A Cornflower Extract Containing N-Feruloylserotonin Reduces Inflammation in Human Skin by Neutralizing CCL17 and CCL22 and Inhibiting COX-2 and 5-LOX.

Thymus and Activation-Regulated Chemokine (TARC/CCL17) and Macrophage-Derived Chemokine (MDC/CCL22) are two key chemokines exerting their biological effect via binding and activating a common receptor CCR4, expressed at the surface of type 2 helper T (Th2) cells. By recruiting Th2 cells in the dermis, CCL17 and CCL22 promote the development of inflammation in atopic skin. The aim of this research was to develop a plant extract whose biological properties, when applied topically, could be beneficial for people with atopic-prone skin. The strategy which was followed consisted in identifying ligands able to neutralize the biological activity of CCL17 and CCL22. Thus, an in silico molecular modeling and a generic screening assay were developed to screen natural molecules binding and blocking these two chemokines. N-Feruloylserotonin was identified as a neutraligand of CCL22 in these experiments. A cornflower extract containing N-feruloylserotonin was selected for further in vitro tests: the gene expression modulation of inflammation biomarkers induced by CCL17 or CCL22 in the presence or absence of this extract was assessed in the HaCaT keratinocyte cell line. Additionally, the same cornflower extract in another vehicle was evaluated in parallel with N-feruloylserotonin for cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) enzymatic cellular inhibition. The cornflower extract was shown to neutralize the two chemokines in vitro, inhibited COX-2 and 5-LOX, and demonstrated anti-inflammatory activities due mainly to the presence of N-feruloylserotonin. Although these findings would need to be confirmed in an in vivo study, the in vitro studies lay the foundation to explain the benefits of the cornflower extract when applied topically to individuals with atopic-prone skin.

CCL22 signaling contributes to sorafenib resistance in hepatitis B virus-associated hepatocellular carcinoma.

The HBV-initiated hepatocellular carcinoma (HCC) frequently develops from or accompanies long-term chronic hepatitis, inflammation, and cirrhosis, and has a poor prognosis. Sorafenib, an orally active multi-kinase inhibitor, currently the most common approved drug for first-line systemic treatment of advanced HCC, only improves overall survival of three months, suggesting the need for new therapeutic strategies. In this study, we identified that sorafenib selectively resisted in immune competent C57BL/6 mice but not nude mice. The chemokines CCL22 and CCL17 were upregulated by sorafenib, which elevated dramatically higher in HBV-associated HCC. Mechanically, sorafenib accelerates CCL22 expression via TNF-α-RIP1-NF-κB signaling pathway. Blocking CCL22 signaling with antagonist C-021 and sorafenib treated in combination can inhibit tumor growth and enhance the antitumor response, whereas no significant differences in tumor burden were observed in nude mice upon addition of C-021. These findings strongly suggest that CCL22 signaling pathway strongly contributes to sorafenib resistance in HBV-associated HCC, indicating a potential therapeutic strategy for immunological chemotherapy complementing first-line agents against HBV-associated HCC.

Azithromycin suppresses Th1- and Th2-related chemokines IP-10/MDC in human monocytic cell line.

BACKGROUND: Cytokines and chemokines play critical roles in the pathogenesis of asthma. Azithromycin, a macrolides, is frequently used in asthmatic children with lower respiratory tract infection and is reported having anti-inflammatory and immunomodulatory effects. However, the effects of azithromycin on the expression of TNF-α, Th1- and Th2-related chemokines, and neutrophil chemoattractant are unknown. We investigated the in vitro effects of azithromycin on the expression of TNF-α, Th1-related chemokine interferon-γ-inducible protein-10 (IP-10/CXCL10), Th2-related chemokine macrophage-derived chemokine (MDC/CCL22) and neutrophil chemoattractant growth-related oncogene-α (GRO-α/CXCL1) in THP-1 cells as a model for human monocytes. METHODS: THP-1 cells were pretreated with various concentrations of azithromycin before Toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS) stimulation. TNF-α, IP-10, MDC and GRO-α were measured by ELISA. Intracellular signaling was investigated by pathway inhibitors and Western blot. RESULT: Azithromycin suppressed MDC and IP-10 expression in LPS-stimulated THP-1 cells. However, azithromycin had no effect LPS-induced TNF-α and GRO-α expression. Western blotting revealed that azithromycin suppressed LPS-induced phosphorylation of mitogen-activated protein kinase (MAPK)-JNK and ERK expression, and also suppressed LPS-induced phosphorylation of nuclear factor (NF) κB-p65 expression. CONCLUSION: Azithromycin suppressed LPS-induced MDC expression via the MAPK-JNK and the NFκB-p65 pathway. Azithromycin also suppressed LPS-induced IP-10 via the MAPK-JNK/ERK and the NFκB-p65 pathway. Azithromycin may benefit asthmatic patients by suppressing chemokines expression.

