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).

Intra‐articular administration of antibiotics in horses: Justifications, risks, reconsideration of use and outcomes

“…The increasing incidence of antibiotic resistance has driven the investigation of novel biological therapies with antimicrobial properties, including platelet-rich plasma lysate and mesenchymal stromal cells. [78][79][80][81][82] Recent literature describes the formation of free-floating biofilm aggregates by Staphylococcus aureus and other gram-positive and -negative isolates that are most frequently reported to be involved in human septic arthritis (Streptococcus zooepidemicus, Escherichia coli and Pseudomonas aeruginosa) in synovial fluid, rendering them more tolerant to antibiotic therapy compared with their planktonic form. 78 The equine model of septic arthritis and availability of equine synovial fluid for use in in vitro assays makes the horse a valuable translational model to study the host-pathogen interactions in human septic arthritis as well, compared with the more difficult to source synovial fluid from healthy human individuals.…”

“…Mesenchymal stromal cells (MSC) exert potent immunomodulatory and antibacterial activities, making them attractive as biological therapies for diverse conditions, including musculoskeletal injuries, wound healing and bacterial infections. 80,82,[91][92][93][94][95][96] Equine MSC were initially demonstrated to inhibit the growth of Escherichia coli and Staphylococcus aureus through antimicrobial peptide secretion and depolarisation of cell membranes. 81 Their application in the treatment of infection is gaining increasing attention and methods to optimise antibacterial activity have been further explored.…”

“…82 Exposure of MSC to TLR-4 agonist lipopolysaccharide (LPS) induced increased expression of cytokines by MSC of all tissue origins, indicating that immunomodulatory properties of MSC may be further upregulated in the presence of bacterial components in infection in vivo. 82 The concept of immune activation has been further investigated by exposing MSC to toll-like and nod-like receptor agonists to enhance antibacterial properties prior to in vivo injection, 80,91 with demonstrated increased bacterial killing and secretion of immunomodulatory cytokines with both human and equine cells in vitro. 80,91 Immune activated cellular therapy (MSC co-cultured with TLR-3 ligand polyinosinic:polycytidylic acid (pIC)) in addition to antibiotic therapy resulted in synergistic enhanced clearance of bacteria in murine models of implant infection and pet dogs with spontaneous multidrug-resistant infections.…”

“…82 The concept of immune activation has been further investigated by exposing MSC to toll-like and nod-like receptor agonists to enhance antibacterial properties prior to in vivo injection, 80,91 with demonstrated increased bacterial killing and secretion of immunomodulatory cytokines with both human and equine cells in vitro. 80,91 Immune activated cellular therapy (MSC co-cultured with TLR-3 ligand polyinosinic:polycytidylic acid (pIC)) in addition to antibiotic therapy resulted in synergistic enhanced clearance of bacteria in murine models of implant infection and pet dogs with spontaneous multidrug-resistant infections. [91][92][93] Case-controlled studies investigating the use of pIC activated MSC in the treatment of equine multidrug-resistant Staphylococcal septic arthritis are ongoing by this group of investigators at the time of this report.…”

Role of Innate Immunity in Initiation and Progression of Osteoarthritis, with Emphasis on Horses

“…Mesenchymal stromal cells (MSC) express immunomodulatory and antimicrobial properties through paracrine recruitment of immune effector cells and antimicrobial peptide secretion. Pre-activation of human, canine and equine MSC with Toll-like receptor (TLR) agonists such as polyinosinic-polycytidylic acid (polyI:C) has been shown to enhance bacterial killing and increase bacterial clearance in rodent Staphylococcal biofilm infection models [ 97 , 98 , 99 ]. This group of collaborators built further on that work to demonstrate that intra-articular administration of TLR3 polyI:C activated mesenchymal stromal cell therapy improved outcomes in treatment of multidrug resistant septic arthritis in an equine model, with reduced quantitative bacterial counts and pro-inflammatory biomarkers in synovial fluid, improved imaging (ultrasound and magnetic resonance imaging) scores and more rapid normalization of clinicopathologic parameters both systemically and in synovial fluid.…”