In vivo sepsis model
This study was approved by the Institutional Animal Care and Use Committee of the author’s institute (CHA University IACUC 230143), in accordance with the National Institute of Health Guidelines. This study was carried out in compliance with the ARRIVE guidelines. Sprague-Dawley rats weighing 270-330 g were used. The rats were housed in a controlled environment (room temperature 20-24˚C) with access to standard food and water ad libitum for 7 days before the experiment.
We used a body weight-adjusted polymicrobial sepsis model in accordance with a previous study (12). We used cecal slurry model. In brief, donor rats were anesthetized with an intramuscular injection of Zoletil (50 mg/kg) and xylazine (10 mg/kg). A midline laparotomy was performed, and the cecum was extruded. A 0.5 cm incision was performed in the antimesenteric surface of the cecum and the cecum was squeezed to expel feces. The donor rats were then euthanized. The feces were collected and weighed, then diluted with 5% dextrose saline at a ratio of 1:3. In sepsis induction, fecal-recipient rats were anesthetized as described above. Thereafter, 0.5 cm midline laparotomy was performed, and fecal slurry was administered into the peritoneal cavity. The fecal slurry was vortexed to obtain a homogeneous suspension before administration into the intraperitoneal cavity. The volume of fecal slurry administered to each animal was adjusted based on the body weight of the recipient rat. We administered subcutaneous fluid resuscitation (30 mL/kg 5% dextrose saline). In addition, imipenem was injected subcutaneously at a dose of 25 mg/kg twice daily for 2 days.
Experiment 1: Endotoxin tolerance (Ex vivo study). Endotoxin tolerance through ex vivo PBMCs/splenocytes 24 hours after sepsis induction was done with stimulation with LPS.
Experiment 2: Omics study. In the omics study, we introduced cecal slurry model with 5.5 mL/kg cecal slurry. The rats were randomly assigned to the study group at 6 h, 24 h, and 5 days. The stratified randomization by weight was performed by an assistant in the experiments. We sacrificed the animals at each allocated time points, and PBMCs was isolated. If the animals could not survive to the allocated time points, they were excluded from the analysis. During the observation period, an employee of the animal research center monitored the animals twice per day. If the animal was doomed to die, the research team would take notice and make the decision for euthanasia.
Experiment 3: Survival study. In the survival study, we introduced two serial doses of cecal slurry model with 5.0 mL/kg cecal slurry at 48-hour interval. L-Ornithine L-Aspartate (LOLA) was injected twice per day for 3 days. It was injected via tail vein at 24 hour/48 hour time points, and intraperitoneal injection at other time points. Survival was monitored every 12 h for 14 days. During the observation period, an employee of the animal research center monitored animals twice per day, and if the animal seemed close to death, they notified the research team, who decided for euthanasia.
Experiment 4: CFU, Blood chemistry, and tissue study. In this study, we administered LOLA twice per day for 2 days. Blood and tissues were harvested 24 h after second fecal slurry administration. The colony forming unit (CFU) in the blood and spleen was counted. We measured serum creatinine, ALT, plasma lactate, glucose, arterial blood gases, and electrolytes.
Experiment 5: Metabolomics study with mitochondrial transplantation. Mitochondria or DPBS were administered 1 h after sepsis induction at a dose of 200 µg via the tail vein as previous study (13). PBMCs/splenocytes were isolated 24 hours after sepsis.
In all experiments, sepsis was verified by observing reduced motor activity, lethargy, shivering, and piloerection (12). We did not think confounders could affect the results with this study design; thus, it was not controlled.
Metabolomics study
Extraction of Animal Sample for Metabolomics
The PBMCs and splenocytes samples were extracted by adding 1 mL of ice-cold 100% methanol and 10 L internal standard solution (2-chlorophenylalanine, 1 mg/mL in water) as described by (14). Frozen spleen (100 mg) were added 100% methanol (1 mL) with 10 L internal standard solution as described by (15). Each mixture samples were homogenized (frequency =30Hz) for 10 min by using a Retsch MM400 mixer mill (Retsch GmbH & Co, Haan, Germany). Then, the sample was centrifuged at 4℃ and 12,000 rpm for 10 minutes. The supernatants were further passed through a 0.2-m polytetrafluoroethylene syringe filter and finally transferred to Eppendorf tubes. The supernatant was completely dried with a speed vacuum machine and stored in a –80℃. Dried extracts were reconstituted with 100% methanol to a final concentration of 10mg/mL for instrument analysis. The samples were again dried using a speed vacuum concentrator prior to a two-staged derivatization step.
