Protective effect of TM6 on LPS-induced acute lung injury in mice

Acute lung injury (ALI) is an acute failure of the respiratory system for which effective treatment is urgently necessary. Previous studies found that several peptides potently inhibited the production of cytokines induced by lipopolysaccharide (LPS). In this study, we synthetized a cell-permeable TIR domain-derived decoy peptide (TM6) and examined its substance for the ability to inhibit TLR signaling in the model of ALI induced by LPS. We demonstrated that TM6 (2.5, 5 and 10 nmol/g) alleviated the histological changes in the lung tissues as well as myeloperoxtidase (MPO) activity, lung W/D ratio, the production of TNF-α, IL-1β and IL-6 induced by LPS. Furthermore, the numbers of total cells, neutrophils and macrophages in the BALF were suppressed by TM6. In vitro, TM6 (5, 10 and 20 µM) inhibited the production of TNF-α, IL-1β and IL-6 in LPS-stimulated alveolar macrophages. Moreover, the activation of Nuclear factor-kappaB (NF-κB) and Mitogen activated protein kinases (MAPK) signaling pathways induced by LPS were also inhibited by TM6. Collectively, our results suggested that TM6 was an effective inhibitor of ALI induced by LPS, and this peptide may very well serve as a future treatment for ALI.

The effect of TM6 on lung wet/dry radio. Mice were pretreated with TM6 (2.5, 5 and 10 nmol/g) and DEX (5 mg/kg) 1 h prior to an i.n. administration of LPS. The lung wet/dry ratio was determined 7 h after LPS challenge. The values presented are the means ± SEM (n = 6). # P < 0.01 is significantly different from the control group; * P < 0.05 and ** P < 0.01 are significantly different from the LPS group.
The effect of TM6 on wet/dry lung ratios. Lung W/D ratios were evaluated 7 h after LPS stimulation.
The lung W/D ratio was markedly higher after LPS administration compared to the control group. However, TM6 (2.5, 5 and 10 nmol/g) significantly reduced the lung W/D ratio induced by LPS. In addition, DEX (5 mg/kg) also reduced the Lung W/D ratios after LPS stimulation (Fig. 2).
The effect of TM6 on myeloperoxtidase (MPO) activity. MPO activity served as a marker of the development of LPS induced ALI. MPO activity was evaluated 7 h after LPS stimulation. The results showed that lung MPO activity was up-regulated after LPS administration compared with control group. TM6 (2.5, 5, and 10 nmol/g) and DEX (5 mg/kg) administration prevented elevated MPO activity after LPS administration (Fig. 5).
The effect of TM6 on cell viability. MTT assay was used to measure the effect of TM6 on the viability of alveolar macrophages. The results showed that TM6 did not affect alveolar macrophages viability at concentrations with 5, 10 and 20 µM (Fig. 6). It suggests that TM6 at the concentrations of 5, 10 and 20 µM did not have any cytotoxic effects on alveolar macrophages.
The effect of TM6 on inflammatory cytokines production in alveolar macrophages. Alveolar macrophages were treated with TM6 (5, 10 and 20 µM) and DEX (1 mM) 1 hour before LPS treatment. 24 hours later, the inflammatory cytokines TNF-α, IL-1β and IL-6 in the supernatant of alveolar macrophages were tested by ELISA. As shown in Fig. 7, after LPS treatment, the production of TNF-α, IL-1β and IL-6 were significantly increased compared with control group. However, TM6 dose-dependently inhibited the production of TNF-α, IL-1β and IL-6 after stimulation with LPS in alveolar macrophages.

