Project description:BackgroundCells of the innate immune system including monocytes and macrophages are the first line of defence against infections and are critical regulators of the inflammatory response. These cells express toll-like receptors (TLRs), innate immune receptors which govern tailored inflammatory gene expression patterns. Monocytes, which produce pro-inflammatory mediators, are readily recruited to the central nervous system (CNS) in neurodegenerative diseases.MethodsThis study explored the expression of receptors (CD11b, TLR2 and TLR4) on circulating monocyte-derived macrophages (MDMs) and peripheral blood mononuclear cells (PBMCs) isolated from healthy elderly adults who we classified as either IQ memory-consistent (high-performing, HP) or IQ memory-discrepant (low-performing, LP).ResultsThe expression of CD11b, TLR4 and TLR2 was increased in MDMs from the LP group when compared to HP cohort. MDMs from both groups responded robustly to treatment with the TLR4 activator, lipopolysaccharide (LPS), in terms of cytokine production. Significantly, MDMs from the LP group displayed hypersensitivity to LPS exposure.InterpretationOverall these findings define differential receptor expression and cytokine profiles that occur in MDMs derived from a cohort of IQ memory-discrepant individuals. These changes are indicative of inflammation and may be involved in the prodromal processes leading to the development of neurodegenerative disease.
Project description:In atherosclerotic arteries, blood monocytes differentiate to macrophages in the presence of growth factors, such as macrophage colony-stimulation factor (M-CSF), and chemokines, such as platelet factor 4 (CXCL4). To compare the gene expression signature of CXCL4-induced macrophages with M-CSF-induced macrophages or macrophages polarized with IFN-gamma/LPS (M1) or IL-4 (M2), we cultured primary human peripheral blood monocytes for 6 d. mRNA expression was measured by Affymetrix gene chips, and differences were analyzed by local pooled error test, profile of complex functionality, and gene set enrichment analysis. Three hundred seventy-five genes were differentially expressed between M-CSF- and CXCL4-induced macrophages; 206 of them overexpressed in CXCL4 macrophages coding for genes implicated in the inflammatory/immune response, Ag processing and presentation, and lipid metabolism. CXCL4-induced macrophages overexpressed some M1 and M2 genes and the corresponding cytokines at the protein level; however, their transcriptome clustered with neither M1 nor M2 transcriptomes. They almost completely lost the ability to phagocytose zymosan beads. Genes linked to atherosclerosis were not consistently upregulated or downregulated. Scavenger receptors showed lower and cholesterol efflux transporters showed higher expression in CXCL4- than M-CSF-induced macrophages, resulting in lower low-density lipoprotein content. We conclude that CXCL4 induces a unique macrophage transcriptome distinct from known macrophage types, defining a new macrophage differentiation that we propose to call M4.
Project description:Macrophages are central mediators of the innate immune system that can be differentiated from monocytes upon exposure to cytokines. While increased cyclic adenosine monophosphate (cAMP) levels are known to inhibit many lipopolysaccharide-elicited macrophage inflammatory responses, the effects of elevated cAMP on monocyte/macrophage differentiation are not as well understood. We show here that during differentiation, cAMP agonists can cause a large increase in the mRNA and protein levels of several of the pro-inflammatory CXCL and CCL chemokines. The cAMP mediator-exchange protein activated by cAMP (Epac) contributes substantially to the increase in these chemokines. These chemokines are known to play an important role in the regulation of immune responses, particularly regarding the pathogenesis of asthma and chronic obstructive pulmonary disorder. We also found that a selective cAMP-degrading phosphodiesterase (PDE) 4 inhibitor can potentiate the chemokine expression elicited by low-dose forskolin or Prostaglandin E2 (PGE(2)). These data suggest that chemokine receptor antagonists administered in conjunction with a PDE4 inhibitor may improve both the efficacy and safety of PDE4-inhibitor therapy for chronic inflammatory disorders.