Gas Chromatography-Time-Of-Flight-Mass Spectrometry Analysis
Gas chromatography-time-of-fight mass spectrometry (GC-TOF-MS) analysis was performed using an Agilent 7890A gas chromatograph system coupled with an Agilent 7693 autosampler (Agilent, Atlanta, GA, USA) as previously as previously described (16). For the GC-TOF-MS analysis, the re-dried sample was oximated with 50 L of methoxyamine hydrochloride in pyridine (20mg/mL) for 90 min at 30℃ using a thermomixer (Eppendorf, Hamburg, Germany). Then, the oximated samples were silylated with 50 L of N-Methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) for 30 min at 37℃, using thermomixer. The pooled quality control (QC) samples were prepared from 10 L blends of each sample.
The derivatized sample (1 L) was injected into the GC-TOF-MS in the splitless mode. The temperatures of the injection and ion source were maintained at 250℃ and 230℃, respectively. The column temperature was sustained at 75℃ for 2 min and then raised to 300℃ at 15℃/min and subsequently maintained for 3 min. The acquisitions were recorded at the rate of 10 scan/s with a mass scan range of 50-1000 m/z. The analytical samples were analyzed in blocks of eight runs followed by an intermittent QC analysis to ensure the data quality and method’s robustness.
UHPLC-LTQ-Orbitrap-MS/MS Analysis
The analysis was performed using a UHPLC system equipped with a Vanquish binary pump H system (Thermo Fisher Scientific, Waltham, MA, USA) coupled with an auto-sampler and column compartment. Chromatographic separation was performed on a Phenomenex KINETEX® C18 Column (100 mm × 2.1 mm, 1.7m; Torrance, CA, USA), and the operational parameters were adapted from a study by Lee et al. (17). To circumvent systematic errors during analysis, the samples were analyzed in random blocks of 8 runs, followed by an intermittent QC sample prepared from pooled blends of each sample extracts.
Data and Statistical Analysis
MS data processing and multivariate statistical analysis were conducted as previously described (16). Raw data were converted to a NetCDF format (*.cdf) using ChromaTOF (version 4.44, LECO). After conversion, the MS data were processed using the Metalign software package (http://www.metalign.nl) to obtain a data matrix containing retention times, accurate masses and normalized peak intensities. The resulting data were exported to Excel (Microsoft, Redmond, WA, USA) for multivariate data analysis. Multivariate data analyses were performed using the SIMCA-P+ software (version 12.0, Umetrics, Umea, Sweden). Principal componenet analysis (PCA) and partial least squares discrimination analysis (PLS-DA) were performed to compare the different experimental groups. The significantly discriminant metabolite with a variable importance in projection (VIP) value exceeding 0.7 at p < 0.05 was obtained using the PLS-DA model.
Significantly different metabolites derived from GC-TOF-MS data were tentatively identified using standard compound retention time and MS fragments. Moreover, we confirmed the MS spectrum data for selected metabolites with in-house libraries and available web databases, including the NIST database (Version 2.0, 2011 FairCom; Gaithersburg, MD, USA), and the Human Metabolome Database (HMDB; http://www.hmdb.ca/).
Transcriptomics study
The collected splenocytes were isolated and total RNA from each sample was extracted using Trizol reagent (Invitrogen), and quality control and quantification were performed by a Bioanalyzer 2100 system (Agilent Technologies, USA) and Nanodrop ND-2000 Spectrophotometer (Thermo Scientific, USA). For RNA-sequencing (RNA-seq), the construction of libraries from total RNAs was performed using the NEBNext Ultra II Directional RNA-Seq Kit (NEW ENGLAND BioLabs, Inc., UK) according to the manufacturer’s instructions. Briefly, the isolated mRNAs were used to ligate the adaptors, and then cDNA was synthesized using reverse-transcriptase with adaptor-specific primers. PCR was performed for library amplification, and subsequently, libraries were checked for quality, quantification, and size distribution using the TapeStation HS D1000 Screen Tape (Agilent Technologies, USA) and using a StepOne Real-Time PCR System (Life Technologies, Inc., USA). High-throughput sequencing was performed as paired-end 101 base pair reads on a NovaSeq 6000 (Illumina, Inc., USA).
