Platelet Serotonin Aggravates Myocardial Ischemia/Reperfusion Injury via Neutrophil Degranulation

Background: Platelets store large amounts of serotonin that they release during thrombus formation or acute inflammation. This facilitates hemostasis and modulates the inflammatory response. Methods: Infarct size, heart function, and inflammatory cell composition were analyzed in mouse models of myocardial reperfusion injury with genetic and pharmacological depletion of platelet serotonin. These studies were complemented by in vitro serotonin stimulation assays of platelets and leukocytes in mice and men, and by measuring plasma serotonin levels and leukocyte activation in patients with acute coronary syndrome. Results: Platelet-derived serotonin induced neutrophil degranulation with release of myeloperoxidase and hydrogen peroxide (H2O2) and increased expression of membrane-bound leukocyte adhesion molecule CD11b, leading to enhanced inflammation in the infarct area and reduced myocardial salvage. In patients hospitalized with acute coronary syndrome, plasmatic serotonin levels correlated with CD11b expression on neutrophils and myeloperoxidase plasma levels. Long-term serotonin reuptake inhibition - reported to protect patients with depression from cardiovascular events - resulted in the depletion of platelet serotonin stores in mice. These mice displayed a reduction in neutrophil degranulation and preserved cardiac function. In line, patients with depression using serotonin reuptake inhibition, presented with suppressed levels of CD11b surface expression on neutrophils and lower myeloperoxidase levels in blood. Conclusions: Taken together, we identify serotonin as a potent therapeutic target in neutrophil-dependent thromboinflammation during myocardial reperfusion injury.Fil: Mauler, Maximilian. No especifíca;Fil: Herr, Nadine. No especifíca;Fil: Schoenichen, Claudia. No especifíca;Fil: Witsch, Thilo. No especifíca;Fil: Marchini, Timoteo Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; ArgentinaFil: Härdtner, Carmen. No especifíca;Fil: Koentges, Christoph. No especifíca;Fil: Kienle, Korbinian. Max Planck Institute Of Immunobiology And Epigenetics; AlemaniaFil: Ollivier, Véronique. Inserm; FranciaFil: Schell, Maximilian. No especifíca;Fil: Dorner, Ludwig. No especifíca;Fil: Wippel, Christopher. No especifíca;Fil: Stallmann, Daniela. No especifíca;Fil: Normann, Claus. No especifíca;Fil: Bugger, Heiko. No especifíca;Fil: Walther, Paul. Universitat Ulm; AlemaniaFil: Wolf, Dennis. La Jolla Institute for Allergy and Immunology; Estados UnidosFil: Ahrens, Ingo. No especifíca;Fil: Lämmermann, Tim. Max Planck Institute Of Immunobiology And Epigenetics; AlemaniaFil: Ho-Tin-Noé, Benoît. Inserm; FranciaFil: Ley, Klaus. La Jolla Institute for Allergy and Immunology; Estados UnidosFil: Bode, Christoph. No especifíca;Fil: Hilgendorf, Ingo. No especifíca;Fil: Duerschmied, Daniel. No especifíca

Functional characterization of the local, pancreatic β-cell renin-angiotensin system

