ABSTRACT
Tumour-associated macrophages (TAMs) support cancer cell survival and suppress anti-tumour immunity. Tumour infiltration by CD163pos TAMs is associated with poor outcome in several human malignancies, including multiple myeloma (MM). Signal transducer and activator of transcription 3 (STAT3) is over-activated in human cancers, and specifically within TAMs activation of STAT3 may induce an immunosuppressive (M2-like) phenotype. Therefore, STAT3-inhibition in TAMs may be a future therapeutic strategy.We investigated TAM markers CD163, CD206, and activated STAT3 (pSTAT3) in patients with MGUS (n = 32) and MM (n = 45), as well as healthy controls (HCs, n = 13).Blood levels of the macrophage biomarkers sCD163 and sCD206, and circulating cytokines, as well as bone marrow mRNA expression of CD163 and CD206, were generally increased in MGUS and MM patients, compared to HCs, but to highly similar levels. By immunohistochemistry, bone marrow levels of pSTAT3 were increased specifically within CD163pos cells in both MGUS and MM patients.In conclusion, macrophage-related inflammatory changes, including activation of STAT3, were present already at the MGUS stage, at similar levels as in MM. Specific increase in pSTAT3 levels within CD163pos cells supports that the CD163 scavenger receptor may be a useful target for future delivery of STAT3-inhibitory drugs to TAMs in MM patients.
Subject(s)
Antigens, CD/biosynthesis , Antigens, Differentiation, Myelomonocytic/biosynthesis , Bone Marrow/metabolism , Macrophages/metabolism , Monoclonal Gammopathy of Undetermined Significance/immunology , Multiple Myeloma/immunology , Receptors, Cell Surface/biosynthesis , STAT3 Transcription Factor/biosynthesis , Aged , Bone Marrow Cells/metabolism , Case-Control Studies , Female , Humans , Immunosuppression Therapy , Immunosuppressive Agents , Inflammation , Male , Middle Aged , Monoclonal Gammopathy of Undetermined Significance/metabolism , Multiple Myeloma/metabolism , Phenotype , Phosphorylation , Prospective StudiesABSTRACT
Human genetic studies have pointed to a prominent role for innate immunity and lipid pathways in immunological and neurodegenerative disorders. Our understanding of the composition and function of immunomodulatory lipid networks in innate immune cells, however, remains incomplete. Here, we show that phospholipase Cγ2 (PLCγ2 or PLCG2)-mutations in which are associated with autoinflammatory disorders and Alzheimer's disease-serves as a principal source of diacylglycerol (DAG) pools that are converted into a cascade of bioactive endocannabinoid and eicosanoid lipids by DAG lipase (DAGL) and monoacylglycerol lipase (MGLL) enzymes in innate immune cells. We show that this lipid network is tonically stimulated by disease-relevant human mutations in PLCγ2, as well as Fc receptor activation in primary human and mouse macrophages. Genetic disruption of PLCγ2 in mouse microglia suppressed DAGL/MGLL-mediated endocannabinoid-eicosanoid cross-talk and also caused widespread transcriptional and proteomic changes, including the reorganization of immune-relevant lipid pathways reflected in reductions in DAGLB and elevations in PLA2G4A. Despite these changes, Plcg2-/- mice showed generally normal proinflammatory cytokine and chemokine responses to lipopolysaccharide treatment, instead displaying a more restricted deficit in microglial activation that included impairments in prostaglandin production and CD68 expression. Our findings enhance the understanding of PLCγ2 function in innate immune cells, delineating a role in cross-talk with endocannabinoid/eicosanoid pathways and modulation of subsets of cellular responses to inflammatory stimuli.
Subject(s)
Eicosanoids/metabolism , Endocannabinoids/metabolism , Immunity, Innate/immunology , Macrophages/immunology , Phospholipase C gamma/metabolism , Animals , Antigens, CD/biosynthesis , Antigens, Differentiation, Myelomonocytic/biosynthesis , COS Cells , Cell Line , Chlorocebus aethiops , Cytokines/immunology , Diglycerides/metabolism , Group IV Phospholipases A2/metabolism , HEK293 Cells , Humans , Inflammation/immunology , Lipopolysaccharides/immunology , Lipoprotein Lipase/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Microglia/immunology , Monoacylglycerol Lipases/metabolism , Phospholipase C gamma/genetics , Prostaglandins/biosynthesis , Receptors, Fc/immunology , Signal Transduction/immunologyABSTRACT
OBJECTIVE: To observe the protective effect of AC-YVAD-CMK on sepsis-induced acute kidney injury in mice and to explore its possible mechanisms primarily. METHODS: Eighteen male C57BL/6 mice were randomly divided into sham-operated group (Control), cecal ligation and puncture group (CLP), and CLP model treated with AC-YVAD-CMK group (AC-YVAD-CMK) (n = 6 in each group). Mice were sacrificed at 24 h after operation, and blood and kidney tissue samples were collected for analyses. Histologic changes were determined microscopically following HE staining. The expression of Ly-6B and CD68 was investigated using immunohistochemistry. Serum concentrations of creatinine (sCR) and blood urea nitrogen (BUN) were measured. Serum levels of interleukin-1ß (IL-1ß), interleukin-18 (IL-18), TNF-α, and interleukin-6 (IL-6) were determined by ELISA. The expressions of Caspas-1, NLRP-1, IL-1ß, and IL-18 in renal tissues were investigated using Western blot. Immunofluorescence staining was used to detect the expression of GSDMD protein in renal tissues. RESULTS: AC-YVAD-CMK treatment significantly alleviates sepsis-induced acute kidney injury, with decreased histological injury in renal tissues, suppresses the accumulation of neutrophils and macrophages in renal tissues, and decreased sCR and BUN level (P < 0.05). Attenuation of sepsis-induced acute kidney injury was due to the prohibited production of inflammatory cytokines and decrease expression of Caspas-1, NLRP-1, IL-1ß, and IL-18 in renal tissues. In addition, AC-YVAD-CMK treatment significantly reduced the expression of GSDMD in renal tissues compared to those observed in controls (P < 0.05). CONCLUSIONS: We demonstrated a marked renoprotective effect of caspase-1-inhibitor AC-YVAD-CMK in a rat model of sepsis by inhibition of pyroptosis.
