Patient study protocol

Patients diagnosed with sepsis in the Department of Critical Care Medicine of Shanghai Ruijin Hospital from July 1st, 2021 to May 31st, 2022 were enrolled. This study was approved by the Ethics Committee of Ruijin hospital (20,200,011). The investigation was based on the institution’s guidelines for human studies and conformed to the ethics guidelines of the declaration of Helsinki. Informed consent was obtained from each participant. The enrollment criteria were as follows: (1) 18–80 years old; (2) adherence to the sepsis 3.0 diagnostic criteria; (3) hospital stay > 24 h. The corresponding exclusion criteria were as follows: (1) discharge or death within 24 h after admission; (2) participation in other clinical research; (3) emergency surgery after admission; and (4) malignant tumor; (5) pregnant patients; (6) lack of necessary clinical data. Finally, a total of 12 healthy volunteers, 29 septic patients were enrolled.

Reagents

2-DG (#S4701), ISRIB (#S0706), Salubrinal (#S2923), GCN2IB (#S8929), C16(#S9668), GSK2656157 (#S7033), and Compound C (#S7840) were purchased from Selleck (shanghai, China). Lipopolysaccharide (LPS, #L2630, E. coli 0111:B4) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse recombinant GDF15 (#10596-GD-025) was purchased from R&D Systems (Minnesota, USA). Pooled mouse ATF4 siRNA (#1. Sense-5’-CUCCCAGAAAGUUUAAUAATT-3’, anti-sense-5’- UUAUUAAACUUUCUGGGAGTT-3’; #2. Sense-5’-GCUGCUUACAUUACUCUAATT-3’, anti-sense-5’-UUAGAGUAAUGUAAGCAGCTT-3’; #. Sense-5’-GUCUCUUAGAUGACUAUCUTT-3’, anti-sense-5’-AGAUAGUCAUCUAAGAGACTT-3’) and pooled mouse GDF15 siRNA (#1. Sense-5’-CUCGAACUCAGAACCAAGUTT-3’, anti-sense-5’-ACUUGGUUCUGAGUUCGAGTT-3’; #2. Sense-5’-GUGGUUCUUAUGCACAGGATT-3’, anti-sense-5’-UCCUGUGCAUAAGAACCACTT-3’; #3. Sense-5’-CUGCUAAUAAAGGUGAGCUTT-3’, anti-sense-5’-AGCUCACCUUUAUUAGCAGTT-3’) were synthesized by GenePharma (Shanghai, China). Antibodies against phosphorylated eIF2α (3597 S), total eIF2α (5324 S), ATF4 (11,815 S), phosphorylated AMPK (2535 S), and AMPK (2532 S) were purchased from cell signaling technology (MA, USA). Antibodies against Glut1(66,290), HK2 (66,974), PFKFB3 (13,123), and PKM2 (60,268) were obtained from Proteintech (Wuhan, China) Antibody against GDF15 (ab105738) was purchased from Abcam (MA, USA).

Cell culture

The alveolar macrophage cell line MH-S (#ZQ0921, ScienCell, CA, USA) were cultured in RPMI 1640 complete medium supplemented with 10% FBS (AU0600), 1% penicillin/streptomycin, 0.05mM β-mercaptoethanol (#ZQ-206, ScienCell) at 37 °C with 95% humidity and 5% CO2. MH-S were pretreated with the indicated inhibitors followed by stimulation with 1 µg/mL LPS for the indicated time periods to imitate inflammation.

siRNA transfection

MH-S were seeded at a density of 50–70%. HiPerFect (301,704, Qiagen, Germany) was used as the transfection reagent according to the manufacturer’s instructions. Cells were treated with different stimuli 48 h after transfection and were harvested for further analysis.

RNA sequencing

Total RNA was extracted from MH-S stimulated with LPS in the absence and presence of 2-DG. The concentration and purity of isolated RNA were measured using a ND-800 spectrophotometer (Thermo Fisher Scientific, DE, USA). The RNA libraries were constructed using a Truseq RNA Library Prep Kit. Sequencing was carried out using a 2 × 150 bp PE configuration. The clean data (reads) were mapped using Hisat2 (version 2.1.0). Then, we performed gene expression analysis using RSEM (version 1.3.1). Differential expression analysis among different groups was conducted using DESeq2, and then |FC| >2 and FDR < 0.05 were determined as thresholds for differentially expressed genes (DEGs). Gene Ontology (GO) analysis was used to determine the significant biological processes of a particular gene set (q < 0.05). The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was used to identify the most significant signaling pathways involved in BMS-303,141-regulated genes (adjusted P < 0.05). The Venn analysis was used to calculate the number of genes in different gene sets.

