Gene Expression Profiles Of Monocytes Research Articles

Trametinib Sensitivity is Defined by a Myeloid Differentiation Profile in Acute Myeloid Leukemia.

Acute myelogenous leukemia (AML) is a common blood cancer marked by heterogeneity in disease and diverse genetic abnormalities. Additional therapies are needed as the 5-year survival remains below 30%. Trametinib is a mitogen-activated extracellular signal-regulated kinase (MEK) inhibitor that is widely used in solid tumors and also in tumors with activating RAS mutations. A subset of patients with AML carry activating RAS mutations; however, a small-scale clinical trial with trametinib showed little efficacy. Here, we sought to identify transcriptomic determinants of trametinib sensitivity in AML. We tested the activity of trametinib against a panel of tumor cells from patients with AML ex vivo and compared this with RNA sequencing (RNA-Seq) data from untreated blasts from the same patient samples. We then used a correlation analysis between gene expression and trametinib sensitivity to identify potential biomarkers predictive of drug response. We found that a subset of AML tumor cells were sensitive to trametinib ex vivo, only a fraction of which (3/10) carried RAS mutations. On the basis of our RNA-Seq analysis we found that markers of trametinib sensitivity are associated with a myeloid differentiation profile that includes high expression of CD14 and CLEC7A (Dectin-1), similar to the gene expression profile of monocytes. Further characterization confirmed that trametinib-sensitive samples display features of monocytic differentiation with high CD14 surface expression and were enriched for the M4 subtypes of the FAB classification. Our study identifies additional molecular markers that can be used with molecular features including RAS status to identify patients with AML that may benefit from trametinib treatment.

The Inflammation Spectrum of Monocytes in Relation to Obesity and Severity of Type 2 Diabetes (T2D), A Case-Control Study

Introduction. Increased monocyte and macrophage inflammatory state and pro-inflammatory cytokine production are linked to type 2 diabetes (T2D). Research design and Methods. This is a case-control study aimed to examine the expression of 23 monocyte genes related to inflammation, adhesion, and repair in individuals with mild (mean HbA1c 7.3%, illness duration 5.6 years) and severe type 2 diabetes (mean HbA1c 8.4%, disease duration 14.2 years) compared also with lean and obese controls. In addition, we determined a set of serum inflammatory cytokines and growth factors. Results. The monocytes of mild T2D patients (who were in general overweight/obese) showed overexpression of a subset of genes related to adhesion (CD9), vascular repair and growth (HGF). The monocytes of the severe T2D patients showed in contrast an upregulation of many of the pro-inflammatory genes, without a significantly increased expression of the repair gene HGF and the adhesion gene CD9. Serum cytokine expression in the severe T2D patients supported the increased inflammatory state of the patients showing high levels of IL-6, IL-1β, and TNF-α. Conclusions. This study, therefore, shows a pro-inflammatory gene expression profile of monocytes of severe T2D patients, while patients with mild T2D did not show such monocyte profile.

Identification of a gene network driving the attenuated response to lipopolysaccharide of monocytes from hypertensive coronary artery disease patients.

The impact of cardiovascular disease (CVD) risk factors, encompassing various biological determinants and unhealthy lifestyles, on the functional dynamics of circulating monocytes-a pivotal cell type in CVD pathophysiology remains elusive. In this study, we aimed to elucidate the influence of CVD risk factors on monocyte transcriptional responses to an infectious stimulus. We conducted a comparative analysis of monocyte gene expression profiles from the CTMM - CIRCULATING CELLS Cohort of coronary artery disease (CAD) patients, at baseline and after lipopolysaccharide (LPS) stimulation. Gene co-expression analysis was used to identify gene modules and their correlations with CVD risk factors, while pivotal transcription factors controlling the hub genes in these modules were identified by regulatory network analyses. The identified gene module was subjected to a drug repurposing screen, utilizing the LINCS L1000 database. Monocyte responsiveness to LPS showed a highly significant, negative correlation with blood pressure levels (ρ< -0.4; P<10-80). We identified a ZNF12/ZBTB43-driven gene module closely linked to diastolic blood pressure, suggesting that monocyte responses to infectious stimuli, such as LPS, are attenuated in CAD patients with elevated diastolic blood pressure. This attenuation appears associated with a dampening of the LPS-induced suppression of oxidative phosphorylation. Finally, we identified the serine-threonine inhibitor MW-STK33-97 as a drug candidate capable of reversing this aberrant LPS response. Monocyte responses to infectious stimuli may be hampered in CAD patients with high diastolic blood pressure and this attenuated inflammatory response may be reversed by the serine-threonine inhibitor MW-STK33-97. Whether the identified gene module is a mere indicator of, or causal factor in diastolic blood pressure and the associated dampened LPS responses remains to be determined.

