RIP3 dependent NLRP3 inflammasome activation is implicated in acute lung injury in mice

Male C57BL/6 mice were purchased from the Experimental Animal Center at Guangzhou University of Chinese Medicine (Guangzhou, China) and were housed under a pathogen-free condition in the Experimental Animal Center at Sun Yat-sen University, Guangzhou, China. The mice were cared for and the experiments were performed in accordance with the National Institutes of Health Guide for Care and Use of Animals. The experiments were approved by the Ethics Committee of Sun Yat-sen University.

LPS-induced ALI model was created as described previously [2]. Briefly, 8-week-old C57BL/6 mice were anesthetized with intraperitoneal ketamine (80 mg/kg) and xylazine (15 mg/kg). Six milligram per kilogram of LPS (Sigma-Aldrich St. Louis, MO, USA) was delivered to the lungs via a 20-gauge angiocath catheter. The control (sham operated) mice were given intratracheal PBS.

GSK872 (Merck Millipore, Damstadt, Germany), a selective RIP3 inhibitor was used. Mice were treated intraperitoneally with or without GSK872 (5 mg/kg) every 24 h while the first injection began at 2 h before LPS administration. Groups of 10 mice were sacrificed 48 h after LPS administration. The bronchoalveolar lavage (BAL) fluid was collected for cell count, protein quantification and enzyme-linked immunosorbent assay (ELISA). Lung tissues were collected for isolation of single cells, histology, ELISA and western blot analysis.

A human monocyte cell line THP-1 was purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cells were cultured at 37 °C, 5% CO₂, RPMI 1640 (Life Technologies, Grand Island, NY, USA) containing 10% fetal calf serum (FCS) (Gibco, McHenry, MD, USA), 100 U/ml penicillin and 100 mg/ml streptomycin. THP-1 cells were primed with 1 μg/ml LPS in the presence or absence of GSK872 (5 mM) for 4 h followed by stimulation with ATP (5 mM) for 1 h. Cell supernatants were collected for detection of IL-1β (R&D Systems, Minneapolis, MN, USA) and IL-18 (Cusabio, Wuhan, China) by ELISA. Cells were subjected to flow cytometry (FACS), western blot and protein interaction analysis.

BAL was performed as previously described [2]. Mice were anesthetized and sacrificed by heart puncture after opening the thoracic cavity. The trachea was exposed and an 18G sterile needle with blunt end was inserted into the trachea through a small semi-excision. PBS was injected and withdrawn for lavage. A total volume of 2.4 ml BAL fluid per mouse was collected. Supernatants were collected for ELISA and total protein analysis. Total cell counts of pelleted cells were determined on a grid hemocytometer. Total protein level was determined by using BCA Protein Assay Kit (Thermo Fisher Scientific, Grand Island, NY, USA) according to the manufacturers’ instructions.

Lung cell isolation was performed as previously described [9]. Lung single cell suspensions were stained with APC-conjugated anti-mouse CD45, PE-conjugated anti-mouse CD103, PerCP-Cy5.5-conjugated anti-mouse CD24 (all from eBioscience, San Diego, CA, USA), Alexa Flour 700-conjugated anti mouse I-A/I-E, BV421-conjugated anti-mouse CD11b (both from Biolegend, San Diego, CA, USA), PE-Cy7-conjugated anti-mouse CD11c, Alexa Flour 647-conjugated anti-mouse siglecF (both from BD Pharmingen, San Diego, CA, USA). Activation of NLRP3 inflammasome was detected as active caspase-1 level using FAM-FLICA Caspase-1 Assay Kit (Immunochemistry Technology, Bloomington, MN, USA). All FACS analyses were performed on a Gallios Flow Cytometer (Gallios, Beckman Coulter, Brea, CA, USA). Alveolar macrophages, interstitial macrophages (IMs), CD11b⁺ monocyte–macrophages/dendritic cells (M–M/DCs) cells and CD103⁺ DCs in lung tissues were isolated using the gating strategy reported by Misharin et al. and Kopf et al. [10, 11].

After sacrifice, lungs were collected and fixed in 10% neutral formalin for 24 h. Lung tissues were embedded in paraffin and sectioned (2 μm), followed by hematoxylin and eosin (HE) staining.

