Deletion of TP signaling in macrophages delays liver repair following APAP-induced liver injury by reducing accumulation of reparative macrophage and production of HGF

Deletion of TP signaling in macrophages delays liver repair following APAP-induced liver injury by reducing accumulation of reparative macrophage and production of HGF | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Deletion of TP signaling in macrophages delays liver repair following APAP-induced liver injury by reducing accumulation of reparative macrophage and production of HGF Mina Tanabe, Kanako Hosono, Atsushi Yamashita, Yoshiya Ito, Masataka Majima, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4078778/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Oct, 2024 Read the published version in Inflammation and Regeneration → Version 1 posted 5 You are reading this latest preprint version Abstract Background Acetaminophen (APAP)-induced liver injury is the most common cause of acute liver failure. Macrophages are key players in liver restoration following APAP-induced liver injury. Thromboxane A 2 (TXA 2 ) and its receptor, thromboxane prostanoid (TP) receptor, have been shown to be involved in tissue repair. However, whether TP signaling plays a role in liver repair after APAP hepatotoxicity by affecting macrophage function remains unclear. Methods Male TP knockout ( TP −/− ) and C57BL/6 wild-type (WT) mice were treated with APAP (300 mg/kg). In addition, macrophage-specific TP-knockout ( TP △mac ) and control WT mice were treated with APAP. We explored changes in liver inflammation, liver repair, and macrophage accumulation in mice treated with APAP. Results Compared with WT mice, TP −/− mice showed aggravated liver injury as indicated by increased levels of alanine transaminase (ALT) and necrotic area as well as delayed liver repair as indicated by decreased expression of proliferating cell nuclear antigen (PCNA). Macrophage deletion exacerbated APAP-induced liver injury and impaired liver repair. Transplantation of TP -deficient bone marrow (BM) cells to WT or TP −/− mice aggravated APAP hepatotoxicity with suppressed accumulation of macrophages, while transplantation of WT-BM cells to WT or TP −/− mice attenuated APAP-induced liver injury with accumulation of macrophages in the injured regions. Macrophage-specific TP −/− mice exacerbated liver injury and delayed liver repair, which was associated with increased pro-inflammatory macrophages and decreased reparative macrophages and hepatocyte growth factor (HGF) expression. HGF treatment mitigated APAP-induced inflammation and promoted liver repair after APAP-induced liver injury. Conclusions Deletion of TP signaling in macrophages delays liver repair following APAP-induced liver injury, which is associated with reduced accumulation of reparative macrophages and the hepatotrophic factor HGF. Specific activation of TP signaling in macrophages may be a potential therapeutic target for liver repair and regeneration after APAP hepatotoxicity. Acetaminophen liver repair thromboxane macrophage Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Acetaminophen (APAP; N-acetyl-p-aminophenol) is a widely used analgesic and antipyretic drug that is considered safe at therapeutic doses. However, APAP overdose induces severe acute liver injury that progresses to acute liver failure [ 1 ]. APAP overdose generates the toxic metabolite N-acetyl-p-benzoquinone imine (NAPQI) in hepatocytes in the centrilobular region of the liver. Excessive NAPQI formation causes mitochondrial damage in hepatocytes and subsequently induces mitochondrial oxidative stress [ 2 ]. The initial NAPQI-induced direct hepatocyte damage results in the release of damage-associated molecular patterns that trigger a sterile inflammatory response [ 3 ]. This response includes the activation of cytokines and the formation of chemokines for the infiltration of immune cells in regions of hepatocyte damage, leading to further aggravation of liver injury in the early phase of APAP toxicity [ 4 , 5 ]. APAP-induced acute liver injury initiates a regenerative response; however, impairment of the resolution of liver inflammation and liver repair induces persistent liver injury, leading to acute liver failure. Aggravation of acute liver injury and inadequate liver recovery and repair cause high mortality in patients with APAP hepatotoxicity. Liver macrophages are important for the resolution of liver damage and recovery from APAP-induced liver injury [ 6 ]. The infiltrating monocytes in the damaged lesions differentiate into monocyte-derived macrophages (MoMFs). MoMFs are divided into two main subpopulations based on their Ly6C expression levels: Ly6C high and Ly6C low . The recruited monocytes expressing Ly6C high differentiate into pro-inflammatory Ly6C high MoMFs (Ly6C high macrophages), and Ly6C high macrophages show pro-inflammatory properties. With the cessation of the inflammatory response, Ly6C high macrophages differentiate into Ly6C low macrophages, which have reparative properties [ 7 ]. The macrophage phenotypic shift from a pro-inflammatory to a reparative phenotype at the site of injury is critical [ 8 ] because Ly6C low reparative macrophages contribute to the resolution of liver inflammation and restoration of damaged tissues induced by APAP overdose [ 9 ]. However, the underlying mechanisms by which liver macrophages contribute to liver repair and regeneration following APAP-induced liver injury remain unknown. Thromboxane (TX) is an arachidonic acid metabolite and a representative prostanoid. TXA 2 is produced by the action of cyclooxygenase and TX synthase (TXS) and exerts its activity via the thromboxane prostanoid (TP) receptor [ 10 ]. TXA 2 /TP signaling plays an important role in platelet aggregation and smooth muscle contraction. As such, TXA 2 is involved in vascular diseases of the heart and brain via thrombosis formation and in bronchial asthma via constriction of the bronchial smooth muscle [ 10 ]. In the liver, TXA 2 /TP signaling contributes to injury elicited by ischemia/reperfusion and endotoxins [ 11 , 12 ]. However, recent evidence indicates that TP signaling promotes tissue repair by stimulating angiogenesis in gastric ulcers [ 13 ] and ischemic hind limbs [ 14 ]. TP signaling is also involved in the resolution of inflammation by enhancing lymphatic drainage through newly formed lymphatic vessels in the mouse tail during secondary lymphedema [ 15 ] and in the diaphragm during chronic peritonitis [ 16 ]. Furthermore, TP signaling plays a role in liver repair after carbon tetrachloride-induced liver injury [ 17 , 18 ]. These findings suggest that TXA 2 /TP signaling promotes liver tissue repair following APAP hepatotoxicity by affecting macrophage function. Therefore, the present study aimed to examine whether TP signaling in macrophages plays a role in liver repair following APAP-induced liver injury in mice. Methods Animals Male thromboxane receptor knockout ( TP −/− ) mice (8 weeks old) were generated as described previously [ 19 ]. Eight-week-old male C57BL/6 wild-type (WT) mice were purchased from CLEA Japan (Tokyo, Japan). TP -floxed mice were generated as previously described [ 16 ]. Mice with Tp deletions in myeloid cells (C57BL/6 background) were generated by crossing TP -floxed homozygous mice with LysM-Cre mice. In this study, LysMCre- Tp -floxed mice were referred to as TP △mac mice, whereas littermate control mice were referred to as control mice. Mice ubiquitously expressing green fluorescent protein (GFP) were kindly provided by Dr. Okabe (Genome Information Research Center, Osaka University, Osaka, Japan). All mice were maintained under controlled humidity (50% ± 5%) and temperature (25°C ± 1°C) with a standard light/dark cycle of 12/12 h and were given ad libitum access to food and water. The experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the Kitasato University School of Medicine (Approval no. 2023-062). All experimental studies were performed in accordance with the institutional guidelines for animal experimentation, based on the Guidelines for Proper Conduct of Animal Experiments published by the Science Council of Japan. Animal procedures The animals were fasted overnight and injected intraperitoneally (i.p.) with 300 mg/kg APAP (Sigma-Aldrich, St. Louis, MO, USA) dissolved in warm pyrogen-free saline (final concentration, 20 mg/mL). At the indicated times, the mice were anesthetized with isoflurane (Pfizer, Manhattan, NY, USA), and blood was drawn from the heart. Liver tissues were collected, a small section of each liver was placed in 10% formaldehyde, and the remainder was immersed in RNAiso Plus reagent (Takara Bio, Shiga, Japan) for further analysis. Following this procedure, the animals were euthanized by cervical dislocation, and death was verified by the lack of heartbeat, respiration, and corneal reflex. Liver injury was determined by measuring serum alanine aminotransferase (ALT) using a Dri-Chem 7000 Chemistry Analyzer System (Fujifilm, Tokyo, Japan). Treatments Ozagrel sodium (Takata Pharmaceutical Co., Saitama, Japan), a TXS inhibitor, or vehicle (phosphate-buffered saline [PBS]) was administered (150 mg/kg, i.p.) [ 13 ] 2 h after APAP treatment. ANG-3777 and HGF mimetics (Selleck Houston, TX, USA) (50 mg/kg) [ 20 ] were dissolved in 0.2 mL PBS containing 4% dimethyl sulfoxide and intraperitoneally administered 2 h and 24 h after APAP administration. Clodronate Clodronate and control liposomes were purchased from FormuMax Scientific (Sunnyvale, CA, USA). Clodronate or control liposomes (100 µL/mouse) were injected intraperitoneally 48 h before APAP administration. Histology and immunohistochemistry Excised liver tissues were fixed immediately with buffered 10% formaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). Sections (3.5 µm thick) were prepared from paraffin-embedded tissues and subjected to either hematoxylin and eosin (H&E) staining or immunostaining. Images of the H&E-stained sections were captured using a microscope (Biozero BZ-700 Series; Keyence, Osaka, Japan). The level of necrosis (as a percentage of the total area) was determined in five fields (100×) from each animal by measuring the necrotic area relative to the entire histological section using the ImageJ software version 1.50i (National Institutes of Health, Bethesda, MD, USA). The results are expressed as