Subject(s)
Acute Kidney Injury/drug therapy , Amino Acid Chloromethyl Ketones/pharmacology , Caspase 1/metabolism , Caspase Inhibitors/pharmacology , Pyroptosis/drug effects , Sepsis/drug therapy , Acute Kidney Injury/metabolism , Animals , Antigens, CD/biosynthesis , Antigens, Differentiation, Myelomonocytic/biosynthesis , Blood Urea Nitrogen , Creatinine , Cytokines/metabolism , Interleukin-18/biosynthesis , Interleukin-1beta/biosynthesis , Interleukin-6/biosynthesis , Kidney/metabolism , Macrophages/metabolism , Male , Mice , Mice, Inbred C57BL , Neutrophils/metabolism , Sepsis/metabolismABSTRACT
OBJECTIVE: The secondary injury caused by RBC autolysis after intracerebral hemorrhage (ICH) can be reduced by increasing the efficiency of microglia (MG)/macrophages (Mø) phagocytizing red blood cells (RBCs). CD47 is an important regulator of MG/Mø phagocytosis. This study aims to clarify whether anti-CD47 antibody administrated into the cisterna magna after ICH can transfer to the hematoma site, promote MG/Mø gathering to phagocytize RBCs and ultimately reduce cell death. METHODS: Forty male Wistar rats were divided into sham, ICH, low-dosage (group A, 0.3 µg), medium-dosage (group B, 0.9 µg) and high-dosage (group C, 1.8 µg) anti-CD47 antibody groups. For the rats in group A, B and C, anti-CD47 antibody solution was administrated into the cisterna magna at 10 min after ICH. Brain tissue was harvested 3 days after the operation. Western blotting was performed to detect the expression of Caspase-3 and Bcl-2. Immunofluorescence was performed to detect the CD68 expression. TUNEL was performed to detect the cell death. RESULTS: The hematoma of the ICH rats was located in the basal ganglia, with a good homogeneity of hematoma volume. Low-dosage anti-CD47 antibody in group A had no effects on the perihematomal CD68 (P = 0.338), Caspase-3 (P = 0.769), Bcl-2 (P = 0.176) expression and cell death (P = 0.698), compared with the ICH group. CD68 and Bcl-2 expression increased and Caspase-3 expression decreased significantly in group B (P < 0.001 for all) and group C (P < 0.001 for all). The increase of CD68 expression in group C was greater than that in group B (P < 0.01) by a large margin, while there was no difference for Bcl-2 (P = 0.908) and Caspase-3 (P = 0.913) expression between the 2 groups. Compared with the ICH group, medium-dosage of anti-CD47 antibody in group B significantly reduced the number of TUNEL-positive cells (P < 0.005), but not for group C (P = 0.311). CONCLUSION: The results suggested that anti-CD47 antibody administration into the cisterna magna in proper dosage (0.9 µg) can effectively reach the hematoma, induce more MG/Møs to gather around the hematoma, and reduce cell death in perihematomal brain tissue. The results of this study has provided a basic theory for improving the efficiency of MG/Mø phagocytizing RBCs and hematoma clearance after ICH by administrating anti-CD47 antibody via the cisterna magna.
Subject(s)
Antibodies, Blocking/therapeutic use , CD47 Antigen/immunology , Cell Death/drug effects , Cerebral Hemorrhage, Traumatic/drug therapy , Cerebral Hemorrhage/drug therapy , Cisterna Magna , Animals , Antibodies, Blocking/administration & dosage , Antigens, CD/biosynthesis , Antigens, Differentiation, Myelomonocytic/biosynthesis , Basal Ganglia/pathology , Caspase 3/metabolism , Dose-Response Relationship, Drug , Hematoma , Male , Microinjections , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Rats , Rats, WistarABSTRACT
We characterised patients with mantle cell lymphoma (MCL) with poor prognosis based on differences in immune infiltration. Different expressions of the tumour cell markers Cyclin D1 and sex-determining region Y-box transcription factor 11 (SOX11), and the immune markers cluster of differentiation 3 (CD3), CD4, CD8, CD25, forkhead box protein P3 (FoxP3), T-box transcription factor TBX21 (T-bet), programmed cell death protein 1 (PD-1), programmed-death ligand 1 (PD-L1) and CD163 were investigated for all-cause mortality in 282 patients with MCL and time-to-progression (TTP) in 106 clinical trial patients. With increasing age, a significantly lower infiltration of CD3+ T lymphocytes was seen. T-cell infiltration was independent of cellular tumour antigen p53 (p53) expression, Ki-67, morphology and frequency of tumour cells. The all-cause mortality was higher in patients with PD-L1-expression above cut-off [hazard ratio (HR) 1·97, 95% confidence interval (CI) 1·18-3·25, adjusted for sex and MCL International Prognostic Index (MIPI)] and a higher frequency of CD163+ cells (continuously, HR 1·51, 95% CI 1·03-2·23, adjusting for age, sex, morphology, Ki-67 and p53). In patients treated within the Nordic Lymphoma Group MCL2/3 trials, TTP was shorter in patients with a higher frequency of FoxP3+ cells (HR 3·22, 95% CI 1·40-7·43) and CD163+ cells (HR 6·09, 95% CI 1·84-20·21), independent of sex and MIPI. When combined a higher frequency of CD163+ macrophages and PD-L1+ cells or high CD163+ macrophages and FoxP3+ regulatory T cells indicated worse outcome independent of established risk factors. The T-cell infiltrate was in turn independent of molecular characteristics of the malignant cells and decreased with age.
