Platelet-Released Factors: Their Role in Viral Disease and Applications for Extracellular Vesicle (EV) Therapy.

Platelets, which are small anuclear cell fragments, play important roles in thrombosis and hemostasis, but also actively release factors that can both suppress and induce viral infections. Platelet-released factors include sCD40L, microvesicles (MVs), and alpha granules that have the capacity to exert either pro-inflammatory or anti-inflammatory effects depending on the virus. These factors are prime targets for use in extracellular vesicle (EV)-based therapy due to their ability to reduce viral infections and exert anti-inflammatory effects. While there are some studies regarding platelet microvesicle-based (PMV-based) therapy, there is still much to learn about PMVs before such therapy can be used. This review provides the background necessary to understand the roles of platelet-released factors, how these factors might be useful in PMV-based therapy, and a critical discussion of current knowledge of platelets and their role in viral diseases.

Upregulation of cytokine signalling in platelets increases risk of thrombophilia in severe COVID-19 patients.

Abnormal coagulation dynamics, including disseminated intravascular coagulopathy, pulmonary embolism, venous thromboembolism and risk of thrombosis are often associated with the severity of COVID-19. However, very little is known about the contribution of platelets in above pathogenesis. In order to decipher the pathophysiology of thrombophilia in COVID-19, we recruited severely ill patients from ICU, based on the above symptoms and higher D-dimer levels, and compared these parameters with their asymptomatic counterparts. Elevated levels of platelet-derived microparticles and platelet-leukocyte aggregates suggested the hyperactivation of platelets in ICU patients. Strikingly, platelet transcriptome analysis showed a greater association of IL-6 and TNF signalling pathways in ICU patients along with higher plasma levels of IL-6 and TNFα. In addition, upregulation of pathways like blood coagulation and hemostasis, as well as inflammation coexisted in platelets of these patients. Further, the increment of necrotic pathway and ROS-metabolic processes in platelets was suggestive of its procoagulant phenotype in ICU patients. This study suggests that higher plasma IL-6 and TNFα may trigger platelet activation and coagulation, and in turn aggravate thrombosis and hypercoagulation in severe COVID-19 patients. Therefore, the elevated IL-6 and TNFα, may serve as potential risk factors for platelet activation and thrombophilia in these patients.

SARS-CoV-2 treatment effects induced by ACE2-expressing microparticles are explained by the oxidized cholesterol-increased endosomal pH of alveolar macrophages.

Exploring the cross-talk between the immune system and advanced biomaterials to treat SARS-CoV-2 infection is a promising strategy. Here, we show that ACE2-overexpressing A549 cell-derived microparticles (AO-MPs) are a potential therapeutic agent against SARS-CoV-2 infection. Intranasally administered AO-MPs dexterously navigate the anatomical and biological features of the lungs to enter the alveoli and are taken up by alveolar macrophages (AMs). Then, AO-MPs increase the endosomal pH but decrease the lysosomal pH in AMs, thus escorting bound SARS-CoV-2 from phago-endosomes to lysosomes for degradation. This pH regulation is attributable to oxidized cholesterol, which is enriched in AO-MPs and translocated to endosomal membranes, thus interfering with proton pumps and impairing endosomal acidification. In addition to promoting viral degradation, AO-MPs also inhibit the proinflammatory phenotype of AMs, leading to increased treatment efficacy in a SARS-CoV-2-infected mouse model without side effects. These findings highlight the potential use of AO-MPs to treat SARS-CoV-2-infected patients and showcase the feasibility of MP therapies for combatting emerging respiratory viruses in the future.

Circulating Microvesicle-Associated Inducible Nitric Oxide Synthase Is a Novel Therapeutic Target to Treat Sepsis: Current Status and Future Considerations.

To determine whether mitigating the harmful effects of circulating microvesicle-associated inducible nitric oxide (MV-A iNOS) in vivo increases the survival of challenged mice in three different mouse models of sepsis, the ability of anti-MV-A iNOS monoclonal antibodies (mAbs) to rescue challenged mice was assessed using three different mouse models of sepsis. The vivarium of a research laboratory Balb/c mice were challenged with an LD80 dose of either lipopolysaccharide (LPS/endotoxin), TNFα, or MV-A iNOS and then treated at various times after the challenge with saline as control or with an anti-MV-A iNOS mAb as a potential immunotherapeutic to treat sepsis. Each group of mice was checked daily for survivors, and Kaplan-Meier survival curves were constructed. Five different murine anti-MV-A iNOS mAbs from our panel of 24 murine anti-MV-A iNOS mAbs were found to rescue some of the challenged mice. All five murine mAbs were used to genetically engineer humanized anti-MV-A iNOS mAbs by inserting the murine complementarity-determining regions (CDRs) into a human IgG1,kappa scaffold and expressing the humanized mAbs in CHO cells. Three humanized anti-MV-A iNOS mAbs were effective at rescuing mice from sepsis in three different animal models of sepsis. The effectiveness of the treatment was both time- and dose-dependent. Humanized anti-MV-A iNOS rHJ mAb could rescue up to 80% of the challenged animals if administered early and at a high dose. Our conclusions are that MV-A iNOS is a novel therapeutic target to treat sepsis; anti-MV-A iNOS mAbs can mitigate the harmful effects of MV-A iNOS; the neutralizing mAb's efficacy is both time- and dose-dependent; and a specifically targeted immunotherapeutic for MV-A iNOS could potentially save tens of thousands of lives annually and could result in improved antibiotic stewardship.

