Cellular Microbiology: Pathogen-Host Cell Molecular Interactions

Interaction of Brucella abortus with Osteoclasts: a Step toward Understanding Osteoarticular Brucellosis and Vaccine Safety

Omar H. Khalaf, Sankar P. Chaki, Daniel G. Garcia-Gonzalez, Larry J. Suva, Dana Gaddy, Angela M. Arenas-Gamboa
Craig R. Roy, Editor
DOI: 10.1128/IAI.00822-19

ABSTRACT

Osteoarticular disease is a frequent complication of human brucellosis. Vaccination remains a critical component of brucellosis control, but there are currently no vaccines for use in humans, and no in vitro models for assessing the safety of candidate vaccines in reference to the development of bone lesions currently exist. While the effect of Brucella infection on osteoblasts has been extensively evaluated, little is known about the consequences of osteoclast infection. Murine bone marrow-derived macrophages were derived into mature osteoclasts and infected with B. abortus 2308, the vaccine strain S19, and attenuated mutants S19vjbR and B. abortus ΔvirB2. While B. abortus 2308 and S19 replicated inside mature osteoclasts, the attenuated mutants were progressively killed, behavior that mimics infection kinetics in macrophages. Interestingly, B. abortus 2308 impaired the growth of osteoclasts without reducing resorptive activity, while osteoclasts infected with B. abortus S19 and S19vjbR were significantly larger and exhibited enhanced resorption. None of the Brucella strains induced apoptosis or stimulated nitric oxide or lactose dehydrogenase production in mature osteoclasts. Finally, infection of macrophages or osteoclast precursors with B. abortus 2308 resulted in generation of smaller osteoclasts with decreased resorptive activity. Overall, Brucella exhibits similar growth characteristics in mature osteoclasts compared to the primary target cell, the macrophage, but is able to impair the maturation and alter the resorptive capacity of these cells. These results suggest that osteoclasts play an important role in osteoarticular brucellosis and could serve as a useful in vitro model for both analyzing host-pathogen interactions and assessing vaccine safety.

INTRODUCTION

Brucellosis is a zoonotic disease caused by a Gram-negative intracellular bacterium of the genus Brucella. Brucella abortus, Brucella melitensis, and Brucella suis are the most pathogenic species to humans, with more than 500,000 new cases reported annually (1). Unfortunately, there is no available vaccine for use in humans, owing in large part to the safety concerns associated with potential residual virulence of live-attenuated vaccines (LAVs). In order for safe vaccines to be designed, a thorough understanding of the host-pathogen interactions resulting in the most common complications in human infection is required (2–4). Human brucellosis is frequently associated with the development of osteoarticular disease, with an incidence ranging from 40% to 80% (5–7). Humans, regardless of age or sex, are susceptible to infection, and the disease can manifest in both acute and chronic forms as peripheral arthritis, sacroiliitis, or spondylitis (6, 8). Importantly, osteoarticular lesions are also reported in natural hosts, as infected dogs commonly develop diskospondylitis, while cattle can exhibit arthritis and hygromas (9–13). An in vitro test could be used as a predictor for vaccine safety, i.e., if the vaccine is not attenuated in osteoclasts, then perhaps it would induce side effects associated with the vaccine.

Bone is a dynamic tissue that constantly undergoes remodeling coordinated by the synchronized activity of three cell types: osteoblasts (bone forming), osteocytes, and osteoclasts (bone resorbing) (14, 15). Although previous studies have demonstrated the ability of B. abortus to invade and replicate within osteoblasts and osteocytes (16–19), the role of osteoclasts in Brucella-induced bone loss has not been explored. Mature osteoclasts are highly specialized bone-resorbing, multinucleated cells of hematopoietic origin. In addition to their resorptive activity, osteoclasts regulate osteoblast precursor differentiation and activity as well as immune cell responses (20). Previous investigations from our laboratory using NOD-SCID IL2rγnull (NSG) mice have demonstrated that infection with B. abortus S19 but not the S19ΔvjbR vaccine candidate induced severe bone resorption with accumulation of myriad bacteria within mature osteoclasts (21). This apparent tropism of Brucella for osteoclasts and the significant amount of bone destruction in the mice, a lesion which is mediated by osteoclast resorptive activity, prompted the investigation of the effect of Brucella infection on these cells. In the present study, we sought to investigate the role of osteoclasts in osteoarticular brucellosis and vaccine safety using an in vitro model.

RESULTS

Characterization of mature osteoclasts derived from murine bone marrow-derived macrophages.An in vitro model of osteoclast differentiation from primary cell culture was used to understand the role of osteoclasts in osteoarticular disease (22–24). Mature osteoclasts are multinucleated bone-resorbing cells derived from the monocyte/macrophage lineage under the control of two main cytokines: macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) (25, 26). In this study, freshly collected mouse bone marrow-derived macrophages (BMDMs) were cultured in alpha minimal essential medium (αMEM) in the presence of M-CSF and RANKL and monitored for their differentiation and maturation starting from 1 to 10 days. Cellular characterization following tartrate-resistant acid phosphatase (TRAP) staining demonstrated that the majority of the cells on day 2 were TRAP-positive (TRAP+) cells with one or two nuclei and were considered osteoclast precursors (pOCs), while the majority of the cells on day 3 were TRAP+ cells with three or more nuclei and were considered multinucleated mature osteoclasts (mOCs) (Fig. 1A and B). Cellular fusion and a gradual increase in the size of osteoclasts were observed from day 2 to day 6 of maturation (Fig. 1A, C, and D). Although no significant changes in the size of osteoclasts were observed beyond day 6, nuclei disappeared gradually beginning on day 8, followed by complete dissolution of a portion of the cells (Fig. 1A). Cellular apoptosis was evident, with nuclear condensation starting on day 6 followed by nuclear fragmentation on days 7 and 8, leaving an empty space in the wake of dead cells by day 10 (Fig. S1 in the supplemental material). The characterization of the kinetics of osteoclast growth and differentiation using this in vitro cellular model permitted us to classify cells on day 2 of macrophage differentiation in the presence of M-CSF and RANKL as osteoclast precursors, while cells on day 3 were classified as mature osteoclasts, for further infection studies.