percentages. Proliferating cell nuclear antigen (PCNA) With regard to PCNA immunohistochemistry, liver sections were stained with a rabbit monoclonal anti-PCNA antibody (Thermo Fisher Scientific, Waltham, MA, USA) at a dilution of 1:200. Immune complexes were detected using Histofine Simple Stain MAX PO (MULTI) (Nichirei, Tokyo, Japan). Images of stained liver sections were captured using a microscope (Biozero BZ-700 Series). The number of PCNA + hepatocytes was counted in five fields (200×) per animal using the ImageJ software. The percentage of PCNA + hepatocytes was then calculated, and the results were expressed as percentages. Immunofluorescence staining Liver tissues from APAP-treated mice were fixed in 4% periodate-lysine-paraformaldehyde overnight at 4°C, transferred to 30% sucrose prepared in 0.1 M phosphate buffer (pH 7.2), and stored at 4°C for 3 days, followed by mounting the liver tissue in Tissue-Tek O.C.T. Compound (Sakura Finetek USA, Inc., Torrance, CA, USA) and stored at − 20°C. Liver tissue sections (8-µm-thick) were cut and blocked with 1% bovine serum albumin in 0.5% Triton X-100 in PBS. The sections were incubated with a rabbit anti-mouse TP (1:100; Cayman Chemical, Ann Arbor, MI, USA), a rabbit anti-mouse TXS(1:100; Bioss, Woburn, MA, USA), a rat anti-mouse CD68 antibody (1:100; Bio-Rad Laboratories, Hercules, CA, USA), a rat anti-mouse CD41 (BioRad Laboratories), and a rabbit anti-mouse HGF (1:100; Proteintech Group, Rosemont, Il, US,) at 4°C overnight. The sections were then incubated with the following secondary antibodies at 4°C for 1 h: Alexa Fluor 488-conjugated donkey anti-rabbit IgG, Alexa Fluor 594-conjugated donkey anti-rabbit IgG, or Alexa Fluor 647-conjugated donkey anti-rabbit IgG (Molecular Probes, Eugene, OR, USA). Nuclei were stained with 4'-6-diamidino-2-phenylindole (DAPI). Images were obtained using a fluorescence microscope (Biozero BZ-700 Series; Keyence, Osaka, Japan). After labeling, five optical fields (×200) per animal were randomly selected, and the number of positive cells was counted. Bone marrow (BM) transplantation BM cells were obtained by flushing the cavities of freshly dissected femurs and tibiae from donor mice (7 weeks old) using PBS. The flushed BM cells from each donor were dispersed by pipetting and resuspended in PBS at a density of 1 × 10 7 cells/mL. Mice were lethally irradiated (9.0 Gy) using an MBR-1505R X-ray irradiator (Hitachi Medico Co., Tokyo, Japan) with a filter (copper, 0.5 mm; aluminum, 2 mm) to monitor the cumulative radiation dose. Donor BM mononuclear cells (1 × 10 6 ) in 200 µL of PBS were transplanted into the tail veins of irradiated mice. Bone marrow transplantations were performed to generate the chimeric mice as follows: WT mice reconstituted with WT BMMs (WT→WT) or TP −/− BMMs ( TP −/− →WT) and TP −/− mice reconstituted with WT BMMs (WT→ TP −/− ) or TP −/− BMMs ( TP −/− → TP −/− ). In a separate experiment, donor BM cells were harvested from GFP + -WT mice, and GFP + -WT BM cells were transplanted into WT mice. The chimeras were treated with APAP for eight weeks after BM transplantation. Measurement of glutathione (GSH) and glutathione disulfide (GSSG) The frozen tissues were homogenized and centrifuged to separate the supernatants. Hepatic GSH and GSSG levels were measured using a spectrophotometric/microplate reader with a GSSG/GSH Quantification Kit (Dojindo Molecular Technologies, Kumamoto, Japan). Measurement of TXB 2 The concentrations of TXB 2 , a stable metabolite of TXA 2 , in liver tissues were measured using a Thromboxane B 2 ELISA Kit (Cayman Chemical, Ann Arbor, MI, USA). Isolation of intrahepatic leukocytes Animals were anesthetized by i.p. injection of mixed anesthetic agents containing 4.0 mg/kg midazolam (Sandoz, a Novartis division, Basel, Switzerland), 0.75 mg/kg medetomidine hydrochloride (Nippon Zenyaku Kogyo, Fukushima, Japan), and 5.0 mg/kg butorphanol (Meiji Seika Pharma, Tokyo, Japan). The liver was perfused with Hank’s balanced salt solution through the portal vein. Excised livers were incubated in Roswell Park Memorial Institute (RPMI) medium containing 0.05% collagenase (Type IV; Sigma Chemical Co., St. Louis, MO, USA) for 20 min at 37°C. The liver homogenates were filtered through a 70 µm cell strainer. Non-parenchymal cells were purified by density-gradient centrifugation on 33% Percoll (GE Healthcare Life Sciences, Piscataway, NJ, USA) as previously reported [ 21 ]. Flow cytometric analyses Isolated non-parenchymal cells were incubated with an anti-mouse CD16/32 antibody (TruStain FcX; BioLegend, San Diego, CA, USA) to block nonspecific binding of the primary mAb. Cells were stained with a combination of the following reagents: PE-conjugated anti-CD45 (30-F11; BioLegend, San Diego, CA, USA), APC/CY7-conjugated anti-Ly6G (1A8; BioLegend), PE/Cy7-conjugated anti-CD11b (M1/70; BioLegend), Brilliant Violet 510-conjugated anti-Ly6C (HK1.4; BioLegend), and anti-F4/80 (BM8; BioLegend). Cells positive for 7-aminoactinomycin D (BioLegend) were excluded from the analysis. Samples were analyzed using a FACSVerse cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Data were analyzed using the Kaluza software v2.1 (Beckman Coulter, Brea, CA, USA). We quantified and presented the number of cells normalized to liver tissue weight (cells/g). RT-qPCR analysis Total RNA was extracted from the liver tissues using RNAiso Plus (Takara Bio). cDNA was constructed with 1 µg of total RNA using the ReverTra Ace qPCR RT Kit (TOYOBO Co., Ltd., Osaka, Japan). Quantitative PCR amplification was performed using the TB Green Premix Ex Taq II (Tli RNase H Plus; Takara Bio, Inc. Shiga, Japan). PCR amplification was performed with the following conditions: 95°C for 10 s, followed by 40 cycles at 95°C for 3 s and 60°C for 20 s. The mRNA expression levels were calculated based on the comparative threshold cycle and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in each sample. The primer sequences are listed in Supplementary Table S1 . Cell preparation and culture To generate BM-derived macrophages, BM cells were isolated from the femurs and tibiae of 8-week-old male mice. BM cells were cultured in 6-well plates (1.0 × 10 6 cells/well) and maintained in RPMI 1640 medium (Gibco, Thermo Scientific, Waltham, MA, USA) containing 10% fetal calf serum and 20 ng/mL macrophage colony-stimulating factor (BioLegend, San Diego, CA, USA), as previously described [ 21 ]. On day 7, BM-derived macrophages were stimulated with lipopolysaccharide (LPS) (10 ng/mL; Sigma-Aldrich, St. Louis, MO, USA) and recombinant murine interferon-gamma (IFN-γ) (20 ng/mL; BioLegend) to polarize toward a pro-inflammatory macrophage phenotype or recombinant murine interleukin (IL)-4 (20 ng/mL; BioLegend) and to polarize toward a reparative phenotype in RPMI 1640 medium for 18 h. Cultured BM-derived macrophages were harvested and homogenized in RNAiso Plus (Takara Bio), and mRNA levels were measured using RT-qPCR. Statistical analysis All results are expressed as the mean ± standard deviation. All statistical analyses were performed using GraphPad Prism, version 8 (GraphPad Software, La Jolla, CA, USA). Data were compared between two groups using unpaired two-tailed Student’s t-tests and between multiple groups using one-way analysis of variance followed by Tukey’s post hoc tests. Statistical significance was set at P < 0.05. Results Exacerbation of APAP-induced hepatotoxicity in TP −/− mice To determine the role of TP signaling in APAP hepatotoxicity, we treated both WT and Tp −/− mice with APAP. Deletion of TP signaling rendered mice highly susceptible to APAP-induced toxicity, as evidenced by increased ALT levels and hepatic necrosis around the central veins (Fig. 1 A, B). The levels of ALT peaked at 6 h after APAP treatment in WT mice. Subsequently, ALT levels were reduced after 48 h. By contrast, the levels of ALT and hepatic necrosis remained high in TP −/− mice. These results indicate that liver injury was enhanced and liver recovery was delayed in TP −/− mice. PCR analysis showed that hepatic levels of Tp increased 6 h after APAP treatment (Fig. 1 C). Hepatic levels of Txs moderately increased in both genotypes 48 h after APAP treatment; however, there was no statistical difference in Txs mRNA levels between the two genotypes. Furthermore, the concentration of TXB 2 , a stable metabolite of TXA 2 , was increased in the livers after APAP administration in WT and TP −/− mice, and no significant difference between the two genotypes was observed (Fig. 1 D). Immunohistochemical staining for TP and TXS demonstrated that TP and TXS were expressed in CD68 + cells, suggesting that TXA 2 synthesized by macrophages bound to TP (Fig. 1 E). Because TP signaling is involved in platelet aggregation, platelet accumulation during APAP hepatotoxicity was evaluated by immunostaining platelets with an anti-CD41 antibody. Immunostaining demonstrated that CD41 + cells were extensively observed in the livers treated with APAP for 48 h; however, there was no statistical difference in the CD41 + area between the two genotypes (Supplementary Fig. 1). Involvement of macrophages in liver repair after APAP-induced liver injury Because macrophages are responsible for liver repair after APAP-induced liver injury, we assessed the accumulation of macrophages during APAP hepatotoxicity. The number of CD68 + cells (macrophages) in WT livers increased 48 h after APAP treatment, and the accumulation of CD68 + cells in WT livers was greater than that in TP -deficient livers (Fig. 2 A). These results suggest that macrophage recruitment was accompanied by liver repair after APAP-induced liver injury. To elucidate the role of macrophage accumulation in the restoration of injury to APAP hepatotoxicity, WT mice were pretreated with clodronate liposomes (CL) to delete macrophages or with control liposomes (Cont). The results showed that the levels of ALT and hepatic necrotic area in CL-treated WT mice 48 h after APAP treatment were higher than those in Cont-treated WT mice (Fig. 2 B), which was consistent with previous reports [ 22 ]. The expression of PCNA, a marker of hepatocyte proliferation, was considerably lower in the CL-treated mice than in the Cont-treated mice. These results suggest that hepatic macrophages are involved in the resolution of liver inflammation and repair after APAP treatment. Deletion of TP signaling