Decoding the intracellular complement system in inflammatory reactions

We have previously demonstrated that intracellular complement, particularly the C5 system, contributes to NLRP3 inflammasome activation upon sensing of danger signals in monocytes and macrophages. However, the exact mechanisms by which intracellular complement regulates this process have not been established. In this reporting period we have identified intracellular stores of C3 split products C3a, C3b and C3c in human macrophages suggesting the presence of a convertase activity inside the macrophages. Interestingly, we observed co-localisation of anti-C3bc and ASC-specks. ASC-specks are hallmarks of inflammasome activation. The C3bc antibody detects a neoepitope which appears on the C3c part of C3b upon C3 cleavage, and which is preserved on the C3c fragment following cleavage of C3b. We have generated and characterized C3, C3aR, C5, C5aR1 knock out (KO) THP1 cells. These are essential research tools for intracellular complement research. Supporting a role of C3 in inflammasome activation, preliminary results from experiments in C3 KO THP-1 cells show reduced cell IL-1beta production upon lipopolysaccharide (LPS) and nigericin treatment. Also other cytokines, like IL-6 and IFNbeta are regulated by C3 in these monocytic cells. Furthermore, preliminary results from THP-1 C5 and C5aR1 KO cells show decreased IL-1beta production and cell death following inflammasome activation in KO cells compared to the wild-type. Infection of the KO cells with M. tuberculosis also caused lower levels of cell death and IL-1beta release compared to wild-type cells. The data demonstrate an important role for intracellular C3, C5 and C5aR1 in mediating inflammatory reactions in human macrophages. Another aspect of the project is to establish the role of Rab11-FIP2 (FIP2) in inflammasome activation and how it controls the trafficking of C5aR1. In order for Rab11 to perform motor functions and transport of endosomes it must interact with FIP2 that acts as an effector protein. FIP2 mediates transports of cargo along actin filaments. We have now generated a lot of data showing that FIP2 is essential for NLRP3 inflammasome assembly and activation and we have also revealed mechanisms underlaying this effect. In the reporting period we have also reconstituted a C5aR1 KO THP-1 cell line with GFP-C5aR1. Our results demonstrate that the GFP-tagged C5aR1 localizes in an expected manner on the plasma membrane and on intracellular compartments like mitochondria. Preliminary data suggest that GFP-C5aR1 is co-localized with FIP2, also on mitochondria. This system will be an important tool for further studies on how FIP2 controls intracellular trafficking and functions of C5aR1.

The liver-derived and plasma-circulating complement system is a key member of the host’s repertoire of pathogen- and damage-associated molecular pattern (PAMP and DAMP) sensors. Pathogen sensing in blood triggers activation of C3 (complement component 3) into C3a and C3b and of C5 into C5a and C5b, by C3 and C5 convertases, respectively. These complement activation fragments together mediate the opsonization and removal of invading microbes, mobilization of immune cells, and induction of a general inflammatory reaction. Recent work has led to the unexpected discovery of a cell-autonomous, intracellularly active complement system, termed the complosome. We have just recently shown that macrophages have an intracellular C5 system and recruit intracellular C5a receptor 1 (C5aR1) for inflammatory responses after sensing of danger signals. Unexpectedly, C5aR1 mediates these inflammatory effects by interacting with C5a on the mitochondria. Our findings raise several new and important questions that need to be addressed. The project will explore intracellular trafficking mechanisms of C5a and C5aR1 that control mitochondrial reactive oxygen species production and inflammasome activation. We aim to find the signalling mechanisms used by C5aR1 on mitochondria, and the role of intracellular complement in bacteria-induced killing of macrophages. It will also be examined if the intracellular C5 system controls cytosolic immune sensing of DNA and RNA. Obtained results will have high general relevance and potential as it provides novel insight into mechanisms of how macrophages respond to sterile and infectious danger.