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

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.


INTRODUCTION
Acute lung injury (ALI) and its severe form of acute respiratory distress syndrome (ARDS) were first described in 1967 (1) and are both known to be common causes of respiratory failure in critically ill patients (2). Pathologically, ALI is often characterized by an uncontrolled acute pulmonary inflammatory response, decreased lung compliance, and impaired gas exchange, ultimately leading to various clinical disorders, such as pneumonia, pulmonary edema, and sepsis (3)(4)(5). Despite advances in understanding of the pathogenesis and treatment of this disease, the morbidity and mortality from ALI/ARDS remain high. It was estimated that the overall mortality rate of ALI/ARDS range 38.5∼41.1%, which significantly depends on various comorbidities (5)(6)(7). A study conducted in Taiwan found that the in-hospital mortality rate increasing from 33.5 to 68.2% was correlated with patients' ages (8). Currently, the treatments of ALI/ARDS available in clinical practice are still limited, which emphasizes the urgent need for the development of novel therapeutic strategies to improve clinical outcomes.
Various inflammation are transmitted through a family of receptors of the innate immune system, known as Toll-like receptors (TLRs), which are known to play essential roles in the pathogenesis of pulmonary inflammation (9)(10)(11)(12). TLR4 is the dominant mammalian receptor for the microbial product, lipopolysaccharide (LPS), a major biologically active component of gram-negative bacterial cell walls, and which is widely used to induce ALI models that are similar to pathological features of ALI in humans by triggering excessive inflammatory mediators (13)(14)(15). Our previous work showed that the somatic nuclear autoantigenic sperm protein (sNASP) is a negative regulator of the TLR4-mediated innate immune response (16,17). In unstimulated cells, it binds tumor necrosis factor (TNF) receptorassociated factor 6 (TRAF6) to avoid excessive inflammation and maintain homeostasis of the innate immune response. Once TLR4 binds to its ligands, sNASP is phosphorylated and released from TRAF6, which ultimately stimulates expressions of proinflammatory cytokines, such as interleukin (IL)-6 and TNFα. In addition, using a cecal ligation and puncture (CLP)-induced sepsis model with dominant-negative sNASP mutant mice, these mutant mice were more susceptible to bacterial infection due to ineffective clearance of bacteria in the lungs after CLP, indicating that the sNASP may be involved in pathological changes in lung inflammation. Those results suggested that sNASP functions as a negative regulator of innate immunity and may contribute to its protective effects against ALI and underling mechanisms.
PEP-1 is a 21-residue peptide carrier that can efficiently transduce proteins into various cells and tissues (18,19). It consists of three domains: (1) a hydrophobic tryptophan-rich motif (KETWWETWWTEW) for targeting cell membranes, (2) a spacer (SQP), and (3) a hydrophilic lysine-rich domain (KKKRKV) for intracellular delivery and peptide solubility. Recently, it was shown that PEP fusion proteins successfully protected against various diseases including lung inflammation and neuronal diseases (20)(21)(22). In this study, we designed a PEP-NASP fusion protein to investigate whether the transduced PEP-NASP protects cells against LPS-induced inflammation. We found that PEP-NASP was efficiently transduced into RAW 264.7 cells through a membrane barrier, and that the transduced PEP-NASP reduced LPS-induced proinflammatory cytokines and nitric oxide (NO) production as well as NLR family pyrin domain containing 3 (NLRP3) inflammasome activation. In vivo, administration of the PEP-NASP markedly attenuated LPS-induced lung injury, with significant reductions in neutrophil infiltration, myeloperoxidase (MPO) activity, and bronchoalveolar lavage fluid (BALF) protein content. Therefore, we suggest that the PEP-NASP fusion protein might be useful as a potential therapeutic agent against ALI.

Mice and LPS-Induced ALI Model
Pathogen-free, 8-week-old male C57BL/6 mice were purchased from the National Laboratory Animal Center (Taipei, Taiwan). Mice were housed in a pathogen-free animal facility at Taipei Medical University (Taipei, Taiwan). Animal protocols described in this study were approved by the animal and ethics review committee of the Laboratory Animal Center at Taipei Medical University.
ALI was induced by intranasal (i.n.) instillation of LPS [5 mg/kg body weight (BW)] as described previously (23,24). LPS was obtained from Sigma-Aldrich (St. Louis, MO, USA). Forty mice were randomly divided into four groups: sham+NASP, sham+PEP-NASP, LPS+NASP, and LPS+PEP-NASP groups. Mice in the sham and LPS groups received i.n. instillation of phosphate-buffered saline (PBS) and LPS (5 mg/kg), respectively, while mice in the LPS+NASP and LPS+PEP-NASP groups received an i.n. injection of NASP or PEP-NASP (0.1 mg/kg) at 1 h before LPS inhalation. The experiment was terminated at 24 h after LPS inhalation, after which lung tissues and BALF were harvested and collected.

