Expression In Blood Monocytes Research Articles

Transcriptomic Context of RUNX3 Expression in Monocytes: A Cross-Sectional Analysis.

The runt-related transcription factor 3 (RUNX3) regulates the differentiation of monocytes and their response to inflammation. However, the transcriptomic context of RUNX3 expression in blood monocytes remains poorly understood. We aim to learn about RUNX3 from its relationships within transcriptomes of bulk CD14+ cells in adults. This study used immunomagnetically sorted CD14+ cell gene expression microarray data from the Multi-Ethnic Study of Atherosclerosis (MESA, n = 1202, GSE56047) and the Correlated Expression and Disease Association Research (CEDAR, n = 281, E-MTAB-6667) cohorts. The data were preprocessed, subjected to RUNX3-focused correlation analyses and random forest modeling, followed by the gene ontology analysis. Immunity-focused differential ratio analysis with intermediary inference (DRAIMI) was used to integrate the data with protein-protein interaction network. Correlation analysis of RUNX3 expression revealed the strongest positive association for EVL (rmean = 0.75, pFDR-MESA = 5.37 × 10-140, pFDR-CEDAR = 5.52 × 10-80), ARHGAP17 (rmean = 0.74, pFDR-MESA = 1.13 × 10-169, pFDR-CEDAR = 9.20 × 10-59), DNMT1 (rmean = 0.74, pFDR-MESA = 1.10 × 10-169, pFDR-CEDAR = 1.67 × 10-58), and CLEC16A (rmean = 0.72, pFDR-MESA = 3.51 × 10-154, pFDR-CEDAR = 2.27 × 10-55), while the top negative correlates were C2ORF76 (rmean = -0.57, pFDR-MESA = 8.70 × 10-94, pFDR-CEDAR = 1.31 × 10-25) and TBC1D7 (rmean = -0.55, pFDR-MESA = 1.36 × 10-69, pFDR-CEDAR = 7.81 × 10-30). The RUNX3-associated transcriptome signature was involved in mRNA metabolism, signal transduction, and the organization of cytoskeleton, chromosomes, and chromatin, which may all accompany mitosis. Transcriptomic context of RUNX3 expression in monocytes hints at its relationship with cell growth, shape maintenance, and aspects of the immune response, including tyrosine kinases.

Allergens elicit upregulated IL‐18 and IL‐18Rα expression in blood monocytes of patients with allergic rhinitis

AbstractInterleukin (IL)‐18 is a potentially important molecule in allergic rhinitis (AR). However, expressions of IL‐18, IL‐18 binding protein isoform a (IL‐18BPa) and IL‐18 receptor alpha (IL‐18Rα) in AR blood monocytes remain obscure. We, therefore, investigated IL‐18, IL‐18BPa and IL‐18Rα expressions in monocytes using flow cytometry, murine AR model and quantitative real‐time polymerase chain reaction (PCR). The results showed that the numbers of IL‐18+ monocytes increased, whereas IL‐18BPa+ monocytes decreased in the peripheral blood of AR patients. It was also observed that Platanus pollen extract provoked elevated expressions of IL‐18 and IL‐18Rα in the monocytes of AR patients. House dust mite extract, Artemisia sieversiana wild extract and Platanus pollen extract enhanced IL‐18Rα protein and mRNA expression in the isolated primary monocytes from AR patients. Using ELISA kits, we observed that the levels of total IL‐18 and free IL‐18 in the plasma of perennial AR (pAR) and seasonal AR (sAR) patients were elevated, and the molar concentration ratio of free IL‐18BPa/free IL‐18 was 16.5 for healthy control subjects and 9.7 for patients with sAR, indicating that IL‐18 likely plays a role in sAR. In the murine AR model, the number of IL‐18Rα+ monocytes increased in the blood, and the number of IL‐18Rα+ macrophages increased in the nasal lavage fluid of WT mice. In conclusion, IL‐18 may serve as a causative factor for AR.

