Immune and inflammatory responses require leukocytes to migrate within and through the vasculature, a process that is facilitated by their capacity to switch to a polarized morphology with asymmetric distribution of receptors. We report that neutrophil polarization within activated venules served to organize a protruding domain that engaged activated platelets present in the bloodstream. The selectin ligand PSGL-1 transduced signals emanating from these interactions, resulting in redistribution of receptors that drive neutrophil migration. Consequently, neutrophils unable to polarize or to transduce signals through PSGL-1 displayed aberrant crawling, and blockade of this domain protected mice against thrombo-inflammatory injury. These results reveal that recruited neutrophils scan for activated platelets, and suggest that their bipolarity allows integration of signals present at both the endothelium and the circulation before inflammation proceeds.

Our understanding of the key players involved in the differential regulation of T-cell responses during inflammation, infection and auto-immunity is fundamental for designing efficient therapeutic strategies against immune diseases. With respect to this, the inhibitory role of the lipid mediator prostaglandin E2 (PGE2) in T-cell immunity has been documented since the 1970s. Studies that ensued investigating the underlying mechanisms substantiated the suppressive function of micromolar concentrations of PGE2 in T-cell activation, proliferation, differentiation and migration. However, the past decade has seen a revolution in this perspective, since nanomolar concentrations of PGE2 have been shown to potentiate Th1 and Th17 responses and aid in T-cell proliferation. The understanding of concentration-specific effects of PGE2 in other cell types, the development of mice deficient in each subtype of the PGE2 receptors (EP receptors) and the delineation of signalling pathways mediated by the EP receptors have enhanced our understanding of PGE2 as an immune-stimulator. PGE2 regulates a multitude of functions in T-cell activation and differentiation and these effects vary depending on the micro-environment of the cell, maturation and activation state of the cell, type of EP receptor involved, local concentration of PGE2 and whether it is a homeostatic or inflammatory scenario. In this review, we compartmentalize the various aspects of this complex relationship of PGE2 with T lymphocytes. Given the importance of this molecule in T-cell activation, we also address the possibility of using EP receptor antagonism as a potential therapeutic approach for some immune disorders.

The β1-adrenergic-receptor (ADRB1) antagonist metoprolol reduces infarct size in acute myocardial infarction (AMI) patients. The prevailing view has been that metoprolol acts mainly on cardiomyocytes. Here, we demonstrate that metoprolol reduces reperfusion injury by targeting the haematopoietic compartment. Metoprolol inhibits neutrophil migration in an ADRB1-dependent manner. Metoprolol acts during early phases of neutrophil recruitment by impairing structural and functional rearrangements needed for productive engagement of circulating platelets, resulting in erratic intravascular dynamics and blunted inflammation. Depletion of neutrophils, ablation of Adrb1 in haematopoietic cells, or blockade of PSGL-1, the receptor involved in neutrophil–platelet interactions, fully abrogated metoprolol's infarct-limiting effects. The association between neutrophil count and microvascular obstruction is abolished in metoprolol-treated AMI patients. Metoprolol inhibits neutrophil–platelet interactions in AMI patients by targeting neutrophils. Identification of the relevant role of ADRB1 in haematopoietic cells during acute injury and the protective role upon its modulation offers potential for developing new therapeutic strategies.

Rationale:The inflammatory processes that initiate and propagate atherosclerosis remain poorly understood, largely because defining the intravascular behavior of immune cells has been technically challenging. Respiratory and pulsatile movements have hampered in vivo visualization of leukocyte accumulation in athero-prone arteries at resolutions achieved in other tissues.Objective: To establish and to validate a method that allows high-resolution imaging of inflammatory leukocytes and platelets within the carotid artery of atherosusceptible mice in vivo. Methods and Results:We have devised a procedure to stabilize the mouse carotid artery mechanically without altering blood dynamics, which dramatically enhances temporal and spatial resolutions using high-speed intravital microscopy in multiple channels of fluorescence. By applying this methodology at different stages of disease progression in atherosusceptible mice, we first validated our approach by assessing the recruitment kinetics of various leukocyte subsets and platelets in athero-prone segments of the carotid artery. The high temporal and spatial resolution allowed the dissection of both the dynamic polarization of and the formation of subcellular domains within adhered leukocytes. We further demonstrate that the secondary capture of activated platelets on the plaque is predominantly mediated by neutrophils. Finally, we couple this procedure with triggered 2-photon microscopy to visualize the 3-dimensional movement of leukocytes in intimate contact with the arterial lumen. Conclusions:

