P21 Novel ex-vivo model of septic arthritis identifies role of neutrophils in joint destruction and identifies a potential biomarker for diagnosis
Authors: McCall Kathryn E , Atherton Caroline , Thudium Christian , Goodyear Carl , Evans Tom , Millar Neal , McInnes Iain 
Published: April 2020

Abstract

Background
Septic arthritis (SA) caused by bacterial species, such as Staphylococcus aureus, has high morbidity and mortality. Currently diagnosis is often prolonged and unreliable, with no suitable near-patient biomarkers available. To generate more reliable biomarkers and to understand pathogenesis we sought to develop a novel ex vivo system to explore the effect of pathogenic Staph. aureus strains in promoting cartilage degradation.

Methods
Human cartilage explants were obtained from femoral heads being surgically removed following trauma. Explants were infected for 48h with 106 cfu bacteria from two Staph. aureus SA-derived patient isolates (28g & 36v strains). In the final 24h of bacterial infection, neutrophils purified from healthy donor blood were added to explant cultures at 3 x 106 cells/well. Chondrocyte viability was assessed using CellTracker green CMFDA and propidium iodide. Images were captured using confocal microscopy (LSM880) and cells counted using Imaris software. Structural damage was measured by glycosaminoglycan (GAG) and a neo-epitope of MMP-mediated degradation of type II collagen (C2M) release. Statistical analysis was performed using GraphPad Prism software.

Results
When cartilage explants were co-cultured with bacteria +/- neutrophils, cell death was significantly increased compared to the negative control or addition of neutrophils alone, (Friedman multiple comparisons test, N = 3, negative control vs. bacteria - neutrophils p &lt;0.05, negative control vs. bacteria + neutrophils p &lt; 0.01). Cartilage breakdown, estimated via GAG release, was induced by Staph. aureus alone, whereas it was significantly enhanced upon neutrophil addition in the final 24h of co-culture (Friedman multiple comparisons test, N = 8, neutrophil vs. neutrophil + bacterial strain 36v p &lt;0.0001, neutrophil vs. neutrophil + bacterial strain 28g p &lt;0.0005). Therefore, it is the combined effect with neutrophils that results in significant cartilage destruction. Although cartilage is damaged with the bacterial infection, shown by GAG release, C2M was only released when the bacteria is co-cultured with neutrophils (Friedman multiple comparisons test, N = 8, 36v vs. 36v + neutrophil p &lt;0.0001, 28g vs. 28g + neutrophil p &lt;0.005). This indicates that C2M is a biomarker that could potentially be used to diagnose septic arthritis as neutrophils infiltrate the joint in response to a bacterial infection.

Conclusion
A co-culture model of septic arthritis has been developed which allows precise examination of the contribution of the host neutrophil response to cartilage damage. We used this to identify a collagen breakdown product as a biomarker of host response to infected cartilage. This novel model will be a valuable tool in understanding the pathology of joint infection and can be used for the identification of future diagnostic biomarkers.

Disclosures
K.E. McCall None. C. Atherton None. C. Thudium None. C. Goodyear Grants/research support; C.G. has received funding for research from Celgene, AstraZeneca, MedAnnex, UCB & Jannsen. T. Evans None. N. Millar Grants/research support; Novartis. I. McInnes Consultancies; I.M. has received consultancies fees from BMS, Abbvie, Lilly, GSK & Pfizer. Grants/research support; I.M received research funding from Calgene, Janssen, Novartis, Boehringer Ingelheim & BMS.