Signature Of Monocytes Research Articles

Critical role of macrophages in checkpoint blockade induced autoimmune diabetes in the NOD mouse

Abstract We confirmed that the PD-1/PD-L1 regulatory pathway is one control mechanism of autoimmune diabetes in NOD mice: administration of anti-PD-1 but not anti-CTLA-4 antibodies rapidly induced diabetes in young mice showing a control phase established very early in the autoimmune process. We confirmed that both CD4 and CD8 T cells and resident macrophages were essential for the PD-1 regulation to take effect. We performed an unbiased single-cell RNA sequencing analysis of islets after anti-PD-1 treatment. We identified a subpopulation of highly activated macrophage showing an IFNγ signature with high level of CXCL9 and PD-L1 and a monocyte signature distinguishable by the markers Ly6c and CCR2. Flow cytometry analysis of islets upon anti-PD-1 treatment identified a predominance of the monocyte derived macrophages compared to the islet resident macrophages. Depleting blood monocytes by clodronate liposomes inhibited the recruitment of the monocyte derived macrophages and prevented the development of acute diabetes induced by anti-PD-1 treatment. To analyze the pathogenicity of this monocyte-derived macrophages, we performed cytotoxicity assays by co-culturing macrophages stimulated with LPS&IFNγ and a β-cell line Min6: the highly activated macrophage killed β-cells in a manner dependent on IFNγ and inducible nitric oxide. In sum, our study shows highly activated macrophage derived from monocytes are cytotoxic for beta cells and promoting diabetes progression. Our findings also indicate that a consequence of checkpoint blockade is the recruitment and activation of cytocidal macrophages derived from monocytes. Targeting monocyte-derived macrophages may prevent some of the checkpoint blockade related immune-adverse effects.

IFN-α Suppresses Myeloid Cytokine Production, Impairing IL-12 Production and the Ability to Support T-Cell Proliferation.

Interferon-α (IFN-α) can suppress production of T-cell polarizing cytokines or induce inhibitory antigen-presenting cells that suppress T-cell activation. Previous studies showed that IFN-α therapy fails to boost virus-specific T-cell immunity in patients with chronic hepatitis B virus infection. Our aim was to determine whether IFN-α exposure alters human antigen-presenting cell function in vivo. We investigated the immunomodulatory effects using peripheral blood mononuclear cells from healthy donors exposed to IFN-α and chronic hepatitis B (CHB) patients starting IFN-α therapy. IFN-α increased HLA-DR, CD80, CD86, and PD-L1 expression on healthy donor monocytes. In contrast to the activated phenotype, IFN-α inhibited Toll-like receptor-induced cytokine production and monocyte-induced T-cell proliferation. In CHB patients, peg-IFN treatment induced an interferon-stimulated gene signature in monocytes and increased HLA-DR, CD80, CD86, and PD-L1 expression. As early as 3 days after CHB patients started treatment, IFN-α inhibited monocyte cytokine production and T-cell stimulation ex vivo. IFN-α-mediated inhibition of IL-12 production, rather than inhibitory receptor expression, was responsible for inhibition of T-cell proliferation. Addition of IL-12 restored T-cell proliferation to baseline levels. Understanding how professional antigen-presenting cells respond to immunomodulation is important for both new innate and adaptive-targeted immunotherapies. NCT00962871.

Human Immunodeficiency Virus (HIV) Infection and Use of Illicit Substances Promote Secretion of Semen Exosomes that Enhance Monocyte Adhesion and Induce Actin Reorganization and Chemotactic Migration.

