ABSTRACT
BACKGROUND: SERPINB2, a biomarker of Type-2 (T2) inflammatory processes, has been described in the context of asthma. Chronic rhinosinusitis with nasal polyps (CRSwNP) is also correlated with T2 inflammation and elevated 15LO1 induced by IL-4/13 in nasal epithelial cells. The aim of this study was to evaluate the expression and location of SERPINB2 in nasal epithelial cells (NECs) and determine whether SERPINB2 regulates 15LO1 and downstream T2 markers in NECs via STAT6 signalling. METHODS: SERPINB2 gene expression in bulk and single-cell RNAseq database was analysed by bioinformatics analysis. SERPINB2, 15LO1 and other T2 markers were evaluated from CRSwNP and HCs NECs. The colocalization of SERPINB2 and 15LO1 was evaluated by immunofluorescence. Fresh NECs were cultured at an air-liquid interface with or without IL-13, SERPINB2 Dicer-substrate short interfering RNAs (DsiRNAs) transfection, exogenous SERPINB2, 15-HETE recombinant protein and pSTAT6 inhibitors. 15LO1, 15-HETE and downstream T2 markers were analysed by qRT-PCR, western blot and ELISA. RESULTS: SERPINB2 expression was increased in eosinophilic nasal polyps compared with that in noneosinophilic nasal polyps and control tissues and positively correlated with 15LO1 and other downstream T2 markers. SERPINB2 was predominantly expressed by epithelial cells in NP tissue and was colocalized with 15LO1. In primary NECs in vitro, SERPINB2 expression was induced by IL-13. Knockdown or overexpression SERPINB2 decreased or enhanced expression of 15LO1 and 15-HETE in NECs, respectively, in a STAT6-dependent manner. SERPINB2 siRNA also inhibited the expression of the 15LO1 downstream genes, such as CCL26, POSTN and NOS2. STAT6 inhibition similarly decreased SERPINB2-induced 15LO1. CONCLUSIONS: SERPINB2 is increased in NP epithelial cells of eosinophilic CRSwNP (eCRSwNP) and contributes to T2 inflammation via STAT6 signalling. SERPINB2 could be considered a novel therapeutic target for eCRSwNP.
Subject(s)
Epithelial Cells , Nasal Polyps , Rhinitis , STAT6 Transcription Factor , Signal Transduction , Sinusitis , Humans , STAT6 Transcription Factor/metabolism , STAT6 Transcription Factor/genetics , Nasal Polyps/metabolism , Nasal Polyps/pathology , Nasal Polyps/immunology , Sinusitis/metabolism , Sinusitis/pathology , Sinusitis/immunology , Rhinitis/metabolism , Rhinitis/pathology , Chronic Disease , Epithelial Cells/metabolism , Plasminogen Activator Inhibitor 2/metabolism , Plasminogen Activator Inhibitor 2/genetics , Female , Male , Chemokine CCL26/metabolism , Chemokine CCL26/genetics , Adult , Middle Aged , Eosinophilia/metabolism , Eosinophilia/pathology , Nasal Mucosa/metabolism , Nasal Mucosa/pathology , Nasal Mucosa/immunology , Gene Expression Regulation , RhinosinusitisABSTRACT
Eosinophil extracellular traps (EETs) are implicated in various eosinophil-associated diseases; however, their role in chronic rhinosinusitis (CRS) remains unclear. In the present study, 57 CRS patients were enrolled, and immunofluorescence was used to analyze EETs in eosinophilic (eCRS) and non-eosinophilic (Non-eCRS) tissues. MSD was used to examine IL-4, IL-5, and IL-13 concentrations in tissue homogenates. Charcot-Leyden crystals (CLCs) protein expression was detected in PMA, PMA+DNase I, and blank control eosinophils using ELISA. Eotaxin-3 mRNA and protein levels were measured in human nasal epithelial cells (HNECs) cultured with EETs, EETs+DNase I, DNase I, and unstimulated eosinophils using PCR and ELISA. EETs were significantly increased in eCRS tissues compared with Non-eCRS (P<0.001), and correlated with VAS and Lund-Mackay CT scores. IL-5 expression was related to EETs formation (r = 0.738, P<0.001). PMA-stimulated eosinophils exhibited higher CLCs protein levels (P<0.01). Co-culturing HNECs with EETs significantly increased eotaxin-3 mRNA and protein levels (P<0.0001, P<0.001) compared with other groups. The study suggests EETs formation is elevated in eCRS patients and is involved in CLCs formation and chemokine secretion, promoting eosinophilic inflammation.
