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Objectives: Lipopolysaccharide (LPS) contributed to the development and progression of type 2 diabetes mellitus (T2D), while TLR4 is reported to mediate the LPS-induced inflammation in macrophages. However, the potential molecular mechanisms for TLR4-mediated macrophages activation in T2D have not yet to be fully clarified., Methods: Type 2 diabetes models in C57BL/6J mice were generated by a combination administration of streptozotocin (STZ) and a high-fat diet (HFD). Cell proportions of M1 and M2 macrophages were analyzed using flow cytometry. Expression profiles of miR-448 and TLR 4 were determined by qRT-PCR and Western blot., Key Findings: LPS/IFN-γ significantly induced M1 polarization in macrophages characterized by the increased levels of TNF-α, IL-6, IL-12, iNOS and decreased levels of TNF-β, CCL-22, IL-10 and Arg-1, with a higher expression of toll-like receptor 4 (TLR4) in vitro. Consistently, T2D mice-derived macrophages had a significantly elevated expression of TLR4 mRNA and decreased expression of miR-448. We further confirmed that miR-448 could inhibit TLR4 expression by targeting the 3'-UTR of TLR4, rescuing the LPS/IFN-γ-induced M1 macrophage polarization., Conclusions: Taken together, our results indicated that decreased miR-448 in diabetic macrophages may contribute to LPS-induced M1 polarization by targeting TLR4, thereby modulating T2D development., (© 2018 Royal Pharmaceutical Society.)

Background: Osteoarthritis (OA) is one of the leading causes of disability worldwide. Previous history of knee injury is a significant risk factor for OA. It has been established that low-level chronic inflammation plays a pivotal role in the onset and pathogenesis of OA. The primary aim of this research was to determine if a history of knee joint injury is associated with systemic inflammation. A secondary aim was to determine if systemic inflammation is related to knee pain and joint structure., Methods: Differences in serum cytokine association networks, knee joint structural changes (MRI), and self-reported pain (i.e., Knee Injury and Osteoarthritis Outcome Score Pain subscale, KOOS PAIN and Intermittent and Constant Osteoarthritis Pain score, ICOAP) between individuals who had sustained a youth (aged 15-26 years) sport-related knee injury 3-10 years previously and age- and sex-matched controls were examined. Proteins of interest were also examined in an OA rat model., Results: Cytokine association networks were found to differ significantly between study groups, yet no significant associations were found between networks and KOOS PAIN or MRI-defined OA. A group of cytokines (MCP1/CCL2, CCL22 and TNFα) were differentially associated with other cytokines between study groups. In a pre-clinical rat OA model, serum CCL22 levels were associated with pain (r = 0.255, p = 0.045) and structural changes to the cartilage. CCL22 expression was also observed in human OA cartilage and furthermore, CCL22 induced apoptosis of isolated human chondrocytes., Discussion: These results suggest that CCL22 may be an early factor in the onset/pathogenic process of cartilage degeneration and/or related to pain OA., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

CCR4 is a chemokine receptor highly expressed by Th2 cells, and regarded as a potential therapeutic target for atopic dermatitis (AD). CCL17 and CCL22 are the CCR4 ligands, and thymic stromal lymphopoietin (TSLP) is shown to promote the expression of CCL17 and CCL22 by dendritic cells. Here, by using dibutyl phthalate (DBP), a TSLP inducer, and a hydrogel patch as a transcutaneous delivery device for ovalbumin, we developed a novel murine AD model and investigated the effect of Compound 22, a CCR4 antagonist. We first found that the mRNA expression of TSLP together with CCL17 and CCL22 was increased in the skins treated with DBP. Furthermore, the topical application of ovalbumin and DBP efficiently and rapidly induced AD-like skin lesions in BALB/c mice, which were characterized by ear swelling accompanied by infiltration of eosinophils, mast cells, and CCR4-expressing Th2 cells in the skin lesions, and elevated total IgE levels in the sera. Using this AD model, we demonstrated that cutaneous administration of Compound 22 inhibited Th2 cell infiltration and ameliorated the AD-like skin lesions. These results suggest that our AD model could be useful for studying new therapeutic strategies. Collectively, CCR4 antagonists may be a promising approach for treating AD., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Aim: CCL22, mainly synthesized by monocyte-derived alternative (M2) macrophages, belongs to the CC family of chemokines and is involved in monocyte migration and recruitment. We have previously investigated CCL22 and histamine in atherosclerosis. Here, we investigated the hypothesis that CCL22 is involved in atherosclerosis, which is influenced by the differentiation of macrophage phenotypes via histamine., Methods: CCL22 expression was investigated in human carotid arteries and coronary arteries with bare metal stents. Ligated carotid arteries of wild-type (C57BL/6J) and apolipoprotein E-deficient mice were also used as atherosclerotic models. The localization and expression of CCL22 and classical (M1)-like and M2-like macrophages in various human and mouse atherosclerotic lesions were investigated by immunohistochemical examination and quantitative real-time polymerase chain reaction. Histamine is expressed in atherosclerosis, and it induces inflammation and immunity. Human- and mice-derived monocytes and macrophages were used to examine the role of histamine in macrophage differentiation and CCL22-expression. Macrophages derived from histamine receptor 1 (H1R)- and 2 (H2R)-knockout (KO) mice were also examined., Results: Atherosclerotic lesions showed a distribution of heterogeneous macrophage phenotypes with M1-like and M2-like macrophage dominant sites. CCL22 was distributed in sparse areas of vascular smooth muscle cells (VSMCs) and associated with M2-like macrophages. Moreover, H2R stimulation was associated with CCL22 expression via M2-like macrophage dominant differentiation., Conclusion: The expression of M1- or M2-like macrophages in atherosclerosis were observed to be dependent on the distribution of VSMCs owing to differences in causal stimuli and the switching of histamine receptors via Th1 or Th2 cytokines. These results suggest that CCL22 may control atherosclerosis.

Macrophages (MΦs) are critical regulators of immune response and serve as a link between innate and acquired immunity. The precise mechanism of involvement of tissue-resident MΦs in the pathogenesis of autoimmune diseases is not clear. Here, using a murine model for Sjögren's syndrome (SS), we investigated the role of tissue-resident MΦs in the onset and development of autoimmunity. Two unique populations of CD11b high and CD11b low resident MΦs were observed in the target tissue of the SS model. Comprehensive gene expression analysis of chemokines revealed effective production of CCL22 by the CD11b high MΦs. CCL22 upregulated the migratory activity of CD4 + T cells by increasing CCR4, a receptor of CCL22, on T cells in the SS model. In addition, CCL22 enhanced IFN-γ production of T cells of the SS model, thereby suggesting that CCL22 may impair the local immune tolerance in the target organ of the SS model. Moreover, administration of anti-CCL22 antibody suppressed autoimmune lesions in the SS model. Finally, histopathological analysis revealed numerous CCL22-producing MΦs in the minor salivary gland tissue specimens of the SS patients. CCL22-producing tissue-resident MΦs may control autoimmune lesions by enhancing T cell response in the SS model. These results suggest that specific chemokines and their receptors may serve as novel therapeutic or diagnostic targets for SS.

The existing therapies of IgA nephropathy are unsatisfying. Acteoside, the main component of Rehmannia glutinosa with anti-inflammatory and anti-immune effects, can improve urinary protein excretion and immune disorder. Th22 cell is involved in IgA nephropathy progression. This study was determined to explore the effect of acteoside on mesangial injury underlying Th22 cell disorder in IgA nephropathy. Serum Th22 cells and urine total protein of patients with IgA nephropathy were measured before and after six months treatment of Rehmannia glutinosa acteoside or valsartan. Chemotactic assay and co-culture assay were performed to investigate the effect of acteoside on Th22 cell chemotaxis and differentiation. The expression of CCL20, CCL22 and CCL27 were analyzed. To explore the effect of acteoside on mesangial cell injury induced by inflammation, IL-1, IL-6, TNF-α and TGF-β1 were tested. Results showed that the proteinuria and Th22 lymphocytosis of patients with IgA nephropathy significantly improved after combination treatment of Rehmannia glutinosa acteoside and valsartan, compared with valsartan monotherapy. In vitro study further demonstrated that acteoside inhibit Th22 cell chemotaxis by suppressing the production of Th22 cell attractive chemokines, i.e., CCL20, CCL22 and CCL27. In addition, acteoside inhibited the Th22 cell proliferation. Co-culture assay proved that acteoside could relieve the overexpression of pro-inflammatory cytokines, and prevent the synthesis of TGF-β1. TGF-β1 level in mesangial cells was positively correlated with the Th22 cell. This research demonstrated that acteoside can alleviate mesangial cell inflammatory injury by modulating Th22 lymphocytes chemotaxis and proliferation.

