Triggering Receptor Expressed on Myeloid Cells-2 (TREM2) Inhibits Steroidogenesis in Adrenocortical Cell by Macrophage-derived Exosomes in Lipopolysaccharide-induced Septic Shock

Abstract Background: Endogenously produced glucocorticoids exhibit immunomodulating properties and are of pivotal importance for sepsis outcome. Uncontrolled activation of the immune-adrenal crosstalk increases the risk of sepsis-related death. Triggering receptor expressed on myeloid cells-2 (TREM2) is richly expressed on macrophages and has been demonstrated to improve outcome of sepsis by enhancing elimination of pathogens. However, the role and mode of action of macrophage TREM2 on adrenocortical steroidogenesis remains unclear in septic shock.Methods: The acute septic shock model was established by intraperitoneally challenging wild-type (WT) and TREM2 knock-out (Trem2-/-) mice with lipopolysaccharide (30 mg/kg). The mice were assessed for TREM2 expression and local inflammation in adrenal gland and synthesis of corticotropin releasing hormone (CRH) and adrenocorticotropic hormone (ACTH) in vivo. Bone marrow-derived macrophages or macrophage-derived exosomes were isolated from WT and Trem2-/- mice and co-cultured with adrenocortical cells. The expression of steroidogenic enzymes and corticosterone production were assessed.Results: Genetic deficiency of TREM2 caused significantly higher corticosterone levels (326.6 ± 73.0 ng/ml in Trem2-/- mice vs. 151.1 ± 58.9 ng/ml in WT mice; p < 0.001) at the early stage of LPS-induced septic shock. While TREM2 deficiency neither increased CRH and ACTH, nor exacerbated the inflammation in adrenocortical tissue during septic shock. Ex vivo study revealed that Trem2-/- macrophages significantly promoted the expression of steroidogenic enzymes and increased production of corticosterone (27.73 ± 1.78 ng/ml in Trem2-/- mice vs. 22.96 ± 1.94 ng/ml in W T mice; p < 0.01). Furthermore, Trem2-/- macrophage-derived exosomes were able to mimic Trem2-/- macrophages in enhancing adrenocortical steroidogenesis. Conclusions: At the early stage of lipopolysaccharide-induced septic shock, macrophage TREM2 inhibited the steroid synthesis and corticosterone production in adrenocortical cells, which may be partially associated with macrophage-derived exosomes.

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DAP12 (KARAP) amplifies inflammation and increases mortality from endotoxemia and septic peritonitis

Journal of Experimental Medicine ◽

10.1084/jem.20050986 ◽

2005 ◽

Vol 202 (3) ◽

pp. 363-369 ◽

Cited By ~ 65

Author(s):

Isaiah R. Turnbull ◽

Jonathan E. McDunn ◽

Toshiyuki Takai ◽

R. Reid Townsend ◽

J. Perren Cobb ◽

...

Keyword(s):

Septic Shock ◽

Cytokine Production ◽

Ex Vivo ◽

Cecal Ligation ◽

Wild Type ◽

Macrophage Response ◽

Cytokine Levels ◽

Septic Peritonitis ◽

Microbial Products

DAP12 (KARAP) is a transmembrane signaling adaptor for a family of innate immunoreceptors that have been shown to activate granulocytes and monocytes/macrophages, amplifying production of inflammatory cytokines. Contrasting with these data, recent studies suggest that DAP12 signaling has an inhibitory role in the macrophage response to microbial products (Hamerman, J.A., N.K. Tchao, C.A. Lowell, and L.L. Lanier. 2005. Nat. Immunol. 6:579–586). To determine the in vivo role for DAP12 signaling in inflammation, we measured the response of wild-type (WT) and DAP12−/− mice to septic shock. We show that DAP12−/− mice have improved survival from both endotoxemia and cecal ligation and puncture–induced septic shock. As compared with WT mice, DAP12−/− mice have decreased plasma cytokine levels and a decreased acute phase response during sepsis, but no defect in the recruitment of cells or bacterial control. In cells isolated after sepsis and stimulated ex vivo, DAP12 signaling augments lipopolysaccharide-mediated cytokine production. These data demonstrate that, during sepsis, DAP12 signaling augments the response to microbial products, amplifying inflammation and contributing to mortality.