Subject(s)
Extracellular Traps , Rhinitis , Rhinosinusitis , Sinusitis , Humans , Eosinophils , Chemokine CCL26/metabolism , Interleukin-5/genetics , Interleukin-5/metabolism , Deoxyribonuclease I/metabolism , RNA, Messenger/metabolismSubject(s)
Heterocyclic Compounds, 3-Ring , Humans , Heterocyclic Compounds, 3-Ring/pharmacology , Th2 Cells/immunology , Th2 Cells/drug effects , Chemokine CCL26/metabolism , Chemokine CCL26/genetics , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolism , Esophagus/drug effects , Esophagus/immunology , Esophagus/metabolism , Cytokines/metabolism , Muscle Contraction/drug effectsABSTRACT
INTRODUCTION: Eotaxin-2 and -3 of the C-C chemokine subfamily function as potent chemoattractant factors for eosinophil recruitment and various immune responses in allergic and inflammatory airway diseases. Mucin 5AC (MUC5AC), a major gel-forming secretory mucin, is overexpressed in airway inflammation. However, the association between mucin secretion and eotaxin-2/3 expression in the upper and lower airway epithelial cells has not been fully elucidated. Therefore, in this study, we investigated the effects of eotaxin-2/3 on MUC5AC expression and its potential signaling mediators. METHODS: We analyzed the effects of eotaxin-2 and -3 on NCI-H292 human airway epithelial cells and primary human nasal epithelial cells (HNEpCs) via reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, and western blotting. Along with immunoblot analyses with specific inhibitors and small interfering RNA (siRNA), we explored the signaling pathway involved in MUC5AC expression following eotaxin-2/3 treatment. RESULTS: In HCI-H292 cells, eotaxin-2/3 activated the mRNA expression and protein production of MUC5AC. A specific inhibitor of C-C motif chemokine receptor 3 (CCR3), SB328437, suppressed eotaxin-2/3-induced MUC5AC expression at both the mRNA and protein levels. Eotaxin-2/3 induced the phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 and p38, whereas pretreatment with a CCR3 inhibitor significantly attenuated this effect. Induction of MUC5AC expression with eotaxin-2/3 was decreased by U0126 and SB203580, specific inhibitors of ERK1/2 and p38 mitogen-activated protein kinase (MAPK), respectively. In addition, cell transfection with ERK1/2 and p38 siRNAs inhibited eotaxin-2/3-induced MUC5AC expression. Moreover, specific inhibitors (SB328437, U0126, and SB203580) attenuated eotaxin-2/3-induced MUC5AC expression in HNEpCs. CONCLUSION: Our results imply that CCR3-mediated ERK1/2 and p38 MAPK are involved in the signal transduction of eotaxin-2/3-induced MUC5AC overexpression.
Subject(s)
Mucin 5AC , p38 Mitogen-Activated Protein Kinases , Humans , p38 Mitogen-Activated Protein Kinases/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Cell Line , Mucin 5AC/genetics , Mucin 5AC/metabolism , Chemokine CCL24/metabolism , Chemokine CCL24/pharmacology , Chemokine CCL26/metabolism , Signal Transduction , Epithelial Cells/metabolism , Receptors, Chemokine/metabolism , RNA, Messenger/metabolismABSTRACT
Rationale: The resolution of inflammation is an active process coordinated by mediators and immune cells to restore tissue homeostasis. However, the mechanisms for resolving eosinophilic allergic lung inflammation triggered by inhaled allergens have not been fully elucidated. Objectives: Our objectives were to investigate the cellular mechanism of tissue-resident macrophages involved in the resolution process of eosinophilic lung inflammation. Methods: For the study, we used the institutional review board-approved protocol for human subsegmental bronchoprovocation with allergen, mouse models for allergic lung inflammation, and novel transgenic mice, including a conditional CCL26 knockout. The samples were analyzed using mass cytometry, single-cell RNA sequencing, and biophysical and immunological analyses. Measurements and Main Results: We compared alveolar macrophage (AM) subsets in the BAL before and after allergen provocation. In response to provocation with inhaled allergens, the subsets of AMs are dynamically changed in humans and mice. In the steady state, the AM subset expressing CX3CR1 is a relatively small fraction in bronchoalveolar space and lung tissue but drastically increases after allergen challenges. This subset presents unique patterns of gene expression compared with classical AMs, expressing high C1q family genes. CX3CR1+ macrophages are activated by airway epithelial cell-derived CCL26 via a receptor-ligand interaction. The binding of CCL26 to the CX3CR1+ receptor induces CX3CR1+ macrophages to secrete C1q, subsequently facilitating the clearance of eosinophils. Furthermore, the depletion of CX3CR1 macrophages or CCL26 in airway epithelial cells delays the resolution of allergic lung inflammation displaying prolonged tissue eosinophilia. Conclusions: These findings indicate that the CCL26-CX3CR1 pathway is pivotal in resolving eosinophilic allergic lung inflammation.
Subject(s)
Alveolitis, Extrinsic Allergic , Hypersensitivity , Pneumonia , Pulmonary Eosinophilia , Humans , Mice , Animals , Complement C1q/metabolism , Lung/metabolism , Macrophages , Allergens , Inflammation/metabolism , Pneumonia/metabolism , Chemokine CCL26/metabolismABSTRACT
AIM: To verify the role of CX3C chemokine ligand 1 - CX3C chemokine receptor 1 (CX3CL1-CX3CR1) pathway in the pathogenesis of primary biliary cholangitis (PBC). To explore whether CCL26, a novel functional ligand to CX3CR1, participates in the immunological mechanism of PBC. METHODS: Fifty-nine PBC patients and 54 healthy controls were recruited. Enzyme-linked immunosorbent assay and flow cytometry were used to measure CX3CL1 and CCL26 concentrations in plasma and CX3CR1 expression on peripheral lymphocytes, respectively. Chemotactic effects of CX3CL1 and CCL26 toward lymphocytes were detected by Transwell cell migration assays. CX3CL1 and CCL26 expressions in liver were assessed by immunohistochemical staining. Effects of CX3CL1 and CCL26 on stimulating cytokine production from lymphocytes were evaluated using intracellular flow cytometry. RESULTS: Significantly elevated CX3CL1 and CCL26 plasma concentration and CX3CR1 expression on CD4+ and CD8+ T cells were noted in PBC patients. CX3CL1 exhibited chemotactic activity toward CD8+ T, natural killer (NK) and NKT cells in a dose-dependent manner while such chemotactic effects were not detected for CCL26. In PBC patients, CX3CL1 and CCL26 were both increasingly expressed in biliary tracts and a concentration gradient of CCL26 in hepatocytes around portal areas was observed. Immobilized CX3CL1 could enhance interferon-γ production from T and NK cells while such effect was not exhibited by soluble CX3CL1 or CCL26. CONCLUSIONS: CCL26 expression is significantly elevated in plasma and biliary duct of PBC patients, yet does not appear to attract CX3CR1-expressing immune cells. CX3CL1-CX3CR1 pathway promotes the infiltration of T, NK and NKT cells into bile ducts and forms a positive feedback loop with T-helper 1 type cytokines in PBC.
