RIG-I and cGAS mediate antimicrobial and inflammatory responses of primary osteoblasts and osteoclasts to Staphylococcus aureus

Abstract

Staphylococcus aureus is the primary causative agent of osteomyelitis, and it is now apparent that osteoblasts and osteoclasts play a significant role in the pathogenesis of such infections. Their responses can either be protective or exacerbate inflammatory bone loss and are mediated by the recognition of microbial motifs by various pattern recognition receptors. We have recently reported that osteoblasts can respond to S. aureus challenge with the production of the type I interferon, interferon-beta, which can reduce the number of viable bacteria harbored within infected cells. In the present study, we demonstrate that S. aureus viability and internalization are necessary for maximal inflammatory cytokine and type I interferon responses of primary bone cells to this pathogen. Importantly, we show that primary murine and human bone cells constitutively express the cytosolic nucleic acid sensors, retinoic acid inducible gene I (RIG-I) and cyclic GMP-AMP synthase (cGAS), and demonstrate that such expression is markedly upregulated following S. aureus infection. The functional status of RIG-I and cGAS in osteoblasts and osteoclasts was confirmed by showing that specific ligands for each can also elevate their expression and induce cytokine responses. We have verified the specificity of such responses using siRNA knockdown or pharmacological inhibition and used these approaches to demonstrate that both sensors play a pivotal role in bone cell responses to infection with clinically relevant strains of S. aureus. Finally, we have begun to establish the biological significance of RIG-I- and cGAS-mediated bone cell responses with the demonstration that their attenuation increases S. aureus burden in infected cells, suggesting a potentially protective role for these sensors in osteomyelitis.IMPORTANCEStaphylococcal osteomyelitis is a severe infection that is often recalcitrant to current treatment strategies. We and others have demonstrated that resident bone cells are not merely passive victims but can respond to bacteria with the production of an array of immune mediators, including type I interferons, that could serve to limit such infections. Here, we demonstrate the functional expression of two cytosolic nucleic acid sensors, retinoic acid inducible gene I and cyclic GMP-AMP synthase, in primary murine and human osteoblasts and murine osteoclasts. We show that these pattern recognition receptors mediate potentially protective bone cell type I interferon responses to Staphylococcus aureus infection.

Keywords: RIG-I; Staphylococcus aureus; cGAS; cytokines; osteoblasts; osteoclasts; type I interferon.

Fig 1

S. aureus viability and internalization are necessary for the maximal stimulation of inflammatory cytokine and type I IFN production by primary murine osteoblasts. Osteoblasts were either uninfected (0) or challenged with viable S. aureus (V), heat-inactivated bacteria (HI), or viable bacteria coated with fibronectin (Fn; 5 µg/mL) at an MOI of 75:1 for 2 h. Panel A: the number of viable bacteria harbored by osteoblasts challenged with live or heat-inactivated S. aureus was assessed by colony counting 8 h post-infection. Panel B: cells were fixed with 4% paraformaldehyde at 2 h following bacterial challenge and processed for immunofluorescent microscopy to label extracellular bacteria (Alexa Fluor 488, green) and intracellular bacteria (Alexa Fluor 647, pink). Arrows indicate intracellular bacteria, while arrowheads indicate extracellular bacteria. Panel C: the number of internalized bacteria was quantified with color thresholding to determine the integrated density using ImageJ at 2 h post-infection. Panel D: at 8 h following viable and inactivated bacterial challenge, osteoblast production of IL-6 and IFN-β was assessed by specific capture ELISA. Panel E: at 8 hours following challenge with uncoated S. aureus or fibronectin-coated S. aureus, osteoblast production of IL-6 and IFN-β was assessed by specific capture ELISA. Asterisks indicate a statistically significant difference compared to unchallenged osteoblasts. Daggers indicate a statistically significant difference compared to cells challenged with viable bacteria (mean ± SEM, n = 3; Student’s t-test, P value < 0.05).

Fig 2

Osteoblasts constitutively express RIG-I and cGAS, and their expression is upregulated following activation associated with S. aureus infection. Primary murine (A and B) and primary human (C and D) osteoblasts were untreated (0) or infected with S. aureus at MOIs of 25:1, 75:1, or 150:1. Panel A: at 4 and 8 h, IL-6 and IFN-β production was assessed in primary murine osteoblasts by specific capture ELISA. Asterisks indicate a statistically significant difference compared to uninfected osteoblasts. Daggers indicate a statistically significant difference compared to similarly challenged cells at 4 h post-infection (mean ± SEM, n = 3; two-way ANOVA, P value < 0.05). Panel B: expression of RIG-I (102 kDa) and cGAS (62 kDa) was assessed at 8 h post-infection by immunoblot analysis. Representative immunoblots and the average expression level of each protein, as determined by densitometric analysis normalized to β-actin levels, are shown. Panel C: at 8 h, IL-6 and IFN-β production was assessed in primary human osteoblasts by specific capture ELISA. Panel D: expression of RIG-I and cGAS was assessed by immunoblot analysis. Representative immunoblots and the average expression level of each protein, as determined by densitometric analysis normalized to β-actin levels, are shown. Asterisks indicate a statistically significant difference compared to uninfected osteoblasts (mean ± SEM, n = 3; one-way ANOVA or Student’s t-test, P value < 0.05).

