of emodin on human TNBC cells and adipocytes were investigated in vivo and in vitro. The TNBC cell lines MDA­MB­231 and MDA­MB­453 were co­cultured with human adipocytes and treated with either emodin or epirubicin. Cell proliferation was assessed by MTT assay and migration and invasion were examined using a wound healing assay and a Transwell assay. interleukin­8, CC­chemokine ligand 5 (CCL5) and insulin­like growth factor­1 levels in the culture supernatants were detected by ELISA. The epithelial­mesenchymal transition (EMT) or metastasis associated markers were determined by western blot analysis. Nude mice fed with a high fat and sugar diet were used investigate the in vivo effect of emodin. The results showed that emodin inhibited TNBC proliferation and invasion more efficiently than epirubicin when co­cultured with adipocytes by downregulating the level of CCL5 in adipocyte supernatants; inhibiting the expression level of protein kinase B (AKT); and activating glycogen synthase kinase­3i (GSK3) and ß­catenin. This led to the suppressed expression of EMT­ and invasion­associated markers, including vimentin, snail, matrix metalloproteinase (MMP)­2 and MMP­9, and upregulation of E­cadherin, contributing to the inhibition of invasion. The in vivo assay showed that emodin inhibited tumor growth, and suppressed the lung and liver metastasis of TNBC cells by decreasing the secretion of CCL5 in mice fed a high fat and sugar diet more efficiently when compared with epirubicin. In conclusion, emodin inhibited the secretion of CCL5 from adipocytes, inhibited the EMT of TNBC cells, and inhibited tumor growth and lung and liver metastasis, which indicated a novel role of emodin in preventing the metastasis of TNBC.

Human Binge Alcohol Intake Inhibits TLR4-MyD88 and TLR4-TRIF Responses but Not the TLR3-TRIF Pathway: HspA1A and PP1 Play Selective Regulatory Roles.

Binge/moderate alcohol suppresses TLR4-MyD88 proinflammatory cytokines; however, alcohol's effects on TLR-TRIF signaling, especially after in vivo exposure in humans, are unclear. We performed a comparative analysis of the TLR4-MyD88, TLR4-TRIF, and TLR3-TRIF pathways in human monocytes following binge alcohol exposure. Mechanistic regulation of TLR-TRIF signaling by binge alcohol was evaluated by analyzing IRF3 and TBK1, upstream regulator protein phosphatase 1 (PP1), and immunoregulatory stress proteins HspA1A and XBP-1 in alcohol-treated human and mouse monocytes/macrophages. Two approaches for alcohol exposure were used: in vivo exposure of primary monocytes in binge alcohol-consuming human volunteers or in vitro exposure of human monocytes/murine macrophages to physiological alcohol concentrations (25-50 mM ethanol), followed by LPS (TLR4) or polyinosinic-polycytidylic acid (TLR3) stimulation ex vivo. In vivo and in vitro binge alcohol exposure significantly inhibited the TLR4-MyD88 cytokines TNF-α and IL-6, as well as the TLR4-TRIF cytokines/chemokines IFN-ß, IP-10, and RANTES, in human monocytes, but not TLR3-TRIF-induced cytokines/chemokines, as detected by quantitative PCR and ELISA. Mechanistic analyses revealed TBK-1-independent inhibition of the TLR4-TRIF effector IRF3 in alcohol-treated macrophages. Although stress protein XBP-1, which is known to regulate IRF3-mediated IFN-ß induction, was not affected by alcohol, HspA1A was induced by in vivo alcohol in human monocytes. Alcohol-induced HspA1A was required for inhibition of TLR4-MyD88 signaling but not TLR4-TRIF cytokines in macrophages. In contrast, inhibition of PP1 prevented alcohol-mediated TLR4-TRIF tolerance in macrophages. Collectively, our results demonstrate that in vivo and in vitro binge alcohol exposure in humans suppresses TLR4-MyD88 and TLR4-TRIF, but not TLR3-TRIF, responses. Whereas alcohol-mediated effects on the PP1-IRF3 axis inhibit the TLR4-TRIF pathway, HspA1A selectively suppresses the TLR4-MyD88 pathway in monocytes/macrophages.

