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Ma huang tang (MHT) is a traditional herbal medicine comprising six medicinal herbs and is used to treat influenza-like illness. However, the effects of MHT on inflammatory skin diseases have not been verified scientifically. We investigated determining the inhibitory effects of MHT against inflammation responses in skin using HaCaT human keratinocyte cells. We found that MHT suppressed production of thymus and activation-regulated chemokine (TARC/CCL17), macrophage-derived chemokine (MDC/CCL22), regulated on activation of normal T-cell expressed and secreted (RANTES/CCL5), and interleukin-8 (IL-8) in tumor necrosis factor-α (TNF-α) and interferon-γ- (IFN-γ-) stimulated HaCaT cells. Consistently, MHT suppressed the mRNA expression of TARC, MDC, RANTES, and IL-8 in TNF-α and IFN-γ-stimulated cells. Additionally, MHT inhibited TNF-α and IFN-γ-stimulated signal transducer and activator of transcription 1 (STAT1) phosphorylation in a dose-dependent manner and nuclear translocation in HaCaT cells. Our finding indicates that MHT inhibits production and expression of inflammatory chemokines in the stimulated keratinocytes by downregulating STAT1 phosphorylation, suggesting that MHT may be a possible therapeutic agent for inflammatory skin diseases.

Objective: The aim of this study was to explore the tape strip sampling technique in the assessment of stratum corneum levels of inflammatory mediators in a clinical trial setting. Methods: Thirty-eight inflammatory mediators were analyzed by a multiplex-assay in the stratum corneum, collected by adhesive tapes before and after 6 weeks of therapy, in mild and moderate atopic dermatitis (AD) patients (n = 90). Treatment was a ceramide- and magnesium-containing emollient. Results: Twenty-four mediators could quantitatively be determined. The Th2 mediators interleukin (IL)-4, IL-13, CCL2 (monocyte chemotactic protein-1), CCL22 (macrophage-derived chemokine), and CCL17 [thymus and activation-regulated chemokine (TARC)] were significantly decreased after therapy as well as IL-1β, IL-2, IL-8 (CXCL8), IL-10, acute-phase protein serum amyloid A, C-reactive protein, and vascular adhesion molecule-1. The decrease of CCL17 and IL-8 was correlated with the decrease in disease severity in a subgroup of moderate AD individuals. Conclusion: Stratum corneum tape stripping offers a minimally invasive approach for studying local levels of immunomodulatory molecules in the skin. CCL17 (TARC) and IL-8 were found to be the most promising biomarkers of AD and might be useful for investigating the course of skin diseases and the effect of local therapy.

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.

Ethnopharmacological relevance Xanthii fructus (XF) is an herb widely used in medicine for the treatment of a variety of inflammatory pathologies. 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). However, the anti-inflammatory mechanisms of XF have not been elucidated in tumor necrosis factor (TNF)-α/interferon (IFN)-γ-stimulated HaCaT cells. The purpose of this study was to investigate the effect of XF on TNF-α/IFN-γ-induced production of TARC/CCL17 and MDC/CCL22 in HaCaT cells. Materials and methods HaCaT cells were stimulated by TNF-α/IFN-γ in the presence of XF. TRAC/CCL17 and MDC/CCL22 productions were monitored by ELISA on the cell culture supernatant and by RT-PCR on total RNA extract. We use immunoblotting to analyze the effect of XF on activation of the NF-κB, STAT1 and MAPK pathways. Results Ethanol extract of XF (EXF) inhibited mRNA expression and production of TARC/CCL17 and MDC/CCL22 induced by TNF-α/IFN-γ in a dose-dependent manner. It also significantly inhibited TNF-α/IFN-γ-induced activation of NF-κB, STAT1 and p38-MAPK. Furthermore, we observed that p38-MAPK contributes to the inhibition of TNF-α/IFN-γ-induced TARC/CCL17 and MDC/CCL22 production by blocking NF-κB and STAT1 activation in HaCaT cells. Conclusions These results demonstrate that developing therapeutic applications XF for the prevention of inflammatory skin diseases are feasible.

Human CMV (HCMV) uses members of the hematopoietic system including neutrophils for dissemination throughout the body. HCMV encodes a viral chemokine, vCXCL-1, that is postulated to attract neutrophils for dissemination within the host. The gene encoding vCXCL-1, UL146, is one of the most variable genes in the HCMV genome. Why HCMV has evolved this hypervariability and how this affects the virus’ dissemination and pathogenesis is unknown. Because the vCXCL-1 hypervariability maps to important binding and activation domains, we hypothesized that vCXCL-1s differentially activate neutrophils, which could contribute to HCMV dissemination, pathogenesis, or both. To test whether these viral chemokines affect neutrophil function, we generated vCXCL-1 proteins from 11 different clades from clinical isolates from infants infected congenitally with HCMV. All vCXCL-1s were able to induce calcium flux at a concentration of 100 nM and integrin expression on human peripheral blood neutrophils, despite differences in affinity for the CXCR1 and CXCR2 receptors. In fact, their affinity for CXCR1 or CXCR2 did not correlate directly with chemotaxis, G protein-dependent and independent (β-arrestin-2) activation, or secondary chemokine (CCL22) expression. Our data suggest that vCXCL-1 polymorphisms affect the binding affinity, receptor usage, and differential peripheral blood neutrophil activation that could contribute to HCMV dissemination and pathogenesis.

We evaluated the utility of chemokine MCP-3 and MDC/CCL22 as molecular adjuvants of DNA vaccines for botulinum neurotoxin serotype A (BoNT/A) in a Balb/c mouse model. Notably, the immunogenicity of the DNA vaccine against BoNT/A was not enhanced using a fusion of the AHc-C antigen with the MCP-3 or MDC/CCL22. Nevertheless, the potency of the DNA vaccine was significantly modulated and enhanced by co-administration of the AHc-C antigen with MCP-3 or MDC/CCL22. This strategy elicited high levels of humoral immune responses and protection against BoNT/A. The enhanced potency was further boosted by co-administration of the AHc-C antigen with both MCP-3 and MDC/CCL22 in Balb/c mice, but not by co-administration of AHc-C antigen with the MCP-3-MDC/CCL22 fusion. Co-immunization with both the MCP-3 and MDC/CCL22 constructs induced the highest levels of humoral immunity and protective potency against BoNT/A. Our results indicated that MCP-3 and MDC/CCL22 are effective molecular adjuvants of the immune responses induced by the AHc-C-expressing DNA vaccine when delivered by co-administration of the individual chemokines, but not when delivered in the form of a chemokine/antigen fusion. Thus, we describe an alternative strategy to the design and optimization of DNA vaccine constructs based on co-administration of the antigen with the chemokine rather than in the form of a chemokine/antigen fusion.

