A Neutrophil Timer Coordinates Immune Defense and Vascular Protection

Neutrophils eliminate pathogens efficiently but can inflict severe damage to the host if they over-activate within blood vessels. It is unclear how immunity solves the dilemma of mounting an efficient anti-microbial defense while preserving vascular health. Here, we identify a neutrophil-intrinsic program that enabled both. The gene Bmal1 regulated expression of the chemokine CXCL2 to induce chemokine receptor CXCR2-dependent diurnal changes in the transcriptional and migratory properties of circulating neutrophils. These diurnal alterations, referred to as neutrophil aging, were antagonized by CXCR4 (C-X-C chemokine receptor type 4) and regulated the outer topology of neutrophils to favor homeostatic egress from blood vessels at night, resulting in boosted anti-microbial activity in tissues. Mice engineered for constitutive neutrophil aging became resistant to infection, but the persistence of intravascular aged neutrophils predisposed them to thrombo-inflammation and death. Thus, diurnal compartmentalization of neutrophils, driven by an internal timer, coordinates immune defense and vascular protection. Neutrophils display circadian oscillations in numbers and phenotype in the circulation. Adrover and colleagues now identify the molecular regulators of neutrophil aging and show that genetic disruption of this process has major consequences in immune cell trafficking, anti-microbial defense, and vascular health.This study was supported by Intramural grants from A∗STAR to L.G.N., BES-2013-065550 to J.M.A., BES-2010-032828 to M.C.-A, and JCI-2012-14147 to L.A.W (all from Ministerio de Economía, Industria y Competitividad; MEIC). Additional MEIC grants were SAF2014-61993-EXP to C.L.-R.; SAF2015-68632-R to M.A.M. and SAF-2013-42920R and SAF2016-79040Rto D.S. D.S. also received 635122-PROCROP H2020 from the European Commission and ERC CoG 725091 from the European Research Council (ERC). ERC AdG 692511 PROVASC from the ERC and SFB1123-A1 from the Deutsche Forschungsgemeinschaft were given to C.W.; MHA VD1.2/81Z1600212 from the German Center for Cardiovascular Research (DZHK) was given to C.W. and O.S.; SFB1123-A6 was given to O.S.; SFB914-B08 was given to O.S. and C.W.; and INST 211/604-2, ZA 428/12-1, and ZA 428/13-1 were given to A.Z. This study was also supported by PI12/00494 from Fondo de Investigaciones Sanitarias (FIS) to C.M.; PI13/01979, Cardiovascular Network grant RD 12/0042/0054, and CIBERCV to B.I.; SAF2015-65607-R, SAF2013-49662-EXP, and PCIN-2014-103 from MEIC; and co-funding by Fondo Europeo de Desarrollo Regional (FEDER) to A.H. The CNIC is supported by the MEIC and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (MEIC award SEV-2015-0505)

Protein kinase D activity controls endothelial nitric oxide synthesis

Vascular endothelial growth factor (VEGF) regulates key functions of the endothelium, such as angiogenesis or vessel repair in processes involving endothelial nitric oxide synthase (eNOS) activation. One of the effector kinases that become activated in endothelial cells upon VEGF treatment is protein kinase D (PKD). Here, we show that PKD phosphorylates eNOS, leading to its activation and a concomitant increase in NO synthesis. Using mass spectrometry, we show that the purified active kinase specifically phosphorylates recombinant eNOS on Ser1179. Treatment of endothelial cells with VEGF or phorbol 12,13-dibutyrate (PDBu) activates PKD and increases eNOS Ser1179 phosphorylation. In addition, pharmacological inhibition of PKD and gene silencing of both PKD1 and PKD2 abrogate VEGF signaling, resulting in a clear diminished migration of endothelial cells in a wound healing assay. Finally, inhibition of PKD in mice results in an almost complete disappearance of the VEGF-induced vasodilatation, as monitored through determination of the diameter of the carotid artery. Hence, our data indicate that PKD is a new regulatory kinase of eNOS in endothelial cells whose activity orchestrates mammalian vascular tone.This work was supported by the Mineco [grant numbers SAF2011-26233 to T.I., BFU2009-10442 and BFU2012-37934 to I.R.-C.]; Comunidad de Madrid [grant number P2010/BMD-2331-Neurodegmodels-CM to T.I.]; and Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Instituto de Salud Carlos III, to T.I. L.S.-R. was funded by research contracts from CIBERNED; C.A.-R. was a recipient of a FPU predoctoral fellowship from Mineco.Peer Reviewe

Beta-3 adrenergic receptor overexpression reverses aortic stenosis-induced heart failure and restores balanced mitochondrial dynamics

