Macrophages are key immune cells present in every tissue in our bodies. They initiate the innate and adaptive immune systems and clear harmful particles by phagocytosis. Particles are recognized by pathogen recognition receptors (PRRs) that trigger internalization of the bound particle into a membranous organelle called the phagosome. Phagosomes mature by fusing with early and late endosomes and finally the lysosome where the internalized cargo is degraded. Toll Like Receptors (TLRs) are the most widely understood group of PRRs. They are integral to the initiation of the innate immune response by recognizing microbial pattern associated molecular patterns (PAMPs) and danger associated molecular patterns (DAMPs) and initiating the pro-inflammatory and type I interferon pathways from the plasma membrane and the endosome, respectively. We have previously shown that IFN-γ stimulation greatly impacts phagosome functions. Here, we wanted to investigate the role of ubiquitin in phagosomal maturation and endosomal TLR signalling in IFN-γ activated macrophages.
Phagosomes were isolated from bone marrow-derived macrophages (BMDMs) and analysed using mass spectrometry, qPCR, western blot, microscopy and flow cytometry.
We have identified the ubiquitin E3 ligase RNF115 as a key regulator of phagosome maturation. RNF115 is recruited to the phagosome and upregulated upon activation with IFN-γ. We show that loss of RNF115 promotes phagosome maturation, indicating that RNF115 is a negative regulator of vesicular trafficking to the lysosome. Furthermore, loss of RNF115 induces an increased pro-inflammatory response to Toll-like receptor (TLR) agonists. We demonstrate increased TLR signalling from the phagosome in RNF115 KO cells and confirm that endosomal and phagosomal maturation is important for the initiation of TLR2 induction of type I IFN from the endo-lysosomal system. Taken together, we have shown that RNF115 plays a role in the regulation of endosomal TLR initiation of the innate immune response in murine macrophages.