Subject(s)
Aging/metabolism , Antigens, CD/biosynthesis , Antigens, Differentiation, Myelomonocytic/biosynthesis , B7-H1 Antigen/biosynthesis , Forkhead Transcription Factors/biosynthesis , Lymphoma, Mantle-Cell , Neoplasm Proteins/biosynthesis , Receptors, Cell Surface/biosynthesis , Aged , Female , Humans , Lymphoma, Mantle-Cell/metabolism , Lymphoma, Mantle-Cell/mortality , Male , Middle Aged , Risk FactorsABSTRACT
OBJECTIVE: Recent studies have indicated that CD47, interacting with SIRP-α, conveys "don't eat me" signal in evasion of tumor cells and serves as a potential target for cancer immunotherapy. The purpose of this study was to investigate the clinical correlation of CD47 and uncover prognostic implications of CD47 and CD68 in non-small cell lung cancer (NSCLC). METHODS: The specimens from 384 patients with completely resected NSCLC were collected for immunohistochemical assays of CD47 and CD68. Cox multivariate proportion hazard analyses were conducted to confirm the independent prognostic value of CD47 and CD68. TCGA database and GSE37745 were used to identify the association between CD47 and immune cells. RESULTS: In 186 pairs of lung cancer and adjacent tissues, the RNA of CD47 was overexpressed in lung cancer tissues (P < 0.001). High expression of CD47 was associated with worse recurrence-free survival in RNA and protein level (P = 0.032 and P < 0.001, respectively). High expression of CD47 was significantly associated with large tumor size (P = 0.004), advanced pathologic TNM stage (P < 0.001), and histology (P = 0.003). Further analyses demonstrated that CD47 and CD68 predicted outcomes of patients independently. In addition, the expression of CD47 correlated with neutrophils, and did not correlated with B cells and CD4 + T cells in the TCGA database and GSE37745. CONCLUSION: Combined use of CD47 and CD68 exhibited excellent performance in predicting survival of patients with NSCLC. CD47 was a potential therapeutic target for immune therapy of lung cancer.
Subject(s)
Antigens, CD/biosynthesis , Antigens, Differentiation, Myelomonocytic/biosynthesis , CD47 Antigen/biosynthesis , Carcinoma, Non-Small-Cell Lung/mortality , Lung Neoplasms/mortality , Aged , Antigens, CD/genetics , Antigens, CD/immunology , Antigens, Differentiation, Myelomonocytic/genetics , Antigens, Differentiation, Myelomonocytic/immunology , Asia/epidemiology , CD47 Antigen/genetics , CD47 Antigen/immunology , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/immunology , Carcinoma, Non-Small-Cell Lung/pathology , Cohort Studies , Female , Humans , Immunohistochemistry , Kaplan-Meier Estimate , Lung Neoplasms/genetics , Lung Neoplasms/immunology , Lung Neoplasms/pathology , Male , Middle Aged , Proportional Hazards Models , RNA/genetics , RNA/metabolismABSTRACT
Hematoma is a crucial factor leading to poor prognosis after intracerebral hemorrhage (ICH). Promoting microglial phagocytosis to enhance hematoma resolution may be an important therapeutic target for recovery after ICH. C-C chemokine receptor 4 (CCR4) is important for regulating immune balance in the central nervous system. However, whether CCR4 activation can attenuate hematoma after ICH remains unknown. We aimed to evaluate whether CCL17 (a specific ligand of CCR4) treatment can promote hematoma resolution through CCR4/ERK/Nrf2/CD163 pathway after ICH. A total of 261 adult male CD1 mice were used. Mice were subjected to intrastriatal injection of autologous blood to induce ICH and randomly assigned to receive recombinant CCL17 (rCCL17) or vehicle which was administered intranasally at 1 h after ICH. To elucidate the underlying mechanism, C021, a selective inhibitor of CCR4 and ML385 and a selective inhibitor of Nrf2 were administered 1 h prior to ICH induction. Clustered regularly interspaced short palindromic repeats (CRISPR) knockout for CD163 was administered by intracerebroventricular injection at 48 h before ICH. Brain edema, short- and long-term neurobehavior evaluation, hematoma volume, hemoglobin content, western blot, and immunofluorescence staining were performed. Endogenous CCL17, CCR4, and CD163 expression increased and peaked at 72 h after ICH. CCR4 was expressed by microglia. CCR4 activation with rCCL17 significantly improved neurobehavioral scores and reduced hematoma volume and brain edema compared with vehicle. Moreover, rCCL17 treatment significantly promoted phosphorylation of ERK1/2, increased the expression Nrf2, and upregulated CD163 expression after ICH. The protective effects of rCCL17 were abolished by administration of C021, ML385, and CD163 CRISPR knockout. This study demonstrated that CCR4 activation with rCCL17 promoted hematoma resolution by increasing CD163 expression and CCR4/ERK/Nrf2 pathway activation after ICH, thereby reducing brain edema and improving neurological function. Overall, our study suggests that CCR4 activation may be a potential therapeutic strategy to attenuate hematoma in early brain injury after ICH.
Subject(s)
Antigens, CD/biosynthesis , Antigens, Differentiation, Myelomonocytic/biosynthesis , Cerebral Hemorrhage/metabolism , Chemokine CCL17/therapeutic use , Hematoma/metabolism , MAP Kinase Signaling System/physiology , NF-E2-Related Factor 2/metabolism , Receptors, CCR4/metabolism , Receptors, Cell Surface/biosynthesis , Animals , Cerebral Hemorrhage/drug therapy , Chemokine CCL17/pharmacology , Hematoma/drug therapy , MAP Kinase Signaling System/drug effects , Male , Mice , Recombinant Proteins/pharmacology , Recombinant Proteins/therapeutic useABSTRACT
Cigarette smoking and opium use are risk factors for coronary artery disease (CAD). It has been known that scavenger receptors such as CD36 and CD68 play critical roles in the pathogenesis of CAD. CD9, as a member of the tetraspanin, has been shown to interact with scavenger receptors. The aim of this study was to investigate the effects of these risk factors on expression levels of CD9, CD36, and CD68 on the THP-1 cell line. The THP-1 cell line treated with cigarette smoke extract (CSE( and opium, both individually and combinatory, in 24 h incubation. The protein and mRNA levels of CD9, CD36, and CD68 were evaluated by flow cytometry and quantitative reverse transcription-Polymerase Chain Reaction (qRT-PCR) techniques, respectively. CD36 and CD68 mRNA and protein expression levels were significantly increased in the cells treated with cigarette smoke extract compared to the control (p<0.001 in mRNA expression levels and p=0.016 and p=0.012 in protein expression levels, respectively). The CSE increased the level of CD9 protein expression compared to the control group (p=0.041) on the human macrophage cell line THP-1. No significant differences were observed in the CD9, CD36, and CD68 gene expression and at the protein levels between opium-treated THP-1 cells and controls. In conclusion, cigarettes by increasing the levels of CD36, CD68, and CD9 can be a risk factor in the development of many inflammatory diseases, including cardiovascular diseases, chronic obstructive pulmonary disease (COPD) and lung carcinoma.
