Infiltration by monocytes of the central nervous system and its role in multiple sclerosis: reflections on therapeutic strategies

Mononuclear macrophage infiltration in the central nervous system is a prominent feature of neuroinflammation. Recent studies on the pathogenesis and progression of multiple sclerosis have highlighted the multiple roles of mononuclear macrophages in the neuroinflammatory process. Monocytes play a significant role in neuroinflammation, and managing neuroinflammation by manipulating peripheral monocytes stands out as an effective strategy for the treatment of multiple sclerosis, leading to improved patient outcomes. This review outlines the steps involved in the entry of myeloid monocytes into the central nervous system that are targets for effective intervention: the activation of bone marrow hematopoiesis, migration of monocytes in the blood, and penetration of the blood–brain barrier by monocytes. Finally, we summarize the different monocyte subpopulations and their effects on the central nervous system based on phenotypic differences. As activated microglia resemble monocyte-derived macrophages, it is important to accurately identify the role of monocyte-derived macrophages in disease. Depending on the roles played by monocyte-derived macrophages at different stages of the disease, several of these processes can be interrupted to limit neuroinflammation and improve patient prognosis. Here, we discuss possible strategies to target monocytes in neurological diseases, focusing on three key aspects of monocyte infiltration into the central nervous system, to provide new ideas for the treatment of neurodegenerative diseases.

Single-cell transcriptome profiling of sepsis identifies HLA-DR^(low)S100A^(high)monocytes with immunosuppressive function

Background Sustained yet intractable immunosuppression is commonly observed in septic patients,resulting in aggravated clinical outcomes.However,due to the substantial heterogeneity within septic patients,precise indicators in deciphering clinical trajectories and immunological alterations for septic patients remain largely lacking.Methods We adopted cross-species,single-cell RNA sequencing(scRNA-seq)analysis based on two published datasets containing circulating immune cell profile of septic patients as well as immune cell atlas of murine model of sepsis.Flow cytometry,laser scanning confocal microscopy(LSCM)imaging and Western blotting were applied to identify the presence of S100A9^(+)monocytes at protein level.To interrogate the immunosuppressive function of this subset,splenic monocytes isolated from septic wild-type or S100a9^(–/–)mice were co-cultured with naive CD4^(+)T cells,followed by proliferative assay.Pharmacological inhibition of S100A9 was implemented using Paquinimod via oral gavage.Results scRNA-seq analysis of human sepsis revealed substantial heterogeneity in monocyte compartments following the onset of sepsis,for which distinct monocyte subsets were enriched in disparate subclusters of septic patients.We identified a unique monocyte subset characterized by high expression of S100A family genes and low expression of human leukocyte antigen DR(HLA-DR),which were prominently enriched in septic patients and might exert immunosuppressive function.By combining single-cell transcriptomics of murine model of sepsis with in vivo experiments,we uncovered a similar subtype of monocyte significantly associated with late sepsis and immunocompromised status of septic mice,corresponding to HLA-DR^(low)S100A^(high)monocytes in human sepsis.Moreover,we found that S100A9^(+)monocytes exhibited profound immunosuppressive function on CD4^(+)T cell immune response and blockade of S100A9 using Paquinimod could partially reverse sepsis-induced immunosuppression.Conclusions This study identifies HLA-DR^(low)S100A^(high)monocytes correlated with immunosuppressive state upon septic challenge,inhibition of which can markedly mitigate sepsis-induced immune depression,thereby providing a novel therapeutic strategy for the management of sepsis.

Essential role of monocytes and macrophages in the progression of acute pancreatitis

Acute pancreatitis(AP) is an inflammatory condition of the pancreas caused by an imbalance in factors involved in maintaining cellular homeostasis.Earliest events in AP occur within acinar cells accompanied by other principal contributors to the inflammatory response i.e.the endothelial cells,immunocytes(granulocytes,monocytes/macrophages,lymphocytes) and neutrophils.Monocytes/macrophages are important inflammatory mediators,involved in the pathophysiology of AP,known to reside in the peritoneal cavity(in the vicinity of the pancreas) and in peripancreatic tissue.Recent studies suggested that impaired clearance of injured acini by macrophages is associated with an altered cytokine reaction which may constitute a basis for progression of AP.This review focuses on the role of monocytes/macrophages in progression of AP and discusses f indings on the inflammatory process involved.