Hyperglycemia and dyslipidemia: Reduced HLA-DR expression in monocyte subpopulations from diabetes patients

Immune dysfunction contributes to the higher risk of communicable and non-communicable diseases among diabetics. HLA-DR expression is a robust marker of immune competence in mononuclear cells, including antigen presentation to CD4 lymphocytes. Given the high prevalence of obesity among diabetics, we evaluated the independent association between hyperglycemia and dyslipidemias with respect to HLA-DR expression in blood monocytes from type 2 diabetes patients. The monocytes from individuals with (n = 16) or without diabetes (n = 25) were phenotyped by flow cytometry to assess the differential expression of HLA-DR on their three subpopulations (classical, intermediate and non-classical monocytes). Diabetes was independently associated with lower HLA-DR expression across all monocyte subpopulations (p < 0.05). Blood triglycerides were associated with further HLA-DR depression (interaction p < 0.002). Cholesterols counterbalanced the reductive effect, with CD36, a receptor for oxidized cholesterol, correlating with HLA-DR (rho = 0.373; p = 0.016). Future studies are warranted to elucidate the complex interactions between hyperglycemia and dyslipidemias on antigen presentation in diabetic monocytes.

The polymorphism rs975484 in the protein arginine methyltransferase 1 gene modulates expression of immune checkpoint genes in hepatocellular carcinoma

Protein arginine methyltransferase 1 (PRMT1) is a key regulator of hepatic immune responses. Recently, we reported that PRMT1 regulates the tumor immune response in hepatocellular carcinoma (HCC). Here we found that PRMT1 expression in human HCC correlates with that of programmed cell death 1 ligand 1 (PD-L1), PD-L2, and other checkpoint genes. PRMT1 deletion in mice reduced PD-L1 and PD-L2 expression in tumors and reduced the efficiency of PD-1 antibody treatment in a diethylnitrosamine-induced HCC mouse model, suggesting that PRMT1 regulates the hepatic immune checkpoint. Mice had reduced PD-L1 and PD-L2 expression when PRMT1 was specifically deleted in tumor cells or macrophages, but PRMT1 deletion in dendritic cells did not alter PD-L1 and PD-L2 expression. rs975484 is a common polymorphism in the human PRMT1 gene promoter, and we found that it alters PRMT1 expression in blood monocytes and tumor-associated macrophages in human HCC. PRMT1 expression was higher in individuals with a GG genotype than in individuals with a CC genotype, and heterozygous carriers had intermediate expression. Luciferase reporter assays indicated that this differential expression is due to an extra C/EBPβ-binding site in the PRMT1 promoter of individuals carrying the minor G allele. The rs975484 genotype also correlated with PRMT1 target expression in HCC. Individuals with the GG genotype had significantly higher levels of the PRMT1 targets PD-L1, PD-L2, and VISTA than those with the CC genotype. We conclude that PRMT1 critically controls immune checkpoints in mice and humans and that the PRMT1 polymorphism rs975484 affects checkpoint gene expression in HCC.

221 - Targeting HDAC2 protects monocytes against metabolic stress-induced priming and dysfunction

Background Monocytes priming and reprogramming induced by metabolic stress is mediated by protein S-glutathionylation and is prevented by overexpression of glutaredoxin 1 (Grx1). Here we tested the hypothesis that inhibition of HDAC2 prevents monocyte dysfunction by inducing Grx1 and preventing metabolic stress-induced protein S-glutathionylation. Methods THP-1 monocytes were incubated with the HDAC inhibitor Scriptaid for up to 72 h and the expression of Grx1, HDAC2 and s-actin, as well as the level of acetylation lysine 9 on H3 (H3K9Ac), were examined by Western blotting. To identify the specific HDAC involved in Grx1 regulation, we used small interfering RNA to knockdown targeting HDAC1, 2 and 3. Bone marrow-derived macrophages (BMDM) were generated from LysMcre+/-HDAC2+/- and LysMcre+/- mice and primed with LDL (100 μg/ml) plus high glucose (25 mM) for 36 h. HDAC2 mRNA levels were quantified by qRT-PCR. Macrophage priming was assessed using the MKP-1 activity assay. Results Scriptaid increased Grx1 protein expression in THP-1 monocytes 1.8-fold after 36 h increased acetylation of H3K9 1.5-fold. Scriptaid also rescued MKP-1 activity and normalized chemotactic activity in primed THP-1 monocytes. Induction of Grx1 was only observed in cells treated with anti-HDAC2 siRNA (1.5-fold increase) but no changes were observed in response to either HDAC1 or HDAC3 knockdown. Grx1 mRNA levels in blood monocytes from HDAC2 deficient mice were 20-fold higher than in monocytes from control mice. BMDM from LysMcre+/-HDAC2+/- mice showed higher MKP-1 activity and Grx1 protein expression than BMDM from LysMCre+/- mice. Furthermore, MKP-1 activity was inhibited by metabolic stress in control BMDM but was maintained in BMDM from LysMCreHDAC2+/- mice. Conclusions Grx1 expression in blood monocytes and monocyte-derived macrophages is regulated by HDAC2. Inhibition of HDAC2 induces Grx1 and protects monocytes from metabolic stress-induced dysfunction, making HDAC2 a novel therapeutic target for chronic inflammatory diseases associated with metabolic disorders.

