1. Cell culture and treatment
Monocyte cell lines RAW264.7 and THP-1 were purchased from the Shanghai Cell Resource Center of the Chinese Academy of Sciences and cultured in RPMI 1640 medium supplemented with 10% Fetal Bovine Serum(FBS)(Biological Industries), 100 IU/mL penicillin (Gibco), and 100 µg/mL streptomycin (Gibco). Human embryonic kidney cell line HEK-293T was obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS (Biological Industries). All cell lines were maintained at 37°C and 5% CO2. RAW264.7 and THP-1 cells were activated by LPS (100 ng/mL) or TNFα (20 ng/mL) for the indicated period. The concentrations of small molecular inhibitors or agonists used were as follows: IKK-16 (3 µM), TPCA-1 (5 µM), PMA (150 nM), CHX (50 ng/mL), SP600125 (10 µM), SCH772984 (10 µM), BAY (10 µM), Rapamycin (10 µM), PP2A inhibitor LB100 (10 µM), Resiquimod (5 µg/mL), and Pam3csk4 (100 ng/mL).
2. Isolation of pMφs and spleen cells
Thioglycollate broth (3% in phosphate-buffered saline; Sigma-Aldrich) was steam-sterilized before use. Each mouse was injected intraperitoneally with 2.5 mL of broth. On the third day, mice were euthanized and enriched peritoneal macrophages were obtained by peritoneal lavage using 8 mL phosphate buffer. Peritoneal macrophages were activated by LPS (100 ng/mL) or TNFα (20 ng/mL). Spleen was collected aseptically, immediately after the euthanasia of mice. Then, the spleen was placed on a sterile 60-mesh stainless-steel sieve above a collector tube, perfused with culture medium, teased apart using micro-dissecting scissors, and gently pushed through the sieve using a syringe plunger. The splenic cell suspension was counted under the microscope by employing a blood-counting cell plate.
3. Animals
Rmpflox/flox was constructed by the Shanghai Southern Model Biology Research Center, as previously described (26). Lyz-Cre mice were purchased from the Jackson Laboratory. All mice were on the genetic background of C57BL/6j and inhabited in a standard condition meeting the criteria of the guidelines of the Naval Medical University Animal Care Facility. In this study, to conveniently record, KO mice mean a mice-specific deficiency of Rmp in myeloid cells (Rmpf/f, Lyz2-Cre+). All animal operating procedures complied with the ethical standards of the Institutional Animal Care and Use Committee of the Naval Medical University. Animal experiments were approved by the Ethics Board of the Eastern Hepatobiliary Surgery Hospital.
4. Plasmids, DNA transfection, and lentivirus infection
Plasmids pCDNA3.1A, pCDNA3.1A-mouse RMP, mouse RMP truncated mutants, mouse RMP S439A mutant, mouse RMP S439E mutant, mouse RMP S369A mutant, human RMP S372A mutant, pCDNA3.1A-mouse TLR4, pCDNA3.1A-mouse IKKβ, mouse IKKβ truncated mutants, mouse IKK-SSEE mutant, pCDNA3.1A-mouse PP1γ, pCDNA3.1A-mouse PP2Aa, pCDNA3.1A-mouse PP2Ab, pCDNA3.1A-mouse PP2Ac were constructed by Biochat Co., Ltd, Shanghai. Plasmids were transfected into cell lines using jetPEI DNA Transfection Reagent (Polyplus), according to the manufacturer’s instructions. The lentivirus-shRNA targeting human RMP was constructed by Genechem Co., Ltd, Shanghai. Viral solutions were added to a cell culture medium containing 4 µg/mL polybrene. After 48 hours of infection, cells were selected using 2 µg/mL puromycin. After that, the RMP expression of cells was detected by RT-PCR or immunoblotting.