in BM-derived macrophages delayed liver repair To examine whether the macrophages accumulated in the injured regions were derived from the BM, we generated chimeric WT mice bearing GFP + BM cells. Immunofluorescence demonstrated that accumulated GFP + cells in the injured regions colocalized with CD68 + cells 48 h after APAP treatment (Fig. 3 A), indicating that the infiltrating macrophages were derived from the BM. To further define the role of TP-expressing BM cells in liver repair after APAP treatment, we performed BM transplantation and assessed ALT levels and hepatic necrosis in response to APAP treatment. Reconstitution of WT mice with TP -deficient BM ( TP −/− →WT mice) resulted in enhancement of APAP-induced liver injury, as indicated by increased levels of ALT (Fig. 3 B) and hepatic necrosis area (Fig. 3 C). The magnitude of liver injury in WT mice transplanted with TP -deficient BM was equivalent to that in TP −/− mice transplanted with TP -deficient BM ( TP −/− → TP −/− mice). By contrast, the levels of ALT and hepatic necrotic area in WT→WT and WT→ TP −/− mice were lower than those in TP −/− →WT and TP −/− → TP −/− mice. The mRNA levels of pro-inflammatory cytokines including Il1b and Il6 in WT→WT and WT→ TP −/− mice were lower than those in TP −/− →WT and TP −/− → TP −/− mice (Fig. 3 D). With respect to liver repair, we determined PCNA expression in hepatocytes (Fig. 3 E). WT→WT and WT→ TP −/− mice exhibited enhanced PCNA expression, while TP −/− →WT and TP −/− → TP −/− mice exhibited reduced PCNA expression. We also determined the recruitment of macrophages to the liver. CD68 + cells in WT→WT and WT→ TP −/− mice were accumulated to the demarcated necrotic regions, while CD68 + cells in TP −/− →WT and TP −/− → TP −/− mice were distributed to the entire liver lobules and were not concentrated in the damaged regions (Fig. 3 F). The numbers of CD68 + cells in WT→WT and WT→ TP −/− mice were higher than those in TP −/− →WT and TP −/− → TP −/− mice. Together, these results suggest that TP signaling in BM cells plays a critical role in the resolution of liver inflammation and repair in APAP-induced liver injury by accumulating macrophages in injured regions. Taken together, these results indicated that the inhibition of TP signaling aggravated APAP-induced liver injury and that TP deficiency in BM cells enhanced liver injury and delayed liver repair after APAP treatment. Additionally, the impaired accumulation of macrophages in injured regions was associated with delayed liver repair following APAP-induced liver injury. Inhibition of TXS aggravated APAP-induced liver injury We further confirmed that TP signaling is involved in liver repair during APAP hepatotoxicity. The TXS inhibitor Ozagrel or vehicle (PBS) was administered 2 h after APAP treatment, and liver injury and repair were assessed 48 h after APAP treatment. Ozagrel treatment sensitized mice to APAP-induced liver injury, as indicated by increased levels of ALT and hepatic necrotic area (Supplementary Fig. 2). Macrophage-specific TP deficiency exacerbated APAP-induced liver injury To confirm that TP signaling in macrophages plays a role in liver repair after APAP treatment, APAP was administered to macrophage-specific TP deficient ( TP △mac ) or control (Controls) mice. Compared with controls, TP △mac mice exhibited sustained liver injury as indicated by increased levels of ALT (Fig. 4 A) and necrotic area (Fig. 4 B), suggesting that liver repair was delayed in TP △mac mice. In addition, the expression of PCNA in TP △mac mice was decreased at 48 h after APAP treatment as compared with controls (Fig. 4 C). Furthermore, changes in hepatic GSH levels, which detoxifies NAPQI via conjugation, were similar in the liver sections of both TP △mac mice and controls (Fig. 4 D). Both mouse models displayed rapid GSH depletion 1 h after APAP treatment, followed by gradual replenishment and recovery to pre-APAP levels within 24 h of APAP treatment. The same was true for the hepatic GSSG levels, which is a marker of oxidative stress. The similar rate of GSH depletion in the early phase of APAP hepatotoxicity between the two genotypes suggested that the difference in hepatotoxicity did not result from APAP metabolic activation. Deficiency of TP signaling in macrophages suppressed accumulation of macrophages in the injured regions To understand the contribution of TP signaling in macrophages to liver injury and repair following APAP treatment, we determined the number of macrophages. The numbers of CD68 + cells in controls was larger than that in TP △mac mice at 48 h after APAP treatment (Fig. 4 E). Immunofluorescence revealed that CD68 + cells accumulated extensively into the necrotic regions of livers from controls, whereas CD68 + cells diffusely distributed to the liver lobules from TP △mac mice (Fig. 4 E). These were accompanied by higher mRNA expression levels of Ccl2, Ccl7 , and Ccl9 in controls than those in TP △mac mice (Fig. 4 F). Loss of TP signaling in macrophages polarized macrophages toward a pro-inflammatory phenotype We further evaluated the phenotypes of infiltrated macrophages during APAP hepatotoxicity using flow cytometry. Flow cytometry analysis demonstrated that the populations of Ly6C high macrophages (pro-inflammatory macrophages) in TP △mac mice 48 h after APAP treatment were larger than those in control mice, while the populations of Ly6C low macrophages (reparative macrophages) in controls were larger than those in TP △mac mice (Fig. 5 A). These results indicated that accelerated liver repair in controls was associated with accumulation of reparative macrophages and that delayed liver repair in TP △mac mice was associated with accumulation of pro-inflammatory macrophages. We also determined the levels of pro- and anti-inflammatory mediators in the liver. The expressions of mRNA encoding pro-inflammatory mediators including Il1b and Il6 in TP △mac mice 48 h after APAP treatment were higher than those in controls (Fig. 5 B). In addition, the expressions of mRNA encoding anti-inflammatory mediators including Mr and Fizz1 in TP △mac mice were lower than those in controls. To further examine whether TP signaling in macrophages polarizes macrophages from a pro-inflammatory macrophage phenotype toward a reparative macrophage phenotype, BM-derived macrophages were stimulated using LPS/IFN-γ or IL-4. The administration of LPS/IFN-γ polarizes BM-macrophages toward a pro-inflammatory phenotype, whereas IL-4 administration polarizes BM-macrophages toward a reparative phenotype [ 21 ]. The levels of mRNA encoding Il1b and Il6 in macrophages from TP △mac mice stimulated with LPS/IFN-γ were enhanced as compared with those from controls (Fig. 5 C). On the other hand, the mRNA expression levels of Mr and Fizz1 were enhanced in macrophages from controls stimulated with IL-4 as compared with those from TP △mac mice. These results suggest that inhibition of TP signaling polarizes macrophages toward a pro-inflammatory phenotype. With respect to other immune cells, including Kuffer cells (KCs), the number of KCs in both genotypes decreased after APAP treatment (Supplementary Fig. 3). By contrast, neutrophils in controls transiently increased at 24 h, and the number of neutrophils at 24 h in controls was higher than that in TP △mac mice. At 48 h, the number of neutrophils in controls declined, whereas that in TP △mac mice was elevated. The number of neutrophils at 48 h in TP △mac mice was higher than that in controls. HGF treatment attenuated APAP-induced liver injury and enhanced liver repair To gain insight into the mechanisms by which TP signaling in macrophages facilitates liver repair after APAP-induced liver injury, we determined the hepatotrophic factor, HGF. mRNA levels of Hgf in TP △mac mice were lower than those in controls (Fig. 6 A). We also examined the localization of HGF. Immunofluorescence revealed that HGF colocalized with CD68 + cells in the injured centrilobular regions of controls 48 h after APAP treatment (Fig. 6 A). To examine the contribution of HGF to promote liver repair, TP △mac mice were given HGF 2 h and 24 h after APAP treatment. Treatment of TP △mac mice with HGF attenuated APAP-induced liver injury as indicated by reduced levels of ALT and hepatic necrotic area (Fig. 6 B) and enhanced liver repair as indicated by increased PCNA + hepatocytes (Fig. 6 C). The number of CD68 + cells in HGF-treated TP △mac mice was higher than that in vehicle-treated TP △mac mice (Fig. 6 D). The levels of mRNA for Il1b and Il6 in HGF-treated TP △mac mice were lower than those in vehicle-treated TP △mac mice, while the levels of mRNA for Mr in HGF-treated TP △mac mice were higher than those in vehicle-treated TP △mac mice (Fig. 6 E). The mRNA levels of Ccl2 , Ccl7 , and Ccl9 in HGF-treated TP △mac mice were higher than those in vehicle-treated TP △mac mice (Fig. 6 E). These results suggest that HGF, probably from macrophages, reduced liver inflammation and facilitated liver repair after APAP treatment. Discussion In the present study, we examined the role of endogenous TP signaling in liver repair following APAP-induced liver injury in mice. Our data showed that the inhibition of TP signaling or TXS aggravated APAP-induced liver inflammation and delayed liver repair following APAP-induced liver injury, which was related to the suppressed accumulation of macrophages in the injured regions. The deficiency of TP signaling in BM-derived macrophages suppresses the accumulation of macrophages in damaged areas during repair. In addition, the inhibition of TP signaling in macrophages prolonged liver inflammation and impaired liver repair, which was accompanied by reduced accumulation of reparative macrophages in the damaged area and downregulated expression of the hepatotrophic factor HGF. Treatment of mice deficient in TP signaling in macrophages with HGF mitigated liver inflammation and facilitated liver repair. These results suggest that the inhibition of TP signaling in macrophages delays liver repair after APAP hepatotoxicity by suppressing the accumulation of reparative macrophages and producing HGF. The processes of liver repair and regeneration after acute liver injury determine the outcomes of patients with APAP hepatotoxicity [ 23 , 24 ]. Although the mechanisms underlying liver repair appear to be complex [ 25 , 26 ], macrophages are key players in liver repair after acute liver