Minocycline/tetracycline is clinically used for the treatment of bullous pemphigoid (BP), and its clinical benefits are superior to those of prednisolone when considering adverse events. Although the clinical benefits of minocycline/tetracycline are well known, its immunosuppressive mechanisms are still unclear. In this study, we investigated the immunomodulatory effects of traditional anti-BP drugs (minocycline, nicotinic acid amide, dexamethasone and cyclosporine) on CD163 + M2 macrophages in vitro, with special focus on the production of CCL18 and CCL22, both of which are produced by CD163 + M2 macrophages in the lesional skin of BP and are increased in the serum of BP patients. Minocycline decreased the production of CCL22, CCL24 and CCL26 as well as CCL2 from M2 macrophages. CCL18 from M2 macrophages was decreased by dexamethasone and cyclosporine, but not decreased by minocycline. These data suggest that the clinical benefit of minocycline is partially explained by its suppressive effects against the production of specific Th2 chemokines from M2 macrophages, which should contribute to the recruitment of Th2 cells and eosinophils in the lesional skin of BP patients., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Chemokines are important signaling molecules in the immune and nervous system. Using a fluorescence reporter mouse model, we demonstrate that the chemokine CCL17, a ligand of the chemokine receptor CCR4, is produced in the murine brain, particularly in a subset of hippocampal CA1 neurons. We found that basal expression of Ccl17 in hippocampal neurons was strongly enhanced by peripheral challenge with lipopolysaccharide (LPS). LPS-mediated induction of Ccl17 in the hippocampus was dependent on local tumor necrosis factor (TNF) signaling, whereas upregulation of Ccl22 required granulocyte-macrophage colony-stimulating factor (GM-CSF). CCL17 deficiency resulted in a diminished microglia density under homeostatic and inflammatory conditions. Further, microglia from naïve Ccl17-deficient mice possessed a reduced cellular volume and a more polarized process tree as assessed by computer-assisted imaging analysis. Regarding the overall branching, cell surface area, and total tree length, the morphology of microglia from naïve Ccl17-deficient mice resembled that of microglia from wild-type mice after LPS stimulation. In line, electrophysiological recordings indicated that CCL17 downmodulates basal synaptic transmission at CA3-CA1 Schaffer collaterals in acute slices from naïve but not LPS-treated animals. Taken together, our data identify CCL17 as a homeostatic and inducible neuromodulatory chemokine affecting the presence and morphology of microglia and synaptic transmission in the hippocampus., (© 2018 Wiley Periodicals, Inc.)

Eosinophil infiltration, a hallmark of allergic asthma, is essential for type 2 immune responses. How the initial eosinophil recruitment is regulated by lung dendritic cell (DC) subsets during the memory stage after allergen challenge is unclear. Here, we show that the initial eosinophil infiltration is dependent on lung cDC1s, which require nitric oxide (NO) produced by inducible NO synthase from lung CD24 - CD11b + DC2s for inducing CCL17 and CCL22 to attract eosinophils. During late phase responses after allergen challenge, lung CD24 + cDC2s inhibit eosinophil recruitment through secretion of TGF-β1, which impairs the expression of CCL17 and CCL22. Our data suggest that different lung antigen-presenting cells modulate lung cDC1-mediated eosinophil recruitment dynamically, through secreting distinct soluble factors during the memory stage of chronic asthma after allergen challenge in the mouse.

Background: Tipping the balance toward regulatory T cells (Tregs) through adoptive cell therapy has shown promise to induce transplantation tolerance. Although such strategy has been explored in many mice organ transplantation studies, less knowledge was available in rat systems. Furthermore, the behaviors of the transferred cells have not been well studied in real-time fashion., Methods: Tregs from naïve LEW rats were purified in two steps with the autoMACS system. Immunosuppression potential of these cells was examined with mixed lymphocyte reaction. Following stimulation by the alloantigen in vitro, the purified Tregs were infused into the recipients of vascularized composite allotransplantation (VCA). Secondary allogeneic skin grafting challenge was performed on the recipients with long-term survived VCA. Live optical imaging was performed to track luciferase-expressing Tregs following infusion to the VCA recipients. Expression of relevant molecules was studied by flow cytometry or quantitative RT-PCR., Results: Rat Tregs were enriched following two-step cell sorting and showed immunosuppressive capacity. Upon infusion into the VCA recipients that have been treated with antilymphocyte serum and short-term Cyclosporin A, the antigen-stimulated Tregs significantly prolonged VCA survival and induced donor-specific tolerance. Tracking of the infused bioluminescent Tregs showed their specific homing to lymph nodes, and then to the VCAs. Following secondary skin grafting, Tregs specifically gathered at the donor-derived skin that was not rejected by the recipient. The in vivo migratory pattern coincided with the altered expression of cell surface molecules of CD62L, CD103, CD134, and CD278, following donor-antigen stimulation. Elevated expression of CCR4 and CCL22 in allograft may also participate in recruiting Tregs for maintenance of VCA survival and promoting donor-specific tolerance., Conclusion: Sorted Tregs induced donor-specific tolerance to VCA in rats. Live cell tracking demonstrated that activated CD4+CD25+FoxP3+ Tregs targeted primarily to the lymph nodes and VCA. The Tregs migrated to the secondary grafted donor skin and contributed to the maintenance of donor-specific tolerance. These behaviors were associated with phenotypic changes induced by donor antigen stimulation. Increased expression of CCR4 and CCL22 in VCA skin may also be relevant., Competing Interests: The authors have declared that no competing interests exist.

In patients with metastatic melanoma, high blood levels of galectin-9 are correlated with worse overall survival and a bias towards a Th2 inflammatory state supportive of tumor growth. Although galectin-9 signaling through TIM3 on T cells has been described, less is known about the interaction of galectin-9 with macrophages. We aimed to determine whether galectin-9 is a binding partner of CD206 on macrophages and whether the result of this interaction is tumor-supportive. It was determined that incubation of CD68 + macrophages with galectin-9 or anti-CD206 blocked target binding and that both CD206 and galectin-9 were detected by immunoprecipitation of cell lysates. CD206 and galectin-9 had a binding affinity of 2.8 × 10 -7  m. Galectin-9 causes CD206 + macrophages to make significantly more FGF2 and monocyte chemoattractant protein (MCP-1), but less macrophage-derived chemokine (MDC). Galectin-9 had no effect on classical monocyte subsets, but caused expansion of the non-classical populations. Lastly, there was a positive correlation between increasing numbers of CD206 macrophages and galectin-9 expression in tumors, and high levels of CD206 macrophages correlated negatively with melanoma survival. These results indicate that galectin-9 binds to CD206 on M2 macrophages, which appear to drive angiogenesis and the production of chemokines that support tumor growth and poor patient prognoses. Targeting this interaction systemically through circulating monocytes may therefore be a novel way to improve local anti-tumor effects by macrophages. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Background: Tonsillitis can promote the progression of IgA nephropathy (IgAN) by aggravating immunopathologic response. Th22 cell disorder is involved in the pathogenesis of IgAN with tonsillitis. This study was determined to explore the possible mechanism of IgAN with tonsillitis underlying Th22 cell chemotaxis response to the effect of CCL20, CCL22, and CCL27., Methods: This research was conducted on 65 subjects including 16 healthy controls (HC group), 5 patients with  renal carcinoma (HTC group) and 44 patients with IgAN between 2015 and 2016. According to clinical symptoms and results of throat swab culture, patients with IgAN were divided into two groups: IgAN with tonsillitis (IgAN + tonsillitis, n = 14) and IgAN patients without tonsillitis (IgAN, n = 30). Distribution of Th22 cells in IgAN patients was determined. The expression of CCL20, CCL22, and CCL27 in both peripheral blood and kidneys of IgAN patients was investigated. Severity of pathological lesions in IgAN patients was analyzed. Coculture assay and transwell assay were performed to explore the impacts of human mesangial cells (HMC) on Th22 cell chemotaxis and Th22 cell local accumulation under hemolytic streptococcus (HS) infection., Results: Th22 cell percentages in IgAN patients increased compared with healthy controls. This increased Th22 cell percentage was positively correlated with the renal lesions of IgAN patients. Correspondingly, the expression of CCL20, CCL22, and CCL27 in renal tissue increased in IgAN patients. Tonsillitis exacerbated these overrepresentations of Th22 cells and chemokines. It was found that HMC could produce CCL20, CCL22, and CCL27. The supernatant of HMC was chemotactic for Th22 cells. This activity of HMC was stimulated by HS infection, whereas treatment of anti-CCL20, anti-CCL22, and anti-CCL27 antibodies partly blocked this chemoattractant effect of HMC., Conclusions: Tonsil infection may aggravate the renal pathological lesions of IgAN by exacerbating Th22 cell accumulation. Our data suggested a collaboration between HMC and Th22 cells in IgAN with tonsillitis underlying the effects of CCL20, CCL22, and CCL27.

Allergic asthma is a complex inflammatory disease that leads to significant healthcare costs and reduction in quality of life. Although many cell types are implicated in the pathogenesis of asthma, CD4 + T-helper cell type 2 (Th2) cells are centrally involved. We previously reported that the asthma phenotype is virtually absent in ovalbumin-sensitized and -challenged mice that lack global expression of β-arrestin (β-arr)-2 and that CD4 + T cells from these mice displayed significantly reduced CCL22-mediated chemotaxis. Because CCL22-mediated activation of CCR4 plays a role in Th2 cell regulation in asthmatic inflammation, we hypothesized that CCR4-mediated migration of CD4 + Th2 cells to the lung in asthma may use β-arr-dependent signaling. To test this hypothesis, we assessed the effect of various signaling inhibitors on CCL22-induced chemotaxis using in vitro-polarized primary CD4 + Th2 cells from β-arr2-knockout and wild-type mice. Our results show, for the first time, that CCL22-induced, CCR4-mediated Th2 cell chemotaxis is dependent, in part, on a β-arr2-dependent signaling pathway. In addition, we show that this chemotactic signaling mechanism involves activation of P-p38 and Rho-associated protein kinase. These findings point to a proinflammatory role for β-arr2-dependent signaling and support β-arr2 as a novel therapeutic target in asthma.

