CCL2 mRNA Expression Research Articles

MO919THE INTERPLAY BETWEEN IL-6 AND IL-17 MIGHT PLAY A SIGNIFICANT ROLE IN CHRONIC ANTIBODY MEDIATED REJECTION IN RENAL ALLOGRAFT RECIPIENTS

Abstract Background and Aims Chronic antibody-mediated rejection (CABMR) plays a critical role in kidney allograft loss and consider among one of the most important barriers that is responsible for late term graft loss. Previously we believed that alloreactive T-cell and de-novo DSA responsible for late term graft loss but recent study suggested that not only immune cell, non-immune cells like fibroblast also plays important role in chronic inflammation and allograft rejection via IL-6 amplifier loop (IL-6+IL-17). The interaction between non-immune tissues/cells and the immune system plays a critical role in chronic inflammation and late graft rejection. In chronic inflammation IL-6 enhance the production of acute phase proteins, T cell Subset differentiation, Maturation of Plasma cells, Generation of cellular and humoral immune responses and Control the transition from acute to chronic inflammation by changing the nature of leucocyte infiltration (from neutrophils to monocyte). We sought to see whether IL-6 and IL-17A mediated synergistic activation of inflammation amplifier is operational in CABMR. Method Recruitment of patients according to Banff 2017criteria and biopsy was taken from consented patients and establishment of fibroblast culture from renal biopsy of patients with CABMR. Fibroblast culture from CABMR patients were cultured to purity and pre stimulated with IL-6 (20ng/ µl), IL-17(50ng/ µl), IL-6 plus IL-17 for 24 hours and culture supernatant were collected for IL-6 ELISA to see synergistic activation. Serum IL-6, MCP1 and CCL20 levels of Healthy control (HC), CABMR and Non-CABMR patients and MCP1, CCL20 level in culture supernatant were measured by ELISA. m-RNA expression of IL-6, MCP1, CCL20 and SOCS3 gene were measured by real time PCR (Syber-green method) One-way ANOVA and Non-parametric Student t tests (two-tailed) were used for the statistical analysis of differences between groups. Results In comparison to IL-6 and IL-17 alone these cytokines synergistically induced more IL-6 production from renal fibroblasts. (Fig 1) also, we found that concentrations of effectors of inflammation amplifiers like IL-6, CCL-20 & MCP-1 in sera were significantly higher in CABMR patients compared to Non rejection patients, while their concentration in culture supernatant was higher when fibroblast cell stimulated with IL-6 and IL-17 together as compared either IL-6 or IL-17 alone. (Fig 2) Gene expression analysis of IL-6, MCP1 and CCL20 was significant higher (p<0.001) with synergistic activation of IL-6 and IL-17 as compared to either IL-6 or IL-17 alone, while SOCS3 gene expression was downregulated. (Fig 3) There was significant reduction in IL-6 concentration in culture supernatant with IL-6 and IL-17 inhibitor together (Fig 4) and m-RNA expression of IL-6 and MCP-1 was significantly reduced. (Fig 5) Conclusion CABMR is perpetuated by inflammation amplifier loop or synergistic induction of IL-6 and IL-17. Inhibition of IL-6 with Anti-IL-6 (Tocilizumab) and IL-17 with Anti-IL-17 reduces the tissue injury marker (IL-6, MCP1, CCL20) and allograft rejection.

Inhibiting adipose tissue M1 cytokine expression decreases DPP4 activity and insulin resistance in a type 2 diabetes mellitus mouse model.

Adipose tissue inflammation is a major cause of the pathogenesis of obesity and comorbidities. To study the involvement of M1/M2 cytokine expression of adipose tissue in the regulatory mechanisms of dipeptidyl peptidase 4 (DPP4) and insulin resistance in diabetes, stromal vascular fractions (SVFs) were purified from inguinal adipose tissue of diabetic (Leprdb/db) and non-diabetic (Lepr+/+) mice followed by analysis of M1/M2 cytokine expression. SVFs of Leprdb/db mice exhibited increased TNF-α, IL-6, IL-1β, CCL2, and DPP4 mRNA expression but decreased IL-10 mRNA expression. Plasma from Leprdb/db mice induced TNF-α, IL-6, IL-1β, CCL2, and DPP4 mRNA expression and plasma from Lepr+/+ mice induced IL-10 mRNA expression in SVFs from Leprdb/db mice. Injection of Lepr+/+ plasma into the adipose tissue of Leprdb/db mice decreased mRNA expression of TNF-α, IL-6, IL-1β, CCL2, and DPP4 and protein expression of pJNK and DPP4 in SVFs, reduced mRNA expression of ICAM, FMO3, IL-1β, iNOS, TNF-α, IL-6, and DPP4 and protein expression of ICAM, FMO3, and DPP4 in liver, and suppressed mRNA expression of TNF-α, IL-6, IL-1β, and DPP4 in Kupffer cells. Plasma from Leprdb/db mice did not induce M1 cytokine expression in SVFs from Leprdb/db-Jnk1-/- mice. Altogether, we demonstrate that diabetes induces M1 but decreases M2 cytokine expression in adipose tissue. Diabetic plasma-induced M1 expression is potentially through pJNK signaling pathways. Non-diabetic plasma reverses M1/M2 cytokine expression, plasma CCL2 levels, DPP4 activity, and Kupffer cell activation in diabetes. Our results suggest M1/M2 cytokine expression in adipose tissue is critical in diabetes-induced DPP4 activity, liver inflammation, and insulin resistance.

