Mice
Mice (strain: C57BL/6; 6-8 weeks old; male) were purchased from Orient Bio Inc. (Seongnam, South Korea). NLRX1 knock-out (NLRX1-/-) mice were kindly provided by Dr. J. P. Ting (University of North Carolina). All animals used were sex-and age-matched, of the same genetic background, and housed under a 12 h dark-light cycle under specific pathogen-free (SPF) conditions. Animals had free access to water and food during the research. Animal experiments were approved by the Institutional Animal Care, Use Committee (IACUC) of the affiliated university (protocol No. 2021-0178; Seoul, Korea), and the study was conducted in compliance with the ARRIVE guidelines.
Oxygen Exposure
WT (NLRX1+/+) and NLRX1 knock-out (NLRX1-/-) mice were exposed to >95% oxygen (Hyperoxia, HO) using cages enclosed in an airtight Plexiglas chamber (57x42x37 cm, JEUNG DO BIO & PLANT Co., Ltd., Seoul, Korea). The pressure inside the chamber was normalized to atmospheric pressure. Oxygen levels were constantly monitored for the duration of the experiment (72 h) using an oxygen analyzer (MaxO2+A, MAXTEC, Salt Lake City, UT, USA.). As controls, sex- and age-matched WT and NLRX1-/- mice were housed in similar conditions under normoxia (room air, RA). All methods were performed in accordance with the relevant guidelines and regulations.
Bronchoalveolar Lavage (BAL) fluid
During sacrificing, mice underwent blunt dissection of the trachea following anesthesia. A small-caliber tube was inserted into the airway; 0.9 ml of phosphate-buffered saline (PBS) was injected into the lungs, was collected, and this collection process was repeated once more so that a total of 1.8 ml BAL was collected from each mouse. The collected BAL fluid was centrifuged at 3,000rpm for 5 min at 4°C, and then separated into cell pellet and supernatant. The cell pellet was dissolved with PBS, mixed in a 1:1 ratio with Trypan Blue Solution, and the cells were counted using a hemocytometer. BAL cells were centrifuged onto slides using a Cytospin centrifuge (Thermo Fisher scientifics, Waltham, MA). Cell differentiation was assessed using a Diff-Quik stain kit (Medion Diagnostics, Fribourg, Switzerland). The supernatant was stored at -80°C and used for various protein analyses and assessment of LDH activity.
Histology and Immunohistochemistry (IHC)
The left lobes of sacrificed mice lungs were fixed in 4% formalin for 3 days and embedded in paraffin. Lung tissues were cut into 5-µm sections and stained using hematoxylin and eosin (H&E) to analyze airway inflammation. To assess immunohistochemistry, sections were deparaffinized twice by washing with xylene for 10 min and rehydrated in 100% (5 min, twice), 95% (5 min), and 70% (5 min) alcohol. The tissues were then heated with retrieval buffer (DAKO, A/S, Glostrup, Denmark) for 20 min in a steamer and allowed cool to room temperature (18-22°C) for 20 min. After washing the sections, they were placed in peroxidase blocking solution (DAKO) for 5 min, and then in protein block solution (DAKO) for 1 h. The NLRX1 antibody (Proteintech, Rosemont, IL) or normal rabbit IgG (Santa Cruz Biotechnology, Inc, Dallas, TX) were prepared as 1:500 dilutions and loaed into sections, followed by incubation at 4°C overnight. The color was developed with DAB solution (DAKO), and the reaction was stopped using deionized water; finally, the immunostained tissue sections were mounted on slides with an aqueous-base mounting medium.
Bicinchoninic acid (BCA) assay
Protein leakage in BAL was evaluated using a PierceTM BCA Protein Assay Kit (Thermo Fisher scientifics, Waltham, MA). The BAL fluid supernatant and standard were dispensed into a 96-well plate in 25 µl aliquots, and 200 µl working reagent was dispensed in each well. The plate was placed in an incubator at 37°C and allowed to react for 30 min. The protein concentration in BAL fluid was measured at an absorbance of 562 nm using a microplate reader (Molecular Devices, CA, USA).
Lactate dehydrogenase (LDH) assay
LDH concentration in BAL fluid was measured using a cytotoxicity detection kit (Roche Applied Science, Mannheim, Germany). We dispensed 100 µl of BAL fluid sample and 100 µl of the reagent mixed with the catalyst and dye solution in a 96-well plate, and this was allowed react at room temperature for 30 min. After the reaction was completed, the reaction was stopped by adding 50 µl of stop solution, and LDH concentration was measured at an absorbance of 492/690 nm using a microplate reader.
