Human tissue samples

The Biobank of LMU’s Department of General, Visceral and Transplant Surgery contributed double-coded liver tissue samples and supported data for this study. The Human Tissue and Cell Research (HTCR) Foundation oversees the operation of this Biobank. The framework of HTCR Foundation [18], which includes obtaining written informed consent from all donors, has been approved by the Bavarian State Medical Association (approval number 11142) in Germany as well as the ethics committee of the Faculty of Medicine at the LMU (approval number 025 − 12). All liver specimens utilized in this study were obtained from the donors devoid of liver diseases such as alcoholism, chronic liver diseases, and obstructive jaundice.

Isolation and culture of primary KCs

Fresh human liver tissues were kept on ice in RPMI 1640 medium (Gibco, Karlsruhe, Germany) and were isolated within 6 hours following surgical removal. To isolate human primary hepatic KCs, density gradient centrifugation with Nycodenz (Axis-Shield, Rodelokka, Norway) was performed as elaborated in our previous study [19, 20]. The purity of isolated KCs was reassessed by staining with markers including CD11b, CD163, and CD68 (Supplementary Fig. S1). The cell yield of isolated KCs was 3 × 106 cells per gram of liver tissue. Freshly isolated primary KCs were seeded in multi-well plates at a concentration of 5 × 105 cells/ml and cultured in RPMI 1640 with 10% fetal calf serum (PAN, Aidenbach, Germany) and 1% Penicillin-Streptomycin (Sigma, St. Louis, USA) at 37 °C in 5% CO2, and the culture medium was changed to a new medium next day.

Culture of THP-1 cell line

THP-1 cell line (American Type Culture Collection, reference number TIB-202TM) was provided by Prof. Peter Nelson. Cells were cultured at a concentration of 5 × 105 cells/ml in RPMI 1640 with 10% fetal calf serum (FCS) and 1% penicillin-streptomycin at 37 °C and 5% CO2. To differentiate THP-1 into adherent macrophages, cells were treated for 48 h with 20 ng/ml phorbol myristate acetate (PMA), and the medium was replaced with a serum-free medium before the stimulation of bacterial products.

Isolation, preparation, and employment of bacterial products

Bacterial strains including E. coli and S. pneumoniae were isolated from patients with spontaneous bacterial peritonitis. The isolates were cultured on Columbia 5% sheep blood media (Becton Dickinson, Heidelberg, Germany) at 37 °C with aeration. Bacterial colonies were carefully removed from the solid medium, being careful not to incorporate any of the media. By pipetting up and down and vortex mixing, the bacterial pellet was resuspended in phosphate-buffered saline (PBS pH 7.4). To eliminate any remaining medium or debris, the cells were washed triple with PBS. The bacterial cell mass was resuspended in PBS buffer and disinfected by heat inactivation after the final washing procedures [21]. By plating the heat-inactivated extracts on a medium and growing them for 48 h at 37 °C, bacterial inactivation was confirmed twice. The protein content of bacterial product solutions was determined in serial dilutions (Bradford), then split into aliquots and refrigerated until use. The bacterial products were diluted in serum-free media to various concentrations (1, 8, 16 g/ml) and then were utilized to stimulate cells. All mentioned microbial strains stimulating cells in this study are their products instead of viable bacteria.

Plasmid and siRNA construction and transfection

The pcDNA3.1-STAT3 vectors were generated by Thermofisher (Waltham, MA USA). The pcDNA3.1-RelB vectors were purchased from VectorBuilder (Chicago, USA). ON-TARGETplus human PLA2G4A siRNA and ON-TARGETplus Non-targeting Control siRNAs were obtained from Dharmacon RNA Technologies (Lafayette, CO, USA). ON-TARGETplus human PLA2G4A siRNA is called SMARTpool including a mixture of 4 siRNA provided as a single reagent and providing advantages in both potency and specificity as the instruction shows. Transfection of the target siRNA (25 nM) and vectors was performed by using Lipofectamine LTX reagent (Invitrogen, CA, USA) based on the manufacturer’s instructions. Cells were incubated for 24 h (for mRNA analysis) or 48 h (for protein analysis).

