S. aureus osteomyelitis alters cortical and trabecular bone remodeling

In order to determine changes in bone remodeling that occur during osteomyelitis, we compared architectural bone parameters between infected and mock infected wild-type (WT) C57BL/6J mice in a post-traumatic model of S. aureus bone infection [6]. We focused our analyses on two distinct anatomical sites of the infected femurs representing the two major architectural types of bone: cortical bone that comprises the mid-region (diaphysis) of the long bone and trabecular bone found in the distal femur (metaphysis and epiphysis) (S1A–S1C Fig). We previously observed that mock infected WT mice display a rapid cortical bone healing response at the surgical site, in which the induced bone defect in the femoral diaphysis is replaced with new bone by 2 weeks post-surgery [6]. In contrast to this sterile cortical bone repair, mice infected with S. aureus develop osteomyelitis, are unable to restore the cortical bone defect, and experience extensive cortical bone loss surrounding the site of inoculation (Fig 1A and 1B). Moreover, S. aureus infected femurs show reactive cortical bone formation surrounding the site of inoculation (Fig 1C) [6].

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 1. S. aureus osteomyelitis alters cortical and trabecular bone remodeling.

(A-H) Mock infection or osteomyelitis was induced in female wild-type C57BL/6J (WT) mice via intraosseous inoculation with PBS or S. aureus. At 14 days post-infection, femurs were harvested, fixed in neutral buffered formalin, and scanned with a μCT50 at 10 μm resolution (n = 5 mice per group). (A) Anteroposterior view of the femur at the inoculation site in representative mock infected and S. aureus infected femurs. (B, C) Cortical bone loss (mm³) (B) and reactive bone formation (mm³) (C) were quantified via μCT. (D) Representative three-dimensional top down (left) and side (right) views of trabecular bone architecture in mock infected and S. aureus infected femurs. (E-H) Trabecular bone indices, including three-dimensional measurements of trabecular bone volume/total volume (BV/TV) (%) (E), trabecular number (Tb.N) (1/mm) (F), trabecular spacing (Tb.Sp) (mm) (G), and trabecular thickness (Tb.Th) (mm) (H) were measured via μCT. After scanning, femurs were decalcified, processed, and embedded in paraffin for histologic sectioning and TRAP staining. (I) Histomorphometric analyses of trabecular bone in the distal femur proximal to the growth plate compared the number of osteoclasts per bone perimeter (N.Oc/B.pm) (1/mm) between mock infected and S. aureus infected femurs (n = 9 mice per group). Symbols represent individual data points from each mouse (Mock = circles; Infected = squares), the top line of each bar represents the mean, and error bars represent standard deviation. Unpaired t-tests were used to compare μCT and histomorphometry measurements between mock infected and S. aureus infected femurs. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

https://doi.org/10.1371/journal.ppat.1007744.g001

S. aureus osteomyelitis induces dramatic alterations in cortical bone surrounding the infectious focus, which was initiated in the middle of the femoral diaphysis. However, trabecular bone, located at the ends of the long bones, is the major site of homeostatic bone remodeling [41]. In order to elucidate how inflammation during osteomyelitis leads to alterations in trabecular bone architecture, we also performed micro-computed tomography (μCT) imaging on trabecular bone in the distal femur. To determine the amount of trabecular bone that was lost during osteomyelitis, we calculated the trabecular bone volume per total volume (BV/TV), which is a standard measure of bone volume and architecture [42]. S. aureus infected femurs exhibited a dramatic loss in trabecular bone, with BV/TV markedly decreased in infected femurs compared to mock infected femurs (Fig 1D and 1E). The observed decrease in BV/TV during infection is reflective of a decline in the number of bony trabeculae, which in turn increases the overall volume of space between trabeculae (Fig 1F and 1G). Trabecular thickness was not significantly reduced in infected relative to mock infected femurs (Fig 1H). Although skeletal histology revealed that the area of the femur encompassing the trabecular bone did not have apparent abscess formation (S1C Fig), viable S. aureus cells were recoverable from the femoral epiphyses encompassing the trabecular bone (S2 Fig). These data collectively reveal that S. aureus osteomyelitis induces changes in bone turnover throughout the entire infected femur, which is reflected in a significant loss of cortical and trabecular bone.

One common mechanism of bone loss is mediated by an increase in the number of bone-resorbing osteoclasts residing on the bone surface. To examine if the inflammation associated with S. aureus osteomyelitis enhances numbers of osteoclasts in vivo, we collected histologic sections of infected femurs for histomorphometry, which enables quantification of the number of osteoclasts and osteoclast resorbing surface relative to intact trabecular bone. Histomorphometric analysis showed an increased number of osteoclasts per bone perimeter (N.Oc/B.pm) in S. aureus infected femurs relative to mock infected femurs, suggesting that enhancement of osteoclastogenesis might be one mechanism underlying trabecular bone loss during osteomyelitis (Fig 1I). Taken together, these data indicate that S. aureus osteomyelitis perturbs normal bone homeostasis to induce pathologic bone remodeling in both cortical and trabecular bone.

Longitudinal cytokine profiling defines the local inflammatory milieu during S. aureus osteomyelitis

Previous studies have shown that toxin-deficient S. aureus strains retain the ability to alter bone remodeling, albeit to a lesser extent than WT S. aureus, implicating inflammation as a potential mediator of dysregulated bone remodeling during osteomyelitis [6, 7]. To characterize the local inflammatory environment during S. aureus osteomyelitis, we conducted longitudinal, multiplexed cytokine profiling of S. aureus and mock infected femurs over the course of 14 days. Relative to mock infected femurs, S. aureus infected femurs have more abundant levels of cardinal pro-inflammatory cytokines including IL-1α, IL-1β, IL-6, and TNFα (Fig 2). While both IL-1α and IL-1β are highly produced in infected femurs, IL-1β had a higher fold change than IL-1α throughout the timecourse when comparing S. aureus infected to mock infected femurs. Furthermore, infected femurs have increased levels of cytokines that support myeloid cell chemotaxis and expansion, including KC (CXCL1), G-CSF, M-CSF, MCP-1 (CCL2), MIP-1α (CCL3), MIP-1β (CCL4), and MIP-2 (CXCL2), compared to mock infected femurs (Fig 2). Cytokine profiling of S. aureus osteomyelitis demonstrated that inflammatory cytokines, chemokines, and growth factors are greatly increased in infected femurs by day 1 and throughout infection.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 2. Longitudinal cytokine profiling defines the local inflammatory milieu during S. aureus osteomyelitis.

