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Background: Immediate activated factor (F)X (FXa) inhibition exerts direct antiplatelet effects in the context of arterial thrombosis but little is known about the impact of long-term therapy on platelet function in ischemic cardiovascular diseases., Objectives: Therefore, we analyzed platelet-derived effects of long-term FXa inhibition in the setting of acute myocardial infarction (AMI) and stroke., Methods: We evaluated the effect of acute versus chronic FXa inhibition on thromboinflammation following AMI and stroke in mice in vivo. Mechanistically, we identified changes in platelet gene expression and proteome under chronic FXa nonvitamin K antagonist oral anticoagulant treatment and characterized its functional consequence on platelet physiology. In a prospectively recruited cohort of patients with AMI, we determined cardiovascular magnetic resonance based cardiac endpoints under FXa nonvitamin K antagonist oral anticoagulant effects on clinical endpoints in a cohort of patients with AMI., Results: Chronic but not acute FXa inhibition reduced cerebral and myocardial infarct size and improved cardiac function 24 hours after AMI in mice. Mechanistically, we identified an attenuated thromboinflammatory response with reduced neutrophil extracellular trap formation in mice and patient samples. Proteome and RNA expression analysis of FXa inhibitor treated patients revealed a reduction of key regulators within the membrane trafficking and secretion machinery hampering platelet α and dense granule release. Subsequent, thromboinflammatory neutrophil extracellular trap density in thrombi isolated from stroke and myocardial infarction patients was reduced. Patients with AMI treated with FXa inhibitors showed decreased infarct size after myocardial infarction compared to patients without anticoagulation treatment., Conclusion: Long-term FXa inhibition induces antithromboinflammatory proteome signatures in platelets, improving infarct size after myocardial infarction and stroke., Competing Interests: Declaration of Competing Interests There are no competing interests to disclose., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening syndrome fueled by uncontrolled mononuclear phagocyte activity, yet the innate immune mechanisms driving HLH pathogenesis remain elusive. Germline gain-of-function (GOF) mutations in CARD11, a pivotal regulator of lymphocyte antigen receptor signaling, cause the lymphoproliferative disease B-cell expansion with NF-κB and T-cell anergy, which is frequently associated with HLH development. Given that CARD11 is physiologically expressed not only in lymphocytes but also in dendritic cells (DCs), we explored whether enforced CARD11 signaling in DCs contributes to immunopathology. We demonstrated that exclusive DC-intrinsic expression of CARD11-GOF in mice was sufficient to induce a lethal autoinflammatory syndrome that mimicked human HLH. Mechanistically, DC-intrinsic CARD11-GOF signaling triggered cell-autonomous inflammatory cytokine production via MALT1 paracaspase engagement. Genetic deletion of Malt1 in CARD11-GOF-expressing animals reversed the hyperinflammatory phenotype. These results highlight the significant role of enforced CARD11/MALT1 signaling in DCs as a contributor to HLH pathology and suggest potential therapeutic strategies for HLH treatment., Competing Interests: Competing interests statement:The authors declare no competing interest.

Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) in the vessel wall regulates blood pressure and cardiovascular hemodynamics. In this study, we generated conditional eNOS knock out (KO) mice characterized by a duplicated/inverted exon 2 flanked with two pairs of loxP regions (eNOS inv/inv ); a Cre-recombinase activity induces cell-specific reactivation of eNOS, as a result of a flipping of the inverted exon 2 (eNOS fl ). This work aimed to test the efficiency of the Cre-mediated cell-specific recombination and the resulting eNOS expression/function. As proof of concept, we crossed eNOS inv/inv mice with DeleterCre pos (DelCre pos ) mice, expressing Cre recombinase in all cells. We generated heterozygous eNOS fl/inv or homozygous eNOS fl/fl mice, and eNOS inv/inv littermate mice. We found that both eNOS fl/fl and eNOS fl/inv mice express eNOS and the overall expression level depends on the number of mutated alleles, while eNOS inv/inv mice did not show any eNOS expression. Vascular endothelial function was restored in eNOS fl/fl and eNOS fl/inv mice, as determined by ACh-dependent vasodilation of aortic rings. Cre-dependent reactivation of eNOS in eNOS fl/fl and eNOS fl/inv mice rescued eNOS inv/inv (phenotypically global eNOS KO) mice from hypertension. These findings demonstrate that eNOS expression is restored in eNOS fl/fl mice at comparable physiological levels of WT mice, and its functional activity is independent on the number of the reactivated alleles. Therefore, eNOS inv/inv mice are a useful model for studying the effects of conditional reactivation of eNOS and gene dosage effects in specific cells for gain-of-function studies., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Our immune system possesses sophisticated mechanisms to cope with invading microorganisms, while pathogens evolve strategies to deal with threats imposed by host immunity. Human plasma protein α1-antitrypsin (AAT) exhibits pleiotropic immune-modulating properties by both preventing immunopathology and improving antimicrobial host defence. Genetic associations suggested a role for AAT in candidemia, the most frequent fungal blood stream infection in intensive care units, yet little is known about how AAT influences interactions between Candida albicans and the immune system. Here, we show that AAT differentially impacts fungal killing by innate phagocytes. We observed that AAT induces fungal transcriptional reprogramming, associated with cell wall remodelling and downregulation of filamentation repressors. At low concentrations, the cell-wall remodelling induced by AAT increased immunogenic β-glucan exposure and consequently improved fungal clearance by monocytes. Contrastingly, higher AAT concentrations led to excessive C. albicans filamentation and thus promoted fungal immune escape from monocytes and macrophages. This underscores that fungal adaptations to the host protein AAT can differentially define the outcome of encounters with innate immune cells, either contributing to improved immune recognition or fungal immune escape.

Objectives: To assess treatment adherence, effectiveness and safety outcomes of patients with chronic lymphocytic leukaemia (CLL) receiving ibrutinib in a real-world setting., Methods: Patients enrolled in REALITY were ≥18 years with a confirmed diagnosis of CLL and were receiving ibrutinib as a first-line (1L), 2L or ≥3L therapy. Treatment retention, adherence, progression-free survival (PFS), overall survival (OS) and time to next therapy were assessed at 1 and 2 years overall, by typology and by cytogenetic subgroups. PFS and OS were analysed using Kaplan-Meier methods., Results: Exactly 302 patients were enrolled across 57 sites in Germany, from January 2017 to July 2021. One-year retention rates were 69.9% overall (primary endpoint), 77.9% for 1L patients, and 77.6%/78.8% for high-risk patients with del17p/TP53. At 2 years, PFS/OS rates were 77.8%/90.7% overall (1L, 82.7%/90.4%), and were consistent across cytogenetic subgroups. PFS rates were higher for 1L versus ≥3L patients. Patients with the low-acceptance/low-control typology at baseline were less likely to retain treatment at 1 year versus the high-acceptance/high-control typology. No new safety signals were observed., Conclusions: The REALITY study provides further evidence of the effectiveness and safety of ibrutinib in patients with CLL in a real-world setting, particularly in earlier treatment lines., (© 2024 The Authors. European Journal of Haematology published by John Wiley & Sons Ltd.)

Medicinal chemistry has discovered thousands of potent protein and lipid kinase inhibitors. These may be developed into therapeutic drugs or chemical probes to study kinase biology. Because of polypharmacology, a large part of the human kinome currently lacks selective chemical probes. To discover such probes, we profiled 1,183 compounds from drug discovery projects in lysates of cancer cell lines using Kinobeads. The resulting 500,000 compound-target interactions are available in ProteomicsDB and we exemplify how this molecular resource may be used. For instance, the data revealed several hundred reasonably selective compounds for 72 kinases. Cellular assays validated GSK986310C as a candidate SYK (spleen tyrosine kinase) probe and X-ray crystallography uncovered the structural basis for the observed selectivity of the CK2 inhibitor GW869516X. Compounds targeting PKN3 were discovered and phosphoproteomics identified substrates that indicate target engagement in cells. We anticipate that this molecular resource will aid research in drug discovery and chemical biology., (© 2023. The Author(s).)