5. Antibodies and reagents
Anti-RMP (11277-1-AP, RRID: AB_10596929), anti-beta-actin (66009-1-Ig, RRID: AB_2687938), and anti-GAPDH (60004-1-Ig, RRID: AB_2107436) were purchased from Proteintech Group, Inc. Anti-HA (AE008, RRID: AB_2770404), anti-His (AE003, RRID: AB_2728734), anti-Myc (AE010, RRID: AB_2770408), and anti-Flag (AE005, RRID: AB_2770401) were purchased from ABclonal, Inc. Anti-P65 (8242, RRID: AB_10859369), anti-p-P65 (Ser536)(3033, RRID: AB_331284), anti-IKKβ (8943, RRID: AB_11024092), anti-p-IKKα/β (Ser176/180)(2697, RRID: AB_2079382), anti-IKKα (2682, RRID: AB_331626), anti-IKKγ (2695, RRID: AB_2124826), anti-IκBα (4814, RRID: AB_390781), anti-p-IκBα (Ser32) (2859, RRID: AB_561111), anti-P38 (9212, RRID: AB_330713), anti-p-p38 (Thr180/182) (9211, RRID: AB_331641), anti-S6 (2217, RRID: AB_331355), anti-p-S6 (Ser235/236) (4858, RRID: AB_916156), anti-ERK1/2 (9102, RRID: AB_330744), anti-p-ERK1/2 (Thr202/204) (9101, RRID: AB_331646), anti-SAPK/JNK (9252, RRID: AB_2250373), anti-p-SAPK/JNK (Thr183/185) (9251, RRID: AB_331659), anti-PP2Aa (2041, RRID: AB_2168121), anti-PP2Ab (2290, RRID: AB_659890), and anti-PP2Ac (2259, RRID: AB_561239) were purchased from Cell Signaling Technology. Mouse IgG (sc-2025, RRID: AB_737182) and PP1γ (sc-515943, RRID: AB_2909495) were purchased from Santa Cruz Biotechnology, Inc. LPS (S1732) was purchased from Beyotime. TNFα (315-01A) was purchased from PeproTech. IKK inhibitors including TPCA-1 (S2824), IKK-16 (S2882), PP2A inhibitor, SP600125 (a JNK inhibitor) (S1460), SCH772984 (an ERK inhibitor) (S7101), MK2206 (an AKT inhibitor) (S1078), rapamycin (an mTOR inhibitor) (S1039), LB100 (a PP2A inhibitor) (S7537) and BAY (a P65 inhibitor) (S2913) were purchased from Selleck. Pam3csk4 (an agonist of TLR1/2) (TP1068) and Resiquimod (an agonist of TLR7/8) (T6965) were purchased from Target Molecule Corp.
6. Immunoblot analysis and co-immunoprecipitation
Whole-cell extracts were collected and lysed in RIPA buffer (Beyotime, 50 mM Tris pH 7.4, 150 mM NaCl, 1% NP40, 0.1% SDS, and 0.5% sodium deoxycholate, 150 mM NaCl, 2 mM EDTA, and 50 mM NaF) supplemented with a cocktail of protease inhibitor (Roche) and phosphatase inhibitors (Sigma-Aldrich). The lysed products were centrifuged at 12,000 rpm (Microfuge 20R, BECKMAN COULTER) for 15 minutes at 4°C to remove precipitates. Primary antibodies used for immunoblot analysis were diluted at 1:1000 with 5% BSA buffer. Immunoblots were performed using specific primary antibodies, followed by a fluorescein-conjugated secondary antibody, and then detected using an Odyssey fluorescence scanner (Li-Cor). Immunoprecipitation was determined according to the instructions of MAg25K/Protein A/G beads (Enriching Biotechnology LTD).
7. Luciferase reporter gene assay
Cells (20,000 cells/well) were plated in 300 µL/well in a 48-well plate. The Renilla (pRL-TK) 10 ng, and pNF-κB-luc 200 ng were co-transfected into cells. Cells were lysed by positive lysis buffer (Promega) and assayed employing the Dual-Luciferase Reporter Assay System (Promega, E1910) after 36 hours according to the manufacturer’s instructions. Luciferase activities were detected by Single Tube Luminometer LB 9507 (Lumat).
