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BackgroundThe IκB kinase (IKK) complex, comprising the two enzymes IKKα and IKKβ, is the main activator of the inflammatory transcription factor NF-κB, which is constitutively active in many cancers. While several connections between NF-κB signaling and the oncogene c-Myc have been shown, functional links between the signaling molecules are still poorly studied.MethodsMolecular interactions were shown by co-immunoprecipitation and FRET microscopy. Phosphorylation of c-Myc was shown by kinases assays and its activity by improved reporter gene systems. CRISPR/Cas9-mediated gene knockout and chemical inhibition were used to block IKK activity. The turnover of c-Myc variants was determined by degradation in presence of cycloheximide and by optical pulse-chase experiments.. Immunofluorescence of mouse prostate tissue and bioinformatics of human datasets were applied to correlate IKKα- and c-Myc levels. Cell proliferation was assessed by EdU incorporation and apoptosis by flow cytometry.ResultsWe show that IKKα and IKKβ bind to c-Myc and phosphorylate it at serines 67/71 within a sequence that is highly conserved. Knockout of IKKα decreased c-Myc-activity and increased its T58-phosphorylation, the target site for GSK3β, triggering polyubiquitination and degradation. c-Myc-mutants mimicking IKK-mediated S67/S71-phosphorylation exhibited slower turnover, higher cell proliferation and lower apoptosis, while the opposite was observed for non-phosphorylatable A67/A71-mutants. A significant positive correlation of c-Myc and IKKα levels was noticed in the prostate epithelium of mice and in a variety of human cancers.ConclusionsOur data imply that IKKα phosphorylates c-Myc on serines-67/71, thereby stabilizing it, leading to increased transcriptional activity, higher proliferation and decreased apoptosis.

Antibody-mediated neutralization of pathogens is a key function of adaptive immunity. Immunoglobulin G (IgG) molecules bind to invading pathogens with high affinity and specificity. Platelets are known to contain IgG in their α-granules1; however, the biological significance of this antibody reservoir is unclear. In addition to their primary role in hemostasis, platelets are active and autonomous participants in host immune responses to bacterial and viral infections.2 Platelets express various receptors, such as Toll-like receptors 2 and 7, for direct responses to viruses, including cytomegalovirus (CMV) and influenza A virus (IAV).3-7 Because of their high sensitivity, rapid responsiveness, and sheer number, platelets function as sentinels and as the first line of defense against invading pathogens. Upon virus recognition, platelets contribute to pathogen clearance by engulfing virus particles and modulating leukocyte effector function.5,8,9 Although platelet-derived antimicrobial peptides restrict bacterial infection,10 the impact of platelet-derived IgG is unknown. Here, we investigated platelet effects on virus neutralization via IgG release and found an unexplored mechanism to potentiate humoral immunity, which might be used for platelet-based drug-delivery therapy.

Background and aims Inflammatory activation of endothelial cells is considered to be the first step in the development of atherosclerosis. Here, we determined the consequences of chronic endothelial activation via the NF-κB activator Ikk2 (Inhibitor of nuclear factor kappa-B kinase 2, Ikk-beta) on the development and progression of atherosclerosis. Methods We established a conditional transgenic mouse model, expressing a tamoxifen-inducible, constitutively active form of Ikk2 exclusively in arterial endothelial cells (caIkk2EC mice) on an ApoE-deficient background. Mice were fed a Western-type diet and endothelial Ikk2 was activated either at early or late stages of atherosclerosis. Results En face preparations of isolated aortas revealed a significant increase in plaque area in caIkk2EC mice at 12 weeks of Western-type diet as compared to ApoE-deficient littermates. This was accompanied by increased infiltration of macrophages and T cells into the lesion. Several chemokine/cytokine and immune cell pathways were significantly upregulated in the aortic transcriptome of caIkk2EC mice. Of note, in mice with established atherosclerosis, activation of endothelial Ikk2 still further accelerated progression of atherosclerosis. This indicates that inflammatory endothelial activation is crucial during all stages of the disease. Conclusions Our results show for the first time that chronic inflammatory activation of arterial endothelial cells accelerates the development and progression of atherosclerosis both at early and late stages of disease development. Thus, pharmacological targeting of endothelial inflammation emerges as a promising treatment approach.