Proteins from lung tissues and THP-1 cells were extracted and analyzed by western blotting as described previously [12]. Total proteins were extracted with cell lysis buffer (Cell Signaling Technology, USA) according to the manufacturer’s instructions. Nuclear and cytosolic proteins were obtained with a commercial nuclear extraction kit (Thermo, USA) according to the manufacturer’s instructions. The primary antibodies used in this study included: mouse anti-NLRP3, mouse anti-caspase-1 (p20) (both from AdipoGen, San Diego, CA, USA), rabbit anti-RIP3, mouse anti-p-RIP3, rabbit anti-MLKL, rabbit anti-p-MLKL (all from Abcam, Cambridge, UK), mouse anti-RIP1 (R&D Systems, Minneapolis, MN, USA), rabbit anti-phospho-p44/42 MAPK kinase (p-ERK), total p44/42 MAPK kinase (t-ERK) and fibrillarin (all from Cell Signaling Technology, Beverly, MA, USA), and rabbit anti-GAPDH and anti-NF-κB p65 antibodies (both from Santa Cruz Biotechnology, Dallas, Texas, USA). HRP conjugated anti-mouse and anti-Rabbit IgG (both from Cell Signaling Technology, Beverly, MA, USA) were used as secondary antibodies. Signals were detected with enhanced chemiluminescence analysis kit (Cell Signaling Technology, Beverly, MA, USA).

THP-1 cells were fixed in 4% paraformaldehyde for 10 min, permeabilized in 0.01% Triton X-100 for 10 min and blocked in 5% BSA for 1 h. For p-RIP3 and p-MLKL staining, cells were incubated with mouse anti-p-RIP3 and rabbit anti-p-MLKL (both from Abcam, Cambridge, UK), overnight at 4 °C and then labeled with secondary anti-mouse antibody conjugated with Alexa Fluor 488 and anti-rabbit antibody conjugated with Alexa Fluor A555 (both from Thermo Fisher Scientific, Grand Island, NY, USA) respectively for 60 min, then mounted in Vectorlabs mounting media with 4′,6-diamidino-2-phenylindole (DAPI). Slides were visualized using a fluorescence microscope LSM 800 (Zeiss, Jena, Germany).

Detection of RIP1–RIP3 and RIP3–NLRP interaction in THP-1 cells were performed using the Duolink^® In situ Detection Reagents (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturers’ instructions. Briefly, THP-1 cells were first incubated with two primary antibodies that recognize target proteins (anti-RIP3 and anti-NLRP3 antibodies or anti-RIP1 and anti-RIP3 antibodies, all from Abcam, Cambridge, UK), and then incubated with a pair of proximity ligation assay (PLA) probes which is composed of species-specific secondary antibodies conjugated to complementary oligonucleotides. In the presence of hybridization solution and ligase, the oligonucleotides form a circle in case of close proximity of proteins. Finally, the polymerase and nucleotides participate to the formation of the rolling circle amplification, which were visualized in green fluorescence.

Immunoprecipitation (IP) was performed using the Thermo Scientific Pierce co-IP kit (Thermo Fisher Scientific, Grand Island, NY, USA) following the manufacturer’s protocol and conducted as previously described [13]. Briefly, THP-1 cells were washed with ice-cold PBS and lysed. The cell lysates were centrifuged, and the supernatant was subjected to IP with rabbit anti-RIP3 coated resin at 4 °C overnight. After the IP, the resin was washed three times and the IP proteins were subsequently analyzed for RIP1, RIP3, and NLRP3 by western blot.

Small interfering RNA duplexes (si-RIP3) targeting the RIP3 (ID11035) (si-RIP3, Ribobio, Guangzhou, China) were synthesized for cell treatment. The THP-1 cells were transfected with 100 nmol siRNA or scramble siRNA and cultured for 48–72 h before stimulation.

Data were presented as mean ± SEM. Statistical analyses were performed using one-way ANOVA. All data were analyzed using SPSS software (version 17.0). p values < 0.05 were considered statistically significant.

Both RIP3 necroptosis pathway and NLRP3 inflammasome pathways were examined in the mouse model of LPS-induced acute lung injury (ALI). RIP3 mediated necroptosis pathway was significantly activated as demonstrated by enhanced expression of p-RIP3, p-MLKL in the lungs of mice with LPS-induced ALI compared with those of mice treated with PBS (Fig. 1a–c). Enhanced protein expression of NLRP3 and caspase-1p20 subunit, which represents the highly active p20/p10 tetrameric forms of processed caspase-1, were also observed in ALI mice. However, there were no significant changes in the expression of caspase-1 (Fig. 1d–f).

Treatment of mice with LPS-induced ALI by a selective RIP3 inhibitor GSK872 resulted in the significant suppression of the activation of necroptosis pathway. Expression of p-RIP3/RIP3 and p-MLKL/MLKL in the lung tissues was significantly downregulated by GSK872 treatment as demonstrated by western blot analysis (Fig. 1a–c). Interestingly, the expression of NLRP3 and caspase-1p20/pro-caspase1 were also significantly inhibited by GSK872 (Fig. 1d–f). IL-1β and IL-18 are important downstream molecular modulated by NLRP3 inflammasome pathway and acts as pivotal inflammatory factors in the LPS-induced ALI. IL-1β and IL-18 expression in lung tissue were apparently enhanced in ALI mice compared with sham mice. The selective RIP3 inhibitor GSK872 significantly inhibited IL-1β and IL-18 production as measured by ELISA (Fig. 1g, h). To further examine the effect of GSK872 on NLRP3 priming, NF-κB p65 and p-ERK were detected by western blot. However, the expression of NF-kB p65 and p-ERK was not changed with treatment of treatment of GSK872 (Additional file 1: Figure S1).