Background: Macrophages play a pivotal role in vascular inflammation and predict cardiovascular complications. Fluorine-19 magnetic resonance imaging ( 19 F MRI) with intravenously applied perfluorocarbon allows a background-free direct quantification of macrophage abundance in experimental vascular disease models in mice. Recently, perfluorooctyl bromide-nanoemulsion (PFOB-NE) was applied to effectively image macrophage infiltration in a pig model of myocardial infarction using clinical MRI scanners. In the present proof-of-concept approach, we aimed to non-invasively image monocyte/macrophage infiltration in response to carotid artery angioplasty in pigs using 19 F MRI to assess early inflammatory response to mechanical injury., Methods: In eight minipigs, two different types of vascular injury were conducted: a mild injury employing balloon oversize angioplasty only (BA, n = 4) and a severe injury provoked by BA in combination with endothelial denudation (BA + ECDN, n = 4). PFOB-NE was administered intravenously three days after injury followed by 1 H and 19 F MRI to assess vascular inflammatory burden at day six. Vascular response to mechanical injury was validated using X-ray angiography, intravascular ultrasound and immunohistology in at least 10 segments per carotid artery., Results: Angioplasty was successfully induced in all eight pigs. Response to injury was characterized by positive remodeling with predominantly adventitial wall thickening and concomitant infiltration of monocytes/macrophages. No severe adverse reactions were observed following PFOB-NE administration. In vivo 19 F signals were only detected in the four pigs following BA + ECDN with a robust signal-to-noise ratio (SNR) of 14.7 ± 4.8. Ex vivo analysis revealed a linear correlation of 19 F SNR to local monocyte/macrophage cell density. Minimum detection limit of infiltrated monocytes/macrophages was estimated at approximately 410 cells/mm 2 ., Conclusions: In this proof-of-concept study, 19 F MRI enabled quantification of monocyte/macrophage infiltration after vascular injury with sufficient sensitivity. This may provide the opportunity to non-invasively monitor vascular inflammation with MRI in patients after angioplasty or even in atherosclerotic plaques., (© 2023. Society for Cardiovascular Magnetic Resonance.)

Although most cancer drugs modulate the activities of cellular pathways by changing posttranslational modifications (PTMs), little is known regarding the extent and the time- and dose-response characteristics of drug-regulated PTMs. In this work, we introduce a proteomic assay called decryptM that quantifies drug-PTM modulation for thousands of PTMs in cells to shed light on target engagement and drug mechanism of action. Examples range from detecting DNA damage by chemotherapeutics, to identifying drug-specific PTM signatures of kinase inhibitors, to demonstrating that rituximab kills CD20-positive B cells by overactivating B cell receptor signaling. DecryptM profiling of 31 cancer drugs in 13 cell lines demonstrates the broad applicability of the approach. The resulting 1.8 million dose-response curves are provided as an interactive molecular resource in ProteomicsDB.

Immune checkpoint inhibitors (ICIs) enhance anticancer immunity by releasing repressive signals into tumor microenvironments (TMEs). To be effective, ICIs require preexisting immunologically "hot" niches for tumor antigen presentation and lymphocyte recruitment. How the mutational landscape of cancer cells shapes these immunological niches remains poorly defined. We found in human and murine colorectal cancer (CRC) models that the superior antitumor immune response of mismatch repair (MMR)-deficient CRC required tumor cell-intrinsic activation of cGAS-STING signaling triggered by genomic instability. Subsequently, we synthetically enforced STING signaling in CRC cells with intact MMR signaling using constitutively active STING variants. Even in MMR-proficient CRC, genetically encoded gain-of-function STING was sufficient to induce cancer cell-intrinsic interferon signaling, local activation of antigen-presenting cells, recruitment of effector lymphocytes, and sensitization of previously "cold" TMEs to ICI therapy in vivo. Thus, our results introduce a rational strategy for modulating cancer cell-intrinsic programs via engineered STING enforcement to sensitize resistant tumors to ICI responsiveness.