Single-Cell RNA Sequencing Reveals Hypo-Responsiveness of T and NK Cells to Interferon Stimulation As an Immune Hallmark in Asymptomatic Waldenstrom's Macroglobulinemia

Introduction Waldenstrom macroglobulinemia (WM) is a rare, indolent non-Hodgkin lymphoplasmacytic lymphoma of the bone marrow (BM), characterized by monoclonal IgM protein production. WM is preceded by IgM monoclonal gammopathy of undetermined significance (IgM MGUS) and smoldering WM (SWM), which are clinically detectable but asymptomatic precursor phases (collectively referred to as asymptomatic WM; AWM). Recent work has revealed significant changes in the immune microenvironment of patients with overt WM, however, less is known about immune dysregulation in patients with early-stage disease. Methods We performed 5' single-cell RNA sequencing (scRNAseq) on BM immune cells from patients with AWM (n=28), healthy donors (HD, n=23), and patients with smoldering multiple myeloma (SMM) (n=27). We also performed an in vitro stimulation experiment using universal Type I interferon (IFN) and subsequent scRNAseq on bone marrow mononuclear cells (BMMCs) and peripheral blood mononuclear cells (PBMCs) from AWM (n=3) and HD (n=3). Results Overall, we annotated 210,019 immune cells, excluding normal B cells. Despite their early stage, AWM patients showed significantly altered BM immune cell composition, including increased proportions of CD56dim NK cells, CD8+ effector memory cells, and CD16 + Monocytes and decreased proportions of pro-inflammatory CD14 + monocytes and activated and interferon (IFN)-stimulated T and NK cells (q&amp;lt;0.2). Low-risk AWM presented similar changes in immune cell composition as intermediate- and high-risk AWM, implying that compositional alterations may be established early in the disease course. Notably, however, the proportion of Tregs was significantly increased in patients with SWM compared to IgM MGUS (p=0.04), suggesting that Tregs may play a role in disease progression. Next, we compared BM immune cell composition between patients with AWM and SMM. In both tumor types, patients showed significantly higher proportions of CD56dim NK cells and CD16 + monocytes compared to healthy donors. However, strikingly, a significant decrease in activated and IFN-stimulated T and NK cells was only observed in AWM, suggesting it may be a hallmark of disease. Comparison of BMMCs and PBMCs from patients with AWM following in vitro culture revealed that the lack of IFN-stimulated cells was systemic-in both BMMCs and PBMCs-although the effect was significantly more pronounced in BMMCs than PBMCs. Interestingly, while in vitro stimulation with Type I IFN resulted in the generation of IFN-stimulated T and NK cells in both HD and AWM, the levels of IFN stimulation did not entirely normalize, reproducing the gradient observed in the control samples without in vitro stimulation: AWM BM &amp;lt; AWM PB &amp;lt; HD BM. To test whether the observed defect was due to a lack of IFN in AWM, we next compared the gene expression profile of monocytes from AWM and HD, which revealed significantly higher activity of the IFN signature in monocytes from AWM compared to HD (p &amp;lt; 10 -5). This suggests that IFN levels in patients are at least not reduced. Collectively, these results indicate that T and NK cells from AWM are hypo-responsive to IFN stimulation, despite the presence of normal or possibly increased IFN in the BM microenvironment. Conclusion Our results indicate that despite the absence of symptoms, patients with AWM already have immune dysregulation in the BM, including the absence of IFN-stimulated T and NK cells, which may provide a rationale for therapeutic intervention. Previous studies have demonstrated clinical responses to therapeutic IFN administration, suggesting that novel formulations with improved therapeutic windows may have a role in treating patients with WM in the future.

Gene expression profiling of monocytes in recent-onset schizophrenia

Immune–related mechanisms have been suggested to be involved in schizophrenia. Various studies have shown changes in monocytes isolated from the blood of schizophrenia patients, including changes in monocyte numbers, as well as altered protein and transcript levels of important markers. However, validation of these findings and understanding how these results are related to immune-related changes in the brain and schizophrenia genetic risk factors, is limited. The goal of this study was to better understand changes observed in monocytes of patients with early-onset schizophrenia. Using RNA sequencing, we analyzed gene expression profiles of monocytes isolated from twenty patients with early-onset schizophrenia and seventeen healthy controls. We validated expression changes of 7 out of 29 genes that were differentially expressed in previous studies including TNFAIP3, DUSP2, and IL6. At a transcriptome-wide level, we found 99 differentially expressed genes. Effect sizes of differentially expressed genes were moderately correlated with differential expression in brain tissue (Pearson’s r = 0.49). Upregulated genes were enriched for genes in NF-κB and LPS signaling pathways. Downregulated genes were enriched for glucocorticoid response pathways. These pathways have been implicated in schizophrenia before and play a role in regulating the activation of myeloid cells. Interestingly, they are also involved in several non-inflammatory processes in the central nervous system, such as neurogenesis and neurotransmission. Future studies are needed to better understand how dysregulation of the NF-κB and glucocorticoid pathways affects inflammatory and non-inflammatory processes in schizophrenia. The fact that dysregulation of these pathways is also seen in brain tissue, provides potential possibilities for biomarker development.