The effects of GSK872 on lung injury induced by LPS were investigated by histological examination with H&E staining. As shown in Fig. 2a, inflammatory cell infiltration was observed in the pulmonary interstitium of mice with LPS-induced ALI and was significantly reduced by GSK872 treatment. Cytospin cells from BAL fluids showed most of the infiltrated cells in the BALFs were neutrophils and macrophages. The numbers of these cells were decreased by GSK 872 treatment (Fig. 2b). To further investigate the effect of GSK872 on lung inflammation, BAL fluids were collected for cell counts, total protein, IL-1β and IL-18 level analysis. In normal control mice, there were very few cells observed in the BAL fluid and almost were alveolar macrophages. Cell number was significantly increased in the BAL fluids from mice with LPS-induced lung injury and neutrophil was the dominated cell type. In contrast, GSK872 treatment significantly reduced total cell number in the BAL fluid (Fig. 2c). Coincident with the cell counts, the total protein, IL-1β and IL-18 level in supernatants of BAL fluids was also elevated in the LPS-induced lung injury group, which was significantly reduced by GSK872 treatment (Fig. 2d–f).

Using the gating strategy as showed in Fig. 3a, CD45⁺I-A⁺CD11b⁺CD24^lo IMs and CD45⁺I-A⁺CD11b^hiCD24⁺ cells (M–M/DCs) were the major subsets of cells in the lung of mice with LPS-induced ALI (Fig. 3a). The percentage of IM and M–M/DC was significantly increased in mice with ALI (Fig. 3b, c). Activated caspase-1 as detected by the AM FLICA Assay was significantly elevated in the IM and M–M/DC of LPS-induced lung injury group. This elevation was inhibited by GSK872 (Fig. 3d).

The above findings showed that IM and M–M/DC were the major sources of NLRP3 and caspase-1. To further investigate the RIP3–NLRP3 interaction in monocytes/macrophages, we used a human monocytic cell line THP-1 for the in vitro studies. Stimulation of THP-1 cells with LPS and ATP significantly elevated the expressions of p-RIP3 and p-MLKL. GSK872 inhibited RIP3 mediated necroptosis pathway as demonstrated by western bolt analysis (Fig. 4a, b). This inhibition was not complete but significant. Further immunofluorescence experiment showed that GSK872 significantly inhibited the expression of p-RIP3 and p-MLKL in LPS and ATP stimulated THP-1 cells (Fig. 4c).

Stimulation of THP-1 cells with both LPS and ATP resulted in upregulation of NLRP3 and caspase-1p20 subunit. Pretreatment with GSK872 resulted in reduced this expression as demonstrated by western blot analysis (Fig. 5a, b). To further confirm the results of western blot, flow cytometry was used to analyze active caspase-1 in THP-1 cells. The results showed that GSK872 significantly reduced the level of active caspase-1 in THP-1 cells (Fig. 5c). The production of IL-1β and IL-18 in the supernatant was also significantly reduced by GSK872 treatment (Fig. 5d).

Interactions between RIP3 and RIP1 and between RIP3 and NLRP3 were examined. RIP1–RIP3 and RIP3–NLRP3 interactions were detected using Duolink^® In situ detection Reagents, in which oligonucleotides were attached to the antibodies of both target proteins to form circular DNA, amplified and detected by fluorescent markers. As shown in Fig. 6a, RIP1–RIP3 and RIP3–NLRP3 interactions exhibiting green fluorescence were enhanced in THP-1 cells treated with LPS and ATP (middle panel of Fig. 6a), which were significantly reduced by GSK872 (lower panel of Fig. 6a). Protein interactions and their inhibition were confirmed by immunoprecipitation analysis. Antibodies to RIP3 brought down both RIP1 and NLRP3 (upper panel of Fig. 6b), documented the interaction between RIP3 with both RIP1 and NLRP3. The interactions between RIP3 and RIP1 and between RIP3 and NLRP3 were enhanced by treating THP-1 with LPS and ATP. GSK872 reduced the co-precipitation among RIP3 with RIP1 and NLRP3.

In order to further document RIP3 involvement in the activation of NLRP3, we knocked down RIP3 mRNA in THP-1 cells by the siRNA technology. As shown in Fig. 7, elevated expression of p-MLKL, NLRP3 and caspase-1p20 in THP-1 cells induced by LPS and ATP was significantly reduced when RIP3 was silenced.