Pharmacological inhibition of ß-catenin/BCL9 interaction overcomes resistance to immune checkpoint blockades by modulating Treg cells.

The Wnt/ß-catenin (ß-cat) pathway plays a critical role in cancer. Using hydrocarbon-stapled peptide technologies, we aim to develop potent, selective inhibitors targeting this pathway by disrupting the interaction of ß-cat with its coactivators B-cell lymphoma 9 (BCL9) and B-cell lymphoma 9-like (B9L). We identified a set of peptides, including hsBCL9CT-24, that robustly inhibits the activity of ß-cat and suppresses cancer cell growth. In animal models, these peptides exhibit potent anti-tumor effects, favorable pharmacokinetic profiles, and minimal toxicities. Markedly, these peptides promote intratumoral infiltration of cytotoxic T cells by reducing regulatory T cells (Treg) and increasing dendritic cells (DCs), therefore sensitizing cancer cells to PD-1 inhibitors. Given the strong correlation between Treg infiltration and APC mutation in colorectal cancers, it indicates our peptides can reactivate anti-cancer immune response suppressed by the oncogenic Wnt pathway. In summary, we report a promising strategy for cancer therapy by pharmacological inhibition of the Wnt/ß-cat signaling.

MiR-34c-5p plays a protective role in chronic obstructive pulmonary disease via targeting CCL22.

Objective: To study the role of miR-34c-5p targeting CCL22 in affecting the progression of chronic obstructive pulmonary disease (COPD). Methods: The dual-luciferase reporter gene assay was applied to verify the targeting relationship of miR-34c-5p and CCL22. The rats were randomly assigned into Control, COPD, COPD + empty plasmids, COPD + agomir, COPD + CCL22 shRNA and COPD + agomir + CCL22 groups. COPD model was built by using cigarette smoke exposure and LPS instillation. After 28 days, the pulmonary function was examined. ELISA method was used to detect TNF-α and IL-8 levels in bronchoalveolar lavage fluid (BALF), HE staining and Masson staining to observe the pathomorphological changes of lung tissues, qRT-PCR and/or Western blot to determine miR-34c-5p and CCL22 levels, and immunohistochemical staining to measure the expression of MMP-9 and TIMP-1. Results: MiR-34c-5p could target CCL22 to down-regulate its expression. Both miR-34c-5p agomir and CCL22 shRNA could reduce breathing frequency (f), airway resistance (RI), and the levels of IL-8 and TNF-α in BALF of COPD rats with increased Cydn (dynamic lung compliance) and PIF (peak inspiratory flow). Besides, the inflammatory cell infiltration, rupture of partial alveolus, enlarged alveolar cavity, and increased deposition of collagen fibers were observed in COPD rat tissues, with rise in mean linear intercept (MLI) and reduction in mean alveolar number (MAN), which could be reversed by miR-34c-5p agomir or CCL22 shRNA. Conclusion: MiR-34c-5p may promote inflammation response and maintain the protease-antiprotease balance via targeting CCL22, which provides scientific basis for the clinical treatment of COPD.

Role of Chemokine Receptor CCR4 and Regulatory T Cells in Wound Healing of Diabetic Mice.

