Human subjects

Healthy individuals were recruited through regular physical examination at The First Affiliated Hospital, College of Medicine, Zhejiang University. The clinical characteristics of LC and LC when accompanied with diabetes are presented in Table 1. All samples excluded HCV infection, Metabolic-dysfunction-associated fatty liver disease (MAFLD), and Metabolic dysfunction-associated steatohepatitis (MASH). The use of human blood was conducted in accordance with the Helsinki Declaration and was approved by the Ethics Committee of The First Affiliated Hospital, College of Medicine, Zhejiang University (IIT2020023A).

Table 1 Clinical characteristics of patientsAnimals model

C57 BL/6 male mice, 6 weeks, purchased from Hangzhou Ziyuan Experimental Animal Technical Company. Mice were intraperitoneally injected with Carbon tetrachloride (CCl4: corn oil = 1: 3, two injections per week, 160 μL/mouse). S100A8/A9 (1 μg/mouse) was injected intravenously at the same time of CCl4 injection. All experiments were performed in accordance with the institutional guidelines from the Animal Care and Use Committee of the Medical College of Zhejiang University (reference number 2021‐007). The mice were killed 24 h after CCl4 injections. The serum, liver, and pancreas were collected for further use.

Cell culture and treatment

The 1.1B4 islet β cell line (Kunming cell bank of Chinese Academy of Sciences, Kunming, China) was cultured in a RPMI 1640 medium (Sigma-Aldrich, Shanghai, China) containing 10% foetal bovine serum (FBS, Gibco, Newcastle, Australia) and 1% penicillin–streptomycin solution (Biological Industries, Shanghai, China). The βTC-6 mouse islet β cell line (Kunming cell bank of Chinese Academy of Sciences, Kunming, China) was cultured in a Dulbecco’s modified Eagle’s medium (Gibco, Newcastle, Australia) containing 10% FBS and 1% penicillin–streptomycin solution. All cells cultured at 37 °C in a 5% CO2 cell incubator.

To evaluate the death form of β cells, pan-caspase inhibitor zVAD-FMK (zVAD, MedChemExpress, Shanghai, China) and MLKL inhibitor Necrosulfonamide (NSA, MedChemExpress, Shanghai, China) were added in the culture medium. Cells were pretreated with zVAD (20 μM), NSA (1 μM), FPS-ZM1 (1 μM), or SB203580 (10 μM) for 30 min before culturing in medium containing 20% plasma from patients with LC, patients with LC and DM, or healthy people.

First, 1 \(\times ^\) 1.1B4 islet β cells were first seeded in a 12-well plate for indirect co-culturing. The 1 \(\times ^\) NK-92 MI cells were seeded in the upper chamber (0.4 μm aperture) 12 h later. The 5 \(\times ^\) 1.1B4 islet β cells were first seeded in the 24-well plate for direct co-culturing. The 5 \(\times ^\) NK-92 MI cells were seeded directly in the wells 12 h later.

Flow cytometry

Immunophenotypic analysis of whole blood or peripheral blood mononuclear cells (PBMCs) was conducted using monoclonal antibodies (mAbs) for the following: PE-Cy7-anti-Human CD56 (Biolegend, California, USA), PE-Cy7-anti-Human CD11b (Biolegend, California, USA), Percp-anti-Human CD45 (BD, NJ, USA), APC-Cy7-anti-Human CD14 (BD, NJ, USA), PE-anti-Human CD1c (Biolegend, California, USA), FITC-anti- Human CD3 (BD, NJ, USA), APC-Cy7-anti-Human HLA-DR (Biolegend, California, USA), PE-anti-Human RAGE (Abcam, Cambridge, UK) PE-anti-Human CD107a (Biolegend, California, USA), and APC-anti-Human NCR1 (Biolegend, California, USA). Intracellular proteins used were APC-anti-Human IFN-γ (BD, NJ, USA). Concentrations of plasma IFN-γ, TNF-α, IL-2, IL-4, IL-10, IL-6, IL-17, and IL-1β were detected using a human Th1/Th2/Th17 subsets detection kit (flow cytometry fluorescence luminescence method) (Jiangxi Cellgene Biotech Co. Ltd, Jiangxi, China). These values were determined based on the manufacturer’s instructions. The fluorochrome-conjugated single cells or cytokines were detected using BD FACS Canto II. The acquired data were analysed using FlowJo 10 or CBA-FCAP Array V3 software.

Propidium iodide (PI) staining

Single cells were stained with antibodies, followed by PI staining (Lianke Biotech, AP101-100-kit, Hangzhou, China) according to the manufacturer’s guidelines. Subsequently, flow cytometry (BD FACS Canto II, USA) was used to detect single cell suspensions. Data acquired were analysed using the FlowJo software.

Enzyme-linked immunosorbent assay

The concentrations of IFN-γ (BioLegend, USA), S100A8/A9 (BioLegend, USA), and insulin (Reddot Biotech, Canada; Abcam, UK) in plasma or cell culture supernatant were examined by enzyme-linked immunosorbent assays (ELISA). As described in a previous study [17], these were detected according to the manufacturer’s instruction.

Quantitative real-time polymerase chain reaction (RT-PCR) analysis

Total RNA was extracted from 1.1B4 and βTC-6 cells using TRIzol reagent (Takara, Kyoto, Japan). SYBR Green PCR Master Mix (Vazyme, Nanjing, China) was used to perform quantitative real-time PCR (qPCR) assays of mRNA. The qPCR conditions were: 95 °C for 30 s, followed by 40 cycles at 95 °C for 10 s, 60 °C for 30 s. Lastly, one PCR cycle was conducted at 95 °C for 15 s, 60 °C for 60 s, and 95 °C for 15 s. Expression levels of target genes were normalised to the expression level of GAPDH (internal control) and calculated based on the comparative cycle threshold (CT) method (2−ΔΔCT).

Western blot analysis

1.1B4 and βTC-6 cells (5 \(\times ^\) cells) were treated with 100 μL RIPA Lysis Buffer (Beyotime Biotechnology, Shanghai, China) containing Halt protease (100 \(\times\), Selleck, HOU, USA) for 30 min at 4 °C, followed by centrifugation at 13,523\(\times\)g at 4 °C for 10 min. The supernatant was collected and loaded onto a 10% polyacrylamide gel for electrophoresis.

Data-independent acquisition mass spectrometry (DIA-MS)

Plasma from patients was used for DIA-MS measurements. The resulting tandem MS/MS data were processed using the DIA-NN (v 1.8) search engine. The fold change (FC) was calculated as the average of the comparisons between biological replicates to determine differentially expressed proteins. The expression of proteins with a fold change > 1.5 and p < 0.05 were considered significant.

Statistical analyses

Data analysis was performed using the Prism 9 software (GraphPad, San Diego, CA, USA). Comparisons between two groups were done using the Student’s t test. Comparisons between more than two groups were done using One-way ANOVA followed by Tukey’s multiple comparison post hoc test. Data are presented as the mean ± S.E.M. and are representative of at least three independent experiments. Statistical significance was set at p < 0.05. *p ≤ 0.05, **p < 0.01, ***p < 0.001, ****p ≤ 0.0001, nsp > 0.05.