Macrophage-derived chemokine, C-C motif chemokine 22 (MDC/CCL22), is one of the inflammatory chemokines that controls the movement of monocytes, monocyte-derived dendritic cells, and natural killer cells. Serum and skin MDC/CCL22 levels are elevated in atopic dermatitis, which suggests that the chemokines produced from keratinocytes are responsible for attracting inflammatory lymphocytes to the skin. A major signaling pathway in the interferon-γ (IFN-γ)-stimulated inflammation response involves the signal transducers and activators of transcription 1 (STAT1). In the present study, we investigated the anti-inflammatory effect of dieckol and its possible action mechanisms in the category of skin inflammation including atopic dermatitis. Dieckol inhibited MDC/CCL22 production induced by IFN-γ (10 ng/mL) in a dose dependent manner. Dieckol (5 and 10 μM) suppressed the phosphorylation and the nuclear translocation of STAT1. These results suggest that dieckol exhibits anti-inflammatory effect via the down-regulation of STAT1 activation.

Regulatory T cells (Tregs) have been shown to regulate the immune response and to control the defense against infection in periapical lesions, but the underlying mechanisms by which Tregs are recruited to these lesions remain unknown. Here we demonstrate that expression of the gene encoding CCL22 (also known as macrophage-derived chemokine), the major chemoattractant that recruits Tregs, is upregulated in periapical tissue during the progression of experimental periapical lesions; this upregulation positively correlated with the number of Tregs that accumulated in the lesions. In terms of mechanism, we determined that lipopolysaccharide (LPS) up-regulates Ccl22 expression in macrophages by suppressing miR-34a. These findings suggest that the LPS-miR-34a-CCL22 axis may contribute to the recruitment of Tregs in periapical lesions, providing a potential therapeutic target for controlling this disease.

Background: Atopic dermatitis (AD) is the most common chronic inflammatory skin disease in infancy with a complex pathology. In adults, the clinical severity of AD has been associated with increases in T helper cell type (Th) 2, Th22, and Th17 serum markers, including high levels of CC chemokine ligand (CCL) 17 and CCL22 chemokines.Objective: To explore the possible association between serum chemokine levels and AD severity in infants with moderate-to-severe AD and elevated immunoglobulin E (IgE).Subjects and methods: Serum samples (n = 41) obtained from a randomized, double-blind, and clinical dietary intervention study were used to study biomarkers in infants with AD. Baseline- and post-intervention samples (4 months) were used, six chemokines and nine ratios thereof were analyzed using Luminex and correlated to AD severity. In the initial study, the infants were randomized to receive extensively hydrolyzed whey-based formula without (control) or with short-chain galacto-oligosaccharides/long-chain fructo-oligosaccharides (9:1) and Bifidobacterium breve M-16V (active).Results: 31 Infants up to 11 months of age, with an objective-SCORAD score (oSCORAD) ≥ 20 and elevated total-IgE and/or specific-IgE levels were included. In time, the median oSCORAD decreased in both groups by -8 (control, p Conclusion: While this study is small and exploratory in nature, these data contribute to immune biomarker profiling and understanding of AD in infants.

Immune molecules, which have been found to be important in tumor microenvironment, seem prospective in tumor therapy, but they are still not effective enough to use in clinical practice. C-C motif chemokine 22 (CCL22) exists in various malignancies and correlates with migration of regulatory T cells, but its clinical significance in gastric cancer is still unclear. In this study, a combined data set of 466 patients with gastric cancer after surgical resection, comprised of a discovery (n = 319) and a validation data set (n = 147), was enrolled. CCL22 expression was assessed by immunohistochemical staining and we evaluated prognostic values of CCL22 staining and clinical outcomes with use of Kaplan-Meier curve and Multivariate Cox regression analysis. Positive CCL22 expression predicted adverse overall survival independent of traditional pathological grade. Multivariate analysis defined CCL22 and TNM stage as two independent prognostic factors for overall survival. Besides, in patients with TNM stage II/III disease, the rate of overall survival was higher among patients with CCL22-positive tumors who were treated with 5-fluorouracil based adjuvant chemotherapy than that among those who were not (P = 0.012, P < 0.001 and P < 0.001, in discovery, validation and combined data set). But for these with CCL22-negative tumors, whether to undergo adjuvant chemotherapy showed no statistical significance (P = 0.595, P = 0.085 and P = 0.252, respectively). To conclude, CCL22 was identified as an independent adverse prognostic immunobiomarker for patients with gastric cancer after surgery, which is associated with tumor-infiltrating immunocytes and could be incorporated into TNM staging system to redefine a high-risk subgroup who were more likely to benefit from 5-fluorouracil based adjuvant chemotherapy.

Sesame is an important oilseed crop, which has been used as a traditional health food to ameliorate the prevention of various diseases. We evaluated the changes in the anti-allergic activities of sesame by bioconversion. SDS-PAGE of non-fermented sesame proteins showed major allergen bands, while that of fermented sesame showed only a few protein bands. Additionally, we investigated the effectiveness of fermented sesame by bioconversion in tumor necrosis factor-α (TNF-α)- and interferon-γ (IFN-γ)-induced HaCaT cells. In HaCaT cells, fermented sesame inhibited the mRNA expression of interleukin-6 (IL-6) and interleukin-1β (IL-1β), thymus and macrophage-derived chemokine (MDC/CCL22), activation-regulated chemokine (TARC/CCL17), and intercellular adhesion molecule-1 (ICAM-1). Moreover, fermented sesame inhibited the activation of nuclear factor-κB (NF-κB) and signal transducer and activator of transcription 1 (STAT1). Fermented sesame exerts anti-allergic effects by suppressing the expression of chemokines and cytokines via blockade of NF-κB and STAT1 activation.

Erythropoietin (EPO) is recognized for neuroprotective and angiogenic effects and has been associated with aging and neovascular age-related macular degeneration (AMD). We hypothesized that systemic EPO facilitates the development of choroidal neovascularization (CNV). Wild type mice expressed murine EPOR (mWtEPOR) in RPE/choroids at baseline and had significantly increased serum EPO after laser treatment. To test the role of EPO signaling, we used human EPOR knock-in mice with the mWtEPOR gene replaced by either the human EPOR gene (hWtEPOR) or a mutated human EPOR gene (hMtEPOR) in a laser-induced choroidal neovascularization (LCNV) model. Loss-of-function hWtEPOR mice have reduced downstream activation, whereas gain-of-function hMtEPOR mice have increased EPOR signaling. Compared to littermate controls (mWtEPOR), hMtEPOR with increased EPOR signaling developed larger CNV lesions. At baseline, hMtEPOR mice had increased numbers of macrophages, greater expression of macrophage markers F4/80 and CD206, and following laser injury, had greater expression of cytokines CCL2, CXCL10, CCL22, IL-6, and IL-10 than mWtEPOR controls. These data support a hypothesis that injury from age- and AMD-related changes in the RPE/choroid leads to choroidal neovascularization through EPOR-mediated cytokine production.

