TBK1 and IKKε act like an OFF switch to limit NLRP3 inflammasome pathway activation

NACHT, LRR, and PYD domains–containing protein 3 (NLRP3) inflammasome activation is beneficial during infection and vaccination but, when uncontrolled, is detrimental and contributes to inflammation-driven pathologies. Hence, discovering endogenous mechanisms that regulate NLRP3 activation is important for disease interventions. Activation of NLRP3 is regulated at the transcriptional level and by posttranslational modifications. Here, we describe a posttranslational phospho-switch that licenses NLRP3 activation in macrophages. The ON switch is controlled by the protein phosphatase 2A (PP2A) downstream of a variety of NLRP3 activators in vitro and in lipopolysaccharide-induced peritonitis in vivo. The OFF switch is regulated by two closely related kinases, TANK-binding kinase 1 (TBK1) and I-kappa-B kinase epsilon (IKKε). Pharmacological inhibition of TBK1 and IKKε, as well as simultaneous deletion of TBK1 and IKKε, but not of either kinase alone, increases NLRP3 activation. In addition, TBK1/IKKε inhibitors counteract the effects of PP2A inhibition on inflammasome activity. We find that, mechanistically, TBK1 interacts with NLRP3 and controls the pathway activity at a site distinct from NLRP3-serine 3, previously reported to be under PP2A control. Mutagenesis of NLRP3 confirms serine 3 as an important phospho-switch site but, surprisingly, reveals that this is not the sole site regulated by either TBK1/IKKε or PP2A, because all retain the control over the NLRP3 pathway even when serine 3 is mutated. Altogether, a model emerges whereby TLR-activated TBK1 and IKKε act like a “parking brake” for NLRP3 activation at the time of priming, while PP2A helps remove this parking brake in the presence of NLRP3 activating signals, such as bacterial pore-forming toxins or endogenous danger signals.

Functional Safety for Developing of Mechatronic Systems – Electric Parking Brake Case Study

The electric parking brake (EPB) system as the complex mechatronic system consists of the actuators that generate the clamping force necessary to hold the vehicle safe, the conventional calipers that convert clamp force into brake torque, electronic hardware with the Electronic Control Unit (ECU), cable harness and switches and especially the control software providing the functions that the driver will experience. Like most of the modern automotive components, the EPB is equipped with embedded electronic systems that include ECU, electronic sensors, signals, bus systems, and coding. Due to the complex application in electrical, electronics and programmable electronics, the need to carry out detailed safety analyses that are focused on the potential risk of malfunction is crucial for automotive systems. This paper describes a possible division of the EPB sub-functions between the supplier the wheel brakes and the supplier which supplying the ECU. Functional safety must be a guarantee with concerning the overall vehicle system. Functional safety is according to the requirements of the ISO 26262 standard and in the context of this paper relates solely to the E/E components (electrical and/or electronic) of the EPB. This paper covers the hazard analysis and risk assessment relevant to the EPB control software, and the derived allocation of ASIL risk levels to the EPB software elements of the functional architecture of the EPB.