Cytokine profiling was performed on homogenates from mock and S. aureus infected femurs from female WT mice. Groups of mock or S. aureus infected mice (n = 3 per time point) were sacrificed on days 1, 4, 7, 10, and 14 post-infection. Relative cytokine levels are represented as Log₂(S. aureus infected/mock infected) to reflect changes in cytokine production between sterile and infected bones. Dark blue shading represents increasing cytokine fold-changes observed in S. aureus infected femurs compared to mock infected femurs. Dark grey shading represents a decreasing cytokine fold-changes in S. aureus infected femurs compared to mock infected femurs.

https://doi.org/10.1371/journal.ppat.1007744.g002

The innate immune signaling adapter MyD88 and IL-1R signaling are critical for the control of bacterial burdens during S. aureus osteomyelitis

A rapid and robust cytokine response to S. aureus in bone led us to focus on identifying host signaling pathways that are responsible for coordinating an innate immune response. Given the central role for the signaling adapter MyD88 in pathogen recognition and induction of innate immune responses, we first sought to determine how MyD88 signaling influences staphylococcal burdens and host morbidity and mortality during osteomyelitis. Myd88^-/- mice have enhanced susceptibility to, and morbidity from, bacterial infection [27, 43, 44]. We therefore inoculated these mice with a range of S. aureus colony forming units (CFUs), from 10⁴−10⁶. Although bacterial inocula up to 10⁶ CFUs did not cause mortality in WT mice, Myd88^-/- mice were exquisitely susceptible to S. aureus osteomyelitis, with mortality observed even at inocula as low as 10⁴ CFUs (Fig 3A). For infected Myd88^-/- mice that met humane endpoints prior to the experimental endpoint or succumbing to disease, bacterial CFUs were enumerated at that time in the femur, liver, and kidneys. At the time of early sacrifice, Myd88^-/- mice had between 10⁷−10⁸ S. aureus CFUs in the femur, kidneys, and liver, which was significantly increased over CFUs recovered from WT mice (S3 Fig). At the experimental endpoint (day 14 post-infection), surviving Myd88^-/- mice not only had significantly elevated bacterial burdens in the infected femur, but also experienced more bacterial dissemination to the kidneys and liver (Fig 3B). Consequently, the inability to prevent systemic bacterial dissemination results in significantly increased mortality in Myd88^-/- mice. Taken together, these data demonstrate a critical role for MyD88-dependent immune responses during S. aureus osteomyelitis.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 3. The innate immune signaling adapter MyD88 is critical for the control of bacterial burdens and systemic dissemination during S. aureus osteomyelitis.

(A-B) Osteomyelitis was established in female WT and Myd88^-/- mice, following infection with varying S. aureus inocula: 10⁶ CFUs (WT n = 5, Myd88^-/- = 6), 10⁵ CFUs (WT n = 10, Myd88^-/- = 12; duplicate experiments), 10⁴ CFUs (WT n = 6, Myd88^-/- = 6). Bacterial burdens were quantified from the infected femur, as well as both kidneys and the liver as a measure of bacterial dissemination. (A) Over the 14-day course of infection, mice were monitored for humane endpoints, and if necessary, mice were euthanized, and mortality was recorded. Log-rank Mantel Cox test was used to compare WT and Myd88^-/- survival curves due to infection mortality for each S. aureus inoculum. ** p < 0.01, ns = not significant. (B) Bacterial burdens were enumerated in surviving mice at day 14 post-infection in duplicate experiments, following inoculation with 10⁵ S. aureus CFUs (WT n = 8, Myd88^-/- n = 6; duplicate experiments). Symbols represent individual data points from each mouse (WT = circles; Myd88^-/- = squares), the top line of each bar represents the mean, and error bars represent standard deviation. Unpaired t-tests were used to compare CFU burdens between WT and Myd88^-/- organ homogenates. * p < 0.05, ns = not significant.

https://doi.org/10.1371/journal.ppat.1007744.g003

Myd88^-/- mice have altered intestinal barrier function and are severely immunocompromised, and therefore may have significant microbiome differences relative to WT mice [45–47]. When considered in concert with recent studies suggesting that the microbiome may regulate bone mass [48–51], these observations prompted us to breed Myd88^-/- and Myd88^+/+ littermate controls from a heterozygous colony and compare these littermate controls for susceptibility to osteomyelitis. We also examined the influence of sex as a biologic variable in these experiments. In line with results from mice bred in separate colonies, significant mortality from osteomyelitis was observed in male Myd88^-/- mice, but not Myd88^+/+ littermate controls (S4A Fig). Of note, at day 14 post-infection, male Myd88^-/- mice had no difference from Myd88^+/+ littermate controls in recovered CFUs from infected femurs (S4B Fig). This observation could indicate sex-dependent differences in osteomyelitis pathogenesis, or alternatively may reflect selection bias from removal of mice that succumbed to infection (S4A Fig). In contrast to male mice, female Myd88^-/- mice exhibited significantly higher bacterial burdens in the infected femur when compared Myd88^+/+ littermate controls (S4C Fig). These experiments confirm that MyD88 is critical for the control of bacterial burdens and systemic dissemination during osteomyelitis independently of any confounding variables associated with separate colony maintenance.