P063 The immunological landscape of intestinal fibrosis in Crohn’s Disease

Abstract Background Patients with Crohn’s disease (CD) often develop strictures, necessitating surgical intervention. The immune pathways expressed in the fibrotic regions, especially in the deeper intestinal layers, are poorly characterized hence hampering therapeutic development for anti-fibrotic agents. We performed a detailed analysis of the immune cell populations in both inflammation and fibrosis in the mucosa and deeper intestinal layers in CD patients. Methods Patients with stricturing CD (n=25) or colorectal cancer (CRC, n=10), undergoing ileocolonic resection were included. The resection specimen of CD patients was macroscopically subdivided into fibrotic, inflammatory but not fibrotic, and unaffected regions by an IBD-experienced histopathologist. The mucosal layer was furthermore separated from the deeper intestinal layers. Immune cells were isolated, followed by flow cytometry. Quantitative real time PCR (qRT-PCR) was performed for the eosinophil chemoattractant CCL11 coding for eotaxin-1 and normalized against the housekeeping genes RPL13a and PPIA. Wilcoxon matched-pairs sign rank test was performed on paired samples while Dunn’s multiple testing was performed for unpaired comparisons. Results Flow cytometric analysis in CD patients showed mainly B cell enrichment in fibrotic tissue compared to the unaffected tissue, both in the mucosa (30.2% vs 20.9% of CD45+ cells, p=0.001) as in deeper layers (34.4% vs 24.1% of CD45+ cells, p=0.01) (figure 1). Mature dendritic cells (mDC), alternatively-activated macrophages and eosinophils were also enriched but in the deeper layers of the fibrotic segment compared to the mucosa (4.3% vs 3.4%, 1.07% vs 0.04% and 2.3% vs 1.5% of CD45+ cells; p = 0.02, 0.008 and 0.02) (figure 2). Moreover, increased eosinophilic presence was also shown in the unaffected CD region compared to the fibrotic area, both in deeper layers (4.0% vs 2.3% of CD45+ cells; p=0.03) as the mucosa (2.4% vs 1.5% of CD45+ cells; p=0.01) as well as in the inflamed deeper layers compared to the superficial layers (3.3% vs 2.1% of CD45+ cells, p=0.04). These data were corroborated by increased CCL11 mRNA expression, in the deeper layers of fibrotic tissue compared to the mucosa (52.3% vs 31.0%, p=0.03). Conclusion Our results suggest a role for innate immune cells in CD complicated with fibrosis. Especially in the deeper layers, eosinophils, mature dendritic cells and alternatively-activated macrophages are implicated. The eosinophilic enrichment in the deeper layers may partly be attributed to an increased eosinophil recruitment by CCL11. Our results offer opportunities for anti-fibrotic drug development in CD.

Deletion of eNOS enhances kidney injury in BTBR ob/ob mice

Background ob/ob mice are a leptin deficient murine model of type 2 diabetes mellitus (T2DM), characterized by hyperphagia, obesity and hyperglycemia. ob/ob mice on a BTBR background have enhanced insulin resistance and mimic the glomerular pathophysiology of diabetic nephropathy (DN) in humans. However, leptin deficiency reduces blood pressure (BP). Since reduced activity of endothelial nitric oxide synthase (eNOS) enhances BP and the progression of kidney disease, we attempted to develop a more robust DN model by introducing eNOS deficiency in BTBR ob/ob mice. Methods BTBR mice heterozygous for both ob and eNOS were bred to generate 6 experimental groups being wild-type for Ob (Con) or ob/ob (Ob) as well as wild-type (WT), heterozygote (HET) or knockout (KO) for eNOS: Con/WT, Con/HET, Con/KO, Ob/WT, Ob/HET & Ob/KO. Systolic BP (automated tail-cuff) and GFR (FITC sinistrin plasma kinetics) were determined in awake mice in weeks 27-30 followed by kidney harvest for RTqPCR analysis of injury markers. Statistics: 2-way ANOVA for Ob and eNOS followed by pairwise comparisons if interaction was significant. 13-24 mice (males plus females)/group for WT and HET, n= 5-8 mice/group for KO. Results In WT, Ob increased blood glucose (BG), body weight (Bw), kidney weight (Kw), GFR, and urinary albumin to creatinine ratio (UACR), and reduced BP vs Con. These responses to Ob were not different in HET. KO increased BP and UACR and reduced Kw and GFR vs WT in both Con and Ob. In Ob, KO reduced BG and Bw vs WT. All effects were independent of gender. In male mice: In WT, Ob increased renal mRNA expression of Ngal, Ccl2, and Timp1, whereas Kim1, Tnfa and Col1a1 were not significantly different vs Con. These responses to Ob were similar in HET. KO increased renal mRNA expression of Ngal, Timp1, Col1a1 vs WT in both Con and Ob; and KO, in addition, also increased Kim1, Ccl2 and Tnfa vs WT in Ob. In female mice: In WT, Ob did not change renal mRNA expression of Ngal, Ccl2, Timp1, and Tnfa and the expression of Kim1 and Col1a1 were actually reduced vs Con. Similar responses to Ob were observed in HET. KO increased renal mRNA expression of Ngal, Ccl2, Timp1, and Col1a1 vs WT in both Con and Ob; and KO in addition increased Tnfa vs WT in Ob. Conclusions Male and female BTBR ob/ob mice at 27-30 weeks of age presented hyperglycemia and increased Bw associated with larger kidneys, glomerular hyperfiltration and a 100x higher UACR vs Con. These effects were associated with modest increases in renal mRNA expression of Ngal, Ccl2, and Timp1 only in male mice, whereas female Ob mice unexpectedly had lesser expression of Kim1 and Col1a1 vs Con. eNOS heterozygosity did not induce robust renal changes vs WT in Con or Ob. In contrast, eNOS KO, which increased BP by 15-25 mmHg, enhanced kidney injury as indicated by lesser Kw and GFR, associated with higher UACR and renal mRNA expression of markers of injury, inflammation and fibrosis, and lower survival rates; these effects of eNOS KO were detectable in Con and more pronounced in Ob.

