MAPK4 deficiency (MAPK4−/−) mice breeding pair in a C57BL/6 background were purchased from The Jackson Laboratory (USA, 027666). Animals were housed under specific pathogen-free conditions at Zunyi Medical University
Raw264.7 cells were purchased from Conservation Genetics CAS Kunming Cell Bank (China, KCB200603YJ), and were cultured in high glucose DEME containing 10% fetal bovine serum at 37 °C in 5% CO2.
Peritoneal macrophage preparation and stimulation
WT and MAPK4−/− mice (7 to 9 week-old) were sacrificed, and the peritoneal cavity was lavaged with cold PBS. The peritoneal cells were collected by centrifugation and seeded in the cell culture plate. Macrophages were allowed to adhere for 6 h, washed with fresh medium to remove unattached cells, and incubated overnight. For the stimulation experiments, macrophages were stimulated with LPS (100 ng/mL) for 24 h, then the cells were collected for the further analysis.
Establishment of ALI Model
WT and MAPK4−/− mice (7 to 9 week-old) were challenged with i.p. injection of 10 mg/kg LPS (Sigma, USA, Escherichia coli 0111:B4) dissolved in sterile PBS as shown in our previous study . Then the body weight and lung weight index (lung weight/body weight) was detected at indicated time.
Immediately after euthanasia, 1 ml aliquots of PBS were slowly infused in the murine lungs through the tracheostomy and then withdrawn gently. This lavage was repeated three times using the same syringe. The collected lavage fluid was stored in a 10 ml tube on ice. The fluid was centrifuged at 1000 rpm and 4 °C for 10 min, and the cell sediment was washed with PBS. The cell-free supernatant was centrifuged again at 14,000 g and 4 °C for 10 min, stored at − 80 °C and used for determination of cytokines content via ELISA. To the pellet, red blood lysis buffer (Solarbio, China, R1010) were used for 15 min and washed with PBS. Next, the pellet was resuspended for analysis.
Lung edema determination
Lungs from mice were excised and completely dried in the oven at 60 °C 24 h for calculation of lung wet/dry ratio.
Histology and immunohistochemistry
Lung tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 4 μm-thick sections. For histology analysis, the lung sections were stained with hematoxylin and eosin (HE). For immunohistochemistry, the lung sections were deparaffinized with xylene and rehydrated in graded ethanol (100% to 70%). To eliminate endogenous peroxidase activity, the slides were treated with 3% H2O2 for 30 min and washed with PBS. Then, the slides were blocked with Goat serum (BOSTER, China, AR1009) for 2 h at room temperature and incubated overnight at 4 °C with corresponding antibody (p-MK5: Biorbyt, UK, orb5579). After washed with PBS, the slides were incubated with secondary antibodies and visualized with DAB kit (Solarbio, China, DA1010). Finally, the slides were counterstained with hematoxylin and analyzed by Olympus microscope. The histopathology of the lung injury was scored quantitatively as previously described . Briefly, five random fields of five dimensions of histology features were evaluated by 5 parts: (A) neutrophils in the alveolar space, (B) neutrophils in the interstitial space, (C) hyaline membranes, (D) proteinaceous debris filling the airspaces, and (E) alveolar septal thickening.
Lung tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 4 μm-thick sections. Briefly, the slides were incubated with corresponding primary antibodies (MAPK4: Proteintech, USA, 26102–1-AP; F4/80: Abcam, UK, ab60343) and secondary antibody (Alexa 647: Cell Signaling Technology, USA, 4414S) after deparaffinization and rehydration. Then, the slides were counterstained with DAPI (Beyotime, China, C1002) and observed by Olympus microscope.
