Spatio-temporal brain invasion pattern of Streptococcus pneumoniae and dynamic changes in the cellular environment in bacteremia-derived meningitis.

Streptococcus pneumoniae (the pneumococcus) is the major cause of bacterial meningitis globally, and pneumococcal meningitis is associated with increased risk of long-term neurological sequelae. These include several sensorimotor functions that are controlled by specific brain regions which, during bacterial meningitis, are damaged by a neuroinflammatory response and the deleterious action of bacterial toxins in the brain. However, little is known about the invasion pattern of the pneumococcus into the brain. Using a bacteremia-derived meningitis mouse model, we combined 3D whole brain imaging with brain microdissection to show that all brain regions were equally affected during disease progression, with the presence of pneumococci closely associated to the microvasculature. In the hippocampus, the invasion provoked microglial activation, while the neurogenic niche showed increased proliferation and migration of neuroblasts. Our results indicate that, even before the outbreak of symptoms, the bacterial load throughout the brain is high and causes neuroinflammation and cell death, a pathological scenario which ultimately leads to a failing regeneration of new neurons.

Microvesicles from Human Adipose Tissue-Derived Mesenchymal Stem Cells as a New Protective Strategy in Osteoarthritic Chondrocytes.

BACKGROUND/AIMS: Chronic inflammation contributes to cartilage degeneration during the progression of osteoarthritis (OA). Adipose tissue-derived mesenchymal stem cells (AD-MSC) show great potential to treat inflammatory and degradative processes in OA and have demonstrated paracrine effects in chondrocytes. In the present work, we have isolated and characterized the extracellular vesicles from human AD-MSC to investigate their role in the chondroprotective actions of these cells. METHODS: AD-MSC were isolated by collagenase treatment from adipose tissue from healthy individuals subjected to abdominal lipectomy surgery. Microvesicles and exosomes were obtained from conditioned medium by filtration and differential centrifugation. Chondrocytes from OA patients were used in primary culture and stimulated with 10 ng/ml interleukin(IL)-1ß in the presence or absence of AD-MSC microvesicles, exosomes or conditioned medium. Protein expression was investigated by ELISA and immunofluorescence, transcription factor-DNA binding by ELISA, gene expression by real-time PCR, prostaglandin E2 (PGE2) by radioimmunoassay, and matrix metalloproteinase (MMP) activity and nitric oxide (NO) production by fluorometry. RESULTS: In OA chondrocytes stimulated with IL-1ß, microvesicles and exosomes reduced the production of inflammatory mediators tumor necrosis factor-α, IL-6, PGE2 and NO. The downregulation of cyclooxygenase-2 and microsomal prostaglandin E synthase-1 would lead to the decreased PGE2 production while the effect on NO could depend on the reduction of inducible nitric oxide synthase expression. Treatment of OA chondrocytes with extracellular vesicles also decreased the release of MMP activity and MMP-13 expression whereas the production of the anti-inflammatory cytokine IL-10 and the expression of collagen II were significantly enhanced. The reduction of inflammatory and catabolic mediators could be the consequence of a lower activation of nuclear factor-κB and activator protein-1. The upregulation of annexin A1 specially in MV may contribute to the anti-inflammatory and chondroprotective effects of AD-MSC. CONCLUSIONS: Our data support the interest of AD-MSC extracellular vesicles to develop new therapeutic approaches in joint conditions.

The single D380 amino acid substitution increases pneumolysin cytotoxicity toward neuronal cells.

Bacterial meningitis, frequently caused by Streptococcus pneumoniae (pneumococcus), represents a substantial global health threat leading to long-term neurological disorders. This study focused on the cholesterol-binding toxin pneumolysin (PLY) released by pneumococci, specifically examining clinical isolates from patients with meningitis and comparing them to the PLY-reference S. pneumoniae strain D39. Clinical isolates exhibit enhanced PLY release, likely due to a significantly higher expression of the autolysin LytA. Notably, the same single amino acid (aa) D380 substitution in the PLY D4 domain present in all clinical isolates significantly enhances cholesterol binding, pore-forming activity, and cytotoxicity toward SH-SY5Y-derived neuronal cells. Scanning electron microscopy of human neuronal cells and patch clamp electrophysiological recordings on mouse brain slices confirm the enhanced neurotoxicity of the PLY variant carrying the single aa substitution. This study highlights how a single aa modification enormously alters PLY cytotoxic potential, emphasizing the importance of PLY as a major cause of the neurological sequelae associated with pneumococcal meningitis.

In Vitro Approaches for the Study of Pneumococcal-Neuronal Interaction and Pathogenesis.

CFU- and confocal microscopy-based in vitro methods to assess pneumococcal adhesion and invasion of relevant human cells, such as neurons, remain a powerful tool to understand the basis of host-pathogen interactions. In recent years, there has been a continuous refinement of confocal detection of human and bacterial cells through the use of specific, fluorochrome-labelled antibodies. Used in combination, these assays provide both the means for quantification and enough flexibility to accommodate specific experimental needs.

Neuronal Damage and Neuroinflammation, a Bridge Between Bacterial Meningitis and Neurodegenerative Diseases.