Functional role of CCL5/RANTES for HCC progression during chronic liver disease.

BACKGROUND & AIMS: During liver inflammation, triggering fibrogenesis and carcinogenesis immune cells play a pivotal role. In the present study we investigated the role of CCL5 in human and in murine models of chronic liver inflammation leading to hepatocellular carcinoma (HCC) development. METHODS: CCL5 expression and its receptors were studied in well-defined patients with chronic liver disease (CLD) and in two murine inflammation based HCC models. The role of CCL5 in inflammation, fibrosis, tumor initiation and progression was analyzed in different cell populations of NEMOΔhepa/CCL5-/- animals and after bone marrow transplantation (BMT). For therapeutic intervention Evasin-4 was injected for 24h or 8weeks. RESULTS: In CLD patients, CCL5 and its receptor CCR5 are overexpressed - an observation confirmed in the Mdr2-/- and NEMOΔhepa model. CCL5 deletion in NEMOΔhepa mice diminished hepatocyte apoptosis, compensatory proliferation and fibrogenesis due to reduced immune cell infiltration. Especially, CD45+/Ly6G+ granulocytes, CD45+/CD11b+/Gr1.1+/F4/80+ pro-inflammatory monocytes, CD4+ and CD8+ T cells were decreased. One year old NEMOΔhepa/CCL5-/- mice displayed smaller and less malignant tumors, characterized by reduced proliferative capacity and less pronounced angiogenesis. We identified hematopoietic cells as the main source of CCL5, while CCL5 deficiency did not sensitise NEMOΔhepa hepatocytes towards TNFα induced apoptosis. Finally, therapeutic intervention with Evasin-4 over a period of 8weeks ameliorated liver disease progression. CONCLUSION: We identified an important role of CCL5 in human and functionally in mice with disease progression, especially HCC development. A novel approach to inhibit CCL5 in vivo thus appears encouraging for patients with CLD. LAY SUMMARY: Our present study identifies the essential role of the chemoattractive cytokine CCL5 for liver disease progression and especially hepatocellular carcinoma development in men and mice. Finally, the inhibition of CCL5 appears to be encouraging for therapy of human chronic liver disease.

Statins Inhibit Fibrillary ß-Amyloid Induced Inflammation in a Model of the Human Blood Brain Barrier.

BACKGROUND: Astrocytes and cerebral endothelial cells are important components of the blood-brain barrier (BBB). Disruption to this barrier through inflammation is a major contributor to Alzheimer's disease (AD) pathology. The amyloid beta (Aß) protein is known to exist in several forms and is a key modulator of AD that is known to cause inflammation and changes to BBB function. While one of these forms, fibrillary Aß (fAß), is known to cause endothelial cell death at the BBB, no studies have looked specifically at its role on inflammation in a model of the human BBB. AIMS: To determine if fAß is inflammatory to the human BBB. As statins have been shown to be anti-inflammatory and protective in AD, we also tested if these could inhibit the inflammatory effect of fAß. METHODS: Using cultured cerebral endothelial cells and astrocytes we determined changes in cytokine release, cell toxicity and barrier function in response to fibrillary ß-amyloid1-42 (fAß1-42) alone and in combination with statins. RESULTS: fAß1-42 induced inflammatory cytokine release from endothelial cells in the absence of cell toxicity. It also induced astrocyte cytokine release and cell death and caused a loss of barrier integrity. Statin treatment inhibited all of these effects. CONCLUSIONS: We conclude that fAß1-42 has both inflammatory and cytotoxic effects on the BBB and the protective effect of statins in AD may in part be through inhibiting these effects.

Tumoral Immune Cell Exploitation in Colorectal Cancer Metastases Can Be Targeted Effectively by Anti-CCR5 Therapy in Cancer Patients.