The systemic renin-angiotensin system (RAS) is an endocrine system that is mainly known to regulate blood pressure, fluid and electrolyte balance as well as volume homeostasis in the body through different active metabolites, the angiotensin (Ang) peptides. In addition, local renin-angiotensin systems have been discovered in various tissues, including the islet of Langerhans. Starting with angiotensinogen, the precursor of all angiotensin peptides which is cleaved into the decapeptide Ang I by renin, the RAS is divided into three axes. The main classical RAS axis is composed of angiotensin converting enzyme (ACE), angiotensin (Ang) II, and the Ang II type 1 receptor (AT1R), whereas the two alternative RAS axes comprise either ACE2, Ang-(1-7) and the receptor Mas or the aminopeptidase N (APN), Ang IV and the insulin-regulated aminopeptidase (IRAP). The activation of the main ACE/Ang II/AT1R RAS axis has been associated with metabolic syndrome, type 2 diabetes mellitus, and islet dysfunction. The detrimental effects resulting from the pathological activation of this axis have been shown to be attenuated or even abolished by the pharmacological inhibition of components of the main RAS axis. However, the impact of the two alternative ACE2/Ang-(1-7)/Mas and APN/Ang IV/IRAP RAS axes on islet function is less well understood. Previous studies mainly focused on the possible protective actions of Ang-(1-7) via the receptor Mas in insulin-sensitive tissues and on well known risk factors of metabolic syndrome (insulin resistance, hyperglycemia, obesity, hypertension and dyslipidemia). Thus, the impact of this axis on β-cell function and, in particular, insulin production and release was examined in the present study. Glucose and fatty acids have been subjects of diabetic research because they are established pathophysiologically relevant features of the metabolic syndrome and are known to harm β cells, phenomena which are referred to as gluco- or lipotoxicity, respectively. The pathophysiologically relevant factors glucose, saturated fatty acid (FA) palmitic acid (PA), and the methyl ester of the omega-3 fatty acid docosahexaenoic acid (DHA-ME) were used in the present study to characterize the local β-cell RAS as well as β-cell function under pathophysiological conditions. Results of the present work demonstrate the expression of selected components of the RAS in isolated murine islets of Langerhans and the rat insulinoma cell line BRIN-BD11 under basal conditions. The alternative ACE2/Ang-(1-7)/Mas and APN/Ang IV/IRAP RAS axes were activated by high glucose in BRIN-BD11 cells after 24 h. Coincidently with these findings insulin production was found to be increased. In contrast, the expression of components of the main ACE/Ang II/AT1R RAS axis and the Ang II type 2 receptor (AT2R) were not affected under the same conditions (Härdtner et al., 2013). Both FAs, PA and DHA-ME were shown to alter the expression of components of the renin-angiotensin system in BRIN-BD11 cells. PA increased the expression of AT1R, the receptor of the main RAS axis, and of AT2R, whereas that of the receptor of the alternative ACE2/Ang-(1-7)/Mas RAS axis, Mas, appeared to be down-regulated at basal low glucose concentrations (5.5 mM). These effects were accompanied by a dose-dependent reduction of the insulin production and secretion. In contrast, DHA-ME augmented the expression of components of the ACE2/Ang-(1-7)/Mas axis and IRAP at low glucose concentrations, an effect which could be partially enhanced under high glucose conditions (25 mM). At basal glucose concentrations DHA-ME reduced the insulin secretion, whereas it was increased under high glucose conditions. However, the insulin mRNA amount remained unaffected by DHA-ME. Additionally, in contrast to glucose and palmitic acid, DHA-ME significantly increased the production of reactive oxygen species, at least hydrogen peroxide after 30 min. Expression alterations of components of the alternative ACE2/Ang-(1-7)/Mas RAS axis by glucose and PA correlated strongly with the corresponding insulin secretion and production. Therefore, an involvement of the ACE2/Ang-(1-7)/Mas RAS axis in the regulation of insulin secretion and production was hypothesized and validated in primary islets of Langerhans of both Mas-deficient and wild-type mice. Islets were exposed to the preferred natural ligand for Mas, Ang-(1-7), or to its pharmacological agonists or antagonists, respectively....Das systemische Renin-Angiotensin-System (RAS) ist ein endokrines System, welches hauptsächlich bekannt dafür ist den Blutdruck, den Wasser- und Salzhaushalt, sowie die Volumenhomöostase