Patients with acute ischemic stroke (AIS) present an increased incidence of systemic inflammatory response syndrome and release of Troponin T coinciding with cardiac dysfunction. The nature of the cardiocirculatory alterations remains obscure as models to investigate systemic interferences of the brain-heart-axis following AIS are sparse. Thus, this study aims to investigate acute cardiocirculatory dysfunction and myocardial injury in mice after reperfused AIS. Ischemic stroke was induced in mice by transient right-sided middle cerebral artery occlusion (tMCAO). Cardiac effects were investigated by electrocardiograms, 3D-echocardiography, magnetic resonance imaging (MRI), invasive conductance catheter measurements, histology, flow-cytometry, and determination of high-sensitive Troponin T (hsTnT). Systemic hemodynamics were recorded and catecholamines and inflammatory markers in circulating blood and myocardial tissue were determined by immuno-assay and flow-cytometry. Twenty-four hours following tMCAO hsTnT was elevated 4-fold compared to controls and predicted long-term survival. In parallel, systolic left ventricular dysfunction occurred with impaired global longitudinal strain, lower blood pressure, reduced stroke volume, and severe bradycardia leading to reduced cardiac output. This was accompanied by a systemic inflammatory response characterized by granulocytosis, lymphopenia, and increased levels of serum-amyloid P and interleukin-6. Within myocardial tissue, MRI relaxometry indicated expansion of extracellular space, most likely due to inflammatory edema and a reduced fluid volume. Accordingly, we found an increased abundance of granulocytes, apoptotic cells, and upregulation of pro-inflammatory cytokines within myocardial tissue following tMCAO. Therefore, reperfused ischemic stroke leads to specific cardiocirculatory alterations that are characterized by acute heart failure with reduced stroke volume, bradycardia, and changes in cardiac tissue and accompanied by systemic and local inflammatory responses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Vornholz, Nienhaus, Gliem, Alter, Henning, Lang, Ezzahoini, Wolff, Clasen, Rassaf, Flögel, Kelm, Gerdes, Jander and Bönner.)

Psoriasis is a chronic inflammatory skin disease arising from poorly defined pathological cross-talk between keratinocytes and the immune system. BCL10 (B cell lymphoma/leukemia 10) and MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) are ubiquitously expressed inflammatory signaling proteins that can interact with the psoriasis susceptibility factor CARD14, but their functions in psoriasis are insufficiently understood. We report that although keratinocyte-intrinsic BCL10/MALT1 deletions completely rescue inflammatory skin pathology triggered by germline Card14 gain-of-function mutation in mice, the BCL10/MALT1 signalosome is unexpectedly not involved in the CARD14-dependent interleukin-17 receptor (IL-17R) proximal pathway. Instead, it plays a more pleiotropic role by amplifying keratinocyte responses to a series of inflammatory cytokines, including IL-17A, IL-1β, and TNF. Moreover, selective keratinocyte-intrinsic activation of BCL10/MALT1 signaling with an artificial engager molecule is sufficient to initiate lymphocyte-mediated psoriasiform skin inflammation, and aberrant BCL10/MALT1 activity is frequently detected in the skin of human sporadic psoriasis. Together, these results establish that BCL10/MALT1 signalosomes can act as initiators and crucial amplifiers of psoriatic skin inflammation and indicate a critical function for this complex in sporadic psoriasis.

