K. kingae strain was isolated from the joint fluid of a patient attended to at the Hospital de Clínicas “José de San Martín”, Buenos Aires, Argentina. It was grown on chocolate agar (Chocolate II Agar; BD, Franklin Lakes, NJ) or Brain Heart Infusion broth (BHI) with 5% of hemoglobin at 37°C with 5% CO₂. The stocks were stored at -80°C in BHI with 30% of glycerol. The bacterial number in culture was estimated by comparing optical densities at 600 nm with a standard curve obtained in our laboratory.

To obtain the standard curve, the spectrophotometer was calibrated using BHI as the blank reference. A single colony of K. kingae was inoculated into 1 ml of BHI and then incubated at 37°C with 5% CO₂ for 48 h. Then, serial dilutions of the inoculum were performed and used to determine the OD600, and another aliquot was plated on chocolate agar plates and incubated overnight at 37°C with 5% CO₂ to determine the colony-forming unit counts.

To prepare the inoculum, cultures were diluted in sterile phosphate-buffered saline (PBS) to the desired bacterial concentration.

To obtain heat-killed K. kingae (HKKk), the bacteria were washed five times for 10 min each in sterile PBS, heat-killed at 60°C for 1 h, aliquoted, and stored at -70°C until they were used. The total absence of K. kingae viability after heat killing was verified by the absence of bacterial growth on chocolate agar.

All experiments were performed in BSL-2 and BSL-3 facilities at the INBIRS.

K. kingae was grown on chocolate agar plates for 72 h, and the bacteria were scraped from plates and harvested by centrifugation at 6,000 × g. The cell-free supernatant was sterilized through a 0.22-µm-pore-size filter. Then, the supernatant was ultracentrifuged at 150,000 × g for 6 h at 4°C to pellet the vesicles. The supernatant was removed, and the pellets were resuspended in PBS with 20% glycerol and 2 mM CaCl₂. The protein concentration was measured using a bicinchoninic acid assay (Pierce). The OMV were stored at -80°C. Between 9 to 14 µg of OMV was obtained from 1 × 10¹⁰ bacteria.

Unless otherwise specified, all experiments were performed at 37°C in 5% CO₂ atmosphere. THP-1 cells (human monocytic cell line) were obtained from the American Type Culture Collection (Manassas, VA) and grown in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (Gibco-BRL, Life Technologies, Grand Island, NY), 100 U/ml of penicillin, and 100 mg/ml of streptomycin (complete medium). To induce maturation, the cells were cultured in the presence of 150 U/ml of 1,25-dihydroxyvitamin D3 (Endogen) for 72 h. For the experiments, 5 × 10⁵ cells/ml were stimulated with HKKk at a final concentration of 1 × 10⁵ and 1 × 10⁶ bacteria/ml or infected with K. kingae at multiplicities of infection of 1 and 10 for 2 h in medium without antibiotics. Then, the cells were extensively washed to remove extracellular bacteria, and the infected cells were maintained for an additional 24 h in a complete medium with 50 μg/ml of gentamicin. The supernatants were harvested and stored at -80°C for the determination of cytokine and osteoclastogenesis experiments. The viability of THP-1 cells after the addition of K. kingae (measured using trypan blue exclusion test) was preserved between 90 to 95% irrespective of the inoculum used.

The secretion of human and mouse IL-1β, IL-6, and TNF-α in the supernatants was quantified with an ELISA kit (BD PharMingen, San Diego, CA, USA) and the production of human and mouse RANKL by an ELISA kit (R&D Systems Minneapolis, MN, USA).