The role of B7 costimulation in benzene immunotoxicity and its potential association with cancer risk

Benzene is a recognized human carcinogen; however, there are still some gaps in the knowledge regarding the mechanism of toxicity of this organic solvent and potential early biomarkers for the damage caused by it. In a previous study, our research group demonstrated that the adhesion molecules of the immune system (B7.1 and B7.2) could be potential biomarkers in the early detection of immunotoxicity caused by benzene exposure. Therefore, this study was developed to deepen the understanding regarding this important topic, aiming to contribute to the comprehension of the benzene toxicity mechanism mediated by B7.1 and B7.2 and its potential association with the risk of carcinogenicity. B7.1 and B7.2 protein expression in blood monocytes and B7.1 and B7.2 gene expression in PBMCs were evaluated. Additionally, complement C3 and C4 levels in serum were measured, as well as p53 gene expression in PBMCs. Seventy-four gas station workers (GSW group) and 71 non-occupationally exposed subjects (NEG) were evaluated. Our results demonstrated decreased levels of B7.1 and B7.2 protein and gene expression in the GSW group compared to the NEG (n = 71) (p < 0.01). Along the same lines, decreased levels of the complement system were observed in the GSW group (p < 0.01), demonstrating the impairment of this immune system pathway as well. Additionally, a reduction was observed in p53 gene expression in the GSA group (p < 0.01). These alterations were associated with both the benzene exposure biomarker evaluated, urinary trans, trans-muconic acid, and with exposure time (p < 0.05). Moreover, strong correlations were observed between the gene expression of p53 vs. B7.1 (r = 0.830; p < 0.001), p53 vs. B7.2 (r = 0.685; p < 0.001), and B7.1 vs. B7.2 (r = 0.702; p < 0.001). Taken together, these results demonstrate that the immune system co-stimulatory molecule pathway is affected by benzene exposure. Also, the decrease in p53 gene expression, even at low exposure levels, reinforces the carcinogenicity effect of benzene in this pathway. Therefore, our results suggest that the promotion of immune evasion together with a decrease in p53 gene expression may play an important role in the benzene toxicity mechanism. However, further and targeted studies are needed to confirm this proposition.

Effect of cigarette smoke on monocyte procoagulant activity: Focus on platelet-derived brain-derived neurotrophic factor (BDNF)

Cigarette smoke (CS) activates platelets, promotes vascular dysfunction, and enhances Tissue Factor (TF) expression in blood monocytes favoring pro-thrombotic states. Brain-derived neurotrophic factor (BDNF), a member of the family of neurotrophins involved in survival, growth, and maturation of neurons, is released by activated platelets (APLTs) and plays a role in the cardiovascular system. The effect of CS on circulating levels of BDNF is controversial and the function of circulating BDNF in atherothrombosis is not fully understood. Here, we have shown that human platelets, treated with an aqueous extract of CS (CSE), released BDNF in a dose-dependent manner. In addition, incubation of human monocytes with BDNF or with the supernatant of platelets activated with CSE increased TF activity by a Tropomyosin receptor kinase B (TrkB)-dependent mechanism. Finally, comparing serum and plasma samples of 12 male never smokers (NS) and 29 male active smokers (AS) we observed a significant increase in microparticle-associated TF activity (MP-TF) as well as BDNF in AS, while in serum, BDNF behaved oppositely. Taken together these findings suggest that platelet-derived BDNF is involved in the regulation of TF activity and that CS plays a role in this pathway by favoring a pro-atherothrombotic state.

