The Chemokine Receptor CCR8 Promotes the Migration of Dendritic Cells into the Lymph Node Parenchyma to Initiate the Allergic Immune Response.

The migration of mature dendritic cells (DCs) into the draining lymph node (dLN) is thought to depend solely on the chemokine receptor CCR7. CD301b+ DCs migrate into the dLN after cutaneous allergen exposure and are required for T helper 2 (Th2) differentiation. We found that CD301b+ DCs poorly upregulated CCR7 expression after allergen exposure and required a second chemokine signal, mediated by CCR8 on CD301b+ DCs and its ligand CCL8, to exit the subcapsular sinus (SCS) and enter the lymph node (LN) parenchyma. After allergen exposure, CD169+SIGN-R1+ macrophages in interfollicular regions produced CCL8, which synergized with CCL21 in a Src-kinase-dependent manner to promote CD301b+ DC migration. In CCR8-deficient mice, CD301b+ DCs remained in the SCS and were unable to enter the LN parenchyma, resulting in defective Th2 differentiation. We have defined a CCR8-dependent stepwise mechanism of DC-subset-specific migration through which LN CD169+SIGN-R1+ macrophages control the polarization of the adaptive immune response.

Chemokine profiling of melanoma-macrophage crosstalk identifies CCL8 and CCL15 as prognostic factors in cutaneous melanoma.

During cancer evolution, tumor cells attract and dynamically interact with monocytes/macrophages. To find biomarkers of disease progression in human melanoma, we used unbiased RNA sequencing and secretome analyses of tumor-macrophage co-cultures. Pathway analysis of genes differentially modulated in human macrophages exposed to melanoma cells revealed a general upregulation of inflammatory hallmark gene sets, particularly chemokines. A selective group of chemokines, including CCL8, CCL15, and CCL20, was actively secreted upon melanoma-macrophage co-culture. Because we previously described the role of CCL20 in melanoma, we focused our study on CCL8 and CCL15 and confirmed that in vitro both chemokines contributed to melanoma survival, proliferation, and 3D invasion through CCR1 signaling. In vivo, both chemokines enhanced primary tumor growth, spontaneous lung metastasis, and circulating tumor cell survival and lung colonization in mouse xenograft models. Finally, we explored the clinical significance of CCL8 and CCL15 expression in human skin melanoma, screening a collection of 67 primary melanoma samples, using multicolor fluorescence and quantitative image analysis of chemokine-chemokine receptor content at the single-cell level. Primary skin melanomas displayed high CCR1 expression, but there was no difference in its level of expression between metastatic and nonmetastatic cases. By contrast, comparative analysis of these two clinically divergent groups showed a highly significant difference in the cancer cell content of CCL8 (p = 0.025) and CCL15 (p < 0.0001). Kaplan-Meier curves showed that a high content of CCL8 or CCL15 in cancer cells correlated with shorter disease-free and overall survival (log-rank test, p < 0.001). Our results highlight the role of CCL8 and CCL15, which are highly induced by melanoma-macrophage interactions in biologically aggressive primary melanomas and could be clinically applicable biomarkers for patient profiling. © 2024 The Pathological Society of Great Britain and Ireland.

Mouse CCL8, a CCR8 agonist, promotes atopic dermatitis by recruiting IL-5+ T(H)2 cells.

Mouse CCL8 is a CC chemokine of the monocyte chemoattractant protein (MCP) family whose biological activity and receptor usage have remained elusive. Here we show that CCL8 is highly expressed in the skin, where it serves as an agonist for the chemokine receptor CCR8 but not for CCR2. This distinguishes CCL8 from all other MCP chemokines. CCL8 responsiveness defined a population of highly differentiated, CCR8-expressing inflammatory T helper type 2 (T(H)2) cells enriched for interleukin (IL)-5. Ccr8- and Ccl8-deficient mice had markedly less eosinophilic inflammation than wild-type or Ccr4-deficient mice in a model of chronic atopic dermatitis. Adoptive transfer studies established CCR8 as a key regulator of T(H)2 cell recruitment into allergen-inflamed skin. In humans, CCR8 expression also defined an IL-5-enriched T(H)2 cell subset. The CCL8-CCR8 chemokine axis is therefore a crucial regulator of T(H)2 cell homing that drives IL-5-mediated chronic allergic inflammation.