Project description:The levels of proinflammatory cytokine or chemokine in blood and cerebrospinal fluid are thought to be one of predictors for clinical severity of enterovirus 71 (EV71) infection, yet the cellular sources or signalling mechanism remain undefined. Here, we focused on the response of human primary monocyte-derived macrophages (MDMs) to EV71 virus and its possible mechanisms.Human primary MDMs were infected by EV71 virus in vitro. Infectivity and viral replication were assayed, and cytokine responses were determined by Cytometric Bead Array(CBA) analysis. The relative changes of Toll-like receptors, retinoic acid-inducible gene I (RIG-I) and melamoma differentiation associated gene 5 (MDA5) mRNA expression were detected by real-time RT-PCR.Effective infection and viral replication were detected in EV71-infected MDMs. The titters of progeny virus released from EV71-infected MDMs gradually increased from 6-h to 48-h point of infection (POI.). Proinflammatory cytokines: IL-1, IL-6, TNF-? but not IFN-? and ? were induced in MDMs by EV71. EV71 infection significantly increased the release of IL-8, IP-10 and RANTES at 12-h or 24-h POI. Upregulation of TLR2, TLR7 and TLR8 mRNA expression rather than TLR3, TLR4, TLR6, TLR9, TLR10, RIG-I, MDA5 were found at different time points in EV71-infected MDMs.Our findings suggested that macrophages are not only the important target cells but also the effectors during EV71 infection, and they may play an important role in the pathogenesis of EV71 infection. And the proinflammatory cytokine and chemokine responses in EV71-infected MDMs may be mediated by the activation of differential pattern of TLRs.
Project description:BackgroundA hallmark of atherosclerosis is its complex pathogenesis, which is dependent on altered cholesterol metabolism and inflammation. Both arms of pathogenesis involve myeloid cells. Monocytes migrating into the arterial walls interact with modified low-density lipoprotein (LDL) particles, accumulate cholesterol and convert into foam cells, which promote plaque formation and also contribute to inflammation by producing proinflammatory cytokines. A number of studies characterized transcriptomics of macrophages following interaction with modified LDL, and revealed alteration of the expression of genes responsible for inflammatory response and cholesterol metabolism. However, it is still unclear how these two processes are related to each other to contribute to atherosclerotic lesion formation.MethodsWe attempted to identify the main mater regulator genes in macrophages treated with atherogenic modified LDL using a bioinformatics approach.ResultsWe found that most of the identified genes were involved in inflammation, and none of them was implicated in cholesterol metabolism. Among the key identified genes were interleukin (IL)-7, IL-7 receptor, IL- 15 and CXCL8.ConclusionOur results indicate that activation of the inflammatory pathway is the primary response of the immune cells to modified LDL, while the lipid metabolism genes may be a secondary response triggered by inflammatory signalling.
Project description:The importance of Chromatin Immunoprecipitation (ChIP) technology has grown exponentially along with an increased interest in epigenetic regulation. The correlation of transcription factors with histone marks is now well established as the center of epigenetic studies; therefore, precise knowledge about histone marks is critical to unravel their molecular function and to understand their role in biological systems. This knowledge constantly accumulates and is provided openly in the expanding hubs of information such as the USCS Genome Browser. Nevertheless, as we gain more knowledge, we realize that the DNA-protein interactions are not driven by a "one size fits all" rule. Also, the diversity of interactions between DNA, histones, and transcriptional regulators is much bigger than previously considered. Besides a detailed protocol of sample preparation for the ChIP assay from primary human monocyte-derived macrophages (MDM) [an acceptable in vitro model for primary, human macrophage cells], we show that differences between various types of cells exist. Furthermore, we can postulate that such variations exist between transformed macrophage-like cell lines and primary macrophages obtained from healthy volunteers. We found that the most efficient fixation time for MDM is 10min. Finally, to perform multiple analytical assays, we showed that even with thorough methodology, the yield of material obtained from primary cells is the major challenge.
