AbstractMacrophages play key roles in innate and adaptive immune systems not only in the response to pathogens but also in tissue homeostasis. They are extremely plastic and recognize external stimuli such as cytokines with substantial changes to the proteome and molecular functions. One major macrophage function altered by cytokine activation is phagocytosis and phagosome maturation, through which macrophages engulf foreign material such as microbes or apoptotic cells to form phagosomes which then fuse with endosome and finally lysosomes, where the particles are finally degraded. My project aims at investigating the phagosome functions regulated in activated macrophages and further exploring the mechanism by which alternative activation regulates phagosome biogenesis. First of all, a comparison of phagosome proteomes of BMDMs and RAW 264.7 cells was performed, suggesting that there are significant differences for a large number of proteins including important receptors such as mannose receptor 1 and Siglec-1. Moreover, BMDM phagosomes mature considerably faster when validated using fluorogenic phagosome function assays.
For the main goal of my project, I have performed a thorough proteomics analysis of the phagosome proteomes of non-activated (RestingMΦ), alternative-activated (IL4 treated, AAMΦ), classical-activated (LPS and IFNγ treated, CAMΦ) and reprogrammed (IL4 activated then LPS and IFNγ treated, ReMΦ) BMDMs. Results indicate that alternative activation leads to phagosomal recruitment of proteins in favour of apoptotic cell clearance, enhanced fusion with lysosomes as well as with parts of the endoplasmic reticulum (ER). Both proteomics and phagosome function assays showed that the phagosome maturation is enhanced in AAMΦ and reduced in CAMΦ and ReMΦ. As side projects, I have also compared phagosome proteomes in IL4 treated, IL13 treated and IL10 treated BMDMs and analysed cellular total proteomes of AAMΦ and RestingMΦ.
Furthermore, proteomic data suggest the specific recruitment of TAK1/MKK7/JNK signalling to the phagosomes in AAMΦ, which was confirmed by immunoblotting and fluorescence microscopy. I uncovered that K63 polyubiquitylation of phagosomal proteins is enhanced in AAMΦ, which is responsible for the translocation of TAK1 complex. In AAMΦ, 55 K63 polyubiquitylation sites on 33 phagosomal proteins were identified, including macrophage scavenger receptor 1 (MSR1/SRA). This receptor was further found to be specifically polyubiquitylated on phagosomes upon alternative activation, and MSR1 activation leads to enhanced JNK activation in AAMΦ. Finally, three hypotheses of the function of JNK pathway on phagosomes were described. Firstly, proteomics reveals a reduction of ER and lipid metabolic proteins to phagosomes by the inhibition of JNK, suggesting that TAK1/MKK7/JNK signalling might regulate phagosomal lipid handling. Secondly, JNK might phosphorylate the lipid-activated transcription factor, PPARγ, to regulate macrophage gene expression in lipid metabolism. Finally, loss of MSR1 impairs oxLDL induced JNK activation and M2-to-M1 shift of macrophages, indicating that MSR1/JNK cascade mediates phenotypic shift of AAMΦ upon lipid laden.
In conclusion, the work in this thesis provides comprehensive characterisation of phagosomal and cellular proteomes in activated BMDMs. Moreover, TAK1/MKK7/JNK signalling was found for the first time to be specially recruited to phagosomes by K63 polyubiquitylation, and 55 novel K63 polyubiquitylation sites on 33 phagosomal proteins were identified, including MSR1. We hypothesise that JNK signalling might regulate lipid metabolism in AAMΦ.
|Date of Award||2015|
|Sponsors||Medical Research Council, AstraZeneca, Boehringer Ingelheim GmbH, GlaxoSmithKline, Janssen Pharmaceutica & Merck KGaA|
|Supervisor||Matthias Trost (Supervisor)|
Phagosome proteomes in activated macrophages
Guo, M. (Author). 2015
Student thesis: Doctoral Thesis › Doctor of Philosophy