In the absence of MyD88 signaling, mice are unable to control S. aureus infection, indicating that upstream receptors that signal through MyD88, including S. aureus-recognizing TLRs and IL-1R, may be important for antibacterial protection. The high levels of IL-1 and IL-1-regulated cytokines present in S. aureus infected femurs led us to investigate the contribution of IL-1R signaling to anti-staphylococcal immunity in bone. We subjected WT and Il1r1^-/- mice to S. aureus osteomyelitis. In contrast to the extreme systemic morbidity observed in MyD88^-/- mice suffering from osteomyelitis, Il1r1^-/- mice had less morbidity when compared to WT mice, in that they lost significantly less weight over the course of infection (Fig 4A). To determine the role of IL-1R and the relative contributions of IL-1 isoforms (IL-1α or IL-1β) to control bacterial burdens in bone, WT, Il1r1^-/-, Il1a^-/-, and Il1b^-/- mice were subjected to S. aureus osteomyelitis. Enumeration of bacterial burdens revealed that Il1r1^-/- mice harbored significantly higher bacterial burdens in infected femurs than WT, Il1a^-/-, and Il1b^-/- mice (Fig 4B). Il1a^-/- and Il1b^-/- mice sustained bacterial burdens that were not significantly different from WT mice (Fig 4B). Unlike MyD88^-/- mice, Il1r1^-/- mice were protected from significant systemic dissemination to the liver or kidneys (Fig 4C). To determine whether differences in WT and Il1r1^-/- strains were due to background genotype or separate colony maintenance, heterozygous Il1r1^+/- mice were bred to generate Il1r1^+/+ and Il1r1^-/- littermate controls. Infection of littermates with 10⁶ S. aureus CFUs confirmed that Il1r1^-/- mice sustained higher bacterial burdens in bone compared to Il1r1^+/+ mice (Fig 4D).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 4. IL-1R signaling contributes to antibacterial immunity in bone.

(A) Percent starting weight was monitored daily in triplicate experiments with 10⁶ S. aureus CFU infection of female WT and Il1r1^-/- mice (WT n = 15, Il1r1^-/- n = 13). (B-C) At day 14 post-infection with 10⁶ S. aureus CFU, bacterial burdens were enumerated from the infected femurs (WT n = 29, Il1r1^-/- n = 20, Il1a^-/- n = 9, Il1b^-/- n = 14; six independent experiments) (B) or kidneys (K) and liver (L) (WT n = 9, Il1r1^-/- n = 7; duplicate experiments) (C) of each mouse. (D) A subsequent experiment with a 10⁶ S. aureus CFU inoculum was conducted in Il1r1^+/+ and ll1r1^-/- mice bred as littermates from a heterozygous colony (Il1r1^+/+ n = 10, ll1r1^-/- n = 9; duplicate experiments). (E) Following a lower 10⁵ S. aureus CFU inoculum, bacterial burdens were enumerated from infected femurs of WT and Il1r1^-/- mice on days 1, 3, 5, 10, and 14 post-infection (n = 3 mice per genotype per time point). On bar graphs, symbols represent individual data points from a single mouse (WT and Il1r1^+/+ = circles; Il1r1^-/- = squares; Il1a^-/- = triangles, Il1b^-/- = diamonds) and the mean is represented by the top line of the bar. On timecourse graphs, symbols represent the mean. On all graphs, error bars represent the standard deviation. Unpaired t-tests compared weight recovery and CFU burdens between WT and Il1r1^-/- mice and between Il1r1^+/+ and Il1r1^-/- femurs. A one-way ANOVA with Tukey’s multiple comparisons test was used to compare bacterial burdens harvested from femurs between mice of all genotypes. For kidney and liver burdens, separate t-tests compared WT and Il1r1^-/- burdens from each organ site. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not significant.

https://doi.org/10.1371/journal.ppat.1007744.g004

We next investigated the kinetics of bacterial clearance between WT and Il1r1^-/- mice. For this analysis we chose a lower S. aureus inoculum of 10⁵ CFUs in an attempt to equilibrate bacterial burdens at day 14 post-infection. In both WT and Il1r1^-/- mice, the initial S. aureus inoculum of 10⁵ CFUs replicates to approximately 10⁷ CFUs by day 1 post-infection (Fig 4E). In WT mice, bacterial burdens decreased by greater than 1 log between days 3 and 5 post-infection. In contrast, bacterial burdens in Il1r1^-/- mice were essentially unchanged through day 5 post-infection, and only declined between days 5 and 10 post-infection. Accordingly, WT and Il1r1^-/- mice had significantly different bacterial burdens at day 5 post-infection with this lower inoculum, even though bacterial burdens were roughly equivalent at the final time point (day 14). These data reveal differences in infection kinetics between WT and Il1r1^-/- mice, and suggest that Il1r1^-/- mice might have a delay in bacterial control during osteomyelitis.

In other S. aureus infection models, IL-1 coordinates neutrophil recruitment and is necessary for sequestration of S. aureus into mature abscesses [27, 30]. We therefore hypothesized that the delay in bacterial clearance in Il1r1^-/- mice subjected to osteomyelitis was related to differences in abscess maturation and neutrophil abundance. To visualize immune cell infiltration and abscess structure, we conducted myeloperoxidase (MPO) staining on histologic sections of infected femurs at day 14 post-infection. Il1r1^-/- mice with osteomyelitis have differential MPO staining in comparison to WT controls, suggesting these mice have disorganized abscess structure (Fig 5A). WT mice have MPO⁺ cells that surround and encompass the abscess, whereas Il1r1^-/- mice show extensive MPO⁺ staining throughout the femur. To assess changes in inflammatory signatures that correspond to differences in infection kinetics, infected femur homogenates from WT and Il1r1^-/- mice were analyzed using multiplexed cytokine analysis. In comparison to WT mice, Il1r1^-/- mice had significantly decreased abundance of neutrophil growth factors G-CSF and GM-CSF and lower levels of the neutrophil chemokine CXCL1 at day 1 post-infection, a timepoint that precedes early bacterial control in WT mice between days 3 and 5 (Figs 5B–5D and 4E). GM-CSF and CXCL1 levels then decline in WT mice by day 5 post-infection. In contrast, Il1r1^-/- mice display significantly higher levels of GM-CSF and CXCL1 at day 5 post-infection when compared to WT mice, prior to the decrease in bacterial burdens that occurs between days 5 and 10 post-infection (Figs 5C, 5D and 4E). Moreover, there are global changes in cytokine abundance when comparing WT and Il1r1^-/- mice (S1 Table). These data suggest that WT mice have an early influx and/or expansion of neutrophils, important for the control of bacterial burdens and normal abscess formation.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 5. Il1r1^-/- mice have altered abscess structure, delayed granulocytic cytokine levels, and lower neutrophil abundance during S. aureus osteomyelitis.

