25‐Hydroxycholesterol protects against acute lung injury via targeting MD‐2

Abstract Acute lung injury (ALI) is mainly caused by uncontrolled inflammatory response, and it remains without effective therapeutic options. 25‐hydroxycholesterol (25HC) has been reported to be a potent regulator of inflammation. The aim of this study was to investigate the effects of 25HC on lipopolysaccharide (LPS)‐induced ALI. C57BL/6 mice were pretreated with 25HC intraperitoneally before intratracheal exposure to LPS. Our results showed that 25HC pretreatment improved survival rate, attenuated the pathological changes of the lung and decreased the release of inflammatory cytokines in mice. Consistently, 25HC reduced expression of Toll‐like receptor (TLR4)‐mediated inflammatory cytokines in vitro. These effects of 25HC were obtained by preventing LPS binding to TLR4 via interaction with myeloid differentiation protein 2 (MD‐2). Crystal structure analysis suggested that 25HC could bind MD‐2 with high affinity into its hydrophobic pocket. Furthermore, LPS‐induced activation of Akt/NF‐κB pathway was partially down‐regulated by 25HC pretreatment. In summary, this study demonstrates that 25HC could inhibit the overwhelming inflammatory response through MD‐2 interaction, which suppresses Akt/NF‐κB signalling pathway. These findings suggest 25HC may be a promising candidate for ALI prevention.

interaction of LPS and MD-2 bridges two TLR4 molecules to induce the multimerization of the LPS-TLR4-MD-2 complex. 12 Activation of the TLR4-MD-2 complex leads to the transcription of nuclear factor (NF)-κB, which triggers the overproduction of proinflammatory cytokines, such as interleukin (IL)-6, tumour necrosis factor α (TNF-α) and IL-8 in the early stage of immune response. 13,14 The cytokine-mediated inflammation prompts the progression of ALI. 15 Oxysterols, oxidized forms of cholesterol, participate in cholesterol homeostasis, immune response and pathogenesis of several diseases such as neurodegenerative diseases and atherosclerosis. 16 25-hydroxycholesterol (25HC), one of the oxysterols, has recently garnered much attention as a potential regulator of innate immunity and inflammation. 17 [18][19][20] Moreover, 25HC has been reported to mediate the activation of NF-κB signalling to induce IL-6 and IL-8 expression. 21,22 A recent study observed that 25HC decreased the production of IL-1β and inflammasome activity, leading to improve recovery from septic shock. 23 These findings suggested that 25HC could be involved in the regulation of inflammatory processes and its function varies based on biological conditions. In this study, we aimed to investigate the effects of 25HC on LPS-treated ALI murine model and RAW264.7 cells. Our study reveals 25HC to possess anti-inflammatory role in both mouse and cell models, which collectively indicate 25HC as a potential therapeutic option for ALI and other acute inflammatory diseases.

| Mouse ALI model
The animal studies were approved by the Ethics Committee of animal experiments at Zhejiang University, and all the processes are in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Pathogen free, female C57BL/6 mice (6-8 weeks old) were obtained from the Animal Center of Slaccas (Shanghai, China). The mice were injected intraperitoneally with 25HC (50 mg/kg) or the vehicle control 6 hours before intratracheal stimulation of LPS (3 mg/kg). The mice treated with vehicle alone were employed as control. Six hours after LPS treatment, the mice were killed to evaluate histological changes and inflammatory cytokine expression. For the mortality study, mice were intratracheally challenged with a lethal dose of LPS (40 mg/kg). The survival rate was recorded every 12 hours until 3 days post-LPS injection.

| Histological analysis
Whole lung tissues of mice were fixed with 4% paraformaldehyde neutral buffer overnight, embedded in paraffin, sectioned at 4 μm thickness and stained with Haematoxylin-Eosin (H&E). Morphometric assessment was conducted under an automatic photo-microscope (Olympus, Tokyo, Japan).

| Collection and analysis of bronchoalveolar lavage fluid (BALF)
BALF was collected through lavaging the lungs using a tracheal cannula with 1 mL of ice-cold PBS for three times. After removing the erythrocytes in the BALF by lysis buffer, the total cell number was counted. 2 × 10 5 cells were smeared on a slide and stained with Giemsa regent (Nanjing Jiancheng Bio-engineering Institute, Nanjing, China) for cell differentiation. BALF protein concentrations were determined using the bicinchoninic acid (BCA) protein assay kit (Beyotime Biotechnology, Beijing, China). LDH activity in BALF was determined with LDH Cytotoxicity Assay Kit (Nanjing Jiancheng Bioengineering Institute).

| Real-time quantitative PCR (RT-qPCR)
Total RNAs were extracted with the Ultrapure RNA kit (Cwbiotech, Beijing, China) and reverse-transcribed to cDNA using cDNA synthe-   Table 1. β-actin was amplified as a housekeeping gene. The relative expression levels of target genes were calculated by applying ΔΔCt (cycle threshold) approach.

