A total of 54 male specific pathogen-free Sprague-Dawley rats weighing 250–300 g (8–10 weeks) were assigned to the Laboratory Animal Center, Fudan University (Shanghai, China) in clean conditions with a controlled temperature of 18–26°C, humidity 40–70%, and 12/12-h light/dark cycle. The animals were given free access to water but restricted to food only for 12 h prior to experimentation. The Animal Ethics Committee of Fudan University approved all experimental protocols. All animals were sacrificed following the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.

The rats received an injection of PBS or LPS (Escherichia coli O55:B5; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany; cat. no. L2637) via the tail vein. A dose of 10 mg/kg of LPS was dissolved in 1 ml PBS. The control animals were injected with 1 ml PBS. After 1, 2, 4, 6, 8, 12, 24 and 48 h (n=6 each), the rats were treated with intraperitoneal injections of 7.5 ml/kg urethane. The wet-to-dry (W/D) weight ratio of the middle lobe of the right lung was measured as previously described (26).

BALF of each experimental rat was collected and was done in the left lung. BALF was centrifuged (600 × g, 10 min at 4°C; LTPA Cytocentrifuge, Experimental Instrument Factory, Academy of Military Medical Sciences, Beijing, China), and cell-free supernatants were stored at −80°C. The cell pellet was diluted in PBS, and total cell number was counted with a hemocytometer. Differential cell counts were done with cytocentrifuge preparations stained with Diff-Quik stain at room temperature for 90 sec. Neutrophil populations were determined by counting 300 cells/sample, and a percentage was calculated based on 6 mice per group. Bronchoalveolar lavage protein concentration was measured in the cell-free supernatant by using a bicinchoninic acid protein assay kit according to the manufacturer's instruction (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

The Shanghai Institutes for Biological Sciences of the Chinese Academy of Science (Shanghai, China) provided the lung macrophage NR8383 cell line from its cell bank. All cells were preserved in humidified air with 5% CO₂ at 37°C. Dulbecco's modified Eagle's medium/F-12K (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. 12100-046) was used to preserve the cells, supplemented with 20% fetal bovine serum (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA; cat. no. SV30087.02), 100 U/ml streptomycin and 100 U/ml penicillin G (Invitrogen; Thermo Fisher Scientific, Inc.; cat no. GB15140-122) with 2.5 g/l NaHCO₃ and 2 mM L-glutamine solution (Sigma-Aldrich; Merck KGaA; cat. no. G7513) at 37°C in humidified air with 5% CO₂.

Sigma-Aldrich (Merck KGaA) provided the siRNA. The sequence of siRNA for rat A20 is: Sense 5′-CCGGCCAATGGTGATGGAAACTGCCCGAGTAGGCAGTTTCCATCACCGTTGGTTTTTG-3′, and antisense 5′-AATTCAAAAACCAATGGTGATGGAAACTGCCCGAGTAGGCAGTTTCCATCACCGTTGG-3′. For siRNA control (scrambled siRNA, SCR), oligos with no matching GeneBank sequence were used: Sense 5′-GCGACGAUCUGCCUAAGAU-3′, and antisense 5′-AUCUUAGGCAGAUCGUCGC-3′. The siRNA oligonucleotide pairs were prepared as 20 µM stocks. For transient transfections, the Lipofectamine™ RNAiMAX kit (Thermo Fisher Scientific, Inc.; cat no. 13778-150) was used on the NR8383 cell line. Following transfection, the cells were left for a further 24 h before they were used for experiments.

Mouse A20 (Tnfaip3; gene ID: NM_009397.3) was cloned from mouse cDNA (Beijing ComWin Biotech Co., Ltd.; Beijing, China) with forward primer (5′-GTTGGCAAAGCATACAACTGAAAGG-3′) and reverse primer (5′-GGCTGTGACGAAGGAAGAGCTTA-3′) under the following conditions: i) 95°C for 2 min; ii) 30 cycles of 95°C for 20 sec, 52°C for 20 sec and 72°C for 60 sec; iii) 72°C for 3 min. The nucleotide sequence was inserted into the pCDH-CMV-MCS-EF1-copGFP vector (System Biosciences, Palo Alto, CA, USA; cat. no. cd511b) at the EcoRI and BamHI restriction sites. The recombinant vector was transfected into the NR8383 cell line using Lipofectamine™ 2000 (Thermo Fisher Scientific, Inc.; cat. no. 11668-019). Following transfection, the cells were treated with LPS (1 µg/ml) at room temperature for 0.5, 1, 2 and 4 h. The cells were then collected for use.

