Resolution of inflammation: an integrated view

O.S. is supported by the NWO (VIDI project 91712303), theDFG (SO876/3-1, SO876/6-1, FOR809, SFB914 TPB08), theGerman-Israeli Foundation, and the Else Kro¨ner FreseniusStiftung. M.P. is supported by the Wellcome Trust (program086867/Z/08), the Arthritis Research UK, the British HeartFoundation (PG/09/060 and PG/11/48/28981) and the MedicalResearch Council

Simvastatin decreases the level of heparin-binding protein in patients with acute lung injury

Background: Heparin-binding protein is released by neutrophils during inflammation and disrupts the integrity of the alveolar and capillary endothelial barrier implicated in the development of acute lung injury and systemic organ failure. We sought to investigate whether oral administration of simvastatin to patients with acute lung injury reduces plasma heparin-binding protein levels and improves intensive care unit outcome. Methods: Blood samples were collected from patients with acute lung injury with 48 h of onset of acute lung injury (day 0), day 3, and day 7. Patients were given placebo or 80 mg simvastatin for up to 14 days. Plasma heparin-binding protein levels from patients with acute lung injury and healthy volunteers were measured by ELISA. Results: Levels of plasma heparin-binding protein were significantly higher in patients with acute lung injury than healthy volunteers on day 0 (p = 0.011). Simvastatin 80 mg administered enterally for 14 days reduced plasma level of heparin-binding protein in patients. Reduced heparin-binding protein was associated with improved intensive care unit survival. Conclusions: A reduction in heparin-binding protein with simvastatin is a potential mechanism by which the statin may modify outcome from acute lung injury

Resolving lipid mediators maresin 1 and resolvin D2 prevent atheroprogression in mice

RATIONALE: Atheroprogression is a consequence of non-resolved inflammation and currently a comprehensive overview of the mechanisms preventing resolution is missing. However, in acute inflammation, resolution is known to be orchestrated by a switch from inflammatory to resolving lipid mediators. Therefore we hypothesized that lesional lipid mediator imbalance favors atheroprogression. OBJECTIVE: To understand the lipid mediator balance during atheroprogression and to establish an interventional strategy based on delivery of resolving lipid mediators. METHODS AND RESULTS: Aortic lipid mediator profiling of aortas from Apoe(-/-) mice fed a high fat diet for four weeks, eight weeks, or four months revealed an expansion of inflammatory lipid mediators, Leukotriene B4 (LTB4) and Prostaglandin E2 (PGE2), and a concomitant decrease of resolving lipid mediators, Resolvin D2 (RvD2) and Maresin 1 (MaR1), during advanced atherosclerosis. Functionally, aortic LTB4 and PGE2 levels correlated with traits of plaque instability while RvD2 and MaR1 levels correlated with signs of plaque stability. In a therapeutic context, repetitive RvD2 and MaR1 delivery prevented atheroprogression as characterized by halted expansion of the necrotic core and accumulation of macrophages along with increased fibrous cap thickness and smooth muscle cell numbers. Mechanistically, RvD2 and MaR1 induced a shift in macrophage profile towards a reparative phenotype which secondarily stimulated collagen synthesis in smooth muscle cells. CONCLUSIONS: We present evidence for the imbalance between inflammatory and resolving lipid mediators during atheroprogression. Delivery of RvD2 and MaR1 successfully prevented atheroprogression suggesting that resolving lipid mediators potentially represent an innovative strategy to resolve arterial inflammation

Atherosclerotic plaque destabilization in Mice: A comparative study

Atherosclerosis-Associated diseases are the main cause ofmortality and morbidity in western societies. The progression of atherosclerosis is a dynamic process evolving from early to advanced lesions thatmay become rupture-prone vulnerable plaques. Acute coronary syndromes are the clinical manifestation of life-Threatening thrombotic events associated with high-risk vulnerable plaques. Hyperlipidemic mouse models have been extensively used in studying the mechanisms controlling initiation and progression of atherosclerosis. However, the understanding of mechanisms leading to atherosclerotic plaque destabilization has been hampered by the lack of proper animalmodelsmimicking this process. Although various mouse models generate atherosclerotic plaques with histological features of human advanced lesions, a consensus model to study atherosclerotic plaque destabilization is still lacking. Hence, we studied the degree and features of plaque vulnerability in different mouse models of atherosclerotic plaque destabilization and find that the model based on the placement of a shear stress modifier in combination with hypercholesterolemia represent with high incidence the most human like lesions compared to the other models

In vivo imaging and quantitative analysis of leukocyte directional migration and polarization in inflamed tissue

Directional migration of transmigrated leukocytes to the site of injury is a central event in the inflammatory response. Here, we present an in vivo chemotaxis assay enabling the visualization and quantitative analysis of subtype-specific directional motility and polarization of leukocytes in their natural 3D microenvironment. Our technique comprises the combination of i) semi-automated in situ microinjection of chemoattractants or bacteria as local chemotactic stimulus, ii) in vivo near-infrared reflected-light oblique transillumination (RLOT) microscopy for the visualization of leukocyte motility and morphology, and iii) in vivo fluorescence microscopy for the visualization of different leukocyte subpopulations or fluorescence-labeled bacteria. Leukocyte motility parameters are quantified off-line in digitized video sequences using computer-assisted single cell tracking. Here, we show that perivenular microinjection of chemoattractants [macrophage inflammatory protein-1alpha (MIP-1alpha/Ccl3), platelet-activating factor (PAF)] or E. coli into the murine cremaster muscle induces target-oriented intravascular adhesion and transmigration as well as polarization and directional interstitial migration of leukocytes towards the locally administered stimuli. Moreover, we describe a crucial role of Rho kinase for the regulation of directional motility and polarization of transmigrated leukocytes in vivo. Finally, combining in vivo RLOT and fluorescence microscopy in Cx3CR1(gfp/gfp) mice (mice exhibiting green fluorescent protein-labeled monocytes), we are able to demonstrate differences in the migratory behavior of monocytes and neutrophils.Taken together, we propose a novel approach for investigating the mechanisms and spatiotemporal dynamics of subtype-specific motility and polarization of leukocytes during their directional interstitial migration in vivo