The Inhibitory Effects of Organosulfide Diallyl Trisulfide on TNF‐α induced CCL2 release through MAPK/MAPK8 and NFқB/IKBKE signaling in TNBC cell lines

Inflammation and immune response play a key role in triple-negative breast cancer (TNBC) progression and metastasis. Previous studies have shown that CCL2 plays a significant role in breast cancer cell signaling and can increase cell proliferation and differentiation, immune suppression, epithelial-to-mesenchymal transition, and angiogenesis. CCL2 expression is modulated via mitogen-activated protein kinase (MAPK) and nuclear factor-kappa beta (NFĶB) signaling pathways. Compounds that can inhibit the release of CCL2 have been shown to prevent cancer-associated inflammatory and pro-oncogenic processes. Diallyl Trisulfide (DATS), an organosulfide found in garlic, has been previously reported to be effective in chemoprevention, apoptosis, and cell cycle arrest in DNA damaged normal breast epithelial cells, breast cancer cells, as well as other cancers. The current study explored the effect of DATS on the genetically different TNBC MDA-MB-231 and MDA-468 cells, stimulated by TNF-α. Cytokine arrays, ELISA, and RT-PCR using individual primers were used. DATS downregulated CCL2 protein expression in MDA-MB-231, but not in MDA-MB-468 cells. These results were confirmed with ELISA analysis that showed a reduction in the expression of CCL2 in the TNF-α stimulated MDA-MB-231 cells treated with DATS. The data also showed that DATS treatment reduced by 50% the mRNA expression of CCL2 in MDA-MB-231cells after a 24-h period. To study a possible mechanism for DATS inhibitory effect over CCL2 expression, this compound's effect on the mRNA expression of IKBKE and MAPK8, which belong to the NFĶB and MAPK signaling pathways, respectively, was also investigated. DATS downregulated both genes in the TNF-α stimulated MDA-MB-231 and MDA-MB-468 cells. In conclusion, this study showed that DATS could downregulate CCL2 expression in the protein and transcription level in MDA-MB-231 cells. A possible mechanism for DATS inhibitory effect may involve the downregulation of IKBKE and MAPK8 mRNA expression. IKBKE gene was reported to be overexpressed in approximately 30% of human breast tumors and promotes cytokine release and pro-survival signaling through the activation of NFĶB and JAK-STAT pathways. Our findings demonstrate that DATS may have potential in breast cancer treatment, slowing down cancer progression by the downregulation of CCL2 expression and that genetically different TNBC cells may respond differently to DATS treatment.

Eotaxin-1/CCL11 is involved in cell migration in rheumatoid arthritis

Eotaxin-1 (CCL11) induces the migration of different leukocyte types by interacting with CCR3. In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) are pathogenic effectors and a major CCR3-expressing cell. The aim of this study was to investigate the expression and function of CCL11 in RA FLS. The expression of CCL11 and CCR3 was evaluated by ELISA, immunofluorescence and quantitative PCR analysis. The CCL11 levels in serum and synovial fluids (SFs) from RA patients were significantly higher than those in serum from healthy controls and SFs from osteoarthritis patients. CCL11 and CCR3 were expressed in the RA synovial tissue lining layers. The secretion of CCL11 in RA FLS-conditioned medium and the mRNA expression of CCL11 and CCR3 were induced by TNF-α. Furthermore, CCL11 induced the mRNA expression of CCL11 and CCR3. Application of a CCR3 antagonist reduced TNF-α-induced CCL11 secretion from RA FLS. CCL11 induced the migration of RA FLS and monocytes. RA FLS migration was decreased by treatment with CCL11 siRNA. The migration of monocytes to medium conditioned with CCL11 siRNA-transfected and TNF-α-stimulated RA FLS was reduced. These data indicate that the self-amplification of CCL11 via CCR3 may play an important role in cell migration in RA.