Subsequent to measurement of the wet weights and examination of the BALF, the lower lobe of the right lung was fixed in 4% formaldehyde at room temperature and paraffin-embedded with the procedure previously described (13), while the upper lobes of the right lung were stored at −80°C. The tissue preparations with paraffin-embedding were cut into 5-µm sections and stained with hematoxylin and eosin to assess the histopathology score. The method of staining and assessment was as previously described (26). Immunostaining of lung tissue was done with 5-µm paraffin sections. Anti-A20 (Cell Signaling Technology, Inc., Danvers, MA, USA; cat. no. 5630) and F4/80 (Abcam, Cambridge, UK; cat. no. ab111101) antibodies were used for staining tissue sections at different time points. Lung tissue was treated twice with fluorescently labeled antibodies; F4/80 (secondary antibody with green fluorescence) and A20 (secondary antibody with red fluorescence), while the nuclei were stained with DAPI. Fluorescent images were captured by a Zeiss LSM 510 META confocal laser-scanning microscope (Carl Zeiss AG, Oberkochen, Germany). The number of A20 and F4/80 positive cells was counted in five randomly selected fields by viewing each slide at a magnification of ×400 and the average number in each group was calculated. All procedures were as previously described (13).

NF-κB DNA binding activity was measured in lung tissue with an ELISA kit from Active Motif (Carlsbad, CA, USA; TransAM^® Flexi NFκB p65; cat. no. 40098) and the result was presented as NF-κB optical density (OD). The procedure was performed according to the manufacturer's protocol. TNF-α (Rat TNF-alpha Quantikine SixPak; R&D Systems, Inc., Minneapolis, MN, USA; cat. no. SRTA00) and IL-1β (Rat IL-1 beta/IL-1F2 Quantikine SixPak; R&D Systems, Inc.; cat. no. SRLB00) levels in the supernatant were measured with ELISA kits. The method was performed according to the manufacturer's protocol.

The procedure for immunoblotting was performed as previously described (27). Proteins were loaded into the lanes of an SDS or tricine-SDS polyacrylamide gel. The proteins were separated and transferred to nitrocellulose membranes (0.45 or 0.22 µm; Schleicher & Schuell; BioScience GmbH, Dassel, Germany). The membranes were blocked with 5% non-fat dry milk in 0.01 M PBS (pH 7.4) and 0.05% Tween-20 (TPBS) at room temperature for 1 h. Subsequently, the membrane was incubated with primary antibodies directed against target proteins overnight at 4°C. The primary antibodies used were: Anti-A20 rabbit monoclonal antibody (A20/TNFAIP3 (D13H3) Rabbit mAb; Cell Signaling Technology, Inc.; Danvers, MA, USA; cat. no. 5630), anti-NF-kB p65 (Abcam; Cambridge, UK; cat. no. ab16502) and GAPDH (R&D Systems, Inc.; cat. no. AF5718). The final dilutions for the primary antibodies were: A20, NF-kB, 1:1,000; and GAPDH, 1:2,000. Following three quick washes in TPBS, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (GE Healthcare, Chicago, IL, USA; cat. no. RPN4301) diluted at 1:5,000 in TPBS for 1 h at 4°C. The enhanced chemiluminescence method (GE Healthcare; cat. no. RPN2106) was used to visualize protein bands. The protein expression was quantified using Quantity One software version 4.62 (Bio-Rad Laboratories Inc., Hercules, CA, USA).

Total RNA was isolated from the lung tissues using TRIzol reagent (Thermo Fisher Scientific Inc.; cat. no. 15596018) and treated with RNase-free DNase I (Roche Applied Science, Penzberg, Germany; cat. no. 10104159001). First-strand cDNA was synthesized using Moloney-murine leukemia virus reverse transcriptase (Promega Corporation, Madison, WI, USA; cat. no. M1701) and oligo-dTs (Promega Corporation; cat. no. C1101), which was performed at 42°C for 1 h followed by 70°C for 15 min. Subsequently, the RevertAid™ First Strand cDNA Synthesis kit (Fermentas; Thermo Fisher Scientific, Inc.) was used for the qPCR, under the following conditions: i) 95°C for 30 sec; ii) 40 cycles of 95°C for 5 sec and 60°C for 30 sec. Quantitative analysis was performed with the Applied Biosystems Prism 7500 Sequence Detection System according to the manufacturer's protocol (Thermo Fisher Scientific, Inc.). The expression levels of target cDNAs were normalized to the endogenous transcription levels of the control and quantification was performed using 2^−ΔΔCq (28). The primer sequences are provided in Table I.