Subject(s)
CD8-Positive T-Lymphocytes , Liver Cirrhosis, Biliary , Humans , Liver Cirrhosis, Biliary/diagnosis , Ligands , Killer Cells, Natural , Cytokines/metabolism , Receptors, Chemokine/metabolism , Chemokine CCL26/metabolismABSTRACT
BACKGROUND: In mice models, eosinophils have been divided into different subpopulations with distinct phenotypes and functions, based on CD62L and CD101 patterns of membrane expression. Limited data are available in humans. OBJECTIVE: To investigate eosinophils subpopulations in peripheral blood (PB) and nasal polyp tissue (NP) from severe eosinophilic asthma (SEA) patients plus concomitant chronic rhinosinusitis with nasal polyps (CRSwNP). METHODS: We recruited 23 SEA patients (14 with CRSwNP); as controls, we enrolled 15 non-severe asthma patients, 15 allergic rhinitis patients without asthma and 15 healthy donors. Eosinophils were isolated from PB and NP and analysed by FACS. Eotaxin-3 and eotaxin-1 mRNA expression in NP tissue was also evaluated. RESULTS: A significantly higher percentage of circulating CD62Llow cells was observed in SEA, as compared with controls, expressing higher levels of CCR3, CD69 and lower levels of CD125 (IL-5R), CRTH2, CD86 and CD28 in comparison with CD62Lbright cells. In NP, eosinophils showed a high proportion of CD62Llow phenotype, significantly greater than that observed in PB. Surface expression of IL-3R, IL-5R, CD69 and CD86 was significantly higher in CD62Llow eosinophils from NP than in those from blood. Moreover, eotaxin-3 mRNA expression positively correlated with the percentage of CD62Llow cells in NP. CONCLUSION: Two different eosinophil subphenotypes can be identified in blood and NP of SEA patients, with a preferential accumulation of CD62Llow inflammatory cells in NP.
Subject(s)
Asthma , Nasal Polyps , Rhinitis , Sinusitis , Humans , Animals , Mice , Eosinophils , Nasal Polyps/complications , Chemokine CCL26/metabolism , Sinusitis/complications , Chronic Disease , RNA, Messenger/metabolismABSTRACT
This study aimed to determine the expression and investigate the role of chemokine 26 (CCL26) in colon cancer cells. The Cancer Genome Atlas (TCGA) analysis, qRT-PCR, and western blotting were used to detect CCL26 expression while the Kaplan-Meier plotter was used to analyze the survival of patients with colon cancer. Cell Counting Kit-8 (CCK-8), colony formation, flow cytometry, and Transwell assays were performed to measure the viability, proliferation, apoptosis, and migration and invasion of colon cancer cells, respectively. The Kyoto Encyclopedia of Genes and Genomes (KEGG) was used to analyze the signaling pathways regulated by CCL26. Western blotting, used to measure protein expression in this study, found overexpression of CCL26 in colon tumors. Low CCL26 levels were associated with better survival of patients as CCL26 siRNAs markedly reduced viability and proliferation, accelerated apoptosis, decreased migration and invasion, enhanced E-cadherin expression, and reduced N-cadherin and vimentin expression in cancer cells. The opposite results were obtained in CCL26-overexpressed colon cancer cells. In addition, CCL26 activated the epithelial-mesenchymal transition (EMT) pathway. CCL26 siRNAs suppressed the expression of tissue inhibitor matrix metalloproteinase 1 (TIMP1), nicotinamide N-methyltransferase (NNMT), and fibromodulin (FMOD), while CCL26 overexpression significantly increased the expression of all. EMT inhibition using the EMT inhibitor C19 eliminated the effect of CCL26 overexpression on colon cancer cells. In summary, CCL26 is involved in colon cancer progression through regulation of the EMT signaling pathway.
Subject(s)
Colonic Neoplasms , Epithelial-Mesenchymal Transition , Humans , Epithelial-Mesenchymal Transition/genetics , Cell Movement/genetics , Cell Proliferation/genetics , Cell Line, Tumor , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , Signal Transduction/genetics , Gene Expression Regulation, Neoplastic , Chemokine CCL26/genetics , Chemokine CCL26/metabolismABSTRACT
Interaction with surrounding healthy cells plays a major role in the growth and metastasis of osteosarcoma. In this study, we hypothesized that humoral factors, which do not require direct contact with cells, are involved in the interaction between osteosarcoma and the surrounding cells. We identified the humoral factor involved in the association between tumor cells and surrounding normal cells using a co-culture model and investigated the significance of our findings. When human osteosarcoma cells (MG63) and human mesenchymal stem cells (hMSCs) were co-cultured and comprehensively analyzed for changes in each culture group, we found that the expression of chemokine (CC motif) ligand 26 (CCL26) was significantly enhanced. We also analyzed the changes in cell proliferation in co-culture, enhanced interaction with administration of recombinant CCL26 (rCCL26), reduced interaction with administration of anti-CCL26 antibodies, changes in invasive and metastatic abilities. CCL26 levels, motility, and invasive capability increased in the co-culture group and the group with added rCCL26, compared to the corresponding values in the MG63 single culture group. In the group with added CCL26 neutralizing antibodies, CCL26 level decreased in both the single and co-culture groups, and motility and invasive ability were also reduced. In a nude mice lung metastasis model, the number of lung metastases increased in the co-culture group and the group with added rCCL26, whereas the number of tumors were suppressed in the group with added neutralizing antibodies compared to those in the MG63 alone. This study identified a possible mechanism by which osteosarcoma cells altered the properties of normal cells to favorably change the microenvironment proximal to tumors and to promote distant metastasis.
