Monocyte IL-6 Research Articles

IL-16 regulates macrophage polarization as a target gene of mir-145-3p

BackgroundInterleukin 16 is an immunomodulatory chemokine that signals through CD4 + T cells, monocytes, macrophages and dendritic cells. Its expression in immune-related cells enhances the antimicrobial effect and inhibits HIV replication in macrophages. However, the role of IL-16 in macrophage polarization is uncertain. Mir-145 was reported to regulate IL-10 expression by targeting histone deacetylase 11 and promotes alternatively activated macrophage (M2) polarization. Mir-145 was also predicted to target IL-16 mRNA. We aimed to explore the roles of IL-16 and mir-145 in macrophage polarization and antimicrobial functions. MethodsTHP1 monocytes were employed in this study, and their cell activity when incubated with different concentrations of IL-16 was evaluated using the CCK-8 cell counting kit. To obtain polarized macrophages, THP-1 cells were induced by IL-4 and IL-13 following PMA incubation (M2 polarized macrophages) or induced by IFN-gamma and LPS (M1 classical macrophage activation). The influence of IL-16 on macrophage phagocytosis was quantified by the amount of chicken red blood cell phagocytized. IL-16, IL-10 and miR-145 expression in THP1 monocytes and induced macrophages was quantified by quantitative PCR. The miR-145 and IL-16 targeting relationship was verified by the dual luciferase reporter assay. The influence of IL-16 and mir-145 on macrophage polarization was evaluated by M1 and M2 macrophage characterized marker gene expression. ResultsThe M0 macrophage subtype was induced by PMA. The M1 and M2 subtypes of macrophage were successfully induced by M1- and M2-specific induction. M1 macrophages express higher levels of IL-16 than M2 macrophages but express lower levels of IL-10 and mir-145 than M2 cells. IL-16 with a concentration up to 150 ng/mL has no influence on THP-1 cell proliferation but improves macrophage phagocytosis ability with the down-expression of IL-10 and up-expression of pro-inflammatory cytokines such as IL-1a and IL-6. Knockdown with its target siRNA is beneficial for macrophage maintenance but reduces phagocytosis ability. Mir-145 specifically targets the IL-16 3’UTR verified by the dual luciferase reporter assay. Mir-145 downregulates IL-16 expression and upregulates IL-10 expression, thereby promoting M2 macrophage polarization. ConclusionIL-16 modulates macrophage polarization through regulating IL-10, IL-1a and IL-6 expression. Mir-145 is involved in M2 macrophage polarization by targeting IL-16 and enhancing IL-10 expression.

Synergistic enhancement of production of proinflammatory cytokines of human peripheral blood monocytes by anti-Sm and anti-RNP antibodies.

The present study was performed to elucidate the roles of serum anti-Sm antibodies in the pathogenesis of systemic lupus erythematosus (SLE). Highly purified peripheral blood monocytes obtained from healthy donors were cultured in the presence of monoclonal anti-Sm antibody (anti-Sm mAb), monoclonal anti-U1-RNP antibody (anti-RNP mAb) or control murine IgG1 or IgG3. After various periods of incubation, levels of IL-6 and TNF-α in the culture supernatants were measured by ELISA and the expression of mRNA for various molecules in monocytes was determined using RT-PCR. Flow cytometry analysis confirmed the bindings of anti-Sm mAb and anti-RNP mAb on viable human monocytes. Both anti-Sm mAb and anti-RNP mAb significantly increased the production of IL-6 and TNF-α of human monocytes in a dose-dependent manner, although the latter was more potent than the former. Of note, anti-Sm mAb synergistically enhanced the production and mRNA expression of IL-6 and TNF-α of human monocytes in the presence of anti-RNP mAb. Notably, anti-RNP mAb, but not anti-Sm mAb, significantly enhanced the mRNA expression of RelA in human monocytes. Finally, anti-Sm mAb still up-regulated the IL-6 production of monocytes in the presence of anti-RNP mAb under the influence of N-acetyl cysteine or pyrrolidine dithiocarbamate that totally abrogated the IL-6 production provoked by anti-Sm mAb alone in the absence of anti-RNP mAb. These results demonstrate that anti-Sm and anti-RNP antibodies synergistically up-regulate the expression of IL-6 and TNF-α in human monocytes. The data also suggest that the effect of anti-Sm in the synergy with anti-RNP might not involve NFkB activation.