Semen exosomes (SE) from HIV-uninfected (HIV−) individuals potently inhibit HIV infection in vitro. However, morphological changes in target cells in response to SE have not been characterized or have the effect of HIV infection or the use of illicit substances, specifically psychostimulants, on the function of SE been elucidated. The objective of this study was to evaluate the effect of HIV infection, psychostimulant use, and both together on SE-mediated regulation of monocyte function. SE were isolated from semen of HIV− and HIV-infected (HIV+) antiretroviral therapy (ART)-naive participants who reported either using or not using psychostimulants. The SE samples were thus designated as HIV−Drug−, HIV−Drug+, HIV+Drug−, and HIV+Drug+. U937 monocytes were treated with different SEs and analyzed for changes in transcriptome, morphometrics, actin reorganization, adhesion, and chemotaxis. HIV infection and/or use of psychostimulants had minimal effects on the physical characteristics of SE. However, different SEs had diverse effects on the messenger RNA signature of monocytes and rapidly induced monocyte adhesion and spreading. SE from HIV infected or psychostimulants users but not HIV−Drug− SE, stimulated actin reorganization, leading to the formation of filopodia-like structures and membrane ruffles containing F-actin and vinculin that in some cases were colocalized. All SE stimulated monocyte chemotaxis to HIV secretome and activated the secretion of matrix metalloproteinases, a phenotype exacerbated by HIV infection and psychostimulant use. SE-directed regulation of cellular morphometrics and chemotaxis depended on the donor clinical status because HIV infection and psychostimulant use altered SE function. Although our inclusion criteria specified the use of cocaine, humans are poly-drug and alcohol users and our study participants used psychostimulants, marijuana, opiates, and alcohol. Thus, it is possible that the effects observed in this study may be due to one of these other substances or due to an interaction between different substances.

Different monocyte phenotypes result in proresolving macrophages in conjugated linoleic acid-induced attenuated progression and regression of atherosclerosis.

Monocytes/macrophages drive progression and regression of atherosclerosis. Conjugated linoleic acid (CLA), an anti-inflammatory lipid, mediates atheroprotective effects. We investigated how CLA alters monocyte/macrophage phenotype during attenuated progression and regression of atherosclerosis. Apolipoprotein E knockout (ApoE-/-) mice were fed a high-fat (60%) high-cholesterol (1%) diet (HFHCD) for 2 wk, followed by 6-wk 1% CLA 80:20 supplementation to investigate disease progression. Simultaneously, ApoE-/- mice were fed a 12-wk HFHCD with/without CLA for the final 4 wk to investigate regression. Aortic lesions were quantified by en face staining. Proteomic analysis, real-time quantitative PCR and flow cytometry were used to interrogate monocyte/macrophage phenotypes. CLA supplementation inhibited atherosclerosis progression coincident with decreased proinflammatory and increased anti-inflammatory macrophages. However, CLA-induced regression was associated with increased proinflammatory monocytes resulting in increased proresolving M2 bone marrow-derived macrophages, splenic macrophages, and dendritic cells in lesion-draining lymph nodes. Proteomic analysis confirmed regulation of a proinflammatory bone marrow response, which was abolished upon macrophage differentiation. Thus, in attenuation and regression of atherosclerosis, regardless of the monocyte signature, during monocyte to macrophage differentiation, proresolving macrophages prevail, mediating vascular repair. This study provides novel mechanistic insight into the monocyte/macrophage phenotypes in halted atherosclerosis progression and regression of atherosclerosis.-Bruen, R., Curley, S., Kajani, S., Lynch, G., O'Reilly, M. E., Dillon, E. T., Fitzsimons, S., Mthunzi, L., McGillicuddy, F. C., Belton, O. Different monocyte phenotypes result in proresolving macrophages in conjugated linoleic acid-induced attenuated progression and regression of atherosclerosis.

Gene expression analysis delineates the potential roles of multiple interferons in systemic lupus erythematosus

A role for interferon (IFN) in systemic lupus erythematosus (SLE) pathogenesis is inferred from the prominent IFN gene signature (IGS), but the major IFN species and its relationship to disease activity are unknown. A bioinformatic approach employing individual IFN species gene signatures to interrogate SLE microarray datasets demonstrates a putative role for numerous IFN species, with prominent expression of IFNB1 and IFNW signatures. In contrast with other SLE-affected organs, the IGS is less prominent in lupus nephritis. SLE patients with active and inactive disease have readily detectable IGS and the IGS changes synchronously with a monocyte signature but not disease activity, and is significantly related to monocyte transcripts. Monocyte over-expression of three times as many IGS transcripts as T and B cells and IGS retention in monocytes, but not T and B cells from inactive SLE patients contribute to the lack of correlation between the IGS and SLE disease activity.