Novel marker for the detection of sickle cell nephropathy: soluble FMS-like tyrosine kinase-1 (sFLT-1)

Given the burden and poor outcome of end-stage renal disease in sickle cell disease (SCD), early markers of sickle cell nephropathy (SN) are desirable. Disordered angiogenesis underlies many complications of SCD. We aimed to determine the relationship between serum FMS-like tyrosine kinase-1 (sFLT-1) and other biomarkers of renal damage for the early diagnosis of SN. Forty-seven SCD patients and 49 healthy controls were enrolled. Microalbuminuria was determined in patient urine samples. Blood samples were tested for sFLT-1, serum creatinine, and various hemolysis and inflammation markers. Peripheral blood monocyte expression of sFLT-1 was measured using real-time polymerase chain reaction (PCR). The serum level of sFLT-1 (pg/ml) in SCD patients was higher than controls (median/range/IQR = 142/ 60-1300/61 pg/ml vs. 125/ 110-187/52 pg/ml, respectively) (p = 0.006). Median (range) of sFLT-1 level was higher in SCD patients with microalbuminuria compared to SCD patients with normoalbuminuria, 185 (140-1300) vs. 125 (60-189) mg/g, respectively) (p = 0.004). There was a significant positive correlation between serum sFLT-1 and microalbuminuria, lactate dehydrogenase (LDH), and indirect bilirubin (r = 0.59, 0.39, 0.30, and p = <0.001, 0.007, 0.041, respectively). sFLT-1 sensitivity in early detection of renal affection in SCD was 93.6%, while specificity was 68.6%. Finally, peripheral blood monocytes (PBM) sFLT-1 expression was significantly higher in SCD patients compared to controls (p = 0.05). sFLT-1 may contribute to pathogenesis of albuminuria in SCD patients and constitute a novel renal biomarker of SN.

Glutaredoxin 2a overexpression in macrophages promotes mitochondrial dysfunction but has little or no effect on atherogenesis in LDL-receptor null mice

AimsReactive oxygen species (ROS)-mediated formation of mixed disulfides between critical cysteine residues in proteins and glutathione, a process referred to as protein S-glutathionylation, can lead to loss of enzymatic activity and protein degradation. Since mitochondria are a major source of ROS and a number of their proteins are susceptible to protein-S-glutathionylation, we examined if overexpression of mitochondrial thioltranferase glutaredoxin 2a (Grx2a) in macrophages of dyslipidemic atherosclerosis-prone mice would prevent mitochondrial dysfunction and protect against atherosclerotic lesion formation. Methods and resultsWe generated transgenic Grx2aMacLDLR−/− mice, which overexpress Grx2a as an EGFP fusion protein under the control of the macrophage-specific CD68 promoter. Transgenic mice and wild type siblings were fed a high fat diet for 14 weeks at which time we assessed mitochondrial bioenergetic function in peritoneal macrophages and atherosclerotic lesion formation. Flow cytometry and Western blot analysis demonstrated transgene expression in blood monocytes and peritoneal macrophages isolated from Grx2aMacLDLR−/− mice, and fluorescence confocal microscopy studies confirmed that Grx2a expression was restricted to the mitochondria of monocytic cells. Live-cell bioenergetic measurements revealed impaired mitochondrial ATP turnover in macrophages isolated from Grx2aMacLDLR−/− mice compared to macrophages isolated from non-transgenic mice. However, despite impaired mitochondrial function in macrophages of Grx2aMacLDLR−/− mice, we observed no significant difference in the severity of atherosclerosis between wildtype and Grx2aMacLDLR−/− mice. ConclusionOur findings suggest that increasing Grx2a activity in macrophage mitochondria disrupts mitochondrial respiration and ATP production, but without affecting the proatherogenic potential of macrophages. Our data suggest that macrophages are resistant against moderate mitochondrial dysfunction and rely on alternative pathways for ATP synthesis to support the energetic requirements.

