Leena Meriläinen Heini Brander Anni Herranen Armin Schwarzbach Leona Gilbert
https://www.sciencedirect.com/science/a ... via%3Dihub
Macrophages are critical to fighting off B. burgdorferi infections in mice and humans , , , . It has been implied that the pleomorphic forms of this bacterium may help in the evasion from the immune system . Our study demonstrated that the induced pleomorphic forms are recognized and engulfed by differentiated macrophages in vitro, however; macrophages engulfed significantly more spirochetes than RBs per cell (Table 1). This result indicates a difference in the uptake of these two forms.
Macrophages form F-actin rich pseudopodia when they interact and phagocytose B. burgdorferi spirochetes , . Actin polymerization required for phagocytosis is mediated by CR3 (αMβ2) and Fcγ receptors . However, it is reported that internalization of unopsonized B. burgdorferi is independent of the Fc receptor . Furthermore, many studies have demonstrated B. burgdorferi internalization without opsonization , , , , . Here, we demonstrated that F-actin participates in the uptake of B. burgdorferi both spirochetes and RBs without opsonization suggesting that F-actin is important also in the Fcγ independent phagocytosis (Fig. 1). In addition, the cytochalasin D significantly diminished the colocalization of F-actin with both bacteria forms supporting this observation (Fig. 1G). Although the cell were synchronized in the beginning of the experiments, the standard deviations (SD) for colocalization were rather high due to the different rate and timing of phagocytosis between the cells.
Coiling phagocytosis is employed by approximately 60–70% of the macrophage cells for engulfment of B. burgdorferi spirochetes . Our results support the previous findings, where coiling phagocytosis is demonstrated for the spirochete uptake (Fig. 1A). However, such an extensive coiling and wrapping was not seen in RBs (Fig. 1B, D). F-actin accumulated in the phagocytosis site of macrophages, surrounded RBs and colocalized with them, nevertheless, within this study it remained unclear whether actual coiling phagocytosis occurred with RBs. RBs are nonmotile and they have completely different morphology compared to spirochetes that might explain why macrophages did not grow such a long and thin pseudopods and coil so profusely. There is also a possibility that spirochetes and RBs are internalized differently in macrophages: while spirochete uptake occurs via coiling phagocytosis, RBs internalization might rather utilize conventional phagocytosis.
After internalization with phagocytosis, bacteria are usually transported via the endocytic pathway to lysosomes and finally processed antigens are presented to T-cells by MHC II class molecules. The colocalization of B. burgdorferi spirochetes with immunolabeled lysosomal proteins , ,  as well as LysoTracker stained acidic compartments  has been previously reported. In those studies, colocalization was observed from early 10 min time points up to 5 h of stimulation. Nevertheless, the quantification of actual colocalization was performed in only one study , where 45% of the spirochetes colocalized with lysosomal endopeptidase enzyme cathepsin L at 10 min time point and 57% after 5 h of incubation. Here, to compare lysosomal processing of spirochetes and RBs in macrophages, colocalization analysis was executed at three different time points. The colocalization of spirochetes and RBs with Lamp2 remained at the same level at all three time points 2 h, 8 h, and 24 h (Fig. 2G). In this study, the colocalization percentage of spirochetes with lysosomes was found smaller than in the previous study . This difference may be due to the different analysis methods: in the previous study colocalization was analyzed using kinetic compartmental analysis, while in this study modern image analysis combined with Costes algorithm for statistical significances was used. Interestingly, RBs colocalized less with lysosomes compared to spirochetes at all time points, and the difference was significant after 24 h post-stimulation (Fig. 2G). This result indicates that spirochetes and RBs are processed differently in macrophages. It is suggested that coiling phagocytosis could lead to a presentation of antigens via MCH I molecules . If spirochetes and RBs are phagocytosed differently, that could explain their different processing in macrophages.
Cytokines are thought to have an important role in Lyme disease pathogenesis. The examination of cytokine profiles induced by B. burgdorferi spirochete and RBs indicated the production of immune-modulating mediators consistent with the cytokine analysis of C3H and C57 mouse bone marrow-derived macrophages stimulated with B. burgdorferi . Cytokines and chemokines expressed in our analysis (Fig. 3) as well as in previous mouse study were G-CSF, GM-CSF, IL-1β, IL-6, CXCL1, CXCL10, MCP-1, MIP-1α, MIP-1β, RANTES and TNF-α. Furthermore, the phagocytosis of spirochetes by human macrophages is reported to increase production of IL-1β, IL-6, IL-10, IFNγ, MCP-1 cytokines and MIP-1α, MIP-1β, TNFα, and CXCL10 chemokines . Anti-inflammatory IL-10 has an important role as a regulator of inflammatory responses in Lyme disease  and IFNγ correlates positively with the Lyme disease severity in humans . However, in this study neither spirochetes nor RBs induced IL-10 or IFNγ expression at a level that could be detected. In addition, we highlighted six cytokines with immunological relevance that has not been fully evaluated in Lyme disease (Fig. 3). The expression of CD54 (Fig. 3D), IL-16 (Fig. 3I), MIF (Fig. 3M) and Serpin E1 (Fig. 3P) indicated that there is an enhancement of adhesion, attraction and activation of immune cells. With respect to IL-8, significant decrease in production compared to media control for both spirochete and RB stimulated macrophages (Fig. 3H) could imply a cell specific response.