Background: In airway epithelium, thymus and activation-regulated chemokine (CCL17) and macrophage-derived chemokine (CCL22) are induced by defective epithelial barriers such as E-cadherin and attract the effector cells of Th2 immunity. However, the association between the epithelial barrier and CCL17 expression has not been studied in chronic rhinosinusitis with nasal polyp (CRSwNP). Thus, we aimed to evaluate the expression of CCL17 and its regulation by Th cytokines in nasal polyp (NP) epithelial cells., Methods: The expression and distribution of CCL17, CCL22, E-cadherin and/or epidermal growth factor receptor (EGFR) were measured using real-time PCR, western blot, and immunohistochemistry and compared between normal ethmoid sinus epithelium and NP epithelium. In addition, the expression level of CCL17 was determined in cultured epithelial cells treated with IL-4, IL-5, IL-13, TNF-α, and IFN-γ., Results: The expression of CCL17 was decreased in the NP epithelium compared to the epithelium of normal ethmoid sinus, whereas the expression of CCL22 was not decreased. E-cadherin was differentially distributed between the epithelium of normal ethmoid sinus and NP epithelium. EGFR was also decreased in NPs. Interestingly, the stimulation of cultured epithelial cells with Th2 cytokines, IL-4 and IL-5, resulted in an upregulation of CCL17 expression only in NP epithelial cells whereas the expression of CCL17 was increased in both normal epithelial cells and NP epithelial cells by Th1 cytokines., Conclusion: Our results suggest that the decreased expression of CCL17 in defective NP epithelium may be closely connected to NP pathogenesis and can be differentially regulated by cytokines in the NP epithelium of patients with CRSwNP.

Atopic dermatitis (AD) is a common inflammatory skin disease. Recent studies have revealed the involvement of T helper (Th)2 cytokines including Interleukin 4 (IL-4) in the pathogenesis of AD. Since epidermal Langerhans cells (LCs) and dermal myeloid dendritic cells (DCs) produce CCL17 and CCL22 that chemoattract Th2 cells, interfering with CCL17 and CCL22 production from LCs and dermal myeloid DCs may be beneficial in the treatment of AD. To investigate this, we stimulated murine bone marrow-derived DCs (BMDCs) with IL-4. IL-4 stimulation produced Ccl17 and Ccl22, which was attenuated by soybean tar Glyteer, a known aryl hydrocarbon receptor (Ahr) activator. Notably, Glyteer treatment blocked the nuclear translocation of Stat6 induced by IL-4 stimulation, suggesting that this treatment impairs the IL-4/Stat6 signaling pathway in BMDCs. Unexpectedly, Glyteer treatment did not potently upregulate the expression of Cyp1a1, a specific Ahr-responsive gene, suggesting that its inhibitory machinery for Ccl17 and Ccl22 expression is likely to operate in an Ahr-independent manner. These findings indicate that Glyteer may exhibit therapeutic potential for AD by downregulating the CCL17 and CCL22 production from DCs in a Th2-deviated microenvironment., Competing Interests: The authors have no conflicts of interest.

Purpose: To determine the role of macrophage polarization on the response of inflammatory breast cancer (IBC) cells to radiation and whether modulation of macrophage plasticity can alter radiation response., Methods and Materials: The human THP-1 monocyte cell line and primary human monocytes isolated from peripheral blood mononuclear cells were differentiated into macrophages and polarized to either an "antitumor" (M1) or a "protumor" (M2) phenotype. These polarized macrophages were co-cultured with IBC cells (SUM149, KPL4, MDA-IBC3, or SUM190) without direct contact for 24 hours, then subjected to irradiation (0, 2, 4, or 6 Gy). Interleukin (IL)4/IL13-induced activation of STAT6 signaling was measured by Western blotting of phospho-STAT6 (Tyr641), and expression of M2 polarization gene markers (CD206, fibronectin, and CCL22) was measured by quantitative polymerase chain reaction., Results: Expression of M2 polarization markers was higher in M2-polarized macrophages after IL4/IL13 treatment than in control (M0) or M1-polarized macrophages. Co-culture of IBC cell lines with M1-polarized THP-1 macrophages mediated radiosensitivity of IBC cells, whereas co-culture with M2-polarized macrophages mediated radioresistance. Phosphopeptide mimetic PM37, targeting the SH2 domain of STAT6, prevented and reversed IL4/IL13-mediated STAT6 phosphorylation (Tyr641) and decreased the expression of M2 polarization markers. Pretreatment of M2-THP1 macrophages with PM37 reduced the radioresistance they induced in IBC cells after co-culture. Targeted proteomics analysis of IBC KPL4 cells using a kinase antibody array revealed induction of protein kinase C zeta (PRKCZ) in these cells only after co-culture with M2-THP1 macrophages, which was prevented by PM37 pretreatment. KPL4 cells with stable short hairpin RNA knockdown of PRKCZ exhibited lower radioresistance after M2-THP1 co-culture., Conclusions: These data suggest that inhibition of M2 polarization of macrophages by PM37 can prevent radioresistance of IBC by down-regulating PRKCZ., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2017 John Wiley & Sons, Ltd.)

Background/purpose: Asthma is a chronic airway inflammatory disease mediated by T-helper (Th)2 cells. Montelukast (trade name Singulair) is a cysteinyl leukotriene receptor antagonist used for asthma treatment. Mirroring Th1-Th2 polarization, two distinct states of macrophages have been recognized: the classically activated (M1) macrophages and the alternatively activated (M2) macrophages. M2 polarization is known to be a response to the Th2 cytokines; however, the effects of montelukast on M2 macrophages have not been well characterized. The aim of the present study was to investigate the effects of montelukast on the expression of cytokines and chemokines in M2-like macrophages, and to explore possible intracellular signaling pathways., Methods: The human monocytic leukemia cell line THP-1 and human monocytes from healthy donors were cultured with interleukin-4 for M2 polarization, and then the cells were pretreated with or without montelukast before lipopolysaccharide (LPS) stimulation. Supernatants were collected to determine interleukin-10, I-309/CCL1, and MDC/CCL22 levels by enzyme-linked immunosorbent assay. Intracellular signaling was investigated using nuclear factor (NF)-κB inhibitors, mitogen-activated protein kinase (MAPK) inhibitors, and western blot analysis., Results: LPS-induced interleukin-10 and I-309/CCL1 expression was significantly suppressed by montelukast in THP-1-derived and human monocyte-derived M2 macrophages after LPS stimulation. MDC/CCL22 expression was only significantly suppressed by montelukast in THP-1-derived M2 macrophages after 48 hours of incubation. In western blot analysis, montelukast was able to suppress LPS-induced MAPK-phospho-p38 and NF-κB-phospho-p65 expression., Conclusion: Montelukast suppressed LPS-induced M2-related cytokines and chemokines in alternatively activated macrophages, and the effects might be mediated through the MAPK-p38 and NF-κB-p65 pathways., (Copyright © 2016. Published by Elsevier B.V.)

We investigated expressions of -CC chemokine ligand 2 (CCL2) and CCL5 in tumor samples from 147 breast cancer (BCa) patients and correlated with transforming growth factor-β (TGF-β) expression. We observed an inverse correlation of TGF-β expression with CCL2, CCL5 expression in early stages of BCa. On contrary, in late stages, CCL2, not CCL5, expression was found to be directly proportional with TGF-β expression. TGF-β stimulated MDA-MB-231 cells to express CCL2, however, downregulated both CCL2 and CCL5 in MCF-7. Interestingly, a significant swing of Th1-Th2 ratio towards Th2 is seen within the primary tumors expressing moderate/high-CCL2-low/negative-CCL5. We observed that CCL2-CCR2 interaction induces monocytes/macrophages to secrete Th2-attracting chemokine CCL22 in vitro. Therefore, CCL2 secreted from the tumor microenvironment may attract and interact with monocytes/macrophages, and favor Th2 accumulation by inducing CCL22 secretion. Study in 4T1-BALB/c BCa mouse model demonstrated significant (p<0.05) decrease in CCL2, CCL5 and CCL22 levels and reduction in lung metastatic nodule numbers upon administering TGF-β inhibitor. These findings collectively indicate that TGF-β regulates CCL2 and CCL5 expression in a stage-dependent manner during BCa progression, which in turn, determines Th1-Th2 balance within the tumor microenvironment., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Background: Mesenchymal stem cells (MSCs) are one of the most promising candidates for the treatment of major neurological disorders. Desirable therapeutic properties of MSCs include reparative and regenerative potential but, despite their proven safety, the efficacy of MSCs remains controversial. Therefore, it is essential to optimise culture protocols to enhance the therapeutic potential of the MSC secretome. Here we aimed to: assess the increase in secretion of cytokines that may induce repair, regeneration, or immunomodulation when cultured in three dimensions; study the effect of interleukin (IL)-1 priming on two- (2D) and three-dimensional (3D) cultures of MSC; and evaluate the potential use of the modified secretome using microglial-MSC co-cultures., Methods: We established a 3D spheroid culture of human MSCs, and compared the secretome in 2D and 3D cultures under primed (IL-1) and unprimed conditions. BV2 microglial cells were stimulated with lipopolysaccharide (LPS) and treated with spheroid conditioned media (CM) or were co-cultured with whole spheroids. Concentrations of secreted cytokines were determined by enzyme-linked immunosorbent assay (ELISA). Protein arrays were used to further evaluate the effect of IL-1 priming in 2D and 3D cultures., Results: 3D culture of MSCs significantly increased secretion of the IL-1 receptor antagonist (IL-1Ra), vascular endothelial growth factor (VEGF), and granulocyte-colony stimulating factor (G-CSF) compared with 2D culture, despite priming treatments with IL-1 being more effective in 2D than in 3D. The addition of CM of 3D-MSCs reduced LPS-induced tumour necrosis factor (TNF)-α secretion from BV2 cells, while the 3D spheroid co-cultured with the BV2 cells induced an increase in IL-6, but had no effect on TNF-α release. Protein arrays indicated that priming treatments trigger a more potent immune profile which is necessary to orchestrate an effective tissue repair. This effect was lost in 3D, partly because of the overexpression of IL-6., Conclusions: Increased secretion of anti-inflammatory markers occurs when MSCs are cultured in 3D, but this specific secretome did not translate into anti-inflammatory effects on LPS-treated BV2 cells in co-culture. These data highlight the importance of optimising priming treatments and culture conditions to maximise the therapeutic potential of MSC spheroids.