Specific function and modulation of teleost monocytes/macrophages: polarization and phagocytosis

Macrophages exist in most tissues and play a variety of functions in vertebrates.Teleost fish species are found in most aquatic environments throughout the world and are quite diverse for a group of vertebrate animals.Due to whole genome duplication and en vironme ntal adaptati on,teleost monocytes/macrophages possess a variety of different functions and modulations compared with those of mammals.A deeper understanding of teleost monocytes/macrophages in the immune system will not only help develop teleost-specific methods of disease prevention but will also help improve our understanding of the various immune mechanisms in mammals.In this review,we summarize the differences in polarizati on and phagocytosis of teleost and mammalian macrophages to improve our understanding of the various immune mechanisms in vertebrates.

Onset of Coronary Heart Disease is Associated with HCMV Infection and Increased CD14^+CD16^+Monocytes in a Population of Weifang,China

Objective To investigate the relationship between human cytomegalovirus(HCMV)infection and peripheral blood CD14+CD16+monocytes in the pathogenesis of coronary heart disease(CHD),and to elucidate the mechanism of pathogenesis in CHD by analyzing the correlation between infection,inflammation,and CHD,to provide a basis for the prevention,evaluation,and treatment of the disease.Methods In total,192 patients with CHD were divided into three groups:latent CHD,angina pectoris,and myocardial infarction.HCMV-IgM and-IgG antibodies were assessed using ELISA;CD14+CD16+monocytes were counted using a five-type automated hematology analyzer;mononuclear cells were assessed using fluorescence-activated cell sorting;and an automatic biochemical analyzer was used to measure the levels of triglyceride,cholesterol,high-and low-density lipoprotein cholesterols,lipoprotein,hs-CRp and Hcy.Results The positive rates of HCMV-IgM and-IgG were significantly higher in the CHD groups than in the control group.HCMV infection affects lipid metabolism to promote immune and inflammatory responses.Conclusion HCMV infection has a specific correlation with the occurrence and development of CHD.The expression of CD14+CD16+mononuclear cells in the CHD group was increased accordingly and correlated with acute HCMV infection.Thus,HCMV antibody as well as peripheral blood CD14+CD16+mononuclear cells can be used to monitor the occurrence and development of CHD.

Systemic immune inflammation index, ratio of lymphocytes to monocytes, lactate dehydrogenase and prognosis of diffuse large Bcell lymphoma patients

BACKGROUND In malignant tumors,inflammation plays a vital role in the development,invasion,and metastasis of cancer cells.Diffuse large B-cell lymphoma(DLBCL),the most common malignant proliferative disease of the lymphatic system,is commonly associated with inflammation.The international prognostic index(IPI),which includes age,lactate dehydrogenase(LDH),number of extranodal lesions,Ann Arbor score,and Eastern Cooperative Oncology Group(ECOG)score,can evaluate the prognosis of DLBCL.However,its use in accurately identifying highrisk patients and guiding treatment is poor.Therefore,it is important to find novel immune markers in predicting the prognosis of DLBCL patients.AIM To determine the association between the systemic immune inflammation index(SII),ratio of lymphocytes to monocytes(LMR),ratio of LMR to LDH(LMR/LDH),and prognosis of patients with DLBCL.METHODS A total of 68 patients diagnosed with DLBCL,treated in our hospital between January 2016 and January 2020,were included.χ2 test,Pearson’s R correlation,Kaplan Meier curves,and Cox proportional risk regression analysis were used.The differences in the SII,LMR,and LMR/LDH among patients with different clinicopathological features were analyzed.The differences in progression-free survival time among patients with different SII,LMR,and LMR/LDH expressions and influencing factors affecting the prognosis of DLBCL patients,were also analyzed.RESULTS The LMR and LMR/LDH in patients with Ann Arbor stage III–IV,ECOG score≥2,and SII,IPI score 2–5 were significantly higher than those of patients with Ann Arbor stage I-II and ECOG score<2(P<0.05).Patients with high SII,LMR,and LMR/LDH had progression-free survival times of 34 mo(95%CI:32.52–38.50),35 mo(95%CI:33.42–36.58)and 35 mo(95%CI:33.49–36.51),respectively,which were significantly lower than those with low SII,LMR,and LMR/LDH(P<0.05);the SII,LMR,and LMR/LDH were positively correlated(P<0.05).Cox proportional risk regression analysis showed that the SII,LMR,and LMR/LDH were influencing factors for the prognosis of DLBCL patients(hazard ratio=1.143,1.665,and 1.704,respectively;P<0.05).CONCLUSION The SII,LMR,and LMR/LDH are related to the clinicopathological features of DLCBL,and they also influence the prognosis of patients with the disease.