Response to infliximab therapy in ulcerative colitis is associated with decreased monocyte activation, reduced CCL2 expression and downregulation of Tenascin C.

The cellular mechanisms leading to infliximab therapy response in patients with ulcerative colitis (UC) are incompletely known. We therefore investigated early effects of infliximab therapy on monocytes and associated chemokines linked to clinical therapy response in UC patients. Blood and biopsies were obtained from anti-TNF therapy-naïve UC patients (n = 43) before (baseline) and during induction therapy with infliximab. Therapy response was evaluated at Week 14. Expression of monocyte activation markers and levels of chemokines in serum and biopsies were determined. Quantitative proteomic analysis was performed in cultured mucosal biopsies, and obtained data was validated in serum. In therapy responders, but not in non-responders, infliximab reduced blood monocyte expression of CD14 and CD86, 2 weeks after therapy commenced, relative to baseline. Serum CCL2 levels were decreased only among therapy responders at Week 2 and Week 14, relative to baseline. These data corresponded with lower levels of CD14, CD86 and CCL2 in intestinal tissue in responders as compared with non-responders at Week 14. Proteomic analysis of cultured biopsies showed that infliximab induced a reduction in Tenascin C that predicted downregulation of CCL2. Therapy responders, but not non-responders, had decreased serum Tenascin C levels at Week 2 and Week 14, relative to baseline. Infliximab therapy response in UC patients is associated with reduced monocyte activation and serum levels of CCL2 2 weeks after therapy commencement. In therapy responders, infliximab influenced Tenascin C, which might be a regulator of CCL2 expression and important for induction of the clinical therapy response.

Abstract 4986: Myeloid-derived suppressor cells as a biomarker of tumor growth and radiosensitivity: Role of hypoxia-inducible arginase-1.

Abstract Introduction: Recent evidence suggests that tumor growth and metastases are sustained by myeloid-derived suppressor cells (MDSC), which over-express arginase-1 (ARG) and cause T-cell suppression. We hypothesize that MDSC impair tumor cell radiosensitivity through ARG-mediated depletion of L-arginine, an essential substrate for the biosynthesis of the endogenous radiosensitizer nitric oxide (NO). Aim: We explored whether MDSC and ARG can be used as biomarkers for tumor growth in experimental mouse models and in the clinical setting of colorectal cancer (CRC). We further examined whether low L-arginine levels are associated with an impaired generation of NO by inducible NO synthase (iNOS) and a reduced radiosensitivity of hypoxic tumor cells. Materials &amp; methods: MDSC (CD11b+Gr-1+) and ARG levels in mouse colon CT26 and mammary 4T1 models were analyzed by flow cytometry (FCM) and RT-PCR. Mice were inoculated I.M. with either CT26 or 4T1 carcinoma cells. After 17 days, MDSC were quantified in the spleen by FCM. In addition, ARG levels were analyzed by RT-PCR and FCM. Similar analyses were performed on human blood MDSC (CD14+HLA-DRlow) derived from CRC patients. To model the hypoxic tumor microenvironment, mouse splenocytes were cultured in 1% oxygen. Activation of iNOS was induced by LPS/IFN-γ and analysed by RT-PCR, Western blot and Griess assay. Radiosensitivity was analysed in a model of metabolic hypoxia by colony formation assay Results: The growth of both CT26 and 4T1 tumors in BALB/c mice was associated with an accumulation of MDSC in the spleen from 2-4% (in naive mice) to 8 and 35% respectively. Freshly isolated splenocytes and MDSC showed low ARG levels. Contrasting, hypoxia-conditioned splenocytes from tumor-bearing mice revealed a drastic transcriptional activation of ARG (up to 525.000-fold), and ARG+ MDSC. On the other hand splenocytes from tumor-free mice did not display ARG activation in hypoxic conditions. The iNOS/NO-mediated radiosensitization of mammary EMT6 tumor cells was strictly dependent on L-arginine levels, with a marginal effect of 1.2-fold at 10-30 μM while approaching a 2.4-fold effect at a physiological concentration of 125 μM. MDSC levels in CRC patients were increased, as compared with healthy donors (8-12% versus 4-5%). Patients with locally advanced CRC had 16-fold higher ARG expression in blood monocytes, as compared to healthy donors. 4 weeks after pre-operative radiotherapy the ARG levels decreased by 2-fold. Conclusions: Although tumor progression is clearly associated with the expansion of MDSC, their hypoxic conditioning appears to be crucial to uncover ARG as a tumor biomarker in vivo. iNOS/NO mediated radiosensitisation of hypoxic tumor cells was drastically impaired at low L-arginine levels which could be depleted by ARG+ MDSC. In a clinical setting, we consider ARG a potential biomarker in locally advanced rectal cancer. Citation Format: Wim Leonard, Inès Dufait, Heng Jiang, Femke Steenbeke, Marieke Vermeersch, Kalun Law, Guy Storme, Joeri Aerts, Valeri Verovski, Mark De Ridder. Myeloid-derived suppressor cells as a biomarker of tumor growth and radiosensitivity: Role of hypoxia-inducible arginase-1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4986. doi:10.1158/1538-7445.AM2013-4986