Increased Gene expression of CCL2/CCR2 axis in bronchial smooth muscles of allergen-challenged mice

PurposeAugmented bronchial smooth muscle (BSM) contraction is a cause of airway hyperresponsiveness (AHR) in asthma. Increasing evidence suggest that C-C motif chemokine 2 (CCL2) modulates smooth muscle contractility by activating its binding partner C-C chemokine receptor type 2 (CCR2). In the present study, changes in the gene expression of CCL2/CCR2 axis were determined in the BSMs of a murine model of allergic asthma. Materials and methodsThe ovalbumin (OA)-sensitized mice were repeatedly challenged with aerosolized OA to induce asthmatic reaction. Twenty-four hours after the last antigen challenge, total RNAs of the main BSM tissues and bronchoalveolar lavage fluids (BALFs) were obtained. ResultsOur published microarray data (GEO accession No. GSE116504) detected changes in gene expression associated with the chemokine signaling pathway (KEGG Map ID: 04062) in BSMs of mice with AHR induced by antigen exposure. Among them, quantitative RT-PCR analyses showed significant increase in mRNA expression of Ccl2 and Ccr2. Analysis of BALFs also revealed a significant increase in Ccl2 protein in the airways of the diseased animals. ConclusionIt is thus possible that, in association with the AHR, the CCL2/CCR2 axis is enhanced in the airways of allergic bronchial asthma.

Downregulation of Chemokine CCL20 Involved in Myeloma Cells Resistant to Elotuzumab and Lenalidomide.

ObjectiveFew studies have focused on investigating resistance mechanisms in myeloma immunotherapy. This study aimed to explore the relevant factor involved in the resistance of Elotuzumab and lenalidomide.MethodsCell models which are resistant to Elotuzumab and lenalidomide were constructed; different expression genes in U266/WT (UW) and resistant UR, UE, and URE cells were detected by using gene expression microarray. RT-qPCR validated CCL20 mRNA expression of four cell lines and patient samples; bioinformatics analysis of CCL20 expressions in NDMM and RRMM; ELISA detected the presence of CCL20 in the plasma of MM patients; constructed UR mouse xenograft model to explore whether or not CCL20 reverse lenalidomide treatment in vivo.ResultsCell models which are resistant to Elotuzumab and lenalidomide (UR, UE, URE) were successfully constructed. CCL20 gene expression decreased in resistant myeloma cell lines and RRMM patients. Furthermore, RRMM patients were found to have lower levels of CCL20 protein in their plasma compared to NDMM. CCL20 increase the sensitivity of drug-resistant myeloma cells to immunomodulatory drugs both in vivo and in vitro.ConclusionThe expression of CCL20 was decreased in lenalidomide and Elotuzumab resistant U266 cells and in RRMM patients. CCL20 could therefore possibly increase the sensitivity of lenalidomide and Elotuzumab.

The Effect of Lonicera Japonica Thunberg on Inflammatory Factor Expression Associated with Atherosclerosis

Objective The purpose of this study was to investigate the effect of Lonicera Japonica Thunberg (LJT) on the inflammatory factor expression associated with atherosclerosis in human umbilical vein endothelial cells (HUVECs). Methods After treatment with LJT in HUVEC which is treated with TNF-α, we measured the expression levels of biomarkers (MCP-1, ICAM-1, VCAM-1, KLF2, and eNOS), mRNA (CCL2, ICAM1, VCAM1, KLF2, and NOS3), and the proteins (MCP-1, ICAM-1, VCAM-1, KLF2, eNOS, ERK, JNK, and p38). Results 1. Compared to the control, LJT significantly reduced MCP-1 and VCAM-1 levels at concentrations of 100, 200, and 400 μg/ml and ICAM-1 expression at 200 and 400 μg/ml compared to the control. It increased KLF2 levels at all three concentrations, but not significantly, while eNOS expression was significantly increased at 400 μg/ml. 2. LJT was seen to significantly reduce the expression of CCL2, ICAM1, and VCAM1 mRNA at concentrations of 100, 200, and 400 μg/ml compared to the control. In contrast, significantly increased KLF2 and NOS3 mRNA levels were observed at 400 μg/ml and at 200 and 400 μg/ml, respectively. 3. Compared to the control, LJT significantly reduced the protein expression of MCP-1 and VCAM-1 at 200 and 400 μg/ml and of ICAM-1 at 400 μg/ml. In addition, it increased both KLF2 and eNOS protein levels at 200 and 400 μg/ml. Although LJT did not have an effect on ERK expression in comparison with the control, it significantly reduced JNK levels at 200 and 400 μg/ml and p38 levels at 400 μg/ml. Conclusions These results suggest that LJT has an effect on the inhibition of inflammatory factor expression associated with atherosclerosis in HUVECs which could contribute to the prevention of cardiovascular and cerebrovascular diseases. Keywords: Lonicera japonica Thunberg, atherosclerosis, inflammation, anti-inflammatory effect

Role of myeloid-derived chemokine CCL5/RANTES at an early stage of atherosclerosis