Cell Culture and Transduction of the PEP-NASP
The mouse macrophage RAW 264.7 cell line (ATCC TIB-71, American Type Culture Collection, Manassas, VA, USA) and embryonic kidney epithelial HEK293 cell line (ATCC CRL-1573) were both cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and 1% penicillin and streptomycin at 37 • C and 5% CO 2 in air as previously described (16).
To determine the dose-dependent transduction ability of the PEP-NASP, cells were grown to 75% confluency for 24 h, and then various concentrations (0.1 ∼ 0.4 µM) of the PEP-NASP were added to the culture medium for 1 h at 37 • C. For timedependent transduction, cells were treated with 0.4 µM of the PEP-NASP for 10 ∼6 0 min. To determine the intracellular stability of the transduced PEP-NASP, cells were treated with 0.4 µM PEP-NASP for 1 h and washed with fresh medium to remove fusion proteins that had not been transduced into cells. Cells were further incubated for 60 h.

Immunoprecipitation (IP) and Western Blotting
Harvested cells were subjected to IP, lysates were resolved on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and a Western blot analysis was performed as previously described (16,25). Signals were detected using the Trident femto Western horseradish peroxidase (HRP) substrate (GTX14698, GeneTex, Taiwan). Images were captured and the density of the bands was measured using the UVP BioSpectrum 815 (Jena, Germany). The detected bands were quantitated using ImageJ image analytical software. Antibodies

Luciferase Assays
Luciferase reporter assays were conducted according to our previous publication (26). RAW 264.7 cells were subcultured in 24-well tissue culture plates and transfected with NF-κB luciferase and pRL-TK Renilla luciferase. After 24 h, cells were transduced with the NASP or PEP-NASP 1 h before LPS stimulation, and then cells were lysed with lysis buffer, and firefly luciferase, and Renilla luciferase activities were determined using the Dual-Glo Luciferase Assay System (E2920, Promega, Madison, WI, USA).

Immunofluorescence (IF) Staining
IF staining was conducted as described previously (27). Cells were maintained on 12-mm glass slides for 24 h and then were stimulated with 1 µg/mL LPS for 30 min in the presence of the NASP or PEP-NASP (0.4 µM) added 1 h before stimulation. Then cells were fixed with 4% paraformaldehyde for 10 min and blocked with Blocking One Histo (06349-64, Nacalai Tesque, Kyoto, Japan) for 1 h, followed by incubation with rabbit anti-NF-κB p65 Alexa Fluor 594 (SC-8008 AF594, Santa Cruz) and mouse anti-His antibodies (34650, Qiagen, USA) overnight at 4 • C. After being washed, cells were incubated with an FITClabeled goat anti-mouse immunoglobulin G (IgG) antibody for 1 h. Nuclear DNA was stained with DAPI for 10 min. Images were captured using confocal microscopy on a Leica TCS SP5 (Wetzlar, Germany).

Histological Analysis
Hematoxylin and eosin (H&E) staining of paraffin-embedded lung tissue sections was performed to determine cellular infiltration. Lung tissues were fixed with 10% buffered formalin for 24 h, embedded in paraffin, and sectioned at 4-µm thickness. After deparaffinization and dehydration, sections were stained with H&E and were observed with an Echo Revolve Fluorescence Microscope (San Diego, CA, USA).

Lung Wet/Dry (W/D) Ratio
The left lung was harvested and weighed to obtain the wet weight. The lung was then placed into a drying oven at 80 • C for 48 h to obtain the dry weight. The W/D weight ratio was calculated to evaluate the degree of lung edema.