8. RNA extraction and RT-PCR
Total RNA was extracted from cells using TRIzol reagent (Invitrogen) and then reverse-transcribed into cDNA using iScript cDNA Synthesis Kit (Bio-Rad). The resulting cDNA was diluted (1:20) for quantitative PCR using SYBR Green SuperMix (Roche) with gene-specific primers. The results were normalized to 18S control. The sequences of the gene-specific primers involved in this study (forward and reverse, respectively) are as follows:
Rmp, 5’-CAAGCAGGCTGTGGGTTTAGT-3’ and 5’-GTGAGCAATTCGTTGTTTTCCTT-3’;
Il-6, 5’-TAGTCCTTCCTACCCCAATTTCC-3’ and 5’-TTGGTCCTTAGCCACTCCTTC − 3’;
Il-1β, 5’-GCAACTGTTCCTGAACTCAACT-3’and 5’-ATCTTTTGGGGTCCGTCAACT − 3’;
Tnfα, 5’-CCCTCACACTCAGATCATCTTCT-3’and 5’-GCTACGACGTGGGCTACAG − 3’;
Ifnγ, 5’-TCTGGAGGAACTGGCAAAAG-3’and 5’-AGTGACAGGCTGGGATGG − 3’;
Ccl2, 5’-TAAAAACCTGGATCGGAACCAAA-3’and 5’-GCATTAGCTTCAGATTTACGGGT-3’;
Ccl5, 5’-GCTGCTTTGCCTACCTCTCC-3’and 5’-TCGAGTGACAAACACGACTGC-3’;
Trail, 5’-ATGATGGTGATTTGCATAGTGCT − 3’ and 5’-AGCTGCTTCATCTCGTTGGTG − 3’;
18S, 5’-CGGCTACCACATCCAAGGAA-3’and 5’-GCTGGAATTACCGCGGCT-3’
9. Cytokine measurement
The concentrations of TNFα, IL-1β, and IL-6 in cellular supernatants or serum were measured using ELISA kits (eBioscience) according to the manufacturer’s instructions. Cellular supernatants were collected and centrifuged at 10,000 rpm (Microfuge 20R, BECKMAN COULTER) for 5 minutes to remove precipitates. Standard concentrations of IL-1β, TNFα, and IL-6 were prepared by serial dilutions of the peak concentration. 20 µL of each sample was added to 80 µL dilution buffer and transferred to the 96-well plate in the ELISA kit. After incubation and washing, 50 µL TMB One-Step Substrate Reagent was added to each well and incubated for 30 minutes in the dark at room temperature. The absolute absorbance value of each sample was obtained by subtracting the optical density readings at 570 nm from readings at 450 nm using a microplate reader (Synergy H1, BioTek).
10. Mass Spectrometry of phosphoproteins
RAW264.7 cells were treated with LPS (100 ng/mL) for 30 minutes. Then, whole cell extracts were collected and lysed to obtain protein samples as described above. After that, each protein sample was incubated with 3 µg anti-RMP antibody and 50 µL MAg25K/Protein A/G beads overnight according to the manufacturer’s instructions. Western blot and Coomassie brilliant blue staining procedures were performed to stain the targeted bands (between 72 and 95 kDa). Further LC-MS/MS analysis was performed on a Q Exactive Mass Spectrometer (ThermoFisher Scientific) that was coupled to Easy nLC (ThermoFisher Scientific) for 60/120/240 min. The digested peptides were loaded onto a reverse phase trap column (ThermoFisher Scientific, Acclaim PepMap100, 100 µm*2 cm, nanoViper C18) connected to the C18-reversed-phase analytical column (ThermoFisher Scientific Easy Column, 10 cm long, 75 µm inner diameter, 3 µm resin) in buffer A (0.1% Formic acid) and separated with a linear gradient of buffer B (84% acetonitrile and 0.1% Formic acid) at a flow rate of 300 nL/min controlled by IntelliFlow technology. The mass spectrometer was operated in positive ion mode. MS data were acquired using a data-dependent top10 method. The MS raw data for each sample were combined and searched using the MaxQuant 1.5.3.17 software for further identification and quantitative analysis.