Real-time PCR
After mice were sacrificed, lung tissues were homogenized with T10 Basic Ultra-Turrax® homogenizer (IKA Labortechnik, Staufen, Germany) and lysed in Trizol reagent (Invitrogen, Charlsbad, CA, USA) according to the manufacturer’s protocol. The relative mRNA expression levels were measured by reverse transcription and real-time PCR; 1 µg of total RNA was synthesized to cDNA by reverse-transcription with ReverTra Ace q PCR RT Master Mix Kit (Toyobo Co., Ltd., Osaka, Japan). Real-time PCR was performed in 20 µl reactions containing 10 µl Power SYBR Green® PCR Master Mix (Applied Biosystems, Foster City, CA, USA), 1 µl cDNA template, 1µl forward primer, 1 µl reverse primer, and deionized water to the desired volume. Quantitative PCR was performed with the StepOnePlusTM Real-Time PCR System (Applied Biosystems, Waltham, MA) according to the manufacturer’s protocol. Primer pairs for real-time PCR were manufactured by MBiotech (Hanam, South Korea). The levels of mRNA were normalized to IPO8. Fold changes were calculated using the 2- Δ ΔCT method.
ELISA
The levels of interleukin-6 (IL-6) and C-C motif chemokine ligand 2 (CCL2) were quantified in BAL fluid by ELISA (R&D systems, Minneapolis, MN, USA) as per manufacturer’s instructions; 96-well plates were each coated with anti-mouse cytokine antibodies overnight at room temperature (18–22 °C). The wells were blocked with PBS containing 1% BSA for 1 h. Dilution standards and samples were then incubated for 2 h. Bound cytokines were detected using incubation with anti-mouse-cytokine antibodies for 2 h. Samples were then washed with streptavidin-horseradish peroxidase for 20 min and with TMB substrate solution (KPL) for a further 20 min. The reaction was stopped using 2 N sulfuric acid, and the colorimetric reactions were read using a microplate reader at 450 nm.
Western blot
Lung tissues were gently homogenized and lysed in RIPA buffer (Thermo fisher scientific, Waltham, MA, USA). The protein concentrations were measured using the Bradford assay. Equal amounts of protein samples were loaded on gel and separated by 8%~12% SDS-PAGE electrophoresis and were then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Membranes were blocked in Tris buffered saline containing 0.1% Tween 20 (TBST) with 5% skim milk and incubated overnight at 4℃ with primary antibodies. After washing with TBST, the membranes were incubated at room temperature with secondary antibodies for 1 h. The protein signal was analyzed using Image J software. Protein samples were normalized to GAPDH. The primary antibodies were used as follows: NLRX1 (Proteintech), and BAX, Cyto C, P-ERK 1/2, T-ERK 1/2, P-JNK, T-JNK, P-p38, T-p38, and GAPDH (all form Cell Signaling Technology).
TUNEL assay
To analyze apoptosis, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay was carried out using an in-situ Cell Death Detection Kit (AP) (Roche Applied Science, Mannheim, Germany) according to the manufacturer’s protocol. TUNEL assay detected the apoptotic cells in lung tissue samples. Pictures were taken using a light microscope at 400× magnification in five random fields for each section and the rate of TUNEL-positive cells was calculated.
Caspase activity
Total protein was extracted from the lungs using RIPA lysis buffer solution and a tissue homogenizer according to the manufacturer’s protocol. In a 96-well plate, 25 µl of Caspase-Glo® 3/7 Reagent (Promega, Madison, WI) and 25 µg of 1 mg/ml protein were added in a 1:1 ratio and incubated for 30 min and luminescence was recorded at 3 h. Caspase-Glo® 8 Assay and Caspase-Glo® 9 Assay protocols were conducted using the same methods as Caspase-Glo® 3/7 Assay.
Statistics
For the animal studies, values were expressed as means ± SEM. Most results were evaluated using Student’s t-test. Cell count results were compared using two-way ANOVA, and survival analysis was corrected for multiple comparisons by the log-rank test. All evaluations were performed using GraphPad Prism (GraphPad Software, Inc., San Diego, CA, USA). In all analyses, p <0.05 was considered statistically significant.