injury, including APAP hepatotoxicity [ 8 ]. Macrophage recruitment is a critical event in the resolution of liver inflammation and restoration of liver function. Our data revealed an extensive accumulation of macrophages in the hepatic necrotic area during the repair phase. The recruited macrophages were derived from the BM to remove necrotic debris and injured hepatocytes. The accumulation of macrophages in injured regions is a prerequisite for proper liver repair [ 21 , 27 , 28 ]. By contrast, macrophages were evenly distributed throughout the livers from TP △mac mice and were not accumulated and concentrated in the damaged regions. These results indicate that the scattered distribution of macrophages is associated with impaired liver repair following APAP-induced acute liver injury. Furthermore, control mice had more infiltrated macrophages than TP △mac mice, suggesting that accumulation of macrophages in the necrotic area would be required for adequate removal of necrotic tissues and consequently for liver recovery and regeneration [ 21 , 29 ]. The importance of macrophages in liver repair after acute liver injury has been highlighted by the deletion of hepatic macrophages using clodronate liposomes [ 22 ]. Pre-elimination of KCs with clodronate liposomes exacerbated APAP-induced liver injury, which is consistent with the present results, demonstrating increased ALT, necrosis and reduced PCNA expression. Macrophage recruitment to injured areas is driven by chemokine production CCL2 is important for the recruitment of MoMFs to promote liver repair following APAP toxicity [ 30 ]. Consistent with this, our data showed that higher levels of CCL2 were associated with macrophage accumulation and recovery from APAP-induced liver injury. Upregulation of CCL2 expression in macrophages is dependent on TP signaling CCL2 is produced by injured hepatocytes in the centrilobular area and recruited by macrophages during APAP hepatotoxicity [ 31 , 32 ]. In addition, deletion of CCR2, a receptor for CCL2, inhibits the accumulation of macrophages and clearance of necrotic cell debris in APAP-treated livers [ 32 ], suggesting that downregulated CCR2 expression might lead to the suppression of phagocytosis of necrotic tissues in APAP-treated livers. Furthermore, a recent study reported that other chemokines, including CCL7 and CCL9, were elevated during APAP-induced liver injury [ 33 ], which is consistent with our results. Macrophages are instrumental in the repair and regeneration processes after acute liver damage [ 34 ]. Pro-inflammatory macrophages peak at 24 h after APAP treatment, and reparative macrophages peak at 48 h [ 9 ], suggesting in situ reprogramming of the macrophage phenotype [ 35 , 36 ]. The current study showed that increased numbers of reparative macrophages were associated with the promotion of liver repair, whereas increased numbers of pro-inflammatory macrophages were associated with the impairment of liver repair. These changes were associated with increased expression of genes related to the pro-inflammatory macrophage phenotype and decreased expression of genes related to the reparative macrophage phenotype in the livers of mice deficient in TP signaling. These results suggest that TP signaling in macrophages is involved in macrophage differentiation to repair damaged liver tissues exposed to APAP overdoses. Additionally, TP signaling in macrophages appears to be involved in macrophage polarization [ 15 ]; however, further studies are required to elucidate the mechanism underlying macrophage polarization via TP signaling. Cells other than MoMFs are likely involved in liver repair following APAP hepatotoxicity [ 37 ]. The current study demonstrated that APAP treatment reduced the number of liver-resident macrophages and KCs in both genotypes. Loss of KCs is a common feature of liver diseases [ 38 ] including APAP toxicity [ 9 , 27 , 33 ]. These results suggest that KCs contribute little to liver repair via TP signaling in macrophages. Neutrophils are another type of immune cells that accumulate in APAP-treated livers. However, the role of neutrophils in the APAP-induced liver injury and repair remains controversial [ 39 ]. The present study demonstrated that control mice had more hepatic neutrophils at 24 h and a smaller hepatic necrotic area at 48 h, whereas mice lacking TP in macrophages had more hepatic neutrophils and more robust hepatic necrosis at 48 h. These results suggest that the accumulation of neutrophils prior to the accumulation of reparative macrophages may induce liver repair, and excessive accumulation of neutrophils in sustained inflammatory livers impairs the resolution of inflammation and delays liver repair [ 40 ]. Thus, the role of neutrophil accumulation in APAP hepatotoxicity appears to be context-dependent. Additionally, it remains unclear why the deletion of TP signaling in macrophages prevented the accumulation of neutrophils in the injured regions induced by APAP treatment. Further studies are necessary to elucidate the underlying mechanisms that regulate the role of neutrophils in APAP hepatotoxicity and their involvement in TP signaling. Hepatic accumulation of platelets is also involved in the progression of APAP-induced liver injury and delays liver repair after APAP treatment [ 41 , 42 ]. Because platelets aggregate through TP signaling, TP signaling in platelets would participate in APAP hepatotoxicity; however, in the present study, the area of platelet did not differ between WT and TP −/− mice, suggesting that TP signaling-induced platelet accumulation did not contribute to APAP-induced liver injury and repair. These results indicate that platelet accumulation and aggregation in the liver are independent of TP signaling. Various trophic factors facilitate liver regeneration and repair after APAP toxicity [ 24 , 26 ]. We found that hepatic HGF levels in control mice were higher than those in TP △mac mice 48 h after APAP treatment, which was consistent with our previous results [ 27 ]. Furthermore, the current study demonstrated that HGF administration attenuated APAP-induced liver injury and facilitated liver repair from the injury. HGF is characterized by pro-proliferative properties. HGF deletion impairs liver regeneration in mice [ 43 ]. In contrast, recombinant HGF appears to confer protection against liver failure by boosting liver regeneration in mice [ 44 ]. Consistent with this, HGF overexpression in BM-derived mesenchymal stem cells facilitates liver repair following liver injury [ 45 ]. In addition, transplantation of HGF-knockout mesenchymal stem cells reduced the survival rate of APAP-treated mice, and HGF administration increased the survival rate during APAP hepatotoxicity, indicating that HGF improved mouse liver failure induced by APAP administration. Macrophages are one of the main sources of HGF during liver regeneration. In liver regeneration induced by partial hepatectomy in mice, accumulated reparative macrophages generate HGF, which contributes to hepatocyte proliferation [ 46 ]. The other possible origins of HGF secretion during APAP hepatotoxicity are likely located in hepatic stellate cells (HSCs) and liver sinusoidal endothelial cells (LSECs) [ 33 ]. In conclusion, our study demonstrates that TP signaling in macrophages facilitates liver repair after APAP overdose. Accumulation of TP-expressing macrophages in injured regions induced by APAP is a critical step in the resolution of liver inflammation and recovery. The activation of TP signaling in macrophages may be a therapeutic target for APAP-induced liver injury and liver recovery. Specific activation of TP signaling in macrophages may be a therapeutic target for liver repair and regeneration after APAP hepatotoxicity. Abbreviations ALT, alanine transaminase; APAP, N-acetyl-p-aminophenol; BM, bone marrow; CCR2, C-C motif chemokine receptor 2; CL, clodronate liposomes; DAPI, 4'-6-diamidino-2-phenylindole; HGF, hepatocyte growth factor; GFP, green fluorescent protein; GSH, glutathione; GSSG, glutathione disulfide; H&E, hematoxylin and eosin; HGF, hepatocyte growth factor; IFN-γ, interferon-gamma; IL, interleukin; KC, Kupffer cell; LPS, lipopolysaccharide; MoMF, monocyte-derived macrophages; NAPQI, N-acetyl-p-benzoquinone imine; PBS, phosphate-buffered saline; PCNA, proliferating cell nuclear antigen; TP, thromboxane prostanoid; TXA 2 , Thromboxane A 2 ; TP, thromboxane prostanoid; TXS, Thromboxane synthase ; WT, wild type. Declarations Acknowledgements We thank Ms. Michiko Ogino and Ms. Kyoko Yoshikawa for technical assistance. Authors’ contributions M.T. designed and performed experiments, analyzed the data, and wrote the article. K.H., A.Y., and Y.I. performed experiments and analyzed the data. M.M. provided the TP-flox mice. S.N. provided the TP-deficient mice. C.K. supervised this study. H.A. .interpreted the data, supervised this study, and edited the article.. All authors have reviewed and approved the final version of the manuscript. Funding This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (22K08856 to YI) and the Takeda Science Foundation (to KH). This study was also supported by the Integrative Research Program of the Graduate School of Medical Science at Kitasato University and the Parents’ Association Grant of Kitasato University School of Medicine. Availability of data and materials All data generated or analyzed during this study are included within the article. Declarations Ethics approval and consent to participate The experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the Kitasato University School of Medicine (Approval no. 2023-062). 