Multifaceted role of SARS-CoV-2 structural proteins in lung injury.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third human coronavirus to cause acute respiratory distress syndrome (ARDS) and contains four structural proteins: spike, envelope, membrane, and nucleocapsid. An increasing number of studies have demonstrated that all four structural proteins of SARS-CoV-2 are capable of causing lung injury, even without the presence of intact virus. Therefore, the topic of SARS-CoV-2 structural protein-evoked lung injury warrants more attention. In the current article, we first synopsize the structural features of SARS-CoV-2 structural proteins. Second, we discuss the mechanisms for structural protein-induced inflammatory responses in vitro. Finally, we list the findings that indicate structural proteins themselves are toxic and sufficient to induce lung injury in vivo. Recognizing mechanisms of lung injury triggered by SARS-CoV-2 structural proteins may facilitate the development of targeted modalities in treating COVID-19.

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.

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.

Human adipose-derived mesenchymal stem cells alleviate obliterative bronchiolitis in a murine model via IDO.

BACKGROUND: Long-term survival of lung transplantation is hindered by the development of obliterative bronchiolitis (OB). Adipose-derived stem cells (ASCs) were documented to have more potent immunosuppressive ability than mesenchymal stem cells (MSCs) from bone marrow and placenta. The goal of our study is to evaluate the effect of repeated administration of ASCs on OB and the involvement of indoleamine 2,3-dioxygenase (IDO) mediating the protective effect of ASCs in a heterotopic tracheal transplantation (HTT) model. METHODS: For studies in vitro, ASCs were treated with interferon-γ (IFN-γ). For in vivo study, tracheas from BALB/c or C57BL/6 donors were transplanted into C57BL/6 recipients to create a HTT model. On days 0, 1, 3, 5, 8, 12, 15, 20 and 25 post-transplant, the allogeneic recipient mice were administered intravenously with phosphate buffered saline, 1 × 106 human ASCs, or 1 × 106 human ASCs plus 1-methyltryptophan (1-MT), an IDO inhibitor. On days 3, 7, 14 and 28, serum, trachea and spleen samples were harvested for analysis. RESULTS: ASCs homed to heterotopic tracheal grafts after infusion. Multiple doses of ASCs significantly increased tracheal IDO levels in allografts. There were significant increases in graft and serum IFN-γ levels in allografts compared with isografts. IFN-γ elevated IDO expression and activity in ASCs in vitro. ASCs alleviated OB in allografts as evidenced by reduced epithelial loss, epithelial apoptosis, and intraluminal obstruction. The effects of ASCs on OB were blocked by 1-MT. 1-MT also blocked the alterations in pro and anti-inflammatory cytokines as well as CD3+ T cell infiltration induced by ASCs. ASCs induced not only splenic levels of CD4+CD25+Foxp3+ regulatory T cells (Treg) but also IL-10 and TGF-ß-producing Treg. Furthermore, IDO inhibition abolished the changes of splenic Treg induced by ASCs. In addition, Treg reduction by cyclophosphamide treatment did not alter the effects of ASCs on tracheal IDO expression in allografts confirming Treg induction is downstream of IDO. CONCLUSIONS: Repeated doses of ASCs are capable of ameliorating OB. ASCs act at least in part via elevating IDO expression. ASCs promote the generation of Treg and suppress T cell infiltration via an IDO-dependent mechanism.

Adipose-derived mesenchymal stem cells modulate CD14++CD16+ expression on monocytes from sepsis patients in vitro via prostaglandin E2.

BACKGROUND: Mesenchymal stem cells (MSCs) have been shown to reduce sepsis-induced inflammation and improve survival in mouse models of sepsis. CD16+ monocytes are proinflammatory and abundant in inflammatory conditions such as sepsis. The primary objective in this exploratory study was to determine the effects of adipose-derived MSCs (ASCs) on three subsets of monocytes from sepsis patients in vitro and to delineate the underlying mechanism. METHODS: This is a prospective cohort study of patients admitted to the medical intensive care unit (ICU) at an academic medical center. The levels of CD14++CD16+, CD14+CD16++, and CD14++CD16- monocytes from 23 patients in the early phase of severe sepsis or septic shock as well as 25 healthy volunteers were determined via flow cytometry after coculture with or without ASCs. To determine the molecular mechanisms, the effects of exogenous prostaglandin E2 (PGE2) and the cyclooxygenase-2 (COX-2) inhibitor NS-398 on monocyte phenotypes and cytokine expression were also examined. RESULTS: Basal levels of CD14++CD16+ but not CD14+CD16++ monocytes were significantly elevated in severe sepsis and septic shock. A positive linear relationship existed between the levels of CD14++CD16+ monocytes and the Acute Physiology and Chronic Health Evaluation (APACHE) II score as well as Sequential Organ Failure Assessment (SOFA) score. Coculture of ASCs with monocytes from sepsis patients for 24 h significantly reduced CD14++CD16+ expression while increasing the CD14++CD16- phenotype. The coculture also significantly elevated PGE2, COX-2, and prostaglandin E2 receptor (EP)4 levels generated from monocytes. Functionally, ASCs reduced the tumor necrosis factor (TNF)-α and increased the interleukin (IL)-10 secretion in monocytes of septic patients. Furthermore, the effects of ASCs on the CD14++CD16+ phenotype and cytokine expression were mimicked by exogenous PGE2 and abolished by the COX-2 inhibitor NS-398. Additionally, ASCs also modified levels of monocyte phenotypes in a mouse model of sepsis. CONCLUSIONS: Levels of CD14++CD16+ monocytes positively correlate with disease severity scores in the early phase of severe sepsis and septic shock. ASCs switch monocytes of sepsis patients from CD14++CD16+ to CD14++CD16- in vitro and modulate the production of inflammatory cytokines. The immunomodulatory effect of ASCs on monocytes is PGE2-dependent. ASCs may exert their therapeutic effect on sepsis via altering monocyte phenotypes and functions.