Discussion
Acute lung injury (ALI), a frequent complication of sepsis, can be caused by many factors, such as sepsis, trauma and bacterial pneumonia 18 . The development of ALI is associated with a large number of inflammatory cells migrating to the lung. Stimulation of these inflammatory cells lead to the releasing of inflammatory mediators. These inflammatory mediators disrupt pulmonary endothelial cells, pulmonary epithelial integrity and finally leads to lung edema 19,20 . Corticosteroids, including DEX, triamcinolone acetonide, flunisolide and so on, have . The effect of TM6 on cytokines production in BALF. Mice were given an intraperitoneal injection of TM6 (2.5, 5 and 10 nmol/g) and DEX (5 mg/kg) 1 h prior to an i.n. administration of LPS. BALF was collected 7 h following LPS challenge to analyze the inflammatory cytokines TNF-α, IL-1β, and IL-6. The values presented are means ± SEM (n = 6). # P < 0.01 is significantly different from the control group; * P < 0.05 and ** P < 0.01 are significantly different from the LPS group.
been widely used to treat inflammatory diseases like acute lung injury. Previous studies reported that treatment with DEX could improve the pathological process in ALI 21 . However, clinical application had suggested that these agents had local or systemic side effects. The reports have been suggested that some cell-permeable peptide can inhibited the development of inflammation in vivo and vitro 12,13 . Our previously studies also demonstrated that the peptide derived from TRAM or TIRAP also protected the development of mastitis induced by LPS in mice 14,15 . In our study, we synthesized a cell-permeable TIR domain of TRAM-derived decoy peptide (TM6) and tested the protective effect of TM6 on LPS-induced ALI in mice. The results showed that the administration of TM6 markedly decreased lung wet/dry ratios, inflammatory cells production, pro-inflammatory cytokines production, and MPO activity. Moreover, TM6 significantly alleviated LPS-induced lung histopathological changes. In vitro, TM6 also inhibited the production of TNF-α, IL-1β and IL-6 and the expression of NF-κB and MAPK. For further research, we also tested the effect of intranasally administered of TM6 in LPS induced ALI. The results suggested that TM6 also inhibited the production of inflammatory cells and pro-inflammation cytokines in BLAF, as well as MPO activity in lung tissues. But the protective effect is not as effective compared with TM6 administered by intraperitoneal injection (data not shown). These findings suggested that TM6 may have potential value as a future therapeutic treatment to prevent ALI.
It is well known that edema is a typical symptom of ALI. Endothelial injury associated with microvascular leakage, was believed to be the main contributor of lung edema 19 . To analyze the magnitude of lung edema, the lung W/D ratio was measured. The results showed that administration of TM6 apparently decreased the lung W/D ratio, which suggested it suppressed the accumulation of serous fluid in lung tissues. From analyzing the lung pathology sections with light microscopy, we also found a resolution in pulmonary edema and most of the histopathological changes. MPO, the influx of a marker of neutrophil into the tissue, has been associated with tissue damage in many diseases 22 . In this study, our results showed that TM6 significantly inhibited LPS-induced MPO activity. These results suggested that TM6 had the ability to protect against LPS-induced ALI.
LPS has been reported to be one of the major factors that induces inflammatory response 23,24 . In our model of ALI, LPS significantly increased the production of inflammatory cytokines including TNF-α, IL-1β and IL-6, which has been shown to be involved in the development of ALI 25,26 . TNF-α is a primary pro-inflammatory factor, and is produced initially during infection 27 . IL-1β, another important cytokine, is one of the crucial mediators in ALI. Moreover, IL-1β is involved in the process of LPS-induced endotoxic shock and caused multiple organ failure 28 . IL-6, another pro-inflammatory cytokine, was considered to be a marker in the LPS-induced inflammatory response 27 . In this study, we measured the production of the cytokines TNF-α, IL-1β and IL-6, both in vivo and vitro. The results showed that TM6 significantly inhibited LPS-induced inflammatory cytokine production both in vivo and in vitro. These results indicated that TM6 has anti-inflammatory property to inhibit the production of cytokines during LPS-induced ALI.
NF-κB and MAPK, served as the major transcription factors, have been reported to play vital roles in the regulation of inflammatory cytokines production 29,30 . Generally, once stimulated, NF-κB activation is initiated by the IκB-α and then NF-κB p65 is translocated to the nucleus. This process may promote the transcription of TNF-α, IL-1β, and IL-6 31 . MAPK family proteins including p38, JNK and ERK, all participate in the production of inflammatory cytokines 32 . To investigate the anti-inflammatory mechanism of TM6 on LPS-induced ALI, we determined the effects of TM6 on NF-κB and MAPK activation. The results showed that LPS-induced NF-κB and MAPK activation were markedly blocked by administration of TM6.
In conclusion, our study showed that TM6 was able to decrease inflammatory cell infiltration into the lung tissues, lung W/D ratio, MPO activity, inflammatory cytokines production, and lung histopathological changes. Moreover, the protective effect of TM6 may be related to attenuation of inflammatory reactions and inhibition of the activation of NF-κB and MAPK signaling pathways. These findings provide powerful evidence that TM6 may be a potential treatment for preventing ALI induced by LPS.

Animals. Adult male Balb/c mice were provided by the Experimental Animal Center of Baiqiuen Medical
College of Jilin University (Changchun, China). The procedures were approved by the Jilin University Animal Care and Use Committee. The mice were supplied with food and water in a constant environment with the temperature at approximately 24 ± 1 °C and 40% to 80% relative humidity. All experiments followed the regulations for the Use and Care of Laboratory Animals Manual published by the US National Institutes of Health. ALI model. Mice were randomly divided into six groups: a control group, an LPS group, TM6 (2.5, 5 and 10 nmol/g) + LPS groups, and a DEX (5 mg/kg) + LPS group. The mice of TM6 (2.5, 5 and 10 nmol/g) + LPS groups were received an intraperitoneal (i.p.) injection of TM6 (2.5, 5 and 10 nmol/g) 1 h before LPS treatment. The mice of DEX + LPS group (5 mg/kg DEX) were received i.p. injections using the same method to avoid Figure 6. The effect of TM6 on cell viability. Alveolar macrophages extract from mice were treatment with different concentrations of TM6 (0-40 µM) in the absence or presence LPS (1 µg/ml) for 24 hours. The cells viability was tested by MTT assay. The values presented are means ± SEM (n = 6). # P < 0.01 is significantly different from the control group; * P < 0.05 and ** P < 0.01 are significantly different from the LPS group. confounding procedural differences between groups. An equal volume of PBS, instead of TM6 or DEX, was given to the LPS group and control group. Then, 1 h later, 10 µg of LPS in 50 µl of PBS was introduced intranasally (i.n.) to produce acute lung injury. Control mice received 50 µl of PBS. All mice were humanely sacrificed 7 h after LPS treatment. Then, the bronchoalveolar lavage fluid (BALF) were collected for subsequent analysis.