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Supplementary Files Supple.doc Cite Share Download PDF Status: Published Journal Publication published 03 Oct, 2024 Read the published version in Inflammation and Regeneration → Version 1 posted Editorial decision: Major revision 30 Apr, 2024 Reviewers agreed at journal 02 Apr, 2024 Reviewers invited by journal 29 Mar, 2024 Editor assigned by journal 12 Mar, 2024 First submitted to journal 11 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4078778","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":285260546,"identity":"412fd47e-2e6d-4d74-b481-53d8037ae843","order_by":0,"name":"Mina Tanabe","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Mina","middleName":"","lastName":"Tanabe","suffix":""},{"id":285260547,"identity":"061f5171-a69d-45be-9500-4cdc75ac0d3d","order_by":1,"name":"Kanako Hosono","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Kanako","middleName":"","lastName":"Hosono","suffix":""},{"id":285260548,"identity":"1ba1a6d4-17be-4aea-ac2e-1ab44713853c","order_by":2,"name":"Atsushi Yamashita","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Atsushi","middleName":"","lastName":"Yamashita","suffix":""},{"id":285260549,"identity":"40e4fcf9-0ce1-464d-b1a1-14b89fe72b93","order_by":3,"name":"Yoshiya Ito","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yoshiya","middleName":"","lastName":"Ito","suffix":""},{"id":285260550,"identity":"eac2ca2a-ae15-416a-b890-1e41934e544d","order_by":4,"name":"Masataka Majima","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Masataka","middleName":"","lastName":"Majima","suffix":""},{"id":285260551,"identity":"20cae5cf-4b53-42a6-bc4e-2297158b53d2","order_by":5,"name":"Shuh Narumiya","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Shuh","middleName":"","lastName":"Narumiya","suffix":""},{"id":285260552,"identity":"19dfb32e-6862-47c4-81dc-cd04f6ce53f5","order_by":6,"name":"Chika Kusano","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Chika","middleName":"","lastName":"Kusano","suffix":""},{"id":285260553,"identity":"88522feb-1fba-4779-bc18-291bd94dcc4b","order_by":7,"name":"Hideki Amano","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFklEQVRIie3Rv2qDQBzA8Z8cmOXAVZf6Cj+XSCHog3T5BcFNKGQpZEhAaBYhq4VCX0GfoMKBWWyyFrro3qVbSjv0xA4JNYZupdx3Ojg+9xdApfqDIQPUljiRQ2JAx5PFEAm/SWv0cwRaAqIjcEz6c0csb9Lr3ZUxIobN3vONh7hgHDwbtKfebS5jfeZk+BLdJTUjomCaljpJEjhL2FIfQcHHVi1J9kysIGIEJXc/OMgBVHiCuO81bqPHjix8u+Qod1kMkbGWYRFlJrUHE1rWETFA9JmVYhClVRMjhZtpXoak3ePGuT11l53I35JPL1qvAmHtJ3P/QogCXm/mtmH2v9hB8nsOlpL/Y1ZnxM+M5NdEpVKp/mVfCZZf0xIl+ggAAAAASUVORK5CYII=","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Hideki","middleName":"","lastName":"Amano","suffix":""}],"badges":[],"createdAt":"2024-03-12 00:15:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4078778/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4078778/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s41232-024-00356-z","type":"published","date":"2024-10-03T15:57:28+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":54018109,"identity":"9733d84e-faa9-4ebf-ac22-f9afbfaf7f7c","added_by":"auto","created_at":"2024-04-03 12:39:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":242381,"visible":true,"origin":"","legend":"\u003cp\u003eDeletion of TP signaling exacerbates APAP-induced liver injury.\u003c/p\u003e\n\u003cp\u003e(A) ALT levels after APAP treatment in WT and \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e−/−\u003c/sup\u003e mice. (B) Representative photos of H\u0026amp;E staining of liver sections from WT and \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e −/−\u003c/sup\u003e mice after APAP treatment. The yellow dotted lines indicate areas of necrosis. CV, central vein; PV, portal vein. Scale bars: 200 μm. Hepatic necrotic area in WT and \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e −/−\u003c/sup\u003e mice after APAP treatment. (C) mRNA levels of \u003cem\u003eTp\u003c/em\u003e and \u003cem\u003eTxs\u003c/em\u003e in the livers from WT and \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e −/−\u003c/sup\u003e mice after APAP treatment. (D) TXB\u003csub\u003e2\u003c/sub\u003e levels in the livers from WT and \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e −/−\u003c/sup\u003e mice after APAP treatment. (E) Double-immunofluorescence staining for TP (red) and CD68 (green) or TXS (red) and CD68 (green). Arrowheads indicate merged cells. CV, central vein. Scale bars: 50 μm.\u003c/p\u003e\n\u003cp\u003eData are expressed as the mean ± SD. ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"OnlineAPAP300TPFig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4078778/v1/6b9a846f6c3c291f29e78482.png"},{"id":54018107,"identity":"5c6509e2-9ad9-4584-ac3c-f43275e6adf9","added_by":"auto","created_at":"2024-04-03 12:39:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":475349,"visible":true,"origin":"","legend":"\u003cp\u003eDeletion of macrophages with clodronate liposomes (CL) aggravates APAP-induced liver injury and delays liver repair.\u003c/p\u003e\n\u003cp\u003e(A) Representative microphotographs of immunostaining of CD68 (red) in the livers from WT and \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e −/−\u003c/sup\u003e mice 48 h after APAP treatment. CV, central vein. Scale bars: 100 μm. The number of CD68\u003csup\u003e+\u003c/sup\u003e cells (macrophages) in the livers from WT and \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e −/−\u003c/sup\u003e mice after APAP treatment. (B) Levels of ALT and hepatic necrotic area 48 h after APAP treatment in WT mice treated with clodronate liposomes (CL) or control liposomes (Cont). Representative images of H\u0026amp;E staining of WT mice treated with CL or Cont. CV, central vein; PV, portal vein. Scale bars: 200 μm. (C) PCNA\u003csup\u003e+\u003c/sup\u003e hepatocytes (%) 48 h after APAP treatment in WT mice treated with CL or Cont. Representative images showing immunohistochemical staining for PCNA in WT mice treated with CL or Cont. PV, portal vein. Scale bars: 100 μm. Data are expressed as the mean ± SD. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"OnlineAPAP300TPFig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4078778/v1/22024b4696c5fcb4e1a6ee25.png"},{"id":54018108,"identity":"2345113c-2c71-407e-9882-703a5d0f98de","added_by":"auto","created_at":"2024-04-03 12:39:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":328859,"visible":true,"origin":"","legend":"\u003cp\u003eTP signaling deficiency in bone marrow cells exacerbates APAP-induced liver injury and delays liver repair.\u003c/p\u003e\n\u003cp\u003e(A) Representative immunofluorescence images of GFP (green) and CD68 (red) in the liver of WT mice bearing GFP\u003csup\u003e+\u003c/sup\u003e bone marrow (BM) cells 48 h after APAP treatment. Yellow arrows indicate double-positive cells. The nuclei were stained with DAPI (blue). CV, central vein. Scale bar: 100 μm. (B) ALT levels 48 h after APAP treatment in BM chimeric mice. (C) Representative images showing H\u0026amp;E staining of liver sections. CV, central vein; PV, portal vein. Scale bars: 200 μm. Hepatic necrotic areas 48 h after APAP treatment in BM chimeric mice. (D) Expression of mRNA encoding \u003cem\u003eIl1b\u003c/em\u003e and \u003cem\u003eIl6\u003c/em\u003e in the liver 48 h after APAP treatment in BM chimeric mice. (E) Representative images showing immunohistochemical staining of PCNA at 48 h after APAP treatment in BM chimeric mice. CV, central vein; PV, portal vein. Scale bars: 100 μm. Percentage of PCNA\u003csup\u003e+ \u003c/sup\u003ehepatocytes in BM chimeric mice. (F) Representative images showing immunofluorescence staining for CD68 48 h after APAP treatment in BM chimeric mice. CV, central vein; PV, portal vein. Scale bars: 100 μm. Number of CD68\u003csup\u003e+ \u003c/sup\u003emacrophages in the BM of chimeric mice. Data are expressed as the mean ± SD. *\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"OnlineAPAP300TPFig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4078778/v1/02b226312c83b9e9ab8fffae.png"},{"id":54018110,"identity":"bf42ae47-17e4-4ece-8dbd-a4904ee2dc8b","added_by":"auto","created_at":"2024-04-03 12:39:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":407315,"visible":true,"origin":"","legend":"\u003cp\u003eDeletion of TP signaling in macrophages delays liver repair after APAP treatment.\u003c/p\u003e\n\u003cp\u003e(A) ALT levels after APAP treatment in Control and \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice (B) Representative photos of H\u0026amp;E staining of liver sections from Control and \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice after APAP treatment. The yellow dotted lines indicate areas of necrosis. CV, central vein; PV, portal vein. Scale bars: 200 μm. Hepatic necrotic area (%) after APAP treatment. (C) Representative images of PCNA immunostaining of liver sections from Control and \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice 48 h after APAP treatment. Percentage of PCNA\u003csup\u003e+\u003c/sup\u003e hepatocytes 48 h after APAP treatment. PV, portal vein. Scale bars: 100 μm. (D) GSH and GSSG concentrations in the livers of Control and \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice after APAP treatment. (E) Immunofluorescence of CD68 (red) in the livers of Control and \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice 48 h after APAP treatment. CV, central vein; PV, portal vein. Scale bars: 100 μm. The numbers of CD68\u003csup\u003e+\u003c/sup\u003e macrophages in livers from Control and \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice after APAP treatment. (F) Expression of genes encoding \u003cem\u003eCcl2\u003c/em\u003e, \u003cem\u003eCcl7\u003c/em\u003e, and \u003cem\u003eCcl9\u003c/em\u003e in the livers of Control and \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice 0 h and 48 h after APAP treatment.\u003c/p\u003e\n\u003cp\u003eData are expressed as the mean ± SD. ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"OnlineAPAP300TPFig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4078778/v1/51d4b5eb8ea1543a4b4ca3a5.png"},{"id":54018113,"identity":"8148df9a-824f-4861-aac9-7c0f3a0814af","added_by":"auto","created_at":"2024-04-03 12:39:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":179909,"visible":true,"origin":"","legend":"\u003cp\u003eDeletion of TP signaling in macrophages polarizes them toward a proinflammatory macrophage phenotype.