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Src Homology 2–containing 5-Inositol Phosphatase (SHIP) Suppresses an Early Stage of Lymphoid Cell Development through Elevated Interleukin-6 Production by Myeloid Cells in Bone Marrow

Journal of Experimental Medicine ◽

10.1084/jem.20031193 ◽

2004 ◽

Vol 199 (2) ◽

pp. 243-254 ◽

Cited By ~ 32

Author(s):

Koji Nakamura ◽

Taku Kouro ◽

Paul W. Kincade ◽

Alexander Malykhin ◽

Kazuhiko Maeda ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Early Stage ◽

Myeloid Cells ◽

Cell Development ◽

Wild Type ◽

Src Homology ◽

B Lineage

The Src homology (SH)2–containing inositol 5-phosphatase (SHIP) negatively regulates a variety of immune responses through inhibitory immune receptors. In SHIP−/− animals, we found that the number of early lymphoid progenitors in the bone marrow was significantly reduced and accompanied by expansion of myeloid cells. We exploited an in vitro system using hematopoietic progenitors that reproduced the in vivo phenotype of SHIP−/− mice. Lineage-negative marrow (Lin−) cells isolated from wild-type mice failed to differentiate into B cells when cocultured with those of SHIP−/− mice. Furthermore, culture supernatants of SHIP−/− Lin− cells suppressed the B lineage expansion of wild-type lineage-negative cells, suggesting the presence of a suppressive cytokine. SHIP−/− Lin− cells contained more IL-6 transcripts than wild-type Lin− cells, and neutralizing anti–IL-6 antibody rescued the B lineage expansion suppressed by the supernatants of SHIP−/− Lin− cells. Finally, we found that addition of recombinant IL-6 to cultures of wild-type Lin− bone marrow cells reproduced the phenotype of SHIP−/− bone marrow cultures: suppression of B cell development and expansion of myeloid cells. The results identify IL-6 as an important regulatory cytokine that can suppress B lineage differentiation and drive excessive myeloid development in bone marrow.

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Adenosine Diphosphate (ADP)-Induced ⍺-Granules Release from Platelets of Native Whole Blood Is Reduced by Ticlopidine but Not by Aspirin or Dipyridamole

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661629 ◽

1986 ◽

Vol 56 (02) ◽

pp. 147-150 ◽

Cited By ~ 18

Author(s):

V Pengo ◽

M Boschello ◽

A Marzari ◽

M Baca ◽

L Schivazappa ◽

...

Keyword(s):

Whole Blood ◽

Light Transmission ◽

Ex Vivo ◽

Early Stage ◽

Shape Change ◽

Adenosine Diphosphate ◽

Dose Dependent ◽

Plateletderived Growth Factor ◽

Platelet Shape

SummaryA brief contact between native whole blood and ADP promotes a dose-dependent release of platelet a-granules without a fall in the platelet number. We assessed the “ex vivo” effect of three widely used antiplatelet drugs, aspirin dipyridamole and ticlopidine, on this system. Aspirin (a single 800 mg dose) and dipyridamole (300 mg/die for four days) had no effect, while ticlopidine (500 mg/die for four days) significantly reduced the a-granules release for an ADP stimulation of 0.4 (p <0.02), 1.2 (p <0.01) and 2 pM (p <0.01). No drug, however, completeley inhibits this early stage of platelet activation. The platelet release of α-granules may be related to platelet shape change of the light transmission aggregometer and may be important “in vivo” by enhancing platelet adhesiveness and by liberating the plateletderived growth factor.

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Differential effect of anesthetics on mucociliary clearance in vivo in mice

Scientific Reports ◽

10.1038/s41598-021-84605-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Kyle S. Feldman ◽

Eunwon Kim ◽

Michael J. Czachowski ◽

Yijen Wu ◽

Cecilia W. Lo ◽

...