RNA extraction, reverse transcription, and real-time polymerase chain reaction (PCR)

Total RNA was extracted using TRIzol (Invitrogen, CA, USA) following the manufacturer’s instructions. Reverse transcription PCR was performed in a Bio-Rad CFX96 real-time system machine (Bio-Rad, Hercules, CA). Complementary deoxyribonucleic acid was synthesized using PrimeScript™ RT Reagent Kit (TaKaRa, Tokyo, Japan). Real-time quantitative PCR was carried out by LightCycler 96 Probes (Startlab, UK) and with FastStart Universal SYBR Green Master Mix (Roche, CA, USA). SYBR primers used in this study are listed in Supplementary Table S1. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a house gene for normalizing data.

Western blotting analysis

Cell lysates were prepared as previously described [22]. Nuclear extraction was prepared using commercial kits from Abcam (Cambridge, UK) according to the manufacturer’s instructions. Protein samples were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (0.40 mm). The sources of primary antibodies and their dilutions are listed in Supplementary Table S2. Western blotting was conducted as previously described [22].

Immunofluorescence staining

Cells were fixed with 4% paraformaldehyde (Roth, Karlsruhe, Germany), then blocked in 5% donkey serum for 30 min before being permeabilized in PBS with 0.5% Triton X-100 for 10 min. Cells were then treated overnight at 4 °C with the primary antibodies (Supplementary Table 2) and with the fluorescent-labeled secondary antibodies for 1 h the next day. Immunofluorescence microscopy (Leica, Wetzlar, Germany) was utilized to capture images.

Luciferase reporter assays for PLA2G4A promoters

The pGL4-basic vector containing the human PLA2G4A gene proximal promoter (–2000 to + 100) and its truncated forms (–1534, − 1373, − 1252, and − 39 to + 100) were generated by Thermofisher, and its mutants (PGL4-PLA2G4A-1252MUT1, 2 and 3) were produced using their primer pairs (Supplementary Table S3). These luciferase reporter constructs (100 ng) were transiently transfected into HKCs using a Lipofectamine LTX reagent. 48 h after transfection, the cells were treated with E. coli (8 µg/ml) for 24 h. The treated cells were lysed using 1x passive lysis buffer, and the luciferase activity was measured with the dual luciferase assay kit (Promega Corp, Madison, WI). The phRL SV40 (2 ng) and the pGL4-basic vector were used as the transfection positive control and negative control, respectively.

Chromatin immunoprecipitation (ChIP) assays

ChIP assays were performed using a commercial ChIP Assay Kit (Millipore, Bedford, MA, USA) in accordance with the manufacturer’s guidelines. Soluble chromatins were prepared from cultivated THP-1 cells rather than HKCs due to their quantity limitations. Chromatin was immunoprecipitated with primary antibodies against STAT3 and RelB (Supplementary Table S2). The primer sequences and the sizes of the amplicon are listed in Supplementary Table S4.

ELISA assays

Cell culture supernatants were obtained from isolated HKCs after 24 h of stimulation with E. coli. TXA2 is extremely unstable in an aqueous solution since it is hydrated to the inactive thromboxane B2 (TXB2) within 30 s. TXB2, the stable breakdown product of TXA2, was therefore quantified in the supernatants of stimulated cells using a TXB2 ELISA kit (Cayman, MI, USA) in accordance with the manufacturer’s instructions.

Statistical analyses

All experiments were replicated at least three times. Results are presented as mean ± standard deviation (SD). Statistical comparisons among experimental groups were analyzed by the unpaired two-tailed Student’s t-test or one-way ANOVA performed using the GraphPad Prism 6 software (La Jolla, CA, USA). A P value less than 0.05 was considered significant. Significant differences among groups are indicated as *p < 0.05, **p < 0.01, and ***p < 0.001.