MyD88-deficient macrophages show comparable binding but reduced uptake of Bb.
The macrophage is an essential cellular element of the human inflammatory response to the LD spirochete (7). Macrophages have also been shown as part of the inflammatory cell infiltrate in heart and joint tissue of mice experimentally infected with Bb (10, 52), and the importance of MyD88 in Bb clearance from mouse tissues has been previously reported (40, 53, 54). It has also been well established that MyD88 enhances phagocytosis of multiple bacterial species by macrophages (28, 32, 34, 35, 55). To better understand the contribution of MyD88 to spirochete binding, uptake and degradation by macrophages, we utilized an ex vivo macrophage model using WT and MyD88−/− BMDMs co-incubated with Bb at MOIs of either 10:1 or 100:1 for 1, 4 or 6 hours. To quantify binding percentages, we imaged macrophages by confocal microscopy and determined the number of cells with spirochetes either attached to the surface or internalized because internalized spirochetes had to bind to macrophages before being taken up (Fig. 1A, yellow and white arrows respectively). We used the same confocal images and total cell numbers to quantify uptake percentages based on the number of cells with internalized spirochetes. The percentages of cells with spirochetes either bound or internalized were comparable between WT and MyD88−/− BMDMs at all three time points irrespective of MOI (Fig. 1B and 1C). While macrophages of both genotypes were able to phagocytose Bb, MyD88−/− BMDMs showed significantly reduced spirochete uptake compared to WT BMDMs at MOI 10:1 (Fig. 1D). Increasing the MOI to 100:1 significantly enhanced uptake in both cell genotypes, but MyD88−/− BMDMs never reached the phagocytic potential of their WT counterparts (Fig. 1E). These results further support the necessity of MyD88 signaling for efficient phagocytosis of Bb, irrespective of contact time with the spirochete.
TLR2, TLR7 and MyD88 are recruited to Bb-containing phagosomes in macrophages.
Once spirochetes are phagocytosed by macrophages, recruitment of TLR and MyD88 proteins to the phagosome is essential to trigger MyD88-dependent signaling cascades (56–59). Importantly, we have demonstrated that in human monocytes TLR2 and TLR8 co-localize to endosomes containing Bb (19). In addition, other investigators have shown a prominent role for TLR7 in the Bb inflammatory response (60). Murine TLR8, unlike murine TLR7 and human TLR8, does not seem to utilize ssRNA as its ligand (61). We therefore next characterized co-localization of TLR2, TLR7 and MyD88 with phagosomes containing Bb in BMDMs. By confocal microscopy, we observed that in WT BMDMs there is colocalization of MyD88 (Fig. 2A), TLR2 (Fig. 2B) and TLR7 (Fig. 2C) with Bb-containing phagosomes. Signals from MyD88 and TLR2 distinctly overlap with Bb GFP signals from phagosomes showing evidence of coiled or degraded spirochetes (Fig. 2A and 2B, graphs), but the intensity of MyD88 or TLR2 signal observed was higher with phagosomes containing degraded spirochetes. We also noted that TLR2 was expressed on the cell membrane and showed colocalization with surface-bound spirochetes (Fig. 2B). The absence of fluorescence in controls with secondary antibody only confirmed that this colocalization was not due to spectral overlap between color channels (Figure S2). TLR7 only showed strong signal with phagosomes containing partially degraded Bb but did not colocalize with surface-bound or recently internalized spirochetes (Fig. 2C). Taken together, these data confirm that endosomal TLR2, TLR7 and MyD88 colocalize to Bb-containing phagosomes to facilitate recognition of bacterial ligands and early response to infection.
Lack of MyD88 does not affect degradation of Bb in the phagosome.