Transcriptomic signatures of monocytes show high similarity between patients with osteoarthritis and rheumatoid arthritis

Transcriptomic signatures of monocytes show high similarity between patients with osteoarthritis and rheumatoid arthritis

Abstract # 3200 IL-6 production during repeated social defeat stress primes monocytes to induce anxiety-like behavior

Interleukin (IL)-6 is elevated in circulation with chronic stress and may contribute to the neurobehavioral complications. We have reported that repeated social defeat (RSD) stress in mice caused recruitment of proinflammatory monocytes to the brain and triggered the onset of anxiety-like behavior. The purpose of this study was to determine the role of IL-6 signaling in the peripheral immune response, neuroinflammation, and anxiety following stress. WT and IL-6KO mice were subjected to RSD. Although monocyte recruitment to the brain following stress was maintained in the IL-6KO mice, anxiety and social avoidance were completely prevented. Nanostring profiling of sorted monocytes from blood (CD11b + Ly6Chi) and brain (CD11b + CD45hi) revealed a unique pattern of immune-related gene expression that was dependent on stress and IL-6. For instance, blood monocytes had a transcriptional signature consistent with priming and glucocorticoid resistance, which was absent in the monocytes from IL-6KO mice after stress. Brain monocytes from WT mice had increased levels of Il-1b, Mmp9, Stat3, Myd88 and Cd14 following stress. This phenotype was absent in brain monocytes from IL-6KO mice. Here, we show the effects of IL-6 on the transcriptional signature of monocytes in circulation and in the brain after stress. Overall, robust increases in circulating IL-6 after stress induced a primed profile in the monocytes, which were recruited to the brain and functioned to augment IL-1-mediated inflammation and anxiety.

Interleukin-6 Induced by Social Stress Promotes a Unique Transcriptional Signature in the Monocytes That Facilitate Anxiety

BackgroundInterleukin-6 (IL-6) is elevated in circulation with chronic stress and may contribute to neurobehavioral complications. We have reported that repeated social defeat stress in mice caused recruitment of proinflammatory monocytes to the brain and triggered the onset of anxiety-like behavior. Therefore, the purpose of this study was to determine the role of IL-6 signaling in the peripheral immune response, neuroinflammation, and anxiety following stress. MethodsWild-type and IL-6 knockout mice were subjected to repeated social defeat, and immune and behavioral parameters were determined 14 hours later. ResultsAlthough monocyte release and recruitment to the brain during stress were maintained in the IL-6 knockout mice, anxiety and social avoidance were prevented. NanoString analysis of fluorescence-activated cell–sorted blood monocytes (CD11b+/Ly6Chi) and brain monocytes (CD11b+/CD45hi) revealed a unique pattern of immune-related gene expression that was dependent on stress and IL-6. For instance, blood monocytes after stress had a transcriptional signature and immune profile consistent with priming, which was attenuated in monocytes from IL-6 knockout stress mice. Moreover, the monocytes recruited to the brain and associated with the development of anxiety had a transcriptional signature (enhanced IL-1β, CD14, Mmp9, Myd88, Ager, and Stat3) that was dependent on IL-6. ConclusionsHere, we show the effects of IL-6 on the transcriptional signature of monocytes in circulation and brain after stress. Overall, robust increases in IL-6 after stress induced a primed profile in monocytes that were recruited to the brain and propagated IL-1-mediated inflammation and anxiety.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Transcriptional profiles of JIA patient blood with subsequent poor response to methotrexate.

The mechanisms that determine the efficacy or inefficacy of MTX in JIA are ill-defined. The objective of this study was to identify a gene expression transcriptional signature associated with poor response to MTX in patients with JIA. RNA sequencing was used to measure gene expression in peripheral blood mononuclear cells collected from 47 patients with JIA prior to MTX treatment and 14 age-matched controls. Differentially expressed baseline genes between responders and non-responders were evaluated. Biological differences between all JIA patients and controls were explored by constructing a signature of differentially expressed genes. Unsupervised clustering and pathway analysis was performed. A signature of 99 differentially expressed genes (Bonferroni-corrected P < 0.05) capturing the biological differences between all JIA patients and controls was identified. Unsupervised clustering of samples based on this list of 99 genes produced subgroups enriched for MTX response status. Comparing this gene signature with reference signatures from sorted cell populations revealed high concordance between the expression signatures of monocytes and of MTX non-responders. CXCL8 (IL-8) was the most significantly differentially expressed gene transcript comparing all JIA patients with controls (Bonferroni-corrected P = 4.12 × 10-10). Variability in clinical response to MTX in JIA patients is associated with differences in gene transcripts modulated in monocytes. These gene expression profiles may provide a basis for biomarkers predictive of treatment response.