(A) Femurs were harvested from female WT and Il1r1^-/- mice (n = 5 per genotype) 14 days after S. aureus infection (10⁶ CFUs). Harvested femurs were fixed in neutral buffered formalin, dehydrated in 70% ethanol, and decalcified in 20% EDTA before embedding in paraffin for sectioning. The infected femurs were sectioned through the medullary cavity and sections from each femur were stained for myeloperoxidase (MPO) to visualize MPO⁺ (brown) neutrophils and gross abscess architecture in WT (left) and Il1r1^-/- (right) sections. Slides were scanned at 20X using a Leica SCN400 Slide Scanner and images were taken at 4X (scale bars = 100 μm) and are representative of n = 5 mice per genotype. Abscesses are indicated by white arrows and a S. aureus microcolony by a grey arrow. (B-D) After infection with 10⁵ S. aureus CFUs, cytokine levels and relative neutrophil abundance were measured from femurs (n = 3 per genotype) harvested at days 1, 3, 5, 10, and 14 post-infection (cytokine analysis, B-D) or days 1, 3, 5, and 14 post-infection (flow cytometry, E). Cytokine levels in pg/mL were corrected for overall protein levels in the femur homogenates as measured using a BCA assay. Depicted are select cytokines G-CSF (B), GM-CSF (C), and CXCL1 (D) from WT (black) and Il1r1^-/- (grey) femurs. (E) Relative neutrophil abundance was measured in bone marrow harvested from infected and contralateral, uninfected femurs of WT and Il1r1^-/- mice. Neutrophils are defined as CD45⁺CD11b⁺Ly6G⁺Ly6C^lo and are reported as % of CD45⁺ immune cells (D). Symbols represent individual data points from each mouse (WT = circles; Il1r1^-/- = squares), horizontal lines or the top of each bar represent the mean, and error bars represent standard deviation. Multiple unpaired t-tests were used to compared pg cytokine/mg protein and neutrophil percentages between WT and Il1r1^-/- mice. * p < 0.05, ** p < 0.01, **** p < 0.0001. If not denoted with asterisks, statistical difference between genotypes was not significant.

https://doi.org/10.1371/journal.ppat.1007744.g005

To monitor neutrophil abundance during the course of osteomyelitis, bone marrow from infected and contralateral, uninfected WT and Il1r1^-/- femurs at various time points after S. aureus infection was analyzed via flow cytometry (S5A–S5F Fig). Neutrophils were identified as CD45⁺CD11b⁺Ly6G⁺Ly6C^lo and reported as the percent of CD45⁺ immune cells. At day 1 post-infection, WT and Il1r1^-/- mice were found to have neutrophils comprising less than 10% of CD45⁺ cells in the bone marrow. By day 3 post-infection, Il1r1^-/- mice have significantly fewer neutrophils in the infected bone marrow compared to WT mice (Fig 5E). Furthermore, differences between relative neutrophil abundance were also observed between WT and Il1r1^-/- mice in the contralateral, uninfected femurs. Neutrophil abundance at day 5 post-infection is comparable between WT and Il1r1^-/- genotypes, but again was significantly decreased in Il1r1^-/- femurs at day 14 post-infection. Therefore, the data suggest that Il1r1^-/- mice with S. aureus osteomyelitis have altered neutrophil responses, indicated by the significant decrease in relative neutrophil abundance at two timepoints post-infection.

S. aureus promotes osteoclastogenesis and pathologic bone loss through IL-1R signaling

WT mice subjected to S. aureus osteomyelitis display significant cortical bone destruction and reactive bone formation at the site of infection, while sustaining alterations in osteoclast number and trabecular bone loss in the distal femur (Fig 1A–1I) [6]. Given the important role of IL-1R signaling in skeletal cell differentiation and function in vitro, we hypothesized that pathologic bone remodeling during S. aureus osteomyelitis is mediated, in part, by IL-1R signaling. To test this hypothesis, we first compared changes in cortical bone remodeling between WT and Il1r1^-/- mice using a lower dose (10⁵ CFUs) S. aureus infection. At the site of infection, Il1r1^-/- mice sustained increased cortical bone loss in comparison to WT mice (Fig 6A and 6B). In areas adjacent to the cortical bone loss, Il1r1^-/- mice had a dramatic increase in new bone formation. In fact, the volume of new bone formation in Il1r1^-/- mice was nearly twice the volume formed in infected WT femurs (Fig 6C). Importantly, these data do not completely control for differences in bacterial burdens at the site of infection, where Il1r1^-/- mice harbor higher bacterial burdens at day 5 post-infection with a lower inocula (Fig 4E). However, Il1r1^-/- mice do equilibrate bacterial burdens to the same level at WT mice by day 14 post-infection (Fig 6D). Consistent with an increase in reactive bone formation, histologic analyses revealed increased callus formation in infected Il1r1^-/- femurs and also demonstrated qualitative differences in callus composition compared to infected WT femurs (Fig 6E–6G). Additionally, the dramatic cortical bone alterations observed in Il1r1^-/- mice were confirmed in littermate controls (S6A and S6B Fig). In contrast to cortical bone remodeling changes during S. aureus infection, μCT analysis revealed no differences in cortical bone remodeling of a sterile bone defect at day 14 post-surgery, where mock infected WT and Il1r1^-/- femurs had no significant differences in cortical bone loss or reactive bone formation (Fig 6H–6J). Collectively, these data indicate that during S. aureus osteomyelitis, Il1r1^-/- mice exhibit significantly altered cortical bone remodeling, with increased reactive bone formation, altered callus architecture, and greater cortical bone loss at the site of infection.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 6. Loss of the IL-1R enhances cortical bone loss and reactive bone formation during S. aureus osteomyelitis.