Subject(s)
Macrophages/drug effects , Macrophages/metabolism , Nicotiana/toxicity , Opium/toxicity , Plant Extracts/toxicity , Smoke/adverse effects , Antigens, CD/biosynthesis , Antigens, CD/drug effects , Antigens, Differentiation, Myelomonocytic/biosynthesis , Antigens, Differentiation, Myelomonocytic/drug effects , CD36 Antigens/biosynthesis , CD36 Antigens/drug effects , Humans , Smoking/adverse effects , THP-1 Cells , Tetraspanin 29/biosynthesis , Tetraspanin 29/drug effects , Tobacco Products/adverse effectsABSTRACT
Dim light at night (dLAN) has become a pervasive part of the modern world, and growing evidence shows its association with increased health risks. Though this link is attributed to a disturbed circadian clock, the underlying mechanisms that can explain how circadian disruption from dLAN causes negative health effects remain unclear. Here, we exposed rats to a light-dark cycle (12:12 h) with low-intensity light at night (~2 lx) for 2 and 5 weeks and explored the steady-state pattern of circulating immune cells and renal immune-related markers, which are well controlled by the circadian clock. After 5 weeks, dLAN impaired the daily variation in several types of white blood cells, especially monocytes and T cells. Two-week dLAN caused a reduction in blood monocytes and altered gene expression of macrophage marker Cd68 and monocyte-attracting chemokine Ccl2 in the kidney. Interestingly, dLAN decreased renal 3-nitrotyrosine levels and resulted in up-regulation of the main endogenous antioxidant pathways, indicating a disturbance in the renal redox balance and an activation of compensatory mechanisms. These effects paralleled the altered renal expression of the molecular clock components and increased plasma corticosterone levels. Together, our results show that chronic exposure to dLAN weakened the circadian control of daily variation of circulating immune cells and disturbed renal immune and redox homeostasis. Consequences of this dLAN-disturbed immune balance on the ability of the immune system to cope with other challenges should by clarified in further studies.
Subject(s)
Circadian Rhythm/immunology , Immune System/radiation effects , Kidney/immunology , Light/adverse effects , Photoperiod , Animals , Antigens, CD/biosynthesis , Antigens, CD/genetics , Antigens, Differentiation, Myelomonocytic/biosynthesis , Antigens, Differentiation, Myelomonocytic/genetics , CLOCK Proteins/biosynthesis , CLOCK Proteins/genetics , Cell Adhesion Molecules/biosynthesis , Cell Adhesion Molecules/genetics , Chemokines/biosynthesis , Chemokines/genetics , Corticosterone/blood , Gene Expression Regulation/radiation effects , Homeostasis/radiation effects , Immunophenotyping , Kidney/metabolism , Kidney Cortex/enzymology , Leukocyte Count , Male , Melatonin/blood , Oxidation-Reduction , Rats , Rats, Wistar , Respiratory Burst , Superoxide Dismutase/analysisABSTRACT
BACKGROUND: The aim of the study was to investigate the relationship between post-myocardial infarction (MI) inflammation and left ventricular (LV) remodeling in a swine model by 18F-fluorodeoxyglucose (FDG) imaging. METHODS: MI was induced in swine by balloon occlusion of the left anterior descending coronary artery. A series of FDG positron emission tomography (PET) images were taken within 2 weeks post-MI, employing a comprehensive strategy to suppress the physiological uptake of cardiomyocytes. Echocardiography was applied to evaluate LV volume, global and regional function. CD68+ macrophage and glucose transporters (GLUT-1, -3 and -4) were investigated by immunostaining. RESULTS: The physiological uptake of myocardium was adequately suppressed in 92.3% of PET scans verified by visual analysis, which was further confirmed by the minimal expression of myocardial GLUT-4. Higher FDG uptake was observed in the infarct than in the remote area and persisted within the observational period of 2 weeks. The FDG uptake of infarcted myocardium on day 1 post-MI was correlated with LV global remodeling, and the FDG uptake of infarcted myocardium on days 1 and 8 post-MI had a trend of correlating with regional remodeling of the infarct area. CONCLUSIONS: We here report a feasible swine model for investigating post-MI inflammation. FDG signal in the infarct area of swine persisted for a longer duration than has been reported in small animals. FDG activity in the infarct area could predict LV remodeling.