One of the hallmarks of atherosclerosis is ongoing accumulation of macrophages in the artery intima beginning at disease onset. Monocyte recruitment contributes to increasing macrophage abundance at early stages of atherosclerosis. Although the chemokine CCL5 (RANTES) has been studied in atherosclerosis, its role in the recruitment of monocytes to early lesions has not been elucidated. We show that expression of Ccl5 mRNA, as well as other ligands of the CCR5 receptor (Ccl3 and Ccl4), is induced in the aortic intima of Ldlr−/− mice 3 weeks after the initiation of cholesterol-rich diet (CRD)-induced hypercholesterolemia. En face immunostaining revealed that CCL5 protein expression is also upregulated at 3 weeks of CRD. Blockade of CCR5 significantly reduced monocyte recruitment to 3-week lesions, suggesting that chemokine signaling through CCR5 is critical. However, we observed that Ccl5-deficiency had no effect on early lesion formation and CCL5-blockade did not affect monocyte recruitment in Ldlr−/− mice. Immunostaining of the lesions in Ldlr−/− mice and reciprocal bone marrow transplantation (BMT) of Ccl5+/+ and Ccl5−/− mice revealed that CCL5 is expressed by both myeloid and endothelial cells. BMT experiments were carried out to determine if CCL5 produced by distinct cells has functions that may be concealed in Ccl5−/−Ldlr−/− mice. We found that hematopoietic cell-derived CCL5 regulates monocyte recruitment and the abundance of intimal macrophages in 3-week lesions of Ldlr−/− mice but plays a minor role in 6-week lesions. Our findings suggest that there is a short window in early lesion formation during which myeloid cell-derived CCL5 has a critical role in monocyte recruitment and macrophage abundance.

Cytokine and Chemokine mRNA Expressions after Mycobacterium tuberculosis-Specific Antigen Stimulation in Whole Blood from Hemodialysis Patients with Latent Tuberculosis Infection

There have been few reports on the kinetics of hemodialyzed (HD) patients’ immune responses in latent tuberculosis infection (LTBI). Therefore, in the present study, messenger ribonucleic acid (mRNA) expression levels of nine immune markers were analyzed to discriminate between HD patients with LTBI and healthy individuals. Nine cytokines and chemokines were screened through relative mRNA expression levels in whole blood samples after stimulation with Mycobacterium tuberculosis (MTB)-specific antigens from HD patients with LTBI (HD/LTBI), HD patients without LTBI, and healthy individuals, and results were compared with the QuantiFERON-TB Gold In-Tube (QFT-GIT) test. We confirmed that the C-C motif chemokine 11 (CCL11) mRNA expression level of the HD/LTBI group was significantly higher than the other two groups. Especially, the CCL11 mRNA expression level of the >0.7 IU/mL group in the QFT-GIT test was significantly higher than the <0.2 IU/mL group in the QFT-GIT test and the 0.2–0.7 IU/mL group in the QFT-GIT test (p = 0.0043). The present study reveals that the relative mRNA expression of CCL11 was statistically different in LTBI based on the current cut-off value (i.e., ≥0.35 IU/mL) and in the >0.7 IU/mL group. These results suggest that CCL11 mRNA expression might be an alternative biomarker for LTBI diagnosis in HD patients.

Aberrantly Expressed Galectin-9 Is Involved in the Immunopathogenesis of Anti-MDA5-Positive Dermatomyositis-Associated Interstitial Lung Disease.

BackgroundDermatomyositis (DM) associated rapidly progressive interstitial lung disease (RP-ILD) has high mortality rate and poor prognosis. Galectin-9 (Gal-9) plays multiple functions in immune regulation. We investigated Gal-9 expression in DM patients and its association with DM-ILD.MethodsA total of 154 idiopathic inflammatory myopathy patients and 30 healthy controls were enrolled in the study. Cross-sectional and longitudinal studies were used to analyze the association between serum Gal-9 levels and clinical features. Enzyme-linked immunosorbent assay and qRT-PCR were used to examine Gal-9 expression in the sera and isolated peripheral blood mononuclear cells (PBMCs) from DM patients. Immunohistochemistry was performed to analyze the expression of Gal-9 and its ligand (T-cell immunoglobulin mucin (Tim)-3 and CD44) in lung tissues from anti-melanoma differentiation-associated gene 5 (MDA5)-positive patients. The effect of Gal-9 on human lung fibroblasts (MRC-5) was investigated in vitro.ResultsSerum Gal-9 levels were significantly higher in DM patients than in immune-mediated necrotizing myopathy patients and healthy controls (all p < 0.001). Higher serum Gal-9 levels were observed in anti-MDA5-positive DM patients than in anti-MDA5-negative DM patients [33.8 (21.9–44.7) vs. 16.2 (10.0–26.9) ng/mL, p < 0.001]. Among the anti-MDA5-positive DM patients, serum Gal-9 levels were associated with RP-ILD severity. Serum Gal-9 levels were significantly correlated with disease activity in anti-MDA5-positive DM patients in both cross-sectional and longitudinal studies. PBMCs isolated from anti-MDA5-positive DM patients (3.7 ± 2.3 ng/mL) produced higher levels of Gal-9 than those from immune-mediated necrotizing myopathy patients (1.1 ± 0.3 ng/mL, p = 0.022) and healthy controls (1.4 ± 1.2 ng/mL, p = 0.045). The mRNA levels of Gal-9 were positively correlated with the levels of type-I interferon-inducible genes MX1 (r = 0.659, p = 0.020) and IFIH1 (r = 0.787, p = 0.002) in PBMCs from anti-MDA5-positive DM patients. Immunohistochemistry revealed increased Gal-9 and Tim-3 expression in the lung tissues of patients with DM and RP-ILD. In vitro stimulation with Gal-9 protein increased CCL2 mRNA expression in MRC-5 fibroblasts.ConclusionsAmong anti-MDA5-positive DM patients, Gal-9 could be a promising biomarker for monitoring disease activity, particularly for RP-ILD severity. Aberrant expression of the Gal-9/Tim-3 axis may be involved in the immunopathogenesis of DM-ILD.