RAW 264.7 cells (mouse macrophage), at a concentration of 5 × 10⁴ cells/well, were cultured onto glass coverslips in 24-well plates for 7 days and cultured in a complete medium containing 30 ng/ml macrophage colony-stimulating factor (M-CSF) and 0.2 ml of culture supernatants from K. kingae-infected THP-1 cells. As positive controls of osteoclast formation, the RAW 264.7 cell cultures received 50 ng/ml murine RANKL. On day 3, the culture medium and all reagents were replaced. To identify osteoclasts, the cells were fixed in 4% paraformaldehyde and stained for tartrate-resistant acid phosphatase (TRAP) (Sigma-Aldrich, St. Louis, MO, USA). TRAP-positive, multinucleated (more than three nuclei) cells were defined as osteoclasts, and the number was determined by microscopic counts. For each well, TRAP-positive multinucleated cells were calculated by counting their number in five microscopic fields (×20).

RAW264.7 cells were infected with K. kingae at multiplicity of infection (MOI) = 1 or stimulated with HKKk (1 × 10⁵ bacteria/ml) in a complete medium without antibiotics containing 30 ng/ml M-CSF. Alternatively, RAW264.7 cells were exposed to the bacteria or HKKk during 2 h, then extensively washed with culture medium to remove extracellular bacteria, and incubated in complete medium containing 30 ng/ml M-CSF with 50 μg/ml of gentamicin. To determine the role of OMV, osteoclast differentiation was performed in the presence of HKKk (1 × 10⁵ bacteria/ml) and different amounts of OMV (10, 1, and 0.1 μg/ml.). The presence of TRAP-positive multinucleated cells was determined as was previously described.

Osteoclast differentiation assay was performed on hydroxyapatite disks (BD BioCoat Osteologic (BD Biosciences, San Diego, CA, USA) for 9 days. The media and all reagents were replaced every day to avoid the acidification of the medium. After culturing with cells, the hydroxyapatite discs were washed with 1 M NH₄OH to remove adherent cells. After rinsing with water, the hydroxyapatite discs were visually examined by light microscopy, and the area of resorption was calculated using the NIH Image J analysis software (http://rsbweb.nih.gov/ij/). The results were expressed as the percentage of the total area of the well that appears resorbed.

Neutralization experiments were performed using anti-TNF-α-neutralizing antibody (clone MAb1) or its isotype control (both from BD Biosciences, San Diego, CA) and anti-IL-1β-neutralizing antibody (clone AS10) or its isotype control (BD Biosciences). In the neutralization experiments with anti-TNF-α antibody or anti-IL-1β antibody, the conditioned medium was preincubated with the corresponding antibody (or isotype control) for 1 h at 37°C before use.

Cell proliferation/viability was measured by MTT colorimetric assay (Sigma-Aldrich, Argentina) at 7 days post-infection or stimulation with HKKk and OMV, according to the instructions of the manufacturer. The absorbance was measured by using a microplate reader at 570 nm. The OD of each well was quantified as a percentage compared with the untreated cells. All experiments were carried out in triplicate.

Statistical analysis was performed with one-way ANOVA. Multiple comparisons between all pairs of groups were made with Tukey’s post-test, and those against two groups were done with Student᾽s t-test and Mann–Whitney test. To determine normality, the Shapiro–Wilk normality test was used. Graphical and statistical analyses were performed with GraphPad Prism 7.0 software. Each experiment was performed in triplicate with different culture preparations on three independent occasions. Data were represented as mean ± SD measured in triplicate from three individual experiments.

It is known that some proinflammatory cytokines could have the ability to promote or inhibit osteoclast differentiation directly or via the RANKL/OPG system (30). Thus, the production of IL-6, TNF-α, IL-1β, and RANKL in culture supernatants from THP-1 monocytes in response to K. kingae infection was examined. Infection of human monocytes with K. kingae performed at MOIs of 1 and 10 elicited the secretion of IL-6, TNF-α, and IL-1β at 24 h post-infection in an inoculum-dependent manner ( Figure 1A ). On the contrary, RANKL secretion was not detected in monocytes infected with K. kingae.