G0S2 regulates innate immunity in Kawasaki disease via lncRNA HSD11B1-AS1.

BackgroundKawasaki disease (KD) is a systemic vasculitis that is currently the most common cause of acquired heart disease in children. However, its etiology remains unknown. Long non-coding RNAs (lncRNAs) contribute to the pathophysiology of various diseases. Few studies have reported the role of lncRNAs in KD inflammation; thus, we investigated the role of lncRNA in KD inflammation.MethodsA total of 50 patients with KD (median age, 19 months; 29 males and 21 females) were enrolled. We conducted cap analysis gene expression sequencing to determine differentially expressed genes in monocytes of the peripheral blood of the subjects.ResultsAbout 21 candidate lncRNA transcripts were identified. The analyses of transcriptome and gene ontology revealed that the immune system was involved in KD. Among these genes, G0/G1 switch gene 2 (G0S2) and its antisense lncRNA, HSD11B1-AS1, were upregulated during the acute phase of KD (P < 0.0001 and <0.0001, respectively). Moreover, G0S2 increased when lipopolysaccharides induced inflammation in THP-1 monocytes, and silencing of G0S2 suppressed the expression of HSD11B1-AS1 and tumor necrosis factor-α.ConclusionsThis study uncovered the crucial role of lncRNAs in innate immunity in acute KD. LncRNA may be a novel target for the diagnosis of KD.Impact This study revealed the whole aspect of the gene expression profile of monocytes of patients with Kawasaki disease (KD) using cap analysis gene expression sequencing and identified KD-specific molecules: G0/G1 switch gene 2 (G0S2) and long non-coding RNA (lncRNA) HSD11B1-AS1.We demonstrated that G0S2 and its antisense HSD11B1-AS1 were associated with inflammation of innate immunity in KD.lncRNA may be a novel key target for the diagnosis of patients with KD.

The Gene Expression Analysis of Peripheral Blood Monocytes From Psoriasis Vulgaris Patients With Different Traditional Chinese Medicine Syndromes.

Psoriasis is chronic skin disease and an important health concern. Traditional Chinese Medicine (TCM) has shown great promise in the treatment of psoriasis. However, the correlation between TCM Syndromes and genomics of psoriasis has not been evaluated. Here, we analyzed gene expression profiling of monocytes from psoriasis vulgaris patients with different TCM syndrome types to reveal the molecular basis of different psoriasis syndromes. Of the 62 cases of psoriasis vulgaris recruited, 16, 23, and 23 cases were of blood-heat syndrome, blood stasis syndrome, and blood-dryness syndrome, respectively; 10 healthy controls were recruited as controls. Affymertix’s Gene Chip ®clariom D gene chip was used to detect the gene expression profile of peripheral blood monocytes collected from recruited individuals. Compared with the healthy control group, 1570 genes were up-regulated and 977 genes were down-regulated in the psoriasis vulgaris patients group; 798 genes and 108 genes were up- and down-regulated in the blood-heat syndrome group respectively; 319 and 433 genes were up- and down-regulated in the blood-dryness syndrome group, respectively; and 502 and 179 genes were up-and down-regulated in the blood-stasis syndrome group. Our analyses indicated not only common differential genes and pathways between psoriasis syndrome groups and healthy controls, but also syndrome-specific genes and pathways. The results of this study link the three syndromes at the gene level and will be useful for clarifying the molecular basis of TCM syndromes of psoriasis. Clinical Trial Registration: (http://www.chictr.org.cn/showproj.aspx?proj=4390), identifier (ChiCTR-TRC-14005185).

Transcriptome Analysis Reveals Docosahexaenoic Acid and α-Linolenic Acid Affect Cholesterol Metabolism and Migration of Monocytes via Common and Distinct Gene Pathways

ObjectivesOne of the earliest events in atherosclerotic plaque formation is the migration of monocytes to damaged blood vessels, and the accumulation of cholesterol in monocyte-derived macrophages. The omega-3 fatty acid docosahexaenoic acid (DHA) is known to inhibit this process. While there is limited evidence suggesting α-linolenic acid (ALA) has a similar effect, ALA has not been directly compared to DHA. The primary objective of this study was to compare the gene expression profiles of monocytes that have been exposed to either ALA or DHA and examine the effect of these fatty acids on monocyte cholesterol content and migration in a cell culture model. MethodsTranscriptome analysis was performed on total mRNA isolated from human THP-1 monocytes treated with ALA, DHA or vehicle for 48 h. Candidate genes identified via fold change and Ingenuity Pathway Analysis were validated by qPCR. Functional assays to measure total cholesterol content and migration were then performed on monocytes treated with ALA or DHA. ResultsTranscriptome analysis identified a series of genes associated with cholesterol metabolism and cell migration altered by ALA and DHA treatment. Changes in mRNA levels for candidate genes were validated by qPCR, with similar expression patterns as in the transcriptome analysis. Based on these data, both fatty acids were predicted to reduce cholesterol synthesis, ALA would increase migration and DHA would have no effect. Functional assays were then performed and revealed that ALA and DHA decreased cholesterol content to a similar extent. Additionally, contrary to our predictions, DHA significantly decreased migration, while ALA had no effect. ConclusionsThe results suggest ALA and DHA may influence monocyte migration through distinct gene pathways, while cholesterol metabolism may be regulated by a common mechanism. Furthermore, only DHA treatment reduced monocyte migration in functional assays, while both fatty acids reduced cholesterol content. Due to the critical role of monocyte migration and cholesterol content in the pathophysiology of atherosclerosis, it may be concluded from this study that both DHA and ALA may exert protective effects involving different mechanisms as they relate to individuals at risk for cardiovascular disease. Funding SourcesCIHR, NSERC