Subject(s)
Bone Neoplasms/metabolism , Chemokine CCL26/metabolism , Osteosarcoma/metabolism , Signal Transduction , Tumor Microenvironment , Antineoplastic Agents, Immunological , Bone Neoplasms/etiology , Bone Neoplasms/pathology , Cell Line, Tumor , Cell Movement , Cell Proliferation , Chemokine CCL26/antagonists & inhibitors , Chemokine CCL26/genetics , Coculture Techniques , Fluorescent Antibody Technique , Gene Expression , Gene Expression Regulation, Neoplastic/drug effects , Humans , Immunohistochemistry , Osteosarcoma/etiology , Osteosarcoma/pathology , Transcriptome , Tumor Microenvironment/genetics , rac GTP-Binding Proteins/metabolism , src-Family Kinases/metabolismABSTRACT
BACKGROUND & AIMS: In upper airway cells, T helper 2 cytokines that signal through interleukin-4 (IL-4) receptor-α have been shown to stimulate eotaxin-3 secretion via a nongastric proton pump (ngH+,K+ATPase). To seek novel targets for eosinophilic esophagitis (EoE) treatments, we evaluated ngH+,K+ATPase expression in EoE squamous cells, and explored molecular pathways involved in eotaxin-3 secretion by IL-4 receptor-α signaling. METHODS: ngH+,K+ATPase expression in EoE cells was evaluated by quantitative real-time polymerase chain reaction and Western blotting. IL-4-stimulated eotaxin-3 secretion was measured by enzyme-linked immunosorbent assay after treatment with omeprazole, SCH 28080 (potassium-competitive acid blocker), ethylene glycol-bis(ß-aminoethyl)-N,N,N',N'-tetraacetoxymethyl ester (calcium chelator), 2-aminoethoxydiphenyl borate (inhibitor of endoplasmic reticulum calcium release), verapamil, and diltiazem (L-type calcium channel inhibitors). Intracellular calcium transients were measured by Fluo-4 fluorescence. Key experiments were confirmed in EoE primary cells and in RNA sequencing datasets from mucosal biopsies of patients with EoE and controls. RESULTS: EoE cells expressed ngH+,K+ATPase messenger RNA and protein. Omeprazole and SCH 28080 decreased IL-4-stimulated eotaxin-3 secretion. IL-4 increased intracellular calcium transients, and IL-4-stimulated eotaxin-3 secretion was blocked by ethylene glycol-bis(ß-aminoethyl)-N,N,N',N'-tetraacetoxymethyl ester, 2-aminoethoxydiphenyl borate, verapamil, and diltiazem. The combination of omeprazole and verapamil suppressed IL-4-stimulated eotaxin-3 secretion more than either agent alone. EoE biopsies expressed higher ngH+,K+ATPase and exhibited more calcium signaling than controls. CONCLUSIONS: EoE cells express a nongastric proton pump that mediates T helper 2 cytokine-stimulated eotaxin-3 secretion. IL-4 induces calcium release from the endoplasmic reticulum and calcium entry via L-type calcium channels, increasing intracellular calcium that contributes to eotaxin-3 secretion by EoE cells. L-type calcium channel inhibitors block T helper 2 cytokine-stimulated eotaxin-3 secretion, suggesting a potential role for these agents in EoE treatment.
Subject(s)
Chemokine CCL26/metabolism , Eosinophilic Esophagitis/metabolism , Eosinophilic Esophagitis/pathology , Epithelial Cells/metabolism , H(+)-K(+)-Exchanging ATPase/genetics , H(+)-K(+)-Exchanging ATPase/metabolism , Biological Transport/drug effects , Boron Compounds/pharmacology , Calcium/metabolism , Calcium Channel Blockers/pharmacology , Cell Line , Diltiazem/pharmacology , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Esophageal Mucosa/metabolism , Esophageal Mucosa/pathology , Famotidine/pharmacology , Female , Histamine H2 Antagonists/pharmacology , Humans , Interleukin-4 Receptor alpha Subunit/metabolism , Male , Omeprazole/pharmacology , Primary Cell Culture , Proton Pump Inhibitors/pharmacology , Proton Pumps/drug effects , Proton Pumps/metabolism , RNA, Messenger/metabolism , Ranitidine/pharmacology , Signal Transduction/drug effects , Th2 Cells/metabolism , Verapamil/pharmacologyABSTRACT
BACKGROUND: Recent studies suggest that alterations in the vaginal microbiome allow for the assessment of the risk for spontaneous preterm birth (PTB), the leading cause of neonatal morbidity and mortality worldwide. However, the associations between the local immune response and the vaginal microbiome are still poorly understood. Herein, we characterize the vaginal host immune-microbiome interactions in women who ultimately underwent PTB and in those who delivered at term. METHODS: Vaginal fluid samples from 52 pregnant women (of whom 18 underwent PTB and 34 delivered at term) were collected between 10 and 32 weeks of gestation in a case-control study. Concentrations of 33 immune mediators were determined using sensitive and specific immunoassays. The previously published 16S rRNA gene sequence and bacterial phylotype data of these subjects were utilized in this study. Linear mixed effects models were utilized to test associations between vaginal immune mediator concentrations and bacterial phylotype relative abundances. RESULTS: 1) In the overall study population, vaginal concentrations of CXCL10, CCL2, CCL3, SLP1 and VEGF negatively correlated with non-Lactobacillus, Community State Type IV (CST IV) members of the vaginal microbiome; 2) CXCL10, in particular, negatively correlated with 15 bacterial phylotypes, most of which are typical members of CST IV, such as Gardnerella vaginalis, Megasphaera spp., and Atopobium vaginae; 3) Gemella spp., also members of CST IV, negatively correlated with vaginal concentrations of VEGF, CCL2, CCL3, SLPI, and CXCL10; 4) when comparing PTB cases to term controls, five soluble immune mediators (CCL26, CCL22, CCL2, CXCL10, and IL-16), especially CCL26, were negatively correlated with five typical members of CST IV: Sneathia sanguinegens, Parvimonas micra, Veillonellaceae, BVAB2, and Gemella spp.; and 5) Sneathia sanguinegens had stronger negative associations with all five soluble immune mediators (CCL26, CCL22, CCL2, CXCL10, and IL-16) in PTB cases than in term controls. CONCLUSIONS: The assessment of vaginal host immune-microbiome interactions revealed that specific soluble immune mediators, mainly CXCL10, negatively correlated with typical members of CST IV of the vaginal microbiome. Sneathia sanguinegens, in particular, had stronger negative associations with different immune mediators, including CXCL10 and CCL26, in women who ultimately underwent PTB compared to those who delivered at term. These findings provide insight into the vaginal host immune-microbiome interactions in normal and complicated pregnancies.