Keyword(s):

Mucociliary Clearance ◽

Defense Mechanism ◽

Ex Vivo ◽

Beat Frequency ◽

Ciliary Beat Frequency ◽

Wild Type ◽

Sulfur Colloid ◽

Ct System

AbstractRespiratory mucociliary clearance (MCC) is a key defense mechanism that functions to entrap and transport inhaled pollutants, particulates, and pathogens away from the lungs. Previous work has identified a number of anesthetics to have cilia depressive effects in vitro. Wild-type C57BL/6 J mice received intra-tracheal installation of 99mTc-Sulfur colloid, and were imaged using a dual-modality SPECT/CT system at 0 and 6 h to measure baseline MCC (n = 8). Mice were challenged for one hour with inhalational 1.5% isoflurane, or intraperitoneal ketamine (100 mg/kg)/xylazine (20 mg/kg), ketamine (0.5 mg/kg)/dexmedetomidine (50 mg/kg), fentanyl (0.2 mg/kg)/1.5% isoflurane, propofol (120 mg/Kg), or fentanyl/midazolam/dexmedetomidine (0.025 mg/kg/2.5 mg/kg/0.25 mg/kg) prior to MCC assessment. The baseline MCC was 6.4%, and was significantly reduced to 3.7% (p = 0.04) and 3.0% (p = 0.01) by ketamine/xylazine and ketamine/dexmedetomidine challenge respectively. Importantly, combinations of drugs containing fentanyl, and propofol in isolation did not significantly depress MCC. Although no change in cilia length or percent ciliation was expected, we tried to correlate ex-vivo tracheal cilia ciliary beat frequency and cilia-generated flow velocities with MCC and found no correlation. Our results indicate that anesthetics containing ketamine (ketamine/xylazine and ketamine/dexmedetomidine) significantly depress MCC, while combinations containing fentanyl (fentanyl/isoflurane, fentanyl/midazolam/dexmedetomidine) and propofol do not. Our method for assessing MCC is reproducible and has utility for studying the effects of other drug combinations.

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Abstract P283: Parkin Mediates Mitophagy in Cardioprotection

Circulation Research ◽

10.1161/res.109.suppl_1.ap283 ◽

2011 ◽

Vol 109 (suppl_1) ◽

Author(s):

Allen M Andres ◽

Chengqun Huang ◽

Eric P Ratliff ◽

Genaro Hernandez ◽

Pamela Lee ◽

...

Keyword(s):

Ex Vivo ◽

Wild Type ◽

Simulated Ischemia ◽

Selective Targeting ◽

Cardioprotective Effects ◽

Mitochondrial Turnover ◽

First Time

Autophagy-dependent mitochondrial turnover in response to cellular stress is necessary for maintaining cellular homeostasis. However, the mechanisms that govern the selective targeting of damaged mitochondria are poorly understood. Parkin, an E3 ubiquitin ligase, has been shown to be essential for the selective clearance of damaged mitochondria. Parkin is expressed in the heart, yet its function has not been investigated in the context of cardioprotection. We previously reported that autophagy is required for cardioprotection by ischemic preconditioning (IPC). In the present study, we used simulated ischemia in vitro and IPC in hearts (in vivo and ex vivo) to investigate the role of Parkin in mediating cardioprotection. In HL-1 cells, simulated ischemia induced Parkin translocation to mitochondria and mitochondrial elimination. Mitochondrial loss was blunted in Atg5-deficient cells, revealing the requirement for autophagy in mitochondrial elimination. Consistent with previous reports implicating p62/SQSTM1 in mitophagy, we found that downregulation of p62 attenuated mitophagy and exacerbated cell death in HL-1 cardiomyocytes subjected to simulated ischemia. While wild type mice showed p62 translocation to mitochondria after IPC, Parkin knockout mice exhibited attenuated translocation of p62 to mitochondria. Importantly, ablation of Parkin in mice abolished the cardioprotective effects of IPC. These results reveal for the first time the crucial role of Parkin and mitophagy in cardioprotection.