RNA-sequencing Identifies Novel Pathways in Sarcoidosis Monocytes

Sarcoidosis is a complex systemic granulomatous disorder of unknown etiology. Genome-wide association studies have not been able to explain a causative role for nucleotide variation in its pathogenesis. The goal of the present study was to identify the gene expression profile and the cellular pathways altered in sarcoidosis monocytes via RNA-sequencing. Peripheral blood monocytes play a role in sarcoidosis inflammation. Therefore, we determined and compared the transcriptional signature of monocytes from peripheral blood from sarcoidosis patients and healthy controls via RNA-sequencing. We found 2,446 differentially expressed (DE) genes between sarcoidosis and healthy control monocytes. Analysis of these DE genes showed enrichment for ribosome, phagocytosis, lysosome, proteasome, oxidative phosphorylation and metabolic pathways. RNA-sequencing identified upregulation of genes involved in phagocytosis and lysosomal pathway in sarcoidosis monocytes, whereas genes involved in proteasome degradation and ribosomal pathways were downregulated. Further studies are needed to investigate the role of specific genes involved in the identified pathways and their possible interaction leading to sarcoidosis pathology.

Gene expression profiles are different in venous and capillary blood: Implications for vaccine studies

BackgroundDetailed analysis of the immunological pathways leading to robust vaccine responses has become possible with the application of systems biology, including transcriptomic analysis. Venous blood is usually obtained for such studies but others have obtained capillary blood (e.g. finger-prick). Capillary samples are practically advantageous, especially in children. MethodsThe aim of this study was to compare gene expression profiles in venous and capillary blood before, 12h and 24h after vaccination with 23-valent pneumococcal polysaccharide or trivalent inactivated seasonal influenza vaccines. ResultsGene expression at baseline was markedly different between venous and capillary samples, with 4940 genes differentially expressed, and followed a different pattern of changes after vaccination. At baseline, multiple pathways were upregulated in venous compared to capillary blood, including transforming growth factor-beta receptor signalling and toll-like receptor cascades. After vaccination with the influenza vaccine, there was enrichment for T and NK cell related signatures in capillary blood, and monocyte signatures in venous blood. By contrast, after vaccination with the pneumococcal vaccination, there was enrichment of dendritic cells, monocytes and interferon related signatures in capillary blood, whilst at 24h there was enrichment for T and NK cell related signatures in venous blood. ConclusionsThese data show differences between venous and capillary gene expression both at baseline, and post vaccination, which may impact on the conclusions regarding immunological mechanisms drawn from studies using these different sampling methodologies.

Epigenetic changes in T-cell and monocyte signatures and production of neurotoxic cytokines in ALS patients.

We have investigated transcriptional and epigenetic differences in peripheral blood mononuclear cells (PBMCs) of monozygotic female twins discordant in the diagnosis of amyotrophic lateral sclerosis (ALS). Exploring DNA methylation differences by reduced representation bisulfite sequencing (RRBS), we determined that, over time, the ALS twin developed higher abundances of the CD14 macrophages and lower abundances of T cells compared to the non-ALS twin. Higher macrophage signature in the ALS twin was also shown by RNA sequencing (RNA-seq). Moreover, the twins differed in the methylome at loci near several genes, including EGFR and TNFRSF11A, and in the pathways related to the tretinoin and H3K27me3 markers. We also tested cytokine production by PBMCs. The ALS twin's PBMCs spontaneously produced IL-6 and TNF-α, whereas PBMCs of the healthy twin produced these cytokines only when stimulated by superoxide dismutase (SOD)-1. These results and flow cytometric detection of CD45 and CD127 suggest the presence of memory T cells in both twins, but effector T cells only in the ALS twin. The ALS twin's PBMC supernatants, but not the healthy twin's, were toxic to rat cortical neurons, and this toxicity was strongly inhibited by an IL-6 receptor antibody (tocilizumab) and less well by TNF-α and IL-1β antibodies. The putative neurotoxicity of IL-6 and TNF-α is in agreement with a high expression of these cytokines on infiltrating macrophages in the ALS spinal cord. We hypothesize that higher macrophage abundance and increased neurotoxic cytokines have a fundamental role in the phenotype and treatment of certain individuals with ALS.-Lam, L., Chin, L., Halder, R. C., Sagong, B., Famenini, S., Sayre, J., Montoya, D., Rubbi L., Pellegrini, M., Fiala, M. Epigenetic changes in T-cell and monocyte signatures and production of neurotoxic cytokines in ALS patients.

