Formyl peptide receptor 2 regulates dendritic cell metabolism and Th17 cell differentiation during neuroinflammation.

Formyl peptide receptor 2 (FPR2) is a receptor for formylated peptides and specific pro-resolving mediators, and is involved in various inflammatory processes. Here, we aimed to elucidate the role of FPR2 in dendritic cell (DC) function and autoimmunity-related central nervous system (CNS) inflammation by using the experimental autoimmune encephalomyelitis (EAE) model. EAE induction was accompanied by increased Fpr2 mRNA expression in the spinal cord. FPR2-deficient (Fpr2 KO) mice displayed delayed onset of EAE compared to wild-type (WT) mice, associated with reduced frequencies of Th17 cells in the inflamed spinal cord at the early stage of the disease. However, FPR2 deficiency did not affect EAE severity after the disease reached its peak. FPR2 deficiency in mature DCs resulted in decreased expression of Th17 polarizing cytokines IL6, IL23p19, IL1ß, and thereby diminished the DC-mediated activation of Th17 cell differentiation. LPS-activated FPR2-deficient DCs showed upregulated Nos2 expression and nitric oxide (NO) production, as well as reduced oxygen consumption rate and impaired mitochondrial function, including decreased mitochondrial superoxide levels, lower mitochondrial membrane potential and diminished expression of genes related to the tricarboxylic acid cycle and genes related to the electron transport chain, as compared to WT DCs. Treatment with a NO inhibitor reversed the reduced Th17 cell differentiation in the presence of FPR2-deficient DCs. Together, by regulating DC metabolism, FPR2 enhances the production of DC-derived Th17-polarizing cytokines and hence Th17 cell differentiation in the context of neuroinflammation.

Neuro-immune crosstalk in hematopoiesis, inflammation, and repair.

Innate immune cells are primary effectors during host defense and in sterile inflammation. Their production in the bone marrow is tightly regulated by growth and niche factors, and their activity at sites of inflammation is orchestrated by a network of alarmins and cytokines. Yet, recent work highlights a significant role of the peripheral nervous system in these processes. Sympathetic neural pathways play a key role in regulating blood cell homeostasis, and sensory neural pathways mediate pro- or anti-inflammatory signaling in a tissue-specific manner. Here, we review emerging evidence of the fine titration of hematopoiesis, leukocyte trafficking, and tissue repair via neuro-immune crosstalk, and how its derailment can accelerate chronic inflammation, as in atherosclerosis.

Targeting extranuclear histones to alleviate acute and chronic inflammation.

Extracellular histones instigate an inflammatory triad - centered on cytotoxicity, immune cell stimulation, and coagulation - ultimately shaping the dynamics and outcome of various inflammatory pathologies. Given the virtual absence of beneficial functions of histones in the extracellular space, in recent years a number of interference strategies have emerged. In this review we summarize pathogenic functions of extracellular histones and highlight current developments of therapeutic interference. Finally, we elaborate on the current status of preclinical attempts to interfere with extracellular histones in the context of a focus on sepsis and cardiovascular diseases, both of which are leading causes of mortality worldwide.

Gasdermin D drives focal crystalline thrombotic microangiopathy by accelerating immunothrombosis and necroinflammation.

ABSTRACT: Thrombotic microangiopathy (TMA) is characterized by immunothrombosis and life-threatening organ failure but the precise underlying mechanism driving its pathogenesis remains elusive. In this study, we hypothesized that gasdermin D (GSDMD), a pore-forming protein that serves as the final downstream effector of the pyroptosis/interleukin-1ß (IL-1ß) pathway, contributes to TMA and its consequences by amplifying neutrophil maturation and subsequent necrosis. Using a murine model of focal crystalline TMA, we found that Gsdmd deficiency ameliorated immunothrombosis, acute tissue injury, and failure. Gsdmd-/- mice exhibited a decrease in mature IL-1ß, as well as in neutrophil maturation, ß2-integrin activation, and recruitment to TMA lesions, in which they formed reduced neutrophil extracellular traps in both arteries and interstitial tissue. The GSDMD inhibitor disulfiram dose-dependently suppressed human neutrophil pyroptosis in response to cholesterol crystals. Experiments with GSDMD-deficient, human-induced, pluripotent stem cell-derived neutrophils confirmed the involvement of GSDMD in neutrophil ß2-integrin activation, maturation, and pyroptosis. Both prophylactic and therapeutic administration of disulfiram protected the mice from focal TMA, acute tissue injury, and failure. Our data identified GSDMD as a key mediator of focal crystalline TMA and its consequences, including ischemic tissue infarction and organ failure. GSDMD could potentially serve as a therapeutic target for the systemic forms of TMA.

Neutrophils - Biology and Diversity.