The immune response influences the clinical course of colorectal cancer (CRC). Analyzing the invasive margin of human CRC liver metastases, we identified a mechanism of immune cell exploitation by tumor cells. While two distinct subsets of myeloid cells induce an influx of T cells into the invasive margin via CXCL9/CXCL10, CCL5 is produced by these T cells and stimulates pro-tumoral effects via CCR5. CCR5 blockade in patient-derived functional in vitro organotypic culture models showed a macrophage repolarization with anti-tumoral effects. These anti-tumoral effects were then confirmed in a phase I trial with a CCR5 antagonist in patients with liver metastases of advanced refractory CRC. Mitigation of tumor-promoting inflammation within the tumor tissue and objective tumor responses in CRC were observed.

Autophagy Inhibition Dysregulates TBK1 Signaling and Promotes Pancreatic Inflammation.

Autophagy promotes tumor progression downstream of oncogenic KRAS, yet also restrains inflammation and dysplasia through mechanisms that remain incompletely characterized. Understanding the basis of this paradox has important implications for the optimal targeting of autophagy in cancer. Using a mouse model of cerulein-induced pancreatitis, we found that loss of autophagy by deletion of Atg5 enhanced activation of the IκB kinase (IKK)-related kinase TBK1 in vivo, associated with increased neutrophil and T-cell infiltration and PD-L1 upregulation. Consistent with this observation, pharmacologic or genetic inhibition of autophagy in pancreatic ductal adenocarcinoma cells, including suppression of the autophagy receptors NDP52 or p62, prolonged TBK1 activation and increased expression of CCL5, IL6, and several other T-cell and neutrophil chemotactic cytokines in vitro Defective autophagy also promoted PD-L1 upregulation, which is particularly pronounced downstream of IFNγ signaling and involves JAK pathway activation. Treatment with the TBK1/IKKε/JAK inhibitor CYT387 (also known as momelotinib) not only inhibits autophagy, but also suppresses this feedback inflammation and reduces PD-L1 expression, limiting KRAS-driven pancreatic dysplasia. These findings could contribute to the dual role of autophagy in oncogenesis and have important consequences for its therapeutic targeting. Cancer Immunol Res; 4(6); 520-30. ©2016 AACR.

Anti-CCR5 Therapy Circumvents Immune Cell Exploitation by Tumors.

CCL5 produced by T cells at invasive margins of colorectal cancer metastases promotes tumor growth.

Structure-Based Design of Peptidic Inhibitors of the Interaction between CC Chemokine Ligand 5 (CCL5) and Human Neutrophil Peptides 1 (HNP1).

Protein-protein interactions (PPIs) are receiving increasing interest, much sparked by the realization that they represent druggable targets. Recently, we successfully developed a peptidic inhibitor, RRYGTSKYQ ("SKY" peptide), that shows high potential in vitro and in vivo to interrupt a PPI between the platelet-borne chemokine CCL5 and the neutrophil-derived granule protein HNP1. This PPI plays a vital role in monocyte adhesion, representing a key mechanism in acute and chronic inflammatory diseases. Here, we present extensive and detailed computational methods applied to develop the SKY peptide. We combined experimentally determined binding affinities (KD) of several orthologs of CCL5 with HNP1 with in silico studies to identify the most likely heterodimeric CCL5-HNP1 complex which was subsequently used as a starting structure to rationally design peptidic inhibitors. Our method represents a fast and simple approach that can be widely applied to determine other protein-protein complexes and moreover to design inhibitors or stabilizers of protein-protein interaction.

Sphingosine-1-phosphate inhibits IL-1-induced expression of C-C motif ligand 5 via c-Fos-dependent suppression of IFN-ß amplification loop.