FIG 1
FIG 1

Differentiation of bone marrow-derived macrophages (BMDMs) into multinucleated mature osteoclasts in vitro. Mouse bone marrow-derived macrophages were cultured in 24-well plates with 20 ng/ml M-CSF and 50 ng/ml RANKL for up to 10 days, and mature osteoclasts were characterized based on TRAP staining and number of nuclei. TRAP+ cells with ≥3 nuclei were considered multinucleated mature osteoclasts (mOCs). (A) Representative brightfield images of TRAP staining during osteoclast maturation, fusion, and growth. Cellular fusion and size increased gradually over time, reaching a maximum on day 6. Although there were no changes in the size of mature osteoclasts between days 6 and 10, the number of pyknotic nuclei increased gradually, leaving an empty space on the dish by day 10. (B) Quantitative image analysis revealed that TRAP+ cells began appearing on day 2 and reached a maximum number by day 3. (C) The number of TRAP+ cells with >20 nuclei reached a maximum on day 6. (D) Quantitative analysis of cell size showed a gradual increase with maximum size reached by day 6 of maturation. Bars represent the mean ± SD; n = 12 wells from three independent experiments. Letters are significantly different from the same group. Asterisks are statistical comparisons performed against day 2. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Brucella invades and replicates inside mature osteoclasts.The kinetics of infection and mechanism behind Brucella replication and survival in macrophages have been extensively studied (27, 28). Here, we wanted to determine if mature osteoclasts were also permissive to infection and replication of different Brucella strains known to have different levels of virulence, including wild-type B. abortus 2308; B. abortus 2308virB2, an attenuated mutant incapable of survival in macrophages; B. abortus S19, a commercially available vaccine for use in cattle; and B. abortus S19ΔvjbR, a vaccine candidate. Following a pilot study of Brucella infection of mature osteoclasts (Fig. S2) at different multiplicities of infection (MOI values of 50, 100 and 500 per cell), downstream experiments were carried out at MOI of 100. When mOCs on day 3 of maturation were infected with the different Brucella strains, no significant differences in bacterial invasion were observed at 3 h postinfection. In contrast, B. abortus 2308 and B. abortus S19 were the only strains able to persist and replicate inside the cells while the attenuated mutants were progressively killed (Fig. 2A, C, and D). A similar phenotype of bacterial survival and replication was observed in BMDMs (Fig. 2B), demonstrating not only that B. abortus is capable of infecting mature osteoclasts, but also that the kinetics of bacterial survival and replication in mature osteoclasts mirrors that seen in the primary target cell, the macrophage (27, 28). Z-sectioning of confocal microscopic images corroborated the bacterial distribution inside different depths of osteoclast cytoplasm (Movies S1 and S2). Comparison of the behavior of the virulent strain B. abortus 2308 and the vaccine strain B. abortus S19, known to be capable of inducing pathology in humans and osteoarthritis in cattle (29, 30), with that of the vaccine candidate S19vjbR also provides evidence that osteoclasts have the potential to serve as an in vitro model for assessing vaccine safety.

FIG 2
FIG 2

B. abortus invades and replicates inside mature osteoclasts (mOCs) and bone marrow-derived macrophages (BMDMs). Mouse BMDMs were cultured in 24-well plates either with 20 ng/ml M-CSF + 50 ng/ml RANKL or with 20 ng/ml M-CSF alone. On day 3, BMDMs and TRAP+ mOCs were infected with B. abortus strains at an MOI of 1:100. Following 2 h of infection, the medium was replaced with a gentamicin-containing medium and incubated for different durations. (A) Invasion and replication of B. abortus strains within mOCs. While all strains invaded mOCs at the same level, B. abortus 2308 and S19 demonstrated replication by 48 h postinfection (hpi); B. abortus ΔvirB2 and S19ΔvjbR were progressively killed. (B) Invasion and replication of B. abortus within BMDMs. Identical infection kinetics were observed in BMDMs. (C) Representative confocal immunofluorescence images showing Brucella colonization and replication (green) inside osteoclasts. Increased numbers of GFP-B. abortus organisms are observed within osteoclasts at 48 h following infection with B. abortus 2308 and S19, coinciding with replication. (D) Quantitative image analysis shows a significantly (P < 0.001) higher level of colonization of B. abortus 2308 and B. abortus S19 than other mutants by 24 h and 48 h of postinfection. Bars represent the mean ± SD; n = 12 wells from four independent experiments. **, P < 0.01; ***, P < 0.001.

B. abortus 2308 impairs the growth of mature osteoclasts.Osteoclast maturation involves the fusion of several mononucleated osteoclast precursors to form multinucleated giant cells. These cells can, in turn, fuse with additional mononucleated or multinucleated precursors in a near-continuous fashion until a size of 100 or more nuclei may be attained (31–33). To determine the effect of Brucella infection on the maturation and growth of mature osteoclasts, these cells were infected with the different bacterial strains on day 3 of maturation and were stained for TRAP. Interestingly, there was no significant difference in the number of TRAP+ mOCs derived following infection with any of the Brucella strains compared to uninfected control cells. However, the size of TRAP+ mOCs infected with wild-type B. abortus 2308 was significantly smaller (P < 0.01) than the size of the uninfected cells or cells infected with different mutant or vaccine strains at 24 h and 48 h postinfection (Fig. 3A, B, and C), suggesting that B. abortus 2308 impairs the fusion and growth of mature osteoclasts.

FIG 3
FIG 3

Wild-type B. abortus 2308 impairs fusion and growth of mature osteoclasts. Mouse bone marrow-derived macrophages (BMDMs) were cultured in 24-well plates with 20 ng/ml M-CSF and 50 ng/ml RANKL. On day 3 of maturation, osteoclasts were infected with B. abortus strains at an MOI of 1:100 and monitored for growth at 3 h, 24 h, and 48 h postinfection. (A) Representative brightfield images of TRAP staining showed increased fusion and growth of osteoclasts by 24 h and 48 h postinfection. Quantitative image analysis revealed no significant changes in the number of TRAP+ mOCs following infection, regardless of strain (B), but a significant (P < 0.01) decrease in the size of mOCs following 24 h and 48 h of infection with wild-type B. abortus 2308 (C). Bars represent the mean ± SD; n = 12 wells from four independent experiments. **, P < 0.01 versus control.

