Mural cell-derived chemokines provide a protective niche to safeguard vascular macrophages and limit chronic inflammation.

Maladaptive, non-resolving inflammation contributes to chronic inflammatory diseases such as atherosclerosis. Because macrophages remove necrotic cells, defective macrophage programs can promote chronic inflammation with persistent tissue injury. Here, we investigated the mechanisms sustaining vascular macrophages. Intravital imaging revealed a spatiotemporal macrophage niche across vascular beds alongside mural cells (MCs)-pericytes and smooth muscle cells. Single-cell transcriptomics, co-culture, and genetic deletion experiments revealed MC-derived expression of the chemokines CCL2 and MIF, which actively preserved macrophage survival and their homeostatic functions. In atherosclerosis, this positioned macrophages in viable plaque areas, away from the necrotic core, and maintained a homeostatic macrophage phenotype. Disruption of this MC-macrophage unit via MC-specific deletion of these chemokines triggered detrimental macrophage relocalizing, exacerbated plaque necrosis, inflammation, and atheroprogression. In line, CCL2 inhibition at advanced stages of atherosclerosis showed detrimental effects. This work presents a MC-driven safeguard toward maintaining the homeostatic vascular macrophage niche.

CD36-triggered cell invasion and persistent tissue colonization by tumor microvesicles during metastasis.

Tumor microvesicles are a peculiar type of extracellular vesicles that circulate in the blood of patients with metastatic cancer. The itineraries and immune cell interactions of tumor microvesicles during the intravascular and extravascular stages of metastasis are largely unknown. We found that the lipid receptor CD36 is a major mediator of the engulfment of pancreatic tumor microvesicles by myeloid immune cells in vitro and critically samples circulating tumor microvesicles by resident liver macrophages in mice in vivo. Direct nanoscopic imaging of individual tumor microvesicles shows that the microvesicles rapidly decay during engulfment whereby their cargo is targeted concomitantly to the plasma membrane and the cytoplasm excluding lysosomal compartments. CD36 also promotes internalization of blood cell (nontumor) microvesicles, which involves endolysosomal pathways. A portion of tumor microvesicles circulating in the liver microcirculation traverses the vessel wall in a CD36-dependent way. Extravasated microvesicles colonize distinct perivascular Ly6C- macrophages for at least 2 wk. Thus, the microvesicles are increasingly integrated into CD36-induced premetastatic cell clusters and enhance development of liver metastasis. Hence, promotion of metastasis by pancreatic tumor microvesicles is associated with CD36-regulated immune cell invasion and extravasation of microvesicles and persistent infiltration of specific tissue macrophages by microvesicle cargo.-Pfeiler, S., Thakur, M., Grünauer, P., Megens, R. T. A., Joshi, U., Coletti, R., Samara, V., Müller-Stoy, G., Ishikawa-Ankerhold, H., Stark, K., Klingl, A., Fröhlich, T., Arnold, G. J., Wörmann, S., Bruns, C. J., Algül, H., Weber, C., Massberg, S., Engelmann, B. CD36-triggered cell invasion and persistent tissue colonization by tumor microvesicles during metastasis.

Coronin 1A, a novel player in integrin biology, controls neutrophil trafficking in innate immunity.

Trafficking of polymorphonuclear neutrophils (PMNs) during inflammation critically depends on the ß2 integrins lymphocyte function-associated antigen 1 (LFA-1) (CD11a/CD18) and macrophage-1 antigen (CD11b/CD18). Here, we identify coronin 1A (Coro1A) as a novel regulator of ß2 integrins that interacts with the cytoplasmic tail of CD18 and is crucial for induction of PMN adhesion and postadhesion events, including adhesion strengthening, spreading, and migration under flow conditions. Transition of PMN rolling to firm adhesion critically depends on Coro1A by regulating the accumulation of high-affinity LFA-1 in focal zones of adherent cells. Defective integrin affinity regulation in the genetic absence of Coro1A impairs leukocyte adhesion and extravasation in inflamed cremaster muscle venules in comparison with control animals. In a Helicobacter pylori mouse infection model, PMN infiltration into the gastric mucosa is dramatically reduced in Coro1A-/- mice, resulting in an attenuated gastric inflammation. Thus, Coro1A represents an important novel player in integrin biology, with key functions in PMN trafficking during innate immunity.

Distinct Pathogenesis of Pancreatic Cancer Microvesicle-Associated Venous Thrombosis Identifies New Antithrombotic Targets In Vivo.

