SARS-CoV-2 infection modifies the transcriptome of the megakaryocytes in the bone marrow.

Megakaryocytes, integral to platelet production, predominantly reside in the bone marrow and undergo regulated fragmentation within sinusoid vessels to release platelets into the bloodstream. Inflammatory states and infections influence megakaryocyte transcription, potentially affecting platelet functionality. Notably, COVID-19 has been associated with altered platelet transcriptomes. In this study, we investigated the hypothesis that SARS-CoV-2 infection could impact the transcriptome of bone marrow megakaryocytes. Utilizing spatial transcriptomics to discriminate subpopulations of megakaryocytes based on proximity to bone marrow sinusoids, we identified approximately 19,000 genes in megakaryocytes. Machine learning techniques revealed that the transcriptome of healthy murine bone marrow megakaryocytes exhibited minimal differences based on proximity to sinusoid vessels. Further, at peak SARS-CoV-2 viremia, when the disease primarily affected the lungs, megakaryocytes were not significantly different from those from healthy mice. Conversely, a significant divergence in the megakaryocyte transcriptome was observed during systemic inflammation, although SARS-CoV-2 RNA was never detected in bone marrow and it was no longer detectable in the lungs. Under these conditions, the megakaryocyte transcriptional landscape was enriched in pathways associated with histone modifications, megakaryocyte differentiation, NETosis, and autoimmunity, which could not be explained by cell proximity to sinusoid vessels. Notably, the type-I interferon signature and calprotectin (S100A8/A9) were not induced in megakaryocytes under any condition. However, inflammatory cytokines induced in the blood and lungs of COVID-19 mice were different from those found in the bone marrow, suggesting a discriminating impact of inflammation on this specific subset of cells. Collectively, our data indicate that a new population of bone marrow megakaryocytes may emerge through COVID-19-related pathogenesis.

Diversity of Megakaryocytes.

Megakaryocytes are commonly known as large, polyploid, bone marrow resident cells that contribute to hemostasis through the production of platelets. Soon after their discovery in the 19th century, megakaryocytes were described in tissue locations other than the bone marrow, specifically in the lungs and the blood circulation. However, the localization of megakaryocytes in the lungs and the contribution of lung megakaryocytes to the general platelet pool has only recently been appreciated. Moreover, the conception of megakaryocytes as uniform cells with the sole purpose of platelet production has been challenged. Here, we review the literature on megakaryocyte cell identity and location with a special focus on recent observations of megakaryocyte subpopulations identified by transcriptomic analyses.

Cytokines and Lipid Mediators of Inflammation in Lungs of SARS-CoV-2 Infected Mice.

Coronavirus disease 19 (COVID-19) is the clinical manifestation of severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) infection. A hallmark of COVID-19 is a lung inflammation characterized by an abundant leukocyte infiltrate, elevated levels of cytokines/chemokines, lipid mediators of inflammation (LMI) and microthrombotic events. Animal models are useful for understanding the pathophysiological events leading to COVID-19. One such animal model is the K18-ACE2 transgenic mice. Despite their importance in inflammation, the study of LMI in lung of SARS-CoV-2 infected K18-ACE2 mice has yet to be studied to our knowledge. Using tandem mass spectrometry, the lung lipidome at different time points of infection was analyzed. Significantly increased LMI included N-oleoyl-serine, N-linoleoyl-glycine, N-oleoyl-alanine, 1/2-linoleoyl-glycerol, 1/2-docosahexaenoyl-glycerol and 12-hydroxy-eicosapenatenoic acid. The levels of prostaglandin (PG) E1, PGF2α, stearoyl-ethanolamide and linoleoyl-ethanolamide were found to be significantly reduced relative to mock-infected mice. Other LMI were present at similar levels (or undetected) in both uninfected and infected mouse lungs. In parallel to LMI measures, transcriptomic and cytokine/chemokine profiling were performed. Viral replication was robust with maximal lung viral loads detected on days 2-3 post-infection. Lung histology revealed leukocyte infiltration starting on day 3 post-infection, which correlated with the presence of high concentrations of several chemokines/cytokines. At early times post-infection, the plasma of infected mice contained highly elevated concentration of D-dimers suggestive of blood clot formation/dissolution. In support, the presence of blood clots in the lung vasculature was observed during infection. RNA-Seq analysis of lung tissues indicate that SARS-CoV-2 infection results in the progressive modulation of several hundred genes, including several inflammatory mediators and genes related to the interferons. Analysis of the lung lipidome indicated modest, yet significant modulation of a minority of lipids. In summary, our study suggests that SARS-CoV-2 infection in humans and mice share common features, such as elevated levels of chemokines in lungs, leukocyte infiltration and increased levels of circulating D-dimers. However, the K18-ACE2 mouse model highlight major differences in terms of LMI being produced in response to SARS-CoV-2 infection. The potential reasons and impact of these differences on the pathology and therapeutic strategies to be employed to treat severe COVID-19 are discussed.