Platelet-derived microparticles generated in vitro resemble circulating vesicles of patients with rheumatoid arthritis and activate monocytes

Patients with rheumatoid arthritis (RA) have increased amount of platelet-derived microparticles (PMPs) positive for citrullinated peptides (CPs) that form immune complexes (PMPs-ICs). Monocytes are important inflammatory mediators that play a role in the clearance of PMPs-ICs. We aimed to generate PMPs-ICs in vitro and determine its effect on monocytes from patients with RA and healthy individuals (HI). PMPs from patients showed platelet markers, mitochondria content, and phosphatidylserine exposure similar to PMPs from HI. However, patients had a higher frequency of IgG+ and CPs+ vesicles than HI. PMPs-ICs generated in vitro were similar to the circulating vesicles of patients with respect to IgG- and CPs-positivity. PMPs-ICs induced pro-inflammatory cytokines and CX3CR1 expression in monocytes from HI, and IL-10 and CD36 upregulation in monocytes from patients. These results suggest that PMPs-ICs induce activation of monocytes, with a pro-inflammatory response in HI and a more tolerant response in cells of patients with RA.

Consistent Compliance with Hydroxyurea and Hematology Measures during L-Glutamine Therapy for Sickle Cell Anemia

Background. Oxidative stress contributes to the pathophysiology of sickle cell anemia (SCA). Oral l-glutamine increases the proportion of reduced NAD in sickle erythrocytes,1 which may decrease oxidative stress and SCA complications.2 Recently, a multicenter, randomized, placebo-controlled, double-blind, phase 3 trial of l-glutamine in SCA reported that the l-glutamine group had fewer pain crises and hospitalizations over 48 weeks of treatment than the placebo arm.3 There was a consistent treatment effect of l-glutamine in subjects who used and did not use hydroxyurea. The purpose of the present analysis was to determine if there was consistent use of hydroxyurea in both arms of the trial and if l-glutamine therapy led to a reduction in hemolysis and an amelioration of anemia.Methods. Because increase in mean corpuscular volume (MCV) is a sensitive indicator of hydroxyurea therapy,4 we used MCV as a surrogate marker of hydroxyurea compliance. We analyzed MCV at 4-week intervals according to hydroxyurea therapy separately in the l-glutamine and placebo arms. We also analyzed hemoglobin concentration and reticulocyte count at 12-week intervals according to experimental treatment with l-glutamine versus placebo as stratified by hydroxyurea use. We imputed missing variables by carrying the last measured value forward. We used the student t-test to compare linear variables, Pearson chi square to compare categorical variables and repeated measures analysis of variance to analyze trends over time.Results. The patients receiving l-glutamine and placebo were well matched (Table 1). During the 48-week trial, the MCV was consistently greater in patients using hydroxyurea than those not using hydroxyurea in both the l-glutamine arm (Figure 1) and the placebo arm (Figure 2). Furthermore, there was no significant change over 48 weeks in the MCV in the patients using hydroxyurea in either arm (P >0.7). In the hydroxyurea group, there were no significant differences according to experimental therapy with l-glutamine versus placebo in hemoglobin concentration or reticulocyte count at 12 weekly intervals. In the group not using hydroxyurea, the hemoglobin concentration was significantly higher at baseline in patients given l-glutamine compared to those given placebo, but this observation did not persist at the subsequent determinations; reticulocyte counts did not differ at any time point (Table 2). We performed many multivariate and repeated measures analyses of hemoglobin concentration and reticulocyte count that adjusted for age, gender and blood transfusions, but did not observe evidence that l-glutamine therapy was associated with an increase in hemoglobin concentration and/or a decline in reticulocyte count.Discussion. Based on the MCV, patients who were assigned to hydroxurea complied with hydroxyurea therapy at baseline and throughout the study; this supports the concept that the beneficial clinical effects of l-glutamine therapy for SCA are additive to those of hydroxyurea. Therapy with l-glutamine did not reduce hemolysis or improve anemia, which parallels observations with therapy with an antibody against the adhesion molecule, P-selectin.5 The mechanism whereby l-glutamine leads to reduced vaso-occlusive complications in SCA requires further investigation. Glutamine levels are lower in sickle erythrocytes compared to erythrocytes from healthy volunteers.6 L-Glutamine reduces intercellular adhesion molecule expression in endothelial cells,7 decreases adhesion of sickle erythrocytes to endothelial cells,8 attenuates the expression of endothelial adhesion molecules and T cell migration markers when the intestines are injured, 9,10 and inhibits IL-6 and TNF-α production in LPS-stimuated monocytes.11 Future research will need to assess the effect of l-glutamine on redox potential, adhesion molecule expression, and inflammatory marker expression in erythrocytes, other circulating blood cells and the vascular endothelium.ReferencesAm J Hematol. 1998;58(2):117-121.IUBMB Life. 2012;64(1):72-80.N Engl J Med. 2018;379(3):226-235.Medicine (Baltimore). 1996;75(6):300-326.N Engl J Med. 2017;376(5):429-439.Blood. 2008;111(1):402-410.J Reprod Med. 2006;51(3):193-198.BMC Blood Disord. 2005;5:4.Eur J Pharmacol. 2010;643(2-3):304-315.Mediators Inflamm. 2014;2014:837107.Cytokine. 2013;62(1):52-57. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Immune Checkpoint Receptors Tim-3 and PD-1 Regulate Monocyte and T Lymphocyte Function in Septic Patients.

