Immunomodulatory Properties of Umbilical Cord Blood-Derived Small Extracellular Vesicles and Their Therapeutic Potential for Inflammatory Skin Disorders.

Umbilical cord blood (UCB) has long been seen as a rich source of naïve cells with strong regenerative potential, likely mediated by paracrine signals. More recently, small extracellular vesicles (sEV), such as exosomes, have been shown to play essential roles in cell-to-cell communication, via the transport of numerous molecules, including small RNAs. Often explored for their potential as biomarkers, sEV are now known to have regenerative and immunomodulating characteristics, particularly if isolated from stem cell-rich tissues. In this study, we aim to characterize the immunomodulating properties of umbilical cord blood mononuclear cell-derived sEV (UCB-MNC-sEV) and explore their therapeutic potential for inflammatory skin diseases. UCB-MNC-sEV were shown to shift macrophages toward an anti-inflammatory phenotype, which in turn exert paracrine effects on fibroblasts, despite previous inflammatory stimuli. Additionally, the incubation of PBMC with UCB-MNC-sEV resulted in a reduction of total CD4+ and CD8+ T-cell proliferation and cytokine release, while specifically supporting the development of regulatory T-cells (Treg), by influencing FOXP3 expression. In a 3D model of psoriatic skin, UCB-MNC-sEV reduced the expression of inflammatory and psoriatic markers IL6, IL8, CXCL10, COX2, S100A7, and DEFB4. In vivo, UCB-MNC-sEV significantly prevented or reversed acanthosis in imiquimod-induced psoriasis, and tendentially increased the number of Treg in skin, without having an overall impact on disease burden. This work provides evidence for the anti-inflammatory and tolerogenic effect of UCB-MNC-sEV, which may be harnessed for the treatment of Th17-driven inflammatory skin diseases, such as psoriasis.

Enhanced ATP release and CD73-mediated adenosine formation sustain adenosine A2A receptor over-activation in a rat model of Parkinson's disease.

BACKGROUND AND PURPOSE: Parkinson's disease (PD) involves an initial loss of striatal dopamine terminals evolving into degeneration of dopamine neurons in substantia nigra (SN), which can be modelled by 6-hydroxydopamine (6-OHDA) administration. Adenosine A2A receptor blockade attenuates PD features in animal models, but the source of the adenosine causing A2A receptor over-activation is unknown. As ATP is a stress signal, we have tested if extracellular catabolism of adenine nucleotides into adenosine (through ecto-5'-nucleotidase or CD73) leads to A2A receptor over-activation in PD. EXPERIMENTAL APPROACH: Effects of blocking CD73 with α,ß-methylene ADP (AOPCP) were assayed in 6-OHDA-treated rats and dopamine-differentiated neuroblastoma SH-SY5Y cells. KEY RESULTS: 6-OHDA increased ATP release and extracellular conversion into adenosine through CD73 up-regulation in SH-SY5Y cells. Removing extracellular adenosine with adenosine deaminase, blocking CD73 with AOPCP, or blocking A2A receptors with SCH58261 were equi-effective in preventing 6-OHDA-induced damage in SH-SY5Y cells. In vivo striatal exposure to 6-OHDA increased ATP release and extracellular formation of adenosine from adenosine nucleotides and up-regulated CD73 and A2A receptors in striatal synaptosomes. Intracerebroventricular administration of AOPCP phenocopied effects of SCH58261, attenuating 6-OHDA-induced (a) increase of contralateral rotations after apomorphine, (b) reduction of dopamine content in striatum and SN, (c) loss of TH staining in striatum and SN, (d) motor dysfunction in the cylinder test, and (e) short-term memory impairment in the object recognition test. CONCLUSION AND IMPLICATIONS: Our data indicate that increased ATP-derived adenosine formation is responsible for A2A receptor over-activation in PD, suggesting CD73 as a new target to manage PD.

Exposure to decabromodiphenyl ether (BDE-209) produces mitochondrial dysfunction in rat liver and cell death.

