Platelet transcription factors license the pro-inflammatory cytokine response of human monocytes.

In humans, blood Classical CD14+ monocytes contribute to host defense by secreting large amounts of pro-inflammatory cytokines. Their aberrant activity causes hyper-inflammation and life-threatening cytokine storms, while dysfunctional monocytes are associated with 'immunoparalysis', a state of immune hypo responsiveness and reduced pro-inflammatory gene expression, predisposing individuals to opportunistic infections. Understanding how monocyte functions are regulated is critical to prevent these harmful outcomes. We reveal platelets' vital role in the pro-inflammatory cytokine responses of human monocytes. Naturally low platelet counts in patients with immune thrombocytopenia or removal of platelets from healthy monocytes result in monocyte immunoparalysis, marked by impaired cytokine response to immune challenge and weakened host defense transcriptional programs. Remarkably, supplementing monocytes with fresh platelets reverses these conditions. We discovered that platelets serve as reservoirs of key cytokine transcription regulators, such as NF-κB and MAPK p38, and pinpointed the enrichment of platelet NF-κB2 in human monocytes by proteomics. Platelets proportionally restore impaired cytokine production in human monocytes lacking MAPK p38α, NF-κB p65, and NF-κB2. We uncovered a vesicle-mediated platelet-monocyte-propagation of inflammatory transcription regulators, positioning platelets as central checkpoints in monocyte inflammation.

Platelet-derived lipids promote insulin secretion of pancreatic ß cells.

Hyperreactive platelets are commonly observed in diabetic patients indicating a potential link between glucose homeostasis and platelet reactivity. This raises the possibility that platelets may play a role in the regulation of metabolism. Pancreatic ß cells are the central regulators of systemic glucose homeostasis. Here, we show that factor(s) derived from ß cells stimulate platelet activity and platelets selectively localize to the vascular endothelium of pancreatic islets. Both depletion of platelets and ablation of major platelet adhesion or activation pathways consistently resulted in impaired glucose tolerance and decreased circulating insulin levels. Furthermore, we found platelet-derived lipid classes to promote insulin secretion and identified 20-Hydroxyeicosatetraenoic acid (20-HETE) as the main factor promoting ß cells function. Finally, we demonstrate that the levels of platelet-derived 20-HETE decline with age and that this parallels with reduced impact of platelets on ß cell function. Our findings identify an unexpected function of platelets in the regulation of insulin secretion and glucose metabolism, which promotes metabolic fitness in young individuals.

Targeting ERK3/MK5 complex for treatment of obesity and diabetes.

Kinases represent one of the largest druggable families of proteins. Importantly, many kinases are aberrantly activated/de-activated in multiple organs during obesity, which contributes to the development of diabetes and associated diseases. Previous results indicate that the complex between Extracellular-regulated kinase 3 (ERK3) and Mitogen-Activated Protein Kinase (MAPK)-activated protein kinase 5 (MK5) suppresses energy dissipation and promotes fatty acids (FAs) output in adipose tissue and, therefore promotes obesity and diabetes. However, the therapeutic potential of targeting this complex at the systemic level has not been fully explored. Here we applied a translational approach to target the ERK3/MK5 complex in mice. Importantly, deletion of ERK3 in the whole body or administration of MK5-specific inhibitor protects against obesity and promotes insulin sensitivity. Finally, we show that the expression of ERK3 and MK5 correlates with the degree of obesity and that ERK3/MK5 complex regulates energy dissipation in human adipocytes. Altogether, we demonstrate that ERK3/MK5 complex can be targeted in vivo to preserve metabolic health and combat obesity and diabetes.

Phenotype and Response to PAMPs of Human Monocyte-Derived Foam Cells Obtained by Long-Term Culture in the Presence of oxLDLs.