Degradation of the spirochete in the phagosome is crucial to expose bacterial ligands for recognition by endosomal TLRs (17). This process, known as phagosome maturation, requires reduction of phagosome pH and fusion with lysosomes (62). Given that both WT and MyD88−/− BMDMs bind and internalize Bb, we next sought to determine if spirochetes are similarly degraded in phagosomes with and without MyD88. Confocal images taken after a 6-hour stimulation at MOI 10:1 showed that both WT and MyD88−/− BMDMs contained degraded GFP + Bb within the cell actin matrix (Fig. 3A and 3B). To assess phagosome maturation, we quantitated recruitment of LAMP-1 to Bb-containing phagosomes by looking at colocalization of LAMP-1 and GFP fluorescence intensity (63). Both WT and MyD88−/− BMDMs showed comparable LAMP-1 and Bb colocalization in phagosomes (Fig. 3A and 3B, graphs). Colocalization between Bb and LAMP-1 was measured in multiple phagosomes in BMDMs from both genotypes and no significant differences were found (Fig. 3C). To confirm MyD88 signaling in response to Bb we also measured cytokine secretion after 1, 4 and 6 hours of incubation with spirochetes. WT BMDMs showed significant increase in IL-6, TNFα and IL-10 secretion in the presence of spirochetes, whereas MyD88−/− BMDMs did not. (Figure S3A-C). Consistent with prior studies by Behera et al (2006), both WT and MyD88−/− BMDMs secrete the macrophage chemokine CCL2 (Figure S3D).
Bb ligand recognition appears to occur solely from within the phagosome.
To test for the presence of bacterial products in the cytosol, we measured cleaved caspase-1, which is indicative of inflammasome activation. Western blot analysis of WT BMDM cell lysates and supernatants showed no activation of caspase-1 by stimulation of Bb alone (Fig. 3D), which is consistent with previously published studies (64). However, in discordance with previous studies (65), we did not see cleavage of IL-1β (Fig. 3D) unless exogenous ATP was added to the stimulation. To further confirm lack of NLRP3 inflammasome activation, we assessed Apoptosis-associated speck-like protein containing a CARD (ASC) in BMDMs stimulated with either Bb or Staphylococcus aureus (Sa) for 30 minutes or 6 hours (Figure S4). As previously reported (66) (Figure S4A and S4C), ASC activation was observed with Sa, but no ASC was observed in BMDMs stimulated with Bb at 30 minutes or 6 hours (Figure S4B and S4D). Thus, recognition of Bb ligands appears to occur solely within the phagosome.
MyD88-dependent signaling causes differential expression of genes in macrophages that promote the inflammatory response.
Our results above show that MyD88 expression in macrophages enhances their capacity to phagocytose spirochetes (Fig. 1). To gain a better understanding of events that occur downstream of signaling by MyD88 which result in this phenotype presentation, we performed RNA-sequencing on WT and MyD88−/− BMDMs stimulated with Bb for 6 hours. This time point was selected based on our data in Fig. 3 showing comparable maturation in both WT and MyD88−/− BMDM phagosomes. We sequenced RNA from WT BMDMs at a MOI of 10:1 and MyD88−/− BMDMs at a MOI of 100:1 for a comparative analysis because the uptake percentages were not significantly different between the two cell phenotypes under these conditions (Fig. 4A). Both WT and MyD88−/− BMDMs showed differentially expressed genes (DEGs) when compared to their respective unstimulated controls. We noted that the number of DEGs in WT BMDMs was much higher than in MyD88−/− BMDMs (2818 genes vs 141 genes respectively) (Fig. 4B). We saw similar numbers of up- and down-regulated DEGs in WT BMDMs (52% and 48%) (Fig. 4B). In the MyD88−/− BMDMs, approximately 83% of the DEGs were up-regulated (Fig. 4B). We classified the DEGs into three categories for further analysis: genes differentially expressed only in WT BMDMs (MyD88-dependent); genes differentially expressed in both WT and MyD88−/− BMDMs (MyD88-independent); and genes that were differentially expressed only in MyD88−/− BMDMs (MyD88-privative) (Fig. 4C).
Similar inflammatory and chemotactic processes are enriched regardless of MyD88-mediated signaling but utilize different regulatory proteins.
