M2-Macrophage-Induced Chronic Inflammation Promotes Reversible Mesenchymal Stromal Cell Senescence and Reduces Their Anti-Fibrotic Properties.

Fibrosis and the associated decline in organ functionality lead to an almost 50% mortality rate in developed countries. Multipotent mesenchymal stromal cells (MSC) were shown to suppress the development and progression of fibrosis through secreted factors including specific non-coding RNAs transferred within extracellular vesicles (EV). However, age-associated chronic inflammation can provoke MSC senescence and change secretome composition, thereby affecting their antifibrotic properties. Alternatively activated macrophages (M2-type) are key players in chronic inflammation that may interact with MSC through paracrine mechanisms and decrease their antifibrotic functions. To confirm this hypothesis, we evaluated the M2-macrophage conditioned medium (CM-M2) effect on human adipose-tissue-derived MSC senescence in vitro. We found that CM-M2, as well as a pro-senescence agent, hydrogen peroxide (H2O2), increased p21+-MSC number and secretion of IL-6 and MCP-1, which are considered main senescence-associated secretory phenotype (SASP) components. Thus, both exposures led to the senescent phenotype acquisition of MSC. EV from both CM-M2 and H2O2-exposed MSC, which showed a decreased effect on the suppression of TGFß-induced fibroblast-to-myofibroblast differentiation compared to EV from control MSC according to αSMA level and the αSMA+-stress fiber reduction. After two weeks of subsequent cultivation under standard conditions, MSC demonstrated a decrease in senescence hallmarks and fibroblast differentiation suppression via EV. These results suggest that M2-macrophage-induced chronic inflammation can reversibly induce MSC senescence, which reduces the MSC's ability to inhibit fibroblast-to-myofibroblast differentiation.

Correlations between biomarkers of senescent cell accumulation at the systemic, tissue and cellular levels in elderly patients.

The aim of the study was to investigate the relationship between established clinical systemic biomarkers of ageing and the development of age-associated diseases and senescent cell biomarkers at tissue and cellular levels. Thirty-eight patients (mean age 70 ± 4.9 years) who were assessed for traditional risk factors for cardiovascular diseases were included. From all patients we obtained biomaterials (peripheral blood, skin, subcutaneous fatty tissue) and isolated different cell types (peripheral blood mononuclear cells (PBMC), fibroblasts (FB) and mesenchymal stem/stromal cells (MSC)). Isolated cells were analyzed using several senescent cells biomarkers such as telomere length and telomerase activity, proliferation rate, cell cycle inhibitor expression (p16 and p21), b-galactosidase activity, gH2AX expression. CD34+ cell content in peripheral blood was determined by flow cytometry. Systemic senescent cell-associated factors (insulin-like growth factor 1 (IGF-1), fibroblast growth factor 21 (FGF-21), osteoprogerin, ferritin, soluble vascular cell adhesion molecule (VCAM-1), intercellular adhesion molecule 1 (ICAM-1)) in peripheral blood as well as senescence-associated secretory phenotype (SASP) components in MSC and FB secretome were evaluated by ELISA. Skin and adipose tissue biopsy samples were analyzed histologically to assess senescent cell markers. A strong significant association of tissue p16 expression with age (r = 0.600, p < 0.001), pulse wave velocity (PWV) (r = 0.394, p = 0.015), vascular cell adhesion molecule (VCAM-1) content (r = 0.312, p = 0.006) in the systemic blood stream and p16 mRNA level in the blood mononuclear cells (MNCs) (r = 0.380, p = 0.046) were confirmed by correlation analysis. Statistically significant correlations were found with indicators of FBs and MSCs proliferation in culture and acquisition of SASP by the cells. Thus, p16 expression in tissues correlated significantly with interleukin-6 (IL-6) (r = 0.485, p < 0.05) and monocyte chemoattractant protein type 1 (MCP-1) (r = 0.372, p < 0.05) secretion by isolated cells. The results of regression analysis confirmed that, regardless of age, the expression of p16 was associated with the proliferation of isolated cells and IL-6 within SASP. Based on these findings, two models have been proposed to predict the level of p16 expression in tissues from the levels of other markers of senescent cell accumulation determined by non-invasive methods and available in clinical practice.

Novel Potential Markers of Myofibroblast Differentiation Revealed by Single-Cell RNA Sequencing Analysis of Mesenchymal Stromal Cells in Profibrotic and Adipogenic Conditions.

Mesenchymal stromal cells (MSCs) are the key regulators of tissue homeostasis and repair after damage. Accumulating evidence indicates the dual contribution of MSCs into the development of fibrosis induced by chronic injury: these cells can suppress the fibrotic process due to paracrine activity, but their promoting role in fibrosis by differentiating into myofibroblasts has also been demonstrated. Many model systems reproducing fibrosis have shown the ability of peroxisome proliferator-activated receptor (PPAR) agonists to reverse myofibroblast differentiation. Thus, the differentiation of multipotent cells into myofibroblasts and adipocytes can be considered as processes that require the activation of opposite patterns of gene expression. To test this hypothesis, we analyzed single cell RNA-Seq transcriptome of human adipose tissue MSCs after stimulation of the myofibroblast or adipogenic differentiation and revealed several genes that changed their expression in a reciprocal manner upon these conditions. We validated the expression of selected genes by RT-PCR, and evaluated the upregulation of several relevant proteins using immunocytochemistry, refining the results obtained by RNA-Seq analysis. We have shown, for the first time, the expression of neurotrimin (NTM), previously studied mainly in the nervous tissue, in human adipose tissue MSCs, and demonstrated its increased gene expression and clustering of membrane receptors upon the stimulation of myofibroblast differentiation. We also showed an increased level of CHD3 (Chromodomain-Helicase-DNA-binding protein 3) in MSCs under profibrotic conditions, while retinol dehydrogenase-10 (RDH10) was detected only in MSCs after adipogenic induction, which contradicted the data of transcriptomic analysis and again highlights the need to validate the data obtained by omics methods. Our findings suggest the further analysis of the potential contribution of neurotrimin and CHD3 in the regulation of myofibroblast differentiation and the development of fibrosis.