Dexamethasone attenuated thoracic aortic aneurysm and dissection in vascular smooth muscle cell Tgfbr2-disrupted mice with CCL8 suppression.

NEW FINDINGS: What is the central question of this study? What is the relationship of chemokine (C-C motif) ligand 8 (CCL8) to thoracic aortic aneurysm and dissection (TAAD) formation in postnatal mice with vascular smooth muscle cell (VSMC) Tgfbr2 disruption, and is dexamethasone a potential treatment? What is the main finding and its importance? CCL8 was associated with the formation of TAAD in VSMC Tgfbr2-disrupted mice. Dexamethasone reduced TAAD formation and inhibited mitogen-activated protein kinase (p-p38) and nuclear factor-κB (p-p65) signalling pathways. CCL8 might be an important promoter of aortic inflammation. Dexamethasone provided potential therapeutic effects in TAAD treatment. ABSTRACT: Aortic inflammation plays a vital role in initiation and progression of thoracic aortic aneurysm and dissection (TAAD). Disturbance of the transforming growth factor-ß (TGF-ß) signalling pathway is believed to be one of the pathogenic mechanisms of TAAD. Initially, Myh11-CreERT2 .Tgfbr2f/f male mice were used to build a TAAD mouse model, and bioinformatic analyses revealed enriched inflammatory signal pathways and upregulated chemokine (C-C motif) ligand 8 (CCL8). So we hypothesized that vascular smooth muscle cell (VSMC) Tgfbr2 disruption in postnatal mice results in aortic inflammation associated with CCL8 secretion. Real-time quantitative PCR and serum enzyme-linked immunosorbent assay (ELISA) results confirmed that CCL8 expression began to increase after VSMC Tgfbr2 disruption. Next, we cultured mouse thoracic aortas ex vivo, and observed that the protein expression of CCL8 in culture supernatants was increased by ELISA. Subsequently, the co-localization of CCL8 with α-smooth muscle actin or CD68 was found to be significantly increased by immunofluorescence. Then, dexamethasone (DEX) was used to treat TAAD in VSMC Tgfbr2-disrupted mice; the results of histochemical, immunofluorescence and immunohistochemical staining indicated that DEX therapy reduced CCL8 secretion, inflammatory cell recruitment, aortic medial thickening, elastic fibre fragmentation, extracellular matrix degradation and contractile apparatus impairment, and thereby ameliorated TAAD formation. Western blotting showed that mitogen-activated protein kinase and nuclear factor-κB signalling pathways in aorta were overactivated after VSMC Tgfbr2 disruption, but inhibited by DEX therapy. Altogether, CCL8 might be an important promoter in TAAD formation of VSMC Tgfbr2-disrupted mice, and DEX provided potential therapeutic effects in TAAD treatment.

Engineered anti-inflammatory peptides inspired by mapping an evasin-chemokine interaction.

Chemokines mediate leukocyte migration and homeostasis and are key targets in inflammatory diseases including atherosclerosis, cytokine storm, and chronic autoimmune disease. Chemokine redundancy and ensuing network robustness has frustrated therapeutic development. Salivary evasins from ticks bind multiple chemokines to overcome redundancy and are effective in several preclinical disease models. Their clinical development has not progressed because of concerns regarding potential immunogenicity, parenteral delivery, and cost. Peptides mimicking protein activity can overcome the perceived limitations of therapeutic proteins. Here we show that peptides possessing multiple chemokine-binding and anti-inflammatory activities can be developed from the chemokine-binding site of an evasin. We used hydrogen-deuterium exchange MS to map the binding interface of the evasin P672 that physically interacts with C-C motif chemokine ligand (CCL) 8 and synthesized a 16-mer peptide (BK1.1) based on this interface region in evasin P672. Fluorescent polarization and native MS approaches showed that BK1.1 binds CCL8, CCL7, and CCL18 and disrupts CCL8 homodimerization. We show that a BK1.1 derivative, BK1.3, has substantially improved ability to disrupt P672 binding to CCL8, CCL2, and CCL3 in an AlphaScreen assay. Using isothermal titration calorimetry, we show that BK1.3 directly binds CCL8. BK1.3 also has substantially improved ability to inhibit CCL8, CCL7, CCL2, and CCL3 chemotactic function in vitro We show that local as well as systemic administration of BK1.3 potently blocks inflammation in vivo Identification and characterization of the chemokine-binding interface of evasins could thus inspire the development of novel anti-inflammatory peptides that therapeutically target the chemokine network in inflammatory diseases.