Increased Toll-Like Receptor 2 Expression in Peptidoglycan-Treated Blood Monocytes Is Associated with Insulin Resistance in Patients with Nondiabetic Rheumatoid Arthritis

The close relationship between increased TLR-2 expression in blood monocytes and insulin resistance in RA patients is shown in this study. Traditional risk factors for metabolic disorders, including the waist circumstance, body mass index (BMI), triglyceride (TG), and ratio of TG to high density lipoprotein (HDL) cholesterol, were closely correlated with HOMA (homoeostasis model assessment) index in patients with nondiabetic RA. Expressions of TLR2 in peripheral blood monocytes, following stimulation with peptidoglycan which is known as a TLR2 agonist, were closely correlated with the HOMA index, TNF-α, and IL-6 concentrations. Accordingly, TLR-2 receptor and its related inflammatory cytokines could be potential therapeutic targets in managing insulin resistance in RA patients.

CD14CD16 monocyte subset levels in heart failure patients.

Our aim was to define the distribution of monocyte subsets in a cohort of congestive heart failure (CHF) patients, to verify whether increased severity of CHF is linked to the expansion of specific monocyte subsets, and finally to investigate the relationship between monocyte subset relative frequencies, laboratory parameters of inflammation, and monocyte ACE expression.Thirty consecutive CHF patients and 26 healthy control subjects were evaluated for peripheral blood monocyte expression of CD14, CD16 and CD143 (ACE) by flow-cytometry, and for endothelial-derived soluble CD146 levels by ELISA. CD14++CD16+ frequency was significantly higher in CHF patients than in Controls (%, median value and IQ) (12.3, 8.7–14.8 vs 5.9, 4.7–6.9, p++CD16+ levels. Frequencies of CD14+CD16+ monocytes were significantly lower in CHF patients as compared to Controls, and negatively correlated with levels of soluble CD146 (r = −0.529; p 0.048).In conclusion, monocytic CD14++CD16+ frequency and CD143 levels are increased and reflect disease status and progressive cardiac deterioration in CHF patients. The CD14+CD16+ subset is depleted in CHF and is linked to endothelial damage in this group of patients.Although the question of whether differences in monocyte CD14CD16 expansion are causal or whether they represent a marker of HF progression which is potentially relevant for risk prediction remains unanswered, we believe that our data represent an important tool for exploring the role of selective inflammatory pathways in CHF progression.

The five transmembrane vasoactive intestinal peptide receptor 1 isoform is expressed in human blood CD14+ monocyte cells

Vasoactive intestinal peptide receptor 1 (VPAC1) is a 7 transmembrane (TM) G protein coupled receptor that couples to Gαs and increases intracellular cAMP levels in response to external stimuli. Recently, a novel VPAC1 isoform was identified in human peripheral blood mononuclear cells (PBMC) that lacks exons 10‐12 to form a 5TM receptor that lacks Gαs coupling, but elicits significant tyrosine phosphorylation when treated with exogenous ligand in ectopically overexpressed CHO cells. VPAC1 is a deactivating factor for peripheral blood monocytes by inhibiting the production of many proinflammatory cytokines, but fails to increase intracellular cAMP levels. We hypothesize dual expression of 5 and 7TM VPAC1 may act antagonistically with regard to intracellular cAMP elevation. Uncovering the expression profile of the 5TM VPAC1 splice variant is a necessary first step at understanding the biological role of this novel receptor isoform. To this end, mRNA was collected from CD14+ cells separated from human PBMC by magnetic beads. First strand cDNA was synthesized with anchored primers and the expression profile of the 7TM and 5TM assessed by RT‐PCR, sequence analysis, and Southern hybridization. Our findings suggest preferential 5TM expression in blood monocytes (CD14+). Research supported by NIH‐KO1 1K01DK064828 and COBRE 2P20RR05566.