Lyme disease, caused by the bacterial spirochete Borrelia burgdorferi, is a tick-borne zoonosis. Lyme neuroborreliosis is a principal manifestation of Lyme disease and its pathogenesis remains incompletely understood. Recent studies have demonstrated that Borrelia burgdorferi lipoproteins caused similar inflammatory effects as exhibited in Lyme neuroborreliosis. Basic membrane protein A (BmpA) is one of the dominant lipoproteins in the Borrelia burgdorferi membrane. In addition, nuclear factor κ-B (NF-κB) modulates the regulation of gene transcription associated with immunity and inflammation; however, in unstimulated cells, NF-κB is combined with the inhibitor of NF-κB (IκB-β). Therefore, it was hypothesized that NF-κB may be associated with BmpA-induced inflammation and the occurrence of Lyme neuroborreliosis. Therefore, the aim of the present study was to investigate the role that NF-κB serves in the signaling pathway of rBmpA-induced inflammatory chemokines. The present study measured the expression levels of NF-κB, IκB-β and inflammatory chemokines following recombinant BmpA (rBmpA) stimulation of murine microglia BV2 cells. Following stimulation with rBmpA, concentrations of pro-inflammatory cytokines including C-X-C motif chemokine 2, C-C motif chemokine (CCL) 5 and CCL22 were determined by ELISA analysis. Reverse transcription-quantitative polymerase chain reaction and western blotting were used to detect the expression levels of NF-κB p65 and IκB-β. The data demonstrated that concentrations of these chemokines in cell supernatants increased significantly following rBmpA stimulation. NF-κB was overexpressed, but IκB-β expression was significantly decreased. In conclusion, these results suggested that NF-κB serves an important stimulatory role in the signaling pathway of rBmpA-induced inflammatory chemokines in BV2 cells.

Toxoplasma gondii is a major source of congenital disease worldwide, but the cellular and molecular factors associated with its vertical transmission are largely unknown. In humans, the placenta forms the key interface between the maternal and fetal compartments and forms the primary barrier that restricts the hematogenous spread of microorganisms. Here, we utilized primary human trophoblast (PHT) cells isolated from full-term placentas and human midgestation chorionic villous explants to determine the mechanisms by which human trophoblasts restrict and respond to T. gondii infection. We show that placental syncytiotrophoblasts, multinucleated cells that are in direct contact with maternal blood, restrict T. gondii infection at two distinct stages of the parasite lytic cycle—at the time of attachment and also during intracellular replication. Utilizing comparative transcriptome sequencing (RNA-seq) transcriptional profiling, we also show that human placental trophoblasts from both the second and third trimesters respond uniquely to T. gondii infection compared to trophoblast cell lines, typified by the upregulation of several immunity-related genes. One of the most differentially induced genes was the chemokine CCL22, which relies on the secretion of a parasite effector(s) either during or after invasion for its induction. Collectively, our findings provide new insights into the mechanisms by which the human placenta restricts the vertical transmission of T. gondii at early and late stages of human pregnancy and demonstrate the existence of at least two interferon-independent pathways that restrict T. gondii access to the fetal compartment., IMPORTANCE Toxoplasma gondii is a major source of congenital disease worldwide and must breach the placental barrier to be transmitted from maternal blood to the developing fetus. The events associated with the vertical transmission of T. gondii are largely unknown. Here, we show that primary human syncytiotrophoblasts, the fetus-derived cells that comprise the primary placental barrier, restrict T. gondii infection at two distinct stages of the parasite life cycle and respond to infection by inducing a unique immunomodulatory transcriptional profile. Collectively, our findings provide important insights into the mechanisms by which human syncytiotrophoblasts restrict T. gondii infection at early and late stages of human pregnancy, identify both permissive and resistant human placental cell types, and identify the placenta-enriched signaling pathways induced in response to infection.

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 naive 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 naive 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 naive 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.

Macrophages undergo activation by pathophysiological stimuli to pro-inflammatory and bactericidal, or wound-healing and anti-inflammatory phenotypes, termed M1 or M2, respectively. Dysregulation of the M1-M2 balance is often associated with inflammatory diseases. Therefore, mechanisms of macrophage polarization may reveal new drug targets. We profiled six compounds with claimed modulatory effects on macrophage polarization using peripheral blood monocyte-derived macrophages. Based on the distinct mRNA or protein expression in macrophages stimulated either with M1 [lipopolysaccharide (LPS) + interferon-g, IFNg] or M2 interleukin-4 (IL-4) stimuli, we selected a combination of M1 (IL1v, tumor necrosis factor-a,TNFa, CC chemokine receptor 7, CCR7 and CD80) and M2 (chemokine (C-C motif) ligand 22, CCL22, CD200R and mannose receptor C type 1, MRC1) markers to monitor drug effects on ""M1 polarization"" or cells ""pre-polarized to M1"". Azithromycin (25-50 mM), tofacitinib (2.5-5 mM), hydroxychloroquine (40 µg/ml) and pioglitazone (15-60 mM) exhibit an anti-inflammatory profile because they downregulated M1 markers and upregulated some M2 markers when given both before and after M1 polarization. Lovastatin given before M1 polarization downregulated M1 marker genes but enhanced the M1 phenotype in macrophages pre-polarized with LPS and IFNg. Methotrexate (1.25-5 mM) did not modulate macrophage polarization. We have, thus, established a test system suitable to identify novel compounds or repurposed drugs that modulate inflammatory macrophage plasticity. Compounds with potential to reduce expression of molecules involved in inflammatory T cell activation (IL-1v, TNFa, CD80), while enhancing production of a major chemokine involved in recruitment of Tregs (CCL22) may be of interest for treating chronic inflammatory diseases.

Summary Background Vitiligo is an autoimmune depigmentation disease, and defects in regulatory T cells (Tregs) have been proposed in the pathogenesis of generalized vitiligo (GV). However, the role of programmed cell death (PD)1+ Tregs has not been studied. Objectives To investigate the status of Tregs, PD1+ Tregs and associated parameters in active GV (aGV) during the first episode of disease attack and to establish the clinical correlation. Methods The percentages of circulating Tregs, PD1+ Tregs and CD3+CD4+PD1+ T cells were evaluated in 50 patients with aGV and 51 controls. Expression levels of FOXP3, TGFB1, CTLA4 and genes for chemokine receptors (CCR4, CCR7) and their ligands (CCL21, CCL22) were quantified in peripheral blood and in lesional, perilesional, nonlesional and normal skin sections. The corresponding proteins were immunolocalized in tissue of aGV. Results The percentage of Tregs was decreased (P = 0·001) and that of PD1+ Tregs increased (P = 0·001) in peripheral blood of patients with aGV compared with controls. The abundance of TGFB1 and CCL21 mRNA was significantly decreased in the peripheral blood of patients with aGV. Significant differences in forkhead box P3, transforming growth factor-β and CCL21 protein expression were found in skin sections. Conclusions Deficiency in Treg frequency and decreased expression of Treg-associated parameters (TGFB and CCL21) suggested a possible defect in Tregs that may alter their suppression function and skin homing in aGV. The increased PD1+ Tregs suggests that the PD1/PD ligand pathway may be involved in aGV and may have a role in Treg exhaustion. Further study is required to delineate the effect of PD1 in regulating Treg function in aGV.