We aim to investigate the effects of Tim-3 and programmed cell death-1 (PD-1) on the monocytes and T lymphocytes in septic patients. Expression of Tim-3 and PD-1 on the CD3, CD4, and CD8 lymphocytes and monocytes was determined using flow cytometry. CBA technique was utilized to determine the expression of cytokines in the lymphocyte supernatant in addition to the IL-10 and TNF-α positivity in monocytes in the presence of Tim-3 and/or PD-1 receptor blockade. Compared with the normal control, significant elevation was observed in the expression of PD-1 on CD3 (P = 0.004), CD4, and CD8 monocytes. Blockade of the Tim-3 signaling pathway contributed to the significant elevation of IL-10 and TNF-α in the supernatant of T lymphocytes in the septic patients, while the PD-1 signaling pathway blockade only triggered the obvious elevation of TNF-α in the T lymphocytes. Blockade of Tim-3 and PD-1 induced the positivity of IL-10- and TNF-α-expressing cells in the peripheral monocytes. Significant changes were noticed in the Tim-3 and PD-1 in the T lymphocytes and monocytes. Blockade of Tim-3 and PD-1 contributed to the function of lymphocytes and monocytes. In the septic process, Tim-3 and PD-1 played crucial roles in the immune response of T lymphocytes and monocytes.

HIV-1 Envelope Glycoproteins Induce the Production of TNF-\u03b1 and IL-10 in Human Monocytes by Activating Calcium Pathway

Human HIV-1 infection leads inevitably to a chronic hyper-immune-activation. However, the nature of the targeted receptors and the pathways involved remain to be fully elucidated. We demonstrate that X4-tropic gp120 induced the production of TNF-α and IL-10 by monocytes through activation of a cell membrane receptor, distinct from the CD4, CXCR4, and MR receptors. Gp120 failed to stimulate IL-10 and TNF-α production by monocytes in Ca2+ free medium. This failure was total for IL-10 and partial for TNF-α. However, IL-10 and TNF-α production was fully restored following the addition of exogenous calcium. Accordingly, addition of BAPTA-AM and cyclosporine-A, fully and partially inhibited IL-10 and TNF-α respectively. The PKA pathway was crucial for IL-10 production but only partially involved in gp120-induced TNF-α. The PLC pathway was partially and equivalently involved in gp120-induced TNF-α and IL-10. Moreover, the inhibition of PI3K, ERK1/2, p38 MAP-kinases and NF-κB pathways totally abolished the production of both cytokines. In conclusion, this study revealed the crucial calcium signaling pathway triggered by HIV-1 gp120 to control the production of these two cytokines: TNF-α and IL-10. The finding could help in the development of a new therapeutic strategy to alleviate the chronic hyper-immune-activation observed in HIV-1 infected patients.

