RAGE Is a Receptor for SARS-CoV-2 N Protein and Mediates N Protein-induced Acute Lung Injury.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (N-protein) increases early in body fluids during infection and has recently been identified as a direct inducer for lung injury. However, the signal mechanism of N-protein in the lung inflammatory response remains poorly understood. The goal of this study was to determine whether RAGE (receptor for advanced glycation endproducts) participated in N-protein-induced acute lung injury. The binding between N-protein and RAGE was examined via assays for protein-protein interaction. To determine the signaling mechanism in vitro, cells were treated with recombinant N-protein and assayed for the activation of the RAGE/MAPK (mitogen-activated protein kinase)/NF-ĸB pathway. RAGE deficiency mice and antagonist were used to study N-protein-induced acute lung injury in vivo. Binding between N-protein and RAGE was confirmed via flow cytometry-based binding assay, surface plasmon resonance, and ELISA. Pull-down and coimmunoprecipitation assays revealed that N-protein bound RAGE via both N-terminal and C-terminal domains. In vitro, N-protein activated the RAGE-ERK1/2-NF-ĸB signaling pathway and induced a proinflammatory response. RAGE deficiency subdued N-protein-induced proinflammatory signaling and response. In vivo, RAGE was upregulated in the BAL and lung tissue after recombinant N-protein insult. RAGE deficiency and small molecule antagonist partially protected mice from N-protein-induced acute lung injury. Our study demonstrated that RAGE is a receptor for N-protein. RAGE is partially responsible for N-protein-induced acute lung injury and has the potential to become a therapeutic target for treating coronavirus disease.

Non-spike and spike-specific memory T cell responses after the third dose of inactivated COVID-19 vaccine.

Background: During the COVID-19 epidemic, vaccination has become the most safe and effective way to prevent severe illness and death. Inactivated vaccines are the most widely used type of COVID-19 vaccines in the world. In contrast to spike-based mRNA/protein COVID-19 vaccines, inactivated vaccines generate antibodies and T cell responses against both spike and non-spike antigens. However, the knowledge of inactivated vaccines in inducing non-spike-specific T cell response is very limited. Methods: In this study, eighteen healthcare volunteers received a homogenous booster (third) dose of the CoronaVac vaccine at least 6 months after the second dose. CD4+ and CD8+ T cell responses against a peptide pool from wild-type (WT) non-spike proteins and spike peptide pools from WT, Delta, and Omicron SARS-CoV-2 were examined before and 1-2 weeks after the booster dose. Results: The booster dose elevated cytokine response in CD4+ and CD8+ T cells as well as expression of cytotoxic marker CD107a in CD8+ T cells in response to non-spike and spike antigens. The frequencies of cytokine-secreting non-spike-specific CD4+ and CD8+ T cells correlated well with those of spike-specific from WT, Delta, and Omicron. Activation-induced markers (AIM) assay also revealed that booster vaccination elicited non-spike-specific CD4+ and CD8+ T cell responses. In addition, booster vaccination produced similar spike-specific AIM+CD4+ and AIM+CD8+ T cell responses to WT, Delta, and Omicron, indicting strong cross-reactivity of functional cellular response between WT and variants. Furthermore, booster vaccination induced effector memory phenotypes of spike-specific and non-spike-specific CD4+ and CD8+ T cells. Conclusions: These data suggest that the booster dose of inactive vaccines broadens both non-spike-specific and spike-specific T cell responses against SARS-CoV-2.

Diagnostic Potential of Plasma Extracellular Vesicle miR-483-3p and Let-7d-3p for Sepsis.

Background: microRNAs (miRNAs) from circulating extracellular vesicles (EVs) have been reported as disease biomarkers. This study aimed to identify the diagnostic value of plasma EV-miRNAs in sepsis. Methods: EVs were separated from the plasma of sepsis patients at admission and healthy controls. The expression of EV-miRNAs was evaluated by microarray and qRT-PCR. Results: A preliminary miRNA microarray of plasma EVs from a discovery cohort of 3 sepsis patients at admission and three healthy controls identified 11 miRNAs with over 2-fold upregulation in sepsis group. Based on this finding, EV samples from a validation cohort of 37 sepsis patients at admission and 25 healthy controls were evaluated for the expression of the 6 miRNAs relating injury and inflammation via qRT-PCR. Elevated expression of miR-483-3p and let-7d-3p was validated in sepsis patients and corroborated in a mouse model of sepsis. miR-483-3p and let-7d-3p levels positively correlated with the disease severity. Additionally, a combination of miR-483-3p and let-7d-3p had diagnostic value for sepsis. Furthermore, bioinformatic analysis and experimental validation showed that miR-483-3p and let-7d-3p target pathways regulating immune response and endothelial function. Conclusion: The present study reveals the potential role of plasma EV-miRNAs in the pathogenesis of sepsis and the utility of combining miR-483-3p and let-7d-3p as biomarkers for early sepsis diagnosis.