RNA-protein interactions with physiological outcomes usually rely on conserved sequences within the RNA element. By contrast, activity of the diverse gamma-interferon-activated inhibitor of translation (GAIT)-elements relies on the conserved RNA folding motifs rather than the conserved sequence motifs. These elements drive the translational silencing of a group of chemokine (CC/CXC) and chemokine receptor (CCR) mRNAs, thereby helping to resolve physiological inflammation. Despite sequence dissimilarity, these RNA elements adopt common secondary structures (as revealed by 2D-1H NMR spectroscopy), providing a basis for their interaction with the RNA-binding GAIT complex. However, many of these elements (e.g. those derived from CCL22, CXCL13, CCR4 and ceruloplasmin (Cp) mRNAs) have substantially different affinities for GAIT complex binding. Toeprinting analysis shows that different positions within the overall conserved GAIT element structure contribute to differential affinities of the GAIT protein complex towards the elements. Thus, heterogeneity of GAIT elements may provide hierarchical fine-tuning of the resolution of inflammation., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background Chemokines facilitate the leukocytes infiltration into the central nervous system (CNS) which is an essential step in the pathogenesis of multiple sclerosis. Ginger has also a broad anti-inflammatory properties. The aim was to evaluate the effects of ginger extract on the expression of CCL20 and CCL22 and their receptors (CCR6 and CCR4, respectively) in experimental autoimmune encephalomyelitis (EAE). Material and Methods Female C57BL/6 mice used for EAE induction by immunization with myelin oligodendroglial glycoprotein. Then, the EAE mice were treated with PBS or ginger extract, from day +3 to +30. At day 31, mice were scarified and the expression of CCL20 and CCL22 and their receptors in the spinal cord measured using real time-PCR. Results The expression of CCL20, CCL22 and CCR4 in the spinal cord of PBS-administrated EAE mice was significantly higher than healthy group (P<0.04, P<0.05 and P<0.02, respectively). In 200- and 300 mg/kg ginger extract-treated EAE mice, the expression of CCL20, CCL22 and CCR4 were significantly reduced as compared with PBS-administrated EAE group (P<0.04, P<0.01 and P<0.002 for 200 mg/kg ginger extract and P<0.01, P<0.005 and P<0.004 for 300 mg/kg ginger extract, respectively). The CCR6 expression in EAE mice treated with 200- or 300 mg/kg ginger extracts was lower than PBS-administrated EAE mice (P<0.01 and P=0.07, respectively). Conclusion Treatment of EAE mice with ginger extract down-regulate the expression of CCL20 and CCL22 and their receptors in EAE mice. The possible therapeutic potential of ginger for treatment of MS can be considered in future investigations., Competing Interests: Conflict of interest: The authors declare that there is no conflict of interests., (© Georg Thieme Verlag KG Stuttgart · New York.)

Vitiligo is a cutaneous autoimmune disease, especially devastating to patients with darker skin tones because of the contrast between unaffected and lesional skin. We studied immune cells infiltrating vitiligo skin and found very few regulatory T cells (Tregs). Vitiligo was not associated with a reduced frequency or function of circulating Tregs. To manipulate Treg function, we used mouse models expressing melanocyte-reactive TCRs, following changes in pelage color. We also isolated splenocytes to measure Treg function and evaluated cutaneous Treg abundance. Even small numbers of Tregs transferred into depigmenting mice could effectively interfere with depigmentation. The same holds true for treatment with rapamycin, readily translatable for use in human patients; such treatment may be well tolerated. Because vitiligo skin is relatively devoid of cells that produce the chemokine CCL22, whereas circulating Tregs express normal levels of its receptor CCR4, we overexpressed Ccl22 in the skin of vitiligo-prone mice to assess the resulting levels of depigmentation. Markedly reduced depigmentation was accompanied by Treg infiltration to the skin. With several options available to support a healthy balance between Tregs and effector T cells, the next challenge will be to render such treatment antigen specific and avoid general immunosuppression., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Lyme neuroborreliosis is a nervous system infectious disease caused by Borrelia burgdorferi (B. burgdorferi). It has been demonstrated that cytokines induced by B. burgdorferi are related to Lyme neuroborreliosis. Microglia is known as a key player in the immune responses that occur within the central nervous system. In response to inflammation, it will be activated and generate cytokines and chemokines. Experiments in vitro cells have showed that B. Burgdorferi membrane protein A (BmpA), a major immunogen of B. Burgdorferi, could induce Lyme arthritis and stimulate human and murine lymphocytes to produce inflammatory cytokines. In our study, the murine microglia BV2 cell line was used as a cell model to explore the stimulating effects of recombinant BmpA (rBmpA); Chemokine chip, ELISA and QPCR technology were used to measure the production of chemokines from microglial cells stimulated by rBmpA. Compared with the negative control group, CXCL2, CCL22, and CCL5 concentrations in the cell supernatant increased significantly after the rBmpA stimulation; the concentration of these chemokines increased with rBmpA concentration increasing; the mRNA expression levels of chemokines (CXCL2, CCL22, and CCL5) in murine BV2 cells increased significantly with 10 μg/mL and 20 μg/mL rBmpA stimulation; CXCL13 was not change after the rBmpA stimulation. Our study shows that chemokines, such as CXCL2, CCL22, and CCL5 were up-regulated by the rBmpA in the BV2 cells. The production of chemokines in Lyme neuroborreliosis may be mainly from microglia cells and the rBmpA may be closely related with the development of Lyme neuroborreliosis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Increased proinflammatory cytokines and chemokines might contribute to infiltration of inflammatory cells and remodeling in airways of asthma. Although these molecules may be associated with asthma, there is lack of systemic evidence showing which and how important these events are in the disease. We aimed to analyze the concentrations of these molecules in the airways and relationships with disease severity and with airway infiltration of inflammatory cells in a large cohort of asthmatics (n = 70, including 37 mild and 33 moderate/severe asthmatics) compared with controls (n = 30)., Methods: Meso scale discovery system and commercial ELISA kits were used to measure the concentrations of proinflammatory cytokines interleukin (IL)-1β; tumor necrosis factor-alpha (TNF-α); IL-6; and IL-17 and CC and CXC chemokines CCL2, CCL4, CCL11, CCL13, CCL17, CCL22, and CCL26 and CXCL8, CXCL9, CXCL10, and CXCL11 in bronchoalveolar lavage fluid of asthmatics and controls., Results: The concentrations of IL-1, TNF-α, IL-6, CXCL8 and CXCL10, and CCL4, CCL11, CCL17, and CCL22 were significantly elevated in asthmatics compared with controls (P < 0.05). The concentrations of TNF-α and CXCL8, but not others, were negatively correlated with severity of disease (lung function forced expiratory volume in 1 s) (TNF-α vs. total: r = -0.359, P= 0.002 vs. moderate/severe: r= -0.541, P= 0.001; CXCL8 vs. total: r = -0.327, P= 0.006 vs. moderate/severe: r = -0.625, P= 0.0001, respectively). In addition, concentrations of these two molecules were also correlated with the absolute numbers of infiltrating eosinophils and neutrophils in asthmatic airways., Conclusions: Increased concentrations of TNF-α and CXCL8 are associated with pathogenesis of asthma. Targeting these molecules might provide an alternative therapeutic for this disease.

Crohn's disease (CD) and ulcerative colitis (UC) are the most widely known types of inflammatory bowel diseases (IBD) and have been paid more attention due to their increasing incidence and a substantial increase in the risk of colorectal cancer (CRC). However, the phenotype and, more importantly, the function in the regulation of mucosal inflammation by different macrophages are poorly understood, even though macrophages constitute a major subset of intestinal myeloid cells. The results firstly showed that the subset of peritoneal CD11b + CD169 + macrophages increased and CCL22 expression level decreased significantly during the DSS-induced colitis. DSS-induced colitis was alleviated in CD169-DTR mice at least partially due to the deletion CD169 + macrophages. Moreover, the CCL22 expression level in peritoneal macrophages from CD169-DTR mice was much higher than that from WT mice with DSS-induced colitis. And, the cell-sorting result revealed that CD11b + CD169 + macrophage cells did not express CCL22 dominantly. Further experiment in vivo demonstrated that treatment with recombinant murine CCL22 (rmCCL22) ameliorated the clinical symptoms of DSS-induced colitis. All these data indicated that macrophage subset of CD11b + CD169 + from peritoneal cavity played critical role probably together with low levels of CCL22 in DSS-induced colitis.