Active infection of mature osteoclasts does not induce significant cell death by either apoptosis or necrosis.To assess the effect of Brucella infection on mature osteoclast survival, the levels of lactate dehydrogenase (LDH), nitric oxide (NO), and cellular apoptosis were analyzed. Measurement of LDH in the culture supernatant is a useful method for detection of necrotic cell death (34, 35). NO plays an essential role in killing intracellular microbes, regulates osteoclastogenesis (36, 37), and can activate apoptotic cell death when produced at high levels (38). Regardless of the bacterial strain, no significant changes in the levels of LDH (0.5% to 4%) or NO production (1.5 to 3.5 μm) by mOCs, as well as by BMDMs, were observed (Fig. S3A to D). Further, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) of fragmented DNA (39) revealed no significant changes in cellular apoptosis over 48 h of infection of mOCs or BMDMs cultured in 96-well plates (Fig. S3E and F), indicating that active Brucella infection of mature osteoclasts, regardless of virulence, does not induce cell death.

Brucella abortus 2308-infected mature osteoclasts resorb calcium matrix at the same level as uninfected cells.Osteoclasts are the only known primary cells of the bone that are able to resorb bone matrix (40, 41). One of the most common methods to assess the matrix degradation property of osteoclasts is the resorption pit assay, which evaluates the ability of cultured osteoclasts to degrade a synthetic calcium phosphate coating (23). We first tested that mature osteoclasts but not macrophages are capable of degrading the calcium matrix (Fig. S4). To assess the functional activity of Brucella-infected mature osteoclasts, BMDMs were cultured in calcium phosphate-coated plates (Corning, MA) in the presence of M-CSF and RANKL. Mature osteoclasts were then infected with the different B. abortus strains on day 3 of maturation and incubated for 48 h to assess the ability of the cells to resorb calcium matrix. There were no significant differences in calcium matrix resorption by cells infected with B. abortus 2308 and B. abortus 2308virB2 from uninfected cells. In contrast, the calcium resorption activity of cells infected with B. abortus S19 and its mutant S19vjbR was significantly higher (P < 0.001) (Fig. 4B and E). Although TRAP staining showed no significant differences in the total number of TRAP+ mOCs between Brucella-infected and uninfected cells (data not shown), there was a tendency of reduced cell size when infected with wild-type Brucella or the VirB2 mutant (Fig. 4C). However, cells infected with B. abortus S19 and B. abortus S19vjbR demonstrated more cellular fusion with the formation of larger giant cells containing a significantly higher number of nuclei than wild-type B. abortus 2308, B. abortus 2308virB2, or control cells (P < 0.001) (Fig. 4A, C, and D). Overall, while highly virulent B. abortus 2308 is able to impair the fusion and growth of mature osteoclasts, it does not impact the ability of these cells to resorb the bone matrix. Additionally, the increased resorptive activity of osteoclasts infected with B. abortus S19 and B. abortus S19vjbR appears to be the result of larger cell size, as previous reports demonstrate (42–44).

FIG 4
FIG 4

B. abortus-infected mature osteoclasts can grow and are capable of calcium matrix resorption. Mouse bone marrow-derived macrophages (BMDMs) were cultured on calcium matrix in the presence of 20 ng/ml M-CSF and 50 ng/ml RANKL. On day 3 of maturation, osteoclasts were infected with B. abortus strains at an MOI of 1:100 and monitored for growth and function 48 h postinfection. (A) Representative brightfield images of TRAP-stained mOCs demonstrate a larger size of mOCs with infection by B. abortus S19 and S19ΔvjbR. (B) Representative brightfield images showing calcium resorption pits (white) by mOCs following 48 h of infection. During infection with B. abortus S19 and S19ΔvjbR, resorption pits appear larger, coinciding with increased cell size. Resorption pits for mOCs infected with B. abortus 2308 and B. abortus ΔvirB2 appear similar to those of uninfected mOCs. Quantitative image analysis of the area of mature osteoclasts following Brucella infection (C), and the number of mOCs based on cellular fusion or nuclei (D). No significant differences in total cell numbers were observed between groups, while mOCs infected with B. abortus S19 and S19ΔvjbR had significantly higher numbers of nuclei. (E) Quantitative analysis of calcium matrix resorption of mOCs infected with Brucella. Measurement of the resorbed area was performed using Fiji software. B. abortus S19 and S19ΔvjbR-infected mOCs resorbed significantly more calcium matrix. Letters are significantly different from the same groups; asterisks are statistical comparisons performed against control or mutant strains. Bars represent the mean ± SD; n = 12 wells from four independent experiments. **, P < 0.01; ***, P < 0.001.

Wild-type B. abortus 2308 infection of BMDMs and osteoclast precursors impairs osteoclastogenesis and calcium matrix resorption.After determining that direct infection of mature osteoclasts by virulent B. abortus 2308 did not impact their resorptive capacity, BMDMs and pOCs were infected to determine if infection at an earlier time point could impact osteoclastogenesis and thereby calcium resorption. As previously mentioned, multinucleated mature osteoclasts are generated from the fusion of precursors belonging to the monocyte/macrophage lineage under the influence of M-CSF and RANKL cytokines, with maturation progressing from macrophages to osteoclast precursors to mature osteoclasts. BMDMs or pOCs plated onto calcium phosphate-coated plates were infected with the different B. abortus strains and incubated in the presence of M-CSF and RANKL for 5 days (120 h). In contrast to what was observed in Brucella-infected mature osteoclasts, TRAP staining of B. abortus 2308-infected cells demonstrated significant reduction (P < 0.01) in the growth of mature osteoclasts derived from infected BMDMs (Fig. 5) or pOCs (Fig. 6) as well as significant reduction (P < 0.01) in calcium matrix resorption compared with uninfected controls. These results indicate an unexpected, direct, and negative impact on osteoclast growth and functional activity when infection occurs at the precursor stage.