SARS-CoV-2 N Protein Induces Acute Lung Injury in Mice via NF-ĸB Activation.

Background: Infection of SARS-CoV-2 may cause acute respiratory syndrome. It has been reported that SARS-CoV-2 nucleocapsid protein (N-protein) presents early in body fluids during infection. The direct involvement of N-protein in lung injury is poorly understood. Methods: Recombinant N-protein was pretreated with polymyxin B, a lipopolysaccharide (LPS)-neutralizing agent. C57BL/6, C3H/HeJ (resistant to LPS), and C3H/HeN (control for C3H/HeJ) mice were exposed to N-protein via intratracheal administration to examine acute lung injury. In vitro, bone marrow-derived macrophages (BMDMs) were cultured with N-protein to study phosphorylation of nuclear factor kappa B (NF-ĸB) p65, macrophage polarization, and expression of proinflammatory cytokines. Results: N-protein produced acute lung injury in C57BL/6 mice, with elevated protein permeability, total cell count, neutrophil infiltration, and proinflammatory cytokines in the bronchioalveolar lavage. N-protein also induced lung injury in both C3H/HeJ and C3H/HeN mice, indicating that the effect could not be attributed to the LPS contamination. N-protein triggered phosphorylation of NF-ĸB p65 in vitro, which was abolished by both N-protein denaturation and treatment with an antibody for N-protein, demonstrating that the effect is N-protein specific. In addition, N-protein promoted M1 macrophage polarization and the expression of proinflammatory cytokines, which was also blocked by N-protein denaturation and antibody for N-protein. Furthermore, N-protein induced NF-ĸB p65 phosphorylation in the lung, while pyrrolidine dithiocarbamate, an NF-ĸB inhibitor, alleviated the effect of N-protein on acute lung injury. Conclusions: SARS-CoV-2 N-protein itself is toxic and induces acute lung injury in mice. Both N-protein and NF-ĸB pathway may be therapeutic targets for treating multi-organ injuries in Coronavirus disease 2019 (COVID-19).

Triggering receptor expressed on myeloid Cells-2 (TREM2) inhibits steroidogenesis in adrenocortical cell by macrophage-derived exosomes in lipopolysaccharide-induced septic shock.

PURPOSE: 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 (LPS, 30 mg/kg). The mice were assessed for TREM2 expression and local inflammation in adrenal gland and for 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 were co-cultured with adrenocortical cells. The expression of steroidogenic enzymes and corticosterone production was assessed. RESULTS: Genetic deficiency of TREM2 caused significantly higher corticosterone levels at the early stage of LPS-induced septic shock; whereas 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. Furthermore, Trem2-/- macrophage-derived exosomes were able to mimic Trem2-/- macrophages in enhancing adrenocortical steroidogenesis. CONCLUSIONS: At the early stage of LPS-induced septic shock, corticosterone biosynthesis can be inhibited by macrophage TREM2 in adrenocortical cells, which might partially associate with macrophage-derived exosomes.

Mesenchymal stem cell-derived extracellular vesicles alter disease outcomes via endorsement of macrophage polarization.

Mesenchymal stem cells (MSCs) are adult stromal cells that reside in virtually all postnatal tissues. Due to their regenerative and immunomodulatory capacities, MSCs have attracted growing attention during the past two decades. MSC-derived extracellular vesicles (MSC-EVs) are able to duplicate the effects of their parental cells by transferring functional proteins and genetic materials to recipient cells without cell-to-cell contact. MSC-EVs also target macrophages, which play an essential role in innate immunity, adaptive immunity, and homeostasis. Recent studies have demonstrated that MSC-EVs reduce M1 polarization and/or promote M2 polarization in a variety of settings. In this review, we discuss the mechanisms of macrophage polarization and roles of MSC-EV-induced macrophage polarization in the outcomes of cardiovascular, pulmonary, digestive, renal, and central nervous system diseases. In conclusion, MSC-EVs may become a viable alternative to MSCs for the treatment of diseases in which inflammation and immunity play a critical role.

Mesenchymal Stem Cell-Derived Extracellular Vesicles Alleviate Acute Lung Injury Via Transfer of miR-27a-3p.