Subject(s)
Fluorodeoxyglucose F18 , Heart Ventricles/diagnostic imaging , Inflammation/diagnostic imaging , Myocardial Infarction/diagnostic imaging , Myocardial Perfusion Imaging/methods , Animals , Antigens, CD/biosynthesis , Antigens, Differentiation, Myelomonocytic/biosynthesis , Coronary Vessels/pathology , Echocardiography , Glucose Transporter Type 1/biosynthesis , Glucose Transporter Type 3/biosynthesis , Glucose Transporter Type 4/biosynthesis , Heart/diagnostic imaging , Image Processing, Computer-Assisted , Muscle Cells/pathology , Myocytes, Cardiac/metabolism , Necrosis , Positron-Emission Tomography , Radiopharmaceuticals , SwineABSTRACT
Signal regulatory protein-α (SIRPα) is a key member of the "do-not-eat-me" signaling pathway, but its biological role and clinical relevance in B-cell NHL is relatively unknown. Using biopsy specimens from follicular lymphoma (FL), we identified three subsets (CD14+SIRPαhi, CD14-SIRPαlow, and CD14-SIRPαneg) of monocyte/macrophages (Mo/MΦ) based on CD14 and SIRPα expression. CD14+SIRPαhi cells expressed common Mo/MΦ markers; exhibited characteristic differentiation, migration, and phagocytosis; and suppressed T-cell function. CD14-SIRPαlow cells expressed fewer typical Mo/MΦ markers; migrated less and phagocytosed tumor cells less efficiently; and stimulated rather than suppressed T-cell function. Interestingly, the CD14-SIRPαneg subset expressed distinct Mo/MΦ markers compared to the other two subsets; had limited ability to migrate and phagocytose; but stimulated T-cell function. When using SIRPα-Fc to block the interaction between SIRPα and CD47, alone or in combination with rituximab, phagocytosis of tumor cells was differentially increased in the three Mo/MΦ subsets. Clinically, increased numbers of CD14+SIRPαhi cells were associated with an inferior survival in FL. In contrast, increased numbers of the CD14-SIRPαlow subset appeared to correlate with a better survival. Taken together, our results show that SIRPα expression delineates unique subsets of intratumoral Mo/MΦs with differing prognostic importance.
Subject(s)
Antigens, Differentiation/metabolism , Lymphoma, Follicular/metabolism , Macrophages/pathology , Monocytes/pathology , Receptors, Immunologic/metabolism , Antigens, CD/biosynthesis , Antigens, CD/immunology , Antigens, Differentiation/biosynthesis , Antigens, Differentiation, Myelomonocytic/biosynthesis , Antigens, Differentiation, Myelomonocytic/immunology , Biomarkers, Tumor/immunology , Biomarkers, Tumor/metabolism , Humans , Lipopolysaccharide Receptors/biosynthesis , Lipopolysaccharide Receptors/immunology , Lymphoma, Follicular/immunology , Lymphoma, Follicular/pathology , Macrophages/immunology , Macrophages/metabolism , Middle Aged , Monocytes/immunology , Monocytes/metabolism , Prognosis , Receptors, Immunologic/biosynthesis , Survival RateABSTRACT
CD163 is a cellular receptor for Porcine reproductive and respiratory syndrome virus (PRRSV). Transgenic expression of CD163 can predispose a variety of PRRSV non-permissive cells to PRRSV infection. These resulting cells can then be used for PRRSV production and the study of PRRSV biology. The PiggyBac (PB) transposon is a non-viral, plasmid-based mobile genetic element that can be used for gene delivery into mammalian cells. In this study, a simple and efficient method for the transfection of the porcine CD163 transgene into an immortalized porcine alveolar macrophage cell line (3D4/21), a non-permissive cell line to PRRSV infection, by PB transposition was demonstrated. The resultant stably transformed 3D4/21/CD163 cells expressed CD163 constitutively and were shown to be fully permissive for PRRSV-2 strains and yielded an excess of 106 TCID50/mL of progeny virus. The PRRSV replicated more efficiently in the 3D4/21/CD163 cells than in Marc-145 cells, and the titers of the progeny PRRSV produced in the 3D4/21/CD163 cells were higher than those produced in Marc-145 cells. This simplified PB transposon-generated PRRSV-2 permissive 3D4/21/CD163 cell line could facilitate PRRSV production and accelerate the study of virus-host interactions in vitro.
Subject(s)
Antigens, CD/biosynthesis , Antigens, Differentiation, Myelomonocytic/biosynthesis , Macrophages, Alveolar/virology , Porcine respiratory and reproductive syndrome virus/growth & development , Receptors, Cell Surface/biosynthesis , Recombinant Proteins/biosynthesis , Virus Cultivation/methods , Animals , Antigens, CD/genetics , Antigens, Differentiation, Myelomonocytic/genetics , Cell Line , Gene Editing , Receptors, Cell Surface/genetics , Recombinant Proteins/genetics , Recombination, Genetic , SwineABSTRACT
Nonalcoholic fatty liver disease (NAFLD) is an emerging global pandemic. Though significant progress has been made in unraveling the pathophysiology of the disease, the role of protein phosphatase 2A (PP2A) and its subsequent inhibition by environmental and genetic factors in NAFLD pathophysiology remains unclear. The present report tests the hypothesis that an exogenous PP2A inhibitor leads to hepatic inflammation and fibrogenesis via an NADPH oxidase 2 (NOX2)-dependent pathway in NAFLD. Results showed that microcystin (MC) administration, a potent PP2A inhibitor found in environmental exposure, led to an exacerbation of NAFLD pathology with increased CD68 immunoreactivity, the release of proinflammatory cytokines, and stellate cell activation, a process that was attenuated in mice that lacked the p47phox gene and miR21 knockout mice. Mechanistically, leptin-primed immortalized Kupffer cells (a mimicked model for an NAFLD condition) treated with apocynin or nitrone spin trap 5,5 dimethyl-1- pyrroline N-oxide (DMPO) had significantly decreased CD68 and decreased miR21 and α-smooth muscle actin levels, suggesting the role of NOX2-dependent reactive oxygen species in miR21-induced Kupffer cell activation and stellate cell pathology. Furthermore, NOX2-dependent peroxynitrite generation was primarily responsible for cellular events observed following MC exposure since incubation with phenylboronic acid attenuated miR21 levels, Kupffer cell activation, and inflammatory cytokine release. Furthermore, blocking of the AKT pathway attenuated PP2A inhibitor-induced NOX2 activation and miR21 upregulation. Taken together, we show that PP2A may have protective roles, and its inhibition exacerbates NAFLD pathology via activating NOX2-dependent peroxynitrite generation, thus increasing miR21-induced pathology.NEW & NOTEWORTHY Protein phosphatase 2A inhibition causes nonalcoholic steatohepatitis (NASH) progression via NADPH oxidase 2. In addition to a novel emchanism of action, we describe a new tool to describe NASH histopathology.