Subject(s)
Black or African American/statistics & numerical data , Microbiota/immunology , Premature Birth/immunology , Vagina/immunology , Adult , Bacteria/classification , Bacteria/genetics , Case-Control Studies , Chemokine CCL26/immunology , Chemokine CCL26/metabolism , Chemokine CXCL10/immunology , Chemokine CXCL10/metabolism , Cohort Studies , Cytokines/immunology , Cytokines/metabolism , Female , Host-Pathogen Interactions/immunology , Humans , Infant, Newborn , Microbiota/genetics , Microbiota/physiology , Pregnancy , Premature Birth/microbiology , RNA, Ribosomal, 16S/genetics , Vagina/microbiology , Young AdultABSTRACT
Nivolumab plus ipilimumab combined therapy is among the most effective therapies for advanced melanoma. However, this therapy is also associated with a high frequency of immune-related adverse events (irAEs). To avoid such severe irAEs caused by additional administration of anti-CTLA4 antibodies, biomarkers to distinguish responders from non-responders among patients treated with anti-PD1 antibodies are important. The purpose of this study is to evaluate the increased serum levels of CCL11, CCL24, and CCL26 as a predictive biomarker for the efficacy of anti-PD1 antibodies in advanced cutaneous melanoma patients. This study analyzed increased serum levels of CCL11, CCL24, and CCL26 in 46 cases of advanced cutaneous melanoma treated with anti-PD1 antibodies. Serum levels on day 42 were compared to baseline (day 0) and analyzed statistically. Receiver operating characteristic curves were established to evaluate the correlation between serum levels of CCL11, CCL24, and CCL26 and efficacy of anti-PD1 antibodies. Increased serum levels of CCL26 correlated significantly with the efficacy of anti-PD1 antibodies. In contrast, no significant correlations were seen between increased serum levels of CCL11 and CCL24 and efficacy of anti-PD1 antibodies. Increased serum levels of CCL26 may be a useful biomarker for identifying those patients with advanced cutaneous melanoma most likely to benefit from anti-melanoma immunotherapy.
Subject(s)
Biomarkers, Tumor/metabolism , Chemokine CCL26/metabolism , Melanoma/diagnosis , Skin Neoplasms/diagnosis , Adult , Aged , Aged, 80 and over , Female , Humans , Male , Melanoma/pathology , Middle Aged , Skin Neoplasms/pathologyABSTRACT
BACKGROUND: Diabetic nephropathy (DN) is the leading cause of end-stage renal disease worldwide. Inflammatory mediators have been implicated in the pathogenesis of DN, thus considered an inflammatory disease. However, further studies are required to assess the renal damage caused by the action of these molecules. Therefore, the objective of this study was to analyze the expression of cytokines and chemokines in renal biopsies from patients with DN and to correlate it with interstitial inflammation and decreased renal function. METHODS: Forty-four native renal biopsies from patients with DN and 23 control cases were selected. In situ expression of eotaxin, MIP-1α (macrophage inflammatory protein-1α), IL-8 (interleukin-8), IL-4, IL-10, TNF-α (tumor necrosis factor-α), TNFR1 (tumor necrosis factor receptor-1), IL-1ß, and IL-6 were evaluated by immunohistochemistry. RESULTS: The DN group showed a significant increase in IL-6 (p < 0.0001), IL-1ß (p < 0.0001), IL-4 (p < 0.0001) and eotaxin (p = 0.0012) expression, and a decrease in TNFR1 (p = 0.0107) and IL-8 (p = 0.0262) expression compared to the control group. However, there were no significant differences in IL-10 (p = 0.4951), TNF-α (p = 0.7534), and MIP-1α (p = 0.3816) expression among groups. Regarding interstitial inflammation, there was a significant increase in IL-6 in scores 0 and 1 compared to score 2 (p = 0.0035), in IL-10 in score 2 compared to score 0 (p = 0.0479), and in eotaxin in score 2 compared to scores 0 and 1 (p < 0.0001), whereas IL-8 (p = 0.0513) and MIP-1α (p = 0.1801) showed no significant differences. There was a tendency for negative correlation between eotaxin and estimated glomerular filtration rate (eGFR) (p = 0.0566). CONCLUSIONS: Our results indicated an increased in situ production of cytokines and chemokines in DN, including IL-6, IL-1ß, IL-4, and eotaxin. It was observed that, possibly, eotaxin may have an important role in the progression of interstitial inflammation in DN and in eGFR decrease of these patients.