Age-related differences in monocyte DNA methylation and immune function in healthy Kenyan adults and children

BackgroundAge-related changes in adaptive and innate immune cells have been associated with a decline in effective immunity and chronic, low-grade inflammation. Epigenetic, transcriptional, and functional changes in monocytes occur with aging, though most studies to date have focused on differences between young adults and the elderly in populations with European ancestry; few data exist regarding changes that occur in circulating monocytes during the first few decades of life or in African populations. We analyzed DNA methylation profiles, cytokine production, and inflammatory gene expression profiles in monocytes from young adults and children from western Kenya.ResultsWe identified several hypo- and hyper-methylated CpG sites in monocytes from Kenyan young adults vs. children that replicated findings in the current literature of differential DNA methylation in monocytes from elderly persons vs. young adults across diverse populations. Differentially methylated CpG sites were also noted in gene regions important to inflammation and innate immune responses. Monocytes from Kenyan young adults vs. children displayed increased production of IL-8, IL-10, and IL-12p70 in response to TLR4 and TLR2/1 stimulation as well as distinct inflammatory gene expression profiles.ConclusionsThese findings complement previous reports of age-related methylation changes in isolated monocytes and provide novel insights into the role of age-associated changes in innate immune functions.

Comparative Transcriptome Analysis Reveals Docosahexaenoic Acid and α-linolenic Acid Mediate Adhesion and Migration of Monocytes Through Distinct Gene PathwayD

Abstract Objectives One of the earliest events in atherosclerotic plaque formation is the adhesion and migration of monocytes to damaged blood vessels. The fish-oil derived omega-3 fatty acid docosahexaenoic acid (DHA) has been shown to inhibit this process. There is limited evidence suggesting α-linolenic acid (ALA) has a similar effect, however, ALA has not been directly compared to DHA. Therefore, the primary objective of this study was to compare the gene expression profiles of monocytes that have been exposed to either ALA or DHA and subsequently examine the effect of these fatty acids on monocyte adhesion in a cell culture model. Methods Whole transcriptome analysis was performed on total mRNA isolated from a human THP-1 monocyte cell line treated with ALA, DHA or vehicle for 48 h. Candidate genes identified via fold change and Ingenuity Pathway Analysis were validated by qPCR. An adhesion assay was subsequently performed on monocytes treated with ALA or DHA to corroborate the predicted outcomes of our analysis. Results Transcriptome analysis identified a series of genes associated with cell adhesion and migration. The change in mRNA levels for each of the candidate genes was validated by qPCR, and in all cases a similar expression pattern was obtained as observed with the gene expression analysis. Based on these data, it was predicted that these processes would be upregulated in response to ALA but not DHA. The functional assay revealed that ALA increased or had no effect on monocyte adhesion, while DHA was found to significantly decrease adhesion. Conclusions The results suggest ALA and DHA influence adhesion and migration through distinct gene pathways. Furthermore, the functional assays indicated that DHA treatment but not ALA reduces adhesion. Due to the critical role of monocyte adhesion and migration in the pathophysiology of atherosclerosis, it may be concluded from this study both DHA and ALA may exert protective effects in different ways as they relate to individuals at risk for cardiovascular disease. Funding Sources Natural Sciences and Engineering Research Council of Canada; Canadian Institutes of Health Research.

Distinct Transcriptional Signatures of Monocytes Treated with α-linolenic Acid and Docosahexaenoic Acid