Non-alcoholic fatty liver disease (NAFLD) is a major health problem and the main cause of liver disease in Western countries. Although NAFLD is strongly associated with obesity and insulin resistance, its pathogenesis remains poorly understood. The disease begins with an excessive accumulation of triglycerides in the liver, which stimulates an inflammatory response. Alternative p38 mitogen-activated kinases (p38c and p38d) have been shown to contribute to inflammation in different diseases. Here we demonstrate that p38d is elevated in livers of obese patients with NAFLD and that mice lacking p38c/d in myeloid cells are resistant to diet-induced fatty liver, hepatic triglyceride accumulation and glucose intolerance. This protective effect is due to defective migration of p38c/d-deficient neutrophils to the damaged liver. We further show that neutrophil infiltration in wild-type mice contributes to steatosis development by means of inflammation and liver metabolic changes. Therefore, p38c and p38d in myeloid cells provide a potential target for NAFLD therapy.

• ESL-1 and PSGL-1 cooperate to mediate E-selectin binding, myeloid homeostasis, and inflammatory cell recruitment.• ESL-1 dominates E-selectin binding and homing of hematopoietic progenitors.Beyond its well-established roles in mediating leukocyte rolling, E-selectin is emerging as a multifunctional receptor capable of inducing integrin activation in neutrophils, and of regulating various biological processes in hematopoietic precursors. Although these effects suggest important homeostatic contributions of this selectin in the immune and hematologic systems, the ligands responsible for transducing these effects in different leukocyte lineages are not well defined. We have characterized mice deficient in E-selectin ligand-1 (ESL-1), or in both P-selectin glycoprotein-1 (PSGL-1) and ESL-1, to explore and compare the contributions of these glycoproteins in immune and hematopoietic cell trafficking. In the steady state, ESL-1 deficiency resulted in a moderate myeloid expansion that became more prominent when both glycoproteins were eliminated. During inflammation, PSGL-1 dominated E-selectin binding, rolling, integrin activation, and extravasation of mature neutrophils, but only the combined deficiency in PSGL-1 and ESL-1 completely abrogated leukocyte recruitment. Surprisingly, we find that the levels of ESL-1 were strongly elevated in hematopoietic progenitor cells. These elevations correlated with a prominent function of ESL-1 for E-selectin binding and for migration of hematopoietic progenitor cells into the bone marrow. Our results uncover dominant roles for ESL-1 in the immature compartment, and a functional shift toward PSGL-1 dependence in mature neutrophils. (Blood. 2013;122(24): 3993-4001)

Understanding the regulation of T-cell responses during inflammation and auto-immunity is fundamental for designing efficient therapeutic strategies against immune diseases. In this regard, prostaglandin E2 (PGE2) is mostly considered a myeloid-derived immunosuppressive molecule. We describe for the first time that T cells secrete PGE2 during T-cell receptor stimulation. In addition, we show that autocrine PGE2 signaling through EP receptors is essential for optimal CD4(+) T-cell activation in vitro and in vivo, and for T helper 1 (Th1) and regulatory T cell differentiation. PGE2 was found to provide additive co-stimulatory signaling through AKT activation. Intravital multiphoton microscopy showed that triggering EP receptors in T cells is also essential for the stability of T cell-dendritic cell (DC) interactions and Th-cell accumulation in draining lymph nodes (LNs) during inflammation. We further demonstrated that blocking EP receptors in T cells during the initial phase of collagen-induced arthritis in mice resulted in a reduction of clinical arthritis. This could be attributable to defective T-cell activation, accompanied by a decline in activated and interferon-γ-producing CD4(+) Th1 cells in draining LNs. In conclusion, we prove that T lymphocytes secret picomolar concentrations of PGE2, which in turn provide additive co-stimulatory signaling, enabling T cells to attain a favorable activation threshold. PGE2 signaling in T cells is also required for maintaining long and stable interactions with DCs within LNs. Blockade of EP receptors in vivo impairs T-cell activation and development of T cell-mediated inflammatory responses. This may have implications in various pathophysiological settings.

Hematopoietic stem cell transplantation or bone marrow transplantation is a common approach to reconstitute the immune system of mice that have been subjected to marrow-ablative doses of radiation. This method can be used in the field of atherosclerosis to assess the contribution of hematopoietic cells of a desired genotype to disease pathogenesis. The engraftment of atherosclerosis-prone mice with donor cells that contain genetic alterations in cells of the innate or adaptive immune system has been invaluable to define the role of multiple gene products in atherosclerosis. Here, we describe the different steps involved in the bone marrow transplantation protocol along with specific guidelines regarding the theoretical and technical details of the procedure.