Functional alterations of myeloid cells during the course of Alzheimer\u2019s disease

BackgroundNeuroinflammation is a hallmark of neurodegenerative disease and a significant component of the pathology of Alzheimer’s disease (AD). Patients present with extensive microgliosis along with elevated pro-inflammatory signaling in the central nervous system and periphery. However, the role of peripheral myeloid cells in mediating and influencing AD pathogenesis remains unresolved.MethodsPeripheral myeloid cells were isolated from peripheral blood of patients with prodromal AD (n = 44), mild AD dementia (n = 25), moderate/severe AD dementia (n = 28), and age-matched controls (n = 54). Patients were evaluated in the clinic for AD severity and categorized using Clinical Dementia Rating (CDR) scale resulting in separation of patients into prodromal AD (CDR0.5) and advancing forms of AD dementia (mild-CDR1 and moderate/severe-CDR2/3). Separation of peripheral myeloid cells into mature monocytes or immature MDSCs permitted the delineation of population changes from flow cytometric analysis, RNA phenotype analysis, and functional studies using T cell suppression assays and monocyte suppression assays.ResultsDuring stages of AD dementia (CDR1 and 2/3) peripheral myeloid cells increase their pro-inflammatory gene expression while at early stages of disease (prodromal AD—CDR0.5) pro-inflammatory gene expression is decreased. MDSCs are increased in prodromal AD compared with controls (16.81% vs 9.53%) and have markedly increased suppressive functions: 42.4% suppression of activated monocyte-produced IL-6 and 78.16% suppression of T cell proliferation. In AD dementia, MDSC populations are reduced with decreased suppression of monocyte IL-6 (5.22%) and T cell proliferation (37.61%); the reduced suppression coincides with increased pro-inflammatory signaling in AD dementia monocytes.ConclusionsPeripheral monocyte gene expression is pro-inflammatory throughout the course of AD, except at the earliest, prodromal stages when pro-inflammatory gene expression is suppressed. This monocyte biphasic response is associated with increased numbers and suppressive functions of MDSCs during the early stages and decreased numbers and suppressive functions in later stages of disease. Prolonging the early protective suppression and reversing the later loss of suppressive activity may offer a novel therapeutic strategy.

Comparative Immunomodulatory Activity of Nigella sativa L. Preparations on Proinflammatory Mediators: A Focus on Asthma.

Introduction: A range of traditional and commercial preparations of NS is frequently used in the treatment of several inflammatory diseases. Often, these preparations have poor preclinical characterization that may lead to variable pharmacological effects.Objective: To assess the in vitro effects of different chemically defined preparations of NS on some asthma-related mediators of inflammation.Methods: Different NS preparations were obtained by either seed extraction with a spectrum of solvents ranging from lipophilic to hydrophilic, or commercial products were collected. The TQ concentration of NS was analyzed by HPLC. Immunomodulatory activity was assessed by the release of mediators (IL-2, IL-6, PGE2) in primary human T-lymphocytes, monocytes, and A549 human lung epithelial cells.Results: Ten distinct NS preparations showed variability in TQ concentration, being highest in the oily preparations extract-7 (2.4% w/w), followed by extract-10 (0.7%w/w). Similarly, the release of mediators was varied, being greatest in extract-7 and 10 via significantly (<0.05) suppressing IL-2, IL-6, and PGE2 in T-lymphocytes as well as IL-6 and PGE2 in monocytes. Also, PGE2 release in A549 cells was significantly enhanced by both extracts.Conclusion: The TQ concentration and in vitro activity were variable among the different NS preparations. TQ-rich oily NS preparations produced potent favorable immunomodulation in asthma inflammation and can be used in future studies.

