Antimicrobial peptides and eicosanoids : roles and interactions in inflammation and host defense

Abstract: Antimicrobial peptides (AMPs) have a broad spectrum of activities and exert their functions by both direct killing of microbes through the interaction with the microbial membrane, and modulation of innate and adaptive immunity of the host. In humans, cathelicidins and defensins represent the major families of AMPs. The only human cathelicidin is LL-37, a cationic, α-helical peptide with 37 amino acids in its sequence. Eicosanoids are a class of oxygenated hydrophobic compounds derived mainly from arachidonic acid (AA). The main pathways of eicosanoid biosynthesis are cyclooxygenase (COX) pathway and lipoxygenase (LOX) pathway, which metabolize AA into bioactive lipid mediators, namely prostaglandins, thromboxanes, leukotrienes and lipoxins. Eicosanoids exert important functions in normal homeostasis as well as in various pathological conditions. In the present thesis, we investigated the roles and interactions of AMPs and eicosanoids in inflammation and host defense towards bacteria, more specifically, in regulation of macrophage functions in innate immunity. Efficient phagocytosis is an important step in the clearance of invading bacteria and host defense. We demonstrate that LL-37 up-regulates the expression of Fcγ receptors (FcγRs) CD64 and CD32, leading to an enhanced phagocytosis capacity of IgG-opsonized bacteria in human macrophages. Using the subcutaneous air pouch model in cathelicidin deficient mice, we further demonstrate the effect of LL-37 on the expression of FcγRs and bacterial phagocytosis in vivo. LL-37 interacts with host cells in many ways. In our study, LL-37 internalization by human macrophages is characterized and we could also demonstrate that macrophages take up LL-37 derived from neutrophils. Further studies show that LL-37 internalization contributes to intracellular bacteria killing by macrophages. Together with the finding that LL-37 enhances bacterial phagocytosis, we conclude that LL-37 enhances the ability of human macrophages to kill bacteria via promoting bacterial phagocytosis, as well as via lysosome accumulation, and ROS production triggered by internalized LL-37. Macrophages can be triggered to produce large amounts of eicosanoids, participating in discrete stages of inflammation. We observe that LL-37 induces a biphasic release of eicosanoids from human macrophages. At early time points (1 h) LTB4 is produced, while induction of COX-2 expression and TXB2 and PGE2 production is observed at a late phase (8 h). The purinergic receptor P2X7R is involved in LL-37 triggered early phase eicosanoid production in human macrophages. Furthermore, LL-37 internalization seems to be required for eicosanoid production. More importantly, we confirm the involvement of cathelicidin in eicosanoid production in vivo. Prostaglandin (PG)E2 is a multifunctional lipid mediator in host defense. Our studies show that PGE2 suppresses the basal level of AMPs in human macrophages, and also VD3-induced expression of cathelicidin. The effect of PGE2 on AMP expression is transduced via EP2/EP4-cAMP-PKA regulated downstream transcription factors CREM/ICER and VDR. Of clinical relevance, we report that PGE2 impairs VD3-induced expression of cathelicidin and concomitant activation of autophagy during Mtb infection, and facilitates intracellular Mtb growth in human macrophages. Collectively, our findings indicate that PGE2 plays deleterious roles in human Mtb infection. Together, the results of the present thesis reveal the modulatory effects of LL-37 and PGE2 in macrophage functions towards bacteria, from multiple perspectives. Moreover, the interactions of AMPs and eicosanoids in macrophages expand our understanding of the inflammatory mediator network and could provide opportunities for future pharmacological intervention in infectious or inflammatory diseases.