OBJECTIVE: Cancer patients are at high risk of developing deep venous thrombosis (DVT) and venous thromboembolism, a leading cause of mortality in this population. However, it is largely unclear how malignant tumors drive the prothrombotic cascade culminating in DVT. APPROACH AND RESULTS: Here, we addressed the pathophysiology of malignant DVT compared with nonmalignant DVT and focused on the role of tumor microvesicles as potential targets to prevent cancer-associated DVT. We show that microvesicles released by pancreatic adenocarcinoma cells (pancreatic tumor-derived microvesicles [pcMV]) boost thrombus formation in a model of flow restriction of the mouse vena cava. This depends on the synergistic activation of coagulation by pcMV and host tissue factor. Unlike nonmalignant DVT, which is initiated and propagated by innate immune cells, thrombosis triggered by pcMV was largely independent of myeloid leukocytes or platelets. Instead, we identified externalization of the phospholipid phosphatidylethanolamine as a major mechanism controlling the prothrombotic activity of pcMV. Disrupting phosphatidylethanolamine-dependent activation of factor X suppressed pcMV-induced DVT without causing changes in hemostasis. CONCLUSIONS: Together, we show here that the pathophysiology of pcMV-associated experimental DVT differs markedly from innate immune cell-promoted nonmalignant DVT and is therefore amenable to distinct antithrombotic strategies. Targeting phosphatidylethanolamine on tumor microvesicles could be a new strategy for prevention of cancer-associated DVT without causing bleeding complications.

Disulfide HMGB1 derived from platelets coordinates venous thrombosis in mice.

Deep venous thrombosis (DVT) is one of the most common cardiovascular diseases, but its pathophysiology remains incompletely understood. Although sterile inflammation has recently been shown to boost coagulation during DVT, the underlying molecular mechanisms are not fully resolved, which could potentially identify new anti-inflammatory approaches to prophylaxis and therapy of DVT. Using a mouse model of venous thrombosis induced by flow reduction in the vena cava inferior, we identified blood-derived high-mobility group box 1 protein (HMGB1), a prototypical mediator of sterile inflammation, to be a master regulator of the prothrombotic cascade involving platelets and myeloid leukocytes fostering occlusive DVT formation. Transfer of platelets into Hmgb1-/- chimeras showed that this cell type is the major source of HMGB1, exposing reduced HMGB1 on their surface upon activation thereby enhancing the recruitment of monocytes. Activated leukocytes in turn support oxidation of HMGB1 unleashing its prothrombotic activity and promoting platelet aggregation. This potentiates the amount of HMGB1 and further nurtures the accumulation and activation of monocytes through receptor for advanced glycation end products (RAGE) and Toll-like receptor 2, leading to local delivery of monocyte-derived tissue factor and cytokines. Moreover, disulfide HMGB1 facilitates formation of prothrombotic neutrophil extracellular traps (NETs) mediated by RAGE, exposing additional HMGB1 on their extracellular DNA strands. Eventually, a vicious circle of coagulation and inflammation is set in motion leading to obstructive DVT formation. Therefore, platelet-derived disulfide HMGB1 is a central mediator of the sterile inflammatory process in venous thrombosis and could be an attractive target for an anti-inflammatory approach for DVT prophylaxis.

Critical role of platelet glycoprotein ibα in arterial remodeling.

OBJECTIVE: Arteriogenesis is strongly dependent on the recruitment of leukocytes, especially monocytes, into the perivascular space of growing collateral vessels. On the basis of previous findings that platelets are central players in inflammatory processes and mediate the recruitment of leukocytes, the aim of this study was to assess the role of platelets in a model of arterial remodeling. APPROACH AND RESULTS: C57Bl6 wild-type mice, IL4-R/Iba mice lacking the extracellular domain of the glycoprotein Ibα (GPIbα) receptor, and mice treated with antibodies to block GPIbα or deplete circulating platelets were studied in peripheral arteriogenesis. Using a novel model of intravital 2-photon and epifluorescence imaging, we visualized and quantified the interaction of platelets with leukocytes and the vascular endothelium in vivo. We found that transient platelet adhesion to the endothelium of collateral vessels was a major event during arteriogenesis and depended on GPIbα. Furthermore, leukocyte recruitment was obviously affected in animals with defective platelet GPIbα function. In IL4-R/Iba mice, transient and firm leukocyte adhesion to the endothelium of collateral vessels, as well as leukocyte accumulation in the perivascular space, were significantly reduced. Furthermore, we detected platelet-leukocyte aggregates within the circulation, which were significantly reduced in IL4-R/Iba animals. Finally, platelet depletion and loss of GPIbα function resulted in poor reperfusion recovery as determined by laser Doppler imaging. CONCLUSIONS: Thus, GPIbα-mediated interactions between platelets and endothelial cells, as well as leukocytes, support innate immune cell recruitment and promote arteriogenesis-establishing platelets as critical players in this process.

The surface chemistry determines the spatio-temporal interaction dynamics of quantum dots in atherosclerotic lesions.

