Plasmid construction

To overexpress USP18 or STING1, their coding sequences were respectively cloned into the pcDNA3.3-HA or pLV2-CMV-FLAG-Puro vector, and the resulting plasmids were designated as HA-USP18 (USP18 OE) and Flag-STING1 (STING1 OE). For ZEB1 and BECN1 overexpression, their coding sequences were inserted into the pCMV-3 × FLAG-SV40-Neo and pLV3-CMV- 3 × FLAG-CopGFP-Puro backbone, respectively, leading to the plasmids of Flag-ZEB1 and Flag-BECN1. For USP18 knockdown, three shRNAs (shUSP18-1, shUSP18-2, and shUSP18-3) were inserted into pLKO.1-puro lentiviral vector and a non-specific shRNA (shNC) was used as the negative control. The His-tagged ubiquitin plasmid (His-Ub) was purchased from Wuhan Miaoling Biotechnology (China). All plasmids were sequenced before use to ensure their sequence accuracy. The lentivirus production and titer determination were performed by Anti-Hela Biotech (China). The virus multiplicity of infection (MOI) for knockdown assays was optimized to 50. Non-viral plasmid transfection was conducted by Lipofectamine 2000. The primer sequences are shown in Table 1.

Table 1 Primers for plasmid constructionGeneration of HKOs

Human pluripotent stem cell (hPSC) H1 cells, cultured to 70% confluence, were digested with TrypLE™ Express (12,605–010, Gibco, USA) for 5 min. The cells were then pelleted, resuspended in complete mTeSR™ 1 medium (85,850, STEMCELL, USA) with 10 μM Y-27632 (MedChemExpress, USA) (50,000 cells/mL). A 2 mL aliquot of the cell suspension was transferred to an ultralow attachment 6-well plate (Corning) and incubated overnight. The following day was designated as Day (D) −1, during which the medium was replaced with mTeSR™ 1 medium without Y-27632. On Day 0 (D0), the cells were cultured in APEL2 medium (05270, STEMCELL) with 8 μM CHIR99021 (HY-10182, MedChemExpress) for three days (D0-D3). This was followed by an induction phase (D4-D6) using APEL2 with 200 ng/mL FGF9 (10,262-HNAE, Sinobiological, China) and 1 μg/mL heparin (60351ES03, Yeasen, China). On Day 7 (D7), the developing HKOs were stimulated with 5 μM CHIR99021 for 1 h. Subsequently, the cells were cultured in APEL2 containing 200 ng/mL FGF9 and 1 μg/mL heparin for an additional three days (D7-D10). Finally, the medium was switched to APEL2 with 1 μg/mL heparin, and the HKOs were harvested and characterized on Day 26 (D26).

Treatment of HKOs

Collect the HKOs into 24-well low-attachment plates. Then 500 μL of complete organoid medium containing 50 μL of a 1: 50 diluted lentiviral suspension (titer = 1 × 107) was added and mixed thoroughly, incubated for 24 h. Afterwards, the fresh culture medium with/without LPS was added for 24 h of cultivation, followed by the subsequent detection. The groupings of HKOs were: Mock, LPS, LPS + shNC, LPS + shUSP18. To conduct the rescue experiment using ferroptosis inhibitors, HKOs were handled with 50 μg/mL LPS and 0.25 μM Ferrostatin-1 (Fer-1) simultaneously for 24 h.

HKO size measurement

The morphology of HKOs was examined using an inverted bright-field microscope (Motic, China) equipped with a digital camera. The diameters of these HKOs were measured using the associated software. For each treatment group, the diameters of six HKOs were analyzed.

ATP concentration measurement

The ATP levels were assessed by a kit (S0027, Beyotime), following the manufacturer’s instruction using a Microplate Luminometer Orion II (Berthold Technologies GmbH, Germany).

