Poster Presentation 29th Australian and New Zealand Bone and Mineral Society Annual Scientific Meeting 2019

Developing a cell line model of osteocyte infection (#178)

Nicholas J Gunn 1 , Asiri R Wijenayaka 1 , Dongqing Yang 1 , Lucian B Solomon 1 , Eugene Roscioli 2 , Stephen P Kidd 3 , Gerald J Atkins 1
  1. Centre for Orthopaedic & Trauma Research, Biomedical Orthopaedic Research Group, Adelaide, SA, Australia
  2. Department of Thoracic Medicine, Adelaide Medical School, Adelaide, SA, Australia
  3. School of Molecular and Biomedical Science, Research Centre for Infectious Disease, Adelaide, SA, Australia

INTRODUCTION

Our group have demonstrated the involvement of osteocytes in the pathogenesis of osteomyelitis associated with periprosthetic joint infection (1). The aim of the current study is to develop a cell line model of osteocyte infection to allow reproducible investigation into the interactions between osteocytes and invasive bacteria and provide a screening assay to test the effects of antimicrobials on these.

METHODS

Osteocyte-like SaOS2 cultures (2) were infected with the methicillin resistant S. aureus strain WCH-SK2 as this was shown previously to become internalised by human primary osteocyte-like cells without causing cell death (1). We analysed a range of parameters including; changes in the expression of a range of genes known to be differentially expressed during infection via qPCR, intracellular localization of the bacteria and their interaction with autophagy via TEM, fluorescent confocal microscopy and western blot and the analysis of bacterial viability and quantity via plate count methods and qPCR.

RESULTS

Intracellular infection was demonstrated within osteocyte-like SaOS2 cells by the culture of bacteria from the lysate in declining numbers until 15 days post-infection (Figure 1). No correlation was seen between colony counts and bacterial presence measured by qPCR, which in concert with declining colony size indicates a transition towards the viable but non-culturable growth phenotype.

The expression of osteocytic markers was not impacted by infection, whilst a significant increase was demonstrated in the expression levels of immune-modulatory cytokines demonstrating an active immune response without compromising the differentiated state.

WCH-SK2 is capable of subverting autophagy by blocking the fusion of phagosomes with lysosomes to evade innate immunity during early internalization.

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Figure 1: Culturability and colony diameter over time of WCH-SK2 within osteocyte-like SaOS2 measured from cultured cellular lysates.

 

CONCLUSIONS

SaOS2 cells differentiated to an osteocyte-like state constitute a biologically relevant model of osteocyte infection with S. aureus.

 

  1. 1: "Novel Insights into Staphylococcus aureus Deep Bone Infections: the Involvement of Osteocytes" -Yang D, et al., MBio. 2018;9(2) 2: "SaOS2 Osteosarcoma Cells as an In Vitro Model for Studying the Transition of Human Osteoblasts to Osteocytes" -Prideaux M, et al., Calcif Tissue Int. 2014;95(2):183-93