RNA extraction and quantitative real time PCR
Total RNA was isolated from mice lungs using RNAiso Plus (TAKARA, Japan, 9108) according to manufacture’s instructions. RNA was quantified and reverse-transcribed according to manufacture’s instructions (TAKARA, Japan, RR037A). SYBR Green-based real time quantitative PCR reactions (TAKARA, Japan, RR820A) and gene specific primers were used. The following primers were used: IL-1β forward: 5′-TGCCACCTTTTGACAGTGATG-3′, reverse: 5′-AAGGTCCACGGGAAAGACAC-3′; IL-6 forward: 5′-GGAAATCGTGGAAATGAG-3′, reverse: 5′-AGGACTCTGGCTTTGTCT-3′; TNF-α forward: 5′-CAGGGGCCACCACGCTCTT C-3′, reverse: 5′-TTTGTGAGTGTGAGGGTCTGG-3′; IL-4 forward: 5′-AACGAGGTCACAGG AGAA-3′, reverse: 5′-CCTTGGAAGCCCTACAGA-3′; IL-10 forward: 5′-TACAGCCGGGAAG ACAATAA-3′, reverse: 5′-AGGAGTCGGTTAGCAGTATG-3′; TGF-β forward: 5′-GGCGGTGC TCGCTTTGTA-3′, reverse: 5′-TCCCGAATGTCTGACGTATTGA-3′; MAPK4 forward: 5′-CCAAAGCATCCCTCAGTTGT-3′, reverse: 5′-CAAGGGGTTGGAAGTCAATG-3′; GAPDH forward: 5′-TCCATGACAACTTTGGCATTG-3′, reverse: 5′-TCACGCCACAGCTTTCCA-3′. Gene expression levels were quantified using Bio-Rad CFX96 detection system (Bio-Rad, USA). With GAPDH was used as internal reference, the expressions of genes were calculated by using the comparative threshold cycle (Ct) method.
DNA extraction and methylation analysis
Genomic DNA was extracted from 12 lungs of control and ALI model mice by using DNeasy Blood and Tissue kit (QIAGEN, Germany, 69504) according to manufacture’s instructions. The quality and quantified were evaluated by gel electrophoresis and a NanoDrop spectrophotometer (Thermo, USA). The genomic DNA from each sample was treated with sodium bisulfite using an EZ DNA methylation kit (Zymo Reasearch, USA). The MassARRAY platform (The Beijing Genomics Institute, China) was used for quantitative analysis of MAPK4 methylation. We used the primers (5′-aggaagagagGGGTGGGTTTTATTAGAGATAGTGG-3′, 5′-cagtaatacgactcactatag-ggagaaggctAATCTAAATCCCAACTAAATAATCCC-3′) to amplify the region of each promoter. Altogether, 35 CpG sites were tested in this region. The spectra methylation ratios of each CpG site were generated by MassARRAY EpiTYPER software (Agena, USA).
Surface markers of series immune cells were detected by flow cytometry (FCM) with Beckman Gallios (Beckman Coulter, USA). FCM was performed on Beckman Gallios with CellQuest Pro software using directly anti-Mouse monoclonal conjugated antibodies against the following markers: F4/80-Percp-Cy5.5 (no.45-4801-82), γδT-APC (no.17-5711-81), NK1.1-APC (no.17-5941-81), CD11c-PE (no.12-0114-82), CD4-PE-Cyanine7 (no.25-0041-82), CD8-Percp-Cy5.5 (no.45-0081-82), CD62L-PE (no.12-0621-81), CD69-APC (no.17-0691-82), Gr-1-PE-Cyanine7 (no.25–5931-82), CD86-APC (no.17-0682-81), MHCII-PE (no.12-5321-81), with corresponding isotype-matched (Thermo Fisher, USA). Cells were stained with corresponding antibodies (1:100) at 4 °C for 30 min, respectively. After washing twice, stained cells were analyzed with a Beckman coulter flow cytometer.
The protein levels of IL-1β, TNF-α, IL-6, IL-4, IL-10 and TGF-β in BAL fluid were detected by ELISA according to manufacture’s instructions, respectively (Thermo Fisher, USA).