C-C Motif Chemokine 8 promotes angiogenesis in vascular endothelial cells.

OBJECTIVES: Angiogenesis is an important progress associated with several pathological situations. Several chemokines have been reported to act as regulators of angiogenesis. The current study aimed to find whether C-C Motif Chemokine 8 is involved in angiogenesis regulation. METHODS: To verify whether C-C Motif Chemokine 8 is related to angiogenesis in plaques, carotid plaques were collected from patients with severe carotid stenosis and analysed using CD31 immunohistochemistry and real-time PCR. To further clarify the relation between C-C Motif Chemokine 8 and angiogenesis, human umbilical vein endothelium cells and human dermal microvascular endothelial cells were treated with C-C Motif Chemokine 8 in the presence or absence of C-C motif chemokine receptor 2-Ab and extracellular regulated MAP kinase 1/2 inhibition (FR180204). Proliferation and migration of human umbilical vein endothelium cells and human dermal microvascular endothelial cells were examined with Cell Counting Kit-8 and Transwell chamber assay, respectively. In vitro angiogenesis stimulated by C-C Motif Chemokine 8 was examined using tube formation assay. Ex vivo and in vivo angiogenesis were assessed by mice aortic ring assay and Matrigel plug assay, respectively. C-C motif chemokine receptors of human umbilical vein endothelium cells were examined with real-time PCR, and C-C motif chemokine receptor 1, C-C motif chemokine receptor 2, extracellular regulated MAP kinase 1/2 and phosphorylation-extracellular regulated MAP kinase 1/2 were examined with western blotting assay. RESULTS: C-C Motif Chemokine 8 was increased in carotid plaques with severe angiogenesis in both RNA and protein level. C-C Motif Chemokine 8 (5 ng/ml) weakly increased human umbilical vein endothelium cell proliferation, but not on human dermal microvascular endothelial cells. Migration and tube formation could be induced by C-C Motif Chemokine 8 in both human umbilical vein endothelium cells and human dermal microvascular endothelial cells. In mice aortic ring assay and Matrigel plug assay, C-C Motif Chemokine 8 could promote angiogenesis compared to vehicle groups. Phosphorylation of extracellular regulated MAP kinase 1/2 was increased with C-C Motif Chemokine 8 stimulation. The migration and tube formation promoted by C-C Motif Chemokine 8 could be largely blocked by C-C motif chemokine receptor 2-Ab or extracellular regulated MAP kinase 1/2 inhibition (FR180204). CONCLUSIONS: C-C Motif Chemokine 8 could promote both in vitro and in vivo angiogenesis. C-C motif chemokine receptor 2 played an important role in the activation of C-C Motif Chemokine 8 and extracellular regulated MAP kinase 1/2 signalling pathway was involved in this mechanism.

Altered Secretome of Diabetic Monocytes Could Negatively Influence Fracture Healing-An In Vitro Study.

Diabetes mellitus is a main risk factor for delayed fracture healing and fracture non-unions. Successful fracture healing requires stimuli from different immune cells, known to be affected in diabetics. Especially, application of mononuclear cells has been proposed to promote wound and fracture healing. Thus, aim was to investigate the effect of pre-/diabetic conditions on mononuclear cell functions essential to promote osteoprogenitor cell function. We here show that pre-/diabetic conditions suppress the expression of chemokines, e.g., CCL2 and CCL8 in osteoprogenitor cells. The associated MCP-1 and MCP-2 were significantly reduced in serum of diabetics. Both MCPs chemoattract mononuclear THP-1 cells. Migration of these cells is suppressed under hyperglycemic conditions, proposing that less mononuclear cells invade the site of fracture in diabetics. Further, we show that the composition of cytokines secreted by mononuclear cells strongly differ between diabetics and controls. Similar is seen in THP-1 cells cultured under hyperinsulinemia or hyperglycemia. The altered secretome reduces the positive effect of the THP-1 cell conditioned medium on migration of osteoprogenitor cells. In summary, our data support that factors secreted by mononuclear cells may support fracture healing by promoting migration of osteoprogenitor cells but suggest that this effect might be reduced in diabetics.