Vascular disease is a progressive inflammatory condition fuelled by an unhealthy lifestyle of physical inactivity, cholesterol-rich diet, and smoking. Together with endogenous factors such as age, gender, and autoimmune status, an unhealthy lifestyle fosters a pro-inflammatory and pro-thrombotic milieu, which can lead to endothelial dysfunction, atherosclerotic plaque formation and vascular obstruction or degradation of the subendothelial matrix. Platelet-leukocyte interplay represents an important feature in this context. Platelets get activated in a pro-inflammatory and pro-thrombotic microenvironment and readily interact with innate and adaptive immune cells alike. Even though platelet affinity for physical cell-cell contact is highest with monocytes/macrophages and neutrophils, platelets also avidly interact with lymphocytes by soluble mediators. Platelet-leukocyte crosstalk regulates essential immune responses, supporting leukocyte recruitment at sites of vascular insult, promoting proliferation and differentiation of leukocytes and enhancing pro-inflammatory effector functions such as cytokine and reactive oxygen production. However, under certain conditions platelet-leukocyte interplay also dampens the inflammatory process. Crosstalk of platelet and leukocytes thus represents a driving force in vascular disease. In this review, we highlight the impact of various risk factors for vascular disease on platelet-leukocyte interactions and discuss the underlying mechanisms of platelet-mediated changes in immune responses and the effect of immune cells on the haemostatic system. As the underlying pathologies differ between vascular diseases, we summarize our current knowledge on platelet-leukocyte interplay in chronic vascular diseases such as abdominal aortic aneurysm, peripheral and coronary artery disease as well as acute vascular diseases such as ischaemic stroke and venous thromboembolism.

Introduction Blood platelets are increasingly recognized as modulators of leukocyte effector functions in various pathologies including acute lung injury (ALI). ALI is a life-threatening disease, caused by damage to the alveolar epi- and endothelium. Excessive accumulation of leukocytes leads to severe lung inflammation, resulting in impaired lung function and hypoxemia. Objective Since leukocyte migration is modulated by activated platelets and phosphatidylinositol 3-kinase (PI3K) signaling is involved in platelet function, we aimed to elucidate the effect of PI3K on platelet-mediated immune responses. Materials and Methods We generated a mouse model with a platelet-specific deletion of p85α, the most important regulatory subunit of the class IA PI3K, and evaluated platelet function and platelet–leukocyte interactions. Moreover, we analyzed the impact of platelet-specific p85α gene deficiency during sterile peritonitis and acid-induced ALI. Results In vitro analyses of platelets revealed that lack of p85α led to decreased downstream signaling and diminished expression of surface activation markers, for example, CD62P and CD63, as well as reduced platelet aggregation. Moreover, platelet PI3K essentially mediated direct interactions of platelets with monocytes and neutrophils. In mice, platelet-specific p85α deficiency prevented leukocyte infiltration into the peritoneum and the bronchoalveolar compartment during sterile peritonitis and ALI, respectively. Additionally, the release of the inflammatory cytokine interleukin-12/23 was diminished in platelet p85α-deficient mice during ALI. In contrast to PI3K, neither overexpression nor depletion of platelet phosphatase and tensin homolog, the endogenous antagonist of PI3K, significantly modulated platelet function. Conclusion Our data indicate a crucial role of platelet PI3K signaling for leukocyte extravasation upon inflammatory stimuli in various diseases models.

Background: Fabry disease is a hereditary genetic defect resulting in reduced activity of the enzyme α-galactosidase-A and the accumulation of globotriaosylceramide (Gb3) in body fluids and cells. Gb3 accumulation was especially reported for the vascular endothelium in several organs. Methods: Three Fabry disease patients were screened using a micro-RNA screen. An in vitro approach in human endothelial cells was used to determine miRNA regulation by Gb3. Results: In a micro-RNA screen of three Fabry patients undergoing enzyme replacement therapy, we found that miRNAs let-7a and let-7d were significantly increased after therapy. We demonstrate in vitro in endothelial cells that Gb3 induced activation of NF-κB and activated downstream targets. In addition, NF-κB activity directly reduced let-7a and let-7d miRNA expression as inhibiting NF-kB nuclear entry abolished the Gb3 effects. Conclusion: We suggest that let-7a and let-7d are potential markers for enzyme activity and inflammation in Fabry disease patients.