Erythorbyl laurate suppresses TNF-α-induced adhesion of monocytes to the vascular endothelium

Abstract Erythorbyl laurate (EL) can be produced via lipase-catalyzed esterification between erythorbic acid and lauric acid. In this study, we evaluate the anti-inflammatory effect of EL in the early stage of atherosclerosis. EL suppressed tumor necrosis factor (TNF)-α-induced monocyte adhesion to vascular endothelial cells and expression of vascular cell adhesion molecule (VCAM)-1 in human umbilical vein endothelial cells (HUVECs). Additionally, EL suppressed TNF-α-induced p65/IκB kinase (IKK)/IκB phosphorylation in HUVECs. Western blot analysis of cytosolic and nuclear cell fractions and immunofluorescence showed that EL suppressed TNF-α-induced translocation of p65 from the cytoplasm to the nucleus. EL also inhibited phosphorylation of extracellular-signal-regulated kinase (ERK) 1/2, p38, and c-Jun N-terminal kinases (JNK) 1/2 in HUVECs. EL suppressed TNF-α-induced phosphorylation of Akt, IRAK1, and TAK1 in HUVECs. Quantitative RT-PCR analysis showed that EL significantly suppressed TNF-α-induced interleukin (IL)1B, IL6, TNFA, and CCL2 mRNA expression in HUVECs. Additionally, oral administration of EL suppressed TNF-α-induced IL6 and TNFA expression in the mouse aorta. EL could represent a promising functional nutrient that can be ingested for the prevention of vascular inflammation via decreased monocyte infiltration to the vascular endothelium and suppression of inflammatory nuclear factor (NF)-κB and mitogen-activated protein kinases (MAPKs) signaling pathways.

Chemokines gene expression in the prefrontal cortex of depressed suicide victims and normal control subjects

Abnormalities of neuroinflammation have been implicated in the pathogenesis of depression and suicide. This is primarily based on the observation that cytokines, which are major inflammatory molecules and play an important role in depression and suicide, are increased in both serum and in postmortem brain of depressed and suicidal subjects. Another class of immune mediators are chemokines which are primarily involved in chemotactic properties and trafficking of immune cells in the central nervous system (CNS). Chemokines also play an important role in CNS function. Whereas chemokines have been studied in the serum of depressed and suicidal patients, their role in brain of depressed or suicidal subjects is relatively unexplored. We studied the gene expression of several chemokines in the prefrontal cortex (PFC) obtained from depressed suicidal (DS) and normal control (NC) subjects. We determined the mRNA expression of several chemokines belonging to CXCL and CCL groups of chemokines using qPCR array technique and qPCR gene expression validation in 24 DS and 24 NC subjects. The postmortem brain samples were obtained from the Maryland Brain Collection. We found that the mRNA expression of chemokines CXCL1, CXCL2, CXCL3 and CCL2 was significantly decreased in the PFC of DS compared with NC subjects. No significant change was observed in CXCL5, CXCL6, CXCL10, CCL8 and CCL19 between DS and NC subjects. Since many of the chemokines are involved in mediating certain important CNS functions, such as neurotrophic effect, neurogenesis, anti-apoptotic growth factor release, modulation of synaptic transmission, brain development and neuronal loss, decreased levels of chemokines can reduce these functions which may be involved in the pathophysiology of depression.

Biomarkers of inflammation and repair in kidney disease progression.

INTRODUCTIONAcute kidney injury and chronic kidney disease (CKD) are common in hospitalized patients. To inform clinical decision making, more accurate information regarding risk of long-term progression to kidney failure is required.METHODSWe enrolled 1538 hospitalized patients in a multicenter, prospective cohort study. Monocyte chemoattractant protein 1 (MCP-1/CCL2), uromodulin (UMOD), and YKL-40 (CHI3L1) were measured in urine samples collected during outpatient follow-up at 3 months. We followed patients for a median of 4.3 years and assessed the relationship between biomarker levels and changes in estimated glomerular filtration rate (eGFR) over time and the development of a composite kidney outcome (CKD incidence, CKD progression, or end-stage renal disease). We paired these clinical studies with investigations in mouse models of renal atrophy and renal repair to further understand the molecular basis of these markers in kidney disease progression.RESULTSHigher MCP-1 and YKL-40 levels were associated with greater eGFR decline and increased incidence of the composite renal outcome, whereas higher UMOD levels were associated with smaller eGFR declines and decreased incidence of the composite kidney outcome. A multimarker score increased prognostic accuracy and reclassification compared with traditional clinical variables alone. The mouse model of renal atrophy showed greater Ccl2 and Chi3l1 mRNA expression in infiltrating macrophages and neutrophils, respectively, and evidence of progressive renal fibrosis compared with the repair model. The repair model showed greater Umod expression in the loop of Henle and correspondingly less fibrosis.CONCLUSIONSBiomarker levels at 3 months after hospitalization identify patients at risk for kidney disease progression.FUNDINGNIH.

