Introduction

The rapid development of antibiotic resistance in human and veterinary medicine necessitates advancement of novel therapeutic strategies to treat infection. Biological therapies (e.g. mesenchymal stromal cells (MSC)) are attractive as they are antimicrobial and immunomodulatory. Direct effects of MSC are mediated by antimicrobial peptide production, while indirect actions are facilitated by the immunomodulatory effect of MSCs on innate immune cells including monocytes and neutrophils.

One mechanism by which MSC play a role in response to inflammation associated with infection is through expression of toll-like receptors (TLR). Previous studies have demonstrated that the antimicrobial and immunomodulatory properties of MSC, including antimicrobial peptide secretion, direct inhibition of bacterial growth, secretion of immunomodulatory cytokines, and neutrophil phagocytosis of bacteria, can be enhanced by stimulation with TLR ligands in vitro. In vitro studies using horse, dog and human MSC demonstrated increased bacterial killing using TLR3 activated MSC. In a murine model of Staphylococcal biofilm infection, MSC cultured with TLR-3 ligand polyinosinic:polycytidylic acid (pIC) demonstrated significantly increased effectiveness in clearing infection compared to non-activated MSC with or without antibiotics, or antibiotics alone. The ability of MSCs to enhance immune function of the host and act synergistically with current therapeutic options is attractive to circumvent the rapid development of resistance inherent in use of new pharmacologic agents.

Content

Purpose:

Objectives were to determine if intra-articular(IA) administration of TLR-3-pIC activated bone-marrow-derived MSC and antibiotics improved clinical parameters and reduced bacterial burden and biomarkers of joint inflammation in equine-modeled of septic arthritis compared to antibiotics alone.

Materials&Methods:

Eight horses were inoculated in one tarsocrural joint with multidrug resistant S.aureus(1x10⁴CFU). Bone-marrow-derived MSC from three donors were activated with TLR-3 agonist polyinosinic:polycytidylic(polyI:C) acid(10µg/mL,2x10⁶MSC/mL,2hours). Recipients received 20x10⁶ pooled allogeneic IA TLR-MSC and vancomycin(100mg) or vancomycin alone. A non-steroidal-anti-inflammatory agent(phenylbutazone 2.2mg/kg intravenously q12h)was administered for the study’s duration alongside antibiotics(gentamicin6.6 mg/kg IV q24h)24hours following infection and continuing to end-term for control horses(d7)or through d10 for treated horses(TH)(end-term was d14).

Inflammation/pain(I/P)scoring, complete blood and bacterial counts, inflammatory biomarkers(IB)in SF and plasma, and imaging were evaluated as outcome parameters. I/P scores of joint circumference, lameness, physical examination, distal limb edema, and joint heat were monitored daily. SF and blood samples were collected on d0,1,4,7,14 following infection, and assessed for bacterial counts, clinicopathologic parameters and IB. Blood samples were assessed for complete blood count, and plasma levels for IB. Radiographs and ultrasound were performed d0,7,14. End-term magnetic resonance imaging, macroscopic joint scoring, quantitative bacterial analysis of SF and synovium, and histology were assessed.

Results:

I/P scores were lower in TH across time points(p=0.0002)and at individual time points (D3,4,p=0.02;D5,6,7,p=0.04). Bacterial counts of SF(D4p=0.03,D7p=0.02)and end-term synovium(p=0.003)were lower with TLR-3 activated MSC-treatments. Complete blood count revealed lower peripheral neutrophil counts in TH(D4p=0.03; D7p=0.06). SF analysis revealed lower total nucleated cell counts(p=0.09), total protein(p=0.08), neutrophils(p=0.005), lactate(p=<0.0001)and higher glucose(p=0.009)in treated versus control D7. Multiplex biomarker analysis of SF revealed lower IL-6 levels across all time points(p=0.02) and at individual time points(D4p=0.03,D7p=0.11)and lower IL-18 levels across time points in TH(p=0.02).

Conclusions:

Intra-articular TLR-3 stimulated MSC injection with vancomycin resulted in lower bacterial counts and improved clinical parameters in multi-drug resistant Staphylococcal septic arthritis compared to antibiotics alone.

Acknowledgments

This study was funded by the Grayson Jockey Club Research Foundation, NIH/NCATS CTSA 5TL1TR002533-02, NIH 5T32OD010437-19, Verdad Foundation, Shipley Foundation and Carolyn Quan and Porter Bennett. The StableLab serum amyloid A testing material was kindly provided by Zoetis.