(A-J) To determine changes in bone remodeling and final bacterial burdens, female mice were infected with S. aureus (10⁵ CFUs: WT n = 10, Il1r1^-/- n = 9; duplicate experiments for bone remodeling; WT n = 9, Il1r1^-/- n = 9; duplicate experiments for CFU enumeration) or mock infected (WT n = 5, Il1r1^-/- n = 5 for bone remodeling) with PBS. Femurs were harvested at day 14 post-infection and scanned using the μCT50. Representative anteroposterior views of S. aureus infected femurs (A) and mock infected femurs (H). μCT three-dimensional analysis of cortical bone loss (mm³) and reactive bone formation (mm³) between S. aureus infected (B, C) and mock infected (I, J) WT and Il1r1^-/- femurs. Bacterial burdens were quantified at day 14 post-infection from WT and Il1r1^-/- femurs (D). (E, F) After μCT analyses, histologic sections of the S. aureus infected femurs were prepared and TRAP-stained. Slides were scanned using a Leica SCN400 Slide Scanner, with representative images shown taken at 0.58X (scale bars = 1 mm) (E) with the region in the black box imaged at 4X (scale bars = 100 μm) (F), with dashed black line demarcating the separation of intact cortical bone (left of dashed line) and the callus (right of dashed line). (G) Tissue IA 2.0 software was used to image callus area of infected femurs at 20X (10⁵ CFUs; n = 5 per genotype). Symbols represent individual data points from each mouse (WT = circles; Il1r1^-/- = squares), the top line of each bar represents the mean, and error bars represent standard deviation. Unpaired t-tests were used to compare CFU burdens, μCT analyses, and Tissue IA 2.0 measurements between WT and Il1r1^-/- mice. ** p < 0.01, **** p < 0.0001.

https://doi.org/10.1371/journal.ppat.1007744.g006

To elucidate the cellular changes driving differences in bone remodeling between WT and Il1r1^-/- mice, we next analyzed trabecular bone remodeling during osteomyelitis. Histomorphometric analysis of trabecular bone was performed in both S. aureus infected femurs and contralateral, uninfected femurs from each genotype. Histomorphometry revealed that the infected femurs from WT mice had significantly lower trabecular BV/TV than contralateral, uninfected femurs (Fig 7A). In contrast, infected femurs from Il1r1^-/- mice showed no significant differences in BV/TV in comparison to the contralateral, uninfected femur, suggesting that these mice were protected from infection-associated trabecular bone loss despite having significantly higher bacterial burdens in the regions encompassing the trabecular bone over time (Fig 7A; S2 Fig). To determine if differences in osteoclast biology might underlie the distinct trabecular bone remodeling parameters of WT and Il1r1^-/- mice, we calculated the numbers of osteoclasts present on trabecular bone surfaces in both infected and contralateral, uninfected femurs. The infected femurs in WT mice displayed greater osteoclast numbers per bone perimeter (N.Oc/B.pm) and osteoclast surface per bone surface (Oc.S/BS) compared to the contralateral, uninfected femurs, correlating with the infection-induced loss of trabecular bone volume (Fig 7A–7C). In contrast, the infected femurs from Il1r1^-/- mice showed no increase in N.Oc/B.pm or Oc.S/BS when compared to the contralateral, uninfected femur (Fig 7B and 7C). These data suggest that S. aureus infection causes enhanced osteoclastogenesis in trabecular bone, which is dependent on intact IL-1R signaling and contributes to bone loss.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 7. IL-1R contributes to infection-induced osteoclastogenesis during S. aureus osteomyelitis.

(A-C) Following S. aureus infection (10⁵ CFUs), female WT and Il1r1^-/- infected femurs (n = 5 per genotype) and contralateral femurs from infected mice (n = 4 per genotype) were harvested, decalcified, processed for sectioning in paraffin, and TRAP-stained for histomorphometric analyses using OsteoMeasure software. Histomorphometric analyses of S. aureus infected femurs and contralateral femurs from infected mice were quantified to calculate the trabecular bone volume/total volume (BV/TV) (%) (A), the number of osteoclasts per bone perimeter (N.Oc/B.pm) (1/mm) (B), and the osteoclast surface per bone surface (Oc.S/BS) (%) (C) in the distal femur. Symbols represent individual data points from each mouse (WT = circles; Il1r1^-/- = squares), the top line of each bar represents the mean, and error bars represent standard deviation. All statistical comparisons used a two-way ANOVA and Fisher’s Least Significant Difference (LSD) test to compare differences in trabecular bone composition of infected and contralateral, uninfected femurs of WT and Il1r1^-/- mice. * p < 0.05, ** p < 0.01, ns = not significant.

https://doi.org/10.1371/journal.ppat.1007744.g007

Histomorphometric analysis revealed that S. aureus infection enhances osteoclastogenesis and trabecular bone loss in an IL-1R-dependent manner. However, bone volume and remodeling are also significantly impacted by osteoblast function. To determine the contribution of osteoblasts toward altered bone homeostasis and trabecular bone loss during staphylococcal osteomyelitis, we measured bone mineralization in the trabecular bone of infected WT and Il1r1^-/- mice. No differences were observed in mineralizing surface, bone formation rate, or mineral apposition rate between WT and Il1r1^-/- mice (S7A–S7C Fig). These data indicate that IL-1R signaling does not drive differences in trabecular osteoblastic function during S. aureus infection, and that the decrease in trabecular BV/TV is not a function of decreased osteoblastic bone formation.