A5429 Relationship between Monocyte Subsets, IL-6 and hs-CRP with the Severity of Coronary Artery Disease in Stable Angina Pectoris Patients

A5429 Relationship between Monocyte Subsets, IL-6 and hs-CRP with the Severity of Coronary Artery Disease in Stable Angina Pectoris Patients

Interleukin-37 Attenuates Bleomycin-Induced Pulmonary Inflammation and Fibrosis in Mice.

Pulmonary fibrosis is a disease with chronic inflammation and excessive collagen deposition for which there is no effective treatments. Interleukin (IL)-37 is a newly identified anti-inflammatory cytokine but its role in pulmonary fibrosis remains unclear. In this study, we investigated the effect of IL-37 on bleomycin-induced pulmonary fibrosis in mice. A lentivirus expressing IL-37 was administered intranasally to bleomycin-induced C57BL/6 mice. We found that IL-37 improved the survival of mice and reduced the body weight loss of mice caused by bleomycin. Furthermore, IL-37 significantly attenuated pulmonary inflammatory infiltration and collagen deposition and decreased the hydroxyproline content in bleomycin-treated mice. Finally, IL-37 treatment inhibited the expression of monocyte chemoattractant protein-1, IL-6, and tumor necrosis factor-α, but increased the expression of interferon-γ in lung tissues from bleomycin-challenged mice. Taken together, these results suggest that in vivo expression of IL-37 is useful in preventing pulmonary fibrosis induced by bleomycin and provides a possible therapeutic approach to pulmonary fibrosis diseases.

Bacteria-induced susceptibility to Candida albicans super-infection in mice via monocyte methyltransferase Setdb2.

Systemic bacterial infections are prone to secondary Candida albicans super-infection. However, the molecular mechanisms involved remain poorly understood. In this study, a model comprising sublethal cecal ligation and puncture plus C.albicans intravenous injection was applied to mimic the situation in super-infection. Compared with mice without systemic bacterial infection, mice with systemic bacterial infection had lower antifungal gene expression (including Il1b, Tnf, Il6, Ifnb, Ifng, Cxcl1, and Ccr2) in monocytes and less inflammatory monocytes and neutrophils infiltrating into the kidney when challenged with C.albicans. Further, lentivirus-mediated Setdb2-knockout and overexpression experiments verified that Setdb2 levels in monocytes correlated negatively with antifungal gene expression and survival rates. Transcriptional repression was probably achieved by Setdb2 through H3 methylation at lysine 9 in promoter regions of these antifungal genes.

Isoprenoid Depletion Increases Cytokine mRNA Levels in Primary Human Monocytes

Mevalonate Kinase Deficiency (MKD) is an autoinflammatory disease caused by mutations in the mevalonate kinase (MVK) gene, which produces an enzyme responsible for the production of isoprenoids. Previous research in various model systems has demonstrated that depletion of isoprenoids leads to inflammasome activation and increased levels of secreted IL‐1B on a post‐translational level. However, patient data indicates that MKD is a multi‐cytokine disease with increased plasma levels of cytokines such as TNF‐alpha, IL‐6, and IFN‐gamma. In order to study the mechansims responsible for these changes, the mevalonate pathway was inhibited with lovastatin in peripheral blood mononulcear cells (PBMCs) and monocytes isolated from the blood of healthy donors followed by stimulation with lipopolysaccharide (LPS) to induce an inflammatory response. Lovastatin treatment resulted in significantly increased levels of TNF‐alpha, IL‐6, IL‐12p40, and IFN‐gamma mRNA in both PBMCs and monocytes following LPS stimulation as compared to control cells. Lovastatin treatment did not alter IL‐1B, CD14, or IL‐18 mRNA levels either before or after LPS stimulation. Addition of mevalonate to the cells treated with lovastatin prevented the increased cytokine mRNA levels indicating that these changes are due to inhibition of the mevalonate pathway. These results indicate that isoprenoid depletion alters the expression of multiple cytokines on the mRNA level in primary human monocytes, which can be used as a model system ex vivo to study these changes.Support or Funding InformationDickinson College Faculty Start‐up and Research &amp; Development Committee FundingThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

CD5L Promotes M2 Macrophage Polarization through Autophagy-Mediated Upregulation of ID3.