Aortic stenosis (AS) is associated with left ventricular (LV) hypertrophy and heart failure (HF). There is a lack of therapies able to prevent/revert AS-induced HF. Beta3 adrenergic receptor (β3AR) signaling is beneficial in several forms of HF. Here, we studied the potential beneficial effect of β3AR overexpression on AS-induced HF. Selective β3AR stimulation had a positive inotropic effect. Transgenic mice constitutively overexpressing human β3AR in the heart (c-hβ3tg) were protected from the development of HF in response to induced AS, and against cardiomyocyte mitochondrial dysfunction (fragmented mitochondria with remodeled cristae and metabolic reprogramming featuring altered substrate use). Similar beneficial effects were observed in wild-type mice inoculated with adeno-associated virus (AAV9) inducing cardiac-specific overexpression of human β3AR before AS induction. Moreover, AAV9-hβ3AR injection into wild-type mice at late disease stages, when cardiac hypertrophy and metabolic reprogramming are already advanced, reversed the HF phenotype and restored balanced mitochondrial dynamics, demonstrating the potential of gene-therapy-mediated β3AR overexpression in AS. Mice with cardiac specific ablation of Yme1l (cYKO), characterized by fragmented mitochondria, showed an increased mortality upon AS challenge. AAV9-hβ3AR injection in these mice before AS induction reverted the fragmented mitochondria phenotype and rescued them from death. In conclusion, our results step out that β3AR overexpression might have translational potential as a therapeutic strategy in AS-induced HF.Sin financiación12.416 JCR (2021) Q1, 13/143 Cardiac & Cardiovascular Systems1.615 SJR (2021) Q1, 49/356 Cardiology and Cardiovascular MedicineNo data IDR 2021UE

NOD1 Activation Induces Cardiac Dysfunction and Modulates Cardiac Fibrosis and Cardiomyocyte Apoptosis

<div><p>The innate immune system is responsible for the initial response of an organism to potentially harmful stressors, pathogens or tissue injury, and accordingly plays an essential role in the pathogenesis of many inflammatory processes, including some cardiovascular diseases. Toll like receptors (TLR) and nucleotide-binding oligomerization domain-like receptors (NLRs) are pattern recognition receptors that play an important role in the induction of innate immune and inflammatory responses. There is a line of evidence supporting that activation of TLRs contributes to the development and progression of cardiovascular diseases but less is known regarding the role of NLRs. Here we demonstrate the presence of the NLR member NOD1 (nucleotide-binding oligomerization domain containing 1) in the murine heart. Activation of NOD1 with the specific agonist C12-iEDAP, but not with the inactive analogue iE-Lys, induces a time- and dose-dependent cardiac dysfunction that occurs concomitantly with cardiac fibrosis and apoptosis. The administration of iEDAP promotes the activation of the NF-κB and TGF-β pathways and induces apoptosis in whole hearts. At the cellular level, both native cardiomyocytes and cardiac fibroblasts expressed NOD1. The NLR activation in cardiomyocytes was associated with NF-κB activation and induction of apoptosis. NOD1 stimulation in fibroblasts was linked to NF-κB activation and to increased expression of pro-fibrotic mediators. The down-regulation of NOD1 by specific siRNAs blunted the effect of iEDAP on the pro-fibrotic TGF-β pathway and cell apoptosis. In conclusion, our report uncovers a new pro-inflammatory target that is expressed in the heart, NOD1. The specific activation of this NLR induces cardiac dysfunction and modulates cardiac fibrosis and cardiomyocyte apoptosis, pathological processes involved in several cardiac diseases such as heart failure.</p> </div

Cardiac parameters collected after M-mode ultrasound evaluation of mice.

<p>Data are means ± SE. HW, heart weight, BW, body weight, HR heart rate, EF, left-ventricle ejection fraction, FS, fractional shortening, LVESD, left-ventricle end-systolic diameter; LVEDD, left-ventricle end-diastolic diameter; SV, systolic volume; DV, diastolic volume;</p>**<p><i>p</i><0.01,</p>***<p><i>p</i><0.001 Vehicle (Veh.) <i>vs.</i> iE (50, 150 or 200 µg iEDAP treated mice for two weeks).</p

Selective activation of NOD1 stimulates NF-κB and TGF-β pathways in cardiac fibroblasts.

<p>(A) Confocal microscopy images of cardiac fibroblasts isolated from mouse hearts stained for NOD1. (B) Isolated murine cardiac fibroblasts were incubated for 15 to 60 min, 24 h or 48 h with 40 µg/ml iE. iE-treatment induces an up-regulation of P-RIP2/RIP2, P-IKK/IKK, P-IκBα/IκBα (15–60 min), NOS2 (24 h) and COX2 (48 h) protein levels. (C) Treatment of fibroblasts with iE for 72 h promoted an increase of TRβ2, P-Smad/Smad, PAI-1 and FGF-2 protein levels. GAPDH was used to normalize all target protein levels. Data are illustrated in histograms as mean±SEM <i>vs.</i> vehicle (100%; n = 3–5 samples).*p<0.05, **p<0.01 and ***p<0.001 <i>vs.</i> vehicle.</p

Murine cardiac tissue expresses NOD1. Specific stimulation of NOD1 induces NF-κB pathway activation.