Role of macrophages and monocytes in hepatitis C virus infections

A number of studies conducted over many years have shown that hepatitis C virus(HCV)can infect a variety of cell types.In vivo infection of monocytes,macrophages,and dendritic cells by HCV has been frequently shown by a number of researchers.These studies have demonstrated replication of HCV by detecting the presence of both negative genomic strands and a variety of non-structural HCV proteins in infected cells.In addition,analyses of genome sequences have also shown that different cell types can harbor different HCV variants.Investigators have also done preliminary studies of which cellular genes are affected by HCV infection,but there have not yet been a sufficient number of these studies to understand the effects of infection on these cells.Analyses of in vitro HCV replication have shown that monocytes,macrophages and dendritic cells can be infected by HCV from patient sera or plasma.These studies suggest that entry and cellular locations may vary between different cell types.Some studies suggest that macrophages may preferentially allow HCV genotype 1 to replicate,but macrophages do not appear to select particular hypervariable regions.Overall,these studies agree with a model where monocytes and macrophages act as an amplification system,in which these cells are infected and show few cytopathic effects,but continuously produce HCV.This allows them to produce virus over an extended time and allows its spread to other cell types.

Caspase-1 activation and mature interleukin-1β release are uncoupled events in monocytes

AIM:To investigate whether caspase-1 activation/intracellular processing of pro-interleukin-1β(pro-IL-1β) and extracellular release of mature IL-1β from activated monocytes are separable events.METHODS:All experiments were performed on fresh or overnight cultured human peripheral blood monocytes(PBMCs) that were isolated from healthy donors.PBMCs were activated by lipopolysaccharide(LPS) stimulation before being treated with Adenosine triphosphate(ATP,1 mmol/L),human α-defensin-5(HD-5,50 μg/mL),and/or nigericin(Nig,30 μmol/L).For each experiment,the culture supernatants were collected separately from the cells.Cell lysates and supernatants were both subject to immunoprecipitation with anti-IL1β antibodies followed by western blot analysis with anti-caspase-1 and anti-IL-1β antibodies.RESULTS:We found that pro-IL-1β was processed to mature IL-1β in LPS-activated fresh and overnight cultured human monocytes in response to ATP stimulation.In the presence of HD-5,this release of IL-1β,but not the processing of pro-IL-1β to IL-1β,was completely inhibited.Similarly,in the presence of HD-5,the release of IL-1β,but not the processing of IL-1β,was significantly inhibited from LPS-activated monocytes stimulated with Nig.Finally,we treated LPS-activated monocytes with ATP and Nig and collected the supernatants.We found that both ATP and Nig stimulation could activate and release cleaved caspase-1 from the monocytes.Interestingly,and contrary to IL-1β processing and release,caspase-1 cleavage and release was not blocked by HD-5.All images are representative of three independent experiments.CONCLUSION:These data suggest that caspase-1 activation/processing of pro-IL-1β by caspase-1 and the release of mature IL-1β from human monocytes are distinct and separable events.

Expression of Monocyte Chemoattractant Protein-1 in Monocytes and Effects of Native and Oxidized Very Low Density Lipoproteins

Monocyte chemoattractant protein-1(MCP-1), a potent chemoattractant, is thought to play an important role in migration of monocytes into atherosclerotic lesions. The present study was designed to investigate the capacity of human peripheral blood monocytes to express MCP-1 and effects of native very low density lipoprotein (VLDL) and oxidized VLDL(OX-VLDL) on the expression. The total RNA was extracted from cultured monocytes, which were exposed to VLDL and OX-VLDL, and the media conditioned by monocytes were collected. MCP-1 mRNA expression was examined by Northern blot analysis. MCP-1 protein in conditioned media was determined by using sandwich ELISA. The results showed that monocytes can express MCP-1 after a 24 h incubation at 37℃，and the expression was markedly increased by a exposure to OX-VLDL, whereas the expression was slightly increased when exposed to VLDL. It suggests that the capacity of monocytes to produce MCP-1 that recruits and activates circulating monocytes may be of considerable importance in atherogenesis, and oxidation of VLDL enhances its potential to promote atherogenesis.