All raw sequence reads were preprocessed to remove bases with low quality and adapter sequences using Trimmomatic (version 0.39) (18). The rat reference genome (in FASTA format), HISAT2 genome index file (Rnor_6.0), and annotation (in GTF format) containing transcript structures were obtained from the Ensembl database (http://www.ensembl.org) (19). The cleaned paired-end reads were aligned to the reference genome using HISAT2 (v2.2.1)(20) with default parameters. The generated BAM (binary alignment map) files from the previous step were further processed with StringTie (v2.2.1) and prepDE (21) to quantify transcript abundances using the transcripts per kilobase million (TPM) and counts per million mapped reads (CPM) normalizations. Differential expression analysis was carried out using edgeR package (v3.38.4) (22). In this study, we defined genes as significantly differentially expressed if they had a greater than 10 read counts in at least one sample and lower than 1% of false discovery rate (FDR; q-value). We performed the enrichment analysis of Gene Ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of DEGs using DAVID (the Database for Annotation Visualization and Integrated Discovery; v2023q4 released; http://david.ncifcrf.gov) functional annotation tool (23).
CFU assay
Blood and spleen samples were used to count bacterial CFUs in cecal slurry model in rats. Thirty milligrams of spleen tissue were homogenized in 500 μL of DPBS and centrifuged at 12,000 rpm for 10 min at 4°C. The cell pellets were resuspended in 1 mL of DPBS (to form stock samples). The stock samples were diluted 1:10 with DPBS. To count blood CFUs, 800 μL of DPBS was added to 200 μL of blood. The samples were spread on TSA agar plates (BD Biosciences) without antibiotics and incubated at 37°C overnight, and the bacterial colonies were counted for analysis.
Intracellular ASP level in splenocytes in in-vivo sepsis model and in-vitro model
Aspartate levels were determined using the Aspartate assay kit (Ab 102512, Abcam, Waltham, MA, USA). Cell lysates were obtained by centrifuging at 14,000 × g for 15 min at 4℃, and the resulting supernatant was collected. Subsequently, lysates were incubated with the enzyme reaction mixture at room temperature, protected from light. The absorbance was measured at 570 nm, and the aspartate concentration was calculated using the following equation:
Aspartate concentration (μM) = sample amount from the standard curve (nmole) / the volume of the sample added to the wells (μL).
Phagocytosis study
The phagocytic activity of PBMCs was assessed using E. coli particles (E2861, Invitrogen, Carlsbad, CA, USA) and an opsonization kit (E2870, Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. PBMCs were incubated with E. coli particles for 1.5 h at 37℃. Subsequently, cells were washed twice with cold DPBS and fixed with 4% paraformaldehyde for 10 min at room temperature. After removing the supernatant, cells were washed twice and resuspended in FACS buffer (0.5% BSA, 2 mM EDTA). Following immediate incubation, cells were analyzed using flow cytometry with CytExpert software (Beckman, Brea, CA, USA).
Serum ALT, BUN, and Creatinine
Serum ALT, BUN, and Creatinine levels were measured using a chemistry analyzer. (Cobas c502, Roche, Basel, Switzerland).
Measuring endotoxin tolerance through ex vivo PBMCs/splenocytes stimulation with LPS
Measuring endotoxin tolerance through ex vivo PBMCs and splenocytes were isolated after induction of sepsis. PBMCs were isolated using the Ficoll-Paque PLUS gradient method (24). Isolated PBMCs were stimulated with LPS to observe and compare the levels of immune paralysis. PBMCs were seeded at a density of 1 × 105 cells/mL in 96-well plates, and 100 ng/mL of LPS (Escherichia coli O26:B6, L2762, Sigma-Aldrich, St.Louis, MO, USA) was added to each well. After 5 h, the culture medium was collected. Splenocytes were isolated and stimulated with LPS to compare immune paralysis (25). Isolated splenocytes were seeded at a density of 5 × 105 cells/mL in 6-well plates, and 1 μg/mL LPS was added to each well. After 5 h, the culture medium was collected.
Plasma glucose, albumin, and electrolyte assay
Plasma glucose, albumin and electrolytes were measured using arterial blood samples via the abdominal artery (ABL90 Flex Plus, Radiometer, Copenhagen, DK). Blood sample was collected using a heparinized syringe.
In vitro Endotoxin tolerance: LPS stimulation model with THP-1 cells
To estimate the immune-enhancing effect of both LOLA and ASP, THP-1 cells (ATCC, Manassas, VA, USA) were seeded at a density of 1.0´105 cells per well in 48-well plates with LPS and either LOLA or ASP. Cells were incubated at 37℃ with 5% CO2. For the endotoxin tolerance model, cells were stimulated with LPS at a concentration of 10 ng/mL for 4h. Following this, LPS was removed, and the cells were allowed to rest for 16 h in fresh RPMI medium (L0498, Biowest, Nuaillé, France). Subsequently, re-stimulation was carried out with the same concentration of LPS for 4h.