Neutrophils, the most abundant white blood cells in the human circulation, play crucial roles in various diseases, including kidney disease. Traditionally viewed as short-lived pro-inflammatory phagocytes that release reactive oxygen species, cytokines and neutrophil extracellular traps, recent studies have revealed their complexity and heterogeneity, thereby challenging this perception. Neutrophils are now recognized as transcriptionally active cells capable of proliferation and reverse migration, displaying phenotypic and functional heterogeneity. They respond to a wide range of signals and deploy various cargo to influence the activity of other cells in the circulation and in tissues. They can regulate the behavior of multiple immune cell types, exhibit innate immune memory, and contribute to both acute and chronic inflammatory responses while also promoting inflammation resolution in a context-dependent manner. Here, we explore the origin and heterogeneity of neutrophils, their functional diversity, and the cues that regulate their effector functions. We also examine their emerging role in infectious and non-infectious diseases with a particular emphasis on kidney disease. Understanding the complex behavior of neutrophils during tissue injury and inflammation may provide novel insights, thereby paving the way for potential therapeutic strategies to manage acute and chronic conditions. By deciphering their multifaceted role, targeted interventions can be developed to address the intricacies of neutrophil-mediated immune responses and improve disease outcomes.

The angiopoietin receptor Tie2 is atheroprotective in arterial endothelium.

Leukocytes and resident cells in the arterial wall contribute to atherosclerosis, especially at sites of disturbed blood flow. Expression of endothelial Tie1 receptor tyrosine kinase is enhanced at these sites, and attenuation of its expression reduces atherosclerotic burden and decreases inflammation. However, Tie2 tyrosine kinase function in atherosclerosis is unknown. Here we provide genetic evidence from humans and from an atherosclerotic mouse model to show that TIE2 is associated with protection from coronary artery disease. We show that deletion of Tie2, or both Tie2 and Tie1, in the arterial endothelium promotes atherosclerosis by increasing Foxo1 nuclear localization, endothelial adhesion molecule expression and accumulation of immune cells. We also show that Tie2 is expressed in a subset of aortic fibroblasts, and its silencing in these cells increases expression of inflammation-related genes. Our findings indicate that unlike Tie1, the Tie2 receptor functions as the dominant endothelial angiopoietin receptor that protects from atherosclerosis.

Antagonistic Roles of Human Platelet Integrin αIIbß3 and Chemokines in Regulating Neutrophil Activation and Fate on Arterial Thrombi Under Flow.

BACKGROUND: Platelets and neutrophils are the first blood cells accumulating at sites of arterial thrombus formation, and both cell types contribute to the pathology of thrombotic events. We aimed to identify key interaction mechanisms between these cells using microfluidic approaches. METHODS: Whole-blood perfusion was performed over a collagen surface at arterial shear rate. Platelet and leukocyte (in majority neutrophil) activation were microscopically visualized using fluorescent markers. The contributions of platelet-adhesive receptors (integrin, P-selectin, CD40L) and chemokines were studied by using inhibitors or antibodies and using blood from patients with GT (Glanzmann thrombasthenia) lacking platelet-expressed αIIbß3. RESULTS: We observed (1) an unknown role of activated platelet integrin αIIbß3 preventing leukocyte adhesion, which was overcome by short-term flow disturbance provoking massive adhesion; (2) that platelet-expressed CD40L controls the crawling pattern and thrombus fidelity of the cells on a thrombus; (3) that continued secretion of platelet substances promotes activation of identified neutrophils, as assessed by (fMLP [N-formylmethionyl-leucyl-phenylalanine, a potent chemotactic agent and leukocyte activator] induced) [Ca2+]i rises and antigen expression; (4) and that platelet-released chemokines activate the adhered cells in the order of CXCL7>CCL5>CXCL4. Furthermore, postsilencing of the platelets in a thrombus suppressed the leukocyte activation. However, the leukocytes on thrombi did no more than limitedly form neutrophil extracellular traps, unless stimulated with phorbol ester or lipopolysaccharide. CONCLUSIONS: Together, these findings reveal a multifaceted regulation of adhesion and activation of neutrophils by platelets in a thrombus, with a balanced role of several platelet-adhesive receptors and a promoting role of platelet-released substances. This multivalent nature of neutrophil-thrombus interactions offers novel prospects for pharmacological intervention.

The RNA editor ADAR2 promotes immune cell trafficking by enhancing endothelial responses to interleukin-6 during sterile inflammation.

