Neutrophils promote venular thrombosis by shaping the rheological environment for platelet aggregation

In advanced inflammatory disease, microvascular thrombosis leads to the interruption of blood supply and provokes ischemic tissue injury. Recently, intravascularly adherent leukocytes have been reported to shape the blood flow in their immediate vascular environment. Whether these rheological effects are relevant for microvascular thrombogenesis remains elusive. Employing multi-channel in vivo microscopy, analyses in microfluidic devices, and computational modeling, we identified a previously unanticipated role of leukocytes for microvascular clot formation in inflamed tissue. For this purpose, neutrophils adhere at distinct sites in the microvasculature where these immune cells effectively promote thrombosis by shaping the rheological environment for platelet aggregation. In contrast to larger (lower-shear) vessels, this process in high-shear microvessels does not require fibrin generation or extracellular trap formation, but involves GPIb alpha-vWF and CD40-CD40L-dependent platelet interactions. Conversely, interference with these cellular interactions substantially compromises microvascular clotting. Thus, leukocytes shape the rheological environment in the inflamed venular microvasculature for platelet aggregation thereby effectively promoting the formation of blood clots. Targeting this specific crosstalk between the immune system and the hemostatic system might be instrumental for the prevention and treatment of microvascular thromboembolic pathologies, which are inaccessible to invasive revascularization strategies

Priming of Anti-tumor Immune Mechanisms by Radiotherapy Is Augmented by Inhibition of Heat Shock Protein 90

Radiotherapy is an essential part of multi-modal cancer therapy. Nevertheless, for certain cancer entities such as colorectal cancer (CRC) the indications of radiotherapy are limited due to anatomical peculiarities and high radiosensitivity of the surrounding normal tissue. The development of molecularly targeted, combined modality approaches may help to overcome these limitations. Preferably, such strategies should not only enhance radiation-induced tumor cell killing and the abrogation of tumor cell clonogenicity, but should also support the stimulation of anti-tumor immune mechanisms – a phenomenon which moved into the center of interest of preclinical and clinical research in radiation oncology within the last decade. The present study focuses on inhibition of heat shock protein 90 (HSP90) whose combination with radiotherapy has previously been reported to exhibit convincing therapeutic synergism in different preclinical cancer models. By employing in vitro and in vivo analyses, we examined if this therapeutic synergism also applies to the priming of anti-tumor immune mechanisms in model systems of CRC. Our results indicate that the combination of HSP90 inhibitor treatment and ionizing irradiation induced apoptosis in colorectal cancer cells with accelerated transit into secondary necrosis in a hyperactive Kras-dependent manner. During secondary necrosis, dying cancer cells released different classes of damage-associated molecular patterns (DAMPs) that stimulated migration and recruitment of monocytic cells in vitro and in vivo. Additionally, these dying cancer cell-derived DAMPs enforced the differentiation of a monocyte-derived antigen presenting cell (APC) phenotype which potently triggered the priming of allogeneic T cell responses in vitro. In summary, HSP90 inhibition – apart from its radiosensitizing potential – obviously enables and supports the initial steps of anti-tumor immune priming upon radiotherapy and thus represents a promising partner for combined modality approaches. The therapeutic performance of such strategies requires further in-depth analyses, especially for but not only limited to CRC

Plasminogen Activator Inhibitor-1 Promotes Neutrophil Infiltration and Tissue Injury on Ischemia–Reperfusion

Objective Ischemia-reperfusion (I/R) injury significantly contributes to organ dysfunction and failure after myocardial infarction, stroke, and transplantation. In addition to its established role in the fibrinolytic system, plasminogen activator inhibitor-1 has recently been implicated in the pathogenesis of I/R injury. The underlying mechanisms remain largely obscure. Approach and Results Using different in vivo microscopy techniques as well as ex vivo analyses and in vitro assays, we identified that plasminogen activator inhibitor-1 rapidly accumulates on microvascular endothelial cells on I/R enabling this protease inhibitor to exhibit previously unrecognized functional properties by inducing an increase in the affinity of 2 integrins in intravascularly rolling neutrophils. These events are mediated through low-density lipoprotein receptor-related protein-1 and mitogen-activated protein kinase-dependent signaling pathways that initiate intravascular adherence of these immune cells to the microvascular endothelium. Subsequent to this process, extravasating neutrophils disrupt endothelial junctions and promote the postischemic microvascular leakage. Conversely, deficiency of plasminogen activator inhibitor-1 effectively reversed leukocyte infiltration, microvascular dysfunction, and tissue injury on experimental I/R without exhibiting side effects on microvascular hemostasis. Conclusions Our experimental data provide novel insights into the nonfibrinolytic properties of the fibrinolytic system and emphasize plasminogen activator inhibitor-1 as a promising target for the prevention and treatment of I/R injury