Platelet activation by SARS-CoV-2 implicates the release of active tissue factor by infected cells.

Platelets are hyperactivated in coronavirus disease 2019 (COVID-19). However, the mechanisms promoting platelet activation by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are not well understood. This may be due to inherent challenges in discriminating the contribution of viral vs host components produced by infected cells. This is particularly true for enveloped viruses and extracellular vesicles (EVs), as they are concomitantly released during infection and share biophysical properties. To study this, we evaluated whether SARS-CoV-2 itself or components derived from SARS-CoV-2-infected human lung epithelial cells could activate isolated platelets from healthy donors. Activation was measured by the surface expression of P-selectin and the activated conformation of integrin αIIbß3, degranulation, aggregation under flow conditions, and the release of EVs. We find that neither SARS-CoV-2 nor purified spike activates platelets. In contrast, tissue factor (TF) produced by infected cells was highly potent at activating platelets. This required trace amounts of plasma containing the coagulation factors FX, FII, and FVII. Robust platelet activation involved thrombin and the activation of protease-activated receptor (PAR)-1 and -4 expressed by platelets. Virions and EVs were identified by electron microscopy. Through size-exclusion chromatography, TF activity was found to be associated with a virus or EVs, which were indistinguishable. Increased TF messenger RNA (mRNA) expression and activity were also found in lungs in a murine model of COVID-19 and plasma of severe COVID-19 patients, respectively. In summary, TF activity from SARS-CoV-2-infected cells activates thrombin, which signals to PARs on platelets. Blockade of molecules in this pathway may interfere with platelet activation and the coagulation characteristic of COVID-19.

Platelet extracellular vesicles in COVID-19: Potential markers and makers.

Platelets and platelet extracellular vesicles (pEV) are at the crossroads of coagulation and immunity. Extracellular vesicles are messengers that not only transmit signals between cells, but also provide information about the status of their cell of origin. Thus, pEVs have potential as both biomarkers of platelet activation and contributors to pathology. Coronavirus Disease-19 (COVID-19), caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a complex disease affecting multiple organs and is characterized by a high degree of inflammation and risk of thrombosis in some patients. In this review, we introduce pEVs as valuable biomarkers in disease with a special focus on their potential as predictors of and contributors to COVID-19.

Extracellular vesicles are associated with C-reactive protein in sepsis.

There is increasing evidence that C-reactive protein (CRP) can mediate inflammatory reactions following the transformation of functionally inert pentameric CRP (pCRP) into its structural isoform pCRP* and into monomeric CRP (mCRP). This conversion can occur on the membranes of apoptotic or activated cells or on extracellular vesicles (EVs) shed from the cell surface. Here, we characterized the association of CRP with EVs in plasma from sepsis patients using flow cytometry, and found highly elevated levels of total EV counts and CRP+ EVs as compared to healthy individuals. We further assessed the ability of PentraSorb CRP, an extracorporeal device for the adsorption of CRP, to deplete free CRP and CRP+ EVs. Treatment of septic plasma with the adsorbent in vitro resulted in almost complete removal of both, free CRP and CRP+ EVs, while total EV counts remained largely unaffected, indicating the detachment of CRP from the EV surface. EVs from septic plasma elicited a release of interleukin-8 from cultured human monocytes, which was significantly reduced by adsorbent treatment prior to EV isolation. Our findings provide evidence that CRP+ EVs exhibit pro-inflammatory characteristics and can contribute to the spreading of inflammation throughout the circulation on top of their pro-coagulant activity.

Natural IgM antibodies inhibit microvesicle-driven coagulation and thrombosis.