Immune cell trafficking constitutes a fundamental component of immunological response to tissue injury, but the contribution of intrinsic RNA nucleotide modifications to this response remains elusive. We report that RNA editor ADAR2 exerts a tissue- and stress-specific regulation of endothelial responses to interleukin-6 (IL-6), which tightly controls leukocyte trafficking in IL-6-inflamed and ischemic tissues. Genetic ablation of ADAR2 from vascular endothelial cells diminished myeloid cell rolling and adhesion on vascular walls and reduced immune cell infiltration within ischemic tissues. ADAR2 was required in the endothelium for the expression of the IL-6 receptor subunit, IL-6 signal transducer (IL6ST; gp130), and subsequently, for IL-6 trans-signaling responses. ADAR2-induced adenosine-to-inosine RNA editing suppressed the Drosha-dependent primary microRNA processing, thereby overwriting the default endothelial transcriptional program to safeguard gp130 expression. This work demonstrates a role for ADAR2 epitranscriptional activity as a checkpoint in IL-6 trans-signaling and immune cell trafficking to sites of tissue injury.

Longitudinal In Vivo Monitoring of Atheroprogression in Hypercholesterolemic Mice Using Photoacoustic Imaging.

BACKGROUND AND AIM: The ability to recognize and monitor atherosclerotic lesion development using noninvasive imaging is crucial in preventive cardiology. The aim of the present study was to establish a protocol for longitudinal monitoring of plaque lipid, collagen, and macrophage burden as well as of endothelial permeability. METHODS AND RESULTS: Photoacoustic signals derived from endogenous or exogenous dyes assessed in vivo, in plaques of albino Apoe -/- mice, correlated with lesion characteristics obtained after histomorphometric and immunofluorescence analyses, thus supporting the validity of our protocol. Using models of atheroprogression and regression, we could apply our imaging protocol to the longitudinal observation of atherosclerotic lesion characteristics in mice. CONCLUSIONS: The present study shows an innovative approach to assess arterial inflammation in a non-invasive fashion, applicable to longitudinal analyses of changes of atherosclerotic lesion composition. Such approach could prove important in the preclinical testing of therapeutic interventions in mice carrying pre-established lesions.

Properties and fate of human mesenchymal stem cells upon miRNA let-7f-promoted recruitment to atherosclerotic plaques.

AIMS: Atherosclerosis is a chronic inflammatory disease of the arteries leading to the formation of atheromatous plaques. Human mesenchymal stem cells (hMSCs) are recruited from the circulation into plaques where in response to their environment they adopt a phenotype with immunomodulatory properties. However, the mechanisms underlying hMSC function in these processes are unclear. Recently, we described that miRNA let-7f controls hMSC invasion guided by inflammatory cytokines and chemokines. Here, we investigated the role of let-7f in hMSC tropism to human atheromas and the effects of the plaque microenvironment on cell fate and release of soluble factors. METHODS AND RESULTS: Incubation of hMSCs with LL-37, an antimicrobial peptide abundantly found in plaques, increased biosynthesis of let-7f and N-formyl peptide receptor 2 (FPR2), enabling chemotactic invasion of the cells towards LL-37, as determined by qRT-PCR, flow cytometry, and cell invasion assay analysis. In an Apoe-/- mouse model of atherosclerosis, circulating hMSCs preferentially adhered to athero-prone endothelium. This property was facilitated by elevated levels of let-7f in the hMSCs, as assayed by ex vivo artery perfusion and two-photon laser scanning microscopy. Exposure of hMSCs to homogenized human atheromatous plaque material considerably induced the production of various cytokines, chemokines, matrix metalloproteinases, and tissue inhibitors of metalloproteinases, as studied by PCR array and western blot analysis. Moreover, exposure to human plaque extracts elicited differentiation of hMSCs into cells of the myogenic lineage, suggesting a potentially plaque-stabilizing effect. CONCLUSIONS: Our findings indicate that let-7f promotes hMSC tropism towards atheromas through the LL-37/FPR2 axis and demonstrate that hMSCs upon contact with human plaque environment develop a potentially athero-protective signature impacting the pathophysiology of atherosclerosis.

Neutrophils in aging and aging-related pathologies.

Over the past millennia, life expectancy has drastically increased. While a mere 25 years during Bronze and Iron ages, life expectancy in many European countries and in Japan is currently above 80 years. Such an increase in life expectancy is a result of improved diet, life style, and medical care. Yet, increased life span and aging also represent the most important non-modifiable risk factors for several pathologies including cardiovascular disease, neurodegenerative diseases, and cancer. In recent years, neutrophils have been implicated in all of these pathologies. Hence, this review provides an overview of how aging impacts neutrophil production and function and conversely how neutrophils drive aging-associated pathologies. Finally, we provide a perspective on how processes of neutrophil-driven pathologies in the context of aging can be targeted therapeutically.

paPAML: An Improved Computational Tool to Explore Selection Pressure on Protein-Coding Sequences.