S. aureus triggers osteoclastogenesis of RANKL-primed myeloid cells through MyD88 and IL-1R signaling

Staphylococcal infection causes bone loss and enhanced osteoclastogenesis in trabecular bone. Accordingly, we hypothesized that secreted bacterial factors might augment osteoclast differentiation. To test this hypothesis, we measured osteoclast differentiation of RANKL-primed myeloid progenitors after stimulation with S. aureus culture supernatant or a vehicle control. To avoid induction of cell death in myeloid cells, we used a S. aureus strain lacking the alpha-type PSMs, which we previously demonstrated are both necessary and sufficient for causing cell death when staphylococcal supernatants are applied to murine bone marrow-derived macrophages (BMMs) [6, 7]. Stimulation of RANKL-primed BMMs with toxin-deficient supernatant resulted in a dramatic increase in mature osteoclasts, as identified as tartrate resistant acid phosphatase positive (TRAP⁺) multinucleated cells, relative to vehicle control in WT cells (Fig 8A). To determine if this bacterial enhancement of osteoclastogenesis was dependent on IL-1R signaling, we performed similar experiments with Myd88^-/- and Il1r1^-/- BMMs. We discovered that Myd88^-/- and Il1r1^-/- cells do not undergo robust S. aureus-mediated osteoclastogenesis, despite being able to differentiate into osteoclasts via canonical RANKL stimulation (Fig 8A, S8A–S8D Fig). Moreover, toxin-deficient S. aureus supernatants caused significantly less osteoclast formation in Myd88^-/- and Il1r1^-/- cells in comparison to WT cells (Fig 8A). We next tested the role of IL-1 blockade on osteoclastogenesis using WT or Il1r1^-/- cells, with or without IL-1R antagonist (IL-1ra) treatment. RANKL pre-commitment of WT cells with simultaneous IL-1ra treatment blunted the generation of osteoclast precursors, leading to 50% fewer S. aureus-stimulated osteoclasts (Fig 8B). This decline in S. aureus-enhanced osteoclastogenesis results in differentiation to a similar level as is observed in Il1r1^-/- osteoclast precursors (Fig 8B). These observations indicate that MyD88 and the IL-1R are required for S. aureus-mediated enhancement of osteoclastogenesis. Therefore, although MyD88 and IL-1R are critical mediators of the anti-staphylococcal immune response, S. aureus infection also elicits osteoclast-mediated bone loss through MyD88 and IL-1R signaling pathways.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 8. MyD88 and IL-1R signaling drive S. aureus enhancement of osteoclast differentiation in vitro.

(A) Osteoclast precursors were generated from primary WT, Myd88^-/-, and Il1r1^-/- BMMs by treatment with 35 ng/mL RANKL treatment for 48 hours. After washing 2X with PBS, fresh media was replenished, and cells were stimulated with vehicle (1% casamino acid-supplemented RPMI) or toxin-deficient S. aureus supernatant (Δpsm) in the absence of ongoing RANKL treatment. All cells were TRAP stained in vitro at day 6 and TRAP⁺ multinucleated cells were manually quantified using OsteoMeasure software (n = 3 wells per condition, representative of duplicate experiments). (B) WT and Il1r1^-/- enriched BMMs were stimulated with 35 ng/mL exogenous RANKL for 2 days before stimulating with Δpsm supernatants for four days. In order to determine the contribution of IL-1 signaling in this assay, vehicle (0.1% low endotoxin BSA) or recombinant murine IL-1ra (1 μg/mL) were added during the 2 days of RANKL pre-commitment (BSA, black bars; IL-1ra, grey bars) or during the 4 days of Δpsm supernatant stimulation (indicated by + under each bar). The top line of each bar represents the mean and error bars represent standard deviation. A repeated measures two-way ANOVA with Dunnett’s multiple comparisons test was performed on cell counts from each genotype, comparing each dose of Δpsm supernatant to the vehicle control, where * p < 0.05, *** p < 0.001, **** p < 0.0001, and if not designated by asterisks is non-significant. A repeated measures two-way ANOVA with Tukey’s multiple comparisons test was performed to compare Myd88^-/- and Il1r1^-/- TRAP⁺ multinucleated cell counts to WT counts within each Δpsm supernatant dose, where # p < 0.05, † p < 0.001, and ‡ p < 0.0001. A three-way ANOVA with Tukey’s multiple comparisons test compared the effects of genotype, IL-1ra treatment during RANKL pre-commitment, and IL-1ra treatment during Δpsm supernatant stimulation. Statistical comparisons to WT cells pre-committed with RANKL + BSA and Δpsm supernatant + BSA (first black bar from the left) are reported as @ p < 0.05, or compared to WT cells pre-committed with RANKL + BSA and Δpsm supernatant + IL-1ra (second black bar from the left) are reported as § p < 0.05. Comparisons between all RANKL + IL-1ra pre-commitment conditions (grey bars) were non-significant.

https://doi.org/10.1371/journal.ppat.1007744.g008

Ethics statement

All experiments involving animals were reviewed and approved by the Institutional Animal Care and Use Committee at Vanderbilt University Medical Center on the animal protocols M12059 and M1800055. All experiments were performed according to NIH guidelines, the Animal Welfare Act, and US Federal law. The murine model of osteomyelitis required inhalational anesthesia with isoflurane (1–5%). Post-operative analgesia (buprenorphine 0.5–0.1 mg/kg) was provided pre-operatively and every 8–12 hours for 48 hours post-infection. Mice were euthanized by CO₂ asphyxiation with secondary confirmation by cervical dislocation and observation of heart rate and breathing.

Animal use

C57BL/6J (Stock #: 000664), Myd88^-/- (Stock #: 009088) and Il1r1^-/- (Stock #: 003245) mice were purchased through The Jackson Laboratory. Il1a^-/- and Il1b^-/- mice were generated as described [85]. WT mice were bred with Myd88^-/- or Il1r1^-/- mice to produce Myd88^+/- or Il1r1^+/- mice, respectively. Heterozygous mice were bred together to create mice carrying knockout (-/-), heterozygous (+/-), or wild-type (+/+) alleles for Myd88 or Il1r1. Mice were bred heterozygously to reduce the confounding influence of microbiome effects associated with genotypes and maintenance of separate mouse colonies. The resulting littermates were earpunched and genotyped through Transnetyx, Inc. (Cordova, TN).

Bacterial strain and growth conditions

All infections were conducted with an erythromycin-sensitive derivative of the USA300 type S. aureus LAC clinical isolate (AH1263) [86]. The toxin-deficient strain LACΔpsmα1–4 (Δpsm) has been previously described and was used for in vitro assays to prevent cell death [6, 87]. Bacteria were routinely grown on tryptic soy agar (TSA) or shaking in tryptic soy broth (TSB) with or without 10 μg/mL erythromycin as detailed previously [6]. To prepare concentrated supernatants, Δpsm was grown overnight in RPMI supplemented with 1% casamino acids [7].