OBJECTIVES: The goal of this study was to determine the role of microRNA transfer in mediating the effects of mesenchymal stem cell-derived extracellular vesicles in acute lung injury. DESIGN: Experimental cell and animal studies. SETTING: University-based research laboratory. SUBJECTS: THP-1 monocytes, bone marrow-derived macrophages, and C57BL/6 mice. INTERVENTIONS: To determine the microRNA transfer in vitro, mesenchymal stem cells and mesenchymal stem cell-derived extracellular vesicles were cultured with THP-1 cells and bone marrow-derived macrophages and then assayed for microRNA expression in the target cells. To examine the role of microRNA transfer in vivo, mesenchymal stem cell-derived extracellular vesicles were administered to mice with lipopolysaccharide-induced lung injury. MEASUREMENTS AND MAIN RESULTS: Mesenchymal stem cell-derived extracellular vesicles were efficiently taken up by macrophages in vitro and in vivo. miR-27a-3p was one of the most highly expressed microRNAs in THP-1 cells in microarray analysis and was transferred from mesenchymal stem cells and mesenchymal stem cell-derived extracellular vesicles to THP-1/bone marrow-derived macrophages. Mesenchymal stem cell-derived extracellular vesicles promoted M2 polarization in bone marrow-derived macrophages, which was inhibited by lentiviral anti-miR-27a-3p transduction. Mesenchymal stem cell-derived extracellular vesicles administered systemically and intratracheally were as effective as mesenchymal stem cells in alleviating acute lung injury, elevating miR-27a-3p levels in alveolar macrophages, and promoting M2 macrophage polarization. Treatment of mesenchymal stem cell-derived extracellular vesicles concurrently decreased alveolar macrophage expression of nuclear factor kappa B subunit 1, a target of miR-27a-3p. Lentiviral transduction of mesenchymal stem cells with anti-miR-27a-3p or knockdown of miR-27a-3p in vivo abolished the effects of mesenchymal stem cell-derived extracellular vesicles on acute lung injury and M2 macrophage polarization. CONCLUSIONS: Mesenchymal stem cell-derived extracellular vesicles mitigate acute lung injury at least partially via transferring miR-27a-3p to alveolar macrophages. miR-27a-3p acts to target NFKB1 and is a crucial regulator of M2 macrophage polarization.

miR-10a in Peripheral Blood Mononuclear Cells Is a Biomarker for Sepsis and Has Anti-Inflammatory Function.

BACKGROUND: Recent literature has reported the use of circulating microRNAs (miRNAs) as biomarkers for sepsis. Immune cells play an essential role in the pathophysiology of sepsis. The aim of this prospective study was to identify miRNAs in peripheral blood mononuclear cells (PBMC) that could differentiate between sepsis and infection based on Sepsis-3 definition. METHODS: A total of 62 patients (41 with sepsis and 21 with infection suffering from pneumonia but without sepsis) and 20 healthy controls were enrolled into the study. PBMC at admission were examined for a panel of 4 miRNAs (miR-10a, miR-17, miR-27a, and miR-125b), which have been documented to participate in inflammatory response in immune cells, via qRT-PCR. Data were validated in a mouse model of sepsis induced via cecal ligation and puncture (CLP) and THP-1 monocytes. RESULTS: miR-10a levels in PBMC at admission were significantly lower in sepsis patients compared with patients with infection and healthy controls. miR-10a levels were negatively correlated with disease severity scores as well as levels for c-reactive protein and procalcitonin. In addition, low miR-10a expression had a diagnostic value for sepsis and a prognostic value for 28-day mortality in receiving operating characteristic analysis. Compared with infection patients and healthy controls, PBMC from sepsis patients also had higher levels of mitogen-activated kinase kinase kinase 7 (MAP3K7), a known target protein of miR-10a and an activator of the NF-κB pathway. In the mouse model of CLP-induced sepsis, miR-10a levels in PBMC were significantly decreased as early as 8 h after CLP. Overexpression of miR-10a in THP-1 cells significantly reduced the expression of MAP3K7 and proinflammatory cytokines including IL-6, TNF-α, and MCP-1. CONCLUSIONS: PBMC miR-10a levels are decreased in sepsis and negatively correlated with the disease severity. Levels of miR-10a could distinguish between sepsis and infection and predict 28-day mortality. miR-10a plays an anti-inflammatory role in the pathogenesis of sepsis.

Functional proteins of mesenchymal stem cell-derived extracellular vesicles.