Polybrominated diphenyl ethers (PBDE) are ubiquitous environmental pollutants. Exposure to these chemicals has been associated with developmental neurotoxicity, endocrine dysfunctions, reproductive disorders, and hepatotoxicity. The widespread use of PBDE as flame retardants has culminated in daily exposure of humans and wildlife to these contaminants and resulted in their banned use. Thus assessment of the potential effects of each PBDE congener on living organisms has become cause for concern. The aim of this study was to (1) examine the effects of decabromodiphenyl ether (BDE)-209 on different functions of HepG2 cells and (2) investigate whether this congener is involved in mitochondrial toxicity. The use of multiple methods was employed to (i) study the influence of BDE-209 on mitochondrial permeability transition (MPT) process in mitochondria isolated from rat liver and (ii) determine the consequential cellular damage. Our results showed that BDE-209 induced matrix swelling related to MPT with 10 µM and ATP depletion with 0.1 µM. In addition, 0.5 µM BDE-209 reduced HepG2 cell viability, produced collapse of membrane potential, but increased levels of reactive oxygen species (ROS) after 48 h incubation. After 24 h with 5 µM treatment elevated levels of ROS, DNA fragmentation and cytochrome c release, accompanied by caspase 9 and caspase 3 activation was noted. Taken together, these results suggest that short-duration exposure (24 or 48 h) to 0.5 µM or 5 µM BDE-209 concentrations diminished HepG2 cell viability due to apoptosis associated with mitochondrial dysfunction.

Adenosine receptors: regulatory players in the preservation of mitochondrial function induced by ischemic preconditioning of rat liver.

Although adenosine A1 receptors (A1R) have been associated to ischemic preconditioning (IPC), direct evidence for their ability to preserve mitochondrial function upon hepatic preconditioning is still missing and could represent a novel strategy to boost the quality of liver transplants. We tested if the A1R antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) prevented IPC in the liver and if the A1R agonist 2-chloro-N6-cyclopentyladenosine (CCPA) might afford a pharmacological preconditioning. Livers underwent a 120 min of 70% warm ischemia and 16 h of reperfusion (I/R), and the IPC group underwent a 5-min ischemic episode followed by a 10-min period of reperfusion before I/R. DPCPX or CCPA was administered intraperitoneally 2 h before IPC or I/R. The control of mitochondrial function emerged as the central element affected by IPC and controlled by endogenous A1R activation. Thus, livers from IPC- or CCPA-treated rats displayed an improved oxidative phosphorylation with higher state 3 respiratory rate, higher respiratory control ratio, increased ATP content, and decreased lag phase. IPC and CCPA also prevented the I/R-induced susceptibility to calcium-induced mitochondrial permeability transition, the rate of reactive oxygen species (ROS) generation, and the decreased mitochondrial content of phospho-Ser9 GSK-3ß. DPCPX abrogated these effects of IPC. These implicate the control of GSK-3ß activity by Akt-mediated Ser9-GSK-3ß phosphorylation preserving the efficiency of oxidative phosphorylation and ROS-mediated cell death in the ability of A1R activation to mimic IPC in the liver. In conclusion, the parallel between IPC and A1R-mediated preconditioning also paves the way to consider a putative therapeutic use of the later in liver transplants.

Evaluation of Polybrominated Diphenyl Ether Toxicity on HepG2 Cells - Hexabrominated Congener (BDE-154) Is Less Toxic than Tetrabrominated Congener (BDE-47).