durch verschiedene, aktive Metabolite, den Angiotensinen, im Körper zu regulieren. Darüber hinaus wurden lokale Renin-Angiotensin-Systeme in verschiedenen Geweben entdeckt, so auch in der Langerhansschen Insel. Ausgehend vom Angiotensinogen, dem Vorläufer aller Angiotensine (Ang), welches durch Renin in das Dekapeptid Ang I gespalten wird, wird das RAS in drei Achsen unterteilt. Die klassische Hauptachse des RAS setzt sich aus dem Angiotensin-konvertierenden Enzym (ACE), Angiotensin (Ang) II und dem Ang II Typ 1 Rezeptor (AT1R) zusammen, wohingegen die zwei alternativen RAS-Achsen entweder aus ACE2, Ang-(1-7) und dem Rezeptor Mas oder der Aminopeptidase N (APN), Ang IV und der Insulin-regulierten Aminopeptidase (IRAP) bestehen. Die Aktivierung der klassischen Hauptachse des RAS, ACE/Ang II/AT1R, wurde mit Metabolischem Syndrom, Diabetes mellitus Typ 2, und Inseldysfunktion in Verbindung gebracht. Die schädlichen Effekte infolge der pathologischen Aktivierung der Hauptachse des RAS konnten durch pharmakologische Inhibitoren verschiedener Komponenten dieser Achse abgeschwächt oder sogar aufgehoben werden. Die Bedeutung der zwei alternativen RAS-Achsen, ACE2/Ang-(1-7)/Mas und APN/Ang IV/IRAP, auf die Inselfunktion ist jedoch weniger gut verstanden. Frühere Untersuchungen konzentrierten sich hauptsächlich auf mögliche protektive Wirkungen von Ang-(1-7) über den Rezeptor Mas in insulinsensitivem Gewebe und auf bekannte Risikofaktoren des Metabolischen Syndroms (Insulinresistenz, Hyperglykämie, Fettleibigkeit, Bluthochdruck und Dyslipidämie). Die Bedeutung dieser Achse auf die β-Zellfunktion und besonders auf die Insulinproduktion und –sekretion wurde deshalb in der vorliegenden Arbeit untersucht. Glukose und Fettsäuren sind Gegenstand der Diabetesforschung, seitdem sie etablierte Pathogenitätsfaktoren des Metabolischen Syndroms darstellen und bekannt dafür sind, β-Zellen schädigen zu können. Diese Phänomene werden als Gluko- bzw. Lipotoxizität bezeichnet. Die pathologisch relevanten Faktoren Glukose, die gesättigte Fettsäure Palmitinsäure (PA) und der Methylester der Omega-3-Fettsäure Docosahexaensäure (DHA-ME) wurden in der vorliegenden Arbeit verwendet, um das lokale β-Zell RAS sowie die β-Zellfunktion unter pathophysiologischen Bedingungen zu charakterisieren. Ergebnisse dieser Arbeit zeigen die Expression ausgewählter Komponenten des RAS in isolierten Langerhansschen Inseln der Maus und der Ratteninsulinoma Zelllinie BRIN-BD11 unter basalen Bedingungen. Die alternativen RAS-Achsen, ACE2/Ang-(1-7)/Mas und APN/Ang IV/IRAP, wurden durch Hochglukose in BRIN-BD11 Zellen nach 24 h aktiviert. Übereinstimmend mit diesen Ergebnissen wurde ein Anstieg der Insulinproduktion festgestellt. Im Gegensatz dazu waren die Expressionen von Komponenten der Hauptachse des RAS, ACE/Ang II/AT1R, und des Ang II Typ 2 Rezeptors (AT2R) unter den gleichen Bedingungen nicht betroffen (Härdtner et al., 2013). Beide Fettsäuren, PA und DHA-ME, modulierten die Expression von Komponenten des RAS in BRIN-BD11 Zellen nach 24 h. Während PA bei basalen, niedrigen Glukosekonzentrationen (5,5 mM) die Expression des AT1R, des Rezeptors der RAS-Hauptachse, sowie des AT2R verstärkte, regulierte es die des Rezeptors der alternativen ACE2/Ang-(1-7)/Mas RAS-Achse, Mas, herunter. Diese Effekte waren begleitet von einer dosisabhängigen Reduktion der Insulinproduktion und -sekretion. Im Gegensatz dazu steigerte DHA-ME die Expression der Komponenten der ACE2/Ang-(1-7)/Mas RAS-Achse und IRAP bei niedrigen Glukosekonzentrationen, ein Effekt, der in Gegenwart hoher Glukosekonzentrationen (25 mM) teilweise noch verstärkt werden konnte. Bei basalen Glukosekonzentrationen reduzierte DHA-ME die Insulinsekretion, wohingegen sie in Gegenwart hoher Glukosekonzentrationen gesteigert wurde. Die Insulin-mRNA-Menge blieb von DHA-ME jedoch unbeeinflusst. Zusätzlich verursachte DHA-ME, im Gegensatz zu Glukose und PA, einen signifikanten Anstieg der Produktion von reaktiven Sauerstoffspezies, auf jeden Fall von Wasserstoffperoxid, nach 30 min. Änderungen der Expression von Komponenten der alternativen ACE2/Ang-(1-7)/Mas RAS-Achse durch Glukose und PA korrelierten stark mit der korrespondierenden Insulinsekretion und -produktion. Daher wurde die Hypothese aufgestellt, dass die ACE2/Ang-(1-7)/Mas RAS-Achse an der Regulation der Insulinsekretion und -produktion beteiligt ist. Die Bedeutung dieser Hypothese wurde in primären Langerhansschen Inseln von Mas-defizienten und wildtypischen Mäusen bestätigt. Inseln wurden mit dem bevorzugten Liganden des Mas-Rezeptors, Ang-(1-7), oder mit dessen pharmakologischen Agonisten bzw. Antagonisten inkubiert...