Bacterial meningitis is an inflammation of the meninges which covers and protects the brain and the spinal cord. Such inflammation is mostly caused by blood-borne bacteria that cross the blood-brain barrier (BBB) and finally invade the brain parenchyma. Pathogens such as Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae are the main etiological causes of bacterial meningitis. After trafficking across the BBB, bacterial pathogens in the brain interact with neurons, the fundamental units of Central Nervous System, and other types of glial cells. Although the specific molecular mechanism behind the interaction between such pathogens with neurons is still under investigation, it is clear that bacterial interaction with neurons and neuroinflammatory responses within the brain leads to neuronal cell death. Furthermore, clinical studies have shown indications of meningitis-caused dementia; and a variety of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and Huntington's disease are characterized by the loss of neurons, which, unlike many other eukaryotic cells, once dead or damaged, they are seldom replaced. The aim of this review article is to provide an overview of the knowledge on how bacterial pathogens in the brain damage neurons through direct and indirect interactions, and how the neuronal damage caused by bacterial pathogen can, in the long-term, influence the onset of neurodegenerative disorders.

Role of peroxiredoxin 6 in the chondroprotective effects of microvesicles from human adipose tissue-derived mesenchymal stem cells.

BACKGROUND: Osteoarthritis (OA) is a joint disease characterized by cartilage degradation, low-grade synovitis and subchondral bone alterations. In the damaged joint, there is a progressive increase of oxidative stress leading to disruption of chondrocyte homeostasis. The modulation of oxidative stress could control the expression of inflammatory and catabolic mediators involved in OA. We have previously demonstrated that extracellular vesicles (EVs) present in the secretome of human mesenchymal stem cells from adipose tissue (AD-MSCs) exert anti-inflammatory and anti-catabolic effects in OA chondrocytes. In the current work, we have investigated whether AD-MSC EVs could regulate oxidative stress in OA chondrocytes as well as the possible contribution of peroxiredoxin 6 (Prdx6). METHODS: Microvesicles (MV) and exosomes (EX) were isolated from AD-MSC conditioned medium by differential centrifugation with size filtration. The size and concentration of EVs were determined by resistive pulse sensing. OA chondrocytes were isolated from knee articular cartilage of advanced OA patients. 4-Hydroxynonenal adducts, IL-6 and MMP-13 were determined by enzyme-linked immunosorbent assay. Expression of Prdx6 and autophagic markers was assessed by immunofluorescence and Western blotting. Prdx6 was downregulated in AD-MSCs by transfection with a specific siRNA. RESULTS: MV and to a lesser extent EX significantly reduced the production of oxidative stress in OA chondrocytes stimulated with IL-1ß. Treatment with MV resulted in a dramatic upregulation of Prdx6. MV also enhanced the expression of autophagy marker LC3B. We downregulated Prdx6 in AD-MSCs by using a specific siRNA and then MV were isolated. These Prdx6-silenced MV failed to modify oxidative stress and the expression of autophagy markers. We also assessed the possible contribution of Prdx6 to the effects of MV on IL-6 and MMP-13 production. The reduction in the levels of both mediators induced by MV was partly reverted after Prdx6 silencing. CONCLUSION: Our results indicate that EVs from AD-MSCs regulate the production of oxidative stress in OA chondrocytes during inflammation. Prdx6 may mediate the antioxidant and protective effects of MV.The translational potential of this article: This study gives insight into the protective properties of EVs from AD-MSCs in OA chondrocytes. Our findings support the development of novel therapies based on EVs to prevent or treat cartilage degradation.

Extracellular vesicles: A new therapeutic strategy for joint conditions.

Extracellular vesicles (EVs) are attracting increasing interest since they might represent a more convenient therapeutic tool with respect to their cells of origin. In the last years much time and effort have been expended to determine the biological properties of EVs from mesenchymal stem cells (MSCs) and other sources. The immunoregulatory, anti-inflammatory and regenerative properties of MSC EVs have been demonstrated in in vitro studies and animal models of rheumatoid arthritis or osteoarthritis. This cell-free approach has been proposed as a possible better alternative to MSC therapy in autoimmune conditions and tissue regeneration. In addition, EVs show great potential as biomarkers of disease or delivery systems for active molecules. The standardization of isolation and characterization methods is a key step for the development of EV research. A better understanding of EV mechanisms of action and efficacy is required to establish the potential therapeutic applications of this new approach in joint conditions.

Extracellular Vesicles from Adipose-Derived Mesenchymal Stem Cells Downregulate Senescence Features in Osteoarthritic Osteoblasts.

Osteoarthritis (OA) affects all articular tissues leading to pain and disability. The dysregulation of bone metabolism may contribute to the progression of this condition. Adipose-derived mesenchymal stem cells (ASC) are attractive candidates in the search of novel strategies for OA treatment and exert anti-inflammatory and cytoprotective effects on cartilage. Chronic inflammation in OA is a relevant factor in the development of cellular senescence and joint degradation. In this study, we extend our previous observations of ASC paracrine effects to study the influence of conditioned medium and extracellular vesicles from ASC on senescence induced by inflammatory stress in OA osteoblasts. Our results in cells stimulated with interleukin- (IL-) 1ß indicate that conditioned medium, microvesicles, and exosomes from ASC downregulate senescence-associated ß-galactosidase activity and the accumulation of γH2AX foci. In addition, they reduced the production of inflammatory mediators, with the highest effect on IL-6 and prostaglandin E2. The control of mitochondrial membrane alterations and oxidative stress may provide a mechanism for the protective effects of ASC in OA osteoblasts. We have also shown that microvesicles and exosomes mediate the paracrine effects of ASC. Our study suggests that correction of abnormal osteoblast metabolism by ASC products may contribute to their protective effects.