Male Sprague-Dawley rats, 8-weeks-old and (weight, 200–220 g), were obtained from Vital River Laboratories Co., Ltd. (Beijing, China). All animals used for experiments were allowed free access to food and water, and housed under specific pathogen-free conditions with a 12 h light/dark cycle in a controlled temperature (20–25°C) and humidity (50±5%) environment. All experimental protocols were approved by the Laboratory Animal Committee of Hebei Medical University, (Shijiazhuang, China). The rats were sacrificed under anesthesia with chloral hydrate (350 mg/kg i.p.), followed by cervical dislocation. All efforts were made to minimize suffering.

A total of 90 rats were randomly divided into three groups (n=12/group): (1) Control group: Received saline by intratracheal instillation (5 ml/kg) under anesthesia using inhaled isoflurane (Boston Biochem, Inc., Cambridge, MA, USA); (2) ALI group: ALI was induced by intratracheal instillation of LPS to induce acute lung injury model in vivo, as previously described (5,19). In brief, rats were anaesthetized and injected intravenously with LPS (5 mg/kg, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). (3) IL-17 mAb group: Rats were injected intraperitoneally with IL-17 monoclonal antibody (100 µg/ml, IL-17 mAb, Clone # 50101; R&D Systems, Inc., Minneapolis, MN, USA). Rats in all groups were sacrificed respectively at 3, 6 and 24 h following injection, 10 rats per group per time point.

At 6 h following LPS or saline challenge, the lung W/D weight ratio was calculated to evaluate the extent of acute pulmonary edema, Five rats per group were anesthetized, harvested and cleaned from blood, weighed to obtain the wet weight, and then placed in an oven for 48 h at 80°C for the measurement of the dry weight. The ratio of the wet weight to dry weight was then calculated (20).

Morphological changes in the lungs were examined by hematoxylin and eosin (H&E) staining. At 6 h and 24 h following the instillation of LPS, rats were euthanized for histological assessment. The right upper lobes were collected, fixed in 10% formalin for 48 h and embedded in paraffin. Sections of fixed embedded tissues (5 µm thick) were cut on a Leica model 2165 rotary microtome (Leica Microsystems GmbH, Wetzlar, Germany), placed on glass slides, deparaffinized and sequentially stained with H&E (Thermo Fisher Scientific, Inc., Waltham, MA, USA) for examination under an optical microscope (BX51; Olympus Corporation, Tokyo, Japan). The extents of histological injury were evaluated by two blinded experienced investigators. The degree of lung injury was graded using a scoring system based on histological features, including edema, congestion and hyperemia, tissue infiltration and neutrophil margination, and the presence of intra-alveolar hemorrhage, debris and cellular hyperplasia. Each feature was graded as either absent=0, mild =1, moderate=2, or severe=3. The total score was calculated for histopathological evaluation of each rat.

BALF was collected by intratracheal intubation as previously described (5). Briefly, after mice were anesthetized, the trachea was exposed and cannulated with a sterile catheter. BAL was performed with a 5 ml 0.9% saline solution. The lavage was repeated twice with saline to recover a total volume of 4–5 ml. Lavage samples were centrifuged at 1,000 × g at 4°C for 10 min. All samples were stored at −80°C to measure chemokine levels by using ELISA kits.

The concentrations of TNF-α, IL-6, IL-1β and IL-17 in BALF were detected using cytokine specific Quantikine ELISA kits (TNF-α, SEA133Ra; IL-6, SEA079Ra; IL-1β, SEA073Ra; IL-17, SEA063Ra; Wuhan USCN Business Co., Ltd., Wuhan, China) according to the manufacturer's instructions. The absorbance was read at 490 nm on an ELISA plate scanner (Molecular Devices, LLC, Sunnyvale, CA, USA). All experiments are performed at least in triplicate samples and results are presented as the mean value. A standard curve using recombinant cytokine was generated for each assay.

Total RNA was extracted from rat lung tissues with Tripure Isolation reagent (Thermo Fisher Scientific, Inc.). The qPCR kits (Takara Biotechnology Co., Ltd., Dalian, China) were used for the qPCR experiment. cDNA was synthesized from total RNA using a SuperScript Reverse Transcriptase kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The primers of PCR were as follows: Rat IL-17 forward, 5′-ATCCCTCAAAGTTCAGTGTGTCC-3′ and reverse, 5′-GGACAATAGAGGAAACGCAGGT-3′; GAPDH forward, 5′-CAAAGTTGTCATGGATGACC-3′ and reverse, 5′-CCATGGAGAAGGCTGGGG-3′. RT-qPCR was performed on a Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.; ABI Prism 7300) instrument with SYBR Premix Ex Taq (Takara Bio, Inc., Otsu, Japan), starting with 1 ng reverse-transcribed total RNA. PCR was performed under the following conditions: 95°C for 10 min, 40 cycles of 95°C for 5 sec and 60°C for 30 sec. GAPDH was used as an internal control. The relative mRNA expression levels were calculated by the 2^−ΔΔCq method (21).