B and T cell acute lymphoblastic leukemia evade chemotherapy at distinct sites in the bone marrow

Persistence of residual disease after induction chemotherapy is a strong predictor of relapse in acute lymphoblastic leukemia (ALL). The bone marrow microenvironment may support treatment escape. Using 3D fluorescence imaging of 10 primary ALL xenografts we identify sites of predilection in the bone marrow for resistance to induction with dexamethasone, vincristine and doxorubicin. We detect B-cell precursor ALL cells predominantly in the perisinusoidal space at early engraftment and after chemotherapy. The spatial distribution of T-ALL cells was more widespread with contacts to endosteum, nestin+ pericytes and sinusoids. Dispersion of T-ALL cells in the bone marrow increased under chemotherapeutic pressure. A subset of slowly dividing ALL cells was transiently detected upon short-term chemotherapy, but not at residual disease after chemotherapy, challenging the notion that ALL cells escape treatment by direct induction of a dormant state in the niche. These lineage-dependent differences point to niche interactions that may be more specifically exploitable to improve treatment

Cytotoxicity of crystals involves RIPK3-MLKL-mediated necroptosis

Crystals cause injury in numerous disorders, and induce inflammation via the NLRP3 inflammasome, however, it remains unclear how crystals induce cell death. Here we report that crystals of calcium oxalate, monosodium urate, calcium pyrophosphate dihydrate and cystine trigger caspase-independent cell death in five different cell types, which is blocked by necrostatin-1. RNA interference for receptor-interacting protein kinase 3 (RIPK3) or mixed lineage kinase domain like (MLKL), two core proteins of the necroptosis pathway, blocks crystal cytotoxicity. Consistent with this, deficiency of RIPK3 or MLKL prevents oxalate crystal-induced acute kidney injury. The related tissue inflammation drives TNF-alpha-related necroptosis. Also in human oxalate crystal-related acute kidney injury, dying tubular cells stain positive for phosphorylated MLKL. Furthermore, necrostatin-1 and necrosulfonamide, an inhibitor for human MLKL suppress crystal-induced cell death in human renal progenitor cells. Together, TNF-alpha/TNFR1, RIPK1, RIPK3 and MLKL are molecular targets to limit crystal-induced cytotoxicity, tissue injury and organ failure

Cytotoxicity of crystals involves RIPK3-MLKL-mediated necroptosis

Crystals cause injury in numerous disorders, and induce inflammation via the NLRP3 inflammasome, however, it remains unclear how crystals induce cell death. Here we report that crystals of calcium oxalate, monosodium urate, calcium pyrophosphate dihydrate and cystine trigger caspase-independent cell death in five different cell types, which is blocked by necrostatin-1. RNA interference for receptor-interacting protein kinase 3 (RIPK3) or mixed lineage kinase domain like (MLKL), two core proteins of the necroptosis pathway, blocks crystal cytotoxicity. Consistent with this, deficiency of RIPK3 or MLKL prevents oxalate crystal-induced acute kidney injury. The related tissue inflammation drives TNF-alpha-related necroptosis. Also in human oxalate crystal-related acute kidney injury, dying tubular cells stain positive for phosphorylated MLKL. Furthermore, necrostatin-1 and necrosulfonamide, an inhibitor for human MLKL suppress crystal-induced cell death in human renal progenitor cells. Together, TNF-alpha/TNFR1, RIPK1, RIPK3 and MLKL are molecular targets to limit crystal-induced cytotoxicity, tissue injury and organ failure