Abstract Objectives Docosahexaenoic acid (DHA) can be obtained directly from the diet or produced by elongation and desaturation of α-linolenic acid (ALA). Both are proposed to reduce inflammation associated with obesity, however, fewer studies have investigated ALA. The objective of this study was to evaluate the gene expression changes in monocytes induced by each fatty acid and to compare the predicted functional outcomes. Methods RNA was extracted from THP-1 monocytes treated with ALA, DHA or vehicle for 48 h and then transcriptomics profiles were assessed by microarray. Multiple tools were used for data interpretation, including fold change analysis, Principal Component Analysis (PCA), Variable Importance Projection (VIP), Ingenuity Pathway Analysis (IPA) and Network Analyst. Results We found that the ALA and DHA treatments produced distinct profiles with many individual genes making small contributions to the separation between groups. Relative to vehicle treatment, many downregulated targets were similarly affected by both ALA and DHA. Several of these downregulated genes are involved in cholesterol synthesis and are regulated by miR-335–5p, a microRNA upregulated by both treatments. Consistently, IPA predicted similar pathways and functions are decreased by ALA and DHA, most notably cholesterol biosynthesis. In contrast, ALA and DHA upregulated unique gene sets and in agreement IPA predicted each treatment would activate distinct pathways and functions. ALA was strongly and uniquely predicted to increase infection responses while only DHA was predicted to increase oxidative phosphorylation. Finally, analysis of the protein-protein interaction network involving the genes modified by each fatty acid treatment allowed us to predict the most functionally important gene targets, which will be tested in future studies. Conclusions These analyses have revealed both unique and overlapping effects of ALA and DHA on the monocyte gene expression profile, providing further evidence that they have distinct bioactivities. Many novel predictions were made and these will form the basis for future studies investigating the effects of ALA and DHA on human physiology. Funding Sources Natural Sciences and Engineering Research Council of Canada; Canadian Institutes of Health Research.

Chitinase 3-like 1 Produced By Fibrocytes Promotes Myelofibrosis

Introduction Monocyte-derived fibrocytes are spindle-shaped fibroblast-like blood cells derived from a sub-population of CD14+ monocytes. Recent studies have shown that fibrocytes play a key role in the pathogenesis of primary myelofibrosis (MF), and these cells have attracted attention as novel therapeutic targets for MF treatment. Using a Romiplostim (Rom)-induced murine MF model, we have previously reported that fibrocyte differentiation, which is directly induced by activating the thorombopoietin receptor signaling pathway, can potentially trigger the progression of MF (Leukemia 2017; 31: 2709). However, the precise molecular mechanism of fibrocyte activity in MF have not been elucidated. To address this question, we compared the RNA sequencing-based gene expression profiles of monocytes and fibrocytes and detected that fibrocytes exhibited a higher level of Chitinase 3-like 1 (CHI3L1) expression than monocytes. CHI3L1, an 18-glycosyl hydrolase-related molecule, is a member of the enzymatically inactive chitinase-like protein family. These proteins are associated with inflammation, tissue remodeling, and organ fibrosis. Therefore, we hypothesized that CHI3L1 produced by fibrocytes is potentially involved in the progression of MF and could be a therapeutic target. In the present study, we evaluated CHI3L1 using serum samples obtained from patients with myeloproliferative neoplasms (MPN) and a Rom-induced murine MF model. Methods Monocytes and fibrocytes (Day 9 and Day 12) were prepared using peripheral blood samples from healthy volunteers. Following total RNA extraction, RNA qualification, and library preparation, a Hiseq4000 (Illumina) system was used for RNA sequencing. ELISA-based analyses were performed using the supernatant of the blood cells to confirm the presence of the CHI3L1 protein. Furthermore, we determined the serum CHI3L1 levels of MPN patients with or without MF using ELISA-based analyses. Overall, 21 patients without MF and 31 patients with MF (MF-1 to MF-3) were enrolled after obtaining written informed consent. Using the Rom-induced murine MF model, we performed in vivo analyses of Chi3l1. Female C57BL/6J mice (8-week-old) were administered with 1 mg/kg Rom once a week for 2 or 3 weeks, following which serum samples, spleens, and bone marrow samples were examined. To eliminate fibrocytes, clodronate liposomes were administered to mice. Chi3l1-deficient (Chi3l1−/−) mice were administered with Rom in the same manner. Following these treatments, RT-PCR and histopathological analyses of bone marrow were conducted. Non-contact co-culture assays of human fibrocytes and fibroblasts were performed using the human fibroblast cell line HS-5 to evaluate the interaction between CHI3L1 and fibroblasts. HS-5 cells were cultured using the supernatant of fibrocytes with or without the neutralizing antibody for CHI3L1 and mRNA expression of HS-5 cells was examined. Results Comparing the RNA sequencing gene expression profiles of monocytes and fibrocytes revealed a significant increase in CHI3L1 expression associated with fibrocyte differentiation. ELISA-based analyses of human monocyte and fibrocyte supernatants confirmed that the secretion of CHI3l1 protein concurred with the amount of mRNA expression (P &lt; 0.01) (a). We demonstrated that CHI3L1 levels were elevated in in serum samples obtained from MPN patients with MF (N = 31) compared with those obtained from patients without MF (N = 21; P = 0.027) (b). High levels of Chi3l1 mRNA expression were observed in the bone marrow of the Rom-induced murine MF model (P &lt; 0.01), which was abrogated by clodronate treatment. Furthermore, compared with wild-type mice, Chi3l1−/− mice showed fewer reticulin fibers in silver-stained bone marrow sections (c) and significantly decreased Col3A1 and Acta2 mRNA expression in the bone marrow (P &lt; 0.05). In addition, co-culture assays demonstrated that HS-5 cells cultured with the supernatant of fibrocytes showed significantly higher levels of COL1A1 and COL3A1 expression (P &lt; 0.05) than that of monocytes and the addition of CHI3L1 neutralizing antibody abrogated these increases (d). Conclusion CHI3L1 is involved in the interaction between fibrocytes and fibroblasts and this molecule exacerbates the progression of bone marrow fibrosis by promoting the production of the extracellular matrix. Therefore, CHI3L1 could be a potential therapeutic target for MF treatment. Figure Disclosures Makishima: Bristol-Myers Squibb: Research Funding. Komatsu:Wako Pure Chemical Industries, Ltd.: Research Funding; Fuso Pharmaceutical Industries, Ltd.: Research Funding; Takeda Pharmaceutical Company Limited: Research Funding, Speakers Bureau; Pharma Essentia: Research Funding, Speakers Bureau; Novartis K.K: Speakers Bureau. Kimura:JSPS KAKENHI: Research Funding.

