PEP-sNASP Peptide Alleviates LPS-Induced Acute Lung Injury Through the TLR4/TRAF6 Axis

Abstract

Acute lung injury (ALI) is a severe inflammatory lung disease associated with macrophages. Somatic nuclear autoantigenic sperm protein (sNASP) is a negative regulator of Toll-like receptor (TLR) signaling that targets tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) in macrophages, which is required to maintain homeostasis of the innate immune response. In the present study, we generated a cell permeable PEP-sNASP peptide using the sNASP protein N-terminal domain, and examined its potential therapeutic effect in a mouse model of ALI induced by the intranasal administration of lipopolysaccharide (LPS) and elucidated the underlying molecular mechanisms in RAW 264.7 cells. In vivo, PEP-sNASP peptide treatment markedly ameliorated pathological injury, reduced the wet/dry (W/D) weight ratio of the lungs and the production of proinflammatory cytokines (interleukin (IL)-1β, IL-6, and TNF-α). In vitro, we demonstrated that when the PEP-sNASP peptide was transduced into RAW 264.7 cells, it bound to TRAF6, which markedly decreased LPS-induced proinflammatory cytokines by inhibiting TRAF6 autoubiquitination, nuclear factor (NF)-κB activation, reactive oxygen species (ROS) and cellular nitric oxide (NO) levels. Furthermore, the PEP-sNASP peptide also inhibited NLR family pyrin domain containing 3 (NLRP3) inflammasome activation. Our results therefore suggest that the PEP-sNASP may provide a potential protein therapy against oxidative stress and pulmonary inflammation via selective TRAF6 signaling.

Home Dust Mites Promote MUC5AC Hyper-Expression by Modulating the sNASP/TRAF6 Axis in the Airway Epithelium

“…Silencing NASP also enhanced NF-κB activity in response to HDM ( Figure 3 B). Since PEP-NASP, as a cell-permeable peptide, can selectively inhibit TLR4-mediated TRAF6/NF-κB signaling pathways [ 27 ], we decided to evaluate whether the transduced PEP-NASP peptide would affect TRAF6 downstream signaling following an HDM stimulation. The results indicated that the treatment of the PEP-NASP peptide, but not the control NASP peptide, diminished the HDM-induced phosphorylation of p38 MAPK and p65 ( Figure 3 C).…”

“…The HDM extract ( D. pteronyssinus/ D. farinae extract mix) was purchased from Citeq (#20.50.85, Groningen, The Netherlands). Preparation of the PEP-NASP and NASP peptides was previously described in [ 27 ]. PEP-NASP or NASP peptides (0.4 μM) were added to the culture medium for 1 h before HDM treatment.…”

“…BALF was harvested according to a previous publication [ 27 ]. Briefly, BALF was obtained by injecting ice-cold PBS using a tracheal catheter.…”

“…NASP was reported as a histone chaperone that regulates histone transportation, cell proliferation, and normal development [ 24 , 25 , 26 ]. In our previous studies, sNASP was found to be a critical element activating TRAF6 downstream signaling during TLR activation [ 27 , 28 ]. Following TLR activation, sNASP is phosphorylated and released from TRAF6 to allow downstream nuclear factor (NF)-κB signaling [ 28 , 29 ].…”

“…Following TLR activation, sNASP is phosphorylated and released from TRAF6 to allow downstream nuclear factor (NF)-κB signaling [ 28 , 29 ]. Transduced cell-permeable PEP-NASP peptides can specifically bind to TRAF6, which significantly reduced TLR4-mediated inflammation by inhibiting TRAF6 autoubiquitination and NF-κB activation [ 27 ]. However, limited information is available on the relationship between mucus hypersecretion and TLR-mediated sNASP/TRAF6 signaling in response to the effects of HDM exposure of the respiratory tract.…”