Evolution is change over time. Although neutral changes promoted by drift effects are most reliable for phylogenetic reconstructions, selection-relevant changes are of only limited use to reconstruct phylogenies. On the other hand, comparative analyses of neutral and selected changes of protein-coding DNA sequences (CDS) retrospectively tell us about episodic constrained, relaxed, and adaptive incidences. The ratio of sites with nonsynonymous (amino acid altering) versus synonymous (not altering) mutations directly measures selection pressure and can be analysed by using the Phylogenetic Analysis by Maximum Likelihood (PAML) software package. We developed a CDS extractor for compiling protein-coding sequences (CDS-extractor) and parallel PAML (paPAML) to simplify, amplify, and accelerate selection analyses via parallel processing, including detection of negatively selected sites. paPAML compiles results of site, branch-site, and branch models and detects site-specific negative selection with the output of a codon list labelling significance values. The tool simplifies selection analyses for casual and inexperienced users and accelerates computing speeds up to the number of allocated computer threads. We then applied paPAML to examine the evolutionary impact on a new GINS Complex Subunit 3 exon, and neutrophil-associated as well as lysin and apolipoprotein genes. Compared with codeml (PAML version 4.9j) and HyPhy (HyPhy FEL version 2.5.26), all paPAML test runs performed with 10 computing threads led to identical selection pressure results, whereas the total selection analysis via paPAML, including all model comparisons, was about 3 to 5 times faster than the longest running codeml model and about 7 to 15 times faster than the entire processing time of these codeml runs.

Endothelial ACKR3 drives atherosclerosis by promoting immune cell adhesion to vascular endothelium.

Atherosclerosis is the foundation of potentially fatal cardiovascular diseases and it is characterized by plaque formation in large arteries. Current treatments aimed at reducing atherosclerotic risk factors still allow room for a large residual risk; therefore, novel therapeutic candidates targeting inflammation are needed. The endothelium is the starting point of vascular inflammation underlying atherosclerosis and we could previously demonstrate that the chemokine axis CXCL12-CXCR4 plays an important role in disease development. However, the role of ACKR3, the alternative and higher affinity receptor for CXCL12 remained to be elucidated. We studied the role of arterial ACKR3 in atherosclerosis using western diet-fed Apoe-/- mice lacking Ackr3 in arterial endothelial as well as smooth muscle cells. We show for the first time that arterial endothelial deficiency of ACKR3 attenuates atherosclerosis as a result of diminished arterial adhesion as well as invasion of immune cells. ACKR3 silencing in inflamed human coronary artery endothelial cells decreased adhesion molecule expression, establishing an initial human validation of ACKR3's role in endothelial adhesion. Concomitantly, ACKR3 silencing downregulated key mediators in the MAPK pathway, such as ERK1/2, as well as the phosphorylation of the NF-kB p65 subunit. Endothelial cells in atherosclerotic lesions also revealed decreased phospho-NF-kB p65 expression in ACKR3-deficient mice. Lack of smooth muscle cell-specific as well as hematopoietic ACKR3 did not impact atherosclerosis in mice. Collectively, our findings indicate that arterial endothelial ACKR3 fuels atherosclerosis by mediating endothelium-immune cell adhesion, most likely through inflammatory MAPK and NF-kB pathways.

Erythroid lineage Jak2V617F expression promotes atherosclerosis through erythrophagocytosis and macrophage ferroptosis.

Elevated hematocrit is associated with cardiovascular risk; however, the causality and mechanisms are unclear. The JAK2V617F (Jak2VF) mutation increases cardiovascular risk in myeloproliferative disorders and in clonal hematopoiesis. Jak2VF mice with elevated WBCs, platelets, and RBCs display accelerated atherosclerosis and macrophage erythrophagocytosis. To investigate whether selective erythroid Jak2VF expression promotes atherosclerosis, we developed hyperlipidemic erythropoietin receptor Cre mice that express Jak2VF in the erythroid lineage (VFEpoR mice). VFEpoR mice without elevated blood cell counts showed increased atherosclerotic plaque necrosis, erythrophagocytosis, and ferroptosis. Selective induction of erythrocytosis with low-dose erythropoietin further exacerbated atherosclerosis with prominent ferroptosis, lipid peroxidation, and endothelial damage. VFEpoR RBCs had reduced antioxidant defenses and increased lipid hydroperoxides. Phagocytosis of human or murine WT or JAK2VF RBCs by WT macrophages induced ferroptosis, which was prevented by the ferroptosis inhibitor liproxstatin-1. Liproxstatin-1 reversed increased atherosclerosis, lipid peroxidation, ferroptosis, and endothelial damage in VFEpoR mice and in Jak2VF chimeric mice simulating clonal hematopoiesis, but had no impact in controls. Erythroid lineage Jak2VF expression led to qualitative and quantitative defects in RBCs that exacerbated atherosclerosis. Phagocytosis of RBCs by plaque macrophages promoted ferroptosis, suggesting a therapeutic target for reducing RBC-mediated cardiovascular risk.