MyD88-dependent mechanisms of inflammation have been well characterized, but little work has been done to understand the drivers of Bb-induced inflammation in the absence of MyD88. To address this issue, we next completed a comprehensive bioinformatics analysis to gain insight into how the DEGs are regulated within Bb-infected macrophages, both in the presence or absence of MyD88. We first identified transcription factors with potential binding sites in the promoter regions of the DEGs for each of the three subsets (66 for MyD88-dependent, 201 for MyD88-independent, and 39 for MyD88-privative). We then identified master regulator proteins upstream of these transcription factors and performed a Gene Ontology (GO) enrichment analysis of each group. Because data shown in Fig. 1 and Figure S3 indicate that in macrophages MyD88 affects both the inflammatory response and uptake of spirochetes, we focused our analysis on identifying whether any master regulators enriched to inflammatory and/or phagocytic biological processes in the MyD88-dependent and -privative conditions. Interestingly, similar inflammatory biological processes enriched to both the MyD88-dependent (including MyD88, Irak2 and Ly96) and MyD88-privative (including Vcam1 and Cxcl2) master regulators (Fig. 4D and 4E), but the individual master regulators involved were different for each subset (Fig. 4E). Importantly, over three times as many master regulators were identified for the MyD88-dependent DEGs than the MyD88-privative DEGs (Fig. 4E), suggesting that MyD88 signaling controls activation of more master regulators in the cell to control expression DEGs and enables the cell to perform unique processes in response to bacterial pathogens such as Bb.
MyD88-privative master regulators are involved in multiple chemotactic biological processes not enriched in WT BMDMs.
We also observed significant overlap between the chemotactic biological processes enriched in MyD88-dependent and MyD88-privative master regulators. However, MyD88-privative master regulators significantly enriched to multiple biological processes involved in chemotaxis that were not enriched in MyD88-dependent master regulators (Figure S5A), suggesting that the lack of MyD88 signaling allows for increased up-regulation of processes to facilitate cell migration into the tissues. The MyD88-privative master regulators involved in these chemotactic processes also enriched to inflammatory processes (Figure S5B and Fig. 4E), suggesting that Bb may trigger other signaling cascades which induce inflammation more skewed to cell recruitment and localization.
MyD88 is a master regulator for transcription factors that control the MyD88-dependent DEGs enriched in uptake processes.
Based on our observation that the presence of MyD88 enhances phagocytosis (Fig. 1), we also analyzed whether any of the MyD88-dependent DEGs enriched to biological processes related to uptake. We identified 164 MyD88-dependent DEGs that enriched to five different biological processes relating to phagocytosis (Actin Filament Polymerization, Regulation of Cell Shape, Actin Cytoskeleton Organization, Cytoskeleton Organization, and Actin Filament Organization). Of particular interest, Daam1 and Fmnl1, encoding two proteins known to play a role in phagocytosis of Bb (15, 37), were differentially expressed in an MyD88-dependent manner. Daam1, which was up-regulated, is a formin protein that bundles actin fibers together to increase stability of coiling pseudopods, which are more adept at capturing the highly motile spirochetes (67). In contrast to Daam1, Fmnl1 was down-regulated in response to Bb. Fmnl1 is also a formin protein that severs actin branches to promote polymerization and increase filopodia protrusion (67). To determine whether MyD88 is a master regulator in any of these processes, we first identified transcription factors that map to promoter regions of the enriched DEGs. Analysis of these transcription factors revealed that Zic1 and Zeb1 have the capacity to bind to the promoter regions of several of the MyD88-dependent DEGs that significantly enriched to processes associated with bacterial uptake (Fig. 5). Zic1 is controlled by the intermediate protein ApoE, which is known to play a role in cholesterol metabolism in macrophages (68) and the absence of ApoE increases Bb burdens in experimentally infected mice (68). We then used OCSANA, a specialized package available in Cytoscape (50) to link MyD88, as a master regulator, with transcription factors that map to DEGs in this specific subset. Based on this information we constructed a network illustrating potential links between MyD88-mediated signaling and up-regulated DEGs that may contribute to enhanced phagocytic capability seen in WT cells. The network (Fig. 5) shows Rhoa, Akt1, Rac1 and Cdc42 as genes that code for proteins which appear as intermediates on the network, meaning that their genes weren’t differentially expressed in our analysis. Daam1 regulates Rhoa activity, which controls Cdc42, Rac1 and Akt1. Cdc42 activates a Rho GTPase, Rhoq, which is up-regulated in response to Bb. Rac1 and Akt1, when translated, both activate multiple proteins whose corresponding genes are also up-regulated, indicating that while the genes for these intermediate proteins aren’t differentially expressed, they are still active in macrophages that have been stimulated with Bb. Taken together, these data suggest that MyD88 signaling upregulates multiple gene products involved in regulating macrophage membrane protrusions. Upregulation of these genes likely contributes to the reorganization of cell machinery that enhances the capability of the WT macrophage to take up spirochetes.