Defects in antigen presenting cell function have been implicated in glioma immunosuppression. We measured peripheral CCL22, a dendritic cell/macrophage derived T cell trafficking chemokine, in sera from 1,208 glioma cases and 976 controls to assess whether it might provide a biomarker of glioma risk, survival and immune dysfunction. Cluster models were used to examine the relationship between CCL22 and glioma risk. Patient survival was assessed using Cox regression models. We also examined the relationship between CCL22 levels and CD4 cell counts, as well as allergy history and IgE levels. CCL22 levels were significantly lower among glioma cases compared with controls (Mean ± SEM: 1.23 ± 0.03 ng/mL in cases vs. 1.60 ± 0.03 ng/mL in controls, p < 0.0001) and this difference remained significant even after controlling for other covariates in the cluster models (highest quartile versus lowest Odds Ratio = 0.21, p < 0.0001). CD4 cell counts were positively correlated with CCL22 in glioma cases (Spearman r(2) = 0.51, p < 0.01) and were significantly lower in cases compared with controls. Higher CCL22 levels were associated with longer survival in all cases combined and in GBM cases (hazard ratio(allcases) = 0.81; 95% CI: 0.72-0.91, p = 0.0003). CCL22 levels were not associated with IgE level or self-reported allergies. Circulating CCL22 levels are related to both glioma risk and survival duration independent of age, histology, grade and IDH mutation status. CCL22 should be considered a marker of immune status with potential prognostic value.

A traditional herbal formula Jakyakgamcho-tang (JYGCT; Paeonia lactiflora and Glycyrrhiza uralensis) has been used for treatment of backache, muscle pain, acute abdominal pain, neuralgia, bronchial asthma, and painful peripheral neuropathy in Oriental medicine. We report on our experiments using the HaCaT human keratinocyte cell line showing that a traditional herbal formula JYGCT has inhibitory effects on inflammatory responses in skin. Stimulation with tumour necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) caused a significant increase in the production of the following chemokines: thymus- and activation-regulated chemokine (TARC)/CCL17; macrophage-derived chemokine (MDC)/CCL22; regulated on activation, normal T-cell expressed and secreted (RANTES)/CCL5; and interleukin-8 (IL-8) in HaCaT cells. By contrast, treatment with JYGCT extract significantly reduced the production of TARC, MDC, RANTES, and IL-8, but caused no cytotoxicity, compared with TNF-α and IFN-γ-treated control cells. Consistently, JYGCT extract downregulated the mRNA expression of TARC, MDC, RANTES, and IL-8 induced by TNF-α and IFN-γ in a dose-dependent manner. In addition, TNF-α and IFN-γ markedly increased the phosphorylation of signal transducer and activator of transcription 1 (STAT1) and the nuclear translocation of nuclear factor kappa B (NF-κB) in HaCaT cells. By contrast, TNF-α and IFN-γ-induced activation of STAT1 and NF-κB activation was inhibited by JYGCT treatment in a dose-dependent manner. Our data indicate that JYGCT attenuates TNF-α and IFN-γ-mediated chemokine production by targeting the STAT1 and NF-κB signalling in keratinocytes. Our findings suggest that JYGCT has potential as a therapeutic drug candidate for the treatment of inflammatory skin diseases.

Summary Filarial infection in humans is initiated when a mosquito deposits third-stage parasite larvae (L3) in the skin. Langerhans cells (LCs) and dermal dendritic cells (DDCs) are the first cells that the parasite encounters, and L3s must evade these highly effective antigen-presenting cells to establish infection. To assess LC and DDC responses to L3 in human skin, we employed three models of increasing physiologic relevance: in vitro-generated LCs, epidermal blister explants and full-thickness human skin sections. In vitro-generated LCs expressed TLR1-10 and robustly produced IL-6 and TNF-α in response to PolyI:C, but pre-exposure to L3s did not alter inflammatory cytokine production or TLR expression. L3s did not modulate expression of LC markers CDH1, CD207, or CD1a, or the regulatory products TSLP or IDO in epidermal explants or in vitro-generated LC. LC, CD14+ DDC, CD1c+ DC and CD141+ DC from human skin sections were analysed by flow cytometry. While PolyI:C potently induced CCL22 production in LC, CD1c+ DC, and CD141+ DC, and IL-10 production in LC, L3s did not modulate the numbers of or cytokine production by any skin DC subset. L3s broadly failed to activate or modulate LCs or DDCs, suggesting filarial larvae expertly evade APC detection in human skin.

Gyejibokryeong-hwan (GJBRH; Keishi-bukuryo-gan in Japan and Guizhi Fuling Wan in China) is a traditional herbal formula comprising five medicinal herbs and is used to treat climacteric syndrome. GJBRH has been shown to exhibit biological activity against diabetes, diabetic nephropathy, atherosclerosis, ischemia, and cancer. However, there is no scientific evidence of its activities against skin inflammation, including atopic dermatitis. We used the HaCaT human keratinocyte cell line to investigate the effects of GJBRH on skin inflammation. No significant cytotoxicity was observed in cells treated with GJBRH up to a concentration of 1000 µg/mL. Exposure to the proinflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) significantly increased HaCaT cell production of the following chemokines: macrophage-derived chemokine (MDC)/CCL22; regulated on activation, normal T-cell expressed and secreted (RANTES)/CCL5; and interleukin-8 (IL-8). In contrast, GJBRH significantly reduced the production of MDC, RANTES, and IL-8 compared with control cells simulated with TNF-α and IFN-γ. Consistently, GJBRH suppressed the mRNA expression of MDC, RANTES, and IL-8 in TNF-α and IFN-γ-treated cells. Treatment with GJBRH markedly inhibited phosphorylation of signal transducer and activator of transcription 1 (STAT1) in HaCaT cells stimulated with TNF-α and IFN-γ. Our findings indicate that GJBRH impairs TNF-α and IFN-γ-mediated inflammatory chemokine production and STAT1 phosphorylation in keratinocytes. We suggest that GJBRH may be a potent therapeutic agent for inflammatory skin disorders.

