HBV suppresses macrophage immune responses by impairing the TCA cycle through the induction of CS/PDHC hyperacetylation

Background: It is now understood that HBV can induce innate and adaptive immune response disorders by affecting immunosuppressive macrophages, resulting in chronic HBV infection. However, the underlying mechanism is not fully understood. Dysregulated protein acetylation can reportedly influence the differentiation and functions of innate immune cells by coordinating metabolic signaling. This study aims to assess whether HBV suppresses macrophage-mediated innate immune responses by affecting protein acetylation and to elucidate the underlying mechanisms of HBV immune escape. Methods: We investigated the effect of HBV on the acetylation levels of human THP-1 macrophages and identified potential targets of acetylation that play a role in glucose metabolism. Metabolic and immune phenotypes of macrophages were analyzed using metabolomic and flow cytometry techniques. Western blot, immunoprecipitation, and immunofluorescence were performed to measure the interactions between deacetylase and acetylated targets. Chronic HBV persistent infected mice were established to evaluate the role of activating the tricarboxylic acid (TCA) cycle in macrophages for HBV clearance. Results: Citrate synthase/pyruvate dehydrogenase complex hyperacetylation in macrophages after HBV stimulation inhibited their enzymatic activities and was associated with impaired TCA cycle and M2-like polarization. HBV downregulated Sirtuin 3 (SIRT3) expression in macrophages by means of the toll-like receptor 2 (TLR2)-NF-κB- peroxisome proliferatoractivated receptor γ coactivator 1α (PGC-1α) axis, resulting in citrate synthase/pyruvate dehydrogenase complex hyperacetylation. In vivo administration of the TCA cycle agonist dichloroacetate inhibited macrophage M2-like polarization and effectively reduced the number of serum HBV DNA copies. Conclusions: HBV-induced citrate synthase/pyruvate dehydrogenase complex hyperacetylation negatively modulates the innate immune response by impairing the TCA cycle of macrophages. This mechanism represents a potential therapeutic target for controlling HBV infection.

Purified PBMCs were maintained in RPMI-1640 containing 10% FBS and 1% antibiotic-antimycotic supplementation.HBV stimulation or NR treatment was processed by the same method as that described for THP-1 cells.For dichloroacetate (DCA) treatment, the culture medium was removed and replaced with complete medium containing 100 μM DCA; the cells were further incubated at 37 °C for 48 h.
Murine primary liver macrophages were isolated as previously described 3 .Briefly, a mouse liver was perfused via the portal vein with 0.05% collagenase in Hank's balanced salt solution (88284, Thermo Fisher, MA, USA).Vascular clamps were applied to stop the arterial and venous blood flow when the liver was filled with collagenase solution.Next, the liver was removed and digested at 42 °C for 10 min.
The obtained suspension was resuspended in DMEM containing 5% FBS.Liver macrophages were purified from the sample via flow cytometric sorting using PE/Cy7 anti-mice F4/80 and APC/Cy7 anti-mice CD11b antibodies.
To examine T cell activation by macrophages treated with DCA, liver macrophages were isolated from HBV mice and then treated with or without DCA for 48h.T cells from spleen were directly co-cultured with macrophages above for another 6h.immunofluorescence staining were performed for macrophages (Anti-Mouse MHC Class II (I-A/I-E), 65122-1-Ig, Proteintech, Wuhan, China) and flow cytometry were performed for T cells.

Targeted metabolomics analysis
The targeted metabolomics analysis was performed by the Shanghai Applied Protein Technology Company (Shanghai, China).For metabolite extraction, cells were collected and washed; the extract solution (acetonitrile: methanol: water = 2:2:1) was then added to each sample and vortexed for 60 s.Thereafter, the samples were kept in an ice-water bath and sonicated twice for 5 min each.Samples were stored at −20 °C for 1 h and centrifuged at 4 200 ×g for 20 min at 4 °C.The supernatant was collected and freeze-dried.
For liquid chromatography-mass spectrometry (LC-MS) analysis, HPLC separation was carried out using an Agilent 1290 series HPLC system (Agilent, CA, USA).The

Analysis of gene expression profiles
Gene expression profiles of liver macrophages from mice with or without HBV infection were downloaded from the GEO database (accession: GSE165250) (https://www.ncbi.nlm.nih.gov/geo/).Differentially expressed genes between the different groups were identified using two-group comparisons.An adjusted P < 0.01 was set as the cut-off criterion.

Tissue section staining
Paraffin-embedded tissue sections were dewaxed, rehydrated and prepared for immunofluorescence, immunohistochemistry, and Sirius red staining, respectively.
Immunofluorescence staining: Antigen retrieval was performed by microwaving the sections in sodium citrate buffer (pH 6.0) for 15 min; endogenous peroxidase was blocked with 0.3% hydrogen peroxide in phosphate-buffered saline (PBS) for 5 min.
Immunohistochemistry staining: Following antigen retrieval and endogenous peroxidase blockage, sections were incubated with primary antibodies against HBsAg (1: 200) overnight at 4 °C, followed by incubation with secondary HRP-conjugated goat anti-mouse IgG antibody (1: 500) for 1 h at room temperature.Color development was performed using the DAB chromogen kit (DA1016, Solarbio, Beijing, China), and counterstained with hematoxylin.
Sirius red staining: Sections were incubated with Sirius red staining solution (S8060, Solarbio, Beijing, China) for 8 min at room temperature and mounted with a neutral resin.The immunohistochemistry and Sirius red staining images were obtained using a DP70 digital microscope camera attached to a IX71 inverted microscope (Olympus, Tokyo, Japan).
GST pull-down assays were performed using a GST-tag protein purification kit (IK-2004, Biolinkedin, Shanghai, China) according to the manufacturer's instruction.
In brief, His-tag CS protein (RPB661Hu01, Cloud-Clone, TX, USA) or His-tag PDHC protein (RPH426Hu01, Cloud-Clone, TX, USA) was incubated with GST-tag SIRT3 protein (RPE913Hu01, Cloud-Clone, TX, USA) or GST control protein (HY-P70270, MedChemExpress, NJ, USA) in binding buffer containing glutathione-agarose beads at room temperature for 2 h with rotation.The bound protein was then wash and immunoblotted with the indicated antibody.

RT-qPCR
Total RNA was extracted using the EasyPure® RNA Kit (ER101-01, TransGen Biotech, Beijing, China), and cDNA was synthesized using the TransScript® mobile phase consisted of 25 mmol/L ammonium acetate and 25 mmol/L ammonia hydroxide in water (pH = 9.75) (A) and acetonitrile (B).The elution gradient was as follows: 0-18.0 min, 90%-40% B; 18.0-18.1 min, 40%-90% B; 18.1-23 min, 90% B.The temperature of the column was maintained at 45 °C.The autosampler temperature was 4 °C, and the injection volume was 2 μL.The repeatability and stability of the system was evaluated by setting the quality control sample-prepared by mixing an equal aliquot of the supernatants from all samples-and injecting into the system every 6 samples throughout the sample cohort.The AB SCIEX QTRAP 5500 (Sciex Applied Biosystems, ON, Canada) was used to acquire mass spectrometry data in negative ion mode during the LC/MS experiment.The ESI source conditions were set as follows: source temperature, 450 °C; ion source Gas1 (Gas1), 45; ion source gas 2 (Gas2), 45; curtain gas (CUR), 30; ion Sapary Voltage Floating (ISVF)-4500 V.The multiple reaction monitoring mode was used for the measurement of ion pairs.Peak chromatographic area and retention time were analyzed with Multiquant software, and the metabolites were accurately identified by correcting the retention times of each sample with those of known metabolite standards.