FIG 5
FIG 5

Wild-type B. abortus 2308 infection of BMDMs reduces osteoclastogenesis and matrix resorption activity. Mouse BMDMs cultured on calcium matrix with 20 ng/ml M-CSF for 48 h were infected with B. abortus strains at an MOI of 1:100. Infected cells were further incubated with 20 ng/ml of M-CSF and 50 ng/ml of RANKL for 120 h to monitor osteoclastogenesis and matrix resorption. (A) Representative brightfield images of TRAP-stained uninfected and infected mOCs. The mOCs derived from macrophages infected with B. abortus 2308 appear subjectively smaller. (B) Representative brightfield images showing calcium resorption pits (white) by mOCs following 120 h of infection. The resorption pits produced by mOCs derived from macrophages infected with B. abortus 2308 appear subjectively smaller. Quantitative image analysis of the total number of TRAP+ mOCs (C), and the number of mOCs based on cellular fusion or number of nuclei (D). A significantly lower number of mOCs were derived from macrophages infected by B. abortus 2308, and these mOCs also showed significantly fewer nuclei. (E) Quantitative analysis of calcium matrix resorption showed a significant decrease in the amount of calcium matrix resorption by cells infected with wild-type B. abortus 2308. Measurement of the resorbed area was performed using Fiji software. Letters are significantly different from the same group; asterisks are statistical comparisons performed against control or mutant strains. Bars represent the mean ± SD; n = 12 wells from four independent experiments. **, P < 0.01.

FIG 6
FIG 6

Wild-type B. abortus 2308 infection of osteoclast precursors (pOC) reduces osteoclast growth and matrix resorption activity. Mouse BMDMs cultured on calcium matrix in the presence of 20 ng/ml M-CSF and 50 ng/ml of RANKL for 48 h (pOC) were infected with B. abortus strains at an MOI of 1:100 to monitor cell growth and matrix resorption after 120 h. (A) Representative brightfield images of TRAP-stained uninfected and infected mOCs show no clear differences in cell size or number. (B) Representative brightfield images showing calcium resorption pits (white) by mOCs following 120 h of infection. Resorption pits produced by mOCs derived from osteoclast precursors infected with B. abortus 2308 appear subjectively smaller. Quantitative image analysis of the total number of TRAP+ mOCs (C), and the number of mOCs based on cellular fusion or number of nuclei (D). While the number of mOCs did not differ significantly between groups, the number of nuclei in mOCs derived from osteoclast precursors infected with B. abortus 2308 showed significantly fewer nuclei. (E) Quantitative analysis of calcium matrix resorption showed a significant decrease in the amount of calcium matrix resorption by cells infected with wild-type B. abortus 2308. Measurement of the resorbed area was performed using Fiji software. Letters are significantly different from the same group; asterisks are statistical comparisons performed against control or mutant strains. Bars represent the mean ± SD; n = 12 wells from four independent experiments. **, P < 0.01.

Brucella-infected osteoblasts fail to drive osteoclastogenesis.As wild-type B. abortus 2308 infection of mature osteoclasts or their precursors did not enhance osteoclast maturation or alter the functional activity of mature osteoclasts, the involvement of other cells associated with the bone may be responsible for the enhanced resorptive activity of osteoclasts seen in Brucella-induced osteoarthritis. Bone homeostasis depends on the functional balances between bone-forming osteoblasts and bone-resorbing osteoclasts. To maintain this homeostasis in the adult skeleton, osteoblasts produce two main cytokines which are necessary to promote osteoclast differentiation and maturation (45). Previous in vitro studies have shown that B. abortus-infected osteoblasts are capable of inducing RANKL expression and secretion (17, 46), suggesting that infected osteoblasts have the potential to stimulate increased osteoclastogenesis.

In this study, murine MC3T3, a standard preosteoblast cell line, was used for the assessment of the impact of osteoblasts on osteoclastogenesis following B. abortus infection. First, MC3T3 preosteoblast cells were allowed to differentiate and mature for various durations (7, 14, and 21 days), and expression of alkaline phosphatase (ALP) and mineralization of bone matrix were used as phenotypic markers to characterize osteoblast maturation (47–50). Alkaline phosphatase activity was first detected on day 7 of osteoblast differentiation. Subsequently, alizarin red S staining of calcium deposition was noted in few cells by day 14 (Fig. 7A), with staining exhibited by 90% to 100% of the cells by day 21, indicating differentiation of mature osteoblasts. MC3T3 osteoblasts infected (MOI of 100) with different Brucella strains on day 21 of maturation demonstrated a similar phenotype of bacterial invasion and replication in BMDMs and mOCs (Fig. 7B) for B. abortus 2308 and S19, as previously reported (16–18). Additionally, the attenuated strains B. abortus ΔvirB2 and the vaccine candidate S19vjbR were able to invade osteoblasts but unable to survive or replicate.

FIG 7
FIG 7

B. abortus invades and replicates inside differentiated MC3T3 osteoblasts. The MC3T3 preosteoblast cell line was cultured in an osteogenic differentiation medium for 21 days to mature. (A) Alizarin red S staining of calcium deposition and alkaline phosphatase activity demonstrate the progressive maturation of MC3T3 cells. (B) Mature osteoblasts (21 days of differentiation) were infected with Brucella, and quantitative analysis of bacterial survival and replication was performed. As observed with macrophages and mOCs, all strains invade osteoblasts at similar rates, while only B. abortus 2308 and S19 demonstrate replication by 48 h.

To evaluate whether B. abortus infection of osteoblasts could enhance osteoclastogenesis, both direct and indirect interaction of infected osteoblasts with osteoclasts was assessed. For direct interaction, BMDMs were cocultured with Brucella-infected osteoblasts, while indirect interaction was performed by exposing BMDMs to the supernatant of Brucella-infected osteoblasts. TRAP staining after 7 days of incubation revealed no influence of MC3T3 mature osteoblast (with or without infection) on BMDM-derived osteoclastogenesis in vitro (Fig. 8).

FIG 8
FIG 8

Brucella-infected and -uninfected MC3T3 osteoblasts fail to drive osteoclastogenesis in vitro. MC3T3 osteoblasts cultured in an osteogenic differentiation medium for 21 days were infected with different B. abortus strains at a concentration of 1:1,000. (A) Cells were incubated for 24 h postinfection and directly cocultured with mouse BMDMs in the presence of osteogenic differentiation medium and 20 ng/ml M-CSF. (B) Alternatively, mouse BMDMs were cultured with culture supernatants from Brucella-infected osteoblasts at different proportions (25% and 50%). After 7 days, cells were stained for TRAP to detect mature osteoclasts. RANKL (1 ng/ml) was used as a positive control for osteoclast formation. Neither infected nor uninfected MC3T3 osteoblasts stimulated osteoclastogenesis as evidenced by lack of TRAP staining at the end of 7 days.