Background/aim: Tumor-associated macrophages (TAMs), together with splenic CD11b + cells, help maintain the tumor microenvironment. The immunomodulatory compound imiquimod (IQM) stimulates innate immune cells, including macrophages, to induce antitumor effects. In order to elucidate the effects of IQM on the tumor microenvironment, we investigated the immunomodulatory effect of IQM during melanoma growth by using the B16F10 melanoma model., Materials and Methods: To elucidate the immunomodulatory effects of IQM on the tumor microenvironment, we isolated CD11b + TAMs and splenic CD11b + cells and evaluated the immunomodulatory effects of IQM, using the B16F10 melanoma model., Results: IQM suppressed B16F10 melanoma growth in parallel with reduction of Foxp3 + regulatory T cells (Tregs) at the tumor site, caused by the down-regulation of CCL22 production by tumor-derived and splenic CD11b + cells. Subsequently, we investigated the antitumor or tumor-loading effects of splenic CD11b + cells on B16F10 melanoma growth in vivo. B16F10 melanoma growth was accelerated by splenic CD11b + cells from untreated mice, but was inhibited by splenic CD11b + cells from IQM-treated mice. Consistent with these results, Foxp3 + Tregs were significantly decreased in tumors of mice implanted with both melanoma and splenic CD11b + cells from topical IQM-treated mice. Furthermore, intratumoral administration of anti-CCL22 antibody inhibited B16F10 melanoma growth by decreasing Treg recruitment at the tumor site., Conclusion: Our results suggest a possible mechanism for the antitumor immune response induced by IQM through tumor-associated macrophages., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Study Question: Do regulatory T cells (Tregs) contribute to angiogenesis in endometriosis?, Summary Answer: High levels of CCL17 and CCL22 cause the recruitment of Tregs, upregulate the immunosuppression of Tregs and, in turn, may promote angiogenesis in endometrial cells in synergy with proinflammatory cytokines., What Is Already Known: The peritoneal fluid of patients with endometriosis has a higher percentage of Tregs than that of normal individuals; however, the regulatory role of Tregs in the disease remains unclear., Study Design, Size, Duration: This study used primary human endometrial stromal cells (ESCs), monocytes (Mo), Tregs and human umbilical vein endothelial cells (HUVECs). All experiments were performed at least three times., Participants/materials, Setting, Methods: The migration of Tregs was evaluated by the transwell migration assay. The activation of extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase and p38 signaling pathways was examined using the In-Cell WesternTM (LI-COR®) western blot analysis system, as well as by traditional western blot analysis. Changes in the expression of CCL22, CCL17, transforming growth factor-beta 1 (TGF-β1), Interleukin (IL)-1β, tumor necrosis factor alpha (TNF-α), IL-8 and vascular endothelial growth factor (VEGF) in cell-culture supernatant were detected by ELISA. We analyzed the Tregs by multicolor flow cytometry to directly test the expression of CCR4, CD4, CD25, Foxp3, CTLA-4, CD39 and CD73., Main Results and the Role of Chance: Our results showed that ESCs-Mo co-culture produced significantly higher levels of CCL22 and CCL17 than ESCs or Mo cultured alone, and that estradiol (E2) or progesterone (P) further promoted this upregulation, demonstrating stronger chemotaxis on Tregs. The co-culture of ESCs with Mo stimulated TGF-β1 secretion by Tregs, which could be inhibited by anti-CCL22 or/and anti-CCL17 neutralizing antibodies (Abs). The expression of CCR4 by Tregs was upregulated in ESCs-Mo co-culture, especially by treatment with E2 and/or P, and this effect could be abolished by anti-CCL22 and/or anti-CCL17-neutralizing Abs. The Treg-ESCs-Mo co-culture treated with E2 (10-8 mol/l) and P (10-8 mol/l) could enhance the immunosuppression of Tregs, as proved by the elevated expression of Foxp3, CTLA-4, CD39 and CD73 on Tregs. ESCs-Mo co-culture could significantly promote the secretion of IL-1β and TNF-α. TGF-β1 from Tregs could activate p38/ERK1/2 signaling pathways in ESCs, and IL-1β and TNF-α produced by ESCs-Mo co-culture had synergistic roles with TGF-β1. TGF-β1 and the proinflammatory cytokines IL-1β or TNF-α could synergistically promote IL-8 and VEGF expression in ESCs via the p38/ERK1/2 signaling pathways. The high levels of IL-8 and VEGF in the supernatant of ESCs stimulated the angiogenesis of HUVECs., Large Scale Data: None., Limitations, Reasons for Caution: This study was only performed in vitro using eutopic ESCs, instead of ectopic cells, from endometriosis patients. Therefore, it is necessary to do further experiments to determine whether Tregs promote angiogenesis in the endometriotic milieu in synergy with proinflammatory cytokines in vivo., Wider Implications of the Findings: Co-targeting Tregs and proinflammatory cytokines may be an effective treatment for endometriosis., Study Funding/competing Interest(s): This study was supported by Ministry of Science and Technology of China 2015CB943300 to L.D.-J.; National Natural Science Foundation of China, item number 81200425 to  W.X.-Q.; National Natural Science Foundation of China, item number 81471548 to L.D.-J.; and The Research Fund for the Doctoral Program of Higher Education of China to W.X.-Q. (20110071120093). The authors have no conflicts of interest to declare., (© The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com)

Objective To analyze the expressions of T cell receptor γ subfamily variable region I, II, and III genes (TRGV I~III) and prognosis-related chemokines and their receptor genes (CCL20, CCR6, CCL17, CCL22 and CCR4) in the peripheral blood of T-cell lymphoma (non-γδ T cell type) patients, and to investigate the correlations between γδ T cells and chemokines/receptors and their relevance to disease prognosis. Methods Peripheral blood samples were collected from 27 patients with T-cell lymphoma (non-γδ T cell type) and 9 healthy individuals as controls. Expressions of TRGV I~III subfamily genes and prognosis-related chemokines/receptors (CCL20/CCR6 and CCL17/CCL22/CCR4) in the peripheral blood of patients with T-cell lymphoma and normal controls were detected by quantitative real-time PCR. Results Compared with the normal controls, the expression levels of TRGV I-III subfamily genes of patients at the stages of initial onset, relapse/refractoriness and complete remission (CR) were significantly higher, and the expression patterns of TRGV subfamilies were changed. The expression levels of CCL22 and CCR4 in initial onset group and relapse/refractory group were significantly higher than that in normal controls, while CCL17 expression was significantly lower than that in normal controls. There were unique correlation patterns in the expressions of TRGV subfamily genes and chemokine/receptor genes in the patients at the stages of initial onset, CR and relapse/refractoriness. There are low expressions of TRGV II subfamilies in newly diagnosed and relapse/refractory T-cell lymphoma patients, and their expressions are elevated after treatment. Conclusion TRGV II subfamilies might be the T cell functional subset against T-cell lymphoma, and might be associated with the outcome of the disease. And the high expression of CCL22/CCR4 might be related to the pathogenesis of T-cell lymphoma.

Previous studies have found that tumor-associated macrophages (TAMs) promote cancer progression. We previously reported that TAMs promote prostate cancer metastasis via activation of the CCL2-CCR2 axis. The CCR4 (receptor of CCL17 and CCL22) expression level in breast cancer was reported to be associated with lung metastasis. The aim of this study was to elucidate the role of CCR2 and CCR4 in prostate cancer progression. CCR2 and CCR4 were expressed in human prostate cancer cell lines and prostate cancer tissues. In vitro co-culture of prostate cancer cells and macrophages resulted in increased CCL2 and CCR2 levels in prostate cancer cells. The addition of CCL2 induced CCL22 and CCR4 production in prostate cancer cells. The migration and invasion of prostate cancer cells via enhanced phosphorylation of Akt were promoted by CCL17 and CCL22. CCR4 may be a potential candidate for molecular-targeted therapy.