Subject(s)
Cytokines/metabolism , Diabetic Nephropathies/metabolism , Kidney/metabolism , Adult , Aged , Aged, 80 and over , Case-Control Studies , Chemokine CCL11/metabolism , Chemokine CCL24/metabolism , Chemokine CCL26/metabolism , Chemokine CCL3/metabolism , Chemokines/metabolism , Diabetic Nephropathies/pathology , Female , Humans , Immunohistochemistry , Interleukin-10/metabolism , Interleukin-1beta/metabolism , Interleukin-4/metabolism , Interleukin-6/metabolism , Interleukin-8/metabolism , Kidney/pathology , Male , Middle Aged , Receptors, Tumor Necrosis Factor, Type I/metabolism , Tumor Necrosis Factor-alpha/metabolism , Young AdultABSTRACT
Atopic dermatitis (AD) is a common inflammatory skin disease predominately related to Type 2 helper T (Th2) immune responses. In this study, we investigated whether piperine is able to improve AD symptoms using a trimellitic anhydride (TMA)-induced AD-like mouse model. Topical treatment with piperine reduced ear swelling (ear thickness and epidermal thickness) induced by TMA exposure. Furthermore, piperine inhibited pro-inflammatory cytokines such as TNF-α and IL-1ß in mouse ears, compared with the TMA-induced AD group. In measuring allergic immune responses in draining lymph nodes (dLNs), we found that IL-4 secretion, GATA3 mRNA level, and STAT6 phosphorylation were suppressed by piperine treatment. In an ex vivo study, piperine also inhibited the phosphorylation of STAT6 on the CD4+ T cells isolated from splenocytes of BALB/c mice, and piperine suppressed IL-4-induced CCL26 mRNA expression and STAT6 phosphorylation in human keratinocytes resulting in the inhibition of infiltration of CCR3+ cells into inflammatory lesions. These results demonstrate that piperine could ameliorate AD symptoms through suppression of Th2-mediated immune responses, including the STAT6/GATA3/IL-4 signaling pathway. Therefore, we suggest that piperine is an excellent candidate as an inhibitor of STAT6 and may help to improve AD symptoms.
Subject(s)
Alkaloids/pharmacology , Anti-Allergic Agents/pharmacology , Benzodioxoles/pharmacology , Dermatitis, Atopic/drug therapy , Piperidines/pharmacology , Polyunsaturated Alkamides/pharmacology , STAT6 Transcription Factor/metabolism , Signal Transduction/drug effects , Th2 Cells/immunology , Alkaloids/therapeutic use , Animals , Anti-Allergic Agents/therapeutic use , Benzodioxoles/therapeutic use , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/immunology , Cells, Cultured , Chemokine CCL26/genetics , Chemokine CCL26/metabolism , Dermatitis, Atopic/chemically induced , Disease Models, Animal , Female , GATA3 Transcription Factor/genetics , GATA3 Transcription Factor/metabolism , Humans , Interleukin-1beta/metabolism , Interleukin-4/metabolism , Keratinocytes/drug effects , Keratinocytes/metabolism , Lymph Nodes/drug effects , Lymph Nodes/immunology , Lymph Nodes/metabolism , Mice , Mice, Inbred BALB C , Phosphorylation , Phthalic Anhydrides/toxicity , Piperidines/therapeutic use , Polyunsaturated Alkamides/therapeutic use , Receptors, CCR3/genetics , Receptors, CCR3/metabolism , Signal Transduction/immunology , Th2 Cells/drug effects , Tumor Necrosis Factor-alpha/metabolismABSTRACT
The human bronchial epithelium is an important barrier tissue that is damaged or pathologically altered in various acute and chronic respiratory conditions. To represent the epithelial component of respiratory disease, it is essential to use a physiologically relevant model of this tissue. The human bronchial epithelium is a highly organized tissue consisting of a number of specialized cell types. Primary human bronchial epithelial cells (HBEC) can be differentiated into a mucociliated tissue in air-liquid interface (ALI) cultures using appropriately supplemented media under optimized growth conditions. We compared the histology, ciliary length, and function, diffusion, and barrier properties of HBEC from donors with no respiratory disease grown in two different media, PneumaCult-ALI or Bronchial Epithelial Differentiation Medium (BEDM). In the former group, HBEC have a more physiological pseudostratified morphology and mucociliary differentiation, including increased epithelial thickness, intracellular expression of airway-specific mucin protein MUC5AC, and total expression of cilia basal-body protein compared with cells from the same donor grown in the other medium. Baseline expression levels of inflammatory mediators, thymic stromal lymphopoietin (TSLP), soluble ST2, and eotaxin-3 were lower in PneumaCult-ALI. Additionally, the physiological cilia beat frequency and electrical barrier properties with transepithelial electrical resistance were significantly different between the two groups. Our study has shown that these primary cell cultures from the same donor grown in the two media possess variable structural and functional characteristics. Therefore, it is important to objectively validate primary epithelial cell cultures before experimentation to ensure they are appropriate to answer a specific scientific question.