NR8383 cells were treated with trypsin, resuspended in PBS and stained with 10 µl F4/80 antibodies (15 min at 37°C) or 10 µl anti-cluster of differentiation CD206 (15 min at 37°C). A20 overexpression and A20 knockdown cells [small interfering (si)RNA for A20] were treated with anti-F4/80 and CD206 antibodies. The anti-F4/80 antibody was labeled with fluorescein isothiocyanate (Abcam; cat. no. ab111101) with green fluorescence, while the anti-CD206 antibody was labeled with allopycocyanin (Abcam; cat. no. ab64693) with red fluorescence. The final dilutions for the antibodies were: Anti-F4/80 antibody, 1:100; and anti-CD206 antibody, 1:3,000. The FL2 and FL1 fluorescence was collected in each experiment by gating and acquiring the cell population using a FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). CD206⁺/F4/80⁺ cells are M2 macrophages while CD206⁻/F4/80⁺ cells are M1 macrophages (29).

Statistical significance was assessed by analysis of variance (ANOVA) and Student's t-test using SPSS software version 22.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 6 (GraphPad Software Inc., La Jolla, CA, USA). One-way ANOVA was used for the analysis of relative quantitative data, with the least significant difference post hoc test. Student's t-test was used for the analysis of independent variables. The data were presented as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

Compared with the control group, the LPS-treated group demonstrated histological features representative of ALI and significant increases in the lung W/D weight ratio, BALF total cell number and BALF protein concentration (P<0.05; data not shown).

Following LPS treatment, the levels of TNF-α and IL-1β, and NF-κB DNA binding activity increased in a time-dependent manner (Fig. 1A-C). At the 2 h time-point, the activity reached a first peak (all P<0.05 compared with the control group). In the following 10 h, the levels of TNF-α and IL-1β, and the NF-κB activity decreased gradually but remained increased compared with the control (all P<0.05). Furthermore, at 24 h, the levels of TNF-α and IL-1β increased again (both P<0.05 compared with the control group).

Following treatment with LPS, the mRNA (data not shown) and protein levels of A20 in the lung tissue increased with time (Fig. 1D) and peaked after 2 h (P<0.05 compared with the control group). Subsequently, the levels gradually decreased but were increased compared with the control group (P<0.05). As demonstrated by immunofluorescence staining, the level of A20 also peaked at 2 h and gradually decreased following treatment with LPS, and A20 was expressed in pulmonary macrophages (Fig. 1E).

To examine the transfection efficiency, the mRNA and protein expression levels of A20 were measured by RT-qPCR and western blotting, respectively. A20 expression was significantly inhibited in the siA20 (A20 small interfering RNA) group compared with the SCR group throughout (Fig. 2A and B). Following induction by LPS, the NF-κB DNA binding activity and protein expression levels increased in the A20-knockdown macrophages; the highest levels were observed at 1 h (Fig. 2C-E). Subsequently, these levels gradually decreased.

The mRNA levels of TNF-α and IL-1β increased in the A20-knockdown macrophages induced by LPS, and they reached peak levels at 1 h (Fig. 3A and B). Subsequently, they gradually reduced. These results were the same as the protein levels of TNF-α and IL-1β measured by ELISA (Fig. 3C and D).

To examine the transfection efficiencies, the mRNA and protein expression levels of A20 were measured by RT-qPCR and western blotting, respectively. When compared with vehicle control (VEC), A20 expression was markedly increased (Fig. 4A and B). NF-κB protein expression increased in VEC macrophages induced by LPS and reached a peak at 1 h. Subsequently, the expression gradually reduced. However, the NF-κB protein expression was significantly inhibited in A20 overexpressing macrophages (Fig. 4C and D).

The mRNA levels of TNF-α and IL-1β were upregulated in the VEC macrophages induced by LPS and were highest at 1 h. (Fig. 5A and B). This result was the same as the levels of TNF-α and IL-1β measured by ELISA (Fig. 5C and D). However, the levels of TNF-α and IL-1β were significantly inhibited in the A20 overexpressing macrophages.

F4/80 and CD206 are expressed in macrophages (27). As presented in Fig. 6, macrophage phenotypes were investigated using FACs. Macrophage phenotypes M2 and M1 were distinguished by FACS using diverse macrophages markers, F4/80 and CD206 in A20-knockdown and A20-overexpressing NR8383 cells. The present data revealed CD206⁺ and F4/80⁺ cells in the A20-overexpressing macrophages in contrast with VEC cells (Fig. 6A and B), and CD206⁺ and F4/80⁺A20-knockdown macrophages compared with SRCs (Fig. 6D and E). The CD206⁺/F4/80⁺ cells (representing M2 macrophages) and the CD206⁻/F4/80⁺ cells (representing M1 macrophages) were assayed by FACS, and it was observed that LPS induced M1 macrophage polarization (Fig. 6C and F). It was concluded that A20-overexpression increased M polarization from the M1 to M2 phenotypes in the NR8383 cells treated with LPS.