Mesenchymal Stem Cell-Educated Macrophages Ameliorate LPS-Induced Systemic Response.

Both bone marrow and adipose-derived mesenchymal stem cells (ASCs) have immunomodulatory effects. The goal of this study was to determine whether ASCs-educated macrophages could directly ameliorate LPS-induced systemic response in a mouse model. Mouse peritoneal macrophages were cocultured with ASCs in a Transwell system for 2 days to educate macrophages. Mice were divided into 5 groups: control, LPS, LPS + ASCs, LPS + untreated macrophages, and LPS + educated macrophages. Educated macrophages decreased lung inflammation, weight loss, pulmonary edema, and inflammatory cytokine response. In vitro, ASCs increased expression of M2 macrophages independent of direct cell-to-cell contact when macrophages were treated with LPS or serum from patients with acute respiratory distress syndrome (ARDS). When macrophages were cultured with serum from ARDS patients who were treated with ASCs or placebo in our previous clinical trial, there was no difference in M2 macrophage levels before and after ASCs treatment indicating a suboptimal response to the treatment protocol. ASCs also reduced the levels of LPS-induced proinflammatory cytokines in vitro which were mimicked by IL-10 and blocked by antibodies for IL-10 and IL-10 receptor supporting the notion that educated macrophages exert their anti-inflammatory effects via IL-10-dependent mechanisms.

Substolides A-G, germacrane sesquiterpenoids from Salvia substolonifera.

Chemical investigation of the whole plants of Salvia substolonifera E.Peter yielded seven germacrane sesquiterpenoids, substolides A-G (1-7), an ethoxylated artifact (8), and two known analogues, 6ß-tigloyloxyglechomafuran (9) and castanin F (10). Four germacrane 8-acetylation derivatives (1a-4a) were obtained by chemical transformation. Their structures and relative or absolute configurations were elucidated by intensive spectroscopic methods, and single-crystal X-ray diffraction analysis. Compounds 1a-4a, and 5-10 were evaluated for their in vitro anti-angiogenic effects. Compounds 7 and 9 significantly inhibited VEGF-induced human umbilical vein endothelial cell (HUVEC) proliferation in vitro, with IC50 values of 16.15 ± 0.19, and 4.03 ± 0.26 µM, respectively. The structure activity relationship of these compounds is discussed.

Mesenchymal Stromal Cells Affect Disease Outcomes via Macrophage Polarization.

Mesenchymal stromal cells (MSCs) are multipotent and self-renewable cells that reside in almost all postnatal tissues. In recent years, many studies have reported the effect of MSCs on the innate and adaptive immune systems. MSCs regulate the proliferation, activation, and effector function of T lymphocytes, professional antigen presenting cells (dendritic cells, macrophages, and B lymphocytes), and NK cells via direct cell-to-cell contact or production of soluble factors including indoleamine 2,3-dioxygenase, prostaglandin E2, tumor necrosis factor-α stimulated gene/protein 6, nitric oxide, and IL-10. MSCs are also able to reprogram macrophages from a proinflammatory M1 phenotype toward an anti-inflammatory M2 phenotype capable of regulating immune response. Because of their capacity for differentiation and immunomodulation, MSCs have been used in many preclinical and clinical studies as possible new therapeutic agents for the treatment of autoimmune, degenerative, and inflammatory diseases. In this review, we discuss the central role of MSCs in macrophage polarization and outcomes of diseases such as wound healing, brain/spinal cord injuries, and diseases of heart, lung, and kidney in animal models.

Two new sesquiterpenoids including a sesquiterpenoid lactam from Curcuma wenyujin.

Two new sesquiterpenoids, namely elema-1,3,7(11),8-tetraen-8,12-lactam (1) and 7ß,8α-dihydroxy-1α,4αH-guai-9,11-dien-5ß,8ß-endoxide (2), together with five known analogs were isolated from the EtOAc extract of the rhizomes of Curcuma wenyujin. Their structures and relative configurations were determined on the basis of spectroscopic methods including 2D-NMR techniques. All compounds were tested for the inhibition of lipopolysaccharide (LPS)-induced nitric oxide (NO) production. Compound 1 showed the significant inhibitory activity with IC50 values of 9.4 µM.