Subject(s)
Culture Media/pharmacology , Epithelial Cells/drug effects , Gene Expression/drug effects , Respiratory Mucosa/drug effects , Air , Bronchi/cytology , Bronchi/metabolism , Cell Differentiation/drug effects , Chemokine CCL26/genetics , Chemokine CCL26/metabolism , Cilia/drug effects , Cilia/metabolism , Culture Media/chemistry , Cytokines/genetics , Cytokines/metabolism , Epithelial Cells/cytology , Epithelial Cells/metabolism , Healthy Volunteers , Humans , Interleukin-1 Receptor-Like 1 Protein/genetics , Interleukin-1 Receptor-Like 1 Protein/metabolism , Models, Biological , Mucin 5AC/genetics , Mucin 5AC/metabolism , Primary Cell Culture , Respiratory Mucosa/cytology , Respiratory Mucosa/metabolismABSTRACT
BACKGROUND: Partial burn injury in older patients is associated with higher rates of morbidity, mortality, and conversion to full thickness burn (Finnerty et al., 2009; Pham et al., 2009). Both human and mouse models demonstrate an altered systemic immune response in older subjects, however less is known about the localized response (Jeschke et al., 2016; Farinas et al., 2018; Mohs et al., 2017). We hypothesized that a mouse model could demonstrate differences in the localized inflammatory response of the old. METHODS: Six old (66 weeks) and young (8 weeks) mice received partial thickness thermal burns. Localized and systemic expression of nine chemokines (TNFalpha, MCP-1, MIP-2, S100A9, EGF, IL-10, RANTES, G-CSF, and EOTAXIN) were evaluated at day 3 after burn using Luminex analysis. Vimentin immunostaining was used to evaluate injury depth. RESULTS: Vimentin staining demonstrated increased burn depth in old mice (449±38µm) as compared to young (166±18µm) (p<0.05). Both groups exhibited increased localized expression of EOTAXIN after burn (p<0.05), however expression in old mice (83.6±6.1pg/ml) was lower than that of young (126.8±18.7pg/ml) (p<0.05). Systemically, however, old mice had increased baseline EOTAXIN expression (1332.40±110.78pg/ml) compared to young (666.12±45.8pg/ml) (p<0.005). CONCLUSIONS: EOTAXIN is one of the primary chemoattractants for selective eosinophilic recruitment and activation. While eosinophils are important for wound healing, a hyperactive eosinophilic response can result in tissue damage. We hypothesize that the increased baseline serum EOTAXIN in the old may prime their hyperactive response, and may contribute to their worse clinical outcomes. Long-term eosinophil activation requires further study, however our findings indicate a role for EOTAXIN and eosinophils in burn response.
Subject(s)
Aging/immunology , Burns/immunology , Chemokine CCL11/immunology , Chemokine CCL24/immunology , Chemokine CCL26/immunology , Eosinophils/immunology , Aging/metabolism , Animals , Burns/metabolism , Burns/pathology , Calgranulin B/immunology , Calgranulin B/metabolism , Chemokine CCL11/metabolism , Chemokine CCL2/immunology , Chemokine CCL2/metabolism , Chemokine CCL24/metabolism , Chemokine CCL26/metabolism , Chemokine CCL5/immunology , Chemokine CCL5/metabolism , Chemokine CXCL2/immunology , Chemokine CXCL2/metabolism , Eosinophils/metabolism , Epidermal Growth Factor/immunology , Epidermal Growth Factor/metabolism , Granulocyte Colony-Stimulating Factor/immunology , Granulocyte Colony-Stimulating Factor/metabolism , Inflammation/immunology , Inflammation/metabolism , Inflammation/pathology , Interleukin-10/immunology , Interleukin-10/metabolism , Mice , Tumor Necrosis Factor-alpha/immunology , Tumor Necrosis Factor-alpha/metabolismSubject(s)
Anti-Asthmatic Agents/therapeutic use , Antibodies, Monoclonal, Humanized/therapeutic use , Asthma/drug therapy , Randomized Controlled Trials as Topic , Th2 Cells/metabolism , Asthma/blood , Asthma/diagnosis , Asthma/immunology , Biomarkers/analysis , Biomarkers/metabolism , Chemokine CCL17/analysis , Chemokine CCL17/metabolism , Chemokine CCL26/analysis , Chemokine CCL26/metabolism , Eosinophils/immunology , Exhalation , Humans , Leukocyte Count , Nitric Oxide/analysis , Th2 Cells/immunology , Treatment OutcomeABSTRACT
BACKGROUND: microRNA (miR)-218-5p is involved in cigarette smoke-induced airway inflammation. In our earlier asthma epithelial miRNA profiling data, miR-218-5p was the top 2 down-regulated miRNA. We hypothesize that miR-218-5p plays a role in asthma airway inflammation. OBJECTIVE: To unveil the role of miR-218-5p and its target gene in asthma airway inflammation. METHODS: We measured miR-218-5p expression in bronchial brushings of asthma patients (n = 50) and healthy controls (n = 15), and analysed the correlations between miR-218-5p expression and airway eosinophilia. We examined whether CTNND2 was a target of miR-218-5p, and the expression of 12 catenin family members in bronchial brushings, in cultured human bronchial epithelial (HBE) cells and BEAS-2B cells. We explored the role of miR-218-5p-CTNND2 pathway using a murine model of allergic airway inflammation. RESULTS: Epithelial miR-218-5p expression was significantly decreased and negatively correlated with eosinophils in induced sputum and bronchial biopsies, and other type 2 biomarkers in asthma patients. We verified that CTNND2 (encoding δ-catenin) was a target of miR-218-5p. Remarkably, CTNND2 was the most significantly up-regulated catenin compared with the other 11 catenin family members in bronchial brushings of asthma patients, IL-13-stimulated HBE and BEAS-2B cells. Moreover, epithelial CTNND2 expression positively correlated with airway eosinophilia in asthma. Airway mmu-miR-218-5p expression was also decreased, and Ctnnd2 expression was increased in a murine model of allergic airway inflammation. Intriguingly, mmu-miR-218-5p overexpression suppressed airway hyperresponsiveness, eosinophilic airway inflammation and Ctnnd2 up-regulation in the mouse model. Finally, perturbation of miR-218-5p or CTNND2 expression significantly altered chemokine CCL26 expression in the cell cultures and the mouse model. CONCLUSIONS AND CLINICAL RELEVANCE: Epithelial miR-218-5p plays a protective role in eosinophilic airway inflammation via targeting CTNND2, a novel catenin in asthma, and suppressing chemokine CCL26 expression.