The neuroinflammation associated with multiple sclerosis involves activation of astrocytes that secrete and respond to inflammatory mediators such as IL-1. IL-1 stimulates expression of many chemokines, including C-C motif ligand (CCL) 5, that recruit immune cells, but it also stimulates sphingosine kinase-1, an enzyme that generates sphingosine-1-phosphate (S1P), a bioactive lipid mediator essential for inflammation. We found that whereas S1P promotes IL-1-induced expression of IL-6, it inhibits IL-1-induced CCL5 expression in astrocytes. This inhibition is mediated by the S1P receptor (S1PR)-2 via an inhibitory G-dependent mechanism. Consistent with this surprising finding, infiltration of macrophages into sites of inflammation increased significantly in S1PR2(-/-) animals. However, activation of NF-κB, IFN regulatory factor-1, and MAPKs, all of which regulate CCL5 expression in response to IL-1, was not diminished by the S1P in astrocytes. Instead, S1PR2 stimulated inositol 1,4,5-trisphosphate-dependent Ca(++) release and Elk-1 phosphorylation and enhanced c-Fos expression. In our study, IL-1 induced the IFNß production that supports CCL5 expression. An intriguing finding was that S1P induced c-Fos-inhibited CCL5 directly and also indirectly through inhibition of the IFN-ß amplification loop. We propose that in addition to S1PR1, which promotes inflammation, S1PR2 mediates opposing inhibitory functions that limit CCL5 expression and diminish the recruitment of immune cells.

Spinal NF-κB and chemokine ligand 5 expression during spinal glial cell activation in a neuropathic pain model.

BACKGROUND: The NF-κB pathway and chemokine (C-C motif) ligand 5 (CCL5) are involved in pain modulation; however, the precise mechanisms of their interactions in chronic neuropathic pain have yet to be established. METHODS: The present study examined the roles of spinal NF-κB and CCL5 in a neuropathic pain model after chronic constriction injury (CCI) surgery. CCI-induced pain facilitation was evaluated using the Plantar and von Frey tests. The changes in NF-κB and CCL5 expression were analyzed by immunohistochemistry and Western blot analyses. RESULTS: Spinal NF-κB and CCL5 expression increased after CCI surgery. Repeated intrathecal infusions of pyrrolidine dithiocarbamate (PDTC, a NF-κB inhibitor) decreased CCL5 expression, inhibited the activation of microglia and astrocytes, and attenuated CCI-induced allodynia and hyperalgesia. Intrathecal injection of a CCL5-neutralizing antibody attenuated CCI-induced pain facilitation and also suppressed spinal glial cell activation after CCI surgery. However, the CCL5-neutralizing antibody did not affect NF-κB expression. Furthermore, selective glial inhibitors, minocycline and fluorocitrate, attenuated the hyperalgesia induced by intrathecal CCL5. CONCLUSIONS: The inhibition of spinal CCL5 expression may provide a new method to prevent and treat nerve injury-induced neuropathic pain.

CCL5/CCR5 axis induces vascular endothelial growth factor-mediated tumor angiogenesis in human osteosarcoma microenvironment.

Chemokines modulate angiogenesis and metastasis that dictate cancer development in tumor microenvironment. Osteosarcoma is the most frequent bone tumor and is characterized by a high metastatic potential. Chemokine CCL5 (previously called RANTES) has been reported to facilitate tumor progression and metastasis. However, the crosstalk between chemokine CCL5 and vascular endothelial growth factor (VEGF) as well as tumor angiogenesis in human osteosarcoma microenvironment has not been well explored. In this study, we found that CCL5 increased VEGF expression and production in human osteosarcoma cells. The conditioned medium (CM) from CCL5-treated osteosarcoma cells significantly induced tube formation and migration of human endothelial progenitor cells. Pretreatment of cells with CCR5 antibody or transfection with CCR5 specific siRNA blocked CCL5-induced VEGF expression and angiogenesis. CCL5/CCR5 axis demonstrably activated protein kinase Cδ (PKCδ), c-Src and hypoxia-inducible factor-1 alpha (HIF-1α) signaling cascades to induce VEGF-dependent angiogenesis. Furthermore, knockdown of CCL5 suppressed VEGF expression and attenuated osteosarcoma CM-induced angiogenesis in vitro and in vivo. CCL5 knockdown dramatically abolished tumor growth and angiogenesis in the osteosarcoma xenograft animal model. Importantly, we demonstrated that the expression of CCL5 and VEGF were correlated with tumor stage according the immunohistochemistry analysis of human osteosarcoma tissues. Taken together, our findings provide evidence that CCL5/CCR5 axis promotes VEGF-dependent tumor angiogenesis in human osteosarcoma microenvironment through PKCδ/c-Src/HIF-1α signaling pathway. CCL5 may represent a potential therapeutic target against human osteosarcoma.