Project description:Historically, the brain has been considered an immune-privileged organ separated from the peripheral immune system by the blood-brain barrier. However, immune responses do occur in the brain in neurological conditions in which the integrity of the blood-brain barrier is compromised, exposing the brain to peripheral antigens and endogenous danger signals. While most of the associated pathological processes occur in the central nervous system, it is now clear that peripheral immune cells, especially mononuclear phagocytes, that infiltrate into the injury site play a key role in modulating the progression of primary brain injury development. As inflammation is a necessary and critical component for the subsequent injury resolution process, understanding the contribution of mononuclear phagocytes on the regulation of inflammatory responses may provide novel approaches for potential therapies. Furthermore, predisposed comorbid conditions at the time of stroke cause the alteration of stroke-induced immune and inflammatory responses and subsequently influence stroke outcome. In this review, we summarize a role for microglia and monocytes/macrophages in acute ischemic stroke in the context of normal and metabolically compromised conditions.
Project description:The highly orchestrated transcriptional and metabolic reprogramming during activation drastically transforms the main functions and physiology of human macrophages across the polarization spectrum. Lipids, for example, can modify protein function by acting remotely as signaling molecules but also locally by altering the physical properties of cellular membranes. These changes play key roles in the functions of highly plastic immune cells due to their involvement in inflammation, immune responses, phagocytosis and wound healing processes. We report an analysis of major membrane lipids of distinct phenotypes of resting (M0), classically activated (M1), alternatively activated (M2a) and deactivated (M2c) human monocyte derived macrophages from different donors. Samples were subjected to supercritical fluid chromatography-ion mobility-mass spectrometry analysis, which allowed separations based on lipid class, facilitating the profiling of their fatty acid composition. Different levels of arachidonic acid mobilization as well as other fatty acid changes were observed for different lipid classes in the distinct polarization phenotypes, suggesting the activation of highly orchestrated and specific enzymatic processes in the biosynthesis of lipid signaling molecules and cell membrane remodeling. Thromboxane A2 production appeared to be a specific marker of M1 polarization. These alterations to the global composition of lipid bi-layer membranes in the cell provide a potential methodology for the definition and determination of cellular and tissue activation states.
Project description:Transgenic Leishmania parasites that encode the murine chemokine monocyte chemoattractant protein 1 (MCP-1) were generated. These parasites transcribed MCP-1 mRNA and secreted MCP-1 protein. Infection of BALB/c, C57BL/6, or MCP-1 knockout (KO) mice with these parasites resulted in minimal lesion development with fewer parasites in the infected foot, lymph node, and spleen compared to wild-type-infected mice. In contrast, transgenic parasites caused substantial lesions with relatively high numbers of parasites in CC chemokine receptor 2 (CCR2) KO mice, indicating that the parasites are viable and healthy and that the lack of lesion development is CCR2 dependent. Prior infection of mice with transgenic parasites offered no protection to subsequent wild-type L. major challenge, suggesting that the transgenic parasites are controlled by an early innate immune response. Consistent with innate immunity, flow cytometry of cells from the ears of mice infected with transgenic parasites revealed an increase in the number of CCR2-positive macrophages by day 7 postinfection. The enumeration of transgenic parasites in ear lesions demonstrated a significant reduction in parasite numbers, which coincided with the increased CCR2-positive macrophage migration. CCR2-positive macrophages isolated from ears of mice infected with transgenic parasites contained virtually no parasites. In vitro studies revealed that optimal parasite killing required the recruitment of CCR2-positive macrophages, followed by stimulation with a combination of both MCP-1 and gamma interferon (IFN-gamma). This work suggests that the parasite-derived MCP-1 can recruit a restrictive population of CCR2-positive macrophages into lesions that can be optimally stimulated by MCP-1 and IFN-gamma to efficiently kill Leishmania parasites.