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on can be a vital pathway that is necessary for the optimal phagocytosis of B. burgdorferi. MyD88 mediated uptake of B. burgdorferi requires the recruitment of Arp2/3 complexes Actin polymerization has been well characterized to be a driving force for the formation and extension of membrane protrusions, which is essential Beta-Lapachone for the effective phagocytosis of microbial organisms. PI3K signaling has been shown to play a crucial role in actin polymerization through activation of Rac. The Rho family members GTPases, Rac1 and CDC42, subsequently recruit Arp2/3 to type the actin complex. To decide whether or not the defect in B. burgdorferi uptake by MyD88 BMDMs was due to a loss of PI3K directed actin polymerization, we examined the localization with the Arp2/3 complex of actin with B. burgdorferi.
The cellular distribution of Arp2/3 complexes was evaluated by using an antibody directed against the 50 kDa Arp3 subunit with the Arp2/3 complex. At 5 min post B. burgdorferi infection, Arp2/3 was discovered clearly associated with contact points where B. burgdorferi had been adhered to the WT cell surface and throughout the whole length of Beta-Lapachone organisms as they are been taken up into WT cells. In contrast, recruitment of Arp2/3 co localized with B. burgdorferi attached to the surface of MyD88 cells was not observed. Similarly, BMDMs treated with the PI3K inhibitor also did not show co localization of Arp2/3 with attached B. burgdorferi. This suggests that MyD88 signaling is very important for the coordination of actin polymerization and efficient recruitment of Arp2/3 necessary for uptake of B. burgdorferi.
These data present further evidence Lomeguatrib that PI3K signaling pathway, by directing cellular distribution of Arp2/3 complexes, is necessary for MyD88 dependent phagocytosis of B. burgdorferi. Discussion A role for MyD88 in distinct aspects of phagocytosis, such as effects on uptake, phagolysosomal maturation, and oxidative killing, has been proposed. In this study, we investigated the mechanisms by which MyD88 participates within the phagocytosis of B. burgdorferi. We have previously shown that MyD88 plays a crucial role in uptake, but not phagolysosomal processing of B. burgdorferi. There have only been a few reports on the role of TLR signaling on the uptake of organisms. A study by Doyle et al. suggested that the role of MyD88 in uptake of organisms occurs through up regulation of particular phagocytic receptors, for example scavenger receptors.
Up regulation of particular Carcinoid Lomeguatrib scavenger receptors such as scavenger receptor A, macrophage receptor with a collagenous structure, and lectin like oxidized low density lipoprotein receptor 1, does happen in response to B. burgdorferi infection. Nonetheless, consistent with the results seen for induction of scavenger receptors by other organisms, up regulation of these receptors by B. burgdorferi appears to happen at a time point soon after uptake with the organism into the cells, suggesting that scavenger receptors will not be big contributors to the early uptake of B. burgdorferi seen in our phagocytic assays. Rather, we've shown that the uptake of B. burgdorferi is mediated by downstream signaling events activated in response to the organism.
We discovered that the role of MyD88 activation in phagocytosis may be replaced by activation with the other big TLR signaling adaptor, TRIF. By pre treating MyD88 cells Beta-Lapachone with a TLR3 ligand, poly I:C, which is able to activate downstream signaling through TRIF without having the involvement of MyD88, we had been able to restore the ability of MyD88 cells to phagocytose B. burgdorferi. The ability to restore phagocytosis with the addition of poly I:C confirms that there is not an intrinsic defect within the ability of MyD88 cells to take up B. burgdorferi and supplies clues as to the possible downstream pathways responsible for controlling phagocytosis of B. burgdorferi.
Activation downstream of TRIF occurs along two big pathways: 1) activation Lomeguatrib of TRAF3, which leads to a subsequent induction of type I interferon and activation of interferon responsive genes and Beta-Lapachone 2) activation of TRAF6 which leads to downstream activation of quite a few signaling pathways and translocation of NFkB. Activation of macrophages by type I and type II IFNs has been shown to improve phagocytic capacity of these cells. Nonetheless, unlike poly I:C, addition of IFN B was unable to restore phagocytosis of B. burgdorferi in MyD88 cells, making it unlikely to be the mechanism by which TRIF activation complements the loss of MyD88. Hence, we focused on pathways directly downstream of TRAF6 also as those that could be activated indirectly as a result of TRAF6 activation. We examined downstream pathways that could be activated by recognition of B. burgdorferi goods such as p38, ERK, JNK, PKC, JAK/STAT and PI3K making use of chemical inhibitors. Of these, only inhibition of PI3K blocked uptake of B. burgdorferi. Lomeguatrib PI3K can be a big regulator for phagocytosis of huge particles. Inhibition of PI3K can block new membrane formation at the web-site of particle internal