CCL8 secreted by tumor-associated macrophages promotes invasion and stemness of glioblastoma cells via ERK1/2 signaling.

Tumor-associated macrophages (TAMs) constitute a large population of glioblastoma and facilitate tumor growth and invasion of tumor cells, but the underlying mechanism remains undefined. In this study, we demonstrate that chemokine (C-C motif) ligand 8 (CCL8) is highly expressed by TAMs and contributes to pseudopodia formation by GBM cells. The presence of CCL8 in the glioma microenvironment promotes progression of tumor cells. Moreover, CCL8 induces invasion and stem-like traits of GBM cells, and CCR1 and CCR5 are the main receptors that mediate CCL8-induced biological behavior. Finally, CCL8 dramatically activates ERK1/2 phosphorylation in GBM cells, and blocking TAM-secreted CCL8 by neutralized antibody significantly decreases invasion of glioma cells. Taken together, our data reveal that CCL8 is a TAM-associated factor to mediate invasion and stemness of GBM, and targeting CCL8 may provide an insight strategy for GBM treatment.

Type I IFN signaling in T regulatory cells modulates chemokine production and myeloid derived suppressor cells trafficking during EAE.

Interferon-ß has therapeutic efficacy in Multiple Sclerosis by reducing disease exacerbations and delaying relapses. Previous studies have suggested that the effects of type I IFN in Experimental Autoimmune Encephalomyelitis (EAE) in mice were targeted to myeloid cells. We used mice with a conditional deletion (cKO) of the type I IFN receptor (IFNAR) in T regulatory (Treg) cells to dissect the role of IFN signaling on Tregs. cKO mice developed severe EAE with an earlier onset than control mice. Although Treg cells from cKO mice were more activated, the activation status and effector cytokine production of CD4+Foxp3- T cells in the draining lymph nodes (dLN) was similar in WT and cKO mice during the priming phase. Production of chemokines (CCL8, CCL9, CCL22) by CD4+Foxp3- T cells and LN resident cells from cKO mice was suppressed. Suppression of chemokine production was accompanied by a substantial reduction of myeloid derived suppressor cells (MDSCs) in the dLN of cKO mice, while generation of MDSCs and recruitment to peripheral organs was comparable. This study demonstrates that signaling by type I IFNs in Tregs reduces their capacity to suppress chemokine production, with resultant alteration of the entire microenvironment of draining lymph nodes leading to enhancement of MDSC homing, and beneficial effects on disease outcome.

Silencing CCL8 inhibited the proliferation and migration of PDGF-BB-stimulated human aortic smooth muscle cells.

C-C motif Chemokine ligand 8 (CCL8) has been found in diseases' pathogenesis. But its molecular mechanism in atherosclerosis (AS) remains to be elucidated. Human aortic smooth muscle cells (HASMCs) were stimulated by PDGF-BB to establish cell model. α-SMA in PDGF-BB-stimulated HASMCs was measured by immunofluorescence staining. Relative gene expressions in PDGF-BB-stimulated HASMCs were detected by quantitative real-time polymerase chain reaction and western blot. HASMCs proliferation, migration, and cell cycle were assessed by cell counting kit-8, wound-healing assay, and flow cytometry. HASMCs viability was increased after PDGF-BB stimulation, with α-SMA downregulation yet CCL8 upregulation. Silencing CCL8 inhibited PDGF-BB-stimulated HASMCs proliferation and migration, and increased cells percentage in G1 phases but decreased those in S phase. Also, silencing CCL8 decreased OPN and cyclinD1 expressions and AKT and ERK1/2 phosphorylation while increased those of α-SMA and Sm22α. However, upregulating CCL8 led to opposite effects, suggesting CCL8 could be an atherosclerosis therapeutic target.

Expression of the Peptidase "Fibroblast Activation Protein" on Decidual Stromal Cells Facilitating Tissue Remodeling.