Apoptotic cell death is one of the main consequences of exposure to brominated flame retardants, including polybrominated diphenyl ethers. However, few of these compounds have had their potential toxicity investigated. BDE-154 is one of the most poorly studied polybrominated diphenyl ether (PBDE) congeners, but its level in the environment and in biological fluids is rising. In addition, its chemical structure differs from the other congeners with well-documented toxicity, so BDE-154 may display a distinct toxicity pattern. This study has evaluated how BDE-154 affects the human hepatoblastoma cell line (HepG2) and has looked into the impact of this congener on human health. In addition, this study has related the effects of BDE-154 with the effects of BDE-47 to clarify the mechanism of PBDE toxicity. The HepG2 cell line was exposed to BDEs for 24 and 48 hr and submitted to assays to examine proliferation, viability, mitochondrial membrane potential, reactive oxygen species accumulation, phosphatidylserine exposure, nuclear fragmentation and evaluation of pro-caspase 3, pro-caspase 9, cytochrome c release, and apoptosis inductor factor release by Western blot analysis. BDE-154 induced mitochondrial damage and led to apoptotic death of HepG2 cells, but these effects were less intense than the effects promoted by BDE-47. Unlike other extensively reported congeners, BDE-154 was only toxic at the higher tested concentrations, whereas BDE-47 cytotoxicity was evident even at lower concentrations. Hence, like the toxicity pattern of other classes of substances such as polychlorinated biphenyls, the toxicity pattern of BDEs also depends on their chemical structure and aromatic substituent.

The Emerging Role of MitomiRs in the Pathophysiology of Human Disease.

microRNAs (miRNAs) are small, single-stranded noncoding RNA molecules involved in posttranscriptional control of gene expression of a wide number of genes. miRNAs align and bind especially to 3'UTR sequences of their target genes and initiate either mRNA degradation or translational repression, resulting in reduced protein levels. miRNAs are now recognized as major players in virtually every biological process. In recent years, the discovery of miRNAs has revolutionized the traditional view of gene expression and our understanding of miRNA biogenesis and function has thereby expanded. The discovery of mitochondrial-located miRNAs raises the issue of the molecular mechanism underlying their translocation from the nucleus to the mitochondria. Studies in different species indicate that it may exist a number of import pathways of nucleus-encoded RNAs to mitochondria, being the most of them largely ATP-dependent. Not only pre-miRNAs, but also mature miRNAs, are present in the mitochondria; these findings have also raised the possibility of mitochondrial miRNA synthesis. Some pre-miRNAs sequences seem to be processed in the mitochondria, giving origin to mature miRNAs, which could be immediately active on the mitochondrial transcripts or exported to the cytosol in order to interfere with genomic-derived mRNA. Thus, the mitochondrial-processed miRNAs are likely to contribute to some posttranscriptional regulation of gene expression related to the mitochondrial functions. Coming from their location, the mitochondria, some miRNAs are currently named as mitomiRs; it refers to those miRNAs that can localize in mitochondria, whether transcribed from the nuclear or, potentially, the mitochondrial genome. When their genomics was analyzed, a number of mitomiRs mapped the nuclear genome at loci relevant to mitochondrial functions or diseases. Current computational analyses, using different algorithms, drive scientists to argue that the mitochondrial genome can harbor sequences that could be a target for several mitomiRs. However, perhaps a more challenging topic concerning mitomiRs is whether the mitochondrial DNA can harbor miRNA sequences, indicating an involvement of mitochondria in small RNA-generating pathways. The identification of populations of miRNAs in the mitochondria pushed scientists in the field to question its biological functions. It is established that miRNAs, originated in the nuclear genome, are exported to cytosol where they are processed and exert their function by inhibiting nuclear genome-derived mRNA. Actually it is also known that some miRNAs are imported into mitochondria where they interact with some mitochondrial genome-derived mRNA molecules. More strikingly, it has also come to light that mitochondrial genome (mtDNA) can originate some miRNA molecules that exert their function directly on mitochondrial transcripts. The links between miRNA deregulation and human disease have been reported in almost all medicine fields. Currently, great efforts are being invested in understanding the involvement of miRNA deregulation in disease and unlocking the mechanisms by which they act. This new field of investigation has revealed the tremendous potential of miRNAs as diagnostic or even as valuable therapeutic tools. miRNAs have recently emerged as key regulators of metabolism. Metabolic syndrome is a systemic disorder that includes a spectrum of abnormalities associated with obesity and type II diabetes. Defects in mitochondrial function, namely related to oxidation of fatty acids, have been linked to diet-induced obesity and the development of insulin resistance in adipose tissue and skeletal muscle. Consistently, obese individuals have mitochondria with compromised bioenergetic capacity. Therefore, increasing interest is being given to the role of miRNAs on metabolic regulation, with relevance on mitochondria and the mechanisms purported for miRNA actions, particularly acting in mitochondria or in mitochondria-related pathways. The involvement of miRNAs in mitochondrial metabolism, mitochondrial oxidative phosphorylation (OXPHOS), electron transport chain (ETC) components, lipid metabolism, and metabolic disorders is becoming more and more comprehended, as well as miRNAs contribution for processes such as mitochondrial dynamics or apoptosis regulation and cancer.