Lung tissues were lysed in total protein cell lysis buffer (Thermo Fisher Scientific, Inc.) supplemented with 5% Proteinase Inhibitor cocktail (Sigma-Aldrich; Merck KGaA), incubated on ice for 30 min, and centrifuged at 15,000 × g for 15 min. Protein concentrations were determined with the bicinchoninic acid protein assay reagents (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Total protein (50 µg) was loaded in each lane, separated using 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes using a wet transfer method at room temperature. Nonspecific binding sites were blocked with 5% BSA for 1 h, then incubated with rabbit anti-rat IL-17 (1:1,000; SC-52567), nuclear factor (NF)-κB (1:1,000; SC-514451), p-extracellular signal-regulated kinase (ERK) 1/2 (1:1,000; SC-136521) and β-actin polyclonal antibody (1:1,000; SC-7210) (all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4°C. The following day, the membranes were incubated with incubated with horseradish peroxidase conjugated secondary antibody (1:5,000, #7074S; Cell Signaling Technology, Inc., Danvers, MA, USA). The immunoreactive bands were visualized with an enhanced chemiluminescence (Bio-Rad Laboratories, Inc., Hercules, CA, USA) reagent. Blots were scanned by densitometry, and integrated density of pixels was quantified using Image Quant software (version, 5.2; Molecular Devices, LLC, Sunnyvale, CA, USA).

All data in the tests and figures were presented as means ± standard deviation. The statistical software SPSS software (version, 13.0; SPSS, Inc., Chicago, IL, USA) was used for data analysis. One-way analysis of variance (ANOVA) followed by Tukey's post hoc test, or a two-tailed unpaired Student's t-test was applied to evaluate statistical significances. Measurements in the intergroup differences at single timepoints were analyzed by an ANOVA, and, if they demonstrated significance, they were further analyzed by the two-tailed unpaired Student's t-test. P<0.05 was considered statistically significant.

In order to confirm that the expression of IL-17 is involved in pathophysiologic process of ALI, the authors detected the mRNA and protein levels of IL-17 in the BALF and lung tissues of a LPS-induced ALI rat model at 3, 6 and 24 h following LPS challenge. As presented in Fig. 1, the protein levels of IL-17 in the BALF and lung tissues were markedly elevated at 3, 6 and 24 h following LPS injection. Similarly, the mRNA expression of IL-17 in the lung tissues of LPS-treated rats was enhanced. The mRNA and protein levels of L-17 peaked at 6 h, and prolonged the lag time of 24 h. These results indicated that the expression of IL-17 may be closely correlated with development of LPS-induced lung injury.

To determine the roles of IL-17 in LPS-induced lung injury, the authors examined the effects of IL-17 mAb on lung injury. The lung histopathology changes at 6 h and 24 h post-ALI are presented in Fig. 2. To observe characteristics of LPS-induced lung injury in rats, LPS treated rats exhibited severe lung injury characterized by inflammation, cell infiltration, interstitial edema, alveolar structural damages and alveolar wall thickening, which was markedly improved by IL-17 mAb treatment, while those phenomena could not be observed in the saline group. A scoring system was used to grade the degree of lung injury, and rats exposed to LPS presented a remarkable increase in lung histologic scores, but the histological scores of IL-17 mAb treated rats were lower than those of ALI group at 6 h (Fig. 2B) and 24 h (Fig. 2C).

Several well-known hallmarks of ALI-induced pulmonary edema, such as BALF exudate volume (Fig. 3A), protein leakage in BALF (Fig. 3B), and lung W/D weight ratio (Fig. 3C) were remarkably increased in LPS challenge rats compared with those in the control group. Importantly, inhibition of IL-17 using IL-17 mAb, significantly alleviated LPS-induced BALF exudate volume, protein leakage and lung W/D weight ratio. These results demonstrated that IL-17 mAb treatment may improve pulmonary edema in LPS-challenged rats.

To confirm the effects of IL-17 mAb treatment on LPS-stimulated expression and release of proinflammatory cytokines and chemokines, the authors measured the levels of IL-17, TNF-α, IL-1β and IL-6 in the BALF of rat by ELISA. As demonstrated in Fig. 4, proinflammatory cytokines including TNF-α, IL-1β and IL-6 were all significantly elevated in BALF in response to LPS treatment. Consistent with this, levels of these proinflammatory factors were dramatically decreased compared with LPS-treated group. These results suggested that IL-17 mAb administration significantly prevented the upregulation of proinflammatory cytokines and chemokines in BALF of LPS challenged rats.

Given the crucial role of IL-17 in the pulmonary inflammation, the authors next sought to investigate the underlying mechanisms of IL-17 involved in the development of ALI. The expression of ERK1/2, p-ERK1/2 in the lung tissues of rats suffering from LPS stimulation was investigated. As demonstrated in Fig. 5, the LPS-treated group presented significant increases in the expression of the p-ERK1/2 protein when compared with the control group. Moreover, the upregulation of p-ERK1/2 protein was reversed by IL-17 mAb treatment. However, the expression of the ERK1/2 protein presented no significant difference between each group. These findings suggested that effects of IL-17 mAb on the features of LPS-induced lung inflammation primarily through inhibition of ERK1/2 pathway activity.

Since NF-κB is known to be a critical transcription factor for inflammation, the molecular mechanisms by which IL-17 mAb administration ameliorates LPS-induced lung inflammation we explored. This was conducted by measuring the levels of NF-κB p65 in the cytoplasm and nucleus. As presented in Fig. 6, a basal level of NF-κB p65 was observed in the cytoplasm and nuclei of lung samples in the control group. The level of NF-κB p65 in nuclear extracts of lung samples was significantly increased following the instillation of LPS compared with control group, this increase was reduced by administration of IL-17 mAb. In contrast, cytosolic NF-κB p65 expression was decreased in the ALI group in comparison with control group, and IL-17 mAb treatment could reversed the decrease in NF-κB p65 protein. Additionally, western blot analyses revealed that the LPS-induced nuclear translocation of NF-κB p65 was restored by administration of IL-17 mAb.