CD5L (CD5 molecule-like) is a secreted glycoprotein that controls key mechanisms in inflammatory responses, with involvement in processes such as infection, atherosclerosis, and cancer. In macrophages, CD5L promotes an anti-inflammatory cytokine profile in response to TLR activation. In the present study, we questioned whether CD5L is able to influence human macrophage plasticity, and drive its polarization toward any specific phenotype. We compared CD5L-induced phenotypic and functional changes to those caused by IFN/LPS, IL4, and IL10 in human monocytes. Phenotypic markers were quantified by RT-qPCR and flow cytometry, and a mathematical algorithm was built for their analysis. Moreover, we compared ROS production, phagocytic capacity, and inflammatory responses to LPS. CD5L drove cells toward a polarization similar to that induced by IL10. Furthermore, IL10- and CD5L-treated macrophages showed increased LC3-II content and colocalization with acidic compartments, thereby pointing to the enhancement of autophagy-dependent processes. Accordingly, siRNA targeting ATG7 in THP1 cells blocked CD5L-induced CD163 and Mer tyrosine kinase mRNA and efferocytosis. In these cells, gene expression profiling and validation indicated the upregulation of the transcription factor ID3 by CD5L through ATG7. In agreement, ID3 silencing reversed polarization by CD5L. Our data point to a significant contribution of CD5L-mediated autophagy to the induction of ID3 and provide the first evidence that CD5L drives macrophage polarization.

Chronic kidney disease induces inflammation by increasing Toll-like receptor-4, cytokine and cathelicidin expression in neutrophils and monocytes

TLR expression in neutrophils and monocytes is associated with increased cytokine synthesis, resulting in increased inflammation. However, the inflammatory pathway related to TLR and cathelicidin expression in these cells from CKD patients is unclear. To evaluate TLR4, cathelicidin, TNF-α, IL-6, IL-10 and MCP-1 expression in neutrophils and monocytes from HD and CKD patients. Blood samples were drawn from 47 CKD and 43 HD patients and 71 age and gender-matched healthy volunteers (CONT). TLR4 was analyzed using flow cytometry. Cathelicidin, TNF-α, IL-6, IL-10 and MCP-1 were analyzed via ELISA.TLR4 expression in neutrophils was higher in HD patients than in stage 3 and 4 CKD patients. In these cells, we observed a positive correlation between TLR4 and cathelicidin, TNF-α, IL-6, IL-10 and MCP-1 levels. In monocytes, TLR4 expression was significantly higher in CKD 3 and 4 groups than in the control and HD groups and positively and negatively correlated with IL-6 and MCP-1 and cathelicidin, respectively. TNF-α, IL-6 and MCP-1 serum levels were higher in HD and CKD patients than in control. Cathelicidin and IL-10 levels were only higher in HD patients. IL-6 serum levels were positively correlated with all cytokines, and cathelicidin was negatively correlated with MCP-1 (r = − 0.35; p < 0.01) and positively correlated with IL-10 (r = 0.37; p = 0.001). These results suggest that a uremic environment induces high TLR4, cathelicidin and cytokine expression and may increase inflammation. Thus, future studies should be conducted to evaluate whether TLR4 and cathelicidin should be targets for anti-inflammatory therapeutic strategies.

ISG15-Induced IL-10 Is a Novel Anti-Inflammatory Myeloid Axis Disrupted during Active Tuberculosis.

IFN-stimulated gene 15 (ISG15) deficiency in humans leads to severe IFNopathies and mycobacterial disease, the latter being previously attributed to its extracellular cytokine-like activity. In this study, we demonstrate a novel role for secreted ISG15 as an IL-10 inducer, unique to primary human monocytes. A balanced ISG15-induced monocyte/IL-10 versus lymphoid/IFN-γ expression, correlating with p38 MAPK and PI3K signaling, was found using targeted in vitro and ex vivo systems analysis of human transcriptomic datasets. The specificity and MAPK/PI3K-dependence of ISG15-induced monocyte IL-10 production was confirmed in vitro using CRISPR/Cas9 knockout and pharmacological inhibitors. Moreover, this ISG15/IL-10 axis was amplified in leprosy but disrupted in human active tuberculosis (TB) patients. Importantly, ISG15 strongly correlated with inflammation and disease severity during active TB, suggesting its potential use as a biomarker, awaiting clinical validation. In conclusion, this study identifies a novel anti-inflammatory ISG15/IL-10 myeloid axis that is disrupted in active TB.

