In the research of rheumatoid arthritis etiology, the idea of a bacterial infection leading to arthritis is becoming increasingly popular [43–45]. Bacterial infections increase the production of NO, and can lead to inflammation. Inflammation then leads to chondrocyte apoptosis [30–35]. In cartilage tissue that is rich in extracellular matrix and with few cells in hard to reach places, clearance of dying cells is especially troublesome and can lead to the development and perpetuation of inflammation, which in turn leads to cartilage and bone degradation [46–48]. The idea of mycoplasmal infections, as the triggering factor of rheumatoid arthritis, has been revisited several times. Still, the role of mycoplasmas that have been detected in synovial fluids and serums of patients suffering from arthropathies remains unclear [49–54]. Factors impeding these studies are many. Because of their obligatory parasitism and complex nutritional demands, mycoplasmas are hard to cultivate. In addition, mycoplasmas adapt to the host in a way that induces subclinical infections that pass with little or no inflammation. Furthermore, mycoplasmal antigens, many of which are enzymes, can affect cells even after bacteria are dead and lysed, and their presence is difficult to detect by certain conventional methods . Mycoplasma species are also well known to cause arthritis in certain animal species, including chickens [1–7]. Experimental infections of chickens with M. synoviae WVU 1853 resulted in thinning of the articular cartilage 25 days post infection . Visible cartilage erosion appeared about two weeks later, presumably due to the presence of increasing numbers of heterophils. It seems likely that M. synoviae could also affect CCH. This assumption is supported by our observations that synovial fluids of chickens infected experimentally into hock joints contained antibodies to CCH and cartilage proteins (Dušanić et al., unpublished observations). This suggests CCH death and tissue destruction, leading to consequent production of autoantibodies against cartilage and CCH proteins. Many Mycoplasma species invade non-phagocytic cells and interfere with apoptosis (Table 1) [9–22]. M. synoviae is capable of invading CCH . The interactions between CCH and M. synoviae are likely to occur via binding of M. synoviae hemagglutinin VlhA to CCH receptors containing Sia (α 2-6) gal and Sia (α 2-3) gal, and their desialylation with M. synoviae neuraminidase [8, 56], Dušanić et al. unpublished observations]. Its effect on cell viability has, however, not been reported before this study.
This is the first report of mycoplasma-induced apoptosis in chondrocytes. The XTT proliferation assay was performed on chicken chondrocytes infected with M. synoviae and a major decrease in cell respiration was noted, suggesting cell death had occurred (Figure 1). Chondrocyte morphology, evaluated under phase contrast microscope and confocal microscope combined with anti-CD44 receptor and propidium iodide staining additionally indicated cytopathology with apoptotic features in chondrocytes infected with M. synoviae (Figures 2 and 3).
RT-qPCR analysis, performed on 15 genes involved in apoptosis (Table 2, Figure 4), involved NO production in apoptosis induction. The Griess assay confirmed secretion of NO to supernates (Figure 6). NO production is closely linked to chondrocyte apoptosis and cartilage loss in human arthropathies such as osteoarthritis and rheumatoid arthritis [30–33]. Also, NO production was shown to be strongly upregulated in chicken macrophages infected with M. synoviae or in contact with its lipoprotein MSPB . Both endogenously and exogenously produced NO is capable of inducing chondrocyte apoptosis, but the analysis here was limited to NO produced by chondrocytes as a result of M. synoviae infection. As expected, Mapk11 (encoding p38B, the major inducer of NO-linked apoptosis) was upregulated as well, 24 h and 48 h after infection. It has been reported that p38 causes upregulation and stimulation of caspase 3 activity, and upregulation of NFκB transcription factor [34, 35]. In our study, upregulation was noticed in genes encoding both caspase 3 and NFκB1, as well as in genes encoding other related apoptotic proteins (Figures 4 and 5).
Increased transcription of caspase genes is the most probable explanation for the formation of apoptotic bodies and cell shrinkage in infected CCH . Although genes encoding endonuclease G, which is released from the mitochondria during apoptosis, and Bak1, a pro-apoptotic mitochondrial protein, were not upregulated, another mitochondrial nuclease, encoded by AIFM1, was strongly upregulated 72 h after infection, indicating mitochondrial involvement (Figures 4 and 5). Occurrence of Fas receptor mediated apoptosis, which has been reported in chondrocytes , was disregarded due to unchanged transcription of both Fas and FASLG during CCH infection with M. synoviae (Additional file 1, Figure S1). Apoptosis inhibitor, encoded by BCL2, was slightly upregulated after 72 h, but so was htrA3, encoding the inhibitor of Bcl2. The expression of another apoptotic inhibitor, encoded by XIAP, remained unaltered (Additional file 1, Figure S1).
Taken together, our results indicate that M. synoviae induces endogenic nitric oxide mediated, caspase 3 and 8 dependent apoptosis in chicken chondrocytes, which involves a loss of mitochondrial function (Figure 5).
5-fluorouracil was used as a positive control of the experiment due to its well established apoptotic effect on other cell lines . Compared to M. synoviae, it induced a stronger and faster apoptotic response in CCH (Figure 4, Additional file 1, Figure S1, Figures 2 and 3). The profile of gene expression modulations indicate a Fas receptor mediated, caspase 3 independent cell death involving mitochondrial inactivation. Interestingly, the Jurkat cell line that was used as a positive control of the viability test shows great susceptibility to M. synoviae infection (Figure 1).
Although live intracellular M. synoviae were re-cultivated in our previous study 24 and 48 h after infection, the percentage of CCH invasion was relatively low (1.2 ± 0.3 for the type strain WVU 1853) . This study demonstrates a high susceptibility to cell death in infected chondrocytes. This suggests that M. synoviae affects CCH both from the outer side of the membrane, probably in a Fas independent manner, and from its location within the cell. It is also possible that mycoplasmas die in cell culture due to their complex nutritional demands, but their antigens continue to stimulate CCH and cause stress. An example of macrophage stimulation to produce NO, IL6 and IL1β by M. synoviae lipoprotein MSPB has been reported previously .
With the exception of caspase 3, patterns of apoptotic gene upregulation were not confirmed on the protein level due to the scarcity of the appropriate specific antibodies to chicken proteins or enzymes involved in apoptotic signal transduction cascades. Even with caspase 3, the antibody used was not specific enough to recognize the cleaved form of the protein (Additional file 2, Figure S2). Still, correlations between demonstrated cytopathologic changes, results of the XTT tests, Griess assay and gene expression data indicate apoptosis and not necrosis as the main mechanism of cell death. As an additional confirmation, CCH infected with M. synoviae were labeled using Annexin V-FITC (AV) and propidium iodide (PI) to discriminate between live (AV-PI-), early apoptotic (AV+PI-), late apoptotic (AV+PI+) and primary/secondary necrotic cells (AV-PI+). The results show an increase in the percentage of cells with phosphatidylserine exposed on the outer membrane layer, to which AV is bound, in the first two days of infection. This was followed by an increase in cells labeled only with PI, indicating secondary necrosis (Additional file 3, Figure S3).
In conclusion, this study provides the first demonstration of mycoplasma-induced apoptosis of chondrocytes. This process may lead to the development of M. synoviae-induced infectious synovitis in the chicken, a disease that in many factors resembles human infectious arthritis. It also supports the hypothesis that mycoplasma-induced arthritic conditions in animals might be useful models for understanding the role of mycoplasmas in similar human diseases. Hence, the study may have value outside the immediate interest of avian arthritis.