The role of interferon regulatory factor 8 for retinal tissue homeostasis and development of choroidal neovascularisation

BACKGROUND: Microglia cells represent the resident innate immune cells of the retina and are important for retinal development and tissue homeostasis. However, dysfunctional microglia can have a negative impact on the structural and functional integrity of the retina under native and pathological conditions. METHODS: In this study, we examined interferon-regulatory factor 8 (Irf8)–deficient mice to determine the transcriptional profile, morphology, and temporospatial distribution of microglia lacking Irf8 and to explore the effects on retinal development, tissue homeostasis, and formation of choroidal neovascularisation (CNV). RESULTS: Our study shows that Irf8-deficient MG exhibit a considerable loss of microglial signature genes accompanied by a severely altered MG morphology. An in-depth characterisation by fundus photography, fluorescein angiography, optical coherence tomography and electroretinography revealed no major retinal abnormalities during steady state. However, in the laser-induced CNV model, Irf8-deficient microglia showed an increased activity of biological processes critical for inflammation and cell adhesion and a reduced MG cell density near the lesions, which was associated with significantly increased CNV lesion size. CONCLUSIONS: Our results suggest that loss of Irf8 in microglia has negligible effects on retinal homeostasis in the steady state. However, under pathological conditions, Irf8 is crucial for the transformation of resident microglia into a reactive phenotype and thus for the suppression of retinal inflammation and CNV formation. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12974-021-02230-y

Myeloid cell-specific Irf5 deficiency stabilizes atherosclerotic plaques in Apoe–/– mice

Objective: Interferon regulatory factor (IRF) 5 is a transcription factor known for promoting M1 type macrophage polarization in vitro. Given the central role of inflammatory macrophages in promoting atherosclerotic plaque progression, we hypothesize that myeloid cell-specific deletion of IRF5 is protective against atherosclerosis. Methods: Female Apoe–/– LysmCre/+ Irf5fl/fl and Apoe −/− Irf5fl/fl mice were fed a high-cholesterol diet for three months. Atherosclerotic plaque size and compositions as well as inflammatory gene expression were analyzed. Mechanistically, IRF5-dependent bone marrow-derived macrophage cytokine profiles were tested under M1 and M2 polarizing conditions. Mixed bone marrow chimeras were generated to determine intrinsic IRF5-dependent effects on macrophage accumulation in atherosclerotic plaques. Results: Myeloid cell-specific Irf5 deficiency blunted LPS/IFNγ-induced inflammatory gene expression in vitro and in the atherosclerotic aorta in vivo. While atherosclerotic lesion size was not reduced in myeloid cell-specific Irf5-deficient Apoe–/– mice, plaque composition was favorably altered, resembling a stable plaque phenotype with reduced macrophage and lipid contents, reduced inflammatory gene expression and increased collagen deposition alongside elevated Mertk and Tgfβ expression. Irf5-deficient macrophages, when directly competing with wild type macrophages in the same mouse, were less prone to accumulate in atherosclerotic lesion, independent of monocyte recruitment. Irf5-deficient monocytes, when exposed to oxidized low density lipoprotein, were less likely to differentiate into macrophage foam cells, and Irf5-deficient macrophages proliferated less in the plaque. Conclusion: Our study provides genetic evidence that selectively altering macrophage polarization induces a stable plaque phenotype in mice