Expression of Hsp72 protein in chronic kidney disease patients

Expression of heat shock protein (Hsp) 72 is one of the major mechanisms acting against cellular injury. It displays a plethora of functions, including a considerable impact on inflammation. The aim of this study was to investigate Hsp72 expression in blood monocytes of patients with chronic kidney disease (CKD). Hsp72 protein level was assessed by flow cytometry in blood monocytes of predialysis, haemodialysis (HD) and continuous peritoneal dialysis patients, and controls. It was followed by evaluation of Hsp72 gene expression in the same cohorts by reverse transcription-polymerase chain reaction. The level of Hsp72 protein was significantly lower in the predialysis (359+/-83 AU) and HD groups (293+/-62 AU) than in controls (405+/-51 AU) (p<0.01 and p<0.001, respectively). The amount of mRNA was significantly lower only in the HD group, compared with controls (0.39+/-0.10 vs 0.48+/-0.10, p<0.01). In the predialysis group, there were negative correlations between Hsp72 protein level and serum creatinine concentration, blood urea nitrogen and C-reactive protein. This study demonstrates that uraemic toxicity decreases expression of Hsp72. Attenuation of Hsp 72 expression in uraemia, found in the present study, could contribute to the inflammatory state, a common complication in CKD patients.

Increased Expression of Monocyte CD44v6 Correlates with the Development of Encephalitis in Rhesus Macaques Infected with Simian Immunodeficiency Virus

In people infected with human immunodeficiency virus type 1 (HIV-1), the accumulation of macrophages in the brain correlates with encephalitis and dementia. We hypothesized that a pattern of surface marker expression in blood monocytes may serve as a marker for central nervous system (CNS) disease. Using the simian immunodeficiency virus (SIV)-rhesus monkey model, we analyzed functionally relevant surface markers on monocytes and macrophages from the blood and brain in animals that did or did not develop SIV encephalitis. At necropsy, multiple markers (CD44v6, CCR2, and CCR5 on blood monocytes and brain microglia and/or macrophages, and CX3CR1 on blood monocytes) allowed us to distinguish animals with encephalitis from those without. Furthermore, the level of expression of CD44v6 on the 2 main populations of blood monocytes--those that express either low or high levels of CD16--was significantly increased in animals with encephalitis. A longitudinal analysis of blood monocyte markers revealed that as early as 28 days after inoculation, CD44v6 staining could distinguish the 2 groups. This provides a potential peripheral biomarker to identify individuals who may develop the HIV-induced CNS disease. Furthermore, given its role in cellular adhesion and as an osteopontin receptor, CD44v6 upregulation on monocytes offers functional clues to the pathogenesis of such complications, and provides a target for preventative and therapeutic measures.

B‐cell Activating Factor of the Tumour Necrosis Factor Family Expression in Blood Monocytes and T Cells from Patients with Primary Sjögren’s Syndrome