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Endothelial glycocalyx thickness and platelet-vessel wall interactions during atherogenesis

Thrombosis and Haemostasis ◽

10.1160/th11-02-0133 ◽

2011 ◽

Vol 106 (11) ◽

pp. 939-946 ◽

Cited By ~ 33

Author(s):

Mirjam oude Egbrink ◽

Viviane Heijnen ◽

Remco Megens ◽

Wim Engels ◽

Hans Vink ◽

...

Keyword(s):

Vessel Wall ◽

Laser Scanning ◽

Ex Vivo ◽

Laser Scanning Microscopy ◽

Endothelial Glycocalyx ◽

Knock Out ◽

Two Photon ◽

Common Carotid Arteries ◽

Wall Interactions

SummaryThe endothelial glycocalyx (EG), the luminal cover of endothelial cells, is considered to be atheroprotective. During atherogenesis, platelets adhere to the vessel wall, possibly triggered by simultaneous EG modulation. It was the objective of this study to investigate both EG thickness and platelet-vessel wall interactions during atherogenesis in the same experimental model. Intravital fluorescence microscopy was used to study platelet-vessel wall interactions in vivo in common carotid arteries and bifurcations of C57bl6/J (B6) and apolipoprotein E knock-out (ApoE-/-) mice (age 7 – 31 weeks). At the same locations, EG thickness was determined ex vivo using two-photon laser scanning microscopy. In ApoE-/- bifurcations the overall median level of adhesion was 48 platelets/mm2 (interquartile range: 16 – 80), which was significantly higher than in B6 bifurcations (0 (0 – 16), p = 0.001). This difference appeared to result from a significant age-dependent increase in ApoE-/- mice, while no such change was observed in B6 mice. At the same time, the EG in ApoE-/- bifurcations was significantly thinner than in B6 bifurcations (2.2 vs. 2.5 μm, respectively; p < 0.05). This resulted from the fact that in B6 bifurcations EG thickness increased with age (from 2.4 μm in young mice to 3.0 μm in aged ones), while in bifurcations of ApoE-/- mice this growth appeared to be absent (2.2 μm at all ages). During atherogenesis, platelet adhesion to the wall of the carotid artery bifurcation increases significantly. At the same location, EG growth with age is hampered. Therefore, glycocalyx-reinforcing strategies could possibly ameliorate atherosclerosis.

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Rationally Designing Simple Rod-Like Amphiphilic NIR-Emissive AIE Probes for Precise In-Vivo Detection of Aβ Fibrils/Plaques at A Super-Early Stage

10.26434/chemrxiv.13699222.v1 ◽

2021 ◽

Author(s):

Yipu Wang ◽

Dong Mei ◽

Xinyi Zhang ◽

Da-Hui Qu ◽

Ju Mei ◽

...

Keyword(s):