INTRODUCTION: Expression of fibroblast activation protein (FAP) has been detected in activated fibroblasts participating in injury response, fibrotic and inflammatory conditions, and tumorigenesis. Human endometrium is equally characterized by rapid tissue remodeling events due to the reproductive tasks comprising the activity of proteolytic enzymes. OBJECTIVE: We therefore hypothesized that FAP-positive fibroblasts could also be involved in physiological processes requiring tissue remodeling, such as decidualization during early pregnancy. METHODS/RESULTS: The expression of FAP was analyzed by immunohistochemistry in frozen sections of decidual tissue from early pregnancy (gestational weeks: 6-12). All tissue samples clearly displayed a strong expression of FAP on the surface of stromal fibroblasts. Additionally, the percentage of FAP-positive fibroblasts freshly isolated from the decidua of the corresponding gestational weeks was calculated by applying FACS analysis. Decidual fibroblasts of different gestational weeks showed a significant decrease in FAP expression between the 6th and 7th weeks of gestation, which was followed by a steady slow reconstitution. By analyzing the expression of cytokines, chemokines, and growth factors of isolated FAP-positive decidual fibroblasts, we detected high levels of monocyte-attracting chemokines (growth-related oncogene alpha and monocyte chemoattractant protein-1 and -2), granulocyte-attracting chemokines (e.g., IL-8), proinflammatory factors (IL-1α and tumor necrosis factor alpha), and angiogenic substances (e.g., vascular endothelial growth factor and IL-8), which all promote an optimal microenvironment for implantation and growth of the conceptus. CONCLUSIONS: Our data demonstrate that the healthy early pregnancy decidua is characterized by a general occurrence of FAP-positive fibroblasts possibly participating in active tissue remodeling during implantation.

An integrative systems approach identifies novel candidates in Marfan syndrome-related pathophysiology.

Marfan syndrome (MFS) is an autosomal dominant genetic disorder caused by mutations in the FBN1 gene. Although many peripheral tissues are affected, aortic complications, such as dilation, dissection and rupture, are the leading causes of MFS-related mortality. Aberrant TGF-beta signalling plays a major role in the pathophysiology of MFS. However, the contributing mechanisms are still poorly understood. Here, we aimed at identifying novel aorta-specific pathways involved in the pathophysiology of MFS. For this purpose, we employed the Fbn1 under-expressing mgR/mgR mouse model of MFS. We performed RNA-sequencing of aortic tissues of 9-week-old mgR/mgR mice compared with wild-type (WT) mice. With a false discovery rate <5%, our analysis revealed 248 genes to be differentially regulated including 20 genes previously unrelated with MFS-related pathology. Among these, we identified Igfbp2, Ccl8, Spp1, Mylk2, Mfap4, Dsp and H19. We confirmed the expression of regulated genes by quantitative real-time PCR. Pathway classification revealed transcript signatures involved in chemokine signalling, cardiac muscle contraction, dilated and hypertrophic cardiomyopathy. Furthermore, our immunoblot analysis of aortic tissues revealed altered regulation of pSmad2 signalling, Perk1/2, Igfbp2, Mfap4, Ccl8 and Mylk2 protein levels in mgR/mgR vs WT mice. Together, our integrative systems approach identified several novel factors associated with MFS-aortic-specific pathophysiology that might offer potential novel therapeutic targets for MFS.

Hyperoside inhibits proinflammatory cytokines in human lung epithelial cells infected with Mycoplasma pneumoniae.

Mycoplasma pneumoniae pneumonia (MPP) is the most common respiratory infection in young children and its incidence has increased worldwide. In this study, high expression of chemokine ligand 5 (CCL5) was observed in the serum of MPP patients, and its expression was positively correlated to DNA of M. pneumoniae (MP-DNA). In vitro, M. pneumoniae (MP) infection to A549 cells induced the expression of CCL5, chemokines receptor 4 (CCR4), nuclear factor-κB (NF-κB) nuclear protein, and phosphorylation of NF-κB-p65 (p-NF-κB-p65), whereas NF-κB cytoplasmic protein was decreased. On the contrary, treatment of hyperoside counteracted the induction of MP infection and promoted the proliferation of MP-infected A549 cells. Similarly, MP-induced IL-8 and TNF-α production was also markedly reduced by hyperoside. And CCR4 inhibitor AZD2098 had a better effect than hyperoside. In addition, CCL5 recombinant protein inhibited the effect of hyperoside to promote IL-8 and TNF-α production and CCR4 expression. These results indicated that CCL5 may be involved in the progression of MPP, and hyperoside was beneficial for MPP probably through CCL5-CCR4 interactions, which may provide a potential effective therapy for MPP.