AIM: To optimize the design of nanoparticles for diagnosis or therapy of vascular diseases, it is mandatory to characterize the determinants of nano-bio interactions in vascular lesions. MATERIALS & METHODS: Using ex vivo and in vivo microscopy, we analyzed the interactive behavior of quantum dots with different surface functionalizations in atherosclerotic lesions of ApoE-deficient mice. RESULTS: We demonstrate that quantum dots with different surface functionalizations exhibit specific interactive behaviors with distinct molecular and cellular components of the injured vessel wall. Moreover, we show a role for fibrinogen in the regulation of the spatio-temporal interaction dynamics in atherosclerotic lesions. CONCLUSION: Our findings emphasize the relevance of surface chemistry-driven nano-bio interactions on the differential in vivo behavior of nanoparticles in diseased tissue.

Dual-Track Clearance of Circulating Bacteria Balances Rapid Restoration of Blood Sterility with Induction of Adaptive Immunity.

Efficient clearance of bacteremia prevents life-threatening disease. Platelet binding to intravascular bacteria, a process involving platelet glycoprotein GPIb and bacterial opsonization with activated complement C3, influences blood clearance and anti-infective immunity. Using intravital microscopy of the bloodstream of mice infected with Listeria monocytogenes, we show that bacterial clearance is not a uniform process but a "dual-track" mechanism consisting of parallel "fast" and "slow" pathways. "Slow clearance" is regulated by time-dependent bacterial opsonization, stochastic platelet binding, and capture of bacteria-platelet-complexes via the complement receptor of the immunoglobulin superfamily, CRIg. The mechanism spares some bacteria from "fast clearance" and rapid destruction in the liver via Kupffer cell scavenger receptors, keeping them available for adaptive immunity induction by splenic CD8α(+) dendritic cells. We consistently find "fast" and "slow" clearance patterns for a broad panel of other Gram+ and Gram- bacteria. Thus, dual-track clearance balances rapid restoration of blood sterility with induction of specific antibacterial immunity.

Modulating CD4+ T cell migration in the postischemic liver: hepatic stellate cells as new therapeutic target?

BACKGROUND: CD4+ T cells play a critical role during hepatic ischemia-reperfusion (I/R) injury although the mechanisms of their migration in the postischemic liver remain unclear. We answered the questions of whether hepatic stellate cells (HSCs) interact with CD4+ T cells during I/R of the liver and whether modulation of HSC activity affects T cell-dependent I/R injury. METHODS: In mice, migration of CD4+ T cells was analyzed in vivo using conventional intravital microscopy and two-photon microscopy. CD4+ T cell-HSC interactions were visualized after infusion of fluorescence-labeled CD4+ T cells into Cx3CR1 mice (mice exhibiting GFP-labeled HSCs) after I/R. Because the activation of HSC is controlled by endocannabinoid receptors, CB-1 and CB-2, the mice received treatment before I/R with the CB-2 agonist JWH-133 to reach HSC depletion or the CB-1 agonist arachidonylcyclopropylamide to activate HSCs. Sinusoidal perfusion and liver transaminases were used as markers of I/R injury. RESULTS: Hepatic I/R induced CD4+ T cell recruitment in sinusoids. More than 25% of adherent CD4+ T cells were colocalized with HSCs during reperfusion, suggesting a direct cell-cell interaction. The HSC deactivation with JWH-133 significantly attenuated the CD4+ T cell recruitment in the postischemic liver and reduced I/R injury as compared to the vehicle-treated group. The HSC hyperactivation by CB-1, however, did not affect T-cell migration and even increased perfusion failure. CONCLUSION: Our in vivo data suggest that CD4+ T cells interact with HSCs on their migration into the hepatic parenchyma, and a depletion or deactivation of HSCs protects the liver from T cell-dependent I/R injury.

Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo.

Deep vein thrombosis (DVT) is a major cause of cardiovascular death. The sequence of events that promote DVT remains obscure, largely as a result of the lack of an appropriate rodent model. We describe a novel mouse model of DVT which reproduces a frequent trigger and resembles the time course, histological features, and clinical presentation of DVT in humans. We demonstrate by intravital two-photon and epifluorescence microscopy that blood monocytes and neutrophils crawling along and adhering to the venous endothelium provide the initiating stimulus for DVT development. Using conditional mutants and bone marrow chimeras, we show that intravascular activation of the extrinsic pathway of coagulation via tissue factor (TF) derived from myeloid leukocytes causes the extensive intraluminal fibrin formation characteristic of DVT. We demonstrate that thrombus-resident neutrophils are indispensable for subsequent DVT propagation by binding factor XII (FXII) and by supporting its activation through the release of neutrophil extracellular traps (NETs). Correspondingly, neutropenia, genetic ablation of FXII, or disintegration of NETs each confers protection against DVT amplification. Platelets associate with innate immune cells via glycoprotein Ibα and contribute to DVT progression by promoting leukocyte recruitment and stimulating neutrophil-dependent coagulation. Hence, we identified a cross talk between monocytes, neutrophils, and platelets responsible for the initiation and amplification of DVT and for inducing its unique clinical features.