Wound healing is a well-coordinated process that involves inflammatory mediators and cellular responses; however, if any disturbances are present during this process, tissue repair is impaired. Chronic wounds are one of the serious long-term complications associated with diabetes mellitus. The chemokine receptor CCR4 and its respective ligands, CCL17 and CCL22, are involved in regulatory T cell recruitment and activation in inflamed skin; however, the role of regulatory T cells in wounds is still not clear. Our aim was to investigate the role of CCR4 and regulatory T cells in cutaneous wound healing in diabetic mice. Alloxan-induced diabetic wild- type mice (diabetic) developed wounds that were difficult to heal, differently from CCR4-/- diabetic mice (CCR4-/- diabetic), and also from anti-CCL17/22 or anti-CD25-injected diabetic mice that presented with accelerated wound healing and fewer regulatory T cells in the wound bed. Consequently, CCR4-/- diabetic mice also presented with alteration on T cells population in the wound and draining lymph nodes; on day 14, these mice also displayed an increase of collagen fiber deposition. Still, cytokine levels were decreased in the wounds of CCR4-/- diabetic mice on day 2. Our data suggest that the receptor CCR4 and regulatory T cells negatively affect wound healing in diabetic mice.

Allopurinol suppresses expression of the regulatory T-cell migration factors TARC/CCL17 and MDC/CCL22 in HaCaT keratinocytes via restriction of nuclear factor-κB activation.

Recent studies have shown that sparse distribution of regulatory T cells (Tregs) in the skin might be involved in the onset of severe cutaneous adverse drug reactions such as Stevens-Johnson syndrome and toxic epidermal necrolysis. Treg migration toward epithelial cells is regulated by certain chemokines, including TARC/CCL17 and MDC/CCL22. In this study, we analyzed the effect of allopurinol (APN), a drug known to cause severe adverse reactions, on the expression of factors affecting Treg migration and the mechanisms involved. APN inhibited the tumor necrosis factor (TNF)-α- and interferon (IFN)-γ-associated expression of TARC/CCL17 and MDC/CCL22 mRNA in HaCaT cells in a dose-dependent manner. Consistent with this, APN also suppressed TNF-α- and IFN-γ-induced production of TARC/CCL17 and MDC/CCL22 proteins and the migration of C-C chemokine receptor type 4-positive cells. Activity of the transcription factors NF-κB and STAT1, which are involved in TARC/CCL17 and MDC/CCL22 expression, was also investigated. APN inhibited activation of NF-κB, but not that of STAT1. Furthermore, it restricted p38 MAPK phosphorylation. These results suggest that APN inhibits TNF-α- and IFN-γ-induced TARC/CCL17 and MDC/CCL22 production through downregulation of p38 MAPK and NF-κB signaling, resulting in the sparse distribution of Tregs in the skin of patients with APN-associated Stevens-Johnson syndrome/toxic epidermal necrolysis.

Fucoidan inhibits CCL22 production through NF-κB pathway in M2 macrophages: a potential therapeutic strategy for cancer.

In tumor microenvironment, macrophages as a polarized M2 population promote tumor progression via releasing multiple cytokines and chemokines. A brown seaweed fucose-rich polysaccharide, fucoidan has antitumor activity and immune modulation through affecting tumor cells and lymphocytes. Here, we focused on the effect of fucoidan on macrophages especially M2 subtype. Our results demonstrated that fucoidan down-regulated partial cytokines and chemokines, especially a M2-type chemokine CCL22. Furthermore, fucoidan inhibited tumor cells migration and CD4+ T lymphocytes, especially Treg cells, recruitment induced by M2 macrophages conditioned medium through suppression of CCL22. Mechanismly, fucoidan inhibited CCL22 via suppressing p65-NF-κB phosphorylation and nuclear translocation. In addition, p38-MAPK and PI3K-AKT also affected the expression of CCL22 through differential modulation of NF-κB transcriptional activity. Taken together, we reveal an interesting result that fucoidan can inhibit tumor cell migration and lymphocytes recruitment by suppressing CCL22 in M2 macrophages via NF-κB-dependent transcription, which may be a novel and promising mechanism for tumor immunotherapy.

Mesenchymal stem cells abrogate experimental asthma by altering dendritic cell function.