Myeloperoxidase (MPO) Activity Assay
Lung tissues were homogenized with MPO assay buffer after collection and then centrifuged at 13,000 g for 10 min at 4 • C.
The supernatant was assayed for MPO activity according to the manufacturer's instructions (K744-100, Biovision, USA). MPO activity was One unit of MPO activity was defined as the quantity of enzyme that converted 1 µmol hydrogen peroxide to water each minute at 37 • C. MPO activity is expressed as units per gram (U/g) wet weight.

Bronchoalveolar Lavage Fluid (BALF) Collection and Analysis
BALF was harvested according to a previous description (28). Briefly, mice were euthanized, and a catheter was installed in the open trachea. Then, 0.8 ml of ice-cold PBS was instilled into the lungs three times, and the recovered BALF was centrifuged at 450 ×g for 10 min. The cell pellet was used to count numbers of total cells, macrophages, and neutrophils counted with a hemocytometer and Wright-Giemsa staining (K1438-30, Biovision, USA).

Statistical Analysis
GraphPad Prism 6 software was used for all statistical analyses by Student's t-test for two groups or a one-way analysis of variance (ANOVA) for three or more groups. Values of p < 0.05 were considered statistically significant.
Transduction of the PEP-NASP Into RAW 264.7 Cells TRAF6 is a crucial mediator that transmits intracellular signals through TLRs to a wide range of immunostimulatory cytokines and chemokines against invading microorganisms (9,30,31). In this study, sNASP residues 1 ∼ 31 fused with PEP-1 (PEP-NASP) were synthesized to determine whether the increase in sNASP by protein transduction had a negative regulatory effect on the LPSinduced inflammatory response in the mouse macrophage RAW 264.7 cell line.
To evaluate the transduction ability of the PEP-NASP, RAW 264.7 cells were incubated with various concentrations (0.1 ∼ 0.4 µM, 1 h) of the PEP-NASP and for various times (10 ∼ 60 min, 0.4 µM). Then protein transduction was analyzed by Western blotting with an anti-His antibody. As shown in Figures 2A,B, the PEP-NASP was successfully transduced into RAW 264.7 cells in dose-and time-dependent manners, whereas the control NASP lacking the PEP-1 domain was not transduced into cells. The intracellular concentration of the transduced PEP-NASP in cells was detected within 10 min and had gradually increased by 60 min (Figure 2B). Since stability is an important factor in protein therapy, we examined the stability of the transduced PEP-NASP at various time periods. Western blotting revealed that the transduced PEP-NASP in RAW 264.7 remained stable until 36 h after treatment, subsequently decomposing over time ( Figure 2C). The viability of RAW 264.7 cells wasn't affected when treated with PEP-NASP, suggesting that PEP-NASP did not cause cytotoxicity in cells (Supplementary Figure 2). In addition, cellular localization of the transduced PEP-NASP in cells was determined by IF staining. PEP-1-NASP fluorescence was distributed in both the nucleus and cytoplasm, but no fluorescent signal was observed in control NASP-transduced cells (Figure 2D). These results demonstrated that the PEP-NASP was efficiently transduced into cells and persisted for 36 h after transduction.

Transduced PEP-NASP Inhibits the LPS-Induced Inflammatory Response Through TRAF6
Our previous report showed that sNASP binds to TRAF6 and inhibits TRAF6 autoubiquitination and its downstream signaling (16); we wanted to evaluate whether the transduced PEP-NASP would affect TRAF6 autoubiquitination following LPS stimulation. Results indicated that transduction of the PEP-NASP, but not the control NASP, diminished LPS-induced TRAF6 autoubiquitination in RAW 264.7 cells (Figure 3A). Next, we investigated whether the PEP-NASP could interact with endogenous TRAF6 during regulation of TLR4 signaling. There was a clear interaction between endogenous TRAF6 and the PEP-NASP, but not the control NASP (Figures 3B,C). IP assays also showed that transduction of the PEP-NASP led to reduced interactions between endogenous sNASP and TRAF6 (Figure 3B). Following LPS stimulation, transduction of the PEP-NASP accelerated the dissociation of the sNASP from TRAF6 (Figure 3C), indicating that the PEP-NASP had a much higher affinity for endogenous TRAF6 than endogenous sNASP. These results suggested that transduction of the PEP-NASP may compete with endogenous sNASP for the TRAF6-binding motif.
Since NF-κB activation is crucial for the LPS/TLR4 signalinginduced inflammatory response in RAW 264.7 cells, we examined the effect of the PEP-NASP against LPS-induced TRAF6 downstream signaling and the nuclear translocation of p65 in RAW 264.7 cells following LPS stimulation. As shown in Figure 4A, LPS-induced phosphorylation of transforming growth factor (TGF)-β activated kinase 1 (TAK1), p38 MAPK, and JNK decreased when the PEP-NASP was transduced into RAW 264.7 cells. Furthermore, the amount of NF-κB p65 in the nucleus markedly increased upon exposure to LPS alone, but the PEP-NASP suppressed the LPS-mediated nuclear translocation of NF-κB p65 (Figure 4B). Transduced PEP-NASP, but not control NASP, was also found to inhibit LPS-mediated NF-κB activation (Figure 4C). In additional, the PEP-NASP significantly decreased the production of the proinflammatory cytokines: TNF-α, IL-6, and IL-1β ( Figure 4D). These results suggested that the transduced PEP-NASP negatively regulates activation of the LPS-triggered TLR4 pathway by regulating TRAF6 autoubiquitination.