DISCUSSION

Brucella-induced osteoarticular damage is a commonly described clinical finding associated with human or animal brucellosis (5, 7, 51–53). The development of live-attenuated vaccines to protect humans against infection has long been hampered by safety concerns regarding the use of live-attenuated vaccines, notably the potential of such vaccines to induce arthritis in livestock (29) and, most importantly, in humans. Due to the incompletely understood mechanisms of osteoarticular brucellosis, it is challenging to predict the potential side effects that can be associated with the administration of live-attenuated vaccines, as some of the most commonly used animal models to study brucellosis do not develop osteoarticular disease in a consistent manner (51, 54, 55). A better understanding of the cellular and molecular events occurring at the joint site during Brucella infection could pave the way for more efficient and inclusive evaluation of safety for new vaccine candidates regarding osteoarticular brucellosis. An in vitro test could be used as a predictor for vaccine safety, i.e., if the vaccine is not attenuated in osteoclasts, then perhaps it would induce side effects associated with the vaccine.

In this study, we sought to investigate the role played by osteoclasts during Brucella infection using different B. abortus strains and vaccine candidates to test whether Brucella would be able to invade, replicate, or change the functional activity of osteoclasts. To the best of our knowledge, this study is the first to investigate the direct interaction between different strains of B. abortus and osteoclasts. Osteoclastogenesis is a complex process requiring different stimuli and cellular molecules to interact without impairment (56, 57). It is well-known that myeloid cells of the monocyte/macrophage lineage differentiate into osteoclast precursors in the presence of the cytokines M-CSF and RANKL (25, 26, 58). Previous studies have demonstrated that multinucleated osteoclasts are formed following fusion of mononuclear preosteoclasts, during which the nuclei enter into the G0 state, which inhibits DNA synthesis and prevents further proliferation of osteoclasts. This allows the osteoclast precursors to gradually grow in size by fusion, although early cellular fusion is known to accelerate cell death (40, 59). In this study, ex vivo differentiation of BMDMs followed a similar pattern of formation of pOCs, mOCs, cellular growth, and apoptotic cell death.

Brucella is known to invade and replicate inside mammalian hosts, and its ability to cause disease relies on its intracellular lifestyle (60). Although in vitro infection of osteoclasts with various types of live bacteria known to cause bone destruction, including Staphylococcus aureus, Mycobacterium tuberculosis, and Cutibacterium acnes, has been studied (41, 61, 62), the direct interaction of Brucella with osteoclasts has never been investigated. A recent study performed by our laboratory has demonstrated severe bone destruction in the tail vertebrae of NSG mice inoculated with B. abortus S19, with accumulation of a large number of bacteria within mature osteoclasts, an outcome which was not observed during infection with the Brucella vaccine candidate S19ΔvjbR (21). In this study, we demonstrated that only B. abortus 2308 and S19 were able to invade and replicate inside mature osteoclasts. In contrast, B. abortus 2308virB2 and S19vjbR mutants, while able to invade, were not capable of replication or sustained survival. This finding demonstrates that B. abortus has a high capacity to invade, survive, and replicate within a wide variety of phagocytic and nonphagocytic host cells, which would allow bacterial cells to escape from host defenses (63).

The ability of Brucella to inhibit phagolysosome fusion in macrophages allows these cells to serve as the bacterium’s predominant replicative niche (63). Typically, smooth strains of Brucella do not induce cell injury and apoptosis in macrophages and instead employ these cells for replication and dissemination (64, 65). We have demonstrated that direct B. abortus 2308 infection of mature osteoclasts impairs early fusion of osteoclasts and reduces cellular growth but, importantly, does not induce cellular death. We noticed differences in cellular growth in noncoated versus calcium phosphate-coated matrices. As mentioned previously, accelerated growth and fusion of osteoclasts in vitro result in premature apoptotic cell death (38, 57). This result therefore suggests that virulent Brucella impairs the growth of osteoclasts following invasion in order to prolong the life of the cell, employing the osteoclast in a strategy of intracellular survival and avoidance of immune detection. Prolonged survival of infected osteoclasts might also contribute to the progressive osteoarticular lesions which frequently occur in human cases of brucellosis. In contrast, attenuated Brucella strains did not impair cellular growth or induce cell death of mature osteoclasts. This, in turn, suggests that the mature osteoclasts infected with attenuated strains might grow faster and undergo apoptosis earlier than those infected with wild-type strains, providing in vitro evidence that such strains would be incapable of inducing osteoarticular disease in a vaccinated host and might therefore be safer. While evidence of cell death was not observed using LDH secretion and TUNEL assays in osteoclasts infected with these attenuated strains over 48 h, it is possible that increased levels of apoptosis might be observed at later time points. Further, we observed a stronger effect on the inhibition of osteoclast maturation if infected in an early stage of maturation, either macrophage or preosteoclast stages.

An imbalance in osteoclast functional activity in bone matrix degradation may lead to bone lysis (61). It has been reported that large osteoclasts are more active, capable of enhanced bone resorption, and more responsive to environmental stimuli (42–44). Physical characterization of Brucella-infected osteoclasts in this study revealed phenotypic variation in cell size due to infection with different bacterial strains. This prompted the analysis of functional activity of infected osteoclasts by assessing the amount of calcium phosphate resorption. Previous in vivo studies have shown that enhanced osteoclastogenesis during wild-type Brucella infection was associated with proinflammatory and pro-osteoclastogenic mediators (41, 54, 55, 66–68). In the present study, mature osteoclasts infected with wild-type B. abortus 2308 exhibited calcium matrix resorption activity at a similar level to uninfected controls, indicating that direct infection of mature osteoclasts is not solely responsible for the high levels of bone resorption observed in Brucella-induced osteoarthritis. Unexpectedly, infection of mature osteoclasts with B. abortus S19 or S19ΔvjbR vaccine strains but not by wild type resulted in larger areas of matrix degradation, corresponding with larger cell size. It is important to note that larger cell size can be a double-edged sword regarding functional activity and cellular survival. Although larger cells are capable of absorbing larger areas of matrix (41–44, 69), such cells would be expected to exhibit significantly shorter survival. The larger cell size and enhanced matrix degradation observed with the B. abortus S19 or S19ΔvjbR vaccine strains might therefore provide additional evidence of safety, as infected osteoclasts would exhibit shortened survival and would be unlikely to contribute to the long-standing and progressive bone loss observed in human Brucella infection. Nevertheless, future studies are required to identify the mechanism behind the increased size and resorptive capacity of mature osteoclasts infected with S19 and S19ΔvjbR strains. The enhanced bone resorption observed in cases of osteoarticular brucellosis appears to be due to chronicity of infection and continuous bone degradation, which could be facilitated by Brucella-infected cells restricting cell growth and enhancing life span.