Background: Achieving long-term cardiac allograft survival without continuous immunosuppression is highly desired in organ transplantation. Studies have shown that Salvia miltiorrhiza, an herb also known as danshen, improves microcirculation and is highly effective in treating coronary heart disease. Our objective is to determine whether tanshinol, an ingredient of danshen, improves cardiac allograft survival., Methods: Fully vascularized heterotopic heart transplantation was performed using BALB/c mice as donors and C57BL/6 mice as recipients, which were then treated with tanshinol and rapamycin. CD4 + FoxP3 + regulatory T cells (Tregs) were quantified by flow analyses, whereas CCL22 was measured by real-time polymerase chain reaction and Western blotting., Results: We found that tanshinol significantly delayed cardiac allograft rejection. It promoted long-term allograft survival induced by rapamycin, a mammalian target-of-rapamycin (mTOR) inhibitor. Tanshinol increased CD4 + FoxP3 + Treg numbers in cardiac allografts, but not spleens and lymph nodes, of recipient mice by enhancing chemokine CCL22 expression in cardiac allografts, especially cardiac dendritic cells. In contrast, rapamycin increased Treg numbers in both lymphoid organs and allografts, suggesting that it generally expands Tregs. Moreover, Tregs induced by rapamycin plus tanshinol were more potent in suppressing T-cell proliferation in vitro than those from untreated recipients. Neutralizing CCL22 hindered CD4 + FoxP3 + Treg migration to cardiac allografts and reversed long-term allograft survival induced by tanshinol plus rapamycin., Conclusions: Tanshinol suppresses cardiac allograft rejection by recruiting CD4 + FoxP3 + Tregs to the graft, whereas rapamycin does so via expanding the Tregs. Thus, tanshinol cooperates with rapamycin to further extend cardiac allograft survival., (Copyright © 2016 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

Background and Objectives: The objective of this study was to evaluate the topical effects of sea buckthorn (SBT) oil on atopic dermatitis (AD)-like lesions in a mouse model generated by repeated topical administration of DNCB in BALB/c mice., Methods: DNCB was applied repeatedly on the dorsal skin of mice to induce AD-like lesions. Following AD induction, SBT oil was applied daily on the dorsal skin for 4 weeks. The severity of skin lesions was examined macroscopically and histologically. We further measured the production of MDC/CCL22 and TARC/CCL17 in IFN-γ/TNF-α activated HaCaT cells., Results: Topically applied SBT oil in DNCB-treated mice ameliorated the severity score of dermatitis, decreased epidermal thickness, reduced spleen and lymph node weights, and prevented mast cell infiltration. In addition, SBT oil suppressed the Th2 chemokines TARC and MDC via dose-dependent inhibition of NF-κB, JAK2/STAT1, and p38-MAPK signaling pathways in IFN-γ/TNF-α-activated HaCaT cells., Conclusion: These results suggest that SBT oil had a beneficial effect on AD-like skin lesions, partially via inhibition of the Th2 chemokines TARC and MDC in inflamed skin., (© 2017 S. Karger AG, Basel.)

Objectives: Here, we hypothesized that Hovenia dulcis branch extract (HDB) and its active constituents ameliorates 2,4-dinitrochlorobenzene-induced atopic dermatitis (AD)-like skin lesions by modulating the T helper Th1/Th2 balance in NC/Nga mice and TNF-α- and IFN-γ-induced production of thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC) in HaCaT cells., Methods: HaCaT cells were stimulated by TNF-α/IFN-γ in the presence of HDB and its constituents. TARC and MDC were measured by ELISA and RT-PCR. For the in-vivo study, oral feeding of HDB was performed for 5 weeks with 2,4-dinitrochlorobenzene (DNCB) treatment every other day. The efficacy of HDB on parameters of DNCB-induced AD was evaluated morphologically, physiologically and immunologically., Key Findings: In-vitro studies showed that HDB and its constituents suppressed TNF-α/IFN-γ-induced production of TARC and MDC in HaCaT cells by inhibiting MAPK signalling. In-vivo studies showed that HDB regulated immunoglobulin (Ig) E and immunoglobulin G2a (IgG2a) levels in serum and the expression of mRNA for Th1- and Th2-related mediators in skin lesions. Histopathological analyses revealed reduced epidermal thickness and reduced infiltration of skin lesions by inflammatory cells., Conclusion: These results suggest that HDB inhibits AD-like skin diseases by regulating Th1 and Th2 responses in NC/Nga mice and in HaCaT cells., (© 2016 Royal Pharmaceutical Society.)

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.

Objective To explore the expressions and co-relationship of immune factors forkhead box p3 (FoxP3),chemokine (C-C motif) ligand 22 (CCL22),tumor necrosis factor receptor superfamily member 40(OX40),and SMAD family member 3 (Smad3) in cervical carcinoma and investigate their immunomodulatory roles in cervical carcinogenesis.Methods Totally 30 cases of cervical carcinoma with adjacent tissues and 20 cases of normal cervix were collected in this study. FoxP3,CCL22,OX40,and Smad3 mRNA expressions were detected by real-time polymerase chain reaction (RT-PCR). Results Compared to normal cervix,the expression levels of FoxP3 and CCL22 mRNA were elevated in neoplastic foci(P=0.000,P=0.002) and tumor periphery (P=0.048,P=0.040).The mRNAs increased modestly in high-grade squamous cell carcinoma focal(P=0.019,P=0.020) and periphery tissue (P=0.023,P=0.031) in comparison with low-grade squamous cell carcinoma. The expression levels of OX40 and Smad3 mRNA were significantly lower in neoplastic foci(P=0.000,P=0.015) than normal cervix. Compared to low-grade squamous cell carcinoma focal and periphery tissue,the mRNAs decreased moderately in high-grade squamous cell carcinoma(P=0.018,P=0.030; P=0.027,P=0.014). In both neoplastic foci and tumor periphery,the mRNA expression level of CCL22 was positively correlated with FoxP3 (r=0.353,P=0.000; r=0.307,P=0.000) but negatively correlated with OX40 (r=-0.288,P=0.031; r=-0.263,P=0.037),while OX40 was positively correlated with Smad3 (r=0.384,P=0.002;r=0.288,P=0.023). The mRNA expressions of FoxP3 and CCL22 were increased in foci and pericarcinous tissues (P=0.024,P=0.039; P=0.032,P=0.034) while Smad3 was decreased in neoplastic foci (P=0.017) in contrast to HPV negative corresponding group. Conclusion FoxP3 and CCL22 expressions increase while OX40 and Smad3 expression decrease at mRNA level in the microenvironment of cervical cancer,which may be associated with such immunological model that the immunosuppressive roles of FoxP3 and CCL22 enhance while the immunity-boosting roles of OX40 and Smad3 are impeded,contributing to the deterioration of immune disequilibrium in local site and cervical cancer carcinogenesis.

In the present study, we aimed to investigate the effects of CC chemokine ligand 22 (CCL22) and interleukin-37 (IL-37) on the proliferation and epithelial-mesenchymal transition (EMT) of non-small cell lung cancer (NSCLC) A549 cells. pDsRed-CCL22 and pEGFP-IL-37 plasmids were constructed. A549 cells were divided into six groups: the control, the pDsRed-N1 blank plasmid, the pEGFP-C1 blank plasmid, the pDsRed-CCL22 plasmid, the pEGFP‑IL-37 plasmid and the pDsRed-CCL22 + pEGFP-IL-37 plasmid group. Expression levels and localization of CCL22 and IL-37 in cells were detected by confocal microscopy. Phase-contrast microscopy was applied for observing cellular morphology. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used for detecting the mRNA levels of vimentin, N-cadherin and E-cadherin, and their protein expression levels were tested using western blotting. Constructed plasmids expressed CCL22 and IL-37, both of which had a co-localization in the cell membrane. MTT assay and cell observation results revealed that CCL22 and IL-37 inhibited the proliferation and EMT process of the A549 cells. The results of RT-qPCR and western blotting revealed that decreased vimentin and N-cadherin mRNA and protein expression levels, and increased E-cadherin mRNA and protein expression levels were found in the pDsRed-CCL22 plasmid, pEGFP-IL-37 plasmid and pDsRed‑CCL22 + pEGFP‑IL-37 plasmid groups when compared with the control, the pDsRed-N1 blank plasmid and the pEGFP-C1 blank plasmid groups (all P<0.05), and decreased vimentin and N-cadherin mRNA and protein expression levels and increased E-cadherin mRNA and protein expression levels were found in the pDsRed‑CCL22 + pEGFP‑IL-37 plasmid group when compared with the pDsRed-CCL22 plasmid and the pEGFP‑IL-37 plasmid groups (all P<0.05). CCL22 and IL-37 with a co-localization in the A549 cells inhibited the proliferation and EMT process in A549 cells. The antitumor effects of CCL22 and IL37 provide a strategy for the treatment of NSCLC.

We examined the inhibitory effect of a histamine 4 receptor (H4R) antagonist (JNJ7777120) on CCL17 and CCL22 chemokine production by human monocyte-derived Langerhans cells (MoLC) in patients with atopic dermatitis (AD) and healthy controls (HC). We confirmed the significantly higher production of both CCL17 and CCL22 in the MoLC of AD patients compared with HC. The H4R antagonist significantly inhibited the production of both CCL17 and CCL22 in the MoLC of AD patients. With regard to TLR2-signaled enhancement, peptidoglycan (PGN)-enhanced production of CCL17 and CCL22 by MoLC was inhibited by the H4R. Immunoblotting analysis demonstrated that phosphorylated p38 mitogen-activated protein kinase was induced by PGN and that this enhancement was attenuated by the application of the H4R antagonist. These data indicate that H4 signaling modulates the production of T-helper 2 chemokine in MoLC and contributes to chronic inflammation in AD patients. Our data suggest a possible novel therapeutic approach using a H4R antagonist in the treatment of patients with AD., (© 2016 Japanese Dermatological Association.)