PEP-NASP Suppresses LPS-Induced ROS/NO Production and NLRP3 Inflammasome Activation
The production of excessive ROS and NO after LPS challenge is an important reflection of the inflammatory process involved in local immune responses. Since iNOS and cyclooxygenase (COX)-2 proteins are associated with NO production, we assessed whether the LPS-induced production of NO mediated by iNOS and COX-2 was regulated by the transduced PEP-NASP. As shown in Figure 5A, expressions of the iNOS and COX-2 proteins significantly increased in LPSstimulated RAW 264.7 cells compared to unstimulated cells. However, the transduced PEP-NASP dose-dependently (0.2 and 0.4 µM) inhibited expressions of iNOS and COX-2 in the presence of LPS (Figure 5A). Similar result was found in ROS production. As shown in Figures 5B,C, ROS production significantly decreased, and GSH, as the major free radical scavenger, level significantly increased in PEP-NASP transduced group compared with the LPS group. These results implied that the PEP-NASP inhibited ROS and NO production by downregulating iNOS and COX-2 expressions.
Our study showed that the PEP-NASP could reduce IL-1β production in LPS-induced RAW 264.7 cells (Figure 4D), and the NLRP3 inflammasome was reported to be essential to turn the inactive precursor (pro-IL-1β) into the active form (IL-1β). Consequently, to elucidate whether its antiinflammatory effect was related to the NLRP3 inflammasome, we further explored the effect of the PEP-NASP on activating the NLRP3 inflammasome. As shown in Figure 5D, LPS challenge significantly enhanced expressions of NLRP3, ASC, and caspase-1 proteins, indicating that the NLRP3 inflammasome was activated by LPS in macrophages. PEP-NASP transduction dramatically blocked the LPS-induced upregulation of NLRP3, ASC, and caspase-1 compared to treatment with LPS alone (Figure 5D). These results indicated that the PEP-NASP might reduce promotion of the NLRP3 inflammasome to inhibit inflammation.

PEP-NASP Transduction Attenuates LPS-Induced Lung Injury in Mice
Next, we investigated the protective effects of the PEP-NASP in a mouse model of ALI induced by the i.n. administration of LPS. Lung histopathology was determined by H&E staining. We found that LPS administration resulted in obvious histopathological lung injury, including pulmonary edema, inflammatory cell infiltration, and alveolar damage, whereas transduction of the PEP-NASP markedly ameliorated these pathological changes ( Figure 6A). There were no obvious pathological changes in the control group ( Figure 6A). Edema is a typical pathological characteristic of ALI lungs. The elevated lung W/D weight indicated edema in LPS-challenged mice ( Figure 6B). In contrast, the transduced PEP-NASP markedly decreased the W/D ratio compared to that in the ALI group ( Figure 6B). These data suggested that the PEP-NASP alleviated the pulmonary pathological injury observed in LPS-treated mice.