GPR55 in B cells limits atherosclerosis development and regulates plasma cell maturation

Dissecting the pathways regulating the adaptive immune response in atherosclerosis is of particular therapeutic interest. Here we report that the lipid G-protein coupled receptor GPR55 is highly expressed by splenic plasma cells (PC), upregulated in mouse spleens during atherogenesis and human unstable or ruptured compared to stable plaques. Gpr55-deficient mice developed larger atherosclerotic plaques with increased necrotic core size compared to their corresponding controls. Lack of GPR55 hyperactivated B cells, disturbed PC maturation and resulted in immunoglobulin (Ig)G overproduction. B cell-specific Gpr55 depletion or adoptive transfer of Gpr55-deficient B cells was sufficient to promote plaque development and elevated IgG titers. In vitro, the endogenous GPR55 ligand lysophsophatidylinositol (LPI) enhanced PC proliferation, whereas GPR55 antagonism blocked PC maturation and increased their mitochondrial content. Collectively, these discoveries provide previously undefined evidence for GPR55 in B cells as a key modulator of the adaptive immune response in atherosclerosis

Deficiency of Endothelial CD40 Induces a Stable Plaque Phenotype and Limits Inflammatory Cell Recruitment to Atherosclerotic Lesions in Mice

Objectives: The co-stimulatory CD40L-CD40 dyad exerts a critical role in atherosclerosis by modulating leukocyte accumulation into developing atherosclerotic plaques. The requirement for cell-type specific expression of both molecules, however, remains elusive. Here, we evaluate the contribution of CD40 expressed on endothelial cells (ECs) in a mouse model of atherosclerosis. Methods and Results: Atherosclerotic plaques of apolipoprotein E-deficient (Apoe -/-) mice and humans displayed increased expression of CD40 on ECs compared with controls. To interrogate the role of CD40 on ECs in atherosclerosis, we induced EC-specific (BmxCre ERT2 -driven) deficiency of CD40 in Apoe -/- mice. After feeding a chow diet for 25 weeks, EC-specific deletion of CD40 (iEC-CD40) ameliorated plaque lipid deposition and lesional macrophage accumulation but increased intimal smooth muscle cell and collagen content, while atherosclerotic lesion size did not change. Leukocyte adhesion to the vessel wall was impaired in iEC-CD40-deficient mice as demonstrated by intravital microscopy. In accord, expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) in the vascular endothelium declined after deletion of CD40. In vitro, antibody-mediated inhibition of human endothelial CD40 significantly abated monocyte adhesion on ECs. Conclusion: Endothelial deficiency of CD40 in mice promotes structural features associated with a stable plaque phenotype in humans and decreases leukocyte adhesion. These results suggest that endothelial-expressed CD40 contributes to inflammatory cell migration and consecutive plaque formation in atherogenesis

Inhibition of macrophage proliferation dominates plaque regression in response to cholesterol lowering

Statins induce plaque regression characterized by reduced macrophage content in humans, but the underlying mechanisms remain speculative. Studying the translational APOE*3-Leiden.CETP mouse model with a humanized lipoprotein metabolism, we find that systemic cholesterol lowering by oral atorvastatin or dietary restriction inhibits monocyte infiltration, and reverses macrophage accumulation in atherosclerotic plaques. Contrary to current believes, none of (1) reduced monocyte influx (studied by cell fate mapping in thorax-shielded irradiation bone marrow chimeras), (2) enhanced macrophage egress (studied by fluorescent bead labeling and transfer), or (3) atorvastatin accumulation in murine or human plaque (assessed by mass spectrometry) could adequately account for the observed loss in macrophage content in plaques that undergo phenotypic regression. Instead, suppression of local proliferation of macrophages dominates phenotypic plaque regression in response to cholesterol lowering: the lower the levels of serum LDL-cholesterol and lipid contents in murine aortic and human carotid artery plaques, the lower the rates of in situ macrophage proliferation. Our study identifies macrophage proliferation as the predominant turnover determinant and an attractive target for inducing plaque regression