Several studies have described that bacterial infection has a dual impact on osteoclastogenesis and functional activity of osteoclasts (41, 62). We demonstrated earlier that B. abortus 2308 impaired the growth of mature osteoclasts. As mature osteoclasts are derived from the monocyte/macrophage lineage, we next investigated the effect of Brucella infection on osteoclast size and activity at an earlier time point, namely, macrophages or osteoclast precursors that were then stimulated with RANKL to become osteoclasts. Our results demonstrated that the vaccine strain S19 and the attenuated mutants did not inhibit osteoclastogenesis and the resultant mature osteoclasts were capable of resorbing calcium matrix at a similar level to controls. However, wild-type B. abortus 2308-infected macrophages or osteoclast precursors exhibited smaller size and smaller areas of calcium resorption. This supports the previous results obtained from infection of mature osteoclasts and provides further evidence that virulent B. abortus delays cellular fusion and growth of infected osteoclasts in an effort to prolong the survival of the cell and thereby maximize the utility of the osteoclast as a replicative niche. Additionally, it can be theorized that due to prolonged survival, Brucella-infected osteoclasts would be able to continue bone resorption and contribute to progressive bone destruction. This strategy would be in direct contrast to other bacterial pathogens capable of inducing bone damage, such as wild-type S. aureus, which results in enhanced osteoclast fusion and calcium matrix resorption following infection and inhibits osteoclastogenesis following infection of osteoclast precursors (41). However, it should be noted that bacteria such as S. aureus are extracellular pathogens and do not prolong host cell life span as a survival strategy. Further, to enhance vaccine safety criteria, the bacterial attenuation property could be accessed simultaneously with changes in cell size and the ability of calcium matrix degradation.

While the effects of Brucella infection on osteoclast growth are intriguing, such cells exist in a complex interaction with numerous other cell types, and the interactions of Brucella with these cells may also play an important role in the development of osteoarticular disease. For instance, bone resorption by mature osteoclasts coincides with bone formation by osteoblasts in a tightly regulated process to maintain the bone homeostasis (17, 70, 71). Several in vitro studies have highlighted the direct impact of B. abortus infection on osteoblast function, including increased secretion of RANKL (17, 46), a cytokine that is critical in driving osteoclast formation and bone remodeling. Under pathological circumstances, RANKL triggers osteoclastogenesis and initiates bone destruction, as observed in a variety of bacterial infections (46, 72, 73).

Since the invasion of Brucella of mature osteoclasts did not enhance osteoclastogenesis, we wanted to see if activation of these cells would occur secondary to invasion of osteoblasts. In order to investigate the additive role of Brucella-infected osteoblasts on osteoclastogenesis, we first demonstrated Brucella replication in differentiated MC3T3 osteoblasts. While wild-type B. abortus 2308 and B. abortus S19 replicated inside osteoblasts, mutant strains, including the vaccine strain candidate S19ΔvjbR, failed to replicate. This not only confirms the ability of virulent Brucella to utilize osteoblasts as an additional replicative environment but also provides further in vitro evidence regarding the attenuation and safety of S19ΔvjbR. When the ability of the infected osteoblasts to influence osteoclastogenesis was next investigated, an in vitro direct coculture of Brucella-infected MC3T3 cells with BMDMs did not stimulate osteoclastogenesis. Additionally, treatment of BMDMs with culture supernatants from B. abortus-infected MC3T3 failed to stimulate osteoclastogenesis. While it is possible that Brucella infection of osteoblasts does not directly drive osteoclastogenesis, we have shown that the addition of 1 ng/ml of RANKL to BMDM culture was sufficient to induce TRAP+ pOC formation. Lack of induction of osteoclastogenesis by MC3T3 cells may be attributed to the insufficient production of RANKL in the in vitro model.

Based on the results obtained in the present study, we hypothesize that intracellular survival and replication of bacteria inside osteoclasts are critical components in the development of osteoarticular brucellosis. Our results imply that Brucella takes advantage of osteoclasts following infection of the mature cells or their precursors and utilizes these cells as an additional environment to replicate by reducing cellular growth and prolonging the life of the cell. By infecting circulating osteoclast precursors, which are abundant in the bloodstream and hematopoietic tissues, B. abortus could avoid immune detection and establish a chronic infection in the bones or joints, ultimately resulting in osteoarthritis. Finally, comparison of wild-type and vaccine strains of B. abortus demonstrates that osteoclasts could serve as a useful in vitro model to assess vaccine safety in terms of potential induction of osteoarticular lesions in the host. Using the in vitro cellular model of osteoclast maturation to study host-pathogen interactions and assess the safety of vaccine candidates, we have provided exciting new opportunities to interrogate the effects of B. abortus infection on bone and beyond.

MATERIALS AND METHODS

Bacterial strains and media.B. abortus 2308, 2308virB2, S19 (NVSL, Ames, IA), and B. abortus S19ΔvjbR (engineered for a previous study) (74) with or without green fluorescent protein (GFP) were used in these experiments. B. abortus strains were grown on tryptic soy agar (TSA) plates (Difco, Becton, Dickinson) at 37°C with 5% (vol/vol) CO2 for 3 days. A single colony was used to inoculate tryptic soy broth (TSB) and was incubated at 37°C overnight. Bacteria were collected by centrifugation and washed twice in phosphate-buffered saline (PBS) (pH 7.2) (Gibco). Bacterial density was measured using a Klett colorimeter and compared against a standard curve. Viable counts were measured by plating serial dilutions of inoculum onto TSA plates and quantifying colonies following 3 to 4 days of incubation at 37°C. All experiments with live bacteria were carried out at biosafety level 3 facilities at the College of Veterinary Medicine and Biomedical Science, Texas A&M University.