qPCR study
Total RNA was extracted from rat splenocytes using TRIzol reagent (15596018, Invitrogen, waltham, MA, USA). Then RNA was reverse transcribed into complementary DNA using RT PreMix, RNase H Minus(dT20) (K-2241, BIONEER, Daejeon, Republic of Korea) according to the manufacturer's instructions. qPCR was performed using qPCR master mix (K-6253, BIONEER, Daejeon, Republic of Korea) with CFX connect Real-Time System (BIORAD, CA, USA). The relative mRNA levels were calculated by the 2−ΔΔCt method with normalization to the reference gene β-actin. The primer used in this study was as follows: Got1; forward: 5’-ACCACGAGTACTTGCCCATC-3’, reverse: 5’-CATCGCCCTAAGAAGTCAGC-3’, Got2; forward: 5’-CCAAGACTTGCGGCTTTGAC-3’, reverse: 5’-CTTTTTCTTCACCACCGCCG-3’, β-actin; forward: 5’-TGTGGATTGGTGGCTCTATC-3’, reverse: 5’-AGAAAGGGTGTAAAACGCAG-3’.
Western blot analysis
The splenocytes were isolated by cell strainer (#352350, Falcon corning, Corning, NY, USA) from rat spleen and lysed by RIPA buffer with a protease inhibitor and phosphatase inhibitor (#1862209 and 1862495, Thermo Fisher Scientific, waltham, MA, USA). Each loading sample has 20 μg protein and was separated by 12% SDS-PAGE. Then the gels were transferred to PVDF membranes (#10600023, cytiva, Amersham, England) and blocked in 5% BSA for 1hr at room temperature. The membranes were incubated with primary antibody (GOT1 Rabbit pAb [1:1000, #A5822, ABclonal Technology, Boston, MA, United States], GOT2 Polyclonal Antibody [1:1000, #PA5-27572, Invitrogen, waltham, MA, USA], β-actin (C4) mouse monoclonal IgG1 [1:1000, #sc-47778, Santa Cruz Biotechnology, Dallas, Texas, USA]) overnight at 4 °C and with secondary antibody (Goat Anti-Rabbit IgG antibody (HRP) [1:10000, #GTX213110-01, GeneTex, Irvine, CA, USA], Mouse IgG antibody (HRP) [1:10000, #GTX213111-01, GeneTex, Irvine, CA, USA]) for 1hr at room temperature. After antibody attaching, the membranes were washed 6 times for 30 minutes with TBST. The protein expressions were visualized using ECL solution (GTB5060, GeneSTAR, Songpa, Seoul, Republic of Korea) with Gel documentation system (G:BOX Chemi XX6, Syngene, Mumbai, India) and the band intensities were quantified by Image J program.
In vitro M1 macrophage study
Cell culture and M1-like macrophage polarization
THP-1 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (S1480, FBS, Biowest, Nuaillé, France) and 1% penicillin-streptomycin (10,000 U/mL, Gibco, Grand Island, NY, USA) in an incubator at 37℃, 5% CO2. For immuno-polarization experiments, THP-1 cells were differentiated by PMA (100 ng/mL, 79346, Sigma-Aldrich, St Louis, MO, USA) for 6 h, followed by the addition of GM-CSF (25 ng/mL, 130-093-865, Miltenyi Biotec, Bergisch Gladbach, Germany), LPS (100 ng/mL, L2762, Sigma-Aldrich, St.Louis, MO, USA), and IFN-y (20 ng/mL, 285-IF, R&D systems, Minneapolis, MN, USA) for 18 h.
Inhibition of GOT1 by AOAA
In our experiment, we utilized two different cell types:
1. M1-like macrophages:
Following differentiation, M1-like macrophages were treated with AOAA (1 mM) for 24h in an incubator at 37℃ with 5% CO2. The treated macrophages were subsequently employed in further experiments.
2. PBMCs:
Isolated PBMCs from rats were cultured in a medium containing 10% FBS and 1% P/S in the presence of AOAA (5 mM), LOLA (100 μg/mL), and ASP (2 mM). The cells were cultured for 20-22h in an incubator at 37℃ with 5% CO2. On the following day, these cultured cells were used in further experiments.
Inhibition of Got1 by siRNA
Got1 siRNA transfection was performed in 1.0×106 M1-like macrophages using Lipofectamine RNAiMAX (Invitrogen, 13778) following the manufacturer's instructions. After a 4 h incubation, 10% FBS was added, and further experiments were conducted following an additional 24 h incubation period. The primer used in this study was as follows: Got1 siRNA; 5’-AACAGGUGCACUUCGAAUUGGUU-3’.