Murine model

Male C57BL/6 mice (6–8 weeks, 20–25 g) were obtained from Charles River (Beijing, China) and randomly divided into experimental groups. The mice were intraperitoneally injected with LPS (5 mg/kg body weight) to induce endotoxemia. In the intervention group, the mice were intraperitoneally injected with mouse recombinant GDF15 (50 ng/kg body weight), salubrinal (1 mg/kg body weight), or 2-DG (500 mg/kg body weight) 1 h before LPS injection. Vehicle control mice were intraperitoneally injected with 100 µL of 0.9% NaCl. 16 h after LPS challenge, the mice were anesthetized and blood samples were taken. Organs were snap frozen or fixed with formalin for further examination. Frozen organs were stored at -80 °C. The protocols for animal experiments were approved by the Animal Ethics Committee of Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (No. 092) and were in line with the International Guidelines for Care and Use of Laboratory Animals (National Academy of Sciences Health Publication No. 85–23, revised in 1996). All animal experiments were conducted according to the principles of laboratory animal care.

Isolation of mouse alveolar macrophages from BALF

After blood collection, mice were sacrificed. The lungs were perfused with PBS using a 20-gauge endotracheal catheter, followed by the collection of the bronchoalveolar lavage fluid (BALF) from lungs. BALF samples were centrifuged at 500 g for 5 min, and the supernatants were stored at -80 °C for further analysis. The pellets were seeded in plate with medium for 3 h and the adherent cells were shown to be alveolar macrophages. The purified alveolar macrophages were used for further analyses.

Western blot assay

The protein samples from cell lysates were separated using SDS-PAGE and transferred to polyvinylidene fluoride membranes (Bio-Rad, #1,620,177, CA, USA). The membranes were blocked using 5% bovine serum albumin (BSA) for 1 h and incubated with primary antibodies overnight at 4 °C. Those were followed by incubation with corresponding secondary antibodies for 1 h at room temperature. The blots were then visualized using a motored molecular imaging system (Tanon, Shanghai, China).

RNA extraction and quantitative RT-PCR

Total RNA was extracted from cell lysates according to the EZB RNA reagent kit protocol, and RNA concentration and purity were measured. After total RNA was reversely transcribed to cDNA using HiScript III RT SuperMix (Vazyme, Nanjing, China). PCR was conducted using Taq Pro Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). Relative mRNA expression was determined using the 2−ΔCT methods relative to the housekeeping gene GAPDH. Data are presented as fold changes of mRNA levels relative to the control groups. The sequences of primers were: IL-6 (5’-ACTTCCATCCAGTTGCCTTCTTGG-3’, 5’-TTAAGCCTCCGACTTGTGAAGTGG-3’), TNFα (5’-GCGACGTGGAACTGGCAGAAG-3’, 5’-GCCACAAGCAGGAATGAGAAGAGG-3’), ATF4 (5’-TCTGCCTTCTCCAGGTGGTTCC-3’, 5’-GCTGCTGTCTTGTTTTGCTCCATC-3’), GDF15 (5’-ATACTCAGTCCAGAGGTGAGAT-3’, 5’-CTTCAGGGGCCTAGTGATG-3’).

Enzyme-linked immunosorbent assay (ELISA)

The concentrations of IL-6 and TNFα in mouse plasma, BALF, and cell supernatants were measured using ELISA kits (MultiSciences Biotechnology, Hangzhou, China) following the manufacturer’s instructions. The concentrations of GDF15 in mouse plasma, BALF, and cell supernatants were measured using the ELISA kit (#MGD150, R&D Systems, MA, USA). Levels of lactate in the supernatants were measured using L-lactate assay kit (Cat#1,200,011,002, Eton Bioscience, CA, USA).

Extracellular acidification rate (ECAR) measurement

ECAR was determined using a XF-96 Extracellular Flux Analyzer (Seahorse Bioscience). 8 × 104 MH-S were plated and incubated overnight in Seahorse XF96 Cell Culture Microplates. 1 h prior to the assay media were changed to bicarbonate-free RPMI supplemented with 2mM glutamine and the plate was kept in a non-carbonated incubator. Measurements were performed under basal conditions and after the sequential addition of final 0.5 µM rotenone & antimycin A and 5mM 2DG.

Statistical analysis

All experiments were independently repeated more than three times. Data were presented as the mean ± SD. All statistical analyses were performed using IBM SPSS Statistical software (26.0, USA), and the experimental results were plotted using Graph Pad Prism software (9.4.1, USA). Any differences between groups were determined using either a two-tailed independent Student’s t-test or one-way analysis of variance (ANOVA) followed with Bonferroni multiple comparison test. P < 0.05 was considered to be statistically significant.