Single-walled carbon nanotubes increase pandemic influenza A H1N1 virus infectivity of lung epithelial cells.

BACKGROUND: Airborne exposure to nanomaterials from unintended occupational or environmental exposures or as a consequence of product use may lead to adverse health effects. Numerous studies have focused on single-walled carbon nanotubes (SWCNTs) and their ability to cause pulmonary injury related to fibrosis, and cancer; however few studies have addressed their impact on infectious agents, particularly viruses that are known for causing severe disease. Here we have demonstrated the ability of pristine SWCNTs of diverse electronic structure to increase the susceptibility of small airway epithelial cells (SAEC) to pandemic influenza A H1N1 infection and discerned potential mechanisms of action driving this response. METHODS: Small airway epithelial cells (SAEC) were exposed to three types of SWCNTs with varying electronic structure (SG65, SG76, CG200) followed by infection with A/Mexico/4108/2009 (pH1N1). Cells were then assayed for viral infectivity by immunofluorescence and viral titers. We quantified mRNA and protein levels of targets involved in inflammation and anti-viral activity (INFß1, IL-8, RANTES/CCL5, IFIT2, IFIT3, ST3GAL4, ST6GAL1, IL-10), localized sialic acid receptors, and assessed mitochondrial function. Hyperspectral imaging analysis was performed to map the SWCNTs and virus particles in fixed SAEC preparations. We additionally performed characterization analysis to monitor SWCNT aggregate size and structure under biological conditions using dynamic light scattering (DLS), static light scattering (SLS). RESULTS: Based on data from viral titer and immunofluorescence assays, we report that pre-treatment of SAEC with SWCNTs significantly enhances viral infectivity that is not dependent on SWCNT electronic structure and aggregate size within the range of 106 nm - 243 nm. We further provide evidence to support that this noted effect on infectivity is not likely due to direct interaction of the virus and nanoparticles, but rather a combination of suppression of pro-inflammatory (RANTES) and anti-viral (IFIT2, IFIT3) gene/protein expression, impaired mitochondrial function and modulation of viral receptors by SWCNTs. CONCLUSIONS: Results of this work reveal the potential for SWCNTs to increase susceptibility to viral infections as a mechanism of adverse effect. These data highlight the importance of investigating the ability of carbon-nanomaterials to modulate the immune system, including impacts on anti-viral mechanisms in lung cells, thereby increasing susceptibility to infectious agents.

Breast cancer cells condition lymphatic endothelial cells within pre-metastatic niches to promote metastasis.

Breast cancer metastasis involves lymphatic dissemination in addition to hematogenous spreading. Although stromal lymphatic vessels (LVs) serve as initial metastatic routes, roles of organ-residing LVs are underinvestigated. Here we show that lymphatic endothelial cells (LECs), a component of LVs within pre-metastatic niches, are conditioned by triple-negative breast cancer (TNBC) cells to accelerate metastasis. LECs within the lungs and lymph nodes, conditioned by tumour-secreted factors, express CCL5 that is not expressed either in normal LECs or in cancer cells, and direct tumour dissemination into these tissues. Moreover, tumour-conditioned LECs promote angiogenesis in these organs, allowing tumour extravasation and colonization. Mechanistically, tumour cell-secreted IL6 causes Stat3 phosphorylation in LECs. This pStat3 induces HIF-1α and VEGF, and a pStat3-pc-Jun-pATF-2 ternary complex induces CCL5 expression in LECs. This study demonstrates anti-metastatic activities of multiple repurposed drugs, blocking a self-reinforcing paracrine loop between breast cancer cells and LECs.