Exposure to dibenzofuran triggers autophagy in lung cells.

Environmental pollutants, such as dioxins and furans, are extremely toxic and related with pulmonary disease development. Exposure of A549 human lung cells to dibenzofuran showed both time- and concentration-dependent decreases in cell proliferation and MTT reduction, but no alterations in cell viability. No differences were observed in the number of apoptotic nuclei, which can be due to the energetic failure caused by dibenzofuran-induced ATP depletion. Moreover, cells in culture exposed to the pollutant showed an increase in the conversion of LC3, a protein involved in the autophagic process. Incubation of A549 lung cells with dibenzofuran caused an increase in Lysotracker Red staining, indicating an increase in lysosomal vacuoles content. These results suggest that exposure to dibenzofuran affects lung mitochondrial phosphorylative function, causing an increase in the population of dysfunctional mitochondria and an impairment in the energetic status maintenance, therefore stimulating autophagy as a possible rescue mechanism in this cell line.

Exposure to dibenzofuran affects lung mitochondrial function in vitro.

Environmental pollutants, such as dioxins and furans, are extremely toxic and related with pulmonary diseases development. Impairment of mitochondrial function has been shown in pollutant-induced hepatic injury, but it has not been addressed in lungs, even though lung mitochondria are primary cellular targets for pollutants-induced toxicity. In isolated lung mitochondria, dibenzofuran significantly increased the lag phase preceding mitochondrial repolarization, suggesting a decrease in the efficiency of the mitochondrial phosphorylative system.

Indirubin-3'-oxime impairs mitochondrial oxidative phosphorylation and prevents mitochondrial permeability transition induction.

Indirubin, a red colored 3,2'-bisindole isomer, is a component of Indigo naturalis and is an active ingredient used in traditional Chinese medicine for the treatment of chronic diseases. The family of indirubin derivatives, such as indirubin-3'-oxime, has been suggested for various therapeutic indications. However, potential toxic interactions such as indirubin effects on mitochondrial bioenergetics are still unknown. This study evaluated the action of indirubin-3'-oxime on the function of isolated rat liver mitochondria contributing to a better understanding of the biochemical mechanisms underlying the multiple effects of indirubin. Indirubin-3'-oxime incubated with isolated rat liver mitochondria, at concentrations above 10microM, significantly depresses the phosphorylation efficiency of mitochondria as inferred from the decrease in the respiratory control and ADP/O ratios, the perturbations in mitochondrial membrane potential and in the phosphorylative cycle induced by ADP. Furthermore, indirubin-3'-oxime at up to 25microM stimulates the rate of state 4 respiration and inhibits state 3 respiration. The increased lag phase of repolarization was associated with a direct inhibition of the mitochondrial ATPase. Indirubin-3'-oxime significantly inhibited the activity of complex II and IV thus explaining the decreased FCCP-stimulated mitochondrial respiration. Mitochondria pre-incubated with indirubin-3'-oxime exhibits decreased susceptibility to calcium-induced mitochondrial permeability transition. This work shows for the first time multiple effects of indirubin-3'-oxime on mitochondrial bioenergetics thus indicating a potential mechanism for indirubin-3'-oxime effects on cell function.