Subject(s)
Enzyme Inhibitors/toxicity , MicroRNAs/metabolism , NADPH Oxidase 2/metabolism , Non-alcoholic Fatty Liver Disease/chemically induced , Non-alcoholic Fatty Liver Disease/metabolism , Protein Phosphatase 2/antagonists & inhibitors , Animals , Antigens, CD/biosynthesis , Antigens, Differentiation, Myelomonocytic/biosynthesis , Cytokines/metabolism , Hepatic Stellate Cells/drug effects , Kupffer Cells/drug effects , Kupffer Cells/metabolism , Liver Cirrhosis/chemically induced , Liver Cirrhosis/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , MicroRNAs/genetics , Microcystins/toxicity , NADPH Oxidase 2/genetics , NADPH Oxidases/metabolism , Peroxynitrous Acid/metabolismABSTRACT
We studied the response of neutrophils, macrophages, and mast cells to local application of silica nanoparticles (10-20 nm). Histological examination of tonsillar postoperative material from 6 patients aged 24-44 years with recurrent tonsillitis was carried out. Irrigation of the tonsillar lacunae was carried out over 5 days before bilateral tonsillectomy: on the right by Polysorb MP suspension (1 g/liter), on the left by saline. The contact of nanoparticles with the mucosa led to a decrease in the number of cells expressing myeloperoxidase (p=0.02) and an increase in the count of CD68+ cells (p=0.04); the count of mast cells remained unchanged. Local use of medical adsorbent based on silica nanoparticles induced changes in cells due to their resorption by the tissue. Positive chemotaxis of CD68+ macrophages revealed in the tonsillar lymphoid tissue attested to stimulation of non-specific immunity and inductive phase of specific immunity. The authors hypothesized that internalization of medical nanoparticles by resident phagocytes of the mucosa could support targeted biodistribution of drugs in the palatine tonsils.
Subject(s)
Macrophages/immunology , Mast Cells/immunology , Neutrophils/immunology , Palatine Tonsil/immunology , Silicon Dioxide/pharmacology , Tonsillitis/drug therapy , Adult , Antigens, CD/biosynthesis , Antigens, Differentiation, Myelomonocytic/biosynthesis , Drug Delivery Systems/methods , Humans , Nanoparticles , Palatine Tonsil/cytology , Peroxidase/biosynthesis , Tonsillectomy , Tonsillitis/surgeryABSTRACT
Multiple Sclerosis (MS) is an autoimmune disease of the central nervous system (CNS), characterized by the infiltration of mononuclear cells into the CNS and a subsequent inflammation of the brain. Monocytes are implicated in disease pathogenesis not only in their function as potential antigen-presenting cells involved in the local reactivation of encephalitogenic T cells but also by independent effector functions contributing to structural damage and disease progression. However, monocytes also have beneficial effects as they can exert anti-inflammatory activity and promote tissue repair. Glucocorticoids (GCs) are widely used to treat acute relapses in MS patients. They act on a variety of cell types but their exact mechanisms of action including their modulation of monocyte function are not fully understood. Here we investigated effects of the therapeutically relevant GC methylprednisolone (MP) on monocytes from healthy individuals and MS patients in vitro and in vivo. The monocyte composition in the blood was different in MS patients compared to healthy individuals, but it was only marginally affected by MP treatment. In contrast, application of MP caused a marked shift toward an anti-inflammatory monocyte phenotype in vitro and in vivo as revealed by an altered gene expression profile. Chemotaxis of monocytes toward CCL2, CCL5, and CX3CL1 was increased in MS patients compared to healthy individuals and further enhanced by MP pulse therapy. Both of these migration-promoting effects were more pronounced in MS patients with an acute relapse than in those with a progressive disease. Interestingly, the pro-migratory GC effect was independent of chemokine receptor levels as exemplified by results obtained for CCR2. Collectively, our findings suggest that GCs polarize monocytes toward an anti-inflammatory phenotype and enhance their migration into the inflamed CNS, endowing them with the capacity to suppress the pathogenic immune response.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Chemotaxis, Leukocyte/drug effects , Methylprednisolone/pharmacology , Monocytes/drug effects , Multiple Sclerosis, Chronic Progressive/drug therapy , Multiple Sclerosis, Relapsing-Remitting/drug therapy , Adult , Aged , Anti-Inflammatory Agents/administration & dosage , Anti-Inflammatory Agents/therapeutic use , Antigens, CD/biosynthesis , Antigens, CD/genetics , Antigens, Differentiation, Myelomonocytic/biosynthesis , Antigens, Differentiation, Myelomonocytic/genetics , Chemokine CCL2/pharmacology , Chemokine CCL2/physiology , Female , Humans , Male , Methylprednisolone/administration & dosage , Methylprednisolone/therapeutic use , Middle Aged , Monocytes/immunology , Multiple Sclerosis, Chronic Progressive/immunology , Multiple Sclerosis, Relapsing-Remitting/immunology , Pulse Therapy, Drug , Receptors, CCR2/biosynthesis , Receptors, CCR2/genetics , Receptors, Cell Surface/biosynthesis , Receptors, Cell Surface/genetics , Receptors, Glucocorticoid/biosynthesis , Receptors, Glucocorticoid/genetics , Young AdultABSTRACT
Background and Purpose- Early erythrolysis in the hematoma contributes to brain injury after intracerebral hemorrhage (ICH). This study investigated the effects of N-acetylheparin, a complement inhibitor, and aurin tricarboxylic acid, a membrane attack complex inhibitor, on early erythrolysis, brain iron deposition, and brain injury in aged rats. Methods- There were 3 parts in the study. First, aged (18 months old) male Fischer 344 rats had an ICH. The time course of erythrolysis in the hematoma was determined by T2* weighted magnetic resonance imaging, and the expression of CD163 was examined. Second, aged rats had an ICH with N-acetylheparin or vehicle. Rats were euthanized at days 1, 3, and 28 after magnetic resonance imaging (T2-, T2*-weighted, and T2* array) and behavioral tests. Brains were used for immunohistochemistry. Third, aged rats had an ICH with avaurin tricarboxylic acid or vehicle. The rats had magnetic resonance imaging and behavioral tests and were euthanized at day 3. Brains were used for immunohistochemistry. Results- Early erythrolysis occurred within the clot in aged F344 rats. There were increased numbers of CD163-positive cells after ICH. Almost all perihematomal CD163-positive cells were microglia/macrophages, while positive neurons were found more distant from the hematoma. Coinjection of N-acetylheparin attenuated erythrolysis, iron accumulation, CD163 expression, microglia activation, brain swelling, and neuronal death in the acute phase, as well as reducing brain atrophy and neurological deficits in the chronic phase. Coinjection of aurin tricarboxylic acid also reduced erythrolysis and ICH-induced brain injury. Conclusions- Inhibiting complement activation resulted in less erythrolysis and brain injury after ICH.