Human monocyte transcriptional profiling identifies IL-18 receptor accessory protein and lactoferrin as novel immune targets in hypertension.

Monocytes play a critical role in hypertension. The purpose of our study was to use an unbiased approach to determine whether hypertensive individuals on conventional therapy exhibit an altered monocyte gene expression profile and to perform validation studies of selected genes to identify novel therapeutic targets for hypertension. Next generation RNA sequencing identified differentially expressed genes in a small discovery cohort of normotensive and hypertensive individuals. Several of these genes were further investigated for association with hypertension in multiple validation cohorts using qRT-PCR, regression analysis, phenome-wide association study and case-control analysis of a missense polymorphism. We identified 60 genes that were significantly differentially expressed in hypertensive monocytes, many of which are related to IL-1β. Uni- and multivariate regression analyses of the expression of these genes with mean arterial pressure (MAP) revealed four genes that significantly correlated with MAP in normotensive and/or hypertensive individuals. Of these, lactoferrin (LTF), peptidoglycan recognition protein 1 and IL-18 receptor accessory protein (IL18RAP) remained significantly elevated in peripheral monocytes of hypertensive individuals in a separate validation cohort. Interestingly, IL18RAP expression associated with MAP in a cohort of African Americans. Furthermore, homozygosity for a missense single nucleotide polymorphism in LTF that decreases antimicrobial function and increases protein levels (rs1126478) was over-represented in patients with hypertension relative to controls (odds ratio 1.16). These data demonstrate that monocytes exhibit enhanced pro-inflammatory gene expression in hypertensive individuals and identify IL18RAP and LTF as potential novel mediators of human hypertension. This article is part of a themed section on Immune Targets in Hypertension. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.12/issuetoc.

No reliable gene expression biomarkers of current or impending neurocognitive impairment in peripheral blood monocytes of persons living with HIV.

Events leading to and propagating neurocognitive impairment (NCI) in HIV-1-infected (HIV+) persons are largely mediated by peripheral blood monocytes. We previously identified expression levels of individual genes and gene networks in peripheral blood monocytes that correlated with neurocognitive functioning in HIV+ adults. Here, we expand upon those findings by examining if gene expression data at baseline is predictive of change in neurocognitive functioning 2years later. We also attempt to validate the original findings in a new sample of HIV+ patients and determine if the findings are HIV specific by including HIV-uninfected (HIV-) participants as a comparison group. At two time points, messenger RNA (mRNA) was isolated from the monocytes of 123 HIV+ and 60 HIV- adults enrolled in the Multicenter AIDS Cohort Study and analyzed with the Illumina HT-12 v4 Expression BeadChip. All participants received baseline and follow-up neurocognitive testing 2years after mRNA analysis. Data were analyzed using standard gene expression analysis and weighted gene co-expression network analysis with correction for multiple testing. Gene sets were analyzed for GO term enrichment. Only weak reproducibility of associations of single genes with neurocognitive functioning was observed, indicating that such measures are unreliable as biomarkers for HIV-related NCI; however, gene networks were generally preserved between time points and largely reproducible, suggesting that these may be more reliable. Several gene networks associated with variables related to HIV infection were found (e.g., MHC I antigen processing, TNF signaling, interferon gamma signaling, and antiviral defense); however, no significant associations were found for neurocognitive function. Furthermore, neither individual gene probes nor gene networks predicted later neurocognitive change. This study did not validate our previous findings and does not support the use of monocyte gene expression profiles as a biomarker for current or future HIV-associated neurocognitive impairment.