<p>(A) Histograms show NOD1 mRNA levels in different mouse tissues. (B) NOD1 protein levels in mouse hearts were analyzed by western-blot. Animals received i.p. 150 µg of iEDAP (iE)/day, a selective agonist of NOD1, or vehicle (Veh.). After 2 weeks of treatment an up-regulation of P-RIP2/RIP2, P-IKK/IKK, P-IκBα/IκBα protein ratio (B), higher p65 binding to κB motifs determined by ELISA (C), and NOS2 and COX2 (D) protein levels were observed in iE treated hearts. NOS2 and COX2 protein values were normalized with GAPDH. Representative blots are shown in the left panels and right panels illustrate the histograms representing the mean (band ratio)±SEM values <i>vs.</i> Veh. (100%); n = 4–6 animals. *p<0.05, ***p<0.001 <i>vs.</i> vehicle.</p

NOD1 activation increases cardiac fibrosis and TGF-β pathway in hearts isolated from iE treated mice.

<p>(A) Histological analysis of the hearts from mice treated with vehicle (Veh.), with 150 µg of iEDAP (iE) or with 150 µg of iE-Lys (NOD1 inactive analogue of iEDAP). Representative whole-heart cross-sections are shown (magnification x20). Myocardial fibrosis was evaluated after staining with Masson’s trichrome. (B) Histograms show the quantitative analysis of subendocardial fibrosis is shown as collagen area <i>vs.</i> total tissue area (100%, mean ± SEM; n = 4 animals per condition). (C) Histograms show type I and III collagen mRNA levels in cardiac tissue from iE and Veh. treated mice. (D) TGF-β signaling was activated in hearts from iE-treated mice. <i>Left panel</i>, representative blots of TRβ1, TRβ2, P-Smad, Smad, PAI-1 and FGF-2 of vehicle and iE treated mouse hearts (150 µg for 2 weeks). <i>Right panel,</i> shows the corresponding histograms representing the mean values <i>vs.</i> vehicle (100%, n = 4–6 animals). GAPDH was used to normalize all the target protein levels. *p<0.05, ***p<0.001 <i>vs.</i> vehicle.</p

Selective stimulation of NOD1 induces cardiac apoptosis.

<p>Animals received i.p. 150 µg/day of iEDAP (iE), 150 µg/day of iE-Lys or vehicle for 2 weeks. (A) TUNEL staining of cells undergoing apoptosis in cardiac tissue sections from vehicle, iE or iE-Lys-treated mice. Light transmission of the preparations indicated that apoptotic cells were predominantly cardiomyocytes. Representative images of TUNEL positive (green) and DAPI (blue) staining (magnification x 40). The percentages of positive TUNEL cells are indicated in the images. (B) Caspase 3, Bax and X-IAP levels determined by Western blot from hearts of vehicle and iE mice. <i>Left panel</i> shows the histograms representing the mean±SEM <i>vs.</i> vehicle (100%). *p<0.05, ***p<0.001 <i>vs.</i> vehicle; n = 4–6 animals.</p

The NOD1 agonist iE induces NF-κB activation and apoptosis in cardiomyocytes and H9c2 cells. Effect of NOD1 ablation with siRNAs.

<p>(A) Native murine cardiomyocytes were incubated for 15 to 60 min, or 48 h with 20 µg/ml of iE. iE treatment induces an up-regulation of P-RIP2/RIP2, P-IKK/IKK, P-IκBα/IκBα (15–60 min) and COX2 (48 h) protein levels. (B) iE induces a decrease in cardiomyocyte viability in a dose-response form. Cardiomyocytes incubated for 2 h with 10–30 µg/ml of iE; staurosporine (Stau, 100 ng/ml) were used as a positive control to induce apoptosis. (C). iE treatment induces an up-regulation of caspase 3 and Bax protein levels and down-regulates the BcL-xL and X-IAP protein levels. (D) iE or Stau administration for 24 h in H9c2 promoted a decrease in viability as determined by MTT activity. NOD1 suppression by siRNA prevented the effect of iE on cell viability. (E) Representative blot of NOD1, caspase 3 and GAPDH obtained in vehicle and iE treated cells, NOD1 siRNAs (siNOD1) and NOD1 siRNA+iE in H9c2 cells. All targets protein levels were normalized by GAPDH. Data are expressed in histograms representing the mean±SEM <i>vs</i>. vehicle (100%); n = 3–5 samples per condition.*p<0.05, **p<0.01 and ***p<0.001 <i>vs.</i> vehicle.</p