Inflammatory modulation in microglia: macropinocytosis for receptor trafficking and signalling

Abstract

As the primary innate immune cells of the central nervous system (CNS), microglia are tasked with protecting the neuronal environment by removing infectious material and cell debris using phagocytic and macropinocytic pathways. Macropinocytosis occurs in both constitutive and induced fashion in these cells, where it serves primarily for fluid phase uptake and tissue surveillance, however it is also a key pathway for the removal of the protein aggregates that accumulate during neurodegeneration. A deeper understanding of this pathway in microglia will enhance efforts to elucidate and address neuroinflammation and neurodegeneration including in Alzheimer’s disease (AD), Parkinson’s disease and other chronic conditions.Like other macrophages, microglial cells express pattern recognition receptors which, when activated, trigger an array of innate immune and inflammatory responses. This project set out to examine Toll-like receptors (TLRs) as one such receptor family, and the signalling pathways that drive and bias transcription of inflammatory cytokines for release. Microglia have dynamic ruffling cell surface membranes which give rise to both macropinosomes and other endosomes that house and support TLR activation and signalling. Studies in our laboratory leading up to this project characterised GTPases, lipid kinases and co-receptors that influence TLR signalling in macrophages. Specifically, a TLR-activated Rab8a-PI3Kg complex was found that modulates Akt and mTOR signalling (Luo et al., 2014). Moreover, the lipoprotein receptor LRP1, was found to be cross-talk activated by TLRs in macrophages. LRP1 recruits the Rab8a-PI3Kg complex for signalling modulation, which biases the cytokine program and constrains inflammation (Luo et al., 2018). Signalling from TLRs and LRP1 occurs in early macropinosomes (Wall et al., 2017, Wall et al., 2019).This project aimed to extend these findings, specifically seeking to establish whether the TLR-LRP1-Rab8a-PI3Kg axis functions in microglia. Since macropinocytosis had not been studied in detail in microglia, this project also set out to demonstrate this fluid-phase uptake pathway and to characterise the macropinosomes and other compartments involved in TLR trafficking and signalling.The BV2 cell line was used for live cell imaging of dextran uptake and for imaging an array of transfected fluorescently tagged fusion proteins. A live cell imaging construct was developed for LRP1 which featured a HaloTag that facilitated differential labelling of endocytic versus surface receptor populations for tracking of LRP1 internalisation. Biochemical approaches were used to examine immune signalling and cytokine profiles of microglial cells challenged with different TLR ligands (LPS, Poly I:C and CpG). Inhibitors were used successfully to implicate Rab8a, PI3Kg and LRP1 in these microglial pathways. Comprehensive comparisons of macropinocytosis, macropinosomes and TLR signalling were made across RAW 264.7 macrophages, BV2 microglial cell lines, as well as primary mouse microglial cells.The intracellular TLR, TLR9, became a focus of this project as its ligand, CpG-DNA was found to be internalised by microglia through macropinocytosis. Through microscopy and pharmacological inhibition of macropinocytosis, this was revealed as a novel site for intracellular TLR ligand-induced activation. The location of TLR9 signalling is controversial and traditionally associated with late endosomes in an acidified environment; whereas, the results obtained here now demonstrate LRP1 and Rab8a–dependent signalling from macropinosomes as a new site of activation. PI3Kg is confirmed here as a regulator of neuroinflammation by exciting live cell imaging showing recruitment of PI3Kg-mediated Akt to the macropinosome. While unmethylated CpG-DNA was thought traditionally to be microbial in origin, it can also be derived from self- and mitochondrial DNA that has been implicated in ageing and associated neuroinflammation and autoimmune disease. TLR9 is thus of increasing interest in the microglia of ageing and diseased CNS. The project findings conclude that TLR9 signals much earlier than previously thought, and these results present the macropinosome as a key signalling site for intracellular TLRs in microglia. These findings highlight new opportunities for constraining disease and ageing-related neuroinflammation initiated from the macropinosome.