Monocyte gene expression in childhood obesity is associated with obesity and complexity of atherosclerosis in adults

Childhood obesity coincides with increased numbers of circulating classical CD14++CD16- and intermediate CD14++CD16+ monocytes. Monocytes are key players in the development and exacerbation of atherosclerosis, which prompts the question as to whether the monocytosis in childhood obesity contributes to atherogenesis over the years. Here, we dissected the monocyte gene expression profile in childhood obesity using an Illumina microarray platform on sorted monocytes of 35 obese children and 16 lean controls. Obese children displayed a distinctive monocyte gene expression profile compared to lean controls. Upon validation with quantitative PCR, we studied the association of the top 5 differentially regulated monocyte genes in childhood obesity with obesity and complexity of coronary atherosclerosis (SYNTAX score) in a cohort of 351 adults at risk for ischemic cardiovascular disease. The downregulation of monocyte IMPDH2 and TMEM134 in childhood obesity was also observed in obese adults. Moreover, downregulation of monocyte TMEM134 was associated with a higher SYNTAX atherosclerosis score in adults. In conclusion, childhood obesity entails monocyte gene expression alterations associated with obesity and enhanced complexity of coronary atherosclerosis in adults.

CD14+CD16+ monocytes are the main target of Zika virus infection in peripheral blood mononuclear cells in a paediatric study in Nicaragua.

The recent Zika pandemic in the Americas is linked to congenital birth defects and Guillain-Barré syndrome. White blood cells (WBCs) play an important role in host immune responses early in arboviral infection. Infected WBCs can also function as 'Trojan horses' and carry viruses into immune-sheltered spaces, including the placenta, testes and brain. Therefore, defining which WBCs are permissive to Zika virus (ZIKV) is critical. Here, we analyse ZIKV infectivity of peripheral blood mononuclear cells (PBMCs) in vitro and from Nicaraguan Zika patients and show CD14+CD16+ monocytes are the main target of infection, with ZIKV replication detected in some dendritic cells. The frequency of CD14+ monocytes was significantly decreased, while the CD14+CD16+ monocyte population was significantly expanded during ZIKV infection compared to uninfected controls. Viral RNA was detected in PBMCs from all patients, but in serum from only a subset, suggesting PBMCs may be a reservoir for ZIKV. In Zika patients, the frequency of infected cells was lower but the percentage of infected CD14+CD16+ monocytes was significantly higher compared to dengue cases. The gene expression profile in monocytes isolated from ZIKV- and dengue virus-infected patients was comparable, except for significant differences in interferon-γ, CXCL12, XCL1, interleukin-6 and interleukin-10 levels. Thus, our study provides a detailed picture of the innate immune profile of ZIKV infection and highlights the important role of monocytes, and CD14+CD16+ monocytes in particular.

The NF-kB regulates the SHP-1 expression in monocytes in congestive heart failure.

It has been shown that functional recovery of patients with acute congestive heart failure (ACHF) after treatment with conventional drugs (CD) is mediated by suppression of inflammation in peripheral blood mononuclear cells. Here, we analyzed gene expression profiles of monocytes from symptomatic ACHF patients (NYHA Class III-IV) before and after pharmacological treatment with CD. The treatment was associated with selective down-regulation of "TNFR signaling" and pro-inflammatory mediators CCL5, MIP-1α receptor, CD14, ITGAM, and significant up-regulation of "TNFR signaling" as evidenced by increase in anti-inflammatory factors including NF-kBIA, TNFAIP3 and SHP-1. In monocyte TNF-alpha-stimulated there is a down-regulation of the phosphatase SHP-1 which induces a significant activation of TAK-1/IKK/NF-kB signaling. These findings suggest that the therapeutic impact of CD treatment in symptomatic ACHF includes negative regulation of the NF-kB signaling in monocytes and the improvement of the SHP-1 activity.

Deficiencies of the T and natural killer cell system in major depressive disorder: T regulatory cell defects are associated with inflammatory monocyte activation

BackgroundIn a previous study, we found an up-regulated inflammatory monocyte gene expression profile in major depressive disorder (MDD) patients aged⩾28years and a down-regulated inflammatory gene expression profile in MDD patients aged<28years. In the same sample of patients, we aimed to investigate immune dysregulation in the lymphocyte arm of the immune system, particularly in the context of the described monocyte (de-)activation states. MethodsFrom deep frozen leukocytes, circulating percentages of monocytes, lymphocytes, B, T, and natural killer (NK) cells, and various functional subsets of T and T helper (Th) cells (Th1, Th2, Th17, and natural T regulatory cells) were measured in N=50 MDD patients and N=58 age- and gender-matched healthy controls (HC). In addition, serum levels of interleukin (IL)-6, sCD25, IL-7, IL-3, SCF, IGF-BP2, and EGF were evaluated. ResultsMDD patients were in general characterized by an impaired maturation of Th2 cells, Th17 cells, and NK cells and by decreased serum levels of IL-7 and sCD25. MDD patients aged⩾28years additionally exhibited decreased percentages of CD4+CD25highFoxP3+ T regulatory cells, next to signs of the above described partial T cell defects. Natural T regulatory cells were inversely associated with the pro-inflammatory state of the monocytes (r=−.311; p=.034) that characterized this patient subgroup. ConclusionsDeficiencies of the NK and T (regulatory) cell system and inflammatory monocyte immune activation co-occur as partly interrelated phenomena within the same MDD patients.