Immunofluorescence (IF)

HKOs were collected and fixed in 4% paraformaldehyde before being processed into 5 μm frozen sections. The sections were washed with PBS and blocked with 1% BSA (BP9700100, Thermo Fisher Scientific, USA) for 1 h at RT. Then, they were incubated overnight with primary antibodies, including anti-human CK19 (10,712–1-AP, Proteintech, 1:500) or anti-human PODXL (RLM0526, Immunoway, 1:500). Afterwards, the sections were washed 3 times with PBST, each wash lasting 10 min. They were then incubated in dark at RT for 1 h with fluorescent secondary antibody solution (RS0011, Immunoway, 1:1000). Afterward, the sections were washed again with PBST, mounted using a DAPI-containing medium, and imaged using LMS 900 confocal microscope.

Hematoxylin and Eosin staining (HE)

Organoids were collected and allowed to stand for 10 min at 4 °C, and the supernatant was removed by centrifugation. Agarose grooves were prepared, the organoid precipitate was aspirated and placed at the bottom of the groove, PBS was added and centrifuged for 5 min, the supernatant was removed, melted agarose was added to the organoid precipitate to coagulate at RT, and then it was fixed at 4 °C overnight. The agarose blocks containing organoid precipitates were treated in 70%, 80%, 90% and 95% ethanol for 1 h, 100% ethanol for 30 min, then placed in xylene for 5 min, treated twice, immersed in wax for 2 h at 60 °C, immersed in wax twice, then embedded into wax blocks and made into paraffin sections. The sections were immersed in xylene. After the wax on the sections was dissolved, the paraffin sections were immersed in absolute ethanol I-II for 5 min, respectively. The paraffin sections were rinsed with absolute ethanol for 20 s, and then the alcohol on the sections was rinsed off with running water. The slices were respectively stained in HE staining solution 1 for 3 min, HE staining solution 2 for 3 s, and HE staining solution 3 for 3 s followed by rapid water washing. Then slices were immersed in 85% ethanol, 95% ethanol, HE staining solution 4, absolute ethanol I-III, n-butyl alcohol, xylene I-II for 3 min, separately, and the slices were quickly dried and sealed with neutral gum.

qPCR

Total RNA was isolated from HKOs using TRIzol and purified with the RaPure Total RNA Kit (R4011-02, Magen Biotech). For cDNA synthesis, 1 μg of RNA was used as a template in conjunction with the All-in-One First-Strand SuperMix (EG15133S, NYBio, China). ChamQ SYBR Color qPCR Master Mix was applied to perform qPCR (Q431-02, Vazyme, China), with 18S ribosomal RNA serving as the endogenous control. Relative levels of gene were determined by 2−ΔΔCT method. Primers are provided in Table 2.

Table 2 qPCR primer informationWestern blot

Total protein was extracted using RIPA buffer with protease inhibitors (Beyotime). Equal amounts of denatured protein (10 μg) were loaded, separated by 10% SDS-PAGE, and then transferred onto PVDF membranes. The membranes were blocked with 5% skim milk (Beyotime) for 1 h, and incubated with primary antibodies, then incubated with the secondary antibody solution for 2 h. Protein signals were detected by ECL solution from Beyotime and visualized with a ChemiDoc imaging system (Bio-Rad). Details of the antibodies used are provided in Table 3.

Table 3 Western blot antibody informationRNA-seq analysis

Total RNA was extracted from mock- and LPS-treated HKOs (approximately 100 organoids per sample) using TRIzol (Invitrogen). Each sample yielded over 1 μg of total RNA, with RNA Integrity Numbers (RIN) of ≥ 9.4. Libraries were prepared by TruSeq Stranded mRNA (PolyA +) kit, and sequenced on Illumina NextSeq 500 platform. Sequencing reads were aligned to the human genome (GRCh38.p13), and DEGs were identified using the R package ‘limma’. Genes with a log2 fold change (Log2FC) ≥ 1 and an adjusted P-value (padj) < 0.05 were considered markedly differentially expressed.