Background Inflammation is a key process during atherosclerotic lesion development and propagation. Recent evidence showed clearly that especially the inhibition of interleukin (IL)-1β reduced atherosclerotic adverse events in human patients. Fatty acid oxidation (FAO) was previously demonstrated to interact with the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) pathway which is required for mature IL-1β secretion. To understand possible anti-inflammatory properties of FAO inhibition, we tested the effect of pharmacological FAO inhibition using the inhibitor for long-chain 3-ketoacyl coenzyme A thiolase trimetazidine on atherosclerotic plaque development and inflammation. Experimental Approach The effect of FAO inhibition was determined in LDL-R−/− male mice on a C57/BL6 background. In vitro effects of trimetazidine treatment were analyzed in human umbilical vein endothelial cells and human monocyte derived macrophages. Key Results We were able to demonstrate that inhibition of FAO reduced atherosclerotic plaque growth. We did not find direct anti-inflammatory properties of trimetazidine in endothelial cells or macrophages in vitro. However, we found that the activation of the NLRP3 system and the secretion of IL-1β were significantly reduced in macrophages after FAO inhibition. These results were confirmed in atherosclerotic lesions of mice treated with trimetazidine as they showed a significant reduction of IL-1β and cleaved caspase-1 in the atherosclerotic lesion as well as of IL-1β and IL-18 in the circulation. Conclusion Overall, we therefore suggest that the main mechanism of reducing inflammation of trimetazidine and FAO inhibition is the reduction of the NLRP-3 activation leading to reduced levels of the proinflammatory cytokine IL-1β.

Thrombospondin-1 (TSP-1) is primarily expressed by platelets and endothelial cells (ECs) and rapidly released upon their activation. It functions in haemostasis as a bridging molecule in platelet aggregation, by promoting platelet adhesion to collagen and by protecting von Willebrand factor strings from degradation. In blood of patients undergoing surgery and in co-cultures of neutrophils with platelets or ECs, we observed proteolysis of the 185 kDa full-length TSP-1 to a 160-kDa isoform. We hypothesized that TSP-1 processing may alter its haemostatic properties. Selective enzyme inhibitors in co-cultures revealed that neutrophil proteases elastase and cathepsin G mediate TSP-1 processing. The cut site of cathepsin G was mapped to TSP-1 amino acids R237/T238 by Edman sequencing. Formation of neutrophil extracellular traps protected TSP-1 from complete degradation and promoted controlled processing to the 160-kDa isoform. Haemostatic properties were tested by platelet aggregation, adhesion, coagulation and string formation under flow. Platelets from TSP-1 deficient mice did not differ from wild-type in platelet aggregation but showed severe impairment of platelet adhesion to collagen and string formation under flow. Reconstitution experiments revealed that the 160-kDa TSP-1 isoform was markedly more potent than the 185-kDa full-length molecule in restoring function. Thus, TSP-1 processing by neutrophil proteases yields a 160-kDa isoform which shows enhanced potency to promote platelet adhesion and string formation. This finding reveals a novel mechanism of neutrophil-mediated thrombus formation and provides first evidence for the impact of TSP-1 proteolysis on its haemostatic properties.

Aim Periodontitis results in platelet activation and enhanced risk for cardiovascular disease. As it is currently unknown whether periodontal treatment reverses platelet hyper-reactivity, we aimed to investigate the role of periodontal treatment on platelet activation. Materials and methods In a prospective controlled therapeutic trial, 52 patients were enrolled and randomly selected for periodontal treatment or monitored without treatment for 3 months. Patient blood was analysed by flow cytometry for platelet activation markers and by light transmission aggregometry for platelet aggregation in response to pro-thrombotic stimuli. Results In this study, platelet activation in the control group aggravated over the observation period of 3 months, whereas patients that underwent periodontal treatment showed unchanged levels of platelet activation, measured by surface expression of CD62P, CD40L, generation of reactive oxygen production, activation of GPIIb/IIIa and fibrinogen binding. Moreover, platelet turnover, measured by platelet RNA content and platelet aggregation in response to collagen, differed significantly between patients that were treated and those who were untreated. Conclusions Subgingival debridement reduces the risk of aggravated platelet activation and therefore might potentially diminish subsequent diseases such as cardiovascular disease in periodontal patients.