Extracellular vesicles (EVs) contain proteins, microRNAs, mRNAs, long non-coding RNAs, and phospholipids, and are a novel mechanism of intercellular communication. It has been proposed that the immunomodulatory and regenerative effects of mesenchymal stem/stromal cells (MSCs) are mainly mediated by soluble paracrine factors and MSC-derived EVs (MSC-EVs). Recent studies suggest that MSC-EVs may serve as a novel and cell-free alternative to whole-cell therapies. The focus of this review is to discuss the functional proteins which facilitate the effects of MSC-EVs. The first section of the review discusses the general functions of EV proteins. Next, we describe the proteomics of MSC-EVs as compared with their parental cells. Then, the review presents the current knowledge that protein contents of MSC-EVs play an essential role in immunomodulation and treatment of various diseases. In summary, functional protein components are at least partially responsible for disease-modulating capacity of MSC-EVs.

Differential effects of extracellular vesicles from aging and young mesenchymal stem cells in acute lung injury.

Old age is a known risk factor for mortality in acute respiratory distress syndrome (ARDS)/acute lung injury. Mesenchymal stem cells (MSCs) possess potent immunomodulatory properties, while aging MSCs have reduced capacity. Recent studies have demonstrated that MSC-derived extracellular vesicles (MSC-EVs) are able to mimic MSCs in alleviating acute lung injury. The goals of this study were to determine whether EVs from young and aging MSCs had differential effects on lipopolysaccharide (LPS)-induced lung injury in young mice and unravel the underlying mechanisms. Our results showed that both aging and young MSC-EVs had similar physical and phenotypical properties. As their parental cells, young MSC-EVs alleviated LPS-induced acute lung injury, while aging MSC-EVs did not exhibit the protective effects. In contrast to young MSC-EVs, aging MSC-EVs failed to alter macrophage phenotypes and reduce macrophage recruitment. In addition, the internalization of aging MSC-EVs by macrophages was significantly lower compared with that of young MSC-EVs. Furthermore, aging and young MSC-EVs differed in levels of several miRNAs relating macrophage polarization. In conclusion, aging and young MSC-EVs have differential effects in alleviating acute lung injury and macrophage polarization, which may be associated with internalization of EVs and their miRNA content.

Mesenchymal stem cell-derived extracellular vesicles affect disease outcomes via transfer of microRNAs.

Mesenchymal stem cells (MSCs) are adult stromal cells with the capacity to differentiate into multiple types of cells. MSCs represent an attractive option in regenerative medicine due to their multifaceted abilities for tissue repair, immunosuppression, and anti-inflammation. Recent studies demonstrate that MSCs exert their effects via paracrine activity, which is at least partially mediated by extracellular vesicles (EVs). MSC-derived EVs (MSC-EVs) could mimic the function of parental MSCs by transferring their components such as DNA, proteins/peptides, mRNA, microRNA (miRNA), lipids, and organelles to recipient cells. In this review, we aim to summarize the mechanism and role of miRNA transfer in mediating the effects of MSC-EVs in the models of human diseases. The first three sections of the review discuss the sorting of miRNAs into EVs, uptake of EVs by target cells, and functional transfer of miRNAs via EVs. Then, we describe the composition of miRNAs in MSC-EVs. Next, we provide the existing evidence that MSC-EVs affect the outcomes of renal, liver, heart, and brain diseases by transferring their miRNA contents. In conclusion, EV-mediated miRNA transfer plays an important role in disease-modulating capacity of MSCs.

Mesenchymal stromal cell-derived extracellular vesicles: regenerative and immunomodulatory effects and potential applications in sepsis.

Mesenchymal stromal (stem) cells (MSCs) have multipotent differentiation capacity and exist in nearly all forms of post-natal organs and tissues. The immunosuppressive and anti-inflammatory properties of MSCs have made them an ideal candidate in the treatment of diseases, such as sepsis, in which inflammation plays a critical role. One of the key mechanisms of MSCs appears to derive from their paracrine activity. Recent studies have demonstrated that MSC-derived extracellular vesicles (MSC-EVs) are at least partially responsible for the paracrine effect. MSC-EVs transfer molecules (such as proteins/peptides, mRNA, microRNA and lipids) with immunoregulatory properties to recipient cells. MSC-EVs have been shown to mimic MSCs in alleviating sepsis and may serve as an alternative to whole cell therapy. Compared with MSCs, MSC-EVs may offer specific advantages due to lower immunogenicity and higher safety profile. The first two sections of the review discuss the preclinical and clinical findings of MSCs in sepsis. Next, we review the characteristics of EVs and MSC-EVs. Then, we summarize the mechanisms of MSC-EVs, including tissue regeneration and immunomodulation. Finally, our review presents the evidences that MSC-EVs are effective in treating models of sepsis. In conclusion, MSC-EVs may have the potential to become a novel therapeutic strategy for sepsis.