Preparation of Brucella abortus strains expressing GFP.B. abortus strains expressing GFP were made by electroporating the pBBR1-MSC6Y plasmid encoding chloramphenicol resistance into different Brucella strains as previously described (75, 76). B. abortus 2308, 2308virB2, S19, and B. abortus S19ΔvjbR were grown in TSB overnight. Bacterial pellets were subsequently washed four times in ice-cold sterile water and resuspended in 0.1 ml of ice-cold sterile water. One microgram of pBBR1-MSC6Y plasmid DNA was mixed with bacteria and incubated for 30 min on ice followed by electroporation at a voltage of 2,500 V, resistance of 200 Ω, and capacitance of 50 μF. Immediately after electroporation, 1 ml of warmed TSB SOC-B medium (6% [wt/vol] tryptic soy broth, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, and 20 mM glucose) was added, and suspensions were incubated at 37°C for 24 h. The following day, bacterial suspensions were plated onto TSA with 5 μg/ml chloramphenicol and incubated at 37°C for 3 days. Single colonies from each bacterial strain were inoculated into 5 ml of TSB with 15 μg/ml chloramphenicol and incubated at 37°C for 24 h. Following microscopic confirmation of fluorescence, 25% glycerol stock of positive clones was made and stored at –80°C for future use.

Cell cultures.Murine bone marrow-derived macrophages (BMDMs) were harvested from the femur and tibia and cultured as previously described with some modifications (77–80). Animal use protocol was approved by the Texas A&M University Institutional Animal Care and Use Committee. Briefly, bone marrow cells collected from the femur and tibia of 4- to 6-week-old female C57BL/6 mice (5 to 7 mice per experiment) were cultured at 1 × 106 cells/ml in an αMEM medium (Life Technologies, Beverly, MA) supplemented with 10% fetal bovine serum (FBS) and 20 ng/ml of recombinant mouse M-CSF (R&D Systems). Following 2 days of incubation at 37°C with 5% CO2, nonadherent cells were discarded, and adherent cells were subcultured as BMDMs. For osteoclast differentiation, BMDMs were cultured (2.5 × 104 cells/well in 24-well culture plates) in the αMEM medium supplemented with 10% FBS, 50 ng/ml of recombinant mouse RANK-L (R&D Systems), and 20 ng/ml of recombinant mouse M-CSF (80). For osteoclast maturation, cells were cultured for different times (1 to 10 days), replacing 50% of the media with fresh media every 2 days. To characterize and identify osteoclasts, cells were fixed in 4% paraformaldehyde and stained for TRAP. TRAP+ cells with 1 to 2 nuclei were classified as osteoclast precursors (pOC), while TRAP+ cells with ≥ 3 nuclei were classified as multinucleated mature osteoclasts (mOCs) (80). Osteoclast precursor infections were performed on day 2, while infection of mature osteoclasts was conducted on day 3 in the presence of 50 ng/ml of recombinant mouse RANK-L and 20 ng/ml of recombinant mouse M-CSF.

The mouse MC3T3-E1 cell line (ATCC CRL-2593) was used for osteoblast studies. MC3T3-E1 cells were seeded in complete growth medium at 1 × 105/well in 24-well tissue culture plates and incubated at 37°C with 5% CO2. After 24 h, the old medium was replaced with αMEM differentiation medium (0.5 ml) supplemented with 10% FBS, 50 μg/ml ascorbic acid, and 4 mM β-glycerol phosphate. The differentiation medium was replaced every other day for 30 days, and osteoblast maturation was confirmed by measuring calcium deposition and alkaline phosphatase activity.

Cellular infection.BMDMs were cultured either in standard tissue culture plates (Genesee Scientific, catalog no. 25-107) or in calcium phosphate-coated plates (Corning Osteo assay surface, product no. 3987) and differentiated into mature osteoclasts in the presence of 50 ng/ml of recombinant mouse RANK-L and 20 ng/ml of recombinant mouse M-CSF. Cells were infected with B. abortus 2308, 2308virB2, S19, and S19ΔvjbR at an MOI of 100. Immediately following infection, plates were centrifuged for 5 min at 200 × g to synchronize infection (81). Following 2 h of incubation at 37°C with 5% CO2 to allow bacterial entry, extracellular bacteria were removed by washing twice and incubating in differentiation medium with 50 μg/ml of gentamicin for 1 h (41). To evaluate bacterial invasion and replication, cells at different time points (3 h, 24 h, and 48 h) were washed twice in PBS and lysed in 0.5% (vol/vol) H2O-Tween 20. Immediately following lysis, lysates were serially diluted (1/10) in PBS and plated on TSA plates. After 3 to 4 days of incubation at 37°C, colonies were enumerated to quantify bacterial CFU. Undifferentiated BMDMs were infected simultaneously to serve as a control.

Infection of the mouse osteoblast MC3T3-E1 cell line was performed on day 21 to assess cellular invasion and survival. Cells were infected with the same B. abortus strains at an MOI of 100. Following cellular infection, plates were centrifuged, incubated, and treated with gentamicin followed by lysis of cells, serial dilution, and plating of suspensions onto TSA plates, as described above.

Direct coculture of Brucella-infected MC3T3-E1 osteoblasts and BMDMs.MC3T3 cells, cultured in a complete osteoblast differentiation medium for 21 days, were infected with B. abortus strains for 2 h at an MOI of 100 or 1,000. At 24 h postinfection, cells were washed twice with warm media and incubated for 7 days following addition of fresh differentiation media containing 2.5 × 104 BMDMs. On day 3, cultured supernatants were replaced with fresh osteogenic differentiation medium supplemented with 20 ng/ml of M-CSF. As positive controls of mOC formation, uninfected MC3T3 cells and BMDMs were cocultured in the presence of 20 ng/ml of M-CSF and various concentrations of RANKL (1, 6.25, and 12 ng/ml) to determine the minimum level (1 ng/ml) of RANKL required for mOC formation in our experiments. To identify mOCs, cells were fixed by 4% paraformaldehyde for 2 h and stained for TRAP.