Inhibition of Electron Transfer Chain
In our experiment, we utilized two different cell types
1. M1-like macrophages:
The cells were treated with specific ETC inhibitors, including ETC inhibitor Ⅰ (Rotenone, R8875, 120 nM), ETC inhibitor Ⅲ (Antimycin A, A8674, 30 nM), and ETC inhibitor Ⅴ (Oligomycin, O4876, 100 nM) for a duration of 24h. All reagents used in the experiment were purchased from Sigma-Aldrich (St Louis, MO, USA).
2. PBMCs:
Isolated PBMCs were cultured in a medium containing 10% FBS and 1% P/S for overnight in an incubator at 37℃ with 5% CO2. On the following day, the cells were treated with ETC inhibitors (Rotenone, Antimycin A, and Oligomycin, each at 20 nM) and co-treated with LOLA (100 μg/mL) and ASP (2 mM) each in the presence or absence for 3h. Subsequently, these cultured cells were used in further experiments.
Human PBMCs study (ASP level and patients’ outcome)
This study was approved by the institutional review board of CHA University Bundang Medical Center (IRB No. 2020-06-042). Human blood samples were obtained from patients with sepsis defined as Sepsis-3. Written informed consent was obtained from all patients prior to sampling.
PBMCs were isolated from human peripheral blood (septic patients and healthy volunteers) using density gradient centrifugation.
Fresh heparinized blood was placed into 50 mL conical centrifuge tube, added an equal volume of room-temperature PBS and mixed by inverting the tube several times. The diluted blood sample was carefully layered onto an equal volume of Ficoll-Paque PLUS (Cytiva, Uppsala, Sweden) and centrifuged at 400 × g for 20 min at 20 °C without brake. The PBMC layer was transferred to another tube and centrifuged at 300 g for 5 min at 20 °C to obtain PBMC pellets. To remove any remaining red blood cells (RBCs), the cell pellet was resuspended in 1 ml RBC lysis buffer for human (J62990, Thermo, USA) for 5 min at room-temperature, and then diluted with 9 ml of PBS. The mixture was centrifuged at 300 g for 5 min at 4 °C and removed the supernatant. Finally, to wash the cell pellet, the pellet was resuspended in 5 ml of PBS and centrifuged at 300 × g for 5 min at 4 °C two times.
Aspartate levels were determined using the Aspartate assay kit (ab102512, Abcam Waltham, MA, USA). Cell lysates were obtained by centrifuging at 14,000 × g for 15 min at 4℃, and the resulting supernatant was collected. Subsequently, lysates were incubated with the enzyme reaction mixture at room temperature, protected from light. The absorbance was measured at 570 nm, and the aspartate concentration was calculated using the following equation:
Aspartate concentration (μM) = sample amount from the standard curve (nmole) / the volume of the sample added to the wells (μL).
Mitochondria isolation
We isolated mitochondria using a previously described method with some modifications (26). In brief, L6 cells (ATCC; CRL-1458, Manassas, VA, USA) were homogenized using a 26 G syringe in SHE buffer (0.25 M sucrose, 20 mM HEPES, 2 mM EGTA, 10 mM KCl, 1.5 mM MgCl2 and 0.1% defatted bovine serum albumin [BSA] with protease inhibitor, pH 7.4) and then centrifuged at 1,500 × g for 5 min to remove cells and cell debris. After centrifugation, the mitochondria-containing supernatant was subsequently centrifuged at 20,000 × g for 10 min at 4°C to obtain mitochondria. For all experiments, the isolated mitochondria were stored at 4°C until use.
Cytokine measurements
The levels of the cytokine TNF-α (rat: ab236712, human: ab181421, Abcam, MA, USA) in the splenocytes, PBMCs and THP-1 cell were measured using ELISA kits according to the manufacturer’s instructions. The optical density at 450 nm was detected by a microplate reader (VersaMax with SoftMax Pro software, Molecular Devices, CA, USA).
Statistical analysis
The Shapiro-Wilk test was performed to determine the normality of the data. Normally distributed data are presented as the mean ± standard deviation and were compared with independent t-tests or One-way ANOVA with post hoc analysis. If the data did not fit a normal distribution, they are presented as the median and interquartile range and were analyzed by Mann-Whitney U test or Kruskal-Wallis test. Survival rates were compared by Kaplan-Meier log-rank test. Correlation was tested using Spearman rank correlation analysis. A p-value of <0.05 was defined as statistically significant. All analyses were performed with Sigma Plot software (SYSTAT Software, San Jose, CA).