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Veterinary surgery : VS2021; 50(4); 858-871; doi: 10.1111/vsu.13628

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

Abstract: 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. Methods: Controlled laboratory study. Methods: 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 × 105 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 × 106 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). Conclusions: TLR-3 pIC MSCs activation was most effective to enhance antibacterial and cytokine responses, which were affected by serum reduction. Conclusions: 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.
© 2021 American College of Veterinary Surgeons.
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Publication Date: 2021-04-02 PubMed ID: 33797775DOI: 10.1111/vsu.13628Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research investigates the impact of stimulating Toll-like and nucleotide-binding oligomerization domain-like receptors in equine mesenchymal stromal cells on their antibacterial properties and ability to immunomodulate. Specific protocols involving different stimulation, cell density, and culture time were tested. The findings suggest that TLR-3 stimulation could be an effective way to enhance their antibacterial properties, which could be beneficial for treating antibiotic-resistant infections.

Purpose and Methodology

  • The focus of the study was to look into the effects of stimulating Toll-like and nucleotide-binding oligomerization domain-like receptor (TLR, NLR) in equine mesenchymal stromal cells (MSCs).
  • The purpose was to observe any changes in the cells’ antibacterial and immunomodulatory properties.
  • The researchers conducted the study in a controlled laboratory environment using MSCs derived from the bone marrow of three horses.
  • The stimulation process involved using TLR (polyinosinic:polycytidylic acid [pIC] and lipopolysaccharide [LPS]) and NLR agonists (γ-d-Glu-mDAP [IE-DAP]).

Experimental Design and Procedures

  • The MSCs were stimulated for 2 hours and placed at 1 x 10 cells/well for 24 hours.
  • The researchers then collected MSC conditioned media (MSC-CM) and evaluated them for the production of antimicrobial peptide cathelicidin/LL-37, their action against multidrug-resistant Staphylococcus aureus and neutrophil phagocytosis.
  • Bacterial growth was measured by counting viable colonies, assessing culture absorbance, and live-dead staining with confocal microscopy imaging.

Results and Further Optimization

  • Post initial comparison, TLR3-agonist pIC protocols were further optimized for bactericidal activity and secretion of interleukin-8 (IL-8), monocyte-chemoattractant-protein (MCP-1), and cathelicidin/LL37.
  • The results showed that the stimulation of MSCs with pIC and IE-DAP promoted an increase in bactericidal activity, as noticeable with reduced viable colony counts.
  • pIC stimulation also suppressed biofilm formation, enhanced neutrophil bacterial phagocytosis, increased MCP-1 secretion, and enhanced cathelicidin/LL-37 production.
  • The effects were more pronounced when the serum concentration was further reduced to 1% and 2.5%.

Conclusions

  • The study concluded that TLR-3 pIC stimulation was the most effective way to enhance antibacterial and cytokine responses in the MSCs.
  • The effects were even more apparent with serum reduction.
  • This suggests that in vitro TLR-3 activation of equine MSCs could be used as a strategy to improve the antibacterial properties of these cells to combat antibiotic-resistant infections.

Cite This Article

APA
Pezzanite LM, Chow L, Johnson V, Griffenhagen GM, Goodrich L, Dow S. (2021). Toll-like receptor activation of equine mesenchymal stromal cells to enhance antibacterial activity and immunomodulatory cytokine secretion. Vet Surg, 50(4), 858-871. https://doi.org/10.1111/vsu.13628

Publication

Veterinary surgery : VS
ISSN: 1532-950X
NlmUniqueID: 8113214
Country: United States
Language: English
Volume: 50
Issue: 4
Pages: 858-871

Researcher Affiliations

Pezzanite, Lynn M
  • Department of Clinical Sciences, College of Veterinary Medicine, Colorado State University, Colorado, USA.
Chow, Lyndah
  • Department of Clinical Sciences, College of Veterinary Medicine, Colorado State University, Colorado, USA.
Johnson, Valerie
  • Department of Clinical Sciences, College of Veterinary Medicine, Colorado State University, Colorado, USA.
Griffenhagen, Gregg M
  • Department of Clinical Sciences, College of Veterinary Medicine, Colorado State University, Colorado, USA.
Goodrich, Laurie
  • Department of Clinical Sciences, College of Veterinary Medicine, Colorado State University, Colorado, USA.
Dow, Steven
  • Department of Clinical Sciences, College of Veterinary Medicine, Colorado State University, Colorado, USA.

MeSH Terms

  • Animals
  • Antimicrobial Cationic Peptides / biosynthesis
  • Biofilms
  • Cytokines / biosynthesis
  • Drug Resistance, Multiple, Bacterial / immunology
  • Horses / immunology
  • Immunomodulation / genetics
  • Mesenchymal Stem Cells / immunology
  • Neutrophils / drug effects
  • Phagocytosis / drug effects
  • Staphylococcus aureus / physiology
  • Toll-Like Receptors / metabolism
  • Cathelicidins

Grant Funding

  • ACVS Zoetis Dual Training Grant
  • Carolyn Quan and Porter Bennett
  • CCTSI NIH/NCATS CTSA TL1TR002533
  • NIH 5T32OD010437-19
  • Shipley Foundation
  • Verdad Foundation

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