| ELISA for LPS binding to MD-2
ELISA was used for assaying LPS binding to rhMD-2 as described before. 24 Briefly, the anti-MD2 antibody was coated in 96-well micro-plates for overnight at 4°C. The plate was washed and blocked F I G U R E 1 25HC pretreatment reduces mortality and lung injury in LPS-induced ALI mice. (A) C57BL/6 mice were received with 25HC (50 mg/kg) or vehicle by intraperitoneal injection 6 h before intratracheal stimulation of LPS (40 mg/kg). Mouse survival rate was observed every 12 hours up to 72 hours (n = 12 mice/group). (B-F) C57BL/6 mice were intraperitoneally received with 25HC (50 mg/kg) or vehicle 6 h before intratracheal administration of LPS (3 mg/kg). Then, the mice were killed 6 h after LPS treatment. (n = 4 mice/group). Representative histopathologic sections of lung tissues by haematoxylin and eosin staining (small panels, ×400, scale bar: 20 μm; big panels, ×100, scale bar: 100 μm) (B). Total protein levels (C), LDH activity (D), counts of the total cells and neutrophils (E) and MPO quantity (F) in BALF were analysed. Quantitative data are shown as mean ± SEM. *P < 0.05 and **P < 0.01

| Fluorescence spectroscopy detection
All the measurements of fluorescence spectra were performed on

| Statistical analysis
Quantitative data are shown as mean ± SEM and calculated on GraphPad Prism 5. Statistical significance among multiple groups was performed using one-way ANOVA test. Mouse survival curve analysis was evaluated by the log rank test. A P value less than 0.05 was considered to be statistically significant.

| 25HC pretreatment reduces mortality and lung injury in LPS-induced ALI mice
To determine the effect of 25HC on the mortality in ALI, mice were

| 25HC inhibits production of proinflammatory cytokines and chemokines in ALI mice
Proinflammatory cytokines and chemokines contribute to inflammatory responses in ALI. 2 In this study, the levels of

| 25HC attenuates LPS-induced activation of Akt and NF-κB pathway
To explore the effect of 25HC on the LPS-initiated TLR4 signalling,

| DISCUSSION
ALI is a life-threatening clinical condition with substantial mortality.
To date, several clinical trials showed ineffectiveness of many pharmacological strategies in reducing ALI-related mortality. 27 Here, we demonstrate that as one of the oxysterols, 25HC has therapeutic effects on LPS-induced ALI via reduction in the inflammatory responses. We further reveal that the underlying anti-inflammatory mechanism of 25HC is mainly based on prevention of LPS binding to TLR4-MD-2 complex by its engagement with the hydrophobic pocket of MD-2, which consequently impairs the activation of downstream Akt/NF-κb signalling and dampens induction of inflammatory cytokines.
As major inflammatory cells in innate immunity, alveolar macrophages are activated by LPS via TLR4-mediated signal transduction, leading to production of proinflammatory cytokines. 28  The oxysterol 25HC is synthesized by cholesterol 25-hydroxylase (Ch25h), which is an interferon-stimulated gene (ISG) strongly up-regulated in macrophages after stimulation by TLR ligands. 19,31,32 25HC has been reported to repress the activation of sterol regulatory element-binding proteins (SREBPs) to interfere with cholesterol biosynthesis. 33 In addition, 25HC could activate orphan nuclear receptors such as liver X receptors (LXRs) and proliferation activator receptor gamma (PPARγ) to regulate lipid metabolism and inflammation. 34 and xanthohumol (4,4′-dihydroxy -5′-isopentenyl-2′ -methoxychalcone), were reported to inhibit TLR4-mediated NF-κB activation via directly antagonizing LPS binding to MD-2. 41,44 Moreover, xanthohumol binding was located in the deep hydrophobic pocket of the MD-2, adjacent to and hydrogen-bonded with Tyr-102. A chalcone derivative L2H21 also showed as a direct inhibitor of MD-2 by binding to Tyr-102 and Arg-90 residues in the hydrophobic pocket of MD-2. 24 Here, we demonstrate that 25HC binds to MD-2 with high affinity and inhibits the interaction between LPS and MD-2. From the molecular docking results, 25HC is predicted to bind to the hydrophobic pocket of MD-2 and in close proximity to the Tyr-102, Tyr-65, Ile-117 and Leu-71 residues, forming a hydrogen bond with Tyr-102.
25HC was previously described as a potential ligand for LXR pathway, the activation of which inhibited proinflammatory genes expression in immune cells. 45,46 The other study showed 25HC decreased nuclear peroxisome proliferation activator receptor γ (PPARγ) and increased nuclear NF-κB levels, which induced release of proinflammatory cytokines. 35 NF-κB is known as a major transcription factor participating in regulation of inflammatory cytokine generation. 47 Phosphoinositide 3-kinase downstream kinase Akt has been shown to be involved in modulating NF-κB activation. 48 Additionally, the MAPK pathway also plays a significant role in TLR4mediated inflammation. 49 Our current data indicate that 25HC interferes with LPS binding to TLR4 complex and inhibits the activation of Akt/NF-κB signal pathway, but does not affect the MAPK pathway. Considering the multiple effects of 25HC, the downstream signalling of TLR4 is likely selectively inhibited by 25HC.
In conclusion, 25HC modulates excessive inflammatory responses and protects against LPS-induced ALI via directly targeting MD-2.
This study offers a therapeutic strategy against ALI through inhibition of MD-2-mediated inflammatory signalling.

ACKNOWLEDG EMENTS
This work was supported by grants from National Natural Science Foundation of China (81770008 and 81370176) and Major Science and Technology Special Project of Zhejiang Province (2014C03033).

CONFLI CT OF INTEREST
The authors declare that they have no conflicts of interest.