Macrophages play an important role in the development of age-related macular degeneration (AMD). In this study, the spatial and temporal changes and the polarization of macrophages in murine laser-induced choroidal neovascularization (CNV) were investigated, and the polarized M1 and M2 biomarkers in the aqueous humors of neovascular AMD (nAMD) patients were studied. Macrophages, the main infiltrating inflammatory cells in CNV lesions, were evidenced by a significant increase in F4/80 mRNA expression and by the infiltration of F4/80+ cells in the lesions and the vicinity of laser-induced CNV. The mRNA expressions of M1-related markers were dramatically upregulated in the early stage, while the M2-related markers were slightly upregulated in the middle stage and sustained until the late stage. The results of immunostaining showed a similar early-but-transient M1 pattern and a delayed-but-sustained M2 pattern in laser-induced CNV. In addition, a higher M2/M1 ratio was found in both the murine models (Arg-1/iNOS and CCL22/CXCL10) and the aqueous humors of nAMD patients (CCL22/CXCL10) than in the controls. Our results suggested that the dynamic patterns of M1 and M2 were different in both the experimental and clinical CNV. The M2 macrophages were predominant and may play a more important role in the development of CNV.

CCR4 is a major chemokine receptor expressed by Treg cells and Th17 cells. While Treg cells are known to suppress antitumor immunity, Th17 cells have recently been shown to enhance the induction of antitumor cytotoxic T lymphocytes. Here, CCR4-deficient mice displayed enhanced tumor growth upon intradermal inoculation of B16-F10 melanoma cells. In CCR4-deficient mice, while IFN-γ+CD8+ effector T cells were decreased in tumor sites, IFN-γ+CD8+ T cells and Th17 cells were decreased in regional lymph nodes. In wild-type mice, CD4+IL-17A+ cells, which were identified as CCR4+CD44+ memory Th17, were found to be clustered around dendritic cells expressing MDC/CCL22, a ligand for CCR4, in regional lymph nodes. Compound 22, a CCR4 antagonist, also enhanced tumor growth and decreased Th17 cells in regional lymph nodes in tumor-bearing mice treated with Dacarbazine. In contrast, CCR6 deficiency did not affect the tumor growth and the numbers of Th17 cells in regional lymph nodes. These findings indicate that CCR4 is critically involved in regional lymph node DC-Th17 cell interactions that are necessary for Th17 cell-mediated induction of antitumor CD8+ effector T cells in mice bearing B16 melanoma., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Studies have long sought specific cytokines that could characterize endometriosis. Either due to variations between study designs regarding the assessment criteria for the cytokine or to low power resulting from small sample size, no factor proved to be sufficiently specific to endometriosis. In other clinical fields, a combination of several markers proved to be more powerful than a single-molecule approach. As well, in the context of endometriosis, simultaneous assessment of several cytokines present in the peritoneal fluid might help in unveiling patho-physiological processes, thus contributing to a better understanding of the condition. Therefore, the objective of this study was to investigate peritoneal fluid cytokines-derived of endometriotic women. For this retrospective case-control study, peritoneal fluid samples were obtained at laparoscopy and assessed by multiplex. Our data showed distinct patterns of peritoneal fluid cytokine concentrations in endometriotic women most notably a marked increase in EGF, FGF-2, IL-1α, MIP-1β, TGFα, PDGF-AA, PDGF-BB, MCP-3, sCD40L, Gro Pan, IL-17α, MDC and Rantes. The overall effect of fertility status revealed a significant difference for only one cytokine, namely MDC. Furthermore, FLT-3L and IP-10 levels were decreased in endometriosis patients, the former in both menstrual cycle phases and the latter in the secretory phase. A significant inverse Pearson correlation (p<0.05) was noted between pro-angiogenic cytokines EGF and FGF and the anti-angiogenic cytokine IP-10 in endometriosis patients at stages III-IV and in the secretory phase. These changes may exacerbate the local peritoneal angiogenic and proliferative reaction observed in women with endometriosis, and contributes to its pathophysiology., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Macrophages and dendritic cells (DCs) in murine spleen are essential for the maintenance of immune homeostasis by elimination of blood-borne foreign particles and organisms. It has been reported that splenic DCs, especially CD8α(+) CD103(+) DCs, are responsible for tolerance to apoptosis-associated antigens. However, the molecular mechanism by which these DCs maintain immune homeostasis by blood-borne apoptotic cell clearance remains elusive. Here, we found that the CCL22/CCR4 axis played a critical role in the maintenance of immune homeostasis during apoptotic cell clearance by splenic CD8α(+) CD103(+) DCs. The present results revealed that systemic administration of apoptotic cells rapidly induced a large number of CCL22 and CCR4(+) regulatory T (Treg) cells in the spleen of C57BL/6J mice. Further study demonstrated that CD8α(+) CD103(+) DCs dominantly produce much higher CCL22 than CD8α(+) CD103(-) DCs. Moreover, the transient deletion of CD8α(+) CD103(+) DCs caused a decrease in CCL22 levels together with CCR4(+) Treg cell percentage. Subsequently, the levels of some pro-inflammatory cytokines, such as interleukin-17 and interferon-γ in the spleen with the absence of CD8α(+) CD103(+) DCs increased in response to the administration of apoptotic cells. Hence, intravenous injection of apoptotic cells induced a subsequent increase in CCL22 expression and CCR4(+) Treg cells, which contribute to the maintenance of immune homeostasis at least partially by splenic CD8α(+) CD103(+) DCs., (© 2016 John Wiley & Sons Ltd.)

Background: Calcineurin inhibitors (CNIs) exhibit remarkable efficacy in atopic dermatitis (AD). Tacrolimus, one type of CNI, is prevalently used to treat AD. AD is a chronic inflammatory disease that exhibits predominant infiltration of T-helper type 2 (Th2) cell in the acute phase and a mixed Th1 and Th0 cell pattern in chronic lesions. Cytokines such as tumor necrosis factor-α (TNF-α), Th2-related chemokines [e.g., macrophage-derived chemokine (MDC)/CCL22 and I-309/CCL1], Th1-related chemokines [e.g., interferon γ-induced protein 10 (IP-10)/CXCL10], and neutrophil chemoattractant growth-related oncogene-α (GRO-α)/CXCL1 are involved in the pathogenesis of AD. However, whether tacrolimus modulates the expression of AD-associated cytokines and chemokines remains unknown. The intracellular mechanisms of tacrolimus are also unclear., Methods: Human monocytic cell line THP-1 cells were pretreated with tacrolimus and stimulated with lipopolysaccharide (LPS). The MDC, I-309, IP-10, GRO-α, and TNF-α concentrations of the cell supernatants were measured using enzyme-linked immunosorbent assay. Intracellular signaling was investigated using the Western blot analysis., Results: Tacrolimus suppressed the expression of MDC, IP-10, I-309, GRO-α, and TNF-α in LPS-stimulated THP-1 cells in a dose- and time-dependent manner. All three mitogen-activated protein kinase (MAPK) inhibitors and the nuclear factor-κB inhibitor suppressed LPS-induced MDC, I-309, and TNF-α expressions in THP-1 cells. Only MAPK inhibitors suppressed LPS-induced expression of IP-10 and GRO-α. Tacrolimus suppressed the LPS-induced phosphorylation of MAPK-extracellular signal-related kinase (ERK)., Conclusion: Tacrolimus suppressed LPS-induced MDC, I-309, IP-10, GRO-α, and TNF-α expressions in monocytes through the MAPK-ERK pathway; thus, tacrolimus may yield therapeutic efficacy by modulating AD-associated cytokines and chemokines., (Copyright © 2014. Published by Elsevier B.V.)

Objective: Although chemokines are critical elements for the selective attraction and activation of various leukocyte subsets in the inflammatory process, there are few findings concerning T helper (Th) 1 or Th2 chemokines in ankylosing spondylitis (AS). This study was designed to determine whether serum levels of chemokines that are preferentially chemotactic for Th1 (IFN-gamma-inducible protein-10, IP-10/CXCL10) and Th2 (thymus and activation regulated chemokine, TARC/CCL17) and (macrophage derived chemokine, MDC/CCL22) cells were elevated and whether they correlated with the clinical features in patients with AS., Methods: Forty-two patients with axial AS and 25 healthy controls were enrolled into the study. Serum levels of chemokines (IP-10, TARC and MDC) and cytokines (IFN-γ, TNF-α and IL-4) were examined using ELISA. The disease activity was evaluated by Ankylosing Spondylitis Disease Activity Score (ASDAS). Serum levels of IgG, IgA, IgM, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were measured., Results: Serum chemokine levels of IP-10, TARC and MDC were significantly higher in patients with AS than those in healthy controls. Serum cytokine levels of IFN-γ, TNF-α were also significantly increased, but the levels of IL-4 were not. Furthermore, IP-10 levels in AS patients correlated with ESP, CRP and ASDAS, while the levels of TARC and MDC did not correlate with these clinic indexes. Correlation analysis between the levels of chemokines and cytokines revealed a positive correlation between IP-10 and TNF-α. The levels of both Th1 and Th2 chemokines decreased under blockade of TNF-α., Conclusion: Our results suggest that both a Th1 chemoattractant IP-10 and Th2 chemoattractants, TARC and MDC, cooperatively play a role in the development of AS., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Purpose: The incidence and severity of allergic asthma is rising, and novel strategies to prevent or treat this disease are needed. This study investigated the effects of different mixtures of non-digestible oligosaccharides combined with Bifidobacterium breve M-16V (BB) on the development of allergic airway inflammation in an animal model for house dust mite (HDM)-induced allergic asthma., Methods: BALB/c mice were sensitized intranasally (i.n.) with HDM and subsequently challenged (i.n.) with PBS or HDM while being fed diets containing different oligosaccharide mixtures in combination with BB or an isocaloric identical control diet. Bronchoalveolar lavage fluid (BALF) inflammatory cell influx, chemokine and cytokine concentrations in lung homogenates and supernatants of ex vivo HDM-restimulated lung cells were analyzed., Results: The HDM-induced influx of eosinophils and lymphocytes was reduced by the diet containing the short-chain and long-chain fructo-oligosaccharides and BB (FFBB). In addition to the HDM-induced cell influx, concentrations of IL-33, CCL17, CCL22, IL-6, IL-13 and IL-5 were increased in supernatants of lung homogenates or BALF and IL-4, IFN-γ and IL-10 were increased in restimulated lung cell suspensions of HDM-allergic mice. The diet containing FFBB reduced IL-6, IFN-γ, IL-4 and IL-10 concentrations, whereas the combination of galacto-oligosaccharides and long-chain fructo-oligosaccharides with BB was less potent in this model., Conclusion: These findings show that synbiotic dietary supplementation can affect respiratory allergic inflammation induced by HDM. The combination of FFBB was most effective in the prevention of HDM-induced airway inflammation in mice.