Furthermore, all of the expressed cytokines, except the IL-23 and MCP-1, were produced in higher levels when cells were stimulated with spirochetes. MCP-1 regulates the migration and infiltration of monocytes, T-cells, and NK-cells, and it potentially has a role in polarization of naïve T cells to Th2 type . MCP-1 expression is required for the development of experimental Lyme arthritis in mice , and it is thought to be associated with other autoimmune diseases as well . The higher expression levels of MCP-1, stimulated by B. burgdorferi RBs, could propose an involvement of the RBs in Lyme arthritis. In this study, RBs demonstrated decreased macrophage phagocytosis, differences in phagocytosis mechanisms and lysosomal processing compared to spirochetes. These differences in the entry and processing could correspond to the lower cytokine and chemokine production in general as well as to different cytokine profiles of spirochetes and RBs. The lower expression of these cytokines by RBs could suggest suppressive immune response against these forms, or immune control dysfunction.
The protein profiling of spirochetes and RBs have performed earlier with RPMI serum starved  and 1 d H2O induced RBs . These studies reported protein spots with molecular weights less than 97 kDa. Here, we observed protein spots ranging from 3.5 kDa to higher than 200 kDa from both spirochetes and RBs (Fig. S1A, Table 2). Our results correspond to the genome data of B. burgdorferi B31 that the bacterium has proteins of molecular weights from 3.3 kDa to 254.2 kDa . However, the 15 proteins detected with increased intensities in RBs were between the molecular weights of 15–40 kDa. Three proteins with higher protein expression in RBs (spots 11, 18 and 19 in Table 2) were determined corresponding to previously reported . In another study  6–16 kDa proteins were examined with mass spectrometry and the differences between spirochetes and 1 d H2O induced RBs were not found. In contrast to their results, we observed two 15 kDa proteins in RBs (spots 24 and 26 in Table 2) with elevated expression when compared to spirochetes. This difference may be due to the different exposure times to H2O, because the longer exposure time used may have decreased the protein expression.
In the previous Western blot analysis , 41 kDa (flagellar protein FlaB) and 46 kDa proteins exhibited less reactivity in 48 h serum starved RBs when probed with sera from two infected monkeys and one Lyme patient. In our study, all tested sera reacted against 41 kDa and 45 kDa antigen. These sizes correspond to very immunogenic antigens flagellar protein (FlaB) and VlsE, respectively, as previously reviewed . However, two patients reacted more against 41 kDa flagella on spirochetes, three against RBs, and three had equal response to both (Table 3). Furthermore, four patients reacted more against RB VlsE. These discrepancies to previous findings may be due to the variability of patients' reactivity against this specific antigen, or in the distinct induction method of RBs. In addition, the number of tested sera was higher in our study.
Interestingly, the bands with a molecular weight of 39, 60, and 66 kDa demonstrated predominantly higher intensity in RBs compared to spirochetes. This supports the 2D PAGE results where smaller intensity spots displayed higher intensity in RBs. The 39, 60, and 66 kDa bands correspond to a laminin binding protein BmpA , heat shock protein GroEL , and p66 , respectively. These bands have been previously reported for B. burgdorferi B31 western blots probed with patients' sera . Nonetheless, their reactivity against RBs has not been demonstrated before. The role of pleomorphic forms in Lyme disease has been previously criticized . Nonetheless, there are in vivo studies that connect RB's to pathogenesis of Lyme disease , , . Obviously, more studies are needed to demonstrate mechanisms how RB's are associated with Lyme disease. Overall, this data implies that patients react differently against B. burgdorferi spirochetes and RBs suggesting that RBs may have a role in Lyme pathogenesis. Different antigenicity between spirochetes and RBs could add value for diagnostic purposes.
The experiment design with in vitro cultured spirochetes and H2O induced RB's may not be fully equivalent to the conditions in vivo. However, these cultured bacteria have similarities when compared to the forms found in vivo. For example, the fluorescent B. burgdorferi strain, used in these experiments, has shown similar movement in situ studies when compared to the spirochetes in vitro . In this previous study  bacteria were adapted to mouse environment in 1% mouse blood before the experiment. Furthermore, this strain contains all the plasmids required for infectivity. H2O RB's present similar morphology in these studies when compared to RBs seen in the histopathological samples of the dogs with myocarditis , and from chicken primary neuronal, and astrocytic cells . In addition, RB's have been demonstrated to have specific staining properties with wheat germ agglutinin both in vitro  and in Langerhans cells in vivo .
Conclusively, these results imply that B. burgdorferi RBs have access to immune cells and have the ability to stimulate an immune response. However, there are differences in phagocytosis and processing of these two pleomorphic B. burgdorferi forms in macrophages. In addition, the immune response differs from spirochetes especially with lower expression of IL-1β, IL-1ra, MIF, MIP-1β and RANTES. Conversely, RBs stimulate a significantly higher expression amount of MCP-1. Spirochetes and RBs have differences in protein expression and they have different antigenic properties as seen in patients IgG responses. These results indicate that RBs may have the ability to induce distinct immune response compared to spirochetes and that they could be associated with different clinical symptoms seen in patients. We suggest that pleomorphic RBs should be included in the Lyme disease diagnostic tools. The recognition and detection of pleomorphic forms are detrimental to the proper diagnosis and treatment of B. burgdorferi infections.