TBK1- and IKKe-mediated control of NLRP3 inflammasome activation and regulation of cell death

Macrophages are equipped with Pattern Recognition Receptors (PRR), such as Toll-like receptors, to detect pathogens or damage signals. Activation of PRRs results in the upregulation of pro-inflammatory cytokines and phagocytic activity to clear the infection but also in a pro-survival programme. If the infection is not cleared and the cell is compromised or invaded by pathogens, intracellular sensors of damage are activated. The NLRP3 inflammasome is one of these intracellular sensors of infection and cell damage. Once activated, it leads to lytic cell death, pyroptosis, and inflammation. During infections and vaccinations, NLRP3 activation is beneficial and vital for a functional response. However, when NLRP3 control is lost due to gain-of-function mutations or in acquired diseases such as gout or atherosclerosis, its premature or excessive activation becomes detrimental and leads to inflammation-driven pathologies.
Therefore, it is important to understand how NLRP3 is normally endogenously controlled to prevent its premature activation. NLRP3 activation is controlled first on the transcriptional level, as its expression is upregulated by PRRs, and then via post-translational modifications (PTM). We discovered one such PTM regulatory “switch”, whereby the phosphatase PP2A turns the NLRP3 “on” upon cell damage or invasion, while the two homologous kinases TBK1 and IKK, activated by PRRs, keep NLRP3 “off” before cell damage or invasion. Blocking the PP2A on-switch, pharmacologically or by genetic deletion, limited NLRP3 activity in vitro and in vivo. Blocking the TBK1/IKKe off-switch, pharmacologically or by genetic deletion, increased NLRP3 activation, and resulted in premature pathway activation, lytic death and an inflammatory response. Mechanistically, one target site under PP2A and TBK1/IKKe control is NLRP3-Serine 3 (S3), but we find that this is not the sole mechanism, as both TBK1/IKKe and PP2A, retained the control over the NLRP3 pathway even when S3 was mutated. We next found that TBK1/IKKe control the NLRP3 pathway by a cell-intrinsic mechanism, where they regulate two processes known to be required for NLRP3 activation. Firstly, TBK1/IKKe limit the activity of kinase IKKB, previously shown to be necessary for NLRP3 activation at endosomal vesicles.
Secondly, TBK1/IKKe are involved in maintaining organelle and endosomal homeostasis. As a result, when TBK1/IKKe activity is blocked, cells accumulate endosomal vesicles, which provide a larger sub-cellular platform for premature NLRP3 activation. Alternative disruption of endosomal homeostasis by deletion of the endosomal effector Rab5 also resulted in premature death, showing the importance of this regulatory pathway to prevent aberrant NLRP3 activation.
Finally, we and others show that TBK1/ IKKe act as a common break that prevents multiple cell death pathways in activated macrophages. The type of death pathway they block depends on the timing and nature of the activating signal. Upon chronic exposure of macrophages to the inflammatory cytokine TNF, TBK1/IKKe prevent cell death by blocking RIPK1-dependent apoptosis and necroptosis. Conversely, upon acute exposure of macrophages to pathogen signals, such as toll-like receptor ligands, or even TNF, in the presence of bacterial toxins, TBK1/IKKe prevent cell death by blocking an NLRP3-dependent pyroptosis. The loss of the TBK1/IKKe break lowers the threshold for NLRP3 activation so that the pathway becomes activated and cells die prematurely even after detection of the NLRP3 activation signal (Signal 2) alone, in the absence of the ‘obligatory’ priming signal (signal 1).
Collectively, we and others describe TBK1 and IKKe as “master regulators” of inflammation and cell death by controlling NLRP3 activation via maintaining endosomal and organelle homeostasis and necroptosis by inhibiting the upstream kinase RIPK1.

Keywords: NLRP3
TBK1
cell death
IKKe
Advisor: Bezbradica Mirkovic, J
Midwood, K