\u003c/p\u003e\n\u003cp\u003e(A) Representative dot plots of liver pro-inflammatory and reparative macrophages in Control and \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice 0 h, 24 h, and 48 h after APAP treatment. After gating out the Ly6G\u003csup\u003e+\u003c/sup\u003e and CD11b\u003csup\u003e+\u003c/sup\u003e cells, the cells were separated into two subsets based on the expression of Ly6C and F4/80. Ly6G\u003csup\u003e-\u003c/sup\u003e/Ly6C\u003csup\u003ehigh\u003c/sup\u003e/F4/80\u003csup\u003eint\u003c/sup\u003e cells were defined as Ly6C\u003csup\u003ehigh\u003c/sup\u003e macrophages (pro-inflammatory macrophages), and Ly6G\u003csup\u003e-\u003c/sup\u003e/Ly6C\u003csup\u003elow\u003c/sup\u003e/F4/80\u003csup\u003eint\u003c/sup\u003e cells were defined as Ly6C\u003csup\u003elow\u003c/sup\u003e macrophages (reparative macrophages). Changes in the numbers of Ly6C\u003csup\u003ehigh\u003c/sup\u003e macrophages and Ly6C\u003csup\u003elow\u003c/sup\u003e macrophages in livers from Control and \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice after APAP treatment are demonstrated. (B) Expression of mRNA encoding genes related to a pro-inflammatory macrophage phenotype (\u003cem\u003eIl1b and Il6\u003c/em\u003e) and genes related to a reparative macrophage phenotype (\u003cem\u003eMr \u003c/em\u003eand\u003cem\u003e Fizz1\u003c/em\u003e) in livers from Control and \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice 0 h and 48 h after APAP treatment. (C) Expression of mRNA encoding genes related to a pro-inflammatory macrophage phenotype (\u003cem\u003eIl1b and Il6\u003c/em\u003e) and genes related to a reparative macrophage phenotype (\u003cem\u003eMr \u003c/em\u003eand\u003cem\u003e Fizz1\u003c/em\u003e) in cultured BM-derived macrophages stimulated with LPS/IFN-γ or IL-4.\u003c/p\u003e\n\u003cp\u003eData are expressed as the mean ± SD. *\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"OnlineAPAP300TPFig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4078778/v1/837a27a30ded4670b611cf50.png"},{"id":54018111,"identity":"e34f5139-3580-4f9b-bb45-db1a378c3978","added_by":"auto","created_at":"2024-04-03 12:39:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":318694,"visible":true,"origin":"","legend":"\u003cp\u003eHGF administration reduced APAP-induced liver injury and facilitated liver repair.\u003c/p\u003e\n\u003cp\u003e(A) Expression of mRNA encoding genes \u003cem\u003eHgf\u003c/em\u003e in the livers of \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e or Control mice 0 h and 48 h after APAP treatment. Double immunofluorescence of CD68 (red) and HGF (green) in Control livers at 48 h. Arrowheads indicate double-positive cells. Scale bar: 50 μm. (B) ALT levels and hepatic necrotic area (%) in \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice treated with HGF or vehicle at 48 h. Representative images of H\u0026amp;E staining of liver sections from \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice treated with HGF or vehicle at 48 h. CV, central vein; PV, portal vein. Scale bars: 200 μm. (C) Representative images of PCNA staining of liver sections from \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice treated with HGF or vehicle at 48 h. CV, central vein; PV, portal vein. Scale bars: 100 μm. PCNA\u003csup\u003e+ \u003c/sup\u003ehepatocytes (%) in \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice treated with HGF or vehicle. (D) Representative immunofluorescence images of CD68 from \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice treated with HGF or vehicle at 48 h. CV, central vein; PV, portal vein. Scale bars: 100 μm. CD68\u003csup\u003e+ \u003c/sup\u003ecells in \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice treated with HGF or vehicle. (E) Expression of mRNA-encoding genes related to a pro-inflammatory macrophage phenotype (\u003cem\u003eIl1b \u003c/em\u003eand\u003cem\u003e Il6\u003c/em\u003e), genes related to a reparative macrophage phenotype (\u003cem\u003eMr \u003c/em\u003eand \u003cem\u003eFizz1\u003c/em\u003e), and genes related to chemokines (\u003cem\u003eCcl2, Ccl7\u003c/em\u003e, and \u003cem\u003eCcl9\u003c/em\u003e) in the livers of \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice treated with HGF or vehicle 48 h after APAP treatment.\u003c/p\u003e\n\u003cp\u003eData are expressed as the mean ± SD. *\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.05, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"OnlineAPAP300TPFig6.png","url":"https://assets-eu.researchsquare.com/files/rs-4078778/v1/87ab404f7e053624355c0bee.png"},{"id":66096795,"identity":"12905d3a-1266-4cf5-90f1-df290376d9c9","added_by":"auto","created_at":"2024-10-07 16:10:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3431732,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4078778/v1/0bf2fe5e-48e8-43fb-a896-8c8d3d718d13.pdf"},{"id":54018112,"identity":"230cbe20-113d-4a94-8063-6bc025d5c93b","added_by":"auto","created_at":"2024-04-03 12:39:46","extension":"doc","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":574968,"visible":true,"origin":"","legend":"","description":"","filename":"Supple.doc","url":"https://assets-eu.researchsquare.com/files/rs-4078778/v1/d835eeaee8c0272404b1a8be.doc"}],"financialInterests":"","formattedTitle":"Deletion of TP signaling in macrophages delays liver repair following APAP-induced liver injury by reducing accumulation of reparative macrophage and production of HGF","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAcetaminophen (APAP; N-acetyl-p-aminophenol) is a widely used analgesic and antipyretic drug that is considered safe at therapeutic doses. However, APAP overdose induces severe acute liver injury that progresses to acute liver failure [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. APAP overdose generates the toxic metabolite N-acetyl-p-benzoquinone imine (NAPQI) in hepatocytes in the centrilobular region of the liver. Excessive NAPQI formation causes mitochondrial damage in hepatocytes and subsequently induces mitochondrial oxidative stress [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The initial NAPQI-induced direct hepatocyte damage results in the release of damage-associated molecular patterns that trigger a sterile inflammatory response [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This response includes the activation of cytokines and the formation of chemokines for the infiltration of immune cells in regions of hepatocyte damage, leading to further aggravation of liver injury in the early phase of APAP toxicity [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. APAP-induced acute liver injury initiates a regenerative response; however, impairment of the resolution of liver inflammation and liver repair induces persistent liver injury, leading to acute liver failure. Aggravation of acute liver injury and inadequate liver recovery and repair cause high mortality in patients with APAP hepatotoxicity.\u003c/p\u003e \u003cp\u003eLiver macrophages are important for the resolution of liver damage and recovery from APAP-induced liver injury [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The infiltrating monocytes in the damaged lesions differentiate into monocyte-derived macrophages (MoMFs). MoMFs are divided into two main subpopulations based on their Ly6C expression levels: Ly6C\u003csup\u003ehigh\u003c/sup\u003e and Ly6C\u003csup\u003elow\u003c/sup\u003e. The recruited monocytes expressing Ly6C\u003csup\u003ehigh\u003c/sup\u003e differentiate into pro-inflammatory Ly6C\u003csup\u003ehigh\u003c/sup\u003e MoMFs (Ly6C\u003csup\u003ehigh\u003c/sup\u003e macrophages), and Ly6C\u003csup\u003ehigh\u003c/sup\u003e macrophages show pro-inflammatory properties. With the cessation of the inflammatory response, Ly6C\u003csup\u003ehigh\u003c/sup\u003e macrophages differentiate into Ly6C\u003csup\u003elow\u003c/sup\u003e macrophages, which have reparative properties [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The macrophage phenotypic shift from a pro-inflammatory to a reparative phenotype at the site of injury is critical [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] because Ly6C\u003csup\u003elow\u003c/sup\u003e reparative macrophages contribute to the resolution of liver inflammation and restoration of damaged tissues induced by APAP overdose [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, the underlying mechanisms by which liver macrophages contribute to liver repair and regeneration following APAP-induced liver injury remain unknown.\u003c/p\u003e \u003cp\u003eThromboxane (TX) is an arachidonic acid metabolite and a representative prostanoid. TXA\u003csub\u003e2\u003c/sub\u003e is produced by the action of cyclooxygenase and TX synthase (TXS) and exerts its activity via the thromboxane prostanoid (TP) receptor [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. TXA\u003csub\u003e2\u003c/sub\u003e/TP signaling plays an important role in platelet aggregation and smooth muscle contraction. As such, TXA\u003csub\u003e2\u003c/sub\u003e is involved in vascular diseases of the heart and brain via thrombosis formation and in bronchial asthma via constriction of the bronchial smooth muscle [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In the liver, TXA\u003csub\u003e2\u003c/sub\u003e/TP signaling contributes to injury elicited by ischemia/reperfusion and endotoxins [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, recent evidence indicates that TP signaling promotes tissue repair by stimulating angiogenesis in gastric ulcers [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and ischemic hind limbs [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. TP signaling is also involved in the resolution of inflammation by enhancing lymphatic drainage through newly formed lymphatic vessels in the mouse tail during secondary lymphedema [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and in the diaphragm during chronic peritonitis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Furthermore, TP signaling plays a role in liver repair after carbon tetrachloride-induced liver injury [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. These findings suggest that TXA\u003csub\u003e2\u003c/sub\u003e/TP signaling promotes liver tissue repair following APAP hepatotoxicity by affecting macrophage function.\u003c/p\u003e \u003cp\u003eTherefore, the present study aimed to examine whether TP signaling in macrophages plays a role in liver repair following APAP-induced liver injury in mice.