In vitiligo, gradual cutaneous depigmentation and cytotoxic T cell activity against melanocytes is accompanied by a paucity of regulatory T cells (Tregs) in vitiligo patient skin, indicating that autoimmune responses are not adequately held in check. Thus we sought a means to repopulate patient skin with Tregs. We hypothesized that enhanced expression of CCL22 can promote Treg skin homing to suppress depigmentation. The mouse Ccl22 gene was cloned into an expression vector and resulting DNA was used for gene gun treatment. Two spontaneous depigmentation models with different kinetics of melanocyte loss were utilized, expressing tyrosinase-reactive and gp100-reactive T cell receptor transgenes. Mice were subjected to 5 gene gun treatments 6 days apart, scanned for depigmentation weekly thereafter and monitored for activation and proliferation of relevant T cells and for Treg infiltration to the skin. Significantly reduced depigmentation 2 weeks after treatment was accompanied by a markedly increased abundance of Tregs in the skin at the expense of melanocyte reactive, TCR transgenic T cells as well as by reduced proliferation and reduced IFN-γ production in response to cognate peptide. Continued treatment may be necessary for sustained, local immunosuppression. These findings suggest that topical CCL22 may be used for the treatment of vitiligo.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). It affects more than two million people worldwide, mainly young adults, and may lead to progressive neurological disability. Chemokines and their receptors have been shown to play critical roles in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a murine disease model induced by active immunization with myelin proteins or transfer of encephalitogenic CD4+ T cells that recapitulates clinical and neuropathological features of MS. Chemokine ligand-receptor interactions orchestrate leukocyte trafficking and influence multiple pathophysiological cellular processes, including antigen presentation and cytokine production by dendritic cells (DCs). The C-C class chemokines 17 (CCL17) and 22 (CCL22) and their C-C chemokine receptor 4 (CCR4) have been shown to play an important role in homeostasis and inflammatory responses. Here, we provide an overview of the involvement of CCR4 and its ligands in CNS autoimmunity. We review key clinical studies of MS together with experimental studies in animals that have demonstrated functional roles of CCR4, CCL17, and CCL22 in EAE pathogenesis. Finally, we discuss the therapeutic potential of newly developed CCR4 antagonists and a humanized anti-CCR4 antibody for treatment of MS.

The focus of this study was to determine which chemokine receptors are present on oral fibroblasts and whether these receptors influence proliferation, migration, and/or the release of wound healing mediators. This information may provide insight into the superior wound healing characteristics of the oral mucosa. The gingiva fibroblasts expressed 12 different chemokine receptors (CCR3, CCR4, CCR6, CCR9, CCR10, CXCR1, CXCR2, CXCR4, CXCR5, CXCR7, CX3CR1, and XCR1), as analyzed by flow cytometry. Fourteen corresponding chemokines (CCL5, CCL15, CCL20, CCL22, CCL25, CCL27, CCL28, CXCL1, CXCL8, CXCL11, CXCL12, CXCL13, CX3CL1, and XCL1) were used to study the activation of these receptors on gingiva fibroblasts. Twelve of these fourteen chemokines stimulated gingiva fibroblast migration (all except for CXCL8 and CXCL12). Five of the chemokines stimulated proliferation (CCL5/CCR3, CCL15/CCR3, CCL22/CCR4, CCL28/CCR3/CCR10, and XCL1/XCR1). Furthermore, CCL28/CCR3/CCR10 and CCL22/CCR4 stimulation increased IL-6 secretion and CCL28/CCR3/CCR10 together with CCL27/CCR10 upregulated HGF secretion. Moreover, TIMP-1 secretion was reduced by CCL15/CCR3. In conclusion, this in-vitro study identifies chemokine receptor-ligand pairs which may be used in future targeted wound healing strategies. In particular, we identified the chemokine receptors CCR3 and CCR4, and the mucosa specific chemokine CCL28, as having an predominant role in oral wound healing by increasing human gingiva fibroblast proliferation, migration, and the secretion of IL-6 and HGF and reducing the secretion of TIMP-1.

The aim of this study was to investigate the modulation of plasma CXCL10, CCL20, CCL22, CCL2, CCL17 and CCL24 levels in HIV-positive patients grouped according to extreme phenotypes of progression to AIDS, and at different stages of HIV infection. HIV-positive individuals with extreme phenotypes of AIDS progression (n = 58) at different clinical stages (chronic individuals, both pre-HAART and under-HAART) and HIV-negative controls (n = 20) were evaluated. Additionally, HIV-positive individuals that initiated HAART with > 350 CD4+ T-cells/mm3 were compared with those who initiated treatment with 350 (initiated HAART with > 350 CD4+ T-cells) (1096 vs. 360.33 pg/mL, p = 0.0101). Normalisation of CXCL10 levels seems to depend on the CD4+ T-cell nadir at HAART initiation. CCL20 levels were higher in chronic SPs, SPs pre-HAART, SPs HAART and RPs HAART compared with the HIV-negative controls, indicating persistent CCL20 expression. In conclusion, our results indicate that CXCL10 levels are a hallmark in the clinical evolution of HIV infection. However, our results must be verified in a study evaluating a larger number of AIDS progressors.

Macrophages are key participants in melanoma growth and survival. In general, macrophages can be classified as M1 or M2 activation phenotypes. Increasing evidence demonstrates that melanoma exosomes also facilitate tumor survival and metastasis. However, the role of melanoma exosomes in directly influencing macrophage function is poorly understood. Herein, we investigated the hypothesis that natural melanoma exosomes might directly influence macrophage polarization. To explore this hypothesis, ELISA, RT-qPCR, and macrophage functional studies were performed in vitro using an established source of melanoma exosomes (B16-F10). ELISA results for melanoma exosome induction of common M1 and M2 cytokines in RAW 264.7 macrophages, revealed that melanoma exosomes do not polarize macrophages exclusively in the M1 or M2 direction. Melanoma exosomes induced the M1 and M2 representative cytokines TNF-α and IL-10 respectively. Further assessment, using an RT-qPCR array with RAW 264.7 and primary macrophages, confirmed and extended the ELISA findings. Upregulation of markers common to both M1 and M2 polarization phenotypes included CCL22, IL-12B, IL-1β, IL-6, i-NOS, and TNF-α. The M2 cytokine TGF-β was upregulated in primary but not RAW 264.7 macrophages. Pro-tumor functions have been attributed to each of these markers. Macrophage functional assays demonstrated a trend toward increased i-NOS (M1) to arginase (M2) activity. Collectively, the results provide the first evidence that melanoma exosomes can induce a mixed M1 and M2 pro-tumor macrophage activation phenotype.