For GO and KEGG enrichment analysis, we utilized the R package ‘clusterProfiler’ (v4.14.4). To identify USPs whose expression is upregulated in LPS-induced HKOs, the DEGs with a Log2FC of > 0.59 and a padj of < 0.05 were selected to compare with 75 known USPs in the human genome.

Lists of ferroptosis markers and drivers were downloaded from FerrDb V2 (http://www.zhounan.org/ferrdb/current/), and their expression profiles were examined in our RNA-seq dataset. The expression matrix of GSE154918 were downloaded from GEO (https://www.ncbi.nlm.nih.gov/geo/). Only sepsis and healthy samples were used in this study. PCA was performed to assess sample homogeneity and consistency. Outlier samples identified through PCA (GSM4683603, GSM4683576 and GSM4683568) were excluded from downstream analyses. Following the removal of outliers, both principal component analysis and Pearson correlation analysis demonstrated high consistency and quality across samples. Differential expression analysis was performed using the methods described above.

MDA, GSH, GSSG, and Fe2+ Levels measurement

HKOs were homogenized and lysed using RIPA buffer, centrifugated at 13,000 rpm for 10 min, the supernatants were collected. The levels of MDA, GSH, GSSG, and Fe2+ were measured using the MDA (BC0020, Solarbio, China), the GSH/GSSG (S0053, Beyotime), and the Fe2+ assay kit (E-BC-K881-M, Elabscience, USA), respectively. OD values were measured using FlexStation 3 microplate reader.

ROS detection

ROS levels were measured using DCF-DA kit (50101ES01, Yeasen). The HKOs were incubated with 10 μM DCF-DA at 37 °C for 20 min in dark, with gentle inversion at intervals. After incubation, the HKOs were washed with serum-free medium to remove unbound DCF-DA, resuspended in PBS, and analyzed using NovoCyte 1300 machine (Agilent, USA).

Co-immunoprecipitation (Co-IP)

For co-IP, protein extracts were incubated with anti-FLAG beads (M8823, Sigma-Aldrich) or anti-HA BeyoMag™ Protein A + G magnetic beads (P2121-2 ml, Beyotime) under gentle agitation at RT for 2 h. After incubation, the beads were collected by centrifugation and washed 3 times with wash buffer to eliminate non-specific protein binding. Following the wash steps, the beads were resuspended in 20 μL of elution buffer, and the proteins bound to the beads were eluted for further analysis by Western blotting.

GST pull-down assay

His-STING1, GST-USP18, and GST proteins were sourced from Anti-Hela Biotech. To prepare the protein mixture, 20 mg of His-STING1 was combined with 20 mg of GST-USP18 or GST protein. An aliquot of the mixture was denatured and centrifuged for the input sample. The remaining mixture was incubated with 20 μL of Glutathione Sepharose 4B beads (GE17-0756–01) for 6 h at 4 °C with gentle inversion. After that, the beads were pelleted and then washed five times with ice-cold PBS, followed by incubation with 80 μL of elution buffer at 95 °C for 10 min. Finally, the mixture was centrifuged, and the resulting supernatant containing the eluted proteins was collected, denatured, and analyzed by Western blotting.

Co-culture of HKOs with THP-1

THP-1 were pretreated with PMA (100 ng/mL) for 24 h to induce differentiation. The HKOs with UPS18 depletion were seeded into the lower chamber and the differentiated THP-1 were seeded into the upper chamber of the Transwell plates. A co-culture system with or without LPS was used to cover the upper and lower chambers for co-culture.

ELISA

ELISA was performed using TNF-α (Elabscience, E-EL-H0109) and IL-1β (E-EL-H0149) kits to detect cytokine concentration. All procedures strictly followed the manufacturer’s guidance.

Statistical analysis

Statistical analysis was performed using GraphPad Prism (v8). Data are expressed as mean ± SD. Each data point in the bar and dot plots represents the mean of biological triplicates and/or the average of technical triplicates. P < 0.05 was considered statistically significant. The methods used to calculate P-values are specified in the figure legend.