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eMale thromboxane receptor knockout (\u003cem\u003eTP\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e) mice (8 weeks old) were generated as described previously [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Eight-week-old male C57BL/6 wild-type (WT) mice were purchased from CLEA Japan (Tokyo, Japan). \u003cem\u003eTP\u003c/em\u003e-floxed mice were generated as previously described [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Mice with \u003cem\u003eTp\u003c/em\u003e deletions in myeloid cells (C57BL/6 background) were generated by crossing \u003cem\u003eTP\u003c/em\u003e-floxed homozygous mice with LysM-Cre mice. In this study, LysMCre-\u003cem\u003eTp\u003c/em\u003e-floxed mice were referred to as \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice, whereas littermate control mice were referred to as control mice. Mice ubiquitously expressing green fluorescent protein (GFP) were kindly provided by Dr. Okabe (Genome Information Research Center, Osaka University, Osaka, Japan). All mice were maintained under controlled humidity (50% \u0026plusmn; 5%) and temperature (25\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C) with a standard light/dark cycle of 12/12 h and were given \u003cem\u003ead libitum\u003c/em\u003e access to food and water.\u003c/p\u003e \u003cp\u003e The experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the Kitasato University School of Medicine (Approval no. 2023-062). All experimental studies were performed in accordance with the institutional guidelines for animal experimentation, based on the Guidelines for Proper Conduct of Animal Experiments published by the Science Council of Japan.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAnimal procedures\u003c/h2\u003e \u003cp\u003eThe animals were fasted overnight and injected intraperitoneally (i.p.) with 300 mg/kg APAP (Sigma-Aldrich, St. Louis, MO, USA) dissolved in warm pyrogen-free saline (final concentration, 20 mg/mL). At the indicated times, the mice were anesthetized with isoflurane (Pfizer, Manhattan, NY, USA), and blood was drawn from the heart. Liver tissues were collected, a small section of each liver was placed in 10% formaldehyde, and the remainder was immersed in RNAiso Plus reagent (Takara Bio, Shiga, Japan) for further analysis. Following this procedure, the animals were euthanized by cervical dislocation, and death was verified by the lack of heartbeat, respiration, and corneal reflex. Liver injury was determined by measuring serum alanine aminotransferase (ALT) using a Dri-Chem 7000 Chemistry Analyzer System (Fujifilm, Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eTreatments\u003c/h2\u003e \u003cp\u003eOzagrel sodium (Takata Pharmaceutical Co., Saitama, Japan), a TXS inhibitor, or vehicle (phosphate-buffered saline [PBS]) was administered (150 mg/kg, i.p.) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] 2 h after APAP treatment. ANG-3777 and HGF mimetics (Selleck Houston, TX, USA) (50 mg/kg) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] were dissolved in 0.2 mL PBS containing 4% dimethyl sulfoxide and intraperitoneally administered 2 h and 24 h after APAP administration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eClodronate\u003c/h2\u003e \u003cp\u003eClodronate and control liposomes were purchased from FormuMax Scientific (Sunnyvale, CA, USA). Clodronate or control liposomes (100 \u0026micro;L/mouse) were injected intraperitoneally 48 h before APAP administration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eHistology and immunohistochemistry\u003c/h2\u003e \u003cp\u003eExcised liver tissues were fixed immediately with buffered 10% formaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). Sections (3.5 \u0026micro;m thick) were prepared from paraffin-embedded tissues and subjected to either hematoxylin and eosin (H\u0026amp;E) staining or immunostaining. Images of the H\u0026amp;E-stained sections were captured using a microscope (Biozero BZ-700 Series; Keyence, Osaka, Japan). The level of necrosis (as a percentage of the total area) was determined in five fields (100\u0026times;) from each animal by measuring the necrotic area relative to the entire histological section using the ImageJ software version 1.50i (National Institutes of Health, Bethesda, MD, USA). The results are expressed as percentages.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eProliferating cell nuclear antigen (PCNA)\u003c/h2\u003e \u003cp\u003eWith regard to PCNA immunohistochemistry, liver sections were stained with a rabbit monoclonal anti-PCNA antibody (Thermo Fisher Scientific, Waltham, MA, USA) at a dilution of 1:200. Immune complexes were detected using Histofine Simple Stain MAX PO (MULTI) (Nichirei, Tokyo, Japan). Images of stained liver sections were captured using a microscope (Biozero BZ-700 Series). The number of PCNA\u003csup\u003e+\u003c/sup\u003e hepatocytes was counted in five fields (200\u0026times;) per animal using the ImageJ software. The percentage of PCNA\u003csup\u003e+\u003c/sup\u003e hepatocytes was then calculated, and the results were expressed as percentages.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e \u003cp\u003eLiver tissues from APAP-treated mice were fixed in 4% periodate-lysine-paraformaldehyde overnight at 4\u0026deg;C, transferred to 30% sucrose prepared in 0.1 M phosphate buffer (pH 7.2), and stored at 4\u0026deg;C for 3 days, followed by mounting the liver tissue in Tissue-Tek O.C.T. Compound (Sakura Finetek USA, Inc., Torrance, CA, USA) and stored at \u0026minus;\u0026thinsp;20\u0026deg;C. Liver tissue sections (8-\u0026micro;m-thick) were cut and blocked with 1% bovine serum albumin in 0.5% Triton X-100 in PBS. The sections were incubated with a rabbit anti-mouse TP (1:100; Cayman Chemical, Ann Arbor, MI, USA), a rabbit anti-mouse TXS(1:100; Bioss, Woburn, MA, USA), a rat anti-mouse CD68 antibody (1:100; Bio-Rad Laboratories, Hercules, CA, USA), a rat anti-mouse CD41 (BioRad Laboratories), and a rabbit anti-mouse HGF (1:100; Proteintech Group, Rosemont, Il, US,) at 4\u0026deg;C overnight. The sections were then incubated with the following secondary antibodies at 4\u0026deg;C for 1 h: Alexa Fluor 488-conjugated donkey anti-rabbit IgG, Alexa Fluor 594-conjugated donkey anti-rabbit IgG, or Alexa Fluor 647-conjugated donkey anti-rabbit IgG (Molecular Probes, Eugene, OR, USA). Nuclei were stained with 4'-6-diamidino-2-phenylindole (DAPI). Images were obtained using a fluorescence microscope (Biozero BZ-700 Series; Keyence, Osaka, Japan). After labeling, five optical fields (\u0026times;200) per animal were randomly selected, and the number of positive cells was counted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eBone marrow (BM) transplantation\u003c/h2\u003e \u003cp\u003eBM cells were obtained by flushing the cavities of freshly dissected femurs and tibiae from donor mice (7 weeks old) using PBS. The flushed BM cells from each donor were dispersed by pipetting and resuspended in PBS at a density of 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells/mL. Mice were lethally irradiated (9.0 Gy) using an MBR-1505R X-ray irradiator (Hitachi Medico Co., Tokyo, Japan) with a filter (copper, 0.5 mm; aluminum, 2 mm) to monitor the cumulative radiation dose. Donor BM mononuclear cells (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e) in 200 \u0026micro;L of PBS were transplanted into the tail veins of irradiated mice. Bone marrow transplantations were performed to generate the chimeric mice as follows: WT mice reconstituted with WT BMMs (WT\u0026rarr;WT) or \u003cem\u003eTP\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e BMMs (\u003cem\u003eTP\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e\u0026rarr;WT) and \u003cem\u003eTP\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice reconstituted with WT BMMs (WT\u0026rarr;\u003cem\u003eTP\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e) or TP\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e BMMs (\u003cem\u003eTP\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e\u0026rarr;\u003cem\u003eTP\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e). In a separate experiment, donor BM cells were harvested from GFP\u003csup\u003e+\u003c/sup\u003e-WT mice, and GFP\u003csup\u003e+\u003c/sup\u003e-WT BM cells were transplanted into WT mice. The chimeras were treated with APAP for eight weeks after BM transplantation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of glutathione (GSH) and glutathione disulfide (GSSG)\u003c/h2\u003e \u003cp\u003eThe frozen tissues were homogenized and centrifuged to separate the supernatants. Hepatic GSH and GSSG levels were measured using a spectrophotometric/microplate reader with a GSSG/GSH Quantification Kit (Dojindo Molecular Technologies, Kumamoto, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of TXB\u003csub\u003e2\u003c/sub\u003e\u003c/h2\u003e \u003cp\u003eThe concentrations of TXB\u003csub\u003e2\u003c/sub\u003e, a stable metabolite of TXA\u003csub\u003e2\u003c/sub\u003e, in liver tissues were measured using a Thromboxane B\u003csub\u003e2\u003c/sub\u003e ELISA Kit (Cayman Chemical, Ann Arbor, MI, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of intrahepatic leukocytes\u003c/h2\u003e \u003cp\u003eAnimals were anesthetized by i.p. injection of mixed anesthetic agents containing 4.0 mg/kg midazolam (Sandoz, a Novartis division, Basel, Switzerland), 0.75 mg/kg medetomidine hydrochloride (Nippon Zenyaku Kogyo, Fukushima, Japan), and 5.0 mg/kg butorphanol (Meiji Seika Pharma, Tokyo, Japan). The liver was perfused with Hank\u0026rsquo;s balanced salt solution through the portal vein. Excised livers were incubated in Roswell Park Memorial Institute (RPMI) medium containing 0.05% collagenase (Type IV; Sigma Chemical Co., St. Louis, MO, USA) for 20 min at 37\u0026deg;C. The liver homogenates were filtered through a 70 \u0026micro;m cell strainer. Non-parenchymal cells were purified by density-gradient centrifugation on 33% Percoll (GE Healthcare Life Sciences, Piscataway, NJ, USA) as previously reported [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometric analyses\u003c/h2\u003e \u003cp\u003eIsolated non-parenchymal cells were incubated with an anti-mouse CD16/32 antibody (TruStain FcX; BioLegend, San Diego, CA, USA) to block nonspecific binding of the primary mAb. Cells were stained with a combination of the following reagents: PE-conjugated anti-CD45 (30-F11; BioLegend, San Diego, CA, USA), APC/CY7-conjugated anti-Ly6G (1A8; BioLegend), PE/Cy7-conjugated anti-CD11b (M1/70; BioLegend), Brilliant Violet 510-conjugated anti-Ly6C (HK1.4; BioLegend), and anti-F4/80 (BM8; BioLegend). Cells positive for 7-aminoactinomycin D (BioLegend) were excluded from the analysis. Samples were analyzed using a FACSVerse cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Data were analyzed using the Kaluza software v2.1 (Beckman Coulter, Brea, CA, USA). We quantified and presented the number of cells normalized to liver tissue weight (cells/g).