To elucidate whether viable bacteria were necessary to induce proinflammatory cytokine secretion, monocytes were stimulated with HKKk. The production of IL-6, TNF-α, and IL-1β was also markedly increased in culture supernatants from monocytes that were stimulated with HKKk when compared with unstimulated cells ( Figure 1B ). Additionally, the levels of cytokines induced by HKKk were comparable to those produced by similar amounts of live bacteria ( Figure 1 ). These results indicate that human monocytes exposed to K. kingae can produce proinflammatory cytokines but do not produce RANKL. The ability of HKKk to produce proinflammatory cytokines also suggests that the inflammatory response could be induced by a structural component of the bacteria.

Osteoclasts originate from the fusion of precursors from monocyte/macrophage lineage in the bone marrow (31, 32) and play a key role in bone resorption. The differentiation process could involve soluble mediators belonging to inflammatory cells, such as monocytes/macrophages, in combination with M-CSF (33, 34). The role of IL-6 in the induction of osteoclast differentiation appears to be controversial and mainly indirectly mediated through osteoblast stimulation (30, 35–37). On the contrary, TNF-α and IL-1β have been implicated in RANKL-independent osteoclast formation (30, 34). To determine if soluble mediators secreted by K. kingae-infected human monocytes could stimulate osteoclast differentiation from RAW 264.7, these cells were stimulated with M-CSF in the presence of culture supernatants from K. kingae-infected human monocytes, and osteoclastogenesis was evaluated by the generation of TRAP-positive multinucleated cells. RANKL was used as a positive control. The production of osteoclast-like cells was generated by culture supernatants from K. kingae-infected human monocytes but not by those from uninfected cells.

To determine the role of TNF-α and IL-1β in the osteoclastogenesis elicited by K. kingae, stimulation experiments were performed as described above but in the presence of an anti-TNF-α- or an anti-IL-1β-neutralizing antibody. The TNF-α blocking antibody—but not the isotype control—completely inhibits osteoclastogenesis induced by culture supernatants from monocytes infected by K. kingae ( Figure 2 ). Osteoclastogenesis was likewise significantly reduced in experiments performed with culture supernatants treated with IL-1β-blocking antibody in which the presence of binucleated cells could also be detected. These results indicate that monocyte-secreted TNF-α and IL-1β induced by K. kingae could be involved in the bone resorption observed in patients.

To better understand the osteoimmunological features involved in K. kingae-induced osteoclastogenesis, experiments were conducted to evaluate the ability of RAW 264.7-derived osteoclast to secrete cytokines in response to culture supernatants from K. kingae-infected THP-1 monocytes in the presence or not of neutralizing antibodies. Our results indicated that the addition of culture supernatants from K. kingae-infected human monocytes to uninfected murine osteoclast precursors induced a significant secretion of TNF-α, IL-1β, and IL-6 compared with unstimulated cells or cells stimulated with culture supernatants from uninfected monocytes. In addition, blocking of TNF-α and IL-1β significantly reduced the ability of culture supernatants from K. kingae-infected human monocytes to induce proinflammatory cytokine secretion, whereas the isotype control had no effect ( Figure 3 ). Considering the species specificity of the antibodies used in the ELISA kit for cytokine measurement, the number of secreted factors present in culture supernatants from stimulated murine osteoclast precursors did not cross-react with human cytokines (not shown). These results indicate that the culture supernatants from K. kingae-infected monocytes activate proinflammatory cytokine production on osteoclast precursors.

To elucidate whether K. kingae infection can generate a microenvironment favoring osteoclastogenesis and bone loss, we have assessed the functional activity of K. kingae-induced osteoclast-like cells by their ability to resorb hydroxyapatite. The K. kingae-infected human monocytes were able to induce significant hydroxyapatite resorption. In contrast, culture supernatants from uninfected cells did not ( Figure 4 ). Additionally, the resorption of hydroxyapatite was significantly inhibited either by anti-TNF-α antibody or by anti-IL-1β antibody but not by the isotype control. These results together indicate that K. kingae-infected monocytes released cytokines, such as TNF-α and IL-1β, that promote functional osteoclast formation.