Mesenchymal stem cells (MSCs) have been investigated in the treatment of numerous autoimmune diseases. However, the immune properties of MSCs on the development of asthma have remained to be fully elucidated. Airway dendritic cells (DCs) have an important role in the pathogenesis of allergic asthma, and disrupting their function may be a novel therapeutic approach. The present study used a mouse model of asthma to demonstrate that transplantation of MSCs suppressed features of asthma by targeting the function of lung myeloid DCs. MSCs suppressed the maturation and migration of lung DCs to the mediastinal lymph nodes, and thereby reducing the allergen-specific T helper type 2 (Th2) response in the nodes. In addition, MSC-treated DCs were less potent in activating naive and effector Th2 cells and the capacity of producing chemokine (C-C motif) ligand 17 (CCL17) and CCL22, which are chemokines attracting Th2 cells, to the airways was reduced. These results supported that MSCs may be used as a potential treatment for asthma.

Sesamin suppresses macrophage-derived chemokine expression in human monocytes via epigenetic regulation.

BACKGROUND: Chemokines play important roles in the pathogenesis of asthmatic inflammation. Sesamin, a class of phytoestrogen isolated from sesame seed Sesamum indicum, is recently regarded as an anti-inflammatory agent. However, the effects of sesamin on asthma-related chemokines are unknown. To this end, we investigated the effects of sesamin on the expression interferon-γ-inducible protein-10 (IP-10/CXCL10), macrophage-derived chemokine (MDC/CCL22), growth-related oncogene-α (GRO-α/CXCL1) and tumor necrosis factor (TNF)-α in human monocytes. METHODS: Cells were pretreated with sesamin before lipopolysaccharide (LPS) stimulation. IP-10, MDC, GRO-α and TNF-α were measured by ELISA. Involved receptors and intracellular signaling were investigated by receptor antagonists, pathway inhibitors, western blotting and chromatin immunoprecipitation. RESULTS: Sesamin suppressed LPS-induced MDC in THP-1 and human primary monocytes. Sesamin suppressed LPS-induced IP-10 in THP-1 cells, but not human primary monocytes. Sesamin had no effects on LPS-induced GRO-α and TNF-α expression in THP-1 and human primary monocytes. The suppressive effect of sesamin on MDC was reversed by the estrogen receptor (ER) and peroxisomal proliferator-activated receptor (PPAR)-α antagonists. Sesamin suppressed LPS-induced phosphorylation of mitogen-activated protein kinase (MAPK)-p38 and nuclear factor kappa B (NFκB)-p65. Sesamin suppressed histone H3/H4 acetylation in the MDC promoter region. CONCLUSION: Sesamin suppressed LPS-induced MDC expression via the ER, the PPAR-α, the MAPK-p38 pathway, the NFκB-p65 pathway and the epigenetic regulation. Sesamin may have therapeutic potential in preventing and treating asthma.

(+)-Nootkatone inhibits tumor necrosis factor α/interferon γ-induced production of chemokines in HaCaT cells.

Chemokines are important mediators of cell migration, and thymus and activation-regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22) are well-known typical inflammatory chemokines involved in atopic dermatitis (AD). (+)-Nootkatone is the major component of Cyperus rotundus. (+)-Nootkatone has antiallergic, anti-inflammatory, and antiplatelet activities. The purpose of this study was to investigate the effect of (+)-nootkatone on tumor necrosis factor α (TNF-α)/interferon γ (IFN-γ)-induced expression of Th2 chemokines in HaCaT cells. We found that (+)-nootkatone inhibited the TNF-α/IFN-γ-induced expression of TARC/CCL17 and MDC/CCL22 mRNA in HaCaT cells. It also significantly inhibited TNF-α/IFN-γ-induced activation of nuclear factor kappa B (NF-κB), p38 mitogen-activated protein kinase (MAPK), and protein kinase Cζ (PKCζ). Furthermore, we showed that PKCζ and p38 MAPK contributed to the inhibition of TNF-α/IFN-γ-induced TARC/CCL17 and MDC/CCL22 expression by blocking IκBα degradation in HaCaT cells. Taken together, these results suggest that (+)-nootkatone may suppress TNF-α/IFN-γ-induced TARC/CCL17 and MDC/CCL22 expression in HaCaT cells by inhibiting of PKCζ and p38 MAPK signaling pathways that lead to activation of NF-κB. We propose that (+)-nootkatone may be a useful therapeutic candidate for inflammatory skin diseases such as AD.

Quercetagetin inhibits macrophage-derived chemokine in HaCaT human keratinocytes via the regulation of signal transducer and activator of transcription 1, suppressor of cytokine signalling 1 and transforming growth factor-ß1.