11. Phos-tag SDS-PAGE
Phosphorylated proteins were separated using conventional Phos-tag SDS-PAGE, as previously reported 27. Phos-tag is a functional molecule that can specifically bind phosphate ions. In Phos-tag SDS-PAGE, a separation gel was prepared using acrylamide containing Mn2+ Phos-tag (FUJIFILM Wako Pure Chemical), according to the manufacturer’s instructions. By electrophoresis, phosphorylated and non-phosphorylated proteins can be separated according to their phosphorylation levels. The separation gel can be used for Coomassie blue staining, Western blotting, and mass spectrometry (MS) experiments.
12. LPS-induced lethal septic shock
For the LPS-induced endotoxemia model, male mice (8–12 weeks old) were injected intraperitoneally with LPS (Escherichia coli 0111:B4, Sigma-Aldrich) at a dose of 25 mg/kg. Control animals were administered with an equal amount of saline. Mice grouping was blind to the model maker and data collector. The survival was monitored and recorded for up to 72 hours. Mice were grouped and euthanized for determination of inflammatory factors in plasma (6 h), or collection of lung tissues for histological evaluation (24 h), respectively. Mouse blood samples were collected and left to set for at least 10 h without anticoagulants at room temperature, and then centrifuged at 3000 rpm/min for 10 minutes. Serum samples were used to measure the concentrations of corresponding cytokines using ELISA kits (eBioscience). Littermates of myeloid-specific Rmp-KO and Rmp-WT mice were grouped and tested.
13. Establishment of CLP-induced sepsis and bacterial load measurement
Male mice, 8–12 weeks old, were completely anesthetized with 1% pentobarbital sodium (Sigma) according to weight (50 µg/g) by intraperitoneal injection before the surgery. After anesthetized, the central abdominal laparotomy was performed with an incision of 1-1.5 cm. 50% of the cecum was ligated with 4 − 0 silk suture, and one penetrable hole was made at the ligated cecum with a 21-G needle. Sham-operated animals underwent neither ligation nor puncture. Littermates of myeloid-specific Rmp-KO and Rmp-WT mice were grouped and tested. Mice grouping was blind to the model maker and data collector. Mice were resuscitated with 500 µL pre-warmed sterile physiological saline by subcutaneous injection after surgery. All mice had free access to food and water after recovery from anesthesia. Mice were checked for their survival state every 6 hours after model construction until the 10th day. Test contents included the presence of heartbeat and mobility. Ascites obtained from tested mice were diluted at 1:1000, and then infiltrated cells in ascites were counted under the microscope by employing a blood cell counting plate. All blood, liver, and lung tissue collection and tissue homogenates were performed using sterile instruments. Serial dilutions were plated onto tryptic soy agar pre-poured petri dishes and incubated aerobically overnight at 37°C.
14. Flow cytometry
Primary BMDM or peritoneal lavage fluids, and spleen homogenates were prepared at 1 × 107/mL in 1.5 mL Eppendorf tubes (final volume of 100 µL). After washing with flow cytometry buffer (PBS with 2% FCS, 2 mM EDTA, and 25 mM HEPES, pH 7.4), cells were incubated with blocking buffer (5% goat serum in flow cytometry buffer) for 15 min at 4°C. 0.5 µg/mL propidium iodide was added to each plate to identify and exclude dead cells. Then, cells were stained with the indicated antibody combinations for 30 min at room temperature. The antibodies used for flow cytometry are purchased from eBioscience and listed as follows: live/dead AmCyan, CD45.2-BV605, Cd11c-APC, Cd11b-PE, F4/80-FITC, Ly6C-pacific blue and Ly6G -Pe-Cy7 (all diluted at 1:100). Then, cells were washed twice with Hanks’ Balanced Salt Solution/Ca/Mg and then transferred into flow testing tubes (FALCON). Unstained cells and single-stained cells were used as control groups. The cells were gated using forward and side scatters on a BD FACS Aria II (BD Biosciences). Raw FCS files were analyzed with the FlowJo software.