Influence of Chemokine N-Terminal Modification on Biased Agonism at the Chemokine Receptor CCR1.

Leukocyte migration, a hallmark of the inflammatory response, is stimulated by the interactions between chemokines, which are expressed in injured or infected tissues, and chemokine receptors, which are G protein-coupled receptors (GPCRs) expressed in the leukocyte plasma membrane. One mechanism for the regulation of chemokine receptor signaling is biased agonism, the ability of different chemokine ligands to preferentially activate different intracellular signaling pathways via the same receptor. To identify features of chemokines that give rise to biased agonism, we studied the activation of the receptor CCR1 by the chemokines CCL7, CCL8, and CCL15(Δ26). We found that, compared to CCL15(Δ26), CCL7 and CCL8 exhibited biased agonism towards cAMP inhibition and away from ß-Arrestin 2 recruitment. Moreover, N-terminal substitution of the CCL15(Δ26) N-terminus with that of CCL7 resulted in a chimera with similar biased agonism to CCL7. Similarly, N-terminal truncation of CCL15(Δ26) also resulted in signaling bias between cAMP inhibition and ß-Arrestin 2 recruitment signals. These results show that the interactions of the chemokine N-terminal region with the receptor transmembrane region play a key role in selecting receptor conformations coupled to specific signaling pathways.

Gene expression profiles of HTR8-S/Vneo cells after changes in ABCA1 expression.

ABCA1 is expressed in placental trophoblasts, such that when the expression level of ABCA1 changes, the function of trophoblasts dramatically changes. However, the mechanism by which ABCA1 affects the function of trophoblast cells remains unclear. Here, we used biochemical and transcriptomic to uncover the potential mechanism of the effect of ABCA1 on trophoblast function. HTR8/SVneo cells were either treated with the agonist T0901317 or transfected with siRNA to regulate ABCA1 expression levels. A human gene expression microarray was used to analyze the expression spectrum of ABCA1. Microarray results were confirmed by Western blotting and RT-PCR. Immunofluorescence allowed detection of the cellular localization of ABCA1, CCL8, CXCL10, CXCL11, and S1PR1 in HTR8/SVneo cells. Co-immunoprecipitation was used to test interactions among these proteins. Concomitant with an increase in ABCA1 expression, S1PR1 expression increased, whereas expression of CCL8, CXCL10, and CXCL11 decreased significantly; opposite effects were observed with a decrease in ABCA1 expression. Thus, changes in ABCA1 expression may lead to changes in downstream gene expression. Whereas the interaction between ABCA1 and S1PR1 was direct, interactions among ABCA1 and CCL8, CXCL10, and CXCL11 were indirect. We propose that, in conjunction with S1PR1, ABCA1 regulates expression levels of CCL8, CXCL10, and CXCL11; this may lead to changes in the immune function of trophoblastic cells. Thus, we suspect that the effect of ABCA1 on trophoblast function may involve many biological processes, molecular function changes, and the activation of multiple signaling pathways.

Anti-inflammatory Properties of Cannabidiol, a Nonpsychotropic Cannabinoid, in Experimental Allergic Contact Dermatitis.

Phytocannabinoids modulate inflammatory responses by regulating the production of cytokines in several experimental models of inflammation. Cannabinoid type-2 (CB2) receptor activation was shown to reduce the production of the monocyte chemotactic protein-2 (MCP-2) chemokine in polyinosinic-polycytidylic acid [poly-(I:C)]-stimulated human keratinocyte (HaCaT) cells, an in vitro model of allergic contact dermatitis (ACD). We investigated if nonpsychotropic cannabinoids, such as cannabidiol (CBD), produced similar effects in this experimental model of ACD. HaCaT cells were stimulated with poly-(I:C), and the release of chemokines and cytokines was measured in the presence of CBD or other phytocannabinoids (such as cannabidiol acid, cannabidivarin, cannabidivarinic acid, cannabichromene, cannabigerol, cannabigerolic acid, cannabigevarin, tetrahydrocannabivarin, and tetrahydrocannabivarinic acid) and antagonists of CB1, CB2, or transient receptor potential vanilloid type-1 (TRPV1) receptors. HaCaT cell viability following phytocannabinoid treatment was also measured. The cellular levels of endocannabinoids [anandamide (AEA), 2-arachidonoylglycerol] and related molecules (palmitoylethanolamide, oleoylethanolamide) were quantified in poly-(I:C)-stimulated HaCaT cells treated with CBD. We show that in poly-(I:C)-stimulated HaCaT cells, CBD elevates the levels of AEA and dose-dependently inhibits poly-(I:C)-induced release of MCP-2, interleukin-6 (IL-6), IL-8, and tumor necrosis factor-α in a manner reversed by CB2 and TRPV1 antagonists 6-iodopravadoline (AM630) and 5'-iodio-resiniferatoxin (I-RTX), respectively, with no cytotoxic effect. This is the first demonstration of the anti-inflammatory properties of CBD in an experimental model of ACD.