Together with regulatory T cells (Tregs), tumor-associated macrophages (TAMs) play roles in maintaining the tumor microenvironment. Although cytotoxic antimelanoma drugs such as dacarbazine (DTIC), nimustine hydrochloride (ACNU) and vincristine (VCR) have been used for the treatment of malignant melanoma as adjuvant therapy in Japan, the detailed mechanisms of their immunomodulatory effects are not fully understood. As the majority of TAMs are alternatively activated M2 macrophages that favour tumor development, the aim of this study was to elucidate the immunomodulatory effects of these reagents on human monocyte-derived M2 macrophages. First, mRNA expressions and protein production of immune checkpoint molecules, PD-L1 and chemokines by CD163+ CD206+ M2 macrophages derived from peripheral blood mononuclear cells were investigated to determine the immunomodulatory effects of DTIC, ACNU, and VCR. DTIC and VCR significantly decreased PD-L1 mRNA expression, which was confirmed by flow cytometry. Moreover, the mRNA expression and production of CCL22 were significantly decreased by DTIC, which suggested that DTIC might suppress the recruitment of Tregs in the tumor site. Furthermore, the decreased expression of PD-L1 and production of CCL22 were validated in vivo, using the B16F10 mouse melanoma model, leading to abrogation of the suppressive function of T-cell proliferation. The present report suggests one of the possible antimelanoma mechanisms of DAV combination chemotherapy for melanoma patients.

Toxoplasma gondii is a major source of congenital disease worldwide, but the cellular and molecular factors associated with its vertical transmission are largely unknown. In humans, the placenta forms the key interface between the maternal and fetal compartments and forms the primary barrier that restricts the hematogenous spread of microorganisms. Here, we utilized primary human trophoblast (PHT) cells isolated from full-term placentas and human mid-gestation chorionic villous explants to determine the mechanisms by which human trophoblasts restrict and respond to T. gondii infection. We show that placental syncytiotrophoblasts, multinucleated cells that are in direct contact with maternal blood, restrict T. gondii infection at distinct stages of the parasite lytic cycle—at the time of attachment and also during intracellular replication. Utilizing comparative RNAseq transcriptional profiling, we also show that human placental trophoblasts at both mid- and late-stages of gestation induce the chemokine CCL22 in response to T. gondii infection, which relies on the secretion of parasite effector(s). Collectively, our findings provide new insights into the mechanisms by which the human placenta restricts the vertical transmission of T. gondii at early and late stages of human pregnancy, and demonstrate the existence of at least two interferon-independent pathways that restrict T. gondii access to the fetal compartment.Significance statementToxoplasma gondii is a major source of congenital disease worldwide and must breach the placental barrier to be transmitted from maternal blood to the developing fetus. The events associated with the vertical transmission of T. gondii are largely unknown. Here, we show that primary human syncytiotrophoblasts, the fetal-derived cells that comprise the primary placental barrier, restrict T. gondii infection at two distinct stages of the parasite life cycle and respond to infection through the induction of the chemokine CCL22. Collectively, our findings provide important insights into the mechanisms by which human syncytiotrophoblasts restrict T. gondii infection at early and late stages of human pregnancy and identify the placental-enriched signaling pathways induced in response to infection.

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.

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.

Background In the damaged tissue of systemic sclerosis (SSc) patients, the presence of immune inflammatory infiltrate, characterized by T-cells and macrophages, represents an important early pathological event in the fibrotic process (1). In the macrophage population, the alternatively activated (M2) macrophages were observed in both peripheral blood and damage tissues of SSc patients and they participate in the fibrotic process exerting profibrotic effects, primarily by the production and release of transforming growth factor β1 (TGFβ1) (1,2). These cells are characterized by the expression of specific phenotype markers, such as mannose receptor (CD206) and scavenger receptors (CD204 and CD163), and the production of specific chemokines (i.e. macrophage derived chemokine, CCL-22) (3). Endothelin-1 (ET1) plays an important role in the fibrotic process of SSc by inducing the profibrotic phenotype in endothelial cells and fibroblasts (4). Objectives To evaluate the ability of ET1 to upregulate the profibrotic gene expression profile characterizing the M2 macrophages in cultured monocytes/macrophages isolated from SSc patients. Methods Human monocytes were isolated from peripheral blood mononuclear cells of 4 SSc patients (mean age 59±11years, 2 females and 2 males, treated with vasodilator drugs) using a monocyte isolation kit. Cultured cells were maintained in RPMI growth medium for 24hrs and then treated with ET1 (100nM) for 6 days or treated for 1hr with ET receptor antagonist (ETA/BRA, bosentan 10μM) before stimulation with ET1. Cultured cells maintained in RPMI growth medium were used as untreated cells. Gene and protein expression of M2 phenotype markers and TGFβ1 were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting (WB). Statistical analysis was carried out using a Mann-Whitney non-parametric test. Results In cultured SSc macrophages, ET1 induced the upregulation of the gene expression of CD206, CD204, CD163, and CCL22 compared to untreated cells. Moreover, ET1 increased the TGFβ1 gene expression compared to untreated cells. ETA/BRA partially contrasted the gene expression of CD204 and CD206. However, ETA/BRA antagonized the ET1-mediated increase in the gene expression of CD163, CCL22 and TGFβ1. WB confirmed the results obtained by qRT-PCR. Conclusions Preliminary results showed the ability of ET1 to stimulate a profibrotic M2 phenotype in cultured human SSc macrophages characterized by the overexpression of TGFβ1, and this process is partially contrasted by the action of ETA/BRA. References Higashi-Kuwata N et al. Arthrit Res Ther. 2010;12:R128. Stifano G et al. Curr Rheumatol Rep. 2016;18:2. Wynn TA et al. Nat Rev. 2013;496:445–55. Cipriani P et al. J Rheumatol. 2015;42:1808–16. Disclosure of Interest None declared

We explore potential dysregulation of macrophage phenotypes in COPD pathogenesis through integrated study of human small airway tissue, bronchoalveolar lavage (BAL) and an experimental murine model of COPD. We evaluated human airway tissue and BAL from healthy controls, normal lung function smokers (NLFS), and COPD subjects. Both small airways and BAL cells were immunohistochemically stained with anti-CD68 for total macrophages and with anti-CD163 for M2, and anti-iNOS for M1 macrophages. Multiplex ELISA measured BAL cytokines. Comparable cigarette smoke-induced experimental COPD mouse model was assessed for relevant mRNA profiles. We found an increase in pro-inflammatory M1s in the small airways of NLFS and COPD compared to controls with a reciprocal decrease in M2 macrophages, which remained unchanged among pathological groups. However, luminal macrophages showed a dominant M2 phenotype in both NLFS and COPD subjects. BAL cytokine skewed towards an M2 profile with increase in CCL22, IL-4, IL-13, and IL-10 in both NLFS and COPDs. The mouse-model of COPD showed similar increase in mRNA for M2 markers. Our finding suggests abnormal macrophage switching in both mucosal and luminal areas of COPD patients, that strongly associated with cytokine balance. There may be potential for beneficial therapeutic cytokine manipulation of macrophage phenotypes in COPD.