Waist Circumference-dependent Peripheral Monocytes Change after Gliclazide Treatment for Chinese Type 2 Diabetic Patients

Gliclazide used for the treatment of type 2 diabetes mellitus（T2DM） stimulates insulin secretion and influences peripheral blood monocytes.The roles of gliclazide in peripheral monocytes of newly diagnosed T2 DM patients were investigated in this study.A total of 105 newly diagnosed T2 DM patients with no history of antihyperglycemic medication were treated with gliclazide-modified release for 16 weeks.The total and differential leukocyte profiles of peripheral blood were measured at baseline and week 16.The peripheral blood monocyte count at week 16 was significantly lower than that at baseline（P=0.019）.Peripheral monocytes level at baseline was positively correlated with waist circumference.After gliclazide treatment,the peripheral monocytes were decreased [（320.09±15.13）×10~6/L vs.（294.19±14.22）×10~6/L] in non-abdominal obesity group,but increased in abdominal obesity group [（344.36±17.24）×10~6/L vs.（351.87±16.93）×10~6/L].Compared with non-abdominal obese patients,abdominal obese patients showed higher Δmonocytes（P=0.046） and Δacute insulin secretion（P=0.049）,but lower ΔHb A1c（P=0.047）.There was significantly positive correlation between Δmonocytes and Δacute insulin secretion（P=0.015）,which disappeared after adjusting for age,waist circumference and dosage at baseline.In conclusion,waist circumference is correlated with peripheral monocyte change after gliclazide treatment in Chinese newly diagnosed T2 DM patients.Peripheral monocytes are decreased in non-abdominal obesity group and increased in abdominal obesity group after gliclazide treatment.

Identification of Monocytes Associated with Severe COVID-19 in the PBMCs of Severely Infected Patients Through Single-Cell Transcriptome Sequencing

Understanding the immunological characteristics of monocytes-including the characteristics associated with fibrosis-in severe coronavirus disease 2019(COVID-19)is crucial for understanding the pathogenic mechanism of the disease and preventing disease severity.In this study,we performed single-cell transcriptomic sequencing of peripheral blood samples collected from six healthy controls and 14 COVID-19samples including severe,moderate,and convalescent samples from three severely/critically ill and four moderately ill patients.We found that the monocytes were strongly remodeled in the severely/critically ill patients with COVID-19,with an increased proportion of monocytes and seriously reduced diversity.In addition,we discovered two novel severe-disease-specific monocyte subsets:Mono 0 and Mono 5.These subsets expressed amphiregulin(AREG),epiregulin(EREG),and cytokine interleukin-18(IL-18)gene,exhibited an enriched erythroblastic leukemia viral oncogene homolog(ErbB)signaling pathway,and appeared to exhibit pro-fibrogenic and pro-inflammation characteristics.We also found metabolic changes in Mono 0 and Mono 5,including increased glycolysis/gluconeogenesis and an increased hypoxia inducible factor-1(HIF-1)signaling pathway.Notably,one pre-severe sample displayed a monocyte atlas similar to that of the severe/critical samples.In conclusion,our study discovered two novel severedisease-specific monocyte subsets as potential predictors and therapeutic targets for severe COVID-19.Overall,this study provides potential predictors for severe disease and therapeutic targets for COVID-19 and thus provides a resource for further studies on COVID-19.

Single-cell RNA sequencing reveals classical monocytes are the major precursors of rat osteoclasts

To dissect which subset of bone marrow monocyte is the major precursor of osteoclast,3-month-old rat bone marrow was obtained for single-cell RNA sequencing.A total of 6091 cells were acquired for detailed analysis,with a median number of 1206 genes detected per cell and 17,959 genes detected in total.A total of 19 cell clusters were recognized,with the main lineages identified as B cells,Granulocytes,Monocytes,T cells,Erythrocytes and Macrophages.Monocytes were further divided into classical monocytes and non-classical monocytes.Compared with non-classical monocytes,classical monocytes highly expressed osteoclast differentiation related genes Mitf,Spi1,Fos and Csf1r.Additionally,biological processes of classical monocytes were related to osteoclast differentiation.qPCR revealed differentially expressed genes of classical monocytes played a role in osteoclast differentiation.In conclusion,classical monocytes were identified as the main precursors of osteoclasts in rats,and may contribute to osteoclast differentiation by regulating S100a4,S100a6,S100a10,Fn1,Vcan and Bcl2a1.The results of this study contribute to the understanding of the origin of osteoclasts and may provide potential biomarkers for early diagnosis of bone metabolic diseases,as well as molecular and cellular targets for clinical intervention in bone metabolic diseases.