Subject(s)
Asthma/genetics , Catenins/genetics , Chemokine CCL26/metabolism , Eosinophilia/genetics , MicroRNAs/genetics , Animals , Asthma/metabolism , Bronchi/metabolism , Case-Control Studies , Cell Line , Cells, Cultured , Chemokine CCL11/metabolism , Chemokine CCL24/metabolism , Eosinophilia/metabolism , Gene Expression , Humans , Mice , Delta CateninABSTRACT
OBJECTIVES: Eosinophilic esophagitis (EoE), a chronic food allergic disease, lacks sensitive and specific peripheral biomarkers. We hypothesized that levels of EoE-related biomarkers captured using a 1-hour minimally invasive Esophageal String Test (EST) would correlate with mucosal eosinophil counts and tissue concentrations of these same biomarkers. We aimed to determine whether a 1-hour EST accurately distinguishes active from inactive EoE or a normal esophagus. METHODS: In a prospective, multisite study, children and adults (ages 7-55 years) undergoing a clinically indicated esophagogastroduodenoscopy performed an EST with an esophageal dwell time of 1 hour. Subjects were divided into 3 groups: active EoE, inactive EoE, and normal esophageal mucosa. Eosinophil-associated protein levels were compared between EST effluents and esophageal biopsy extracts. Statistical modeling was performed to select biomarkers that best correlated with and predicted eosinophilic inflammation. RESULTS: One hundred thirty-four subjects (74 children, 60 adults) with active EoE (n = 62), inactive EoE (n = 37), and patient controls with a normal esophagus (n = 35) completed the study. EST-captured eosinophil-associated biomarkers correlated significantly with peak eosinophils/high-power field, endoscopic visual scoring, and the same proteins extracted from mucosal biopsies. Statistical modeling, using combined eotaxin-3 and major basic protein-1 concentrations, led to the development of EoE scores that distinguished subjects with active EoE from inactive EoE or normal esophagi. Eighty-seven percent of children, 95% of parents, and 92% of adults preferred the EST over endoscopy if it provided similar information. DISCUSSION: The 1-hour EST accurately distinguishes active from inactive EoE in children and adults and may facilitate monitoring of disease activity in a safe and minimally invasive fashion.
Subject(s)
Eosinophilic Esophagitis/diagnosis , Eosinophils , Esophageal Mucosa/cytology , Esophagus/cytology , Adolescent , Adult , Biomarkers/analysis , Biomarkers/metabolism , Biopsy , Chemokine CCL24/analysis , Chemokine CCL24/metabolism , Chemokine CCL26/analysis , Chemokine CCL26/metabolism , Child , Endoscopy, Digestive System , Eosinophilic Esophagitis/pathology , Esophageal Mucosa/diagnostic imaging , Esophageal Mucosa/pathology , Esophagus/diagnostic imaging , Esophagus/pathology , Feasibility Studies , Female , Humans , Leukocyte Count , Male , Middle Aged , Prospective Studies , Young AdultABSTRACT
BACKGROUND: Interleukin (IL)-17A is involved in the pathogenesis of allergic rhinitis (AR). Increased expression of IL-17A is correlated with disease severity and nasal eosinophilia. However, the molecular mechanisms by which IL-17A contributes to T-helper 2 cytokine IL-13-driven pathology in AR remain unclear. We sought to obtain mechanistic insight into how IL-17A and IL-13 regulate the epithelial production of eotaxin-3 representing eosinophilic inflammation in AR. METHODS: Human nasal epithelial cells (HNECs) from AR patients were cultured and stimulated with IL-17A, IL-13, or IL-17A and IL-13. Phosphorylated signal transducer activator of transcription 6 (p-STAT6) and suppressor of cytokine signaling 1 (SOCS1) in HNECs were assayed using Western blotting. Immunocytochemistry was used to determine p-STAT6-positive expression in the cells. Eotaxin-3 expression in the cells and culture supernatants was evaluated using real-time polymerase chain reaction and enzyme-linked immunosorbent assays. RESULTS: Stimulation with IL-13 alone induced STAT6 phosphorylation and promoted p-STAT6 nuclear translocation, leading to eotaxin-3 production by HNECs. These effects were further enhanced by cotreatment with IL-13 and IL-17A, whereas IL-17A alone had no impact on STAT6 or eotaxin-3 expression. Incubation with IL-17A or IL-13 increased the level of SOCS1 protein in the cells, whereas the addition of IL-17A attenuated IL-13-induced SOCS1 expression. CONCLUSION: IL-17A potentiated IL-13-driven STAT6 activation through the downregulation of SOCS1 expression, leading to enhancement of eotaxin-3 production by HNECs. These factors contributed to eosinophilic inflammation in AR.