Cholesterol-laden, foam macrophages constitute the most characteristic component of human atherosclerotic plaques. Persistent uptake of oxLDLs results in accumulation of lipid bodies inside the cells and determines their phenotype and subsequent functions. In this work, we describe the phenotype of human monocyte-derived foam cells obtained by differentiation in the constant presence of oxLDLs for 30 days (prolonged-hMDFCs). Although neither the total cellular nor the cell surface expression of Toll-like receptors (TLR) was regulated by oxLDLs, the prolonged-hMDFCs changed dramatically their responsiveness to TLR ligands and inactivated bacteria. Using multiplex technology, we observed an acute decline in cytokine and chemokine production after surface and endosomal TLR stimulation with the exception of TLR2/6 triggering with agonists Pam2CSK4 and MALP-2. We also noted significant reduction of some surface receptors which can have accessory function in recognition of particulate antigens (CD47, CD81, and CD11b). In contrast, the prolonged-hMDFCs responded to inflammasome activation by LPS/nigericin with extensive, necrotic type cell death, which was partially independent of caspase-1. This pyroptosis-like cell death was aggravated by necrostatin-1 and rapamycin. These findings identify a potential contribution of mature foam cells to inflammatory status by increasing the immunogenic cell death burden. The observed cross-talk between foam cell death pathways may lead to recognition of a potential new marker for atherosclerosis disease severity. Overall, our study demonstrates that, in contrast to other cellular models of foam cells, the prolonged-hMDFCs acquire a functional phenotype which may help understanding the role of foam cells in the pathogenesis of atherosclerosis.

Regulation of DNA replication within the immunoglobulin heavy-chain locus during B cell commitment.

The temporal order of replication of mammalian chromosomes appears to be linked to their functional organization, but the process that establishes and modifies this order during cell differentiation remains largely unknown. Here, we studied how the replication of the Igh locus initiates, progresses, and terminates in bone marrow pro-B cells undergoing B cell commitment. We show that many aspects of DNA replication can be quantitatively explained by a mechanism involving the stochastic firing of origins (across the S phase and the Igh locus) and extensive variations in their firing rate (along the locus). The firing rate of origins shows a high degree of coordination across Igh domains that span tens to hundreds of kilobases, a phenomenon not observed in simple eukaryotes. Differences in domain sizes and firing rates determine the temporal order of replication. During B cell commitment, the expression of the B-cell-specific factor Pax5 sharply alters the temporal order of replication by modifying the rate of origin firing within various Igh domains (particularly those containing Pax5 binding sites). We propose that, within the Igh C(H)-3'RR domain, Pax5 is responsible for both establishing and maintaining high rates of origin firing, mostly by controlling events downstream of the assembly of pre-replication complexes.

Determining the replication dynamics of specific gene loci by single-molecule analysis of replicated DNA.

In metazoans, development and cell differentiation are known to affect various aspects of chromosomal organization at developmentally regulated gene loci (e.g., nuclear localization, locus accessibility, chromatin modifications, etc.). Recent evidence also indicates that cell differentiation can have dramatic effects on DNA replication initiation and replication fork progression, at several of these loci. Hence, origin selection and activation are no longer viewed as constitutive events, but as actively regulated processes, the deregulation of which could potentially affect gene expression and genomic stability at particular chromosomal locations. For these reasons, the study of DNA replication across large genomic regions has recently received increasing attention. Here we describe an assay based on the analysis of single DNA molecules that can be used to determine the steady-state distribution of replication forks across large genomic regions. This assay can be used to study how DNA replication initiates, progresses, pauses, and terminates at single-copy loci in mammalian cells.

Saccharomyces cerevisiae phospholipase C regulates transcription of Msn2p-dependent stress-responsive genes.

Phosphatidylinositol phosphates are involved in signal transduction, cytoskeletal organization, and membrane trafficking. Inositol polyphosphates, produced from phosphatidylinositol phosphates by the phospholipase C-dependent pathway, regulate chromatin remodeling. We used genome-wide expression analysis to further investigate the roles of Plc1p (phosphoinositide-specific phospholipase C in Saccharomyces cerevisiae) and inositol polyphosphates in transcriptional regulation. Plc1p contributes to the regulation of approximately 2% of yeast genes in cells grown in rich medium. Most of these genes are induced by nutrient limitation and other environmental stresses and are derepressed in plc1 Delta cells. Surprisingly, genes regulated by Plc1p do not correlate with gene sets regulated by Swi/Snf or RSC chromatin remodeling complexes but show correlation with genes controlled by Msn2p. Our results suggest that the increased expression of stress-responsive genes in plc1 Delta cells is mediated by decreased cyclic AMP synthesis and protein kinase A (PKA)-mediated phosphorylation of Msn2p and increased binding of Msn2p to stress-responsive promoters. Accordingly, plc1 Delta cells display other phenotypes characteristic of cells with decreased PKA activity. Our results are consistent with a model in which Plc1p acts together with the membrane receptor Gpr1p and associated G(alpha) protein Gpa2p in a pathway separate from Ras1p/Ras2p and converging on PKA.