IN VITRO ANTI-HEPATOMA EFFECTS OF MONOCYTES AND KUPFFER CELLS ISOLATED FROM HEPATOMA PATIENTS AFTER TREATMENT WITH BIOLOGICAL IMMUNE STIMULANTS

Monocytes (MC), lymphocytes (LC) and Kupffer cells (KC) were isolated respectively from blood and surgical liver samples of patients suffering from he-patocellular carcinoma (HCC). 13 patients were given BCG, mixed bacterium vaccine (MBV) and human white blood cell interferon (IFN), the other 3 patients were not treated with any biological immune stimulants (BIS) and served as controls. The cytosta-tic and cytotoxic effects of MC and KC on human hepatoma SMMC-7721 (TC) were assayed in vitro and the numbers of T total (Tt), T helper (Th) and T suppressor (Ts) cells were counted using CD monoclonal antibody immunofluorescence. The results were as follows: (1) On the 7th day after the first administration of BIS, the cytostatic and cytotoxic effects of MC on TC showed obvious increase over pre-administration. The activity of BIS was 1 ?5 times as high as that in the controls. (2) After 3 administrations, the cytostatic effect of MC on TC increased to the normal level (84%), while the controls remained as before (45%). (3) On the 7th day after first administration, cytostatic and cytotoxic effects of KC on TC were 0.5 and 1 times higher respectively than those of the controls. (4) The numbers of Tt and Th of patients given BIS increased continuously; on the contrary Ts decreased in number. These results indicate that combined use of BCG, MBV and IFN can actively enhance the immune anti-hepatoma function of patients suffering from HCC.

Malarial pigment does not induce MMP-2 and TIMP-2 protein release by human monocytes

Dear editor, In the recent years growing evidence on the involvement of human matrix metalloproteinases(MMPs) and tissue inhibitors of metalloproteinases(TIMPs) in cerebral malaria (CM) has been reported[1]and a role for malarial pigment haemozoin(HZ) has been proposed[2,3].In a recent work my group showed that in human microvascular endothelial

IMPAIRED AUTOLOGOUS MIXED LYMPHOCYTE REACTION CORRELATED WITH DECREASED EXPRESSION OF HLA-Ⅱ ANTIGENS ON MONOCYTES IN PATIENTS WITH MYELOID LEUKEMIA

The proliferative response of T-cells to autolo-gous non-T-cells is referred to as the autologous mixed lymphocyte reaction (AMLR). Recent studies have suggested that AMLR represents a mechanism of immune regulation in vivo. We investigated AMLR in patients with acute- and chronic myeloid leukemia (AML and CML). AMLR was found to be significantly depressed (P<0.001) in AML patients (n=17, cpm=532±95) and CML patients (n=13, cpm=688±99) when compared with that of their healthy HLA-identical siblings serving as controls (n=17, cpm=4152±619 and n=13 cpm=4086±421, respectively). In order to understand the cellular basis of the defective AMLR in patients with AML end CML, we performed mitogen-treated T-cell cultures analysis of T-cell subsets and HLA-Ⅱ antigen detection on monocytes. The results indicated that the defect of AMLR in patients resided at the stimulator monocyte level rather than at the responder T-cell level. Enumeration of monocytes reactive with monoclonal antibody Tu22, which recognizes determinants of HLA-DQ, demonstrated that ML patients had a significantly decreased (P<0.091) number of circulating Tu22+ monocytes when compared with normal controls. These studies suggest that a deficiency of HLA-DQ+ monocytes contributes to the depression of AMLR in ML and possibly underlies the abnormalities of immune response present in this disease.