Neutrophil depletion reduces edema formation and tissue loss following traumatic brain injury in mice

Background: Brain edema as a result of secondary injury following traumatic brain injury (TBI) is a major clinical concern. Neutrophils are known to cause increased vascular permeability leading to edema formation in peripheral tissue, but their role in the pathology following TBI remains unclear. Methods: In this study we used controlled cortical impact (CCI) as a model for TBI and investigated the role of neutrophils in the response to injury. The outcome of mice that were depleted of neutrophils using an anti-Gr-1 antibody was compared to that in mice with intact neutrophil count. The effect of neutrophil depletion on blood-brain barrier function was assessed by Evan's blue dye extravasation, and analysis of brain water content was used as a measurement of brain edema formation (24 and 48 hours after CCI). Lesion volume was measured 7 and 14 days after CCI. Immunohistochemistry was used to assess cell death, using a marker for cleaved caspase-3 at 24 hours after injury, and microglial/macrophage activation 7 days after CCI. Data were analyzed using Mann-Whitney test for non-parametric data. Results: Neutrophil depletion did not significantly affect Evan's blue extravasation at any time-point after CCI. However, neutrophil-depleted mice exhibited a decreased water content both at 24 and 48 hours after CCI indicating reduced edema formation. Furthermore, brain tissue loss was attenuated in neutropenic mice at 7 and 14 days after injury. Additionally, these mice had a significantly reduced number of activated microglia/macrophages 7 days after CCI, and of cleaved caspase-3 positive cells 24 h after injury. Conclusion: Our results suggest that neutrophils are involved in the edema formation, but not the extravasation of large proteins, as well as contributing to cell death and tissue loss following TBI in mice

Protein C Inhibitor—A Novel Antimicrobial Agent

Protein C inhibitor (PCI) is a heparin-binding serine proteinase inhibitor belonging to the family of serpin proteins. Here we describe that PCI exerts broad antimicrobial activity against bacterial pathogens. This ability is mediated by the interaction of PCI with lipid membranes, which subsequently leads to their permeabilization. As shown by negative staining electron microscopy, treatment of Escherichia coli or Streptococcus pyogenes bacteria with PCI triggers membrane disruption followed by the efflux of bacterial cytosolic contents and bacterial killing. The antimicrobial activity of PCI is located to the heparin-binding site of the protein and a peptide spanning this region was found to mimic the antimicrobial activity of PCI, without causing lysis or membrane destruction of eukaryotic cells. Finally, we show that platelets can assemble PCI on their surface upon activation. As platelets are recruited to the site of a bacterial infection, these results may explain our finding that PCI levels are increased in tissue biopsies from patients suffering from necrotizing fasciitis caused by S. pyogenes. Taken together, our data describe a new function for PCI in innate immunity

Dendritic Cells Take up and Present Antigens from Viable and Apoptotic Polymorphonuclear Leukocytes

Dendritic cells (DC) are endowed with the ability to cross-present antigens from other cell types to cognate T cells. DC are poised to meet polymorphonuclear leukocytes (PMNs) as a result of being co-attracted by interleukin-8 (IL-8), for instance as produced by tumor cells or infected tissue. Human monocyte-derived and mouse bone marrow-derived DC can readily internalize viable or UV-irradiated PMNs. Such internalization was abrogated at 4°C and partly inhibited by anti-CD18 mAb. In mice, DC which had internalized PMNs containing electroporated ovalbumin (OVA) protein, were able to cross-present the antigen to CD8 (OT-1) and CD4 (OT-2) TCR-transgenic T cells. Moreover, in humans, tumor cell debris is internalized by PMNs and the tumor-cell material can be subsequently taken up from the immunomagnetically re-isolated PMNs by DC. Importantly, if human neutrophils had endocytosed bacteria, they were able to trigger the maturation program of the DC. Moreover, when mouse PMNs with E. coli in their interior are co-injected in the foot pad with DC, many DC loaded with fluorescent material from the PMNs reach draining lymph nodes. Using CT26 (H-2d) mouse tumor cells, it was observed that if tumor cells are intracellularly loaded with OVA protein and UV-irradiated, they become phagocytic prey of H-2d PMNs. If such PMNs, that cannot present antigens to OT-1 T cells, are immunomagnetically re-isolated and phagocytosed by H-2b DC, such DC productively cross-present OVA antigen determinants to OT-1 T cells. Cross-presentation to adoptively transferred OT-1 lymphocytes at draining lymph nodes also take place when OVA-loaded PMNs (H-2d) are coinjected in the footpad of mice with autologous DC (H-2b). In summary, our results indicate that antigens phagocytosed by short-lived PMNs can be in turn internalized and productively cross-presented by DC