Abatacept blocks anti-citrullinated protein antibody and rheumatoid factor\xa0mediated cytokine production in human macrophages in IDO-dependent manner

BackgroundThe anti-inflammatory effect of abatacept is most pronounced in patients with high-titer autoantibodies (including anticitrullinated protein antibodies [ACPA] and rheumatoid factor [RF]). Considering that autoantibodies trigger inflammatory cytokine production by monocytes and that abatacept binds to monocytes, influencing their functional state, we hypothesized that abatacept may effectively inhibit the production of several different cytokines by ACPA- or RF-challenged monocytes.MethodsPeripheral blood CD68+ monocytes stimulated with macrophage colony-stimulating factor for 24 h were exposed to random immunoglobulin G alone (negative control), purified ACPA, purified RF, or lipopolysaccharide (positive control) in cell culture plates coated with citrullinated vimentin (to allow ACPA immune complex formation). Stimulations were done in the presence or absence of abatacept or tumor necrosis factor (TNF) antibody (adalimumab) with or without indoleamine 2,3-dioxygenase (IDO) inhibitor 1-methyl-d-tryptophan. Supernatants were analyzed for key proinflammatory cytokines TNF-α, interleukin (IL)-1β, IL-6, IL-8, and chemokine (C-C motif) ligand 2 (CCL2) after 24 h.ResultsExposure to ACPA or RF significantly induced the production of TNF-α (20-fold and 27-fold, respectively), IL-1β (each 4-fold), IL-6 (12-fold and 11-fold, respectively), IL-8 (43-fold and 30-fold, respectively), and CCL2 (each 4-fold) in human monocytes. Abatacept inhibited this autoantibody-mediated upregulation of cytokines, reducing TNF-α by > 75%, IL-1β by > 65%, IL-6 and IL-8 by > 80%, and CCL2 by > 60%. In contrast, a TNF inhibitor did not influence autoantibody-induced proinflammatory cytokine production. IDO inhibition reversed the effect of abatacept and again permitted the induction of cytokine production by ACPA and RF.ConclusionsThese data show that abatacept interferes with autoantibody-mediated cytokine production by monocytes through induction of IDO. This inhibitory effect on the production of several effector cytokines in RA may explain the fast anti-inflammatory effect of abatacept as well as its preferential efficacy in patients with high-titer ACPA and RF.

CRT-500.01 Relationship Between Monocyte Subsets, Il-6 and Hs-crp with the Severity of Coronary Artery Disease in Stable Angina Pectoris Patients

Monocytes are crucially involved in all stages of atherogenesis as cellular drivers of vascular inflammation hallmarking atherosclerotic disease.CD16+monocytes are pro-inflammatory cells, whose proportion is related to the occurrence of coronary artery disease (CAD), intma-media thickness and plaque

Innate immunity recovers earlier than acquired immunity during severe postoperative immunosuppression.

Background: Postoperative immune suppression, particularly a loss of cell-mediated immunity, is commonly seen after surgery and is associated with worse outcome, i.e. delayed wound healing, infections, sepsis, multiple-organ failure and cancer recurrence. However, the recovery of immune cells focusing on differences between innate and acquired immunity during severe postoperative immunosuppression is not investigated. Methods: In this retrospective randomized controlled trial (RCT) subgroup analysis, 10 postoperatively immune suppressed patients after esophageal or pancreatic resection were analyzed. Innate and acquired immune cells, the expression of human leukocyte antigen-D related on monocytes (mHLA-DR), lipopolysaccharide (LPS)-induced monocytic TNF-α and IL-10 secretion ex vivo, Concanavalin A (Con A)-induced IFN-γ, TNF-α, IL-2, IL-4, IL-5 and IL-10 release were measured preoperatively (od) until day 5 after surgery (pod5). Recovery of immune cells was defined by a significant decrease respectively increase after a significant postoperative alteration. Statistical analyses were performed using nonparametric statistical procedures. Results: Postoperative alterations of innate immune cells recovered on pod2 (eosinophils), pod3 (neutrophils) and pod5 (mHLA-DR, monocytic TNF-α and IL-10 secretion), whereas alterations of acquired immune cells (lymphocytes, T cells, T helper cells, and cytotoxic T cells) did not recover until pod5. Peripheral blood T cells showed an impaired production of the T helper (Th) 1 cytokine IFN-γ upon Con A stimulation on pod1, while Th2 specific cytokine release did not change until pod5. Conclusions: Innate immunity recovered earlier than acquired immunity during severe postoperative immunosuppression. Furthermore, we found a more anti- than pro-inflammatory T cell function on the first day after surgery, while T cell counts decreased.