Changes of NLRP1-ASC-Caspase-1 signaling pathways in peripheral blood monocytes from patients with atrial fibrillation

Objective:To detect the expression of(Peripheral blood mononuclear cells,PBMCs)NLRP3 signal pathway in peripheral blood monocytes of patients with chronic heart failure,and to explore the expression of NLRP3 signal pathway and its induced inflammatory response in PBMCs of patients with different types of chronic heart failure.Methods:patients with chronic heart failure(NYHAⅡ~Ⅳ),Ⅱ(nude 20),Ⅲ(nude 20)andⅣ(nude 20)admitted to our hospital from 2019 to 2020 were selected,and 20 normal subjects were selected as the control group.The peripheral venous blood of all subjects was collected,and the plasma and monocytes were extracted respectively.The monocytes were identified by magnetic beads sorting.The mRNA and protein expression levels of NLRP3,ASC and Caspase-1 in PBMCs were detected by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)and Western blot,and the level of interleukin-1β(IL-1β)in plasma was detected by enzyme-linked immunosorbent assay((ELISA)).Results:compared with the normal control group,the expression of NLRP3,ASC,Caspase-1 protein and mRNA in PBMCs of patients with gradeⅡ,ⅢandⅣincreased.Compared with patients with gradeⅡ,the expression of these indexes increased in patients with gradeⅢandⅣ.Compared with patients with gradeⅢ,the expression of these indexes increased in patients with gradeⅣ.Compared with the normal control group,the plasma levels of IL-1βin patients with gradeⅡ,ⅢandⅣwere higher than those in patients with gradeⅡ,ⅢandⅣ(P<0.05).The expression of these indexes in patients with gradeⅢandⅣwas higher than that in patients with gradeⅢ(P<0.05).Conclusion:the results suggest that NLRP3-ASC-Caspase-1 signal pathway may cause chronic inflammation in patients with heart failure and play a role in the progression of chronic heart failure.

Activation of human monocytes/macrophages by OHR/AVR118 promotes both pro- and Anti-Inflammatory phenotypes

The immunomodulator OHR/AVR118 has been shown to increase IL-8 and MCP-1 secretion from non-activated human monocytes and U937 pro-monocytic cells, but to decrease MCP-1 secretion from LPS-activated monocytes, suggesting its effect depends on immune cell environment and/or activation state. We therefore assessed the effect of OHR/AVR118 on cytokine secretion by human PBMCs and adherent monocytes. OHR/AVR118 increased IL-6, IL-1β, and TNF-α secretion byPHA/IL-2-primed PBMCs, but did not alter IL-12 secretion. In contrast, treatment of LPS-activated monocytes decreased TNF-α and IL-12, increased IL-6, but did not alter IL-1β, secretion. To further show that the effect of OHR/AVR118 depends on cellular environment, we monitored U937 differentiation towards mature macrophages in the presence of drug. OHR/AVR118 promoted a pro-inflammatory response in PMA-activated cells, as demonstrated by increased expression of the maturation markers CD86, CD32, and CD87 and by increased IL-8, MCP-1, and GM-CSF secretion. In undifferentiated U937 cells, OHR/AVR118 did not alter phagocytosis of opsonized S. aureus and IL-10 secretion. Whereas, after activation, OHR/AVR118 induced an anti-inflammatory phenotype, as indicated by reduced phagocytosis and increased IL-10 secretion. Overall, these findings suggest that OHR/AVR118 has a dual action on monocyte/macrophage function depending on cellular activation state, resulting in either further activation or suppression.

Differential Effects of Alternative Glycoforms of IgG on Human Monocytes and Macrophages: Sialylated IgG Induces Novel Expression Signatures of Cell Surface Markers, Cytokines, and Chemokines

The effector functions elicited by the fragment crystallizable (Fc) region of immunoglobulin G (IgG) antibodies are subject to variation by the presence of terminal sialic acid (Sia) residues at asparagine-297 (Asn-297). We have previously shown that the sialic acid-containing (Sia+) fraction of intravenous immune globulin (IVIG) influences cell surface marker expression and cytokine/ chemokine secretion during the differentiation and maturation of human dendritic cells (DC). The present study examined the effects of Sia+ IgG on human peripheral blood mononuclear cell (PBMC)-derived monocyte and macrophage surface marker expression and cytokine/chemokine secretion. Sia+ IgG induced increased expression of CD80 and dendritic cell immunoreceptor (DCIR) on monocytes, whereas the expression of HLA-DR was decreased. In addition, the production of IL-6, TNFα, IL-1β, and CXCL1 by monocytes was profoundly increased by treatment with Sia+ IgG. Sia+ IgG also increased the expression of cell surface markers associated with macrophage polarization (e.g. CD40 and CD206) on monocytes. In macrophage-colony stimulating factor (MCSF) generated macrophages, Sia+ IgG induced increased production of numerous cytokines/ chemokines including IL-6, TNFα, CXCL1, and IL-10, and the expression of the macrophage surface marker CD163. Our data extended prior observations of Sia+ IgG on DC function and showed that Sia+ IgG was able to differentially modulate multiple pathways in monocytes and macrophages. Our data indicate that the Sia+ fraction of IVIG possesses the ability to influence inflammatory processes in multiple immune cell types and induces novel signatures in cell surface marker expression and cytokine/chemokine production.