Suppression of IL-17A-induced CCL20 production by cytokine inducible SH2-containing protein 1 in epidermal keratinocytes

BackgroundLesions of atopic dermatitis have fewer Th17 cells than those of psoriasis, resulting in frequent skin infections. Expression of CCL20, a chemokine that is important for recruiting Th17 cells, is suppressed in the lesions of atopic dermatitis. We previously reported that IL-4 induces the expression of cytokine-inducible SH2-containing protein 1 (CIS1), a member of the CIS/SOCS family, in epidermal keratinocytes. ObjectiveTo investigate whether CIS1 influences CCL20 production in epidermal keratinocytes. MethodsExpression of CIS1 was examined in atopic dermatitis skin and in cultured keratinocytes. The effects of overexpression of CIS1 on CCL20 production by IL-17A, and on signaling pathways inhibited by CIS1, were assessed in vitro. ResultsExpression of CIS1 was enhanced in the basal layer of the lesional epidermis of skin with atopic dermatitis. When CIS1 was expressed in keratinocytes using adenoviral vectors, IL-17A-induced CCL20 expression, but not HBD2 or S100A7 expression, was significantly suppressed. TNF-α/IL-1-induced CCL20 production was not altered by CIS1. Overexpression of CIS1 attenuated IL-17A-induced ERK phosphorylation. ERK phosphorylation was mediated by the Act1 and Src family kinase pathways. CIS1 overexpression suppressed Src phosphorylation. Among the Src family kinases, the Yes kinase may have an important role because knockdown of Yes in epidermal keratinocytes resulted in suppression of ERK phosphorylation and CCL20 mRNA expression by IL-17A. ConclusionCIS1 induced by Th2 cytokines has the ability to change the response of epidermal keratinocytes to IL-17A by suppression of Src family kinases.

The differentiation of prehypertrophic into hypertrophic chondrocytes drives an OA-remodeling program and IL-34 expression

We hypothesize that chondrocytes from the deepest articular cartilage layer are pivotal in maintaining cartilage integrity and that the modification of their prehypertrophic phenotype to a hypertrophic phenotype will drive cartilage degradation in osteoarthritis. Murine immature articular chondrocytes (iMACs) were successively cultured into three different culture media to induce a progressive hypertrophic differentiation. Chondrocyte were phenotypically characterized by whole-genome microarray analysis. The expression of IL-34 and its receptors PTPRZ1 and CSF1R in chondrocytes and in human osteoarthritis tissues was assessed by RT-qPCR, ELISA and immunohistochemistry. The expression of bone remodeling and angiogenesis factors and the cell response to IL-1β and IL-34 were investigated by RT-qPCR and ELISA. Whole-genome microarray analysis showed that iMACs, prehypertrophic and hypertrophic chondrocytes each displayed a specific phenotype. IL-1β induced a stronger catabolic effect in prehypertrophic chondrocytes than in iMACs. Hypertrophic differentiation of prehypertrophic chondrocytes increased Bmp-2 (95%CI [0.78; 1.98]), Bmp-4 (95%CI [0.89; 1.59]), Cxcl12 (95%CI [2.19; 5.41]), CCL2 (95%CI [3.59; 11.86]), Mmp 3 (95%CI [10.29; 32.14]) and Vegf mRNA expression (95%CI [0.20; 1.74]). Microarray analysis identified IL-34, PTPRZ1 and CSFR1 as being strongly overexpressed in hypertrophic chondrocytes. IL-34 was released by human osteoarthritis cartilage; its receptors were expressed in human osteoarthritis tissues. IL-34 stimulated CCL2 and MMP13 in osteoblasts and hypertrophic chondrocytes but not in iMACs or prehypertrophic chondrocytes. Our results identify prehypertrophic chondrocytes as being potentially pivotal in the control of cartilage and subchondral bone integrity. Their differentiation into hypertrophic chondrocytes initiates a remodeling program in which IL-34 may be involved.

BRAFV595E Mutation Associates CCL17 Expression and Regulatory T Cell Recruitment in Urothelial Carcinoma of Dogs.