Post-traumatic osteomyelitis infection

The murine model of osteomyelitis was performed as described previously [6, 7]. AH1263 was sub-cultured from an overnight culture, grown for 3 hours, and then adjusted in PBS to a concentration of approximately 1x10⁶ CFUs in 2 μl PBS, unless diluted 1:10 or 1:100 to deliver inoculum doses of 1x10⁵ or 1x10⁴ CFUs, respectively. Osteomyelitis was induced in 5- to 8-week old male and female mice following the introduction of a unicortical bone defect using a 21G needle, into which 2 μl of bacterial suspension or PBS (mock infection) was injected into the intermedullary canal. Muscle fascia and skin were closed with sutures and mice were given buprenorphine analgesic every 12 hours for 48 hours, with daily monitoring until the experimental end point. Mice were euthanized if they met human endpoints, including inability to ambulate, inability to eat or drink, loss of greater than 20% body weight, and/or hunched posture.

Micro-computed tomography (μCT) of cortical and trabecular bone

Femurs were harvested 14 days post-infection and fixed for 48 hours in neutral buffered formalin at 4°C. Bones were scanned using a μCT50 (Scanco Medical, Switzerland) and analyzed with μCT Tomography V6.3–4 software (Scanco USA, Inc., Wayne, PA). To expand previous μCT50 analyses that assessed only the cortical bone of the femoral diaphysis [6], here the diaphysis and distal epiphysis of each femur were visualized in the scout-view radiographs and imaged with 10.0 μm voxel size at 70 kV, 200 μA, and an integration time of 350 ms in a 10.24 mm view. Each imaging scan resulted in 1088 slices (10.88 mm) of the femur that included the diaphysis surrounding the inoculation site, trabecular bone in the distal femur, and excluded the proximal epiphysis. Three-dimensional volumetric analyses were conducted by contouring transverse image slices in the region of interest. The diaphysis of each femur was comprised of 818 image slices. These image slices were used to quantify cortical bone destruction (mm³) and reactive bone formation (mm³) surrounding the cortical bone inoculation site as described previously [6]. Trabecular bone measurements were obtained in the distal femur by advancing proximally past the growth plate 30 slices. 101 slices were analyzed with an inclusive contour drawn along the endosteal surface to include trabeculae and exclude the cortical bone. Trabecular bone volume per total volume (%), trabecular number (1/mm), trabecular thickness (mm), and trabecular spacing (mm) were determined by segmentation of the image with a lower threshold of 329 mg HA/ccm, sigma 1.3, and support 1.

Bone histology and histomorphometric analysis of osteoclasts in trabecular bone

After μCT imaging, femurs were decalcified for three days in 20% EDTA at 4°C. Decalcified bones were processed and embedded in paraffin before sectioning at 4μm thickness through the infectious nidus and bone marrow cavity using a Leica RM2255 microtome. Sectioned femurs were stained with a modified hematoxylin and eosin (H&E) that included orange G and phloxine for enhanced bone contrast, tartrate-resistant acid phosphatase (TRAP) stain with hematoxylin counterstain, or 3,3’-diaminobenzidine (DAB) immunohistochemistry to detect myeloperoxidase (MPO). OsteoMeasure software (OsteoMetrics, Inc., Decatur, GA) was used to manually analyze TRAP-stained histologic sections at a region of interest encompassing the trabeculae proximal to the growth plate in the distal femur. Osteoclast number, osteoclast surface, and bone perimeter were calculated and reported per ASBMR standards [42]. A Leica SCN400 Slide Scanner was used to scan stained femur sections in brightfield at 20X. Images were uploaded to and imaged with the Digital Imaging Hub (Leica Biosystems, Buffalo Grove, IL) and Tissue Image Analysis 2.0 (Tissue IA 2.0) (Leica Microsystems, Buffalo Grove, IL) was used to analyze callus area of infected femurs at 20X.

Determination of bone formation rate with double calcein labelling

WT and Il1r1^-/- mice were intraperitoneally injected with 20 mg/kg calcein on days 8 and 12 post-infection with 10⁵ CFUs. Femurs were subsequently harvested, formalin fixed, and dehydrated prior to embedding in poly(methyl methacrylate) for sectioning, and counterstained with toluidine blue. Fluorescent labels were identified as single- or double-labeled surface. Fluorescent labels and trabecular bone were traced in the distal femur using OsteoMeasure software, and the mineralizing surface per bone surface (MS/BS), mineral apposition rate (MAR), and bone formation rate per bone surface (BFR/BS) were calculated per ASBMR standards [42].

CFU enumeration

At various time points post-infection, tissues were harvested and homogenized using a BulletBlender and NAVY lysis tubes (Next Advance, Inc., Averill Park, NY) at 4°C. To enumerate bacterial CFUs in infected femurs, the whole femur or only the regions encompassing the trabecular bone (i.e. metaphyses and epiphyses) were homogenized in PBS. To maximize cytokine signals in femur homogenates, CelLytic Buffer MT Cell Lysis Reagent (Sigma, Saint Louis, MO) was substituted for PBS to specifically lyse mammalian cells. Livers and kidneys were homogenized in PBS. Femur and organ homogenates were vortexed, serially diluted in PBS, and plated on TSA for bacterial enumeration. Femur homogenates lysed in CelLytic Buffer MT and PBS showed no difference in recoverable bacterial burdens.

Multiplexed cytokine detection

Following homogenization, femur homogenates were centrifuged at 4000 x g for 5 minutes to remove debris and the supernatant was stored at -80°C for subsequent analysis using Milliplex MAP multiplex magnetic bead-based antibody detection kits (EMD Millipore, Billerica, MA) according to the manufacturer’s protocols. Cytokine quantification from bone homogenates was performed using the 32-plex Mouse Cytokine/Chemokine Magnetic Bead Panel (MCYTMAG-70K-PX32) on the FLEXMAP 3D instrument. The quality controls for IL-13 failed, and these data were excluded.