BACKGROUND: Inflammatory chemokines, such as macrophage-derived chemokine (MDC/CCL22), are elevated in the serum and lesioned skin of patients with atopic dermatitis (AD), and are ligands for C-C chemokine receptor 4, which is predominantly expressed on T helper 2 lymphocytes, basophils and natural killer cells. We have previously reported that quercetagetin has an inhibitory activity on inflammatory chemokines, which is induced by interferon (IFN)-γ and tumour necrosis factor (TNF)-α, occurring via inhibition of the signal transducer and activator of transcription 1 (STAT1) signal. OBJECTIVES: To investigate the specific mechanisms of quercetagetin on the STAT1 signal. METHODS: We confirmed the inhibitory activity of quercetagetin on MDC and STAT1 in HaCaT keratinocytes. The interaction between STAT1 and IFN-γR1 was investigated using immunoprecipitation. The small interfering RNA approach was used to investigate the role of suppressor of cytokine signalling 1 (SOCS1) and transforming growth factor (TGF)-ß1 induced by quercetagetin. RESULTS: Quercetagetin inhibited the expression of MDC at both the protein and mRNA levels in IFN-γ- and TNF-α-stimulated HaCaT human keratinocytes. Moreover, quercetagetin inhibited the phosphorylation of STAT1 through upregulation of SOCS1. Increased expression of SOCS1 disrupted the binding of STAT1 to IFN-γR1. Furthermore, quercetagetin augmented the expression of TGF-ß1, which is known to modulate the immune response and inflammation. CONCLUSIONS: These results suggest that quercetagetin may be a potent inhibitor of the STAT1 signal, which could be a new molecular target for anti-inflammatory treatment, and may thus have therapeutic applications as an immune modulator in inflammatory diseases such as AD.

Casuarinin suppresses TARC/CCL17 and MDC/CCL22 production via blockade of NF-κB and STAT1 activation in HaCaT cells.

Casuarinin is a naturally occurring tannin that is isolated from the leaves of Hippophae rhamnoides. It has been shown to have anti-oxidant, anti-cancer, anti-viral, and anti-inflammatory activities. The aim of this study was to investigate the possible mechanism by which casuarinin inhibits TNF-α/IFN-γ-induced Th2 chemokines expression in the human keratinocytes cell line HaCaT. We found that casuarinin suppressed TNF-α/IFN-γ-induced expression of TARC and MDC mRNA and protein in HaCaT cells. Casuarinin significantly inhibited TNF-α/IFN-γ-induced activation of NF-κB, STAT1, and p38 MAPK. Furthermore, we observed that p38 MAPK contributes to inhibition of TNF-α/IFN-γ-induced TARC and MDC production by blocking NF-κB and STAT1 activation in HaCaT cells. Taken together, these results suggest that casuarinin may exert anti-inflammatory responses by suppressing TNF-α/IFN-γ-induced expression of TARC and MDC via blockage of p38 MAPK activation and subsequent activation of NF-κB and STAT1. We propose that it could therefore be used as a therapeutic agent against inflammatory skin diseases.

Fluvastatin inhibits expression of the chemokine MDC/CCL22 induced by interferon-gamma in HaCaT cells, a human keratinocyte cell line.

BACKGROUND AND PURPOSE: The macrophage-derived chemokine (MDC/CCL22) is a prototypic Th2-type chemokine intimately involved in Th2-skewed allergic diseases, such as atopic dermatitis and asthma. The statins (3-hydroxy-3-methyl glutaryl coenzyme A reductase inhibitors) have been demonstrated to relieve allergic inflammation. However, the immunological effects and mechanisms of statins against atopic dermatitis remain unknown, at least in vitro. This study aimed to define how different statins affect MDC expression in HaCaT cells, a human keratinocyte cell line. EXPERIMENTAL APPROACH: To measure the effects of statins on MDC expression in HaCaT cells, we used a cell viability assay, reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay and Western blotting analyses. KEY RESULTS: Fluvastatin, but not atorvastatin or simvastatin, inhibited MDC expression induced by interferon (IFN)-gamma and NF-kappaB activation. A NF-kappaB inhibitor, but not a STAT1 inhibitor, suppressed MDC expression in HaCaT cells. Further, inhibition of p38 mitogen-activated protein kinases (MAPKs) significantly suppressed IFN-gamma-induced MDC expression and NF-kappaB activation. Interestingly, fluvastatin suppressed IFN-gamma-induced NF-kappaB activation in parallel with p38 MAPK phosphorylation. CONCLUSIONS AND IMPLICATIONS: These results indicate that fluvastatin inhibited expression of the CC chemokine MDC induced by IFN-gamma in HaCaT cells, by inhibiting NF-kappaB activation via the p38 MAPK pathway. This blockade of a Th2 chemokine by fluvastatin may suppress the infiltration of Th2 cells into skin lesions and lessen the skin inflammation seen in atopic dermatitis, suggesting a potential therapeutic use of fluvastatin for this condition.