Thrombosis and its associated complications are a major cause of morbidity and mortality worldwide. Microvesicles (MVs), a class of extracellular vesicles, are increasingly recognized as mediators of coagulation and biomarkers of thrombotic risk. Thus, identifying factors targeting MV-driven coagulation may help in the development of novel antithrombotic treatments. We have previously identified a subset of circulating MVs that is characterized by the presence of oxidation-specific epitopes and bound by natural immunoglobulin M (IgM) antibodies targeting these structures. This study investigated whether natural IgM antibodies, which are known to have important anti-inflammatory housekeeping functions, inhibit the procoagulatory properties of MVs. We found that the extent of plasma coagulation is inversely associated with the levels of both free and MV-bound endogenous IgM. Moreover, the oxidation epitope-specific natural IgM antibody LR04, which recognizes malondialdehyde adducts, reduced MV-dependent plasmatic coagulation and whole blood clotting without affecting thrombocyte aggregation. Intravenous injection of LR04 protected mice from MV-induced pulmonary thrombosis. Of note, LR04 competed the binding of coagulation factor X/Xa to MVs, providing a mechanistic explanation for its anticoagulatory effect. Thus, our data identify natural IgM antibodies as hitherto unknown modulators of MV-induced coagulation in vitro and in vivo and their prognostic and therapeutic potential in the management of thrombosis.

Platelet Extracellular Vesicles: Beyond the Blood.

Extracellular vesicles (EVs) are a means of cell-to-cell communication and can facilitate the exchange of a broad array of molecules between adjacent or distant cells. Platelets are anucleate cells derived from megakaryocytes and are primarily known for their role in maintaining hemostasis and vascular integrity. Upon activation by a variety of agonists, platelets readily generate EVs, which were initially identified as procoagulant particles. However, as both platelets and their EVs are abundant in blood, the role of platelet EVs in hemostasis may be redundant. Moreover, findings have challenged the significance of platelet-derived EVs in coagulation. Looking beyond hemostasis, platelet EV cargo is incredibly diverse and can include lipids, proteins, nucleic acids, and organelles involved in numerous other biological processes. Furthermore, while platelets cannot cross tissue barriers, their EVs can enter lymph, bone marrow, and synovial fluid. This allows for the transfer of platelet-derived content to cellular recipients and organs inaccessible to platelets. This review highlights the importance of platelet-derived EVs in physiological and pathological conditions beyond hemostasis.

Platelets Can Associate with SARS-Cov-2 RNA and Are Hyperactivated in COVID-19.

Rationale: In addition to the overwhelming lung inflammation that prevails in COVID-19, hypercoagulation and thrombosis contribute to the lethality of subjects infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Platelets are chiefly implicated in thrombosis. Moreover, they can interact with viruses and are an important source of inflammatory mediators. While a lower platelet count is associated with severity and mortality, little is known about platelet function during COVID-19. Objective: To evaluate the contribution of platelets to inflammation and thrombosis in COVID-19 patients. Methods and Results: Blood was collected from 115 consecutive COVID-19 patients presenting non-severe (n=71) and severe (n=44) respiratory symptoms. We document the presence of SARS-CoV-2 RNA associated with platelets of COVID-19 patients. Exhaustive assessment of cytokines in plasma and in platelets revealed the modulation of platelet-associated cytokine levels in both non-severe and severe COVID-19 patients, pointing to a direct contribution of platelets to the plasmatic cytokine load. Moreover, we demonstrate that platelets release their alpha- and dense-granule contents in both non-severe and severe forms of COVID-19. In comparison to concentrations measured in healthy volunteers, phosphatidylserine-exposing platelet extracellular vesicles were increased in non-severe, but not in severe cases of COVID-19. Levels of D-dimers, a marker of thrombosis, failed to correlate with any measured indicators of platelet activation. Functionally, platelets were hyperactivated in COVID-19 subjects presenting non-severe and severe symptoms, with aggregation occurring at suboptimal thrombin concentrations. Furthermore, platelets adhered more efficiently onto collagen-coated surfaces under flow conditions. Conclusions: Taken together, the data suggest that platelets are at the frontline of COVID-19 pathogenesis, as they release various sets of molecules through the different stages of the disease. Platelets may thus have the potential to contribute to the overwhelming thrombo-inflammation in COVID-19, and the inhibition of pathways related to platelet activation may improve the outcomes during COVID-19.

Ikk2-mediated inflammatory activation of arterial endothelial cells promotes the development and progression of atherosclerosis.