Subject(s)
Aurintricarboxylic Acid/therapeutic use , Brain Injuries/blood , Brain Injuries/drug therapy , Complement Inactivating Agents/therapeutic use , Hemolysis , Heparin/analogs & derivatives , Intracranial Hemorrhages/blood , Intracranial Hemorrhages/drug therapy , Ischemic Attack, Transient/drug therapy , Aging , Animals , Antigens, CD/biosynthesis , Antigens, Differentiation, Myelomonocytic/biosynthesis , Brain Edema/prevention & control , Erythrocytes , Heparin/therapeutic use , Macrophage Activation , Male , Microglia , Rats , Rats, Inbred F344 , Receptors, Cell Surface/biosynthesisABSTRACT
BACKGROUND: This study aimed to investigate the protective effect of 5-aminolevulinic acid (5-ALA) and sodium ferrous citrate (SFC) against binge alcohol-induced gut leakiness and inflammatory liver disease in HIV transgenic (TG) rats. METHODS: TG rats were treated with 3 consecutive doses of binge ethanol (EtOH) with or without 5-ALA/SFC. Blood and liver tissue samples were collected at 6 hours following the last dose of EtOH. RESULTS: Compared with the wild-type (WT) rats, the TG rats showed increased sensitivity to alcohol-mediated inflammation, as evidenced by the significantly elevated levels of serum endotoxin, AST, ALT, ED1, and ED2 staining in liver. In contrast, 5-ALA/SFC improved the above biochemical and histochemical profiles. 5-ALA/SFC also attenuated the up-regulated mRNA expression of leptin and CCL2. Furthermore, down-regulated intestinal ZO-1 protein expression was also inhibited by 5-ALA/SFC. Moreover, the expressions of HO-1, HO-2, Sirt1, and related signal transduction molecules in liver were increased by 5-ALA/SFC. These results demonstrated that 5-ALA/SFC treatment ameliorated binge alcohol exposure liver injury in a rat model of HIV-infected patients by reducing macrophage activation and expression of inflammatory cytokines/chemokines, and by inducing HO-1, HO-2, and Sirt1 expression. CONCLUSIONS: Taken together, these findings suggested that treatment with 5-ALA/SFC has a potential therapeutic effect for binge alcohol exposure liver injury in HIV-infected patients.
Subject(s)
Aminolevulinic Acid/pharmacology , Binge Drinking/physiopathology , Ethanol/adverse effects , Hepatitis/prevention & control , Intestines/physiopathology , Permeability/drug effects , Alanine Transaminase/blood , Animals , Antigens, CD/biosynthesis , Antigens, CD/immunology , Antigens, Differentiation, Myelomonocytic/biosynthesis , Antigens, Differentiation, Myelomonocytic/immunology , Aspartate Aminotransferases/blood , Cell Adhesion Molecules, Neuronal/genetics , Citric Acid , Endotoxins/blood , Enterobacteriaceae Infections/microbiology , Ferrous Compounds , HIV Infections/complications , HIV-1/genetics , Heme Oxygenase (Decyclizing)/biosynthesis , Heme Oxygenase-1/biosynthesis , Hepatitis/blood , Hepatitis/complications , Inflammation Mediators/metabolism , Lipopolysaccharides/blood , Liver/metabolism , Rats , Rats, Transgenic , Receptors, Cell Surface/biosynthesis , Receptors, Cell Surface/immunology , Sirtuin 1/biosynthesis , Stem Cells , Triglycerides/metabolism , Zonula Occludens-1 Protein/biosynthesis , gag Gene Products, Human Immunodeficiency Virus/geneticsABSTRACT
Activation states of immune cells (among them, the so-called pro- or anti-inflammatory states) influence the pathogenesis of multiple sclerosis (MS). The neuropeptide calcitonin gene-related peptide (CGRP) can exert a pro- or anti-inflammatory role in a context-dependent manner. In mice CGRP was found to attenuate the development of experimental autoimmune encephalomyelitis (EAE, a common MS animal model). We analyzed CGRP effects on the expression of cytokines and markers of activation states, as well as its intracellular cascade, following intrathecal administration during EAE immunization. Real Time quantitative-PCR (RT-PCR) showed that IL-1beta and IL-6 (associated to a pro-inflammatory state in EAE), but also Ym1 (also known as Chil3), Arg1 and CD163 (associated to an anti-inflammatory state in EAE) were decreased in the encephalon (devoid of cerebellum). In the cerebellum itself, IL-1beta and Ym1 were decreased. TNF-alpha (associated to a pro-inflammatory state in EAE), but also IL-10 (associated to another type of anti-inflammatory state) and BDNF were unchanged in these two regions. No changes were detected in the spinal cord. Additional tendencies toward a change (as revealed by RT-PCR) were again decreases: IL-10 in the encephalon and Arg1 in the spinal cord. CGRP decreased percentage of Ym1+/CD68+ immunoreactive cells and cell density of infiltrates in the cervical spinal cord pia mater. Instead, Ym1 in the underlying parenchyma and at thoracic and lumbar levels, as well as Arg1, were unchanged. In cultured microglia the neuropeptide decreased Ym1, but not Arg1, immunoreactivity. Inducible NOS (iNOS) was unchanged in spinal cord microglia and astrocytes. The neuropeptide increased the activation of ERK1/2 in the astrocytes of the spinal cord and in culture, but did not influence the activation of ERK1/2 or p38 in the spinal cord microglia. Finally, in areas adjacent to infiltration sites CGRP-treated microglia showed a larger ramification radius. In conclusion, CGRP-induced EAE amelioration was associated to a concomitant, context-dependent decrease in the expression of markers belonging to both pro- or anti-inflammatory activation states of immune cells. It can be hypothesized that CGRP-induced EAE attenuation is obtained through a novel mechanism that promotes down-regulation of immune cell activation that facilitates the establishment of a beneficial environment in EAE provided possibly also by other factors.