Essentials Androgen deprivation increases the rate of venous thromboembolism in prostate cancer patients. We characterized androgen receptor-mediated tissue factor regulation in prostate epithelial cells. Androgen receptor is dampening tissue factor expression in prostate epithelial cells. Androgen deprivation could enhance tissue factor expression and raise venous thromboembolism rates. Summary Background Prostate cancer is one of the leading causes of cancer death in men. Advanced prostate cancer is usually treated by androgen deprivation therapy (ADT), which is aimed at reducing circulating testosterone levels to reduce cancer growth. There is growing evidence that ADT can increase the rate of venous thromboembolism (VTE) in prostate cancer patients. The tissue factor (TF) gene is one of the most important mediators of coagulation and VTE, but, so far, there are limited data on androgen receptor (AR)-mediated TF gene expression. Objectives To characterize AR-mediated TF regulation in vitro and in vivo. Methods We used the androgen-dependent prostate cancer cell lines LNCaP and MyC-CaP to test whether TF expression is regulated by AR. Furthermore, we cloned the TF gene promoter into a luciferase reporter vector to identify the transcription factor-binding sites that mediate TF regulation downstream of AR. Finally, we used castration experiments in mice to characterize AR-mediated TF regulation in vivo. Results TF is directly regulated by AR. In LNCaP cells, nuclear factor-κB signaling and EGR1 mediate TF expression. By using castration experiments in mice, we could detect upregulation of TF and early growth response protein 1 mRNA and protein expression in prostate epithelial cells. Conclusion AR is crucial for dampening TF expression, which could be important for increased TF expression and TF-positive microvesicle release in androgen-deprived prostate cancer patients.

Platelets, besides their specialised role in haemostasis and atherothrombosis, actively modulate innate and adaptive immune responses with crucial roles in immune surveillance, inflammation and host defence during infection. An important prerequisite for platelet-mediated changes of immune functions involves direct engagement with different types of leukocytes. Indeed, increased platelet-leukocyte aggregates (PLAs) within the circulation and/or locally at the site of inflammation represent markers of many thrombo-inflammatory diseases, such as cardiovascular diseases, acute lung injury, renal and cerebral inflammation. Therefore, measurement of PLAs could provide an attractive and easily accessible prognostic and/or diagnostic tool for many diseases. To measure PLAs in different (patho-)physiological settings in human and animal models flow cytometric and microscopic approaches have been applied. These techniques represent complementary tools to study different aspects relating to the involvement of leukocyte subtypes and molecules, as well as location of PLAs within tissues, dynamics of their interactions and/or dynamic changes in leukocyte and platelet behaviour. This review summarises various approaches to measure and interpret PLAs and discusses potential experimental factors influencing platelet binding to leukocytes. Furthermore, we summarise insights gained from studies regarding the underlying mechanism of platelet-leukocyte interactions and discuss implications of these interactions in health and disease.

Platelets are small anucleate cells that release a plethora of molecules to ensure functional hemostasis. It has been reported that IκB kinase 2 (IKK2), the central enzyme of the inflammatory NF-κB pathway, is involved in platelet activation, because megakaryocyte/platelet-specific deletion of exons 6 and 7 of IKK2 resulted in platelet degranulation defects and prolonged bleeding. We aimed to investigate the role of IKK2 in platelet physiology in more detail, using a platelet-specific IKK2 knockout via excision of exon 3, which makes up the active site of the enzyme. We verified the deletion on genomic and transcriptional levels in megakaryocytes and were not able to detect any residual IKK2 protein; however, platelets from these mice did not show any functional impairment in vivo or in vitro. Bleeding time and thrombus formation were not affected in platelet-specific IKK2-knockout mice. Moreover, platelet aggregation, glycoprotein GPIIb/IIIa activation, and degranulation were unaltered. These observations were confirmed by pharmacological inhibition of IKK2 with TPCA-1 and BMS-345541, which did not affect activation of murine or human platelets over a wide concentration range. Altogether, our results imply that IKK2 is not essential for platelet function.

In response to injury, adult Schwann cells (SCs) re-enter the cell cycle, change their expression profile, and exert repair functions important for wound healing and the re-growth of axons. While this phenotypical instability of SCs is essential for nerve regeneration, it has also been implicated in cancer progression and de-myelinating neuropathies. Thus, SCs became an important research tool to study the molecular mechanisms involved in repair and disease and to identify targets for therapeutic intervention. A high purity of isolated SC cultures used for experimentation must be demonstrated to exclude that novel findings are derived from a contaminating fibroblasts population. In addition, information about the SC proliferation status is an important parameter to be determined in response to different treatments. The evaluation of SC purity and proliferation, however, usually depends on the time consuming, manual assessment of immunofluorescence stainings or comes with the sacrifice of a large amount of SCs for flow cytometry analysis. We here show that rat SC culture derived cytospins stained for SC marker SOX10, proliferation marker EdU, intermediate filament vimentin and DAPI allowed the determination of SC identity and proliferation by requiring only a small number of cells. Furthermore, the CellProfiler software was used to develop an automated image analysis pipeline that quantified SCs and proliferating SCs from the obtained immunofluorescence images. By comparing the results of total cell count, SC purity and SC proliferation rate between manual counting and the CellProfiler output, we demonstrated applicability and reliability of the established pipeline. In conclusion, we here combined the cytospin technique, a multi-colour immunofluorescence staining panel, and an automated image analysis pipeline to enable the quantification of SC purity and SC proliferation from small cell aliquots. This procedure represents a solid read-out to simplify and standardize the quantification of primary SC culture purity and proliferation.