International audience; We previously identified the chemokine receptor CCR4 as part of the molecular signature of melanoma brain metastasis. The aim of this study was to determine the functional significance of CCR4 in melanoma brain metastasis. We show that CCR4 is more highly expressed by brain metastasizing melanoma cells than by local cutaneous cells from the same melanoma. Moreover, we found that the expression of CCR4 is significantly higher in paired clinical specimens of melanoma metastases than in samples of primary tumors from the same patients. Notably, the expression of the CCR4 ligands, Ccl22 and Ccl17 is upregulated at the earliest stages of brain metastasis, and precedes the infiltration of melanoma cells to the brain. In-vitro, CCL17 induced migration and transendothelial migration of melanoma cells. Functionally, human melanoma cells over-expressing CCR4 were more tumorigenic and produced a higher load of spontaneous brain micrometastasis than control cells. Blocking CCR4 with a small molecule CCR4 antagonist in-vivo, reduced the tumorigenicity and micrometastasis formation of melanoma cells. Taken together, these findings implicate CCR4 as a driver of melanoma brain metastasis.

ObjectivesThe previously observed inverse association between hog farming and risk of lung cancer in the Agricultural Health Study (AHS) has been attributed to endotoxin exposure, the levels of which are particularly high in industrial hog confinement facilities. We conducted an investigation to explore the potential biological mechanisms underlying this association, as well as other immunological changes associated with hog farming.MethodsSerum immune marker levels were measured using a multiplexed bead-based assay in 61 active hog farmers and 61 controls matched on age, phlebotomy date and raising cattle. Both groups comprised non-smoking male AHS participants from Iowa. We compared natural log-transformed marker levels between hog farmers and controls using multivariate linear regression models.ResultsCirculating levels of macrophage-derived chemokine (CCL22), a chemokine previously implicated in lung carcinogenesis, were reduced among hog farmers (17% decrease; 95% CI −28% to −4%), in particular for those with the largest operations (>6000 hogs: 26% decrease; 95% CI −39% to −10%; ptrend=0.002). We also found that hog farmers had elevated levels of other immune markers, including macrophage inflammatory protein-3 alpha (MIP-3A/CCL20; 111% increase, 95% CI 19% to 273%), basic fibroblast growth factor (FGF-2; 93% increase, 95% CI 10% to 240%) and soluble interleukin-4 receptor (12% increase, 95% CI 1% to 25%), with particularly strong associations for MIP-3A/CCL20 and FGF-2 in winter.ConclusionsThese results provide insights into potential immunomodulatory mechanisms through which endotoxin or other exposures associated with hog farming may influence lung cancer risk, and warrant further investigation with more detailed bioaerosol exposure assessment.

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.

Objectives Targeting the granulocyte-macrophage colony-stimulating factor (GM-CSF) pathway holds great potential in the treatment of inflammatory diseases. Mavrilimumab, a human monoclonal GM-CSF receptor-α antibody, has demonstrated clinical efficacy in RA. Our current study aimed to elucidate mechanisms of action and identify peripheral biomarkers associated with therapeutic responses of GM-CSF antagonism in RA. Methods A 24-week placebo (PBO)-controlled trial was conducted in 305 RA patients who received mavrilimumab (30, 100 or 150 mg) or PBO once every 2 weeks. Serum biomarkers and whole blood gene expression profiles were measured by protein immunoassay and whole genome microarray. Results Mavrilimumab treatment induced significant down-regulation of type IV collagen formation marker (P4NP 7S), macrophage-derived chemokine (CCL22), IL-2 receptor α and IL-6 compared with PBO. Both early and sustained reduction of P4NP 7S was associated with clinical response to 150 mg mavrilimumab treatment. Gene expression analyses demonstrated reduced expression of transcripts enriched in macrophage and IL-22/IL-17 signalling pathways after GM-CSF blockade therapy. Myeloid and T cell-associated transcripts were suppressed in mavrilimumab-treated ACR20 responders but not non-responders. While CCL22 and IL-6 down-regulation may reflect a direct effect of GM-CSFR blockade on the production of pro-inflammatory mediators by myeloid cells, the suppression of IL-2 receptor α and IL-17/IL-22 associated transcripts suggests an indirect suppressive effect of mavrilimumab on T cell activation. Conclusion Our results demonstrated association of peripheral biomarker changes with therapeutic response to mavrilimumab in RA patients. The sustained efficacy of mavrilimumab in RA may result from both direct effects on myeloid cells and indirect effects on T cell activation after GM-CSFR blockade.

Chemokine receptors regulate cell migration and homing. They belong to the rhodopsin-like family of GPCRs. Their ancestor genes emerged in the early stages of vertebrate evolution. Since then, the family has been greatly expanded through whole and segmental genome duplication events. During evolution, many amino acid changes have been introduced in individual chemokine receptors, but certain motifs and residues are highly conserved. Previously, we proposed a nomenclature system of the vertebrate chemokine receptors based on their evolutionary history and phylogenetic analyses. With the use of this classification system, we are now able to confidently assign the species orthologs of vertebrate chemokine receptors. Here, we systematically analyze conserved motifs and residues of each group of orthologous chemokine receptors that may play important roles in their signaling and biologic functions. Our present analysis may provide useful information on how individual chemokine receptors are activated upon ligand binding.

The impact of environmental stressors on the magnitude of specific chemokine gene expression was examined in mouse bone marrow–derived macrophages stimulated through various TLRs. Levels of TLR-stimulated CXCL1 and CXCL2 but not CXCL10 or CCL5 mRNAs were selectively enhanced (>10-fold) in stressed macrophages. The amplification was also manifested for other proinflammatory cytokines, including TNF-α, IL-1α, and IL-6. Responses through TLR3 and TLR4 exhibited the greatest sensitivity, reflecting a requirement for Toll/IL-IR domain–containing adaptor-inducing IFN-β (TRIF), the adaptor protein selectively associated with these TLRs. IFN regulatory factor 3, a transcription factor that is downstream of TLR4/TRIF signaling, was not required for sensitivity to stress-induced chemokine amplification. c/EBP homologous protein and X box binding protein 1 have been reported to enhance inflammatory cytokine responses but are not required for amplification of TLR3/4-induced CXCL1 expression. Rather, receptor-interacting protein kinase 1, a kinase also linked with TLR3/4/TRIF signaling, is required and involves a stress-dependent increase in its abundance and ubiquitination. Whereas NF-κB activation is necessary for TLR-induced chemokine gene transcription, this factor does not appear to be the primary mechanistic target of environmental stress. The application of stress also enhanced chemokine expression in macrophages infiltrating the peritoneal cavity but was not observed in the resident peritoneal cells or in the liver. These findings identify novel mechanisms for modulating the magnitude and duration of selective TLR-induced chemokine and cytokine gene expression and further establish the importance of cell stress pathways in coordinating the outcomes of cellular and tissue injury.