Atopic dermatitis (AD) is a chronic skin disease that affects millions of people worldwide. Keratinocytes and macrophages are two cells types that play a pivotal role in the development of AD. These cells produced different chemokines and cytokines, especially thymus and activation-regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22), as well as nitric oxide (NO) through inducible nitric oxide synthase (iNOS) and COX2 in response to stimulation by TNF-α/IFN-γ and lipopolysaccharide (LPS) respectively. These mediators are thought to be crucial regulators of the pathogenesis of AD. Although several natural compounds to treat AD have been studied, the effect of Rg5:Rk1 from Panax ginseng (P. ginseng) on AD has not yet been investigated. In this study, we evaluated the inhibitory effect of Rg5:Rk1 on TNF-α/IFN-γ stimulated keratinocytes (HaCaT cells) and LPS-stimulated macrophages (RAW 264.7 cells). Enzyme-linked immunosorbent assay (ELISA) data showed that pretreatment of HaCaT cells with Rg5:Rk1 significantly reduced the TNF-α/IFN-γ-induced increase in TARC/CCL17 expression in a dose-dependent manner. In addition, Rg5:Rk1 decreased LPS-mediated nitric oxide (NO) and reactive oxygen species (ROS) production in RAW 264.7 cells. A considerable reduction in messenger RNA (mRNA) expression of the aforementioned AD mediators was also observed. Pretreatment with Rg5:Rk1 attenuated the TNF-α/IFN-γ-induced phosphorylation of p38 MAPK, STAT1, and NF-κB/IKKβ in HaCaT cells. Together, these findings suggest that ginsenoside Rg5:Rk1 may have a potential anti-AD effect by suppressing NF-κB/p38 MAPK/STAT1 signaling.

Monocytes/macrophages (MOs/MΦs) are suggested to be crucial for skeletal muscle repair and remodeling. This has been attributed to their proangiogenic potential, secretion of growth factors, and clearance of tissue debris. Skeletal muscle injury increases the number of MΦs in the tissue, and their importance for muscle regeneration has been supported by studies demonstrating that depletion of MOs/MΦs greatly impairs repair after muscle injury. Whether noninjurious exercise leads to induced expression of chemoattractants for MOs/MΦs is poorly investigated. To this end, we analyzed the expression of CX3CL1 (fractalkine), CCL2 (MCP-1), and CCL22 (MDC) in human skeletal muscle after a bout of exercise, all of which are established MO/MΦ chemotactic factors that are expressed by human myoblasts. Muscle biopsies from the musculus vastus lateralis were obtained up to 24 h after 1 h of cycle exercise in healthy individuals and in age-matched nonexercised controls. CX3CL1 increased at both the mRNA and protein level in human skeletal muscle after one bout of exercise. It was not possible to distinguish changes in CCL2 or CCL22 mRNA levels between biopsy vs. exercise effects, and the expression of CCL22 was very low. CX3CL1 mainly localized to the skeletal muscle endothelium, and it increased in human umbilical vein endothelial cells stimulated with tissue fluid from exercised muscle. CX3CL1 increased the expression of proinflammatory and proangiogenic factors in THP-1 monocytes (a human acute monocytic leukemia cell line) and in human primary myoblasts and myotubes. Altogether, this suggests that CX3CL1 participates in cross-talk mechanisms between endothelium and other muscle tissue cells and may promote a shift in the microenvironment toward a more regenerative milieu., (Copyright © 2016 the American Physiological Society.)

Fibromyalgia (FM) is a syndrome characterized by widespread chronic pain and is associated with elevated systemic inflammatory biomarkers, and an elevated innate cellular response. The aim of this study was to determine if fibromyalgia patients have altered ability to release pro-inflammatory chemokines by isolated neutrophils and monocytes. The study participants were women diagnosed with FM (n = 6) and a control group of healthy women (HW) (n = 6). Supernatant concentrations of eotaxin (CCL11), human macrophage-derived chemokine (MDC) (CCL22) and growth regulated-oncogene (GRO-α) (CXCL1) released by both monocytes and neutrophils either resting or stimulated by LPS were determined by ELISA and compared between the FM and HW groups. Both resting and activated monocytes from FM patients released more eotaxin, MDC and GRO-α than those from HW. However, there were no significant differences in the release of chemokines from neutrophils of FM patients and the ones from healthy women. In conclusion, monocytes from women with FM are deregulated, releasing higher amounts of eotaxin, MDC and GRO-α than healthy individuals. This fact does not occur in neutrophils from women with FM., (© The Author(s) 2015.)

Approximately 10% of gastric carcinomas are associated with Epstein-Barr virus (EBV) and are defined as EBV-associated gastric carcinomas (EBVaGCs). EBVaGCs are known to be accompanied by massive CD8(+) cytotoxic T cell (CTL) infiltration; however, adoptive cellular immunotherapy based on EBV-specific CD8(+) CTLs has been explored with limited success. Because regulatory T cells (Tregs) are regarded as a critical hurdle in anti-tumour immunity, we assessed the distribution of Tregs in 45 cases of EBVaGC and 45 cases of EBV-negative gastric carcinoma (EBVnGC) with matched clinicopathological parameters by immunohistochemistry. We showed that Tregs were significantly increased in EBVaGC compared to EBVnGC (15.92 ± 11.45/HPF vs. 8.45 ± 6.16/HPF, p = 0.001). In addition, we explored the accumulation mechanisms of Tregs in EBVaGC by using EBV (+) gastric carcinoma cell lines SNU719 and GT39 as ex vivo models. When peripheral blood mononuclear cells (PBMCs) were co-cultured with EBV (+) gastric carcinoma cell lines, the Treg frequency increased, and they underwent phenotypic and functional changes. The enhanced recruitment by CCL22 produced by EBVaGC cells, the decreased emigration due to CCR7 downregulation on the Treg surface, the higher proliferation rate, and the lower apoptosis rate of Tregs at tumour sites may promote the accumulation of Tregs in EBVaGC.

Background: Sanguisorba officinalis L. (SOL) is a perennial plant widely distributed in Asia, its roots are well-known as a traditional herbal medicine to treat burns, chronic intestinal infections, scalds, and inflammation in Korea. Also, the roots of SOL are used for treatment of many types of allergic skin diseases, including urticarial, eczema, and allergic dermatitis., Purpose: In this study we investigated the underlying mechanism of anti-inflammatory effect of an ethanol extract of SOL roots (ESOL)., Study Design: The ability of ESOL to inhibit inflammatory skin disorder was tested in human keratinocyte HaCaT cells., Methods: Viability test using MTT assay were used to determine non-cytotoxic concentrations of ESOL on HaCaT cells. ESOL-mediated inhibition of the tumor necrosis factor (TNF)-α/interferon (IFN)-γ-induced production of pro-inflammatory chemokines-such as macrophage-derived chemokine (MDC), regulated on activation, normal T-cell expressed and secreted (RANTES), interleukin (IL)-8, and thymus and activation regulated chemokine (TARC)-at the mRNA level was determined by real-time reverse transcription-polymerase chain reaction (RT-PCR). The ability of ESOL to reduce the expression of pro-inflammatory marker proteins was evaluated by Western blot analysis and immunocytochemistry., Results: ESOL reduced the production of MDC, RANTES, IL-8, and TARC in HaCaT cells stimulated with TNF-α/IFN-γ at both protein and mRNA levels. ESOL also suppressed the phosphorylation of signal transducer and activator of transcription (STAT)-1, extracellular signal-regulated kinase (ERK), and inhibited both nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-alpha (IκB-α) degradation and the nuclear translocation of NF-κB/p65. ESOL exerts anti-inflammatory effects by suppressing the expression of TNF-α/IFN-γ-stimulated chemokines and pro-inflammatory molecules via a blockade NF-κB, STAT-1, and ERK activation., Conclusion: Our results suggest the preventive potential of ESOL as a herbal medicine for the treatment of inflammatory skin diseases., (Copyright © 2015 Elsevier GmbH. All rights reserved.)