Induction of protective interferon-β responses in murine osteoblasts following Staphylococcus aureus infection

Abstract

Introduction: The refractory and recurrent nature of chronic staphylococcal osteomyelitis may be due, at least in part, to the ability of Staphylococcus aureus to invade and persist within bone-forming osteoblasts. However, osteoblasts are now recognized to respond to S. aureus infection and produce numerous immune mediators and bone regulatory factors that can shape the host response. Type I interferons (IFNs) are best known for their antiviral effects, but it is becoming apparent that they impact host susceptibility to a wide range of pathogens including S. aureus.

Methods: Here, we have assessed the local expression of IFN-β by specific capture ELISA in an established in vivo mouse model of staphylococcal osteomyelitis. RNA Tag-Seq analysis, specific capture ELISAs, and/or immunoblot analyses, were then used to assess the expression of type I IFNs and select IFN stimulated genes (ISGs) in S. aureus infected primary murine osteoblasts. The effect of IFN-β on intracellular S. aureus burden was assessed in vitro following recombinant cytokine treatment by serial colony counts of liberated bacteria.

Results: We report the presence of markedly elevated IFN-β levels in infected bone tissue in a mouse model of staphylococcal osteomyelitis. RNA Tag-Seq analysis of S. aureus infected osteoblasts showed enrichment of genes associated with type I IFN signaling and ISGs, and elevated expression of mRNA encoding IFN-β and ISG products. IFN-β production was confirmed with the demonstration that S. aureus induces its rapid and robust release by osteoblasts in a dose-dependent manner. Furthermore, we showed increased protein expression of the ISG products IFIT1 and IFIT3 by infected osteoblasts and demonstrate that this occurs secondary to the release of IFN-β by these cells. Finally, we have determined that exposure of S. aureus-infected osteoblasts to IFN-β markedly reduces the number of viable bacteria harbored by these cells.

Discussion: Together, these findings indicate an ability of osteoblasts to respond to bacteria by producing IFN-β that can act in an autocrine and/or paracrine manner to elicit ISG expression and mitigate S. aureus infection.

Keywords: Staphylococcus aureus; interferon stimulated genes; interferon-beta; osteoblasts; osteomyelitis.

Figure 1

Murine osteoblasts produce IFN-β in response to S. aureus infection. (A) IFN-β levels in infected femurs (SA) and uninfected contralateral femurs (0) in a mouse model of staphylococcal osteomyelitis at 3 days post infection. Levels were determined by specific capture ELISA, normalized to bone weight, and shown as the mean ± SEM. Asterisk indicates a statistically significant difference from the uninfected femur (p < 0.05; n = 6). (B,C) Primary murine osteoblasts were uninfected (0) or S. aureus (SA) challenged (MOI of 75 or 150:1). At 4 h, mRNA expression was assessed by RNA Tag-Seq analysis and shown as a heatmap (B), and IFN-β protein release was assessed at 4, 6, and 8 h by specific capture ELISA (C). Data is shown as mean ± SEM (n = 9). Asterisks indicate a statistically significant difference from time-matched uninfected cells and daggers indicate a significant difference from cells at 4 h for that treatment. (D) Upregulation of genes associated with PRR and type I IFN signaling adapted from KEGG pathway analysis of RNA Tag-Seq data conducted using ShinyGO 0.76. Differentially expressed genes in these pathways are indicated in red.

Figure 2

Murine osteoblasts express ISGs in response to S. aureus. Osteoblasts were uninfected (0) or S. aureus challenged (25, 50, 75, or 150:1). (A) At 8 (left) and 20 (right) hours, IFIT1 expression was determined by immunoblot analysis. As a positive control, cells were treated with recombinant IFN-β (1 ng/mL). A representative immunoblot is shown and average band intensities were determined and normalized to β-actin expression (n = 3). Asterisks indicate a statistically significant difference from uninfected/untreated cells (p < 0.05). (B,C) At 8 h, IFIT3 levels were determined by immunoblot analysis (B) and specific capture ELISA (C). A representative immunoblot is shown and average band intensities were determined and normalized to β-actin (n = 6–9), and ELISA data is shown as mean ± SEM (n = 4). Asterisks indicate a statistically significant difference from uninfected cells (p < 0.05). (D) At 8 h, PLSCR1 expression was determined by immunoblot analysis. For comparison purposes, expression in osteoblasts treated with recombinant IFN-β (1 ng/mL) is shown. A representative immunoblot is shown and average band intensities were determined and normalized to β-actin (n = 5–11).

Figure 3

Osteoblast-derived IFN-β mediates ISG expression following S. aureus challenge and IFN-β can limit bacterial burden in infected cells. (A) Osteoblasts were untreated/uninfected, treated with recombinant IFN-β (1 ng/mL), or S. aureus challenged at MOI of 75 and 150:1 (SA 75 and SA 150, respectively), in the presence or absence of IFNAR blocking antibody (αIFNAR) or isotype control antibody (IC). At 8 h, protein isolates were taken and IFIT1 expression determined by immunoblot analysis. A representative immunoblot is shown and average band intensities were determined and normalized to β-actin expression (n = 4). Data is shown as the mean ± SEM (n = 4) and asterisks indicate a statistically significant difference from similarly stimulated cells treated with isotype control antibody (p < 0.05). (B) Osteoblasts (2 × 10⁶ cells) were infected with S. aureus (MOI of 75:1) in the absence or presence of recombinant IFN-β (1 ng/mL). At 2, 6, 24, and 48-h following infection, cells were lysed in antibiotic-free media and the number of viable intracellular bacteria were determined by colony count. Data is shown as the mean ±SEM (n = 6) and asterisks indicate a statistically significant difference from untreated cells (p < 0.05).