MCP2 activates NF-κB signaling pathway promoting the migration and invasion of ESCC cells.

MCP2, aliased CCL8, has been suggested to be implicated in the metastasis of cancer cells; however, no direct evidence has been established in esophageal squamous cell carcinoma (ESCC). In our present study, to investigate the role MCP2 played in the metastasis of ESCC cells; in vitro cell co-culture system was established. Wound-healing and Transwell assays were used to evaluate the migratory and invasive variation of ESCC cells before and after treatment with recombinant human MCP2. It was shown that MCP2 was able to activate the NF-κB signaling pathway inducing the epithelial-mesenchymal transition (EMT) and promoting the migration and invasion of ESCC cells in vitro. Our study provides an alternative working mechanism for M2 macrophage mediated the metastasis in tumor microenvironment in ESCC.

The PA-interacting host protein nucleolin acts as an antiviral factor during highly pathogenic H5N1 avian influenza virus infection.

Polymerase acidic (PA) protein is a multifunctional regulator of influenza A virus (IAV) replication and pathogenesis. In a previous study, we reported that nucleolin (NCL) is a novel PA-interacting host protein. In this study, we further explored the role of NCL during highly pathogenic H5N1 avian influenza virus infection. We found that depletion of endogenous NCL in mammalian cells by siRNA targeting during H5N1 infection resulted in significantly increased viral polymerase activity, elevated viral mRNA, cRNA and vRNA synthesis, accelerated viral replication, and enhanced apoptosis and necrosis. Moreover, siRNA silencing of NCL significantly exacerbated the inflammatory response, resulting in increased secretion of IL-6, TNF-α, TNF-ß, CCL-4, CCL-8, IFN-α, IFN-ß and IFN-γ. Conversely, overexpression of NCL significantly decreased IAV replication. Collectively, these data show that NCL acts as a novel potential antiviral factor during H5N1 infection. Further studies exploring the antiviral mechanisms of NCL may accelerate the development of new anti-influenza drugs.

Functional expression of CCL8 and its interaction with chemokine receptor CCR3.

BACKGROUND: Chemokines and their cognate receptors play important role in the control of leukocyte chemotaxis, HIV entry and other inflammatory diseases. Developing an effcient method to investigate the functional expression of chemokines and its interactions with specific receptors will be helpful to asses the structural and functional characteristics as well as the design of new approach to therapeutic intervention. RESULTS: By making systematic optimization study of expression conditions, soluble and functional production of chemokine C-C motif ligand 8 (CCL8) in Escherichia coli (E. coli) has been achieved with approx. 1.5 mg protein/l culture. Quartz crystal microbalance (QCM) analysis exhibited that the purified CCL8 could bind with C-C chemokine receptor type 3 (CCR3) with dissociation equilibrium constant (K D) as 1.2 × 10-7 M in vitro. Obvious internalization of CCR3 in vivo could be detected in 1 h when exposed to 100 nM of CCL8. Compared with chemokine C-C motif ligand 11 (CCL11) and chemokine C-C motif ligand 24 (CCL24), a weaker chemotactic effect of CCR3 expressing cells was observed when induced by CCL8 with same concentration. CONCLUSION: This study delivers a simple and applicable way to produce functional chemokines in E. coli. The results clearly confirms that CCL8 can interact with chemokine receptor CCR3, therefore, it is promising area to develop drugs for the treatment of related diseases.