Histopathological evaluation. Histopathological analysis proceeded via H & E staining and light
microscopy. Lung tissues were fully fixed with 10% buffered formalin for approximately 1 week, followed by paraffin-embedding and eosin staining. Histopathological changes of the lungs were examined with a light microscope (Olympus, Japan). The scores of histological changes in the lungs were evaluated as previously described 33 . Each animal is the average score of the above four criteria, including thickening of alveolar walls and epithelium, the numbers of infiltration cell, increase in peribronchial, and perivascular cuff area. The histological changes were scored 0 to 5.
Lung wet/dry ratio. The lung lobe tissues were extracted and weighted to obtain the wet weight. Then, the tissues were incubated in 80 °C for 48 h to obtain the dry weight. Finally, the lung W/D ratio was calculated. Figure 7. The effect of TM6 on inflammatory cytokines production in alveolar macrophages. Alveolar macrophages were treated with TM6 (5, 10 and 20 µM) and DEX (1 mM) 1 hour prior to stimulation with LPS. 24 hours later, the suspension was collected to test the levels of TNF-α, IL-1β, and IL-6. The values presented are means ± SEM (n = 6). # P < 0.01 is significantly different from the control group; * P < 0.05 and ** P < 0.01 are significantly different from the LPS group.
Scientific RepoRts | 7: 572 | DOI:10.1038/s41598-017-00551-8 Inflammatory cell numbers of BALF. BALF was collected 7 h after ALI was induced by LPS. After centrifugation (3000 rpm at 4 °C) for 10 min, the supernatants were collected and used to analyze the production of inflammatory cells.
Pulmonary myeloperoxidase activity analysis. MPO activity reflects accumulation of macrophages and neutrophils in the lung tissues. The MPO activity in the lung tissues was measured by MPO kit according to the manufacturer's instructions.
Cell culture and treatment. Mice alveolar macrophages were isolated and cultured as previously described 34 .
In brief, the mice were killed and the lungs were lavaged with PBS. The fluids were collected and centrifuged at 1000 g for 10 min at 4 °C. Then, the supernatants were removed and the cells were resuspended with RPMI 1640 media. The cells were plated in well plate and cultured at 37 °C in 5% CO 2 incubator for 4 hours. Then the cells were washed once a day for 3 days. The nonadherent cells were removed and attached cells were cultured in RPMI 1640 media with 10% fetal calf serum. The media was changed once every day. In the study, the mice alveolar macrophages were preincubated with or without TM6 (5, 10 and 20 µM) for 1 hour before stimulation with LPS (1 µg/ml).
Cell viability assays. Cell viability was tested by MTT assay. The alveolar macrophages were plated at a density of 4 × 10 5 cells/ml in 96-well plates at 37 °C. 1 hour later, the cells were treated with TM6 at the dose with 0-40 µM and then cultured for another 1 hour. Next, the cells were treatment with LPS for 18 hour, followed by treatment with MTT (5 mg/ml). The cells were incubated for an additional 4 hours. Then, the supernatants were removed and DMSO (150 µl/well) were added to each well. The OD was read at 570 nm on a microplate reader.
Supernatant samples were used to analyze the production of TNF-α, IL-1β and IL-6 using special ELISA kits according to the instructions provided by the manufacturer.
Western blot analysis. The proteins from the alveolar macrophages were extracted with mammal protein extraction reagent (M-PER) according to the manufacturer's instructions. Equal amounts of protein (30 µg) were subjected to SDS-PAGE on 10% gels and transferred to PVDF membranes. The membranes were blocked with 3% BSA at room temperature on a rotary shaker for 2 h. Incubation with specific primary antibodies (1:1000) in TBS-T was performed overnight at 4 °C. Following washing, the membranes were incubated with secondary antibody for 1 h. The proteins bands were detected using an enhanced chemiluminescence western blotting detection kit. Figure 8. The effect of TM6 on NF-κB pathway. The effects of TM6 on the expression of the NF-κB pathway that was induced by LPS. Alveolar macrophages were pretreated with TM6 (5, 10 and 20 µM) for 1 hour and then treated with LPS for 1 hour. NF-κB protein samples were analyzed by western blot with specific antibodies. β-actin was used as a control. The values presented are the means ± SEM of three independent experiments, # P < 0.01 is significantly different from the control group; * P < 0.05 and ** P < 0.01 are significantly different from the LPS group. Figure 9. The effect of TM6 on MAPK pathway. Alveolar macrophages were pretreated with TM6 (5, 10 and 20 µM) for 1 hour and then treated with LPS for 1 hour. MAPK protein samples were analyzed by western blot with specific antibodies. β-actin was used as a control. The values presented are the means ± SEM of three independent experiments, # P < 0.01 is significantly different from the control group; * P < 0.05 and ** P < 0.01 are significantly different from the LPS group.