Abstract
The early innate immune response is a critical component of host defense, driving the activation of early inflammatory signaling and cytokine responses that lead to cellular activation, cellular recruitment, and microbicidal activity to aid in the clearance of invading microbes and the resolution of inflammatory states. The detection of microbes and initiation of an innate immune response occurs through pattern recognition receptors (PRRs). It is unlikely that innate immune cells are presented with a single inflammatory stimulus during infection. Instead, the host is primed to detect several microbe associated molecular patterns (MAMPs) through the use of many PRRs, a feature thought to provide further specificity to the cellular response. Thus, the engagement of PRRs and the initiation of an immune response is a collaborative and coordinated process.
Brain angiogenesis inhibitor 1 (BAI1) is an adhesion heterotrimeric G-protein coupled receptor (GPCR) expressed in various tissues and cell types including myeloid lineage cells. In addition to negatively regulating neovascularization in brain tumor models, it was previously characterized as a phagocytic receptor for apoptotic cells. More recently, BAI1 has been shown to mediate the recognition and internalization of Gram-negative bacteria through an interaction with bacterial lipopolysaccharide (LPS). Prior to this study, the role of BAI1 during the recognition and response to Gram-negative bacteria in macrophages was largely unknown. The work presented in this thesis provides advances to our understanding of the ligand specificity of the TSR domains, the impact of BAI1-mediated phagocytosis on the fate of the internalized microbe, and the interaction of BAI1 with inflammatory signaling and transcriptional responses. BAI1, through the five TSR domains in the extracellular region, recognizes the negatively charged phosphorylated L-glycero-d-manno-heptose sugars in the inner core oligosaccharide of LPS in the Gram-negative bacterial outer membrane. This suggests that BAI1-mediated detection is broadly relevant for bacterial recognition, as the phosphorylation of the inner core oligosaccharide is critical for membrane stability and is conserved across many Gram-negative bacterial species, commensals and pathogens alike.
The fate of a microbe upon contact with a host cell is determined by the local inflammatory environment and the route of cellular entry. The role and impact of BAI1-mediated recognition in the immune response of macrophages was previously undetermined. Here, we show that BAI1-driven Rac activation promotes the phagocyte NADPH oxidase-dependent ROS response, resulting in oxidative bacterial killing in vitro, and protection in an in vivo bacterial challenge model. This provides mechanistic insight into how upstream signals from non-opsonic phagocytic receptors, like BAI1, couple to the activation of critical microbicidal machinery in the context of several representative Gram-negative microbes.
We further characterized the impact of BAI1 on innate inflammatory signaling pathways and assessed the means of receptor crosstalk and interaction. We found that BAI1 selectively promotes intracellular signaling and transcriptional responses of toll-like receptor (TLR) 4 by enhancing the downstream phosphorylation and activation of TBK1 and IRF3. TRIF-dependent type-I IFN-β, IL-10, and CCL5 induction were all reduced in macrophages lacking BAI1, indicating a selective role for BAI1 in critical early innate responses that drive local cellular activation and regulate inflammatory responses. Moreover, TLR4 and BAI1 physically associate in a manner dependent upon an interaction with the cytoplasmic region of BAI1, and the spatial interaction between Gram-negative bacteria and TLR4 is enhanced by BAI1 co-expression. The direct mechanisms of this interaction remain to be explored, but likely involve BAI1-dependent signaling that modulates either the local recruitment or activation of TLR4 signaling partners.
Collectively, this highlights the critical and unique specificity and function of BAI1 during TLR4-driven bacterial recognition and early innate responses and suggests that the phagosome serves as a multifunctional and heterogeneous organelle that is distinct from other innate signaling compartments within macrophages.