Cytokine levels from femurs homogenized in 500 μl volume were read as pg/mL. Femur homogenates reported as relative values were homogenized in PBS, whereas cytokines values corrected for total protein were homogenized in CelLytic Buffer MT to maximize cytokine signals. Total protein (mg/mL) was quantified using the Pierce BCA Protein Assay Kit (ThermoFisher Scientific, Waltham, MA) per manufacturer’s directions. Infected Il1r1^-/- femurs were up to two times larger than WT infected femurs and four times larger than mock infected WT and Il1r1^-/- femurs. Cytokine levels are therefore reported as pg cytokine/mg protein to control for femur size differences between infected Il1r1^-/- and WT femurs.

Flow cytometry

Following S. aureus infection (10⁵ CFUs), femurs from WT and Il1r1^-/- mice were harvested at 1, 3, 5, and 14 days post-infection. Whole bone marrow (WBM) was flushed through a 70 μm nylon cell strainer (Falcon, Corning, New York) and red blood cells (RBCs) were lysed using the Ammonium Chloride Potassium (ACK) Lysing Buffer (Lonza, Walkersville, MD). Bone marrow (BM) mononuclear cells were counted and 1 million cells were plated per well and washed in PBS supplemented with 3% FBS and 0.1% sodium azide (FACS buffer). Cells were incubated with Anti-CD16/CD32 (Biolegend, 1:100, clone 93, San Diego, CA) to block non-specific antibody staining. Cells were then incubated with a mixture of murine-specific cell surface antibodies on ice, including Anti-Ly6G-PE (Biolegend, 1:3200, clone 1A8), Anti-Ly6C-PE-Dazzle 594 (Biolegend, 1:1600, clone HK1.4), Anti-CD68-PE-Cy7 (Biolegend, 1:100, clone FA-11), Anti-CD11b-APC (Tonbo 1:4800, clone M1/70, San Diego, CA), and Anti-CD45-AlexaFluor 700 (Biolegend, 1:400, clone 30-F11). Cells were washed two times in FACS buffer, resuspended in 2% paraformaldehyde solution, and run on a 3-laser BD LSRII flow cytometer the following day. Single BM cells were identified from successive gates, including side scatter-area by forward scatter-area (SSC-A x FSC-A), forward scatter area by height (FSC-A x FSC-H), and side scatter area by height (SSC-A x SSC-H). Next, CD45⁺ cells, CD11b⁺ cells, and Ly6G⁺LyC^lo cells (neutrophils) were gated sequentially.

Osteoclastogenesis assays

WBM was flushed from femurs of 8- to 13-week old male mice using α-MEM media. Following RBC lysis, WBM was resuspended in a 90% FBS and 10% DMSO solution and frozen in liquid nitrogen until thawed for use. BMMs were enriched by plating 8 to 13 million cells per 10 cm dish in α-MEM, 10% FBS, 1X Penicillin/Streptomycin (P/S), and 100 ng/mL recombinant murine M-CSF (R&D Systems, Minneapolis, MN, 416-ML) for 4 days. Non-adherent cells were removed, and adherent cells were washed with PBS, scraped into fresh media, and counted prior to plating. Enriched BMMs were plated at a density of 50,000 cells/well in 96-well plates, and media (α-MEM, 10% FBS, 1X P/S) was supplemented 1:20 with CMG14-12 as an M-CSF source [88]. Osteoclastogenesis assays were performed with RANKL-primed osteoclast precursors, which were generated by plating BMMs in 35 ng/mL recombinant murine RANKL (R&D Systems, Minneapolis, MN, 462-TR) for 2 days. Prior to stimulation, RANKL-primed osteoclast precursors were washed twice with PBS. RANKL-primed osteoclast precursors were stimulated with either a vehicle control (1% casamino acid-supplemented RPMI) or Δpsm supernatant. M-CSF was supplemented into the media containing each stimulation. To test the specific role of IL-1R inhibition on S. aureus-enhanced osteoclast differentiation, osteoclastogenesis assays in WT and Il1r1^-/- cells were conducted with the addition of a vehicle control (0.1% low endotoxin BSA) or 1 μg/mL recombinant murine IL-1ra (Novus Biologicals, LLC, Littleton, CO, NBP2-35105) during the 2 days of RANKL pre-commitment or during the 4 days of Δpsm supernatant stimulation. On day 6 in culture, all RPMI- and S. aureus-stimulated osteoclastogenesis assays were fixed with a 4% formaldehyde and 0.05% Triton X-100 solution in PBS (10 minutes) and 1:1 acetone:ethanol (1 minute), before TRAP staining with reagents from the Acid Phosphatase, Leukocyte (TRAP) Kit (Sigma, Saint Louis, MO, 378A). In control osteoclastogenesis assays without S. aureus supernatant stimulation, cells were stimulated at the time of plating with 1:20 CMG14-12 and 35ng/mL RANKL. Fresh media, CMG14-12, and RANKL were replenished on days 4 and 6 in culture, with cells TRAP stained on day 7. OsteoMeasure was used to manually quantify mature osteoclasts, identified as TRAP⁺ multinucleated cells.

Statistical analysis

Data analysis and statistical tests were conducted using Graph Pad Prism software. Unpaired t-tests were used to compare CFU burdens, measurements of bone architecture using μCT and histology, cytokine levels, neutrophil abundance, and TRAP⁺ multinucleated cell counts when two groups were being compared. Log-rank Mantel Cox tests compared survival curves between genotypes for each S. aureus inoculum. A one-way ANOVA with Tukey’s multiple comparison test compared CFU burdens from femurs between multiple genotypes. A two-way ANOVA was used with Fisher’s Least Significant Difference (LSD) test to compare the effects of genotype and infection status between histomorphometry measurements. A repeated measures two-way ANOVA with Tukey’s multiple comparisons test was used to compare TRAP⁺ multinucleated cell counts between genotype at each Δpsm supernatant dose. Repeated measures two-way ANOVAs with Dunnett’s multiple comparisons test were conducted on TRAP⁺ cell counts from each genotype, to compare Δpsm supernatant dosage effects. A three-way ANOVA with Tukey’s multiple comparisons test was used to compare cell genotype, IL-1ra pre-treatment, and IL-1ra treatment alongside Δpsm supernatant stimulation. P values of less than 0.05 were considered statistically significant. Details on number of data points, experimental replicates, calculated standard deviation, and statistical significance for each experiment are described in figure legends.