Background: Current paradigms suggest that nitric oxide (NO) produced by endothelial cells (ECs) through endothelial nitric oxide synthase (eNOS) in the vessel wall is the primary regulator of blood flow and blood pressure. However, red blood cells (RBCs) also carry a catalytically active eNOS, but its role is controversial and remains undefined. This study aimed to elucidate the functional significance of RBC eNOS compared with EC eNOS for vascular hemodynamics and nitric oxide metabolism., Methods: We generated tissue-specific loss- and gain-of-function models for eNOS by using cell-specific Cre-induced gene inactivation or reactivation. We created 2 founder lines carrying a floxed eNOS (eNOS flox/flox ) for Cre-inducible knockout (KO), and gene construct with an inactivated floxed/inverted exon (eNOS inv/inv ) for a Cre-inducible knock-in (KI), which respectively allow targeted deletion or reactivation of eNOS in erythroid cells (RBC eNOS KO or RBC eNOS KI mice) or in ECs (EC eNOS KO or EC eNOS KI mice). Vascular function, hemodynamics, and nitric oxide metabolism were compared ex vivo and in vivo., Results: The EC eNOS KOs exhibited significantly impaired aortic dilatory responses to acetylcholine, loss of flow-mediated dilation, and increased systolic and diastolic blood pressure. RBC eNOS KO mice showed no alterations in acetylcholine-mediated dilation or flow-mediated dilation but were hypertensive. Treatment with the nitric oxide synthase inhibitor N -nitro-l-arginine methyl ester further increased blood pressure in RBC eNOS KOs, demonstrating that eNOS in both ECs and RBCs contributes to blood pressure regulation. Although both EC eNOS KOs and RBC eNOS KOs had lower plasma nitrite and nitrate concentrations, the levels of bound NO in RBCs were lower in RBC eNOS KOs than in EC eNOS KOs. Reactivation of eNOS in ECs or RBCs rescues the hypertensive phenotype of the eNOS γ -nitro-l-arginine methyl ester further increased blood pressure in RBC eNOS KOs, demonstrating that eNOS in both ECs and RBCs contributes to blood pressure regulation. Although both EC eNOS KOs and RBC eNOS KOs had lower plasma nitrite and nitrate concentrations, the levels of bound NO in RBCs were lower in RBC eNOS KOs than in EC eNOS KOs. Reactivation of eNOS in ECs or RBCs rescues the hypertensive phenotype of the eNOS inv/inv mice, whereas the levels of bound NO were restored only in RBC eNOS KI mice., Conclusions: These data reveal that eNOS in ECs and RBCs contribute independently to blood pressure homeostasis.

Caspase recruitment domain protein 9 (CARD9) forms essential signaling complexes in the innate immune system that integrate cues from C-type lectin receptors and specific intracellular pattern recognition receptors. These CARD9-mediated signals are pivotal for host defense against fungi, and they mediate immunity against certain bacteria, viruses and parasites. Furthermore, CARD9-regulated pathways are involved in sterile inflammatory responses critical for immune homeostasis and can control pro- and antitumor immunity in cancer microenvironments. Consequently, multiple genetic alterations of human CARD9 are connected to primary immunodeficiencies or prevalent inflammatory disorders in patients. This review will summarize our current understanding of CARD9 signaling in the innate immune system, its physiological and pathological functions and their implications for human immune-mediated diseases.

Myeloid cells contribute to tumor progression, but how the constellation of receptors they express regulates their functions within the tumor microenvironment (TME) is unclear. We demonstrate that Fcmr (Toso), the putative receptor for soluble IgM, modulates myeloid cell responses to cancer. In a syngeneic melanoma model, Fcmr ablation in myeloid cells suppressed tumor growth and extended mouse survival. Fcmr deficiency increased myeloid cell population density in this malignancy and enhanced anti-tumor immunity. Single-cell RNA sequencing of Fcmr-deficient tumor-associated mononuclear phagocytes revealed a unique subset with enhanced antigen processing/presenting properties. Conversely, Fcmr activity negatively regulated the activation and migratory capacity of myeloid cells in vivo, and T cell activation by bone marrow-derived dendritic cells in vitro. Therapeutic targeting of Fcmr during oncogenesis decreased tumor growth when used as a single agent or in combination with anti-PD-1. Thus, Fcmr regulates myeloid cell activation within the TME and may be a potential therapeutic target.