Lung tissues were homogenized in ice-clod lysis buffer (KeyGEN BioTECH, China, KGP2100) according to manufacture’s instructions. Equal amounts of protein were separated by 10% SDS-PAGE and protein were transferred onto polyvinyldifluoride membranes. Membranes were incubated with 5% skim milk in PBS for 1 h. Immunoblotting was performed using mAbs to MAPK4 (Abcam, UK, ab96816), AKT (Cell Signaling Technology, USA, 4691), p-AKT (Cell Signaling Technology, USA, 4060), ERK1/2 (Cell Signaling Technology, USA, 4695), p-ERK1/2 (Cell Signaling Technology, USA, 4370), p-NF-κB (Cell Signaling Technology, USA, 3039S), p-JNK (Cell Signaling Technology, USA, 4668), p-p38 MAPK (Cell Signaling Technology, USA, 4511S), MK5 (Cell Signaling Technology, USA, 7419S), p-MK5 (Biorbyt, UK, orb5579) and GAPDH (Cell Signaling Technology, UK, 5174). Membranes were washed in PBST and subsequently incubated with a secondary anti-rabbit antibody conjugated to HRP (Cell Signaling Technology, USA, 7074S). The signal was detected and analyzed using Bio-Rad ChemiDoc MP Imaging System (Bio-Rad, USA). GAPDH was used as internal reference.
Series versions of truncated MAPK4 promoter (NCBI, NC_000084.6 74064925 to 74067228) were synthesized and cloned into pGL3.0 basic vector between KpnI and MluI sites (Gene Create, China, GS1-1905109). And we synthesized MAPK4-shRNA (forward: 5′-GATCCGCAAGGGTTATCTGTCAGAAGGGTTGTTCAAGAGACAACCCTTCTGACAGATAACCCTTGTTTTTTACGCGTG-3′, reverse: 5′-AATTCACGCGTAAAAAACAAGGGTTATCTGTCAGAAGGGTTGTC TCTTGAACAACCCTTCTGACAGATAACCCTTGCG-3′) and control group (forward:5′-GATCCGCAAATTGGTCTGACTGGAAGGGTTGTTCAAGAGACAACCCTTCCAGTCAGACCAATTTGTTTTTTACGCGTG-3′, reverse: 5′-AATTCACGCGTAAAAAACAAATTGGTCTGACTG GAAGGGTTGTCTCTTGAACAACCCTTCCAGTCAGACCAATTTGCG-3′), then cloned into pLVX-shRNA1 vector between BamHI and EcoRI sites. These vectors were extracted by using EndoFree Plasmid Maxi Kit (QIAGEN, Germany, 12,123). After verified by DNA sequencing, these vectors were used for further study.
Transfection and luciferase reporter assay
Raw264.7 cells were transfected with series of truncated MAPK4 promoter vectors by using Lipofectamine 3000 reagent (Invitrogen, USA, L3000015) according to manufacture’s instruction. After 24 h, the cells were detected for Luciferase activity according to the manufacture’s instruction (Promega, USA).
At 2 days prior to LPS, MAPK4-shRNA was used to inhibit MAPK4 expression, while a control shRNA was used as a control group. A 10ug vector with transfection reagent (Engreen, UK, 18668-11-2) was dripped into nasal cavity. After 2 days treatment with MAPK4-shRNA or MAPK4-control, mice were challenged with 10 mg/kg LPS by intraperitoneal injection. Then, the following 2 days, mice were treated with MAPK4-shRNA or MAPK4-control. Finally, the lung tissues were collected in day 5.
Nuclear proteins were extracted from the lung tissues of control or ALI mice by using Nuclear and Cytoplasmic Extraction Regents (Thermo Fisher, USA, 78,833) according to manufacture’s instruction. The biotinylated and un-biotinylated probes (NFKB1: forward:5′-GAGCTCCACATCGCGACATCCCTTCCTCAAGGAC-3′, reverse: 5′- CTCGAGGTGTAGCGCTGTAGGGAAGGAGTTCCTG-3′; NR3C1: forward:5′-GTTGCTGGGGCTCCAGCTGTCCCCGCCGCAGCA-3′, reverse: 5′-CAACGACCCCGAGGTCGACAGGGGCGGCGTCGT-3′) were synthesized (Sangon, China). The electrophoretic mobility shift assays for each transcription factor were performed using Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Fisher, USA, 89880) according to manufacture’s instruction.
Statistical analysis was performed using GraphPad Prism 5 software. 1-way ANOVA followed by Bonferroni’s post-hoc was applied for multiple comparisons and student’s t-test was used when two conditions were compared. P < 0.05 was considered statistically significant and two-sided tests were performed. All data are shown as a mean ± standard error of the mean (SEM). Survival was evaluated by the Kaplan–Meier method.