Osteocytes Serve as a Reservoir for Intracellular Persisting Staphylococcus aureus Due to the Lack of Defense Mechanisms

Abstract

Chronic staphylococcal osteomyelitis can persist for long time periods causing bone destruction. The ability of Staphylococcus aureus to develop chronic infections is linked to its capacity to invade and replicate within osteoblasts and osteocytes and to switch to a dormant phenotype called small colony variants. Recently, osteocytes were described as a main reservoir for this pathogen in bone tissue. However, the mechanisms involved in the persistence of S. aureus within these cells are still unknown. Here, we investigated the interaction between S. aureus and osteoblasts or osteocytes during infection. While osteoblasts are able to induce a strong antimicrobial response and eliminate intracellular S. aureus, osteocytes trigger signals to recruit immune cells and enhance inflammation but fail an efficient antimicrobial activity to clear the bacterial infection. Moreover, we found that extracellular signals from osteocytes enhance intracellular bacterial clearance by osteoblasts. Even though both cell types express Toll-like receptor (TLR) 2, the main TLR responsible for S. aureus detection, only osteoblasts were able to increase TLR2 expression after infection. Additionally, proteomic analysis indicates that reduced intracellular bacterial killing activity in osteocytes is related to low antimicrobial peptide expression. Nevertheless, high levels of lipid mediators and cytokines were secreted by osteocytes, suggesting that they can contribute to inflammation. Taken together, our results demonstrate that osteocytes contribute to severe inflammation observed in osteomyelitis and represent the main niche for S. aureus persistence due to their poor capacity for intracellular antimicrobial response.

Keywords: S. aureus; chronic osteomyelitis; osteoblasts; osteocytes; persistence.

Figure 1

Staphylococcus aureus predominantly persists in osteocytes than osteoblasts. (A) Intracellular bacteria (log CFU/10⁶ cells) recovered from infected osteoblasts and osteocytes at different time points during long-term persistence. Osteoblasts LS1; n = 6, osteocytes LS1; n = 7, osteoblasts SH1000; n = 6 and osteocytes SH1000; n = 3; (B) SCV formation (log SCVs/10⁶ cells) on day 7 for S. aureus LS1 and SH1000 strains in osteoblasts and osteocytes. (C) SCV formation on day 7 on blood agar plates. S. aureus LS1 strain within; (i) osteoblasts and (ii) osteocytes. Staphylococcus aureus SH1000 strain within; (iii) osteoblasts; and (iv) osteocytes; n = 3. Differences were analyzed by using two-way and one-way ANOVA, respectively, with Tukey’s multiple comparison test; *p < 0.05, **p < 0.01, and ****p < 0.0001. The bars and whiskers represent the means ±SD of independent experiments.

Figure 2

Elevated persistence of Staphylococcusaureus occurs in osteocytes during co-cultivation with osteoblasts. (A) Intracellular bacteria (log CFU/106 cells) recovered from infected osteoblasts and osteocytes with S. aureus LS1 strain at different time points and both possible orientations in transwell experiments (co-cultivation); (B) SCV (log SCVs/106 cells) formation of S. aureus LS1 strain for both possible orientations. Statistical analysis was performed using two-way ANOVA with Tukey’s multiple comparison test; **p < 0.01, ***p < 0.001, and ****p < 0.0001. The bars and whiskers represent the means ±SD of different independent experiments; n = 3.

Figure 3

Osteocytes trigger osteoblasts to eliminate intracellular bacteria but not vice versa. (A) Model of infection. (B) Infected osteoblast (Ob) or osteocytes (Oc) cultured with control CM or infected CM were lysed and the numbers of intracellular bacteria were quantified by serial dilutions on blood agar plates (log CFU/106 cells). Statistical analysis was performed using the unpaired t-test for each pair; **p < 0.01. The bars and whiskers represent the means ±SD of different independent experiments; n = 6, n = 4, n = 4, and n = 5, respectively. CM, conditioned medium.

Figure 4

TLR2 expression is not altered in infected osteocytes. Influence of Staphylococcus aureus on osteoblasts and osteocytes surface TLR2 expression. Expression of TLR2 among living cells was analyzed by flow cytometry. The mean fluorescence intensity (MFI) of TLR2 was determined. Statistics are calculated with raw data (MFI), infected vs. uninfected and a statistical analysis was performed using the unpaired t-test for each pair unpaired t-test; *p < 0.05. The bars and whiskers represent the means ±SD of different independent experiments; n = 3. −: control cells and +: infected cells.

Figure 5

Proteomic analysis of osteoblasts and osteocytes infected with the Staphylococcus aureus LS1 strain. At 24 h post infection, the supernatants of infected and non-infected cells were collected and analyzed by mass spectrometry. (A) Principal component analysis (PCA) score plot comparing metabolic profiles of infected and non-infected osteoblasts and osteocytes. (B–D) Volcano plot (q-value vs. log₂ fold change) for differentially expressed proteins: (B) Infected vs. non-infected osteoblasts. (C) Infected vs. non-infected osteocytes. (D) Infected osteoblasts vs. infected osteocytes. The X axis represents −log10Q value (values of p < 0.05); the Y axis represents log₂ values of protein fold changes. See details in Supplementary Tables S3–5; n = 3.

Figure 6

Antimicrobial response and cytotoxicity induced by Staphylococcus aureus in osteoblasts and osteocytes. (A) Secretion of LL-37 was measured by ELISA in osteoblasts and osteocytes cell free supernatants, 24 h after infection (ng/10⁶cells). Uninfected values were subtracted from infected values; n = 3. (B) % Cell death induced by S. aureus in osteoblasts and osteocytes at different doses (MOIs). Twenty-four hours post infection, cell death was measured by flow cytometry by staining cells with propidium iodine; n = 5. Statistical analysis was performed using the unpaired t-test and two-way ANOVA with Sidak’s multiple comparison test; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.001. The bars and whiskers represent the means ±SD of different independent experiments.

Figure 7

Inflammatory responses are more pronounced in infected osteocytes than in osteoblasts. (A) Infected cells were incubated for 24 h and IL-6 was measured by ELISA in cell free supernatants. Uninfected values were subtracted from infected values (pg/10⁶cells); n = 3. (B) Infected cells were incubated for 24 h. Next, extracted LMs were analyzed by UPLC-MS–MS (pg/10⁶cells); n = 6. Statistical analysis was performed using unpaired t-test with Welch’s correction: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. The bars and whiskers represent the means ±SEM of different independent experiments. −: control cells and +: infected cells.

Figure 8

Inhibition of TLR2 does not affect bacterial persistence. (A,B) Osteoblasts (A) and osteocytes (B) were treated with 50 μM MMG-11 or not and infected with S. aureus LS1. Intracellular bacteria were quantified after 24 h post infection. (C,D) LL-37 was measured after 24 post infection from the supernatants from treated and non-treated osteoblasts (C) and osteocytes (D). Statistical analysis was performed using unpaired t-test. The bars and whiskers represent the means ±SD of different independent experiments n = 3.