MicroRNA-204-3p inhibits lipopolysaccharide-induced cytokines in familial Mediterranean fever via the phosphoinositide 3-kinase γ pathway.

We sought to identify the microRNA (miRNA) profile and potential biomarkers in FMF and to clarify their gene targets to elucidate the pathogenesis of FMF. We performed an miRNA microarray using serum from FMF patients in attack and in remission. We then examined the expression of miRNAs in macrophages derived from THP-1 cells stimulated with toll-like receptor (TLR) ligands. Macrophages derived from THP-1 cells transfected with pre-miRNA were stimulated with lipopolysaccharides (LPSs) for the quantification of inflammatory cytokine production. To identify the target genes, we overexpressed their miRNA and performed a complementary DNA microarray. Transfection with reporter construct and the precursor miRNA was performed to confirm the suppression of target mRNA. We found that miR-204-3p was greatly decreased in the serum from FMF patients in attack. The expression of miR-204-3p was suppressed by LPS stimulation in the macrophages derived from THP-1 cells and the inhibition of miR-204-3p significantly induced the production of TLR4-related cytokines. The bioinformatic analysis showed that miR-204-3p is predicted to target genes implicated in the TLR pathway through the regulation of PI3Kγ signalling. The reporter assay revealed that miR-204-3p directly suppressed the luciferase activity of 3'-UTR of PIK3CG reporter construct. The inhibition of PI3Kγ resulted in decreased amounts of IL-6 and IL-12p40 in monocytes from FMF patients. These data suggest that serum miR-204-3p has potential as a useful biomarker in FMF patients and that miR-204-3p serves as a suppressor of inflammatory cytokine production in FMF by targeting the PI3Kγ pathway.

Activation of the Innate Immune System by Treponema denticola Periplasmic Flagella through Toll-Like Receptor 2.

Treponema denticola is an indigenous oral spirochete that inhabits the gingival sulcus or periodontal pocket. Increased numbers of oral treponemes within this environment are associated with localized periodontal inflammation, and they are also part of an anaerobic polymicrobial consortium responsible for endodontic infections. Previous studies have indicated that T. denticola stimulates the innate immune system through Toll-like receptor 2 (TLR2); however, the pathogen-associated molecular patterns (PAMPs) responsible for T. denticola activation of the innate immune system are currently not well defined. In this study, we investigated the role played by T. denticola periplasmic flagella (PF), unique motility organelles of spirochetes, in stimulating an innate immune response. Wild-type T. denticola stimulated the production of the cytokines tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), IL-6, IL-10, and IL-12 by monocytes from human peripheral blood mononuclear cells, while its isogenic nonmotile mutant lacking PF resulted in significantly diminished cytokine stimulation. In addition, highly purified PF were able to dose dependently stimulate cytokine TNF-α, IL-1β, IL-6, IL-10, and IL-12 production in human monocytes. Wild-type T. denticola and the purified PF triggered activation of NF-κB through TLR2, as determined using a variety of TLR-transfected human embryonic 293 cell lines, while the PF-deficient mutants lacked the ability to stimulate, and the complemented PF-positive T. denticola strain restored the activation. These findings suggest that T. denticola stimulates the innate immune system in a TLR2-dependent fashion and that PF are a key bacterial component involved in this process.