Chemokines act mainly in guiding leukocyte migration along the endothelium. Apart from angiogenesis or neuronal survival, chemokines are involved in damage and repair of brain tissue after ischemic stroke. We studied the presence of chemokines directly in neurons and brain blood vessels that were obtained by means of laser microdissection from human ischemic brains. Using multiple ELISA Searchlight® array we evaluated nine chemokines (CCL1−5, CCL11, CCL17, CCL22, and CXCL8) in microdissected samples. We found higher levels of CCL1 and CCL2 in neurons than in vessels; CCL5 and CCL22 were decreased in the infarcted areas. The same ELISA array was performed in plasma samples from stroke patients. We explored the temporal profile of circulating chemokines from admission to 90 days after the cerebrovascular event, and found that only CCL22 showed significant changes along time and that these changes negatively correlated with neurological severity. When neurological outcome was assessed in the hyperacute phase of stroke no associations were found. From our study, we can conclude that these chemokines do not perform a clear role of outcome biomarkers. Further studies are necessary to assess which mechanisms underlie the association of chemokines with the neurological state at distinct time points since the differences found here could be reflecting the dual role of chemokines in neuroinflammation.

Many observational epidemiologic studies suggest an association between exercise and colon cancer risk. The mechanisms contributing to a preventative effect of exercise on colon cancer are complex and multifaceted. Altered immune system function is one possible mechanism that has been largely unexplored. Therefore, the purpose of this study was to examine the effects of exercise on markers associated with macrophages and select T cell populations in a mouse model of intestinal tumorigenesis and to relate this to polyp characteristics. Male Apc(Min/+) mice were randomly assigned to either sedentary (Sed) or exercise (Ex) treatment (n=6-9/group). The exercise treatment consisted of treadmill running for 1 h/day and 6 days a week at 15 m/min from 4 until 16 weeks of age. Intestinal polyps were counted and categorized by size. Mucosal tissue was analyzed for mRNA expression of overall macrophages (F4/80), for genes associated with M1 (IL-12, IL-23 and Nos2) and M2 (CD206, IL-10, IL-4, CCL17, CCL22 and Arg-1) macrophages and the macrophage chemoattractants MCP-1, fetuin A and CXCL14. Markers for cytotoxic T cells (CTLs) and regulatory T cells were also examined by measuring mRNA expression of CD8 and Foxp3, respectively. While there was no significant difference in overall polyp number between the groups (Sed, 23.3±4.3; and Ex, 16.5±4.3), Ex did have a reduction in the number of large polyps (Sed, 6.1±1.1; and Ex, 3.0±0.6) (P

Rhinovirus (RV) is responsible for the majority of virus-induced asthma exacerbations. We showed previously that RV infection of ovalbumin-sensitized and -challenged BALB/c mice induces production of type 2 cytokines from M2-polarized macrophages. In the present study, we sought to determine the mechanism of RV-induced cytokine expression. We infected bone marrow-derived macrophages (BMMs) from BALB/c mice with RV serotype 1B, a minor group virus that infects mouse cells. Selected cultures were pretreated with IL-4, a type 2 cytokine increased in allergic asthma. RV infection of untreated cells increased messenger RNA and protein expression of the M1 cytokines TNF-α, CXCL1, and IL-6 but failed to induce expression of the M2 cytokines CCL22 and CCL24. Cells pretreated with IL-4 showed decreased expression of M1 cytokines but increased expression of Ym-1, Arg-1 (M2 markers), CCL22, and CCL24. Infection with ultraviolet (UV)-irradiated, replication-deficient RV elicited similar cytokine responses, suggesting that the outcome is replication independent. Consistent with this, viral RNA copy number did not increase in RV-treated BMMs or bronchoalveolar macrophages. RV-induced cytokine expression was not affected when cells were pretreated with cytochalasin D, suggesting that viral endocytosis is not required for the response. Finally, RV-induced cytokine expression and viral attachment were abolished in BMMs from myeloid differentiation factor 88 and Toll-like receptor (TLR)2 KO mice, suggesting a specific requirement of TLR2. We conclude that RV elicits a proinflammatory cytokine response in BMMs through a cell-surface-mediated, TLR2-dependent mechanism that does not require viral endocytosis or replication.

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.

Introduction Tumor-macrophage (Mφ) crosstalk has been extensively studied and both cytokine networks and cell-cell interactions at the tumour site directly impact therapeutic outcome. Tumour modelling brings up tools that can be explored to uncover targets for therapeutic intervention. We developed a model system based on the co-culture of lung tumour spheroids, fibroblasts and Mφ, which can emulate aspects of tumour microenvironment dynamics. In co-culture, Mφ infiltrate the tumour mass and display tumour promoting features, e.g. secretion of M2-like cytokines, among which CCL24 showed the highest upregulation. In a tumoral context, CCL24 was shown to be upregulated in primary tumours and liver metastasis of colorectal cancer and contributes to hepatocellular carcinoma malignancy. Therefore, this study aims to investigate this chemokine for its possible role in NSCLC invasion and metastasis. Material and methods Three lung cancer cell lines (NCI-H157, NCI-H1650, NCI-H1437), representing tumour stages with different invasion potential and EMT scores, and 3 sources of fibroblasts (cancer-associated – CAF, normal – NF, human dermal fibroblasts – hDF) were supplemented with increasing concentrations of CCL24. Gene expression of EMT markers, transwell invasion and EDU proliferation assays were performed. Results and discussions In the model, a tumor-supportive secretory profile was observed, with identification of cytokines associated with M2-like Mφ polarisation (G-CSF, IL4, IL13, IL10). Hierarchical clustering analysis identified CXCL13, CCL22, CCL23 and CCL24 as cytokines specifically upregulated in triple cultures. CCL24 supplementation resulted in an increase in the invasion potential of lung cancer cell lines (up to 40%), most evident in non-invasive cell line NCI-H1437. This was concomitant with an upregulation of EMT markers Slug and Snail (up to 5-fold) in NCI-H1437 cells. hDF showed a marked increase in the invasion potential (up to 2-fold), while for CAF and NF the increase was around 20%. No effects in proliferation were observed. Conclusion This data suggests that CCL24 promotes the invasion but not the proliferation of lung cancer cell lines, which could be linked to an EMT process in non-invasive cell lines presenting an epithelial phenotype. We are currently investigating the downstream effectors, namely upregulation of MMPs and angiogenesis stimulating factors. We acknowledge the IMI Joint Undertaking (grant agreement no.115188) and FCT (iNOVA4Health—UID/Multi/04462/2013, SFRH/BD/52202/2013).