Abstract

Background: Nuclear factor κB (NF-κB) activity is a central component of inflammatory and innate immune responses, which plays a crucial role in sepsis. The inhibition of NF-κB signaling and the IκB kinase (IKK) complex is important for understanding the control of innate immunity and regulating the progress of sepsis.

Methods: We constructed transgenic mouse strains (Rmp^f/f; Lyz2-Cre⁺), and then established lipopolysaccharide (LPS), cecal ligation and perforation (CLP)-induced sepsis models. Hematoxylin-eosin (HE) staining, ELISA, and flow cytometry assay were employed to evaluate the sepsis-related damage and the activation of the inflammatory-related signaling pathway. In vitro, differential expression of RMP cell lines and primary macrophage isolated from transgenic mice were utilized to assess the activation of the NF-κB signaling pathway by Western blot (WB), reverse transcription-polymerase chain reaction (RT-PCR), and ELISA tests. Co?immunoprecipitation (Co-IP), WB, GST-pulldown, phosphorylation mass spectrometry, surface plasmon resonance (SPR), and IKK activity detection assay were employed to investigate the underlying molecular mechanism by which RMP restrains IKK-NF-κB pathway.

Results: We identified RNA polymerase II subunit 5 (RPB5)-mediating protein (RMP) as an inhibitor of the IKK complex, which thus inhibited NF-κB signaling in macrophages. In resting macrophages, RMP was directly bound to the kinase domain of IKKβ and inhibited its activity by recruiting protein phosphatase 2?A (PP2A) to the IKK complex. When mouse macrophages were treated with LPS, a Toll-like receptor 4 (TLR4) agonist that stimulates NF-κB signaling, RMP was phosphorylated by IKKβ at Ser⁴³⁹ and dissociated from the IKK complex, which further activated NF-κB signaling. Macrophage-specific deletion of Rmp reduced survival in mice due to an increased inflammatory response in experimental models of sepsis.

Conclusions: RMP inhibits TLR4-induced NF-κB activation and exerts homeostatic control of innate immunity, and may be promising as a therapeutic target in the limiting of NF-κB signaling and attenuating sepsis-related damage.