Suppressive effects of Bifidobacterium longum on the production of Th2-attracting chemokines induced with T cell-antigen-presenting cell interactions.

In human trials, Bifidobacterium longum BB536 alleviates subjective symptoms of Japanese cedar pollinosis, an IgE-mediated type I allergy caused by exposure to Japanese cedar, and significantly suppresses the increase of plasma thymus- and activation-regulated chemokine (TARC) associated with pollen dispersion. In the present study, we investigated the suppressive effects of BB536 on the production of T helper type 2 (Th2)-attracting chemokines, such as TARC and macrophage-derived chemokine (MDC), together with the mechanisms of their production. Murine splenocytes were cultured with heat-killed BB536, and the levels of Th2-attracting chemokines in the supernatants were measured. TARC and MDC were produced in cultures without stimulation, and the production was significantly suppressed by BB536. These chemokines were produced by antigen-presenting cells (APCs) of splenocytes stimulated with an anti-CD40 antibody. Furthermore, TARC production was induced with granulocyte macrophage colony-stimulating factor that was produced by T cells and dendritic cells. BB536 suppressed MDC production induced with the anti-CD40 antibody by APCs from the spleen, mesenteric lymph nodes (MLNs) and Peyer's patches, and it suppressed TARC production by APCs from the spleen and MLNs. These results indicate that BB536 suppresses the production of Th2-attracting chemokines induced by the T cell-APC interaction, suggesting a novel mechanism for alleviating symptoms of allergic disorders by probiotics.

Tax-inducible production of CC chemokine ligand 22 by human T cell leukemia virus type 1 (HTLV-1)-infected T cells promotes preferential transmission of HTLV-1 to CCR4-expressing CD4+ T cells.

Adult T cell leukemia is a mature CD4+ T cell malignancy which predominantly expresses CCR4 and is etiologically associated with human T cell leukemia virus type 1 (HTLV-1). Because HTLV-1 transmission depends on close cell-cell contacts, HTLV-1-infected T cells may preferentially interact with CCR4+CD4+ T cells for efficient viral transmission. In terms of gene expression and protein secretion, we found a strong correlation between HTLV-1 Tax oncoprotein and CCL22, a CCR4 ligand, in HTLV-1-infected T cells. Transient Tax expression in an HTLV-1-negative T cell line activated the CCL22 promoter and induced CCL22. Additionally, tax gene knockdown by small interference RNA reduced CCL22 expression in the infected T cells. These findings indicate that CCL22 is a cellular target gene of Tax. In chemotaxis assays, the culture supernatants of HTLV-1-infected T cells selectively attracted CCR4+CD4+ T cells in PBMCs. This was blocked by pretreating the supernatants with anti-CCL22 Ab or PBMCs with a synthetic CCR4 antagonist. In coculture experiments, primary CCR4+CD4+ T cells significantly adhered to Tax-expressing cells. This adhesion was blocked by the CCR4 antagonist or pertussis toxin. Interestingly, CCR4 was redistributed to the contact region, and in some cases, this was accompanied by a polarized microtubule-organizing center, which is an indicator of virological synapse formation, in the infected T cells. Finally, anti-CCL22 Ab treatment also blocked HTLV-1 transmission to primary CD4+ T cells in coculture experiments with HTLV-1 producer cells. Thus, HTLV-1-infected T cells produce CCL22 through Tax and selectively interact with CCR4+CD4+ T cells, resulting in preferential transmission of HTLV-1 to CCR4+CD4+ T cells.