Inhibition of matrix metalloproteinase-9 secretion by dimethyl sulfoxide and cyclic adenosine monophosphate in human monocytes

BACKGROUND Matrix metalloproteinases(MMPs),including MMP-9,are an integral part of the immune response and are upregulated in response to a variety of stimuli.New details continue to emerge concerning the mechanistic and regulatory pathways that mediate MMP-9 secretion.There is significant evidence for regulation of inflammation by dimethyl sulfoxide(DMSO)and 3',5'-cyclic adenosine monophosphate(cAMP),thus investigation of how these two molecules may regulate both MMP-9 and tumor necrosis factorα(TNFα)secretion by human monocytes was of high interest.The hypothesis tested in this study was that DMSO and cAMP regulate MMP-9 and TNFαsecretion by distinct mechanisms.AIM To investigate the regulation of lipopolysaccharide(LPS)-stimulated MMP-9 and tumor necrosis factorαsecretion in THP-1 human monocytes by dimethyl sulfoxide and cAMP.METHODS The paper describes a basic research study using THP-1 human monocyte cells.All experiments were conducted at the University of Missouri-St.Louis in the Department of Chemistry and Biochemistry.Human monocyte cells were grown,cultured,and prepared for experiments in the University of Missouri-St.Louis Cell Culture Facility as per accepted guidelines.Cells were treated with LPS for selected exposure times and the conditioned medium was collected for analysis of MMP-9 and TNFαproduction.Inhibitors including DMSO,cAMP regulators,and anti-TNFαantibody were added to the cells prior to LPS treatment.MMP-9 secretion was analyzed by gel electrophoresis/western blot and quantitated by ImageJ software.TNFαsecretion was analyzed by enzyme-linked immuno sorbent assay.All data is presented as the average and standard error for at least 3 trials.Statistical analysis was done using a two-tailed paired Student t-test.P values less than 0.05 were considered significant and designated as such in the Figures.LPS and cAMP regulators were from Sigma-Aldrich,MMP-9 standard and antibody and TNFαantibodies were from R&D Systems,and amyloid-βpeptide was from rPeptide.RESULTS In our investigation of MMP-9 secretion from THP-1 human monocytes,we made the following findings.Inclusion of DMSO in the cell treatment inhibited LPSinduced MMP-9,but not TNFα,secretion.Inclusion of DMSO in the cell treatment at different concentrations inhibited LPS-induced MMP-9 secretion in a dosedependent fashion.A cell-permeable cAMP analog,dibutyryl cAMP,inhibited both LPS-induced MMP-9 and TNFαsecretion.Pretreatment of the cells with the adenylyl cyclase activator forskolin inhibited LPS-induced MMP-9 and TNFαsecretion.Pretreatment of the cells with the general cAMP phosphodiesterase inhibitor IBMX reduced LPS-induced MMP-9 and TNFαin a dose-dependent fashion.Pre-treatment of monocytes with an anti-TNFαantibody blocked LPSinduced MMP-9 and TNFαsecretion.Amyloid-βpeptide induced MMP-9 secretion,which occurred much later than TNFαsecretion.The latter two findings strongly suggested an upstream role for TNFαin mediating LPS-stimulate MMP-9 secretion.CONCLUSION The cumulative data indicated that MMP-9 secretion was a distinct process from TNFαsecretion and occurred downstream.First,DMSO inhibited MMP-9,but not TNFα,suggesting that the MMP-9 secretion process was selectively altered.Second,cAMP inhibited both MMP-9 and TNFαwith a similar potency,but at different monocyte cell exposure time points.The pattern of cAMP inhibition for these two molecules suggested that MMP-9 secretion lies downstream of TNFαand that TNFαmay a key component of the pathway leading to MMP-9 secretion.This temporal relationship fit a model whereby early TNFαsecretion directly led to later MMP-9 secretion.Lastly,antibody-blocking of TNFαdiminished MMP-9 secretion,suggesting a direct link between TNFαsecretion and MMP-9 secretion.