Platelets Guide Leukocytes to Their Sites of Extravasation

Effective immune responses require the directed migration of leukocytes from the vasculature to the site of injury or infection. How immune cells "find" their site of extravasation remains largely obscure. Here, we identified a previously unrecognized role of platelets as pathfinders guiding leukocytes to their exit points in the microvasculature: upon onset of inflammation, circulating platelets were found to immediately adhere at distinct sites in venular microvessels enabling these cellular blood components to capture neutrophils and, in turn, inflammatory monocytes via CD40-CD40L-dependent interactions. In this cellular crosstalk, ligation of PSGL-1 by P-selectin leads to ERK1/2 MAPK-dependent conformational changes of leukocyte integrins, which promote the successive extravasation of neutrophils and monocytes to the perivascular tissue. Conversely, blockade of this cellular partnership resulted in misguided, inefficient leukocyte responses. Our experimental data uncover a platelet-directed, spatiotemporally organized, multicellular crosstalk that is essential for effective trafficking of leukocytes to the site of inflammation

Endothelial expression of adhesion and signaling molecules, composition of the vascular wall, and shear rates in the venular microvasculature.

<p>Representative confocal microscopy images of ICAM-1/CD54, VCAM-1/CD106, ICAM-2/CD102, PECAM-1/CD31, JAM-A, and CCL2 expression in venular endothelial cells of the cremaster muscle of WT mice before (open dots) and after (filled dots) stimulation with CCL2 (<b>A</b>; scale bar 50 μm). Panels show quantitative expression levels of these proteins in dependency of the venular diameter as well as the corresponding venular shear rates (mean ± SEM; <i>n</i> = 3–4 per group; #<i>p</i> < 0.05 versus PBS). Panels (<b>B</b>) show representative images and quantitative data for the number of perivascular macrophages, pericytes, and collagen IV LERs of venular microvessels in dependency of the venular diameter (scale bar 50 μm; mean ± SEM; <i>n</i> = 3 per group).</p

Consequences of interactions of iMOs with platelets and neutrophils for the extravasation of iMOs.

<p>Interactions of intravascularly adherent neutrophils and iMOs were analyzed by multichannel in vivo microscopy. Panel shows quantitative data for interactions (>30 s) between intravascularly adherent neutrophils and iMOs in animals receiving blocking mAbs directed against CD62L or PSGL-1/CD162, or istoype control antibodies (<b>A</b>; mean ± SEM; <i>n</i> = 4 per group; *<i>p</i> < 0.05 versus isotype control; scale bar 10 μm). Binding of ICAM-1/CD54 or VCAM-1/CD106 to iMOs isolated from the peripheral blood of WT mice was assessed upon exposure to recombinant murine CD40L/CD154, P-selectin/CD62P, L-selectin/CD62L, or PSGL-1/CD162 by flow cytometry (<b>B</b>; mean ± SEM; <i>n</i> = 4–6 per group; #<i>p</i> < 0.05 versus unstimulated; *<i>p</i> < 0.05 versus isotype control or vehicle). P-selectin-elicited binding of ICAM-1/CD54 to murine iMOs was quantified upon blockade of PSGL-1/CD162 or inhibition of MAPK (<b>B</b>, upper panels). Conformational changes of β2 integrins in human iMOs were analyzed by using conformation-specific mAbs (mean ± SEM; <i>n</i> = 4 per group; #<i>p</i> < 0.05 versus unstimulated; <b>B,</b> lower panels). Using multichannel in vivo microscopy on the CCL2-stimulated cremaster muscle of CX₃CR1^GFP/+ mice, intravascular rolling flux and firm adherence of iMOs were analyzed. Extravasation of iMOs was evaluated by using a peritonitis assay. Panels (<b>C</b>) show quantitative data for CCL2-challenged animals receiving blocking mAbs directed against Mac-1/CD11b, LFA-1/CD11a, VLA-4/CD49d, ICAM-1/CD54, ICAM-2/CD102, VCAM-1/CD106, PECAM-1/CD31, or JAM-A, or isotype control antibodies (mean ± SEM; <i>n</i> = 4–6 per group; *<i>p</i> < 0.05 versus isotype control).</p