PEP-NASP Negatively Regulates LPS-Induced Production of Inflammatory Cytokines in the Lungs
Expressions of inflammatory cytokines (TNF-α, IL-6, and IL-1β) in lung tissues were determined using a qPCR and ELISA. We found that mRNA expressions in lung tissues and protein expressions of TNF-α, IL-6, and IL-1β in BALF had dramatically increased in the LPS group (Figures 7A,B). By contrast, transduction of the PEP-NASP resulted in significant decreases in expressions of these cytokines (Figures 7A,B). These data indicated that the PEP-NASP negatively regulated expressions of inflammatory cytokines in LPS-induced lung injury. We next explored the neutrophil and macrophage percentage changes in total cells by Wright-Giemsa staining. As shown in Figures 8C,D, percentages of neutrophils and macrophages had both significantly increased at 24 h after LPS challenge, while pretreatment with the PEP-NASP fusion protein reduced the percentage of neutrophils by 70% and macrophages by 28% compared to the LPS group. In addition, we also found that PEP-NASP fusion protein decreased the ROS production and increased the levels of GSH in mice of ALI (Figures 8E,F). These findings indicated that the transduced PEP-NASP suppressed ROS production and the infiltration of inflammatory cells into the lungs induced by LPS.

DISCUSSION
ALI, which is mainly characterized by pulmonary inflammation, is a common clinical disease in humans, and the mortality remains high at 30% ∼ 40% in most studies (32,33). Our previous study suggested that the sNASP had the ability to negatively regulate TLR signaling through targeting TRAF6 (16,17). In a polymicrobial sepsis model, sNASP-mutant mice had defective bacterial clearance from the lungs due to marked reductions in IL-1β, TNF-α, and IFN-γ production associated with poor adhesion molecule expression and leukocyte recruitment, and defective phagolysosome formation. In this study, we demonstrated, for the first time, that administration of the PEP-NASP fusion protein effectively protected mice against LPS-induced ALI, with significantly attenuated pulmonary polymorphonuclear (PMN) infiltration, decreased lung MPO activity, suppressed inflammation, reduced BALF protein content, and ameliorated lung pathological changes. Moreover, we showed that the PEP-NASP alleviated the LPSinduced inflammatory response by inhibiting activation of the TLR/TRAF6 signaling pathway in macrophages in vitro. Thus, our data indicated that the PEP-NASP fusion protein conferred protection against lung injury induced by LPS.
Inflammation is a defense mechanism against exogenous pathogens, and it has been implicated in various aggressive and excessive inflammatory responses leading to serious tissue damage (34)(35)(36). Development of ALI is due to uncontrolled inflammation, which is connected to the overproduction of various inflammatory mediators released by diverse immune cells, such as endothelial cells, neutrophils, and macrophages. LPS, a major constituent of gram-negative bacterial cells, is a ligand of TLR4 that induces inflammatory syndromes, such as multiple organ injury and sepsis, and is generally recognized as a key factor in inducing ALI (35)(36)(37). Challenge of macrophages with LPS can produce high levels of NO, subsequently leading to proinflammatory and destructive effects mainly caused by iNOS (38,39). In this study, we found that the PEP-NASP inhibited expressions of iNOS and COX-2 in LPS-stimulated macrophages. As is known, overproduction of inflammatory cytokines drives both COX-2 and iNOS gene expressions in macrophages, causing NO release (40). NO is a highly reactive free radical that participates in many inflammatory processes (41). Studies showed that iNOS and COX-2 gene expressions markedly increased during ALI induced by LPS (42). These potential effects of the PEP-NASP on reducing NO suggest an anti-reactive oxygen species (ROS) role of the PEP-NASP in bacterium-induced inflammation.
TRAF6 is a key adaptor for TLR4/NF-κB signaling in regulating innate and adaptive immunity (43,44). Increasing evidence indicates that the TLR4/TRAF6 pathway is considered to be involved in the process of inflammation-related ALI (45)(46)(47). For example, Ding et al. and Liu et al. both demonstrated that the inhibition of TRAF6 alleviated the severity of inflammation and lung injury in an ALI mouse model (45,47). Inhibition of TRAF6 ubiquitination can attenuate TRAF6-mediated NF-κB activation and proinflammatory cytokine expressions (45,46,48). In recent years, many bioactive ingredients extracted from herbs have been reported to effectively ameliorate ALI via different mechanisms. For example, glycyrrhizic acid attenuates LPS-induced ALI via modulation of the PI3K/AKT/mTOR pathway (49); picrasma quassiodes attenuates LPS-induced pulmonary inflammation and