We investigated B-cell activating factor of the tumour necrosis factor family (BAFF) level in peripheral blood mononuclear cells (PBMCs), monocytes and T cells from patients with primary Sjögren's syndrome (pSS) and controls both ex vivo and in vitro after cytokine stimulation. PBMCs, monocytes and T cells were isolated from 15 patients with pSS and 17 controls. Cells were cultured alone or with interferon (IFN)alpha, IFNgamma and interleukin 10 (IL-10). T cells were stimulated with phytohaemagglutin and anti-CD3. BAFF protein was assessed by enzyme-linked immunosorbent assay. Ex vivo, no difference was observed in BAFF mRNA level in PBMCs and monocytes from patients and controls. Blood monocytes were the main cell type secreting BAFF both in patients and controls. In vitro, after IFNalpha stimulation, BAFF mRNA level was significantly higher in cells from patients than from controls (63.8 versus 20.7, P = 0.03). T cells from patients secreted a higher level of BAFF protein than those from healthy donor cells (17.4 versus 2.9 pg/ml, respectively, P = 0.04) but at a lower level than that from monocytes. Stimulation of T cells did not change BAFF secretion level. The induction of Th17 cells showed no increased BAFF expression. In conclusion, similar to epithelial cells, blood monocytes in patients with pSS show increased production of BAFF under IFNalpha, which confirms the involvement of IFNalpha in pSS. BAFF expression is also increased in blood T cells of such patients, independently of T-cell stimulation.

Oxidized low-density lipoprotein-induced expression of ABCA1 in blood monocytes precedes coronary atherosclerosis and is associated with plaque complexity in hypercholesterolemic pigs.

Elevated oxidized low-density lipoprotein (oxLDL) is associated with atherosclerosis and high cardiovascular risk. Previously, we identified 18 genes in coronary plaque macrophages of hypercholesterolemic pigs that correlated with plaque oxLDL. To determine which of these genes were differentially expressed in blood monocytes and correlated with blood and plaque oxLDL and with plaque complexity. RNA expression in monocytes of 27 hypercholesterolemic and 12 control pigs was analyzed with quantitative real-time polymerase chain reaction. Five of 12 genes with detectable expression in monocytes were overexpressed (at P < 0.01 level) in blood monocytes of hypercholesterolemic pigs: ABCA1, SCD, IRF1, SDC2, and TLR2. ABCA1 RNA expression in blood monocytes correlated with blood oxLDL, and its RNA and protein expression was increased prior to atherosclerotic plaque formation. Higher expression of ABCA1 in monocytes was associated with higher plaque complexity and higher plaque oxLDL. Immunostaining of coronary plaques showed the association of ABCA1 with macrophages, lipids, and oxLDL; ABCA1 protein correlated with plaque oxLDL (R(2) = 0.66; P < 0.0001). In THP-1 monocytes, oxLDL induced ABCA1 expression. OxLDL-induced foam cell generation in THP-1 and human monocyte-derived macrophages was associated with a further increase of ABCA1 expression. The increase of ABCA1 in monocytes in association with blood oxLDL prior to atherosclerotic lesion formation and the association of higher ABCA1 with higher plaque complexity suggests that ABCA1 is an early biomarker of atherosclerosis. Studies in humans are warranted.

Positive regulation of tumor necrosis factor-α by ganglioside GM3 through Akt in mouse melanoma B16 cells

GM3 has been shown to suppress TNFα expression in blood monocytes. However, we found that GM3 and TNFα were expressed in parallel in mouse melanoma B16 cells that were transfected with UDP-Gal:glucosylceramide β-1,4-galactosyltransferase cDNA in a sense or antisense direction or CMP-NeuAc:lactosylceramide α-2,3-sialyltransferase siRNA. TNFα expression was increased by addition of GM3 to the B16 transfectants and decreased after treatment with d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of glucosylceramide synthesis. These results clearly indicate that GM3 positively regulates TNFα expression in B16 cells. Phosphoinositide 3-kinase inhibitors, wortmannin and LY294,002, suppressed TNFα expression and Akt phosphorylation. GM3 was shown to increase phosphorylation of Akt in B16 cells and the B16-derived transfectants. Treatment of B16 cells with siRNA targeted to Akt1/2 resulted in TNFα suppression, indicating that Akt plays an important role in regulation of TNFα expression. Suppression of Akt1/2 rendered cells insensitive to GM3, suggesting that the GM3 signal may be transduced via Akt.

Reduced Expression of Toll-Like Receptor 2 on Peripheral Monocytes in Patients with Chronic Hepatitis B

Persistent infection with hepatitis B virus (HBV) likely depends on viral inhibition of host defenses. We report that chronic hepatitis B e antigen-positive HBV infection is associated with a significant reduction in peripheral blood monocyte expression of Toll-like receptor 2, a key component of innate immunity, thereby providing a mechanism by which wild-type HBV may establish persistent infection.