15. Histology and injury evaluation of lung
Lung tissues were fixed in situ using 4% paraformaldehyde and then paraffin-embedded. Lung sections (4 µm thick) were stained with H&E and imaged using the Aperio ImageScope Viewer. Assessment of lung injury was previously described 28. In brief, lung injury was assessed in four aspects, namely alveolar congestion, edema, cellular infiltrates, and widening of alveolar septa. Each aspect was given a score of 0 to 2 on the scale of severity. As a result, the total score of lung injury ranged from 0 to 8. The evaluation of lung injury was blindly conducted by two independent pathologists.
16. GST pull-down assay.
The proteins including GST, GST-mouse RMP-WT, GST-mouse RMP-S439A, and His-mouse IKKβ were constructed and purified by AtaGenix. 5 µg GST fusion proteins plus 50 µL glutathione beads (Boppard) were added to a purified protein of IKKβ in vitro. After incubation with gentle rotation overnight at 4°C, beads were washed three times with cell lysis buffer, and proteins were eluted with 2 × SDS loading buffer and detected by immunoblot.
17. ADP-Glo Kinase Assay
ADP-Glo Kinase kit was purchased from Promega. ADP-Glo Kinase Assay was conducted according to the manufacturer’s instructions. Purified His-tagged IKKβ (0.5 µg), GST-RMP WT (1 µg) or GST-RMP S439A (1 µg), ATP (25 µM), and protease inhibitors were mixed with 50 µL kinase buffer. Mixtures were added to 96-well plates and incubated for 60 minutes at room temperature. Then, 50 µL of ADP-Glo Reagent was added into each well and incubated for 40 minutes at room temperature. Subsequently, 100 µL of Kinase Detection Reagent was added to each well. After incubation for 30 minutes at room temperature, samples underwent luminescence detection using a microplate reader (Synergy H1, BioTek).
18. Immunofluorescence staining
RAW264.7 cell was treated with DMSO or LPS (30 min, 3 h). Then, the cells for immunostaining were fixed in 4% methanol-free formaldehyde at room temperature for 20 min and penetrated with 0.2% Triton X-100 for 20 min at 4°C. After rinsing with PBS three times, cells were blocked with 10% goat serum for 1 h at room temperature and incubated with primary antibodies (RMP, 1:100; IKKβ, 1:100) overnight at 4°C. After washing three times with PBS, the cells were incubated with secondary antibodies conjugated with Alexa 488 or Alexa 568 (1:100) at room temperature for 1 h. After immunostaining, the cells were briefly stained with Hoechst to display the cell nuclei. Finally, images were visualized and photographed using a Leica confocal microscope.
19. Surface plasmon resonance analysis
Interactions between the GST-RMP WT, GST-RMP S439A, Flag-PP2Ab, and His-IKKβ proteins were analyzed using the OpenSPR system (Nicoya) at 25°C. Recombinant His-IKKβ protein was immobilized on a sensor chip (NTA, Nicoya) using an amine coupling kit (Nicoya). Final His-IKKβ immobilized levels were typically ~ 16000 RU. Subsequently, GST, GST-RMP WT, GST-RMP S439A, and Flag-PP2Ab were injected as analytes at various concentrations. PBS-P (10 mM phosphate buffer with 2.7 mM KCl and 137 mM NaCl, 0.05% Surfactant P20, pH 4.5) was used as the running buffer. For binding studies, analytes were applied at corresponding concentrations in the running buffer at a flow rate of 20 µL/min with a contact time of 240 s and a dissociation time of 480 s. Chip platforms were washed with the running buffer and 50% DMSO. Data were analyzed with TraceDrawer (Ridgeview Instruments ab, Sweden) by curve fitting using a 1:1 binding model.
20. Statistical analysis
All grouped data were statistically analyzed by GraphPad Prism software (version 7.0) and presented as bar plots in the figures. Data were presented as mean ± SEM. An unpaired Student’s t-test (two-tailed) was used to define the statistical significance between the two groups. One-way ANOVA was used to define the statistical significance in multiple groups of variables with more than three different managements. Kaplan-Meier survival curves were generated using GraphPad Prism software (version 7.0), and a log-rank test was performed to assess statistical significance. Asterisks presented P values in figures and P < 0.05 was considered statistically significant