High Performance ◽

Ex Vivo ◽

Early Stage ◽

Signal To Noise Ratio ◽

High Signal ◽

In Vivo Detection ◽

Different Strains ◽

Aβ Fibrils

With increase of social aging, Alzheimer's disease (AD) has been one of the serious diseases threatening human health. The occurrence of Aβ fibrils or plaques is recognized as the hallmark of AD. Currently, optical imaging has stood out to be a promising technique for the imaging of Aβ fibrils/plaques and the diagnosis of AD. However, restricted by their poor blood-brain barrier (BBB) penetrability, short-wavelength excitation and emission, and aggregation-caused quenching (ACQ) effect, the clinically used gold-standard optical probes such as <a>thioflavin</a> T (ThT) and thioflavin S (ThS), are not effective enough in the early diagnosis of AD in vivo. Herein, we put forward an “all-in-one” design principle and demonstrate its feasibility in developing high-performance fluorescent probes which are specific to Aβ fibrils/plaques and promising for super-early in-vivo diagnosis of AD. As a proof of concept, a simple rod-like amphiphilic NIR fluorescent AIEgen, i.e., AIE-CNPy-AD, is developed by taking the specificity, BBB penetration ability, deep-tissue penetration capacity, high signal-to-noise ratio (SNR) into consideration. AIE-CNPy-AD is constituted by connecting the electron-donating and accepting moieties through single bonds and tagging with a propanesulfonate tail, giving rise to the NIR fluorescence, aggregation-induced emission (AIE) effect, amphiphilicity, and rod-like structure, which in turn result in high binding-affinity and excellent specificity to Aβ fibrils/plaques, satisfactory ability to penetrate BBB and deep tissues, ultrahigh SNR and sensitivity, and high-fidelity imaging capability. In-vitro, ex-vivo, and in-vivo <a>identifying of Aβ fibrils/plaques</a> in different strains of mice indicate that AIE-CNPy-AD holds the universality to the detection of Aβ fibrils/plaques. It is noteworthy that AIE-CNPy-AD is even able to trace the small and sparsely distributed Aβ fibrils/plaques in very young AD model mice such as 4-month-old APP/PS1 mice which are reported to be the youngest mice to have Aβ deposits in brains, suggesting its great potential in diagnosis and intervention of AD at a super-early stage.

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Loss of Sirt3 accelerates arterial thrombosis by increasing formation of neutrophil extracellular traps and plasma tissue factor activity

Cardiovascular Research ◽

10.1093/cvr/cvy036 ◽

2018 ◽

Vol 114 (8) ◽

pp. 1178-1188 ◽

Cited By ~ 14

Author(s):

Daniel S Gaul ◽

Julien Weber ◽

Lambertus J van Tits ◽

Susanna Sluka ◽

Lisa Pasterk ◽

...

Keyword(s):

Oxidative Stress ◽

Myocardial Infarction ◽

Bone Marrow ◽

Arterial Thrombosis ◽

Ex Vivo ◽

Neutrophil Extracellular Traps ◽

Wild Type ◽

Rotational Thromboelastometry ◽

Extracellular Traps

AbstractAimsSirtuin 3 (Sirt3) is a mitochondrial, nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase that reduces oxidative stress by activation of superoxide dismutase 2 (SOD2). Oxidative stress enhances arterial thrombosis. This study investigated the effects of genetic Sirt3 deletion on arterial thrombosis in mice in an inflammatory setting and assessed the clinical relevance of these findings in patients with ST-elevation myocardial infarction (STEMI).Methods and resultsUsing a laser-induced carotid thrombosis model with lipopolysaccharide (LPS) challenge, in vivo time to thrombotic occlusion in Sirt3−/− mice (n = 6) was reduced by half compared to Sirt3+/+ wild-type (n = 8, P < 0.01) controls. Ex vivo analyses of whole blood using rotational thromboelastometry revealed accelerated clot formation and increased clot stability in Sirt3−/− compared to wild-type blood. rotational thromboelastometry of cell-depleted plasma showed accelerated clotting initiation in Sirt3−/− mice, whereas overall clot formation and firmness remained unaffected. Ex vivo LPS-induced neutrophil extracellular trap formation was increased in Sirt3−/− bone marrow-derived neutrophils. Plasma tissue factor (TF) levels and activity were elevated in Sirt3−/− mice, whereas plasma levels of other coagulation factors and TF expression in arterial walls remained unchanged. SOD2 expression in bone marrow -derived Sirt3−/− neutrophils was reduced. In STEMI patients, transcriptional levels of Sirt3 and its target SOD2 were lower in CD14+ leukocytes compared with healthy donors (n = 10 each, P < 0.01).ConclusionsSirt3 loss-of-function enhances experimental thrombosis in vivo via an increase of neutrophil extracellular traps and elevation of TF suggesting thrombo-protective effects of endogenous Sirt3. Acute coronary thrombosis in STEMI patients is associated with lower expression levels of SIRT3 and SOD2 in CD14+ leukocytes. Therefore, enhancing SIRT3 activity by pan-sirtuin activating NAD+-boosters may provide a novel therapeutic target to prevent or treat thrombotic arterial occlusion in myocardial infarction or stroke.