Abstract 153: High neuropilin-1 expression on monocytes is positively associated with trastuzumab-mediated antibody-dependent cellular cytotoxicity of the HER2-overexpressing breast cancer cell line

Abstract Purpose Neuropilin-1 (Nrp1) was initially characterized as a guide for migrating cells and axons in developing nervous systems and is essential for the precise formation of neurons and vasculature. However, it was recently reported to also play an important role in the immune system. This study aimed to investigate the role of Nrp1 on monocytes in trastuzumab-mediated antibody-dependent cellular cytotoxicity (ADCC) targeting the HER2-positive human breast cancer cell line SKBR3. In addition, we evaluated the gene expression profile of monocytes expressing high and low levels of Nrp1. Methods Peripheral blood mononuclear cells from healthy volunteers were prepared using BD Vacutainer CPT Cell Preparation Tubes. The cells were stained with fluorescein isothiocyanate-conjugated anti-CD14 and allophycocyanin-conjugated anti-Nrp1 antibodies, and Nrp1high and Nrp1low monocyte subsets were sorted using FACSAria. These cells were used as effector cells in a conventional ADCC assay with SKBR3 as target cells and trastuzumab as the antibody. Target cell cytotoxicity was measured using the LDH cytotoxic test kit. The effector cell to target cell ratios of 20:1 and 10:1 were used. Gene expression analysis was performed by Affymetrix GeneChip microarrays. All study protocols were approved by the Ethics Committee for Clinical Research, Kyoto University Hospital, Kyoto, Japan (authorization number G424). Results The LDH cytotoxic test revealed significantly higher target cancer cell cytotoxicity in sorted Nrp1high monocytes than in Nrp1low monocytes (75% vs. 45%, p&amp;lt;0.01). Trastuzumab-mediated ADCC was significantly suppressed by Nrp1 neutralization antibodies in a dose-dependent manner (Control arm 14.3%, Treatment arm 6%; p&amp;lt;0.01). Gene expression analysis revealed that 308 genes were up-regulated in the Nrp1high population, including monocyte-specific chemokine receptor and hematopoietic transcriptional factor. Conclusion These results suggest that determination of Nrp1 expression can be used to classify monocytes as populations with and without cellular cytotoxic activity. Analysis of Nrp1 expression levels in monocytes can therefore be used to identify HER2-positive breast cancer patients for whom trastuzumab therapy will be less effective. Citation Format: Kosuke Kawaguchi, Eiji Suzuki, Masao Kawashima, Masakazu Toi. High neuropilin-1 expression on monocytes is positively associated with trastuzumab-mediated antibody-dependent cellular cytotoxicity of the HER2-overexpressing breast cancer cell line. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 153. doi:10.1158/1538-7445.AM2014-153

Thymosin α1: a novel therapeutic option for patients with refractory chronic purulent rhinosinusitis

Background Chronic purulent rhinosinusitis (CPR) is an inflammatory disorder of the nose and paranasal sinuses of unknown cause. Despite various available medical and surgical treatment options still 5 to 10% of patients remain refractory. Immune deficiency is one of the underlying risk factors for CPR and previous studies demonstrated defects in monocyte chemotaxis. Subsequent treatment with the thymic hormone preparation thymostimulin led to in vitro restoration of monocyte chemotaxis and significant clinical improvement in patients. However, thymostimulin became unavailable in recent years. In the present study we evaluated the effects of the thymic peptide thymosin a1 on monocytes from CPR patients as well as aberrant gene expression profiles in these monocytes in order to further elucidate the pathogenesis of CPR. Method Monocytes were isolated from 16 patients with CPR and 13 healthy volunteers. Monocyte polarization was assessed using the Cianciolo and Snyderman monocyte polarization assay and the effects of thymosin a 1o n monocyte polarization were evaluated. Furthermore, by Affymetrix whole-genome gene expression profiling and Q-PCR analysis we analyzed aberrant gene expression profiles in monocytes from CPR patients. Results In 4 out of 16 CPR patients we found diminished monocyte polarization (45%, sd 8%) when compared to healthy volunteers (58%, sd 12,5%)(p=0.078). More interestingly, in vitro treatment with thymosin a1 significantly restored monocyte polarization in these patients (64% sd 10%, p=0.029). In the “poor polarizing” monocytes we found aberrant expression of genes involved in pathways of inflammation, chemotaxis and cell migration. Conclusion Patients with CPR show diminished monocyte polarization in vitro that could be restored by thymosin a1. Moreover, these monocytes show an aberrant gene expression profile. We hypothesize that thymosin a 1m ay be ap romising agent in treatment of refractory CPR patients. These effects may be mediated through interference with pathways involving the aberrantly expressed genes.