Fig 3

Intracellular administration of nucleic acid ligands for RIG-I or cGAS elicits murine osteoblast immune responses. Panel A: osteoblasts were transfected with B-DNA (B; 1 µg/mL), 5′ triphosphate double-stranded RNA (3P; 2 µg/mL), polyinosinic–polycytidylic acid (pIC; 1 µg/mL), or Y-DNA (Y; 2.5 µg/mL) complexed with Lipofectamine 2000 (L2K) or were treated with L2K alone or challenged with lipopolysaccharide (LPS; 10 ng/mL). At 8 h post-transfection, expression of RIG-I (102 kDa) and cGAS (62 kDa) was assessed by immunoblot analysis. Representative immunoblots and the average expression level of each protein, as determined by densitometric analysis normalized to β-actin levels, are shown. Asterisks indicate a statistically significant difference compared to osteoblasts treated with L2K alone (mean ± SEM, n = 2–4; Student’s t-test, P value < 0.05). Panel B: osteoblasts were transfected with siRNA (15 nM) directed against RIG-I, cGAS, or both using RNAiMAX or exposed to control siRNA (Control). At 8 h, RIG-I and cGAS expressions were assessed by immunoblot analysis. Panel C: cells were transfected with siRNA (15 nM) directed against RIG-I, cGAS, both, or control siRNA prior to intracellular challenge with B-DNA (B; 1 µg/mL), 5′ triphosphate double stranded RNA (3P; 2 µg/mL), polyinosinic−polycytidylic acid (pIC; 1 µg/mL), or Y-DNA (Y; 2.5 µg/mL) complexed with L2K or were treated with L2K alone or challenged with LPS (10 ng/mL). At 8 h, production of IL-6 and IFN-β was assessed with specific capture ELISA. Asterisks indicate significance compared to the L2K treatment alone. Daggers represent significance compared to the similarly stimulated control siRNA-treated group (mean ± SEM, n = 3–6; two-way ANOVA with Šídák’s multiple comparison test, P value < 0.05).

Fig 4

RIG-I and cGAS mediate the inflammatory and anti-microbial IFN responses of murine osteoblasts to S. aureus infection. Osteoblasts were transfected with siRNA (10 nM) directed against RIG-I, cGAS, or RNA polymerase III (RP3) or control siRNA (Control) using RNAiMAX. These cells were then uninfected (0) or challenged with S. aureus (MOI of 75:1). At 8 h post-infection, RIG-I (102 kDa), cGAS (62 kDa), and RP3 (165 kDa) expressions were quantified by immunoblot analysis (A). Representative immunoblots and the average expression level of each protein, as determined by densitometric analysis normalized to β-actin levels, are shown. Asterisks indicate a significant decrease compared to the corresponding control siRNA-treated group (mean ± SEM, n = 3–6; one-way ANOVA, P value < 0.05). In addition, IL-6 and IFN-β production by these cells was assessed by specific capture ELISA, and intracellular bacterial viability was assessed by colony counting 24 h post-infection (B). Asterisks indicate a significant increase compared to uninfected cells. Daggers represent a significant decrease compared to the similarly infected control siRNA-treated group (mean ± SEM, n = 3–6; two-way ANOVA with Šídák’s multiple comparison test and Student’s t-test, P value < 0.05).

Fig 5

RIG-I and cGAS mediate the inflammatory and anti-microbial IFN responses of primary murine and human osteoblasts to challenge with clinically relevant S. aureus strains. Primary murine (A) and human (B) were either untreated or treated with a TBK1/IKKε inhibitor (BX795; 2 µM) for 4 h prior to challenge with S. aureus strains (MOI of 75:1) UAMS1, HFH29568, and TCH 1516 or Pam3CSK4 (P3C: 10 ng/mL). At 8 h post-infection, IL-6 and IFN-β production was assessed with specific capture ELISAs. Intracellular bacterial viability was assessed by colony counting at 24 h post-infection. Asterisks indicate significance compared to uninfected cells. Daggers indicate significance compared to the similarly infected but untreated group (mean ± SEM, n = 3; two-way ANOVA with Šídák’s multiple comparison test, P value < 0.05).

Fig 6

Activated osteoclasts express RIG-I and cGAS, and these sensors similarly mediate the anti-microbial IFN responses of these cells to S. aureus infection. Panels A and B: bone marrow-derived primary murine osteoclasts were untreated (0) or infected with S. aureus at MOIs of 25:1, 75:1, or 150:1. At 8 h, production of IFN-β was determined by specific capture ELISA (A), and expressions of RIG-I (102 kDa) and cGAS (62 kDa) were assessed by immunoblot analysis (B). Representative immunoblots and the average expression level of each protein, as determined by densitometric analysis normalized to β-actin levels, are shown. Asterisks indicate a significant increase compared to unchallenged cells. Panel C: osteoclasts were transfected with B-DNA (B; 0.5 µg/mL), 5′ triphosphate double-stranded RNA (3P; 1 µg/mL), polyinosinic–polycytidylic acid (pIC; 0.5 µg/mL), or Y-DNA (Y; 1 µg/mL) complexed with L2K or were treated with L2K alone or challenged with LPS (5 ng/mL). At 8 h post-transfection, expressions of RIG-I and cGAS were assessed by immunoblot analysis. Representative immunoblots and the average expression level of each, as determined by densitometric analysis normalized to β-actin levels, are shown. Asterisks indicate a statistically significant difference compared to osteoclasts treated with L2K alone (mean ± SEM, n = 3–4; Student’s t-test, P value < 0.05). Panels D and E: osteoclasts were either untreated or treated with a TBK1/IKKε inhibitor (BX795; 1 µM) for 2 h prior to challenge with RIG-I or cGAS nucleic acid agonists, S. aureus infection (MOI of 75), or Pam3CSK4 (P3C: 10 ng/mL). At 8 h, IFN-β production was assessed by specific capture ELISA, and viable intracellular bacterial burden was assessed at 24 h post-infection by colony counting. Asterisks indicate a significance compared to unchallenged cells. Daggers represent significance compared to the corresponding untreated group (mean ± SEM, n = 3; two-way ANOVA with Šídák’s multiple comparison test and Student’s t-test, P value < 0.05).