OMV from K. kingae have been previously implicated in the ability to induce cytokine production from osteoblast and synovial fibroblasts (4). Therefore, experiments were conducted to assess whether stimulation with K. kingae-released OMV can modulate the cytokine responses of human monocytes. To this end, cytokine production was measured in culture supernatants from THP-1 cells exposed to 0.1, 1, and 10 μg/ml of OMV. Our results indicated that THP-1 cells exposed to OMV during 4 and 24 h did not induce IL-1β, TNF-α, and IL-6 secretion ( Figure 5 ).

OMV have a range of immunomodulatory outcomes to stimulate or suppress immune cell responses through their direct effects on host cells (38). Therefore, experiments were conducted to assess whether pre-exposure with K. kingae-derived OMV could modulate cytokine release from human monocytes in response to K. kingae infection. As shown in Figure 5 , the level of TNF-α, IL-1β, and IL-6 released from THP-1 cells pre-treated with OMV was significantly lower than from untreated cells. Such inhibitory effects of OMV appear to be dose dependent ( Figure 5C ). In conjunction, these results indicated that OMV from K. kingae could inhibit proinflammatory response monocytes to K. kingae infection.

Our results indicate that OMV are crucial for inflammatory responses induced by K. kingae. To assess the role of OMV in subsequent osteoclastogenesis induction in response to K. kingae infection, osteoclast differentiation was performed in the presence of M-CSF and culture supernatants from K. kingae-infected monocytes. Osteoclastogenesis was induced by culture supernatants obtained from K. kingae-infected human monocytes, but it was abrogated when cultured human monocytes were exposed to OMV before K. kingae infection ( Figure 6 ). Additionally, culture supernatants from uninfected monocytes treated with OMV were unable to induce osteoclastogenesis ( Figure 6 ). Collectively, these results indicate that cytokine response from human monocytes to K. kingae exposure may be conditioned by the OMV coexistence, even modulating its effects on osteoclastogenesis.

Live bacteria could contribute to OMV release during in vitro infection experiments. To measure the OMV capability to downregulate the osteoclastogenesis, human monocytes were incubated with different doses of OMV prior to HKKk exposure, and then the supernatants were collected for osteoclastogenesis experiments.

As shown in Figure 7 , the osteoclastogenesis was lower as the OMV dose was higher. In concordance, when monocytes were incubated with OMV before HKKk treatment, the secretion of IL-1β, TNF-α, and IL-6 was significantly lower than cells not pretreated with OMV. These results together indicate that K. kingae infection could have an opposite action on osteoclast differentiation which is dependent on the bacteria–OMV ratio.

The osteoarticular damage associated to K. kingae infection may also involve direct pathogenic mechanisms. To elucidate this contribution to osteoclastogenesis, the following experiments were carried out. First, cultured cells were infected with live K. kingae or stimulated with HKKk and maintained in the presence of M-CSF, without antibiotics. Second, the cultured cells were challenged as previously but during 2 h and then were washed and maintained in the presence of antibiotics. In both conditions, K. kingae infection was able to induce osteoclast differentiation ( Figure 8 ). However, when osteoclast differentiation was performed in the absence of antibiotics, the number of osteoclasts from cells treated with HKKk was significantly higher than those produced by live bacteria. The RAW 264.7 cell viability of K. kingae-infected ones or from those treated with HKKk was well preserved (80–90%). These results show that K. kingae can directly promote osteoclastogenesis beyond its viability. Paradoxically, the osteoclastogenic capability of live bacteria is lower than that of the heat-killed ones, thus insinuating that viable bacteria-released factor/s may downregulate it.

Since HKKk induces higher levels of osteoclastogenesis than live bacteria, we have examined the role of OMV in such osteoclast differentiation. To this aim, RAW264.7 cells were pretreated with different doses of OMV and then treated with HKKk. The OMV diminished osteoclastogenesis in a concentration-dependent manner ( Figure 9 ), but they were unable to induce osteoclastogenesis (not shown). These results together insinuate that K. kingae-mediated osteoclastogenesis may be counteracted directly by their OMV in a dose-dependent manner. The viability of RAW 264.7 cells exposed to HKKk and treated with OMV was preserved (80–90%).