Association between cetuximab response and differential immunologic gene expression signatures in colorectal cancer metastases.

558 Background: Different immune cell infiltrates into colorectal cancer (CRC) tumors are associated with different prognoses. Tumor-associated macrophages contribute to immune evasion and accelerated tumor progression. Conversely, tumor infiltrating lymphocytes at the invasive margin of CRC liver metastases are associated with improved outcomes with chemotherapy. Cetuximab is an IgG1 monoclonal antibody against epidermal growth factor receptor (EGFR) and stimulates antibody-dependent cellular cytotoxicity (ADCC) in vitro. However, it is unclear in humans if response to cetuximab is modulated by the immune response. We hypothesized that different immune patterns detected in gene expression profiles of CRC metastases are associated with different responses to cetuximab. Methods: We retrieved gene expression data from biopsies of metastases from 80 refractory CRC patients treated with cetuximab monotherapy (GEO GSE5851). Samples were dichotomized by cetuximab response as having either disease control (DC) or progressive disease (PD). We performed gene set enrichment analysis (GSEA) with GenePattern 3.9.4 using gene sets of immunologic signatures obtained from the Molecular Signatures Database v5.0. Results: Among the 68 patients with response annotated, 25 had DC and 43 had PD. In the PD cohort, 59/1910 immunologic gene sets had false discovery rate (FDR) &lt; 0.1. Notably, multiple gene sets upregulated in monocyte signatures were associated with PD. Also, gene sets consistent with PD1-ligated T cells compared to control activated T cells (FDR = 0.052) or IL4-treated CD4 T cells compared to controls (FDR = 0.087) were associated with PD. Conclusions: Cetuximab-resistant patients tended to have baseline increased expression of gene signatures reflective of monocytic infiltrates, consistent with also having increased expression of the IL4-treated T-cell signature. Cetuximab resistance was also associated with increased expression of the PD1-ligated T cell signature. These preliminary findings support further evaluation of the effect of differential immune infiltrates in prognosis of metastatic CRC treated with cetuximab.

The interferon type I signature is present in systemic sclerosis before overt fibrosis and might contribute to its pathogenesis through high BAFF gene expression and high collagen synthesis

BackgroundInterferon (IFN) signature has been reported in definite systemic sclerosis (SSc) but it has not been characterised in early SSc (EaSSc). We aim at characterising IFN type I signature in...

Tumour-educated circulating monocytes are powerful candidate biomarkers for diagnosis and disease follow-up of colorectal cancer

ObjectiveCancer immunology is a growing field of research whose aim is to develop innovative therapies and diagnostic tests. Starting from the hypothesis that immune cells promptly respond to harmful stimuli,...

Role of monocyte chemoattractant protein-1 (MCP-1) in atherosclerosis: Signature of monocytes and macrophages

The monocyte chemoattractant protein-1 (MCP-1/CCL2) is a member of the C-C chemokine family, and a potent chemotactic factor for monocytes. MCP-1 is believed to be identical to JE, a gene whose expression is induced in mouse fibroblasts by platelet-derived growth factor. Two SNPs of CCL2, namely, G-927C and A-2578G, were found to be associated with carotid intima-media thickness, which reflects generalized atherosclerosis and is predictive of future vascular events. Monocyte chemoattractant protein-1 (MCP-1) is the first discovered and most extensively studied CC chemokine, and the amount of studies on its role in the etiologies of atherosclerosis-related diseases have increased exponentially during recent years. This review attempted to provide a perspective of the history, regulatory mechanisms, functions, and therapeutic strategies of this chemokine. The highlights of this review include the roles of MCP-1 in the development of atherosclerosis, cardiovascular diseases, and dyslipidemia. Therapies that specifically or non-specifically inhibit MCP-1 overproduction have been summarized.