Subject(s)
Arginase/antagonists & inhibitors , Calcitonin Gene-Related Peptide/therapeutic use , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Interleukin-1beta/antagonists & inhibitors , Interleukin-6/antagonists & inhibitors , Lectins/antagonists & inhibitors , Receptors, Cell Surface/antagonists & inhibitors , beta-N-Acetylhexosaminidases/antagonists & inhibitors , Animals , Antigens, CD/biosynthesis , Antigens, CD/genetics , Antigens, Differentiation, Myelomonocytic/biosynthesis , Antigens, Differentiation, Myelomonocytic/genetics , Arginase/biosynthesis , Arginase/genetics , Biomarkers/metabolism , Brain/drug effects , Brain/metabolism , Calcitonin Gene-Related Peptide/pharmacology , Cells, Cultured , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/metabolism , Female , Gene Expression , Interleukin-1beta/biosynthesis , Interleukin-1beta/genetics , Interleukin-6/biosynthesis , Interleukin-6/genetics , Lectins/biosynthesis , Lectins/genetics , Mice , Mice, Inbred C57BL , Receptors, Cell Surface/biosynthesis , Receptors, Cell Surface/genetics , beta-N-Acetylhexosaminidases/biosynthesis , beta-N-Acetylhexosaminidases/geneticsABSTRACT
Multiple system atrophy (MSA) is an adult-onset neurodegenerative disease characterized by aggregation of α-synuclein in oligodendrocytes to form glial cytoplasmic inclusions. According to the distribution of neurodegeneration, MSA is subtyped as striatonigral degeneration (SND), olivopontocerebellar atrophy (OPCA), or as combination of these 2 (mixed MSA). In the current study, we aimed to investigate regional microglial populations and gene expression in the 3 different MSA subtypes. Microscopy with microglial marker Iba-1 combined with either proinflammatory marker CD68 or anti-inflammatory marker Arginase-1 was analyzed in control, SND, and OPCA cases (n = 5) using paraffin embedded sections. Western immunoblotting and cytokine array were used to determine protein expression in MSA and control brain regions. Gene expression was investigated using the NanoString nCounter Human Inflammation panel v2 mRNA Expression Assay. Analysis of neuropathological subtypes of MSA demonstrated a significant increase in microglia in the substantia nigra of OPCA cases. There was no difference in the microglial activation state in any region. Cytokine expression in MSA was comparable with controls. Decreased expression of CX3CL1 precursor protein and significantly greater CX3CR1 protein was found in MSA. NanoString analysis revealed the >2-fold greater expression of ARG1, MASP1, NOX4, PTGDR2, and C6 in MSA.
Subject(s)
Brain/pathology , Inflammation/genetics , Inflammation/pathology , Multiple System Atrophy/genetics , Multiple System Atrophy/pathology , Antigens, CD/biosynthesis , Antigens, CD/genetics , Antigens, Differentiation, Myelomonocytic/biosynthesis , Antigens, Differentiation, Myelomonocytic/genetics , Arginase/biosynthesis , Arginase/genetics , Calcium-Binding Proteins , Chemokines/analysis , Chemokines/biosynthesis , Corpus Striatum/pathology , Cytokines/analysis , Cytokines/biosynthesis , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , Gene Expression Regulation/genetics , Humans , Immunohistochemistry , Microfilament Proteins , Microglia/pathology , Olivopontocerebellar Atrophies/pathology , Substantia Nigra/pathologyABSTRACT
PURPOSE: Cancer cell fusion with macrophages results in highly tumorigenic hybrids that acquire genetic and phenotypic characteristics from both maternal cells. Macrophage traits, exemplified by CD163 expression, in tumor cells are associated with advanced stages and poor prognosis in breast cancer (BC). In vitro data suggest that cancer cells expressing CD163 acquire radioresistance. METHODS: Tissue microarray was constructed from primary BC obtained from 83 patients treated with breast-conserving surgery, 50% having received postoperative radiotherapy (RT) and none of the patients had lymph node or distant metastasis. Immunostaining of CD163 in cancer cells and macrophage infiltration (MI) in tumor stroma were evaluated. Macrophage:MCF-7 hybrids were generated by spontaneous in vitro cell fusion. After irradiation (0, 2.5 and 5 Gy γ-radiation), both hybrids and their maternal MCF-7 cells were examined by clonogenic survival. RESULTS: CD163-expression by cancer cells was significantly associated with MI and clinicopathological data. Patients with CD163-positive tumors had significantly shorter disease-free survival (DFS) after RT. In vitro generated macrophage:MCF-7 hybrids developed radioresistance and exhibited better survival and colony forming ability after radiation compared to maternal MCF-7 cancer cells. CONCLUSIONS: Our results suggest that macrophage phenotype in tumor cells results in radioresistance in breast cancer and shorter DFS after radiotherapy.