Stimulation with conditioned medium.Culture supernatants from Brucella-infected mature MC3T3-E1 osteoblasts at an MOI of 100:1 and 1,000:1 were aspirated at 24 h postinfection, passed through a 0.22-μm filter, and used as a conditioned medium to stimulate uninfected BMDMs for 7 days. Conditioned media were used at either 25% or 50% concentration in the presence of 20 ng/ml of M-CSF, and on day 3, all media were replaced. As positive controls of mOC formation, M-CSF and different concentrations of RANKL were used as described above. After 7 days of incubation, media were aspirated, and cells were fixed in 4% paraformaldehyde for 2 h at room temperature and stained for TRAP.

Determination of nitrite oxide concentrations.The level of nitric oxide (NO) was determined by measuring the released NO metabolites (nitrites) using modified Griess reagent (catalog no. G4410, Sigma) at 3 h, 24 h, and 48 h postinfection. Briefly, 100 μl of Griess reagent was mixed with 100 μl of culture supernatant in a 96-well plate and incubated for 15 min, and spectrophotometric measurement was performed at 540 nm. Nitrite concentration was measured against a freshly made NaNO2 standard curve.

Determination of cell death.LDH released into culture supernatants of infected and uninfected cells at 3 h, 24 h, and 48 h postinfection was determined using the CytoTox 96 nonradioactive cytotoxicity assay kit (catalog no. G1780, Promega) according to the manufacturer’s instructions (81). Cell cytotoxicity was expressed as the percentage of LDH release, which was calculated using the following formula: percentage of LDH release =100 × (test LDH release − spontaneous release)/(maximum release − spontaneous release).

Apoptosis assays.BMDMs were cultured at 5 × 103/well in 96-well tissue culture plates (Genesee Scientific, catalog no. 25-109) and differentiated into mature osteoclasts in the presence of 50 ng/ml of recombinant mouse RANKL and 20 ng/ml of recombinant mouse M-CSF. Cells were infected with B. abortus 2308, 2308ΔvirB2, S19, and S19ΔvjbR at an MOI of 100. Immediately following infection, plates were centrifuged, incubated, and treated with gentamicin, as described above.

Apoptotic cells were detected using terminal deoxynucleotidyl transferase (TdT) in situ apoptosis detection kit-DAB (catalog no. 4810-30-K; R&D Systems).

Osteoclast resorption assay.BMDMs cultured in calcium phosphate-coated plates (Corning, MA) were differentiated into mature osteoclasts and infected on day 3. Following 48 h of infection, culture supernatants were aspirated, and 10% bleach was added for 5 min to remove the cells. The wells were washed twice in distilled water (dH2O) and dried at room temperature. Resorption pits were observed under a brightfield microscope, and images were quantified using Fiji (https://imagej.net/Fiji/Downloads). TRAP staining was simultaneously performed in unbleached cells to characterize mature osteoclasts and correlate with pit formation.

TRAP staining.TRAP staining was performed as follows: uninfected and Brucella-infected cells were washed three times with PBS, then fixed with 4% paraformaldehyde for 2 h at room temperature. Fixative was aspirated, and the cells were washed three time with PBS and incubated with TRAP staining solution (0.504% sodium acetate, 0.232% glacial acetic acid, 1.412% potassium sodium tartrate tetrahydrate, 4.16% sodium nitrite, 2 N HCl, 4% pararosaniline solution, and 10% Naphthol AS-BI solution) for 30 to 45 min at 37°C until desired staining intensity was achieved. Subsequently, the TRAP staining solution was removed, cells were washed three times with distilled water, and the plates were left upside down to dry before counting TRAP+ mature osteoclasts having ≥3 nuclei per cell.

Confocal microscopy.BMDMs seeded at a concentration of 2.5 × 104 cells/well in 24-well black microplates (Ibidi, catalog no. 82406) in the presence of 50 ng/ml of recombinant mouse RANKL and 20 ng/ml of recombinant mouse M-CSF were infected with GFP-B. abortus strains for 3 h, 24 h, and 48 h and subsequently fixed in 4% paraformaldehyde. Cells were permeabilized in 0.25% Triton X-100 in PBS for 5 min, washed twice with PBS, and stained for F-actin using Texas Red-X phalloidin (catalog no. T7471, Thermo Fisher Scientific, USA) at 1:300 for overnight at 4°C. Following washing in PBS, cell nuclei were stained using Hoechst 33342 solution (catalog no. 62249, Thermo Fisher Scientific, USA) for 10 min. Washed cells stored in PBS were imaged and analyzed using a Zeiss 780 confocal microscope.

Alizarin red S and alkaline phosphatase staining.To assess calcium deposition, alizarin red staining was used. MC3T3-E1 cells were seeded at 1 × 105 per well onto 24-well tissue culture plates. On days 7, 14, and 21 of differentiation, cells were washed in PBS, fixed in 4% paraformaldehyde, stained in 2% (wt/vol) alizarin red S, and visualized by light microscopy (17). At the same time points, ALP staining of osteoblasts was performed using ALP kit (catalog no. 86R-1KT, Sigma-Aldrich) according to the manufacturer’s instructions.

Statistical analysis.Each experiment was repeated at least three times, and statistical analysis was performed using one-way and two-way analysis of variance (ANOVA), followed by Tukey’s range test using GraphPad Prism software, version 4.0. Data are represented as means ± standard deviations (SDs). A P value of <0.05 was considered significant.

ACKNOWLEDGMENTS

This work is supported by a faculty startup grant from Texas A&M University and grant KO1TW009981 from NIH to A.M.A.-G. A research scholarship to O.H.K. was provided by the College of Veterinary Medicine, University of Baghdad, Iraq.

We thank R. Barhoumi for confocal microscopy and L. W. Stranahan for additional reading and editing of the manuscript.

FOOTNOTES

    • Received 27 October 2019.
    • Returned for modification 5 December 2019.
    • Accepted 2 January 2020.
    • Accepted manuscript posted online 13 January 2020.

  • Supplemental material is available online only.

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KEYWORDS

Brucella abortus
calcium matrix resorption
macrophages
mature osteoclasts
osteoarticular brucellosis
osteoclast precursors
vaccines

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