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Potential caveats of putative microglia-specific markers for assessment of age-related cerebrovascular neuroinflammation

Journal of Neuroinflammation ◽

10.1186/s12974-020-02019-5 ◽

2020 ◽

Vol 17 (1) ◽

Author(s):

Pedram Honarpisheh ◽

Juneyoung Lee ◽

Anik Banerjee ◽

Maria P. Blasco-Conesa ◽

Parisa Honarpisheh ◽

...

Keyword(s):

Flow Cytometry ◽

Ex Vivo ◽

Myeloid Cells ◽

Amyloid Angiopathy ◽

Future Studies ◽

Age Related ◽

In Vivo Animal Models ◽

Intermediate Expression ◽

The Brain

Abstract Background The ability to distinguish resident microglia from infiltrating myeloid cells by flow cytometry-based surface phenotyping is an important technique for examining age-related neuroinflammation. The most commonly used surface markers for the identification of microglia include CD45 (low-intermediate expression), CD11b, Tmem119, and P2RY12. Methods In this study, we examined changes in expression levels of these putative microglia markers in in vivo animal models of stroke, cerebral amyloid angiopathy (CAA), and aging as well as in an ex vivo LPS-induced inflammation model. Results We demonstrate that Tmem119 and P2RY12 expression is evident within both CD45int and CD45high myeloid populations in models of stroke, CAA, and aging. Interestingly, LPS stimulation of FACS-sorted adult microglia suggested that these brain-resident myeloid cells can upregulate CD45 and downregulate Tmem119 and P2RY12, making them indistinguishable from peripherally derived myeloid populations. Importantly, our findings show that these changes in the molecular signatures of microglia can occur without a contribution from the other brain-resident or peripherally sourced immune cells. Conclusion We recommend future studies approach microglia identification by flow cytometry with caution, particularly in the absence of the use of a combination of markers validated for the specific neuroinflammation model of interest. The subpopulation of resident microglia residing within the “infiltrating myeloid” population, albeit small, may be functionally important in maintaining immune vigilance in the brain thus should not be overlooked in neuroimmunological studies.

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Targeted inactivation of Gαi does not alter cardiac function or β-adrenergic sensitivity

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2001.280.2.h569 ◽

2001 ◽

Vol 280 (2) ◽

pp. H569-H575 ◽

Cited By ~ 22

Author(s):

Mohit Jain ◽

Chee Chew Lim ◽

Kohzo Nagata ◽

Vannessa M. Davis ◽

David S. Milstone ◽

...

Keyword(s):

Cardiac Function ◽

Ventricular Myocytes ◽

Ex Vivo ◽

Cardiac Response ◽

Similar Degree ◽

Wild Type ◽

Isolated Hearts ◽

Targeted Inactivation ◽

Contractile Performance

Inhibitory Gαi protein increases in the myocardium during hypertrophy and has been associated with β-adrenergic receptor (β-AR) desensitization, contractile dysfunction, and progression of cardiac disease. The role of Gαi proteins in mediating basal cardiac function and β-AR response in nonpathological myocardium, however, is uncertain. Transgenic mice with targeted inactivation of Gαi2 or Gαi3 were examined for in vivo cardiac function with the use of conscious echocardiography and for ex vivo cardiac response to inotropic stimulation with the use of Langendorff blood-perfused isolated hearts and adult ventricular cardiomyocytes. Echocardiography revealed that percent fractional shortening and heart rate were similar among wild-type, Gαi2 -null, and Gαi3 -null mice. Comparable baseline diastolic and contractile performance was also observed in isolated hearts and isolated ventricular myocytes from wild-type mice and mice lacking Gαi proteins. Isoproterenol infusion enhanced diastolic and contractile performance to a similar degree in wild-type, Gαi2 -null, and Gαi3 -null mice. These data demonstrate no observable role for inhibitory G proteins in mediating basal cardiac function or sensitivity to β-AR stimulation in nonpathological myocardium.

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