Abstract TAC242: Kinin B1 Receptor as the Activation Signal in TLR4 Primed NLRP3 Inflammasome Activation

Introduction: The NLRP3 inflammasome is a central driver of inflammation and is activated through a two-step process involving a priming phase followed by an activation signal. While toll like receptor 4 (TLR4) mediated priming is well established, endogenous secondary signals capable of inflammasome assembly in the hypertensive brain is unclear. We previously showed that TLR4 interacts with kinin B1 receptor (B1R) in the brain during hypertension, however, whether B1R activation can promote inflammasome assembly is unknown. We hypothesize that B1R upregulation serves as the activation signal following TLR4 priming to drive full NLRP3 inflammasome activation in the brain. Approach: To assess the role of B1R in inflammasome activation, C57Bl/6NJ wild-type (WT) and B1R knockout (B1RKO) mice were administered angiotensin II (Ang II, 600 ng/kg/min) or saline via osmotic minipumps for 28 days (n=5 mice/group). To determine receptor specific contributions to inflammasome activation, microglia from neonatal WT mice were exposed to LPS (100 ng/ml, TLR4 agonist), LDABK (300 nM, B1R agonist), or their combination with selective inhibition of TLR4 (10 µM, TAK242), B1R (10 µM, SSR240612), or NLRP3 (10 µM, MCC950) (n=3-5 independent cultures/group). Results: Ang II-infused WT mice had elevated NLRP3 activation (p=0.0253), caspase-1 (p=0.0421), IL-1β (p=0.0370) and IL-18 (p=0.0023) in the brain compared to saline controls. B1RKO mice with Ang II were protected from these responses. In microglia, LPS and LDABK alone increased TLR4 (LPS p=0.0002, LDABK p=0.0204) and NLRP3 (LPS p=0.0013, LDABK p=0.0310) but did not activate the inflammasome. However, co-treatment led to increased caspase-1 (p=0.0336) and cytokine release (IL-18 p=0.0021, IL-1β p=0.0030), indicative of full inflammasome activation. These effects were abrogated by MCC950 (IL-1β p=0.009, IL-18 p=0.004, caspase-1 p=0.0717), confirming NLRP3 involvement. TLR4 inhibition attenuated priming (p=0.0073), whereas B1R blockade inhibited inflammasome activation without altering NLRP3 expression (IL-1β p=0.0011, IL-18 p=0.0052, caspase-1 p=0.0055, NLRP3 p=0.7433), supporting a distinct role for B1R as the activation stimulus. Conclusion: This data highlights a novel TLR4-B1R-NLRP3 signaling axis, where B1R functions as an activation signal for inflammasome assembly following TLR4 priming. This proinflammatory axis may be a potential target to disrupt maladaptive innate immune responses in neurogenic hypertension.