Toll‐like receptor activation of equine mesenchymal stromal cells to enhance antibacterial activity and immunomodulatory cytokine secretion

Abstract Objective To evaluate effects of Toll‐like and nucleotide‐binding oligomerization domain (NOD)‐like receptor (TLR, NLR) ligand stimulation of equine mesenchymal stromal cells (MSCs) on antibacterial and immunomodulatory properties in vitro. Study Design Controlled laboratory study. Sample Population Equine bone‐marrow‐derived MSCs (three horses). Methods MSCs were stimulated with TLR (polyinosinic:polycytidylic acid pIC and lipopolysaccharide LPS) and NLR agonists (γ‐ d ‐Glu‐mDAP IE‐DAP) for 2 h, and plated at 1 × 10 5 cells/well 24 h. MSC‐conditioned media (MSC‐CM) were collected and assessed for antimicrobial peptide cathelicidin/LL‐37 production, bactericidal action against multidrug‐resistant planktonic and biofilm Staphylococcus aureus and neutrophil phagocytosis. Bacterial growth was measured by plating bacteria and counting viable colonies, reading culture absorbance, and live‐dead staining with confocal microscopy imaging. Following initial comparison of activating stimuli, TLR3‐agonist pIC protocols (cell density during activation and plating, culture time, %serum) were further optimized for bactericidal activity and secretion of interleukin‐8 (IL‐8), monocyte‐chemoattractant‐protein (MCP‐1), and cathelicidin/LL37. Results MSCs stimulation with pIC ( p = .004) and IE‐DAP ( p = .03) promoted increased bactericidal activity, evidenced by reduced viable planktonic colony counts. PIC stimulation (2 × 10 6 cells/ml, 2 h, 10 μg/ml) further suppressed biofilm formation ( p = .001), enhanced neutrophil bacterial phagocytosis ( p = .009), increased MCP‐1 secretion ( p < .0001), and enhanced cathelicidin/LL‐37 production, which was apparent when serum concentration in media was reduced to 1% ( p = .01) and 2.5% ( p = .05). Conclusion TLR‐3 pIC MSCs activation was most effective to enhance antibacterial and cytokine responses, which were affected by serum reduction. Clinical Significance In vitro TLR‐3 activation of equine MSCs tested here may be a strategy to improve antibacterial properties of MSCs to treat antibiotic‐resistant infections.