Priming With Toll-Like Receptor 3 Agonist Poly(I:C) Enhances Content of Innate Immune Defense Proteins but Not MicroRNAs in Human Mesenchymal Stem Cell-Derived Extracellular Vesicles

Abstract

Mesenchymal stem cells (MSCs) help fight infection by promoting direct bacterial killing or indirectly by modulating the acute phase response, thereby decreasing tissue injury. Recent evidence suggests that extracellular vesicles (EVs) released from MSCs retain antimicrobial characteristics that may be enhanced by pretreatment of parent MSCs with the toll-like receptor 3 (TLR3) agonist poly(I:C). Our aim was to determine whether poly(I:C) priming can modify EV content of miRNAs and/or proteins to gain insight into the molecular mechanisms of their enhanced antimicrobial function. Human bone marrow-derived MSCs were cultured with or without 1 μg/ml poly(I:C) for 1 h and then conditioned media was collected after 64 h of culture in EV-depleted media. Mass spectrometry and small RNA next-generation sequencing were performed to compare proteomic and miRNA profiles. Poly(I:C) priming resulted in 49 upregulated EV proteins, with 21 known to be important in host defense and innate immunity. In contrast, EV miRNA content was not significantly altered. Functional annotation clustering analysis revealed enrichment in biological processes and pathways including negative regulation of endopeptidase activity, acute phase, complement and coagulation cascades, innate immunity, immune response, and Staphylococcus aureus infection. Several antimicrobial peptides identified in EVs remained unaltered by poly(I:C) priming, including dermcidin, lactoferrin, lipocalin 1, lysozyme C, neutrophil defensin 1, S100A7 (psoriasin), S100A8/A9 (calprotectin), and histone H4. Although TLR3 activation of MSCs improves the proteomic profile of EVs, further investigation is needed to determine the relative importance of particular functional EV proteins and their activated signaling pathways following EV interaction with immune cells.

Mesenchymal Stem Cell-Derived Exosome Therapy of Microbial Diseases: From Bench to Bed

“…In this review, we mainly focus on MSC-Exos, but studies on MSC-EVs or MSC-MVs are also included in consideration of comprehensiveness. Proteomic ( Pierce and Kurata, 2021 ), metabolic, lipidomic ( Showalter et al, 2019 ), and miRNA-sequence analysis ( Shao et al, 2017 ; Zhao et al, 2019a ) and experimental studies have indicated that MSC-Exos inherit similar biological properties from their parent cells, in aspect of immunomodulation ( Willis et al, 2018 ), tissue repair promotion ( Shao et al, 2017 ) and homing capacity ( Shao et al, 2017 ; Guo et al, 2019 ), which are important properties for treatments of microbial diseases. What is more, exosome therapy is superior to stem cell therapy in biosafety.…”

“…In addition, protein profiling of MSC-EVs reveals that exosomal proteins are related to biological process such as innate immunity, antimicrobial, host-virus interaction, cellular oxidant detoxification, and complement and coagulation cascades. Several AMPs were identified, including dermcidin, lactoferrin, lipocalin 1, lysozyme C, neutrophil defensin 1, S100A7 (psoriasin), S100A8/A9 (calprotectin), and histone H4 ( Pierce and Kurata, 2021 ). AMPs partially account for the antimicrobial effects of MSCs’ secretome, which may also work in terms of MSC-Exos ( Alcayaga-Miranda et al, 2017 ).…”

“…The activation of toll-like receptors (TLRs)/pathogen-associated molecular patterns (PAMPs) signaling pathway plays an important role in stimulating immune responses and tissue repair in MSCs ( Shirjang et al, 2017 ). PAMPs, such as LPS ( Ti et al, 2015 ), or synthetic ligands, such as Poly (I:C; Pierce and Kurata, 2021 ), can interact with TLRs in MSCs to initiate downstream pathways to enhance the antimicrobial and immunomodulatory proteomic profile of secreted EVs, which can promote M2 polarization and enhance pathogen phagocytosis of macrophages/monocytes. Similar enhancements in EVs’ biological properties can be acquired by preconditioning with pro-inflammatory cytokines, TNF-α ( Nakao et al, 2021 ) and IFN-γ ( Varkouhi et al, 2019 ).…”

Exosomes as New Biomarkers and Drug Delivery Tools for the Prevention and Treatment of Various Diseases: Current Perspectives

“…MSCs, exosomes secreted by these cells can be used as complementary antimicrobial agents, as a substitute for or in combination with antibiotics under specific physiological conditions or specific priming conditions [146]. In particular, antimicrobial properties are associated with the paracrine of several antimicrobial peptides (AMP), which have a wide range of antimicrobial properties, as well as specific extracellular vesicle (EV) secretion, including the release of immunomodulatory factors MSCs that retain antimicrobial properties [147] and are considered safer than parental cell administration [148]. EVS as a cell-free agent and/or drug carrier may have therapeutic effects for sepsis [148] and may be developed as a superior drug delivery vehicle [149].…”

Microfluidic Tools for Enhanced Characterization of Therapeutic Stem Cells and Prediction of Their Potential Antimicrobial Secretome

“…MSC features include the detection of infection signals, the ability to migrate toward the infection site, and on-demand secretion of combinations of antimicrobial peptides (AMPs) [16]. The EVs released from MSCs retain antimicrobial characteristics [13] and are considered to be safer than parent cell administration [17]. The EVs as cell-free agents and/or drug carriers may have therapeutic functions in sepsis [18] and may evolve into superior drug delivery tools.…”