sNASP inhibits TLR signaling to regulate immune response in sepsis
Feng-Ming Yang, … , Hui-Ming Chang, Edward T.H. Yeh
Feng-Ming Yang, … , Hui-Ming Chang, Edward T.H. Yeh
Published May 7, 2018
Citation Information: J Clin Invest. 2018;128(6):2459-2472. https://doi.org/10.1172/JCI95720.
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Research Article Immunology Inflammation

sNASP inhibits TLR signaling to regulate immune response in sepsis

Abstract

Many Toll-like receptors (TLRs) signal through TNF receptor–associated factor 6 (TRAF6) to activate innate immune responses. Here, we show that somatic nuclear autoantigenic sperm protein (sNASP) binds to TRAF6 to prevent TRAF6 autoubiquitination in unstimulated macrophages. Following LPS stimulation, a complex consisting of sNASP, TRAF6, IRAK4, and casein kinase 2 (CK2) is formed. CK2 phosphorylates sNASP at serine 158, allowing sNASP to dissociate from TRAF6. Free TRAF6 is then autoubiquitinated, followed by activation of downstream signaling pathways. In sNasp S158A knockin (S158A-KI) mice, LPS-treated macrophages could not phosphorylate sNASP, which remained bound to TRAF6. S158A-KI mice were more susceptible to sepsis due to a marked reduction in IL-1β, TNF-α, and IFN-γ production accompanied by an inability to clear bacteria and recruit leukocytes. Furthermore, phosphorylation-regulated release of sNASP from TRAF6 is observed following activation of TLR-1, -2, -4, -5, and -6. Thus, sNASP is a negative regulator of TLR signaling to modulate the innate immune response.

Authors

Feng-Ming Yang, Yong Zuo, Wei Zhou, Chuan Xia, Bumsuk Hahm, Mark Sullivan, Jinke Cheng, Hui-Ming Chang, Edward T.H. Yeh

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Figure 1

sNASP inhibits TLR4-induced NF-κB activation through TRAF6.

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sNASP inhibits TLR4-induced NF-κB activation through TRAF6. (A) Immunopr...
(A) Immunoprecipitation (IP) of Flag-TRAF6 (with anti-Flag agarose) from HEK293 cells transiently transfected with Flag-TRAF6 plus GFP-tNASP (+), GFP-tNASP (+), or empty vector (–), followed by immunoblotting (IB) with antibody against Flag or GFP. TCL IB was done with anti-Flag. TCL, total cell lysates. (B) IP of endogenous TRAF6 (with anti-TRAF6) or endogenous sNASP (with anti-NASP) or IgG from THP-1 cells, followed by IB with antibody against TRAF6 or NASP. TCL IB was done with anti-NASP and anti-TRAF6. (C) IP of Flag-TRAF6 (with anti-Flag agarose) in HEK293 cells transfected with Flag-tagged TRAF6 and hemagglutinin-tagged ubiquitin (HA-Ub) in the presence (+) or absence (–) of vector encoding GFP-tagged sNASP, and IB with anti-Flag followed by IB with antibody against Flag or Ub. TCL IB was done with anti-GFP and β-actin. (D) IB of indicated antibodies in LPS-stimulated Raw264.7 cells transduced with empty vector (EV) or GFP-tagged sNASP or siNASP. (E) Luciferase activity in HEK293 cells transfected with a luciferase reporter vector driven by an NF-κB–responsive promoter, plus EV or vector encoding TRAF6 and increasing concentrations of a vector encoding sNASP. Results were standardized to EV (set as 1). Data are mean ± SE for each group. **P < 0.01 (Student’s t test). Data represent a minimum of 3 independent experiments.