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.
Highlights
Mesenchymal stromal cells (MSCs) are multipotent cells isolated from bone marrow, adipose tissue, umbilical cord, and other tissues that are widely used in translational applications (De Castro et al, 2019)
Transmission electron microscopy (TEM) analysis showed the typical cup-shaped morphology of the Mesenchymal stromal (stem) cells (MSCs)-extracellular vesicles (EVs) with a central depression characteristic of EVs isolated by ultracentrifugation under TEM (Figure 1A)
Functional gene ontology (GO) enrichment analysis of proteins identified by mass spectrometry using DAVID software revealed that approximately 79.7% of identified proteins were associated with the GO term extracellular exosome (Benjamini p value 2.78 × 10−156; Figure 1B)
Summary
Mesenchymal stromal (stem) cells (MSCs) are multipotent cells isolated from bone marrow, adipose tissue, umbilical cord, and other tissues that are widely used in translational applications (De Castro et al, 2019). MSCs have been shown to benefit wound healing by promoting tissue regeneration and increasing the recruitment of macrophages and endothelial cells into the wound and by possessing immunomodulatory and antimicrobial activity against both Gram-negative and Gram-positive pathogens (AlcayagaMiranda et al, 2017; Marrazzo et al, 2019). MSCs help fight infection by promoting direct bacterial killing or indirectly by modulating the acute phase response, thereby decreasing tissue injury (Marrazzo et al, 2019). MSCs have been shown to secrete antimicrobial peptides (AMPs) such as cathelicidin (hCAP-18/LL-37), beta-defensin 2, lipocalin 2, and keratinocyte growth factor (Krasnodembskaya et al, 2010; Lee et al, 2013; Sung et al, 2016). Biofilms are produced when a cluster of organisms attach to a surface and secrete an extracellular polysaccharide matrix that serves as a protective barrier against conventional antibiotics and host defenses (Khatoon et al, 2018)
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