pulmonary edema by regulating the iNOS, HO-1, NF-κB and MAPK signaling (50); and magnoflorine exerts anti-inflammatory effects against LPSinduced ALI through regulation of TLR4-mediated MAPK and NF-κB signaling pathways (51). Moreover, isoalantolactone, a traditional Chinese herbal medicine, was reported to improve pulmonary inflammation and permeability through inhibiting TRAF6 ubiquitination (45). Our findings indicated that the PEP-NASP prevented TRAF6 ubiquitination by directly interacting with TRAF6 in RAW 264.7 cells. Transduction of the PEP-NASP also negatively regulates TRAF6-downstream TAK1/p38/JNK and NF-κB activation. LPS-induced proinflammatory cytokines also decreased by the PEP-NASP in lung tissue in vivo and in macrophages in vitro. We also showed that the PEP-NASP inhibited activation of the NLRP3 inflammasome in RAW 264.7 cells. These results suggested that the PEP-NASP exerts its antiinflammatory effects through suppressing TRAF6. Whether PEP-NASP regulate multiple levels of cellular process in response to LPS-induced inflammation is needed to be investigated in future studies.
Macrophages as important inflammatory cells were reported to attract neutrophil infiltration into the lungs and induce lung tissue damage (52). Meanwhile, clinical studies indicated that augmentation of the proinflammatory cytokines TNF-α, IL-1β, and IL-6 has a major role in the development of inflammatory diseases, including ALI (53). These cytokines amplify lung inflammation in ALI patients and were correlated with severity and poor outcomes, such as high protein pulmonary edema and respiratory failure (54,55). The lung W/D ratio is an indicator of pulmonary edema. We found that LPS induced a higher lung W/D ratio than that of control mice, and administration of the PEP-NASP ameliorated the degree of pulmonary edema due to LPS. Higher levels of total proteins induced by LPS in BALF were also inhibited by the PEP-NASP. In addition, due to cytokines, neutrophils infiltrated into lung tissues, leading to the production of proinflammatory cytokines, which are secreted by activated macrophages in ALI patients and influence pulmonary gas exchange (56,57). Inhibition of these proinflammatory cytokines provides important guidance for treating ALI. Our in vivo analysis performed in the ALI mouse model showed that the PEP-NASP suppressed the release of proinflammatory cytokines, including TNF-α, IL-1β, and IL-6, in lung tissues and BALF. These effects of the PEP-NASP on macrophages also revealed that the PEP-NASP might attenuate LPS-induced ALI via inhibiting cytokine production in macrophages. MPO is an enzyme stored in cytoplasmic granules of neutrophils, and its activity reflects the infiltration of neutrophils into lung tissues (58). We found that the PEP-NASP significantly decreased the activity of MPO induced by LPS and attenuated neutrophil infiltration into the lungs in LPS-induced ALI, indicating that the PEP-NASP can suppress neutrophil recruitment into lung tissues.
Studies have also found that the expression of TRAR6 was increased in lung tissues in mice with ALI (59)(60)(61). Clinically, TRAF6 variant (rs4755453) was significantly associated with susceptibility to sepsis-induced ALI in Chinese Han population (59). Therefore, it suggests that genetic variants in TRAF6 could influence TRAF6 mRNA stability and subsequently the production of inflammatory cytokines, which directly impacted lung tissue injury. The results of this study indicated the potent therapeutic effects of the novel peptide PEP-NASP against oxidative stress and pulmonary inflammation via selective TRAF6 signaling. Thus, PEP-NASP may exert potent therapeutic effects targeting genetic variants in TLR signaling, especially focused on high expression of TRAF6 population. We are planning to investigate the mechanism of PEP-NASP beyond genetic variants (rs4755453) in TRAF6 to further explore the clinical application.
In summary, we demonstrated for the first time that the human NASP, a negative inflammatory mediator, with its Nterminal fused to the PEP-1 peptide (PEP-NASP), can be efficiently transduced in vivo and in vitro. We also revealed that the transduced PEP-NASP has anti-inflammatory and antioxidant effects which ameliorated lung injury by regulating inflammatory responses and oxidative stress. The mechanisms for the protective role of the PEP-NASP are associated with the inhibition of TLR4-mediated TRAF6/NF-κB signaling pathways. Thus, our success in the protein transduction of PEP-NASP may provide a new therapeutic agent for protecting against inflammation-associated lung diseases, like ALI.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The animal study was reviewed and approved by the Animal and Ethics Review Committee of the Laboratory Animal Center at Taipei Medical University.