Baseline Gene Expression Signatures in Monocytes from Multiple Sclerosis Patients Treated with Interferon-beta

BackgroundA relatively large proportion of relapsing-remitting multiple sclerosis (RRMS) patients do not respond to interferon-beta (IFNb) treatment. In previous studies with peripheral blood mononuclear cells (PBMC), we identified a subgroup of IFNb non-responders that was characterized by a baseline over-expression of type I IFN inducible genes. Additional mechanistic experiments carried out in IFNb non-responders suggested a selective alteration of the type I IFN signaling pathway in the population of blood monocytes. Here, we aimed (i) to investigate whether the type I IFN signaling pathway is up-regulated in isolated monocytes from IFNb non-responders at baseline; and (ii) to search for additional biological pathways in this cell population that may be implicated in the response to IFNb treatment.MethodsTwenty RRMS patients classified according to their clinical response to IFNb treatment and 10 healthy controls were included in the study. Monocytes were purified from PBMC obtained before treatment by cell sorting and the gene expression profiling was determined with oligonucleotide microarrays.Results and discussionPurified monocytes from IFNb non-responders were characterized by an over-expression of type I IFN responsive genes, which confirms the type I IFN signature in monocytes suggested from previous studies. Other relevant signaling pathways that were up-regulated in IFNb non-responders were related with the mitochondrial function and processes such as protein synthesis and antigen presentation, and together with the type I IFN signaling pathway, may also be playing roles in the response to IFNb.

Origin and function of the repair macrophage in the murine sterile wound

Experiments were performed to define the phenotype and origin of repair macrophages (mφ). Wound cells were isolated from 1 to 14 days following subcutaneous insertion of sterile polyvinyl alcohol sponges in mice and examined for monocyte/mφ markers by flow cytometry. F4/80+Ly6Chi cells with an inflammatory monocyte signature appeared early, followed by accumulation of F4/80+Ly6Clow/int repair‐like mφ. Ly6Chi wound monocytes expressed CD64 but not the mφ marker Mer tyrosine kinase (MerTK), similar to blood monocyte precursors. In contrast to blood monocytes, wound monocytes upregulated CD14 and had a proinflammatory cytokine production profile. F4/80+Ly6Clow/int mφ were CD64+MerTK+ and produced cytokines associated with repair including VEGF and TGF‐β. Examination of CX3CR1‐GFP mice and selective labeling of circulating monocyte subsets demonstrated that Ly6Chi monocytes preferentially migrated from the blood to the wound. Analysis of cells from adoptively transferred sponges suggested that Ly6Chi monocytes mature within the wound to become Ly6Clowmφ. Overall, these studies functionally and phenotypically distinguished wound monocyte/mφ subsets and identified MerTK as a potential marker for repair mφ during sterile inflammation. Research supported by R01 GM042859, K08 GM079227 and the RIH Department of Surgery.

Down-regulation of inflammation-protective microRNAs 146a and 212 in monocytes of patients with postpartum psychosis

Postpartum psychosis (PP) is thought to belong to the bipolar spectrum. Recently we described an immune activation signature in monocytes of patients with PP using gene expression profiling. Immune activation genes are regulated by microRNAs (miRNAs). We therefore profiled miRNA expression in monocytes of PP patients to identify differentially expressed miRNAs between PP and the healthy state. In a profiling study we carried out miRNA profiling using TaqMan array human microRNA A cards v2.0 and monocytes of 8 PP patients. Data were analyzed against monocytes of healthy postpartum women (CP). Nine miRNAs were selected and tested using individual Q-PCR in a larger validation study on monocytes of 20 PP patients, 20 CP and 20 healthy non-postpartum women (HC). In the validation study miR-146a expression was significantly down-regulated in the monocytes of first onset PP patients as compared to CP and HC; miR-212 expression was significantly down-regulated in PP patients with prior bipolar disorder. In silico miR-146a targeted 4 genes of the previously described monocyte activation signature in bipolar disorder; miR-212 targeted 2 of such genes. In a correlation study decreased expression of miR-146a in monocytes was related to decreased natural T regulator cells in PP patients; decreased miR-212 was correlated to increased Adrenomedulin and decreased IL-6 expression in monocytes and to higher Th2 cell levels. This study identified changes in miR-146a and -212 expression in PP. Since these miRNAs are linked to inflammation, the study strengthens the view that PP is an inflammation-like condition.