Regulatory T cells may serve as targets in cancer immunotherapy. A previous study showed that the chemokine CCL17 and the receptor CCR4 play roles in regulatory T cell recruitment in canine urothelial carcinoma. In this article, we show that the BRAFV595E mutation is associated with tumor-produced CCL17 and regulatory T cell infiltration in dogs with urothelial carcinoma. In comparison with healthy dogs, dogs with urothelial carcinoma showed increased CCL17 mRNA expression in the bladder and elevated CCL17 protein concentration in urine. Immunohistochemistry showed increased levels of Foxp3+ regulatory T cells in the tumor tissues of urothelial carcinoma. The density of Foxp3+ regulatory T cells was positively correlated with CCL17 concentration in urine, indicating that CCL17 is involved in regulatory T cell recruitment. Moreover, tumor-infiltrating regulatory T cells and urine CCL17 concentration were associated with poor prognosis in dogs with urothelial carcinoma. The number of tumor-infiltrating regulatory T cells, CCL17 mRNA expression, and urine CCL17 concentration in cases with BRAFV595E mutation were higher than those in cases with wild-type BRAF. In vitro, high CCL17 production was detected in a canine urothelial carcinoma cell line with BRAFV595E mutation but not in an urothelial carcinoma cell line with wild-type BRAF. Dabrafenib, a BRAF inhibitor, decreased CCL17 production in the cell line with BRAFV595E mutation. These results suggest that BRAFV595E mutation induced CCL17 production and contributed to regulatory T cell recruitment in canine urothelial carcinoma.

Empagliflozin Inhibits Basal and IL-1β-Mediated MCP-1/CCL2 and Endothelin-1 Expression in Human Proximal Tubular Cells.

SGLT2 inhibitors (SGLT2i) slow the progression of chronic kidney disease; however, evidence for the underlying molecular mechanisms is scarce. We investigated SGLT2i-mediated effects on differential gene expression in two independent human proximal tubular cell (HPTC) lines (HK-2 and RPTEC/TERT1) at the mRNA and protein levels under normoglycemic conditions, utilizing IL-1β as a pro-inflammatory mediator. Microarray hybridization identified 259 genes that were uniformly upregulated by IL-1β (10 mg/mL) and downregulated by empagliflozin (Empa) (500 nM) after 24 h of stimulation in two independent HPTC lines (n = 2, each). The functional annotation of these genes identified eight pathway clusters. Among 12 genes annotated to the highest ranked cluster (enrichment score, 3.51), monocyte chemoattractant protein-1/CC-chemokine ligand 2 (MCP-1/CCL2) and endothelin-1 (ET-1) were selected for verification at mRNA and protein levels based on their established involvement in the early pathogenesis of chronic kidney disease: IL-1β upregulated basal MCP-1/CCL2 (15- and 19-fold) and ET-1 (3- and 8-fold) mRNA expression, while Empa downregulated basal MCP-1/CCL2 (0.6- and 0.5-fold) and ET-1 (0.3- and 0.2-fold) mRNA expression as early as 1 h after stimulation and for at least 24 h in HK-2 and RPTEC/TERT1 cells, respectively. The co-administration of Empa inhibited IL-1β-mediated MCP-1/CCL2 (0.2-fold, each) and ET-1 (0.2-fold, each) mRNA expression as early as 1 h after ligand stimulation and for at least 24 h in both HPTC lines, respectively. This inhibitory effect of Empa on basal and IL-1β-mediated MCP-1/CCL2 and ET-1 mRNA expression was corroborated at the protein level. Our study presents novel evidence for the interference of SGLT2 inhibition with tubular inflammatory response mechanisms under normoglycemic conditions that might account for SGLT2i-mediated nephroprotection.

Possible Roles of Proinflammatory Signaling in Keratinocytes Through Aryl Hydrocarbon Receptor Ligands for the Development of Squamous Cell Carcinoma.

Aryl hydrocarbon receptor (AhR) provides a deeper insight into the pathogenesis of cutaneous squamous cell carcinoma (cSCC). AhR ligands, such as 6-formylindolo[3,2-b] carbazole (FICZ), and 7,12-Dimethylbenz[a]anthracene (DMBA), constitute major substrates for the cytochrome P450 (CYP) family, and influence the expression of various cytokine genes, including IL-17 and IL-23-related genes via the AhR. On the other hand, proinflammatory cytokines could drive tumor progression through the TRAF-ERK5 signaling pathway in cSCC. From the above findings, we hypothesized that AhR ligands might enhance the mRNA expression of proinflammatory cytokines via the AhR, leading to the development of cSCC. The purpose of this study was to investigate (1) the immunomodulatory effects of FICZ and DMBA on normal human keratinocytes (NHKCs), focusing on IL-17, and related cytokines/chemokines (IL-23, IL-36γ, and CCL20), (2) the expression of these factors in AhR-dependent pathways using a two-stage chemically induced skin carcinogenesis mouse model, and (3) the expression of these factors in lesion-affected skin in cSCC. Both FICZ and DMBA augmented the expression of CYP1A1, p19, CCL20, and IL-36γ mRNA in NHKCs in vitro. Moreover, the mRNA expression of these proinflammatory factors, as well as IL-17, in mouse cSCC is significantly decreased in the AhR-(fl/fl) Krt5-(Cre) mice compared to wild type mice, leading to a decrease in the number of developed cSCC lesions. Furthermore, CCL20, IL-23, as well as IL-17, are detected in the lesion-affected skin of cSCC patients. Our study demonstrates a possible mechanism for the development of cSCC involving AhR-mediated signaling by epidermal keratinocytes and recruitment of Th17 cells.