BACKGROUND AND AIMS: Inflammatory activation of endothelial cells is considered to be the first step in the development of atherosclerosis. Here, we determined the consequences of chronic endothelial activation via the NF-κB activator Ikk2 (Inhibitor of nuclear factor kappa-B kinase 2, Ikk-beta) on the development and progression of atherosclerosis. METHODS: We established a conditional transgenic mouse model, expressing a tamoxifen-inducible, constitutively active form of Ikk2 exclusively in arterial endothelial cells (caIkk2EC mice) on an ApoE-deficient background. Mice were fed a Western-type diet and endothelial Ikk2 was activated either at early or late stages of atherosclerosis. RESULTS: En face preparations of isolated aortas revealed a significant increase in plaque area in caIkk2EC mice at 12 weeks of Western-type diet as compared to ApoE-deficient littermates. This was accompanied by increased infiltration of macrophages and T cells into the lesion. Several chemokine/cytokine and immune cell pathways were significantly upregulated in the aortic transcriptome of caIkk2EC mice. Of note, in mice with established atherosclerosis, activation of endothelial Ikk2 still further accelerated progression of atherosclerosis. This indicates that inflammatory endothelial activation is crucial during all stages of the disease. CONCLUSIONS: Our results show for the first time that chronic inflammatory activation of arterial endothelial cells accelerates the development and progression of atherosclerosis both at early and late stages of disease development. Thus, pharmacological targeting of endothelial inflammation emerges as a promising treatment approach.

Mitochondria Are a Subset of Extracellular Vesicles Released by Activated Monocytes and Induce Type I IFN and TNF Responses in Endothelial Cells.

RATIONALE: Extracellular vesicles, including microvesicles, are increasingly recognized as important mediators in cardiovascular disease. The cargo and surface proteins they carry are considered to define their biological activity, including their inflammatory properties. Monocyte to endothelial cell signaling is a prerequisite for the propagation of inflammatory responses. However, the contribution of microvesicles in this process is poorly understood. OBJECTIVE: To elucidate the mechanisms by which microvesicles derived from activated monocytic cells exert inflammatory effects on endothelial cells. METHODS AND RESULTS: LPS (lipopolysaccharide)-stimulated monocytic cells release free mitochondria and microvesicles with mitochondrial content as demonstrated by flow cytometry, quantitative polymerase chain reaction, Western Blot, and transmission electron microscopy. Using RNAseq analysis and quantitative reverse transcription-polymerase chain reaction, we demonstrated that both mitochondria directly isolated from and microvesicles released by LPS-activated monocytic cells, as well as circulating microvesicles isolated from volunteers receiving low-dose LPS-injections, induce type I IFN (interferon), and TNF (tumor necrosis factor) responses in endothelial cells. Depletion of free mitochondria significantly reduced the ability of these microvesicles to induce type I IFN and TNF-dependent genes. We identified mitochondria-associated TNFα and RNA from stressed mitochondria as major inducers of these responses. Finally, we demonstrated that the proinflammatory potential of microvesicles and directly isolated mitochondria were drastically reduced when they were derived from monocytic cells with nonrespiring mitochondria or monocytic cells cultured in the presence of pyruvate or the mitochondrial reactive oxygen species scavenger MitoTEMPO. CONCLUSIONS: Mitochondria and mitochondria embedded in microvesicles constitute a major subset of extracellular vesicles released by activated monocytes, and their proinflammatory activity on endothelial cells is determined by the activation status of their parental cells. Thus, mitochondria may represent critical intercellular mediators in cardiovascular disease and other inflammatory settings associated with type I IFN and TNF signaling.

Hyponatremia and V2 vasopressin receptor upregulation: a result of HSP90 inhibition.

PURPOSE: Small-molecule inhibitors of heat-shock protein 90 (HSP90) have been under development as chemotherapeutic agents. The adverse events reported from early clinical trials included hyponatremia. Given the limited number of patients enrolled, the number of hyponatremia incidents was remarkable and repeatedly, the event was judged as severe. Inappropriate V2 vasopressin receptor stimulation is an established cause of hyponatremia. We explored the hypothesis that HSP90 inhibition produces hypersensitivity to vasopressin by upregulating V2-receptors. METHODS: Experiments were carried out in cell culture using HEK293 cells with heterologous expression of the human V2-receptor and HELA cells with an endogenous V2-receptor complement. We tested the effect of HSP90 inhibition by three structurally unrelated compounds (alvespimycin, luminespib, radicicol) and asserted its specificity in cells depleted of cytosolic HSP90 (by RNA interference). Assays encompassed surface V2-receptor density and vasopressin-stimulated formation of cyclic AMP (cAMP). RESULTS: The results demonstrate a twofold increase in cell-surface receptor density following pre-incubation with each of the HSP90 inhibitors. The effect had a concentration-dependence consistent with the individual potencies to inhibit HSP90. Similarly, depletion of cytosolic HSP90 increased surface-receptor density and at the same time, reduced the inhibitor effect. Upregulated V2-receptors were fully functional; hence, in culture treated with an HSP90 inhibitor, addition of vasopressin resulted in higher levels of cAMP than in controls. CONCLUSION: Since formation of cAMP is the first signalling step in raising water permeability of the collecting duct epithelia, we suggest that V2-receptor upregulation generates hypersensitivity to vasopressin linking HSP90 inhibition to the development of hyponatremia.