Beyond their traditional role in haemostasis and thrombosis, platelets are increasingly recognised as immune modulatory cells. Activated platelets and platelet-derived microparticles can bind to leukocytes, which stimulates mutual activation and results in rapid, local release of platelet-derived cytokines. Thereby platelets modulate leukocyte effector functions and contribute to inflammatory and immune responses to injury or infection. Platelets enhance leukocyte extravasation, differentiation and cytokine release. Platelet-neutrophil interactions boost oxidative burst, neutrophil extracellular trap formation and phagocytosis and play an important role in host defence. Platelet interactions with monocytes propagate their differentiation into macrophages, modulate cytokine release and attenuate macrophage functions. Depending on the underlying pathology, platelets can enhance or diminish leukocyte cytokine production, indicating that platelet-leukocyte interactions represent a fine balanced system to restrict excessive inflammation during infection. In atherosclerosis, platelet interaction with neutrophils, monocytes and dendritic cells accelerates key steps of atherogenesis by promoting leukocyte extravasation and foam cell formation. Platelet-leukocyte interactions at sites of atherosclerotic lesions destabilise atherosclerotic plaques and promote plaque rupture. Leukocytes in turn also modulate platelet function and production, which either results in enhanced platelet destruction or increased platelet production. This review aims to summarise the key effects of platelet-leukocyte interactions in inflammation, infection and atherosclerosis.

Transfusion of platelet concentrates represents an important treatment for various bleeding complications. However, the short half-life and frequent contaminations with bacteria restrict the availability of platelet concentrates and raise a clear demand for platelets generated ex vivo. Therefore, in vitro platelet generation from megakaryocytes represents an important research topic. A vital step for this process represents accurate analysis of thrombopoiesis and proplatelet formation, which is usually conducted manually. We aimed to develop a novel method for automated classification and analysis of proplatelet-forming megakaryocytes in vitro. After fluorescent labelling of surface and nucleus, MKs were automatically categorized and analysed with a novel pipeline of the open source software CellProfiler. Our new workflow is able to detect and quantify four subtypes of megakaryocytes undergoing thrombopoiesis: proplatelet-forming, spreading, pseudopodia-forming and terminally differentiated, anucleated megakaryocytes. Furthermore, we were able to characterize the inhibitory effect of dasatinib on thrombopoiesis in more detail. Our new workflow enabled rapid, unbiased, quantitative and qualitative in-depth analysis of proplatelet formation based on morphological characteristics. Clinicians and basic researchers alike will benefit from this novel technique that allows reliable and unbiased quantification of proplatelet formation. It thereby provides a valuable tool for the development of methods to generate platelets ex vivo and to detect effects of drugs on megakaryocyte differentiation.

Idiopathic pulmonary fibrosis (IPF) is a life-threatening disease with limited treatment options. Additionally, the lack of a complete understanding of underlying immunological mechanisms underscores the importance of discovering novel options for therapeutic intervention. Since the PI3K/PTEN pathway in myeloid cells influences their effector functions, we wanted to elucidate how sustained PI3K activity induced by cell-type specific genetic deficiency of its antagonist PTEN modulates IPF, in a murine model of bleomycin-induced pulmonary fibrosis (BIPF). We found that myeloid PTEN deficient mice (PTEN(MyKO)), after induction of BIPF, exhibit increased TGF-β1 activation, mRNA expression of pro-collagens and lysyl oxidase as well as augmented collagen deposition compared to wild-type littermates, leading to enhanced morbidity and decreased survival. Analysis of alveolar lavage and lung cell composition revealed that PTEN(MyKO) mice exhibit reduced numbers of macrophages and T-cells in response to bleomycin, indicating an impaired recruitment function. Interestingly, we found dysregulated macrophage polarization as well as elevated expression and release of the pro-fibrotic cytokines IL-6 and TNF-α in PTEN(MyKO) mice during BIPF. This might point to an uncontrolled wound healing response in which the inflammatory as well as tissue repair mechanisms proceed in parallel, thereby preventing resolution and at the same time promoting extensive fibrosis.