Skjesol, A. | Search bioRxiv

bioRxiv2025

Rab11FIP2 controls NLRP3 inflammasome activation through Rab11b

Membrane trafficking through the trans-Golgi network has recently been shown to guide activation of the NLRP3 inflammasome. The GTPases Rab11a and Rab11b, and their effector molecule Rab11-FIP2, are regulators of endosome trafficking and retrograde transport. Rab11-FIP2 binds phosphatidylinositol species including PI4P, enriched in the trans-Golgi network and peripheral endosomes following NLRP3 inflammasome activation. We here demonstrate that Rab11-FIP2 and Rab11b, but not Rab11a, control caspase-1 mediated cleavage of pro-IL-1{beta} and GSDMD, and pyroptotic cell death in human macrophages. Rab11-FIP2 also controlled LPS stimulated IKK{beta} activation by TAK1 and IKK{beta} mediated NLRP3 translocation to the trans-Golgi network. Furthermore, we show that NLRP3 bound Rab11-FIP2 via its KMKK motif and that Rab11-FIP2 interacts with NLRP3 via its N-terminal C2-domain. The formation of PI4P positive endosomes and ASC-specks were also controlled by Rab11-FIP2. Collectively our results demonstrate that Rab11-FIP2 and Rab11b control NLRP3 inflammasome activation on early endosomes in human macrophages.

immunology↗

bioRxiv2025

A novel TIRAP-MyD88 inhibitor blocks TLR7- and TLR8-induced type I IFN responses

Endosomal toll-like receptors TLR7 and TLR8 are critical sensors of microbial RNA that initiate antiviral and antibacterial immune responses through type I interferon (IFN) and proinflammatory cytokine production. While TIRAP is traditionally associated with plasma membrane TLR signaling, recent evidence suggests it also contributes to signaling via endosomal TLRs. Here, we examined the role of TIRAP in TLR7/8 signaling using P7-Pen, a novel SLAMF1-derived peptide that disrupts the TIRAP-MyD88 interaction. In primary human monocytes and a whole blood model, P7-Pen inhibited TLR7- and TLR8-induced expression and secretion of IRF5-regulated cytokines IFN{beta}, IL-12p40, and IL-12p70, without effect on TNF or IL-6. Mechanistically, P7-Pen blocked TIRAP recruitment to the TLR8-MyD88 complex, leading to reduced late-stage IRAK1 activation, Akt and IKK/{beta} phosphorylation, and downstream IRF5 dimerization and nuclear translocation. Inhibition of Staphylococcus aureus-induced cytokine production by P7-Pen was associated with reduced bacterial phagocytosis, impairing endosomal delivery of bacterial RNA. Notably, P7-Pen failed to inhibit murine TLR7 responses, which correlated with a lack of TIRAP recruitment to MyD88 in mouse macrophages following TLR7 ligand stimulation, highlighting species-specific differences in TLR signaling mechanisms. These findings support a noncanonical role for TIRAP in regulating IRF5-dependent signaling downstream of human TLR7 and TLR8, and demonstrate that selective disruption of TIRAP recruitment by a SLAMF1-derived peptide effectively attenuates IFN{beta} production. This strategy may hold therapeutic potential in diseases characterized by dysregulated type I IFN responses, such as systemic lupus erythematosus and chronic infections.

immunology↗