Characterization of Natural IgM Antibodies Recognizing Oxidation-Specific Epitopes on Circulating Microvesicles.

Natural IgM antibodies specific for oxidation-specific epitopes (OSEs) have been found to bind to circulating microvesicles (MVs), also known as microparticles. This chapter describes how endogenous natural IgM antibodies bound to circulating MV subsets can be characterized on the one hand, and how monoclonal natural IgM antibodies can be used to characterize subsets of circulating MVs on the other hand.

Malondialdehyde epitopes are sterile mediators of hepatic inflammation in hypercholesterolemic mice.

Diet-related health issues such as nonalcoholic fatty liver disease and cardiovascular disorders are known to have a major inflammatory component. However, the exact pathways linking diet-induced changes (e.g., hyperlipidemia) and the ensuing inflammation have remained elusive so far. We identified biological processes related to innate immunity and oxidative stress as prime response pathways in livers of low-density lipoprotein receptor-deficient mice on a Western-type diet using RNA sequencing and in silico functional analyses of transcriptome data. The observed changes were independent of the presence of microbiota and thus indicative of a role for sterile triggers. We further show that malondialdehyde (MDA) epitopes, products of lipid peroxidation and markers for enhanced oxidative stress, are detectable in hepatic inflammation predominantly on dying cells and stimulate cytokine secretion as well as leukocyte recruitment in vitro and in vivo. MDA-induced cytokine secretion in vitro was dependent on the presence of the scavenger receptors CD36 and MSR1. Moreover, in vivo neutralization of endogenously generated MDA epitopes by intravenous injection of a specific MDA antibody results in decreased hepatic inflammation in low-density lipoprotein receptor-deficient mice on a Western-type diet. CONCLUSION: Accumulation of MDA epitopes plays a major role during diet-induced hepatic inflammation and can be ameliorated by administration of an anti-MDA antibody. (Hepatology 2017;65:1181-1195).

Two types of muscarinic acetylcholine receptors in Drosophila and other arthropods.

Muscarinic acetylcholine receptors (mAChRs) play a central role in the mammalian nervous system. These receptors are G protein-coupled receptors (GPCRs), which are activated by the agonists acetylcholine and muscarine, and blocked by a variety of antagonists. Mammals have five mAChRs (m1-m5). In this study, we cloned two structurally related GPCRs from the fruit fly Drosophila melanogaster, which, after expression in Chinese hamster ovary cells, proved to be muscarinic acetylcholine receptors. One mAChR (the A-type; encoded by gene CG4356) is activated by acetylcholine (EC50, 5 × 10(-8) M) and muscarine (EC50, 6 × 10(-8) M) and blocked by the classical mAChR antagonists atropine, scopolamine, and 3-quinuclidinyl-benzilate (QNB), while the other (the B-type; encoded by gene CG7918) is also activated by acetylcholine, but has a 1,000-fold lower sensitivity to muscarine, and is not blocked by the antagonists. A- and B-type mAChRs were also cloned and functionally characterized from the red flour beetle Tribolium castaneum. Recently, Haga et al. (Nature 2012, 482: 547-551) published the crystal structure of the human m2 mAChR, revealing 14 amino acid residues forming the binding pocket for QNB. These residues are identical between the human m2 and the D. melanogaster and T. castaneum A-type mAChRs, while many of them are different between the human m2 and the B-type receptors. Using bioinformatics, one orthologue of the A-type and one of the B-type mAChRs could also be found in all other arthropods with a sequenced genome. Protostomes, such as arthropods, and deuterostomes, such as mammals and other vertebrates, belong to two evolutionarily distinct lineages of animal evolution that split about 700 million years ago. We found that animals that originated before this split, such as cnidarians (Hydra), had two A-type mAChRs. From these data we propose a model for the evolution of mAChRs.