Exosomes/microvesicles target SARS-CoV-2 via innate and RNA-induced immunity with PIWI-piRNA system.

Murine neural stem cells (NSCs) were recently shown to release piRNA-containing exosomes/microvesicles (Ex/Mv) for exerting antiviral immunity, but it remains unknown if these Ex/Mv could target SARS-CoV-2 and whether the PIWI-piRNA system is important for these antiviral actions. Here, using in vitro infection models, we show that hypothalamic NSCs (htNSCs) Ex/Mv provided an innate immunity protection against SARS-CoV-2. Importantly, enhanced antiviral actions were achieved by using induced Ex/Mv that were derived from induced htNSCs through twice being exposed to several RNA fragments of SARS-CoV-2 genome, a process that was designed not to involve protein translation of these RNA fragments. The increased antiviral effects of these induced Ex/Mv were associated with increased expression of piRNA species some of which could predictably target SARS-CoV-2 genome. Knockout of piRNA-interacting protein PIWIL2 in htNSCs led to reductions in both innate and induced antiviral effects of Ex/Mv in targeting SARS-CoV-2. Taken together, this study demonstrates a case suggesting Ex/Mv from certain cell types have innate and adaptive immunity against SARS-CoV-2, and the PIWI-piRNA system is important for these antiviral actions.

Binding of phosphatidylserine-positive microparticles by PBMCs classifies disease severity in COVID-19 patients.

Infection with SARS-CoV-2 is associated with thromboinflammation, involving thrombotic and inflammatory responses, in many COVID-19 patients. In addition, immune dysfunction occurs in patients characterised by T cell exhaustion and severe lymphopenia. We investigated the distribution of phosphatidylserine (PS), a marker of dying cells, activated platelets and platelet-derived microparticles (PMP), during the clinical course of COVID-19. We found an unexpectedly high amount of blood cells loaded with PS+ PMPs for weeks after the initial COVID-19 diagnosis. Elevated frequencies of PS+ PMP+ PBMCs correlated strongly with increasing disease severity. As a marker, PS outperformed established laboratory markers for inflammation, leucocyte composition and coagulation, currently used for COVID-19 clinical scoring. PS+ PMPs preferentially bound to CD8+ T cells with gene expression signatures of proliferating effector rather than memory T cells. As PS+ PMPs carried programmed death-ligand 1 (PD-L1), they may affect T cell expansion or function. Our data provide a novel marker for disease severity and show that PS, which can trigger the blood coagulation cascade, the complement system, and inflammation, resides on activated immune cells. Therefore, PS may serve as a beacon to attract thromboinflammatory processes towards lymphocytes and cause immune dysfunction in COVID-19.

COVID-19 Infection and Circulating Microparticles-Reviewing Evidence as Microthrombogenic Risk Factor for Cerebral Small Vessel Disease.

Severe acute respiratory syndrome corona virus-2 (SARS-CoV-2) due to novel coronavirus disease 2019 (COVID-19) has affected the global society in numerous unprecedented ways, with considerable morbidity and mortality. Both direct and indirect consequences from COVID-19 infection are recognized to give rise to cardio- and cerebrovascular complications. Despite current limited knowledge on COVID-19 pathogenesis, inflammation, endothelial dysfunction, and coagulopathy appear to play critical roles in COVID-19-associated cerebrovascular disease (CVD). One of the major subtypes of CVD is cerebral small vessel disease (CSVD) which represents a spectrum of pathological processes of various etiologies affecting the brain microcirculation that can trigger subsequent neuroinflammation and neurodegeneration. Prevalent with aging, CSVD is a recognized risk factor for stroke, vascular dementia, and Alzheimer's disease. In the background of COVID-19 infection, the heightened cellular activations from inflammations and oxidative stress may result in elevated levels of microthrombogenic extracellular-derived circulating microparticles (MPs). Consequently, MPs could act as pro-coagulant risk factor that may serve as microthrombi for the vulnerable microcirculation in the brain leading to CSVD manifestations. This review aims to appraise the accumulating body of evidence on the plausible impact of COVID-19 infection on the formation of microthrombogenic MPs that could lead to microthrombosis in CSVD manifestations, including occult CSVD which may last well beyond the pandemic era.

Cell-mimicking nanodecoys neutralize SARS-CoV-2 and mitigate lung injury in a non-human primate model of COVID-19.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has grown into a global pandemic, and only a few antiviral treatments have been approved to date. Angiotensin-converting enzyme 2 (ACE2) plays a fundamental role in SARS-CoV-2 pathogenesis because it allows viral entry into host cells. Here we show that ACE2 nanodecoys derived from human lung spheroid cells (LSCs) can bind and neutralize SARS-CoV-2 and protect the host lung cells from infection. In mice, these LSC-nanodecoys were delivered via inhalation therapy and resided in the lungs for over 72 h post-delivery. Furthermore, inhalation of the LSC-nanodecoys accelerated clearance of SARS-CoV-2 mimics from the lungs, with no observed toxicity. In cynomolgus macaques challenged with live SARS-CoV-2, four doses of these nanodecoys delivered by inhalation promoted viral clearance and reduced lung injury. Our results suggest that LSC-nanodecoys can serve as a potential therapeutic agent for treating COVID-19.