Application Prospects of FTIR Spectroscopy and CLSM to Monitor the Drugs Interaction with Bacteria Cells Localized in Macrophages for Diagnosis and Treatment Control of Respiratory Diseases.

Visualization of the interaction of drugs with biological cells creates new approaches to improving the bioavailability, selectivity, and effectiveness of drugs. The use of CLSM and FTIR spectroscopy to study the interactions of antibacterial drugs with latent bacterial cells localized in macrophages create prospects to solve the problems of multidrug resistance (MDR) and severe cases. Here, the mechanism of rifampicin penetration into E. coli bacterial cells was studied by tracking the changes in the characteristic peaks of cell wall components and intracellular proteins. However, the effectiveness of the drug is determined not only by penetration, but also by efflux of the drugs molecules from the bacterial cells. Here, the efflux effect was studied and visualized using FTIR spectroscopy, as well as CLSM imaging. We have shown that because of efflux inhibition, eugenol acting as an adjuvant for rifampicin showed a significant (more than three times) increase in the antibiotic penetration and the maintenance of its intracellular concentration in E. coli (up to 72 h in a concentration of more than 2 µg/mL). In addition, optical methods have been applied to study the systems containing bacteria localized inside of macrophages (model of the latent form), where the availability of bacteria for antibiotics is reduced. Polyethylenimine grafted with cyclodextrin carrying trimannoside vector molecules was developed as a drug delivery system for macrophages. Such ligands were absorbed by CD206+ macrophages by 60-70% versus 10-15% for ligands with a non-specific galactose label. Owing to presence of ligands with trimannoside vectors, the increase in antibiotic concentration inside macrophages, and thus, its accumulation into dormant bacteria, is observed. In the future, the developed FTIR+CLSM techniques would be applicable for the diagnosis of bacterial infections and the adjustment of therapy strategies.

Mannosylated Systems for Targeted Delivery of Antibacterial Drugs to Activated Macrophages.

Macrophages are a promising target for drug delivery to influence macrophage-associated processes in the body, namely due to the presence of resistant microorganisms in macrophages. In this work, a series of mannosylated carriers based on mannan, polyethylenimine (PEI) and cyclodextrin (CD) was synthesized. The molecular architecture was studied using FTIR and 1H NMR spectroscopy. The particle size, from small 10-50 nm to large 500 nm, depending on the type of carrier, is potentially applicable for the creation of various medicinal forms: intravenous, oral and inhalation. Non-specific capture by cells with a simultaneous increase in selectivity to CD206+ macrophages was achieved. ConA was used as a model mannose receptor, binding galactosylated (CD206 non-specific) carriers with constants of the order of 104 M-1 and mannosylated conjugates of 106-107 M-1. The results of such primary "ConA-screening" of ligands are in a good agreement in terms of the comparative effectiveness of the interaction of ligands with the CD206+ macrophages: non-specific (up to 10%) absorption of highly charged and small particles; weakly specific uptake of galactosylated polymers (up to 50%); and high affine capture (more than 70-80%) of the ligands with grafted trimannoside was demonstrated using the cytometry method. Double and multi-complexes of antibacterials (moxifloxacin with its adjuvants from the class of terpenoids) were proposed as enhanced forms against resistant pathogens. In vivo pharmacokinetic experiments have shown that polymeric carriers significantly improve the efficiency of the antibiotic: the half-life of moxifloxacin is increased by 2-3 times in conjugate-loaded forms, bio-distribution to the lungs in the first hours after administration of the drug is noticeably greater, and, after 4 h of observation, free moxifloxacin was practically removed from the lungs of rats. Although, in polymer systems, its content is significant-1.2 µg/g. Moreover, the importance of the covalent crosslinking carrier with mannose label was demonstrated. Thus, this paper describes experimental, scientifically based methods of targeted drug delivery to macrophages to create enhanced medicinal forms.

Platelet-Derived Growth Factor Induces SASP-Associated Gene Expression in Human Multipotent Mesenchymal Stromal Cells but Does Not Promote Cell Senescence.

Activation of multipotent mesenchymal stromal cells (MSCs) is a central part of tissue response to damage. Platelet-derived growth factor (PDGF-BB), which is abundantly released in the damaged area, potently stimulates the proliferation and migration of MSCs. Recent evidence indicates that tissue injury is associated with the accumulation of senescent cells, including ones of MSC origin. Therefore, we hypothesized that PDGF-BB induces MSC senescence. To evaluate mechanisms of early activation of MSCs by PDGF-BB, we performed transcriptome profiling of human MSCs isolated from adipose tissue after exposure to PDGF-BB for 12 and 24 h. We demonstrated that PDGF-BB induced the expression of several genes encoding the components of senescence-associated secretory phenotype (SASP) in MSCs such as plasminogen activator inhibitor-1 (PAI-1), urokinase-type plasminogen activator and its receptor (uPA and uPAR), and some matrix metalloproteases. However, further experimental validation of transcriptomic data clearly indicated that PDGF-BB exerted mitogenic and pro-migratory effects on MSCs, and augmented their pro-angiogenic activity, but did not significantly stimulate MSC senescence.