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRT-qPCR analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from the liver tissues using RNAiso Plus (Takara Bio). cDNA was constructed with 1 \u0026micro;g of total RNA using the ReverTra Ace qPCR RT Kit (TOYOBO Co., Ltd., Osaka, Japan). Quantitative PCR amplification was performed using the TB Green Premix Ex Taq II (Tli RNase H Plus; Takara Bio, Inc. Shiga, Japan). PCR amplification was performed with the following conditions: 95\u0026deg;C for 10 s, followed by 40 cycles at 95\u0026deg;C for 3 s and 60\u0026deg;C for 20 s. The mRNA expression levels were calculated based on the comparative threshold cycle and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in each sample. The primer sequences are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCell preparation and culture\u003c/h2\u003e \u003cp\u003eTo generate BM-derived macrophages, BM cells were isolated from the femurs and tibiae of 8-week-old male mice. BM cells were cultured in 6-well plates (1.0 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/well) and maintained in RPMI 1640 medium (Gibco, Thermo Scientific, Waltham, MA, USA) containing 10% fetal calf serum and 20 ng/mL macrophage colony-stimulating factor (BioLegend, San Diego, CA, USA), as previously described [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. On day 7, BM-derived macrophages were stimulated with lipopolysaccharide (LPS) (10 ng/mL; Sigma-Aldrich, St. Louis, MO, USA) and recombinant murine interferon-gamma (IFN-γ) (20 ng/mL; BioLegend) to polarize toward a pro-inflammatory macrophage phenotype or recombinant murine interleukin (IL)-4 (20 ng/mL; BioLegend) and to polarize toward a reparative phenotype in RPMI 1640 medium for 18 h. Cultured BM-derived macrophages were harvested and homogenized in RNAiso Plus (Takara Bio), and mRNA levels were measured using RT-qPCR.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll results are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. All statistical analyses were performed using GraphPad Prism, version 8 (GraphPad Software, La Jolla, CA, USA). Data were compared between two groups using unpaired two-tailed Student\u0026rsquo;s t-tests and between multiple groups using one-way analysis of variance followed by Tukey\u0026rsquo;s post hoc tests. Statistical significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eExacerbation of APAP-induced hepatotoxicity in TP\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice\u003c/h2\u003e \u003cp\u003eTo determine the role of TP signaling in APAP hepatotoxicity, we treated both WT and \u003cem\u003eTp\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice with APAP. Deletion of TP signaling rendered mice highly susceptible to APAP-induced toxicity, as evidenced by increased ALT levels and hepatic necrosis around the central veins (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). The levels of ALT peaked at 6 h after APAP treatment in WT mice. Subsequently, ALT levels were reduced after 48 h. By contrast, the levels of ALT and hepatic necrosis remained high in \u003cem\u003eTP\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. These results indicate that liver injury was enhanced and liver recovery was delayed in \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. PCR analysis showed that hepatic levels of \u003cem\u003eTp\u003c/em\u003e increased 6 h after APAP treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Hepatic levels of \u003cem\u003eTxs\u003c/em\u003e moderately increased in both genotypes 48 h after APAP treatment; however, there was no statistical difference in \u003cem\u003eTxs\u003c/em\u003e mRNA levels between the two genotypes. Furthermore, the concentration of TXB\u003csub\u003e2\u003c/sub\u003e, a stable metabolite of TXA\u003csub\u003e2\u003c/sub\u003e, was increased in the livers after APAP administration in WT and \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, and no significant difference between the two genotypes was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Immunohistochemical staining for TP and TXS demonstrated that TP and TXS were expressed in CD68\u003csup\u003e+\u003c/sup\u003e cells, suggesting that TXA\u003csub\u003e2\u003c/sub\u003e synthesized by macrophages bound to TP (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBecause TP signaling is involved in platelet aggregation, platelet accumulation during APAP hepatotoxicity was evaluated by immunostaining platelets with an anti-CD41 antibody. Immunostaining demonstrated that CD41\u003csup\u003e+\u003c/sup\u003e cells were extensively observed in the livers treated with APAP for 48 h; however, there was no statistical difference in the CD41\u003csup\u003e+\u003c/sup\u003e area between the two genotypes (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eInvolvement of macrophages in liver repair after APAP-induced liver injury\u003c/h2\u003e \u003cp\u003eBecause macrophages are responsible for liver repair after APAP-induced liver injury, we assessed the accumulation of macrophages during APAP hepatotoxicity. The number of CD68\u003csup\u003e+\u003c/sup\u003e cells (macrophages) in WT livers increased 48 h after APAP treatment, and the accumulation of CD68\u003csup\u003e+\u003c/sup\u003e cells in WT livers was greater than that in \u003cem\u003eTP\u003c/em\u003e-deficient livers (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). These results suggest that macrophage recruitment was accompanied by liver repair after APAP-induced liver injury.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo elucidate the role of macrophage accumulation in the restoration of injury to APAP hepatotoxicity, WT mice were pretreated with clodronate liposomes (CL) to delete macrophages or with control liposomes (Cont). The results showed that the levels of ALT and hepatic necrotic area in CL-treated WT mice 48 h after APAP treatment were higher than those in Cont-treated WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), which was consistent with previous reports [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The expression of PCNA, a marker of hepatocyte proliferation, was considerably lower in the CL-treated mice than in the Cont-treated mice. These results suggest that hepatic macrophages are involved in the resolution of liver inflammation and repair after APAP treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eDeletion of TP signaling in BM-derived macrophages delayed liver repair\u003c/h2\u003e \u003cp\u003eTo examine whether the macrophages accumulated in the injured regions were derived from the BM, we generated chimeric WT mice bearing GFP\u003csup\u003e+\u003c/sup\u003e BM cells. Immunofluorescence demonstrated that accumulated GFP\u003csup\u003e+\u003c/sup\u003e cells in the injured regions colocalized with CD68\u003csup\u003e+\u003c/sup\u003e cells 48 h after APAP treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), indicating that the infiltrating macrophages were derived from the BM. To further define the role of TP-expressing BM cells in liver repair after APAP treatment, we performed BM transplantation and assessed ALT levels and hepatic necrosis in response to APAP treatment. Reconstitution of WT mice with \u003cem\u003eTP\u003c/em\u003e-deficient BM (\u003cem\u003eTP\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e\u0026rarr;WT mice) resulted in enhancement of APAP-induced liver injury, as indicated by increased levels of ALT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) and hepatic necrosis area (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The magnitude of liver injury in WT mice transplanted with \u003cem\u003eTP\u003c/em\u003e-deficient BM was equivalent to that in \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice transplanted with \u003cem\u003eTP\u003c/em\u003e-deficient BM (\u003cem\u003eTP\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e\u0026rarr;\u003cem\u003eTP\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice). By contrast, the levels of ALT and hepatic necrotic area in WT\u0026rarr;WT and WT\u0026rarr;\u003cem\u003eTP\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were lower than those in \u003cem\u003eTP\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e\u0026rarr;WT and \u003cem\u003eTP\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e\u0026rarr;\u003cem\u003eTP\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. The mRNA levels of pro-inflammatory cytokines including \u003cem\u003eIl1b\u003c/em\u003e and \u003cem\u003eIl6\u003c/em\u003e in WT\u0026rarr;WT and WT\u0026rarr;\u003cem\u003eTP\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were lower than those in \u003cem\u003eTP\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e\u0026rarr;WT and \u003cem\u003eTP\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e\u0026rarr;\u003cem\u003eTP\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). With respect to liver repair, we determined PCNA expression in hepatocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). WT\u0026rarr;WT and WT\u0026rarr;\u003cem\u003eTP\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice exhibited enhanced PCNA expression, while \u003cem\u003eTP\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e\u0026rarr;WT and \u003cem\u003eTP\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e\u0026rarr;\u003cem\u003eTP\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice exhibited reduced PCNA expression. We also determined the recruitment of macrophages to the liver. CD68\u003csup\u003e+\u003c/sup\u003e cells in WT\u0026rarr;WT and WT\u0026rarr;\u003cem\u003eTP\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were accumulated to the demarcated necrotic regions, while CD68\u003csup\u003e+\u003c/sup\u003e cells in \u003cem\u003eTP\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e\u0026rarr;WT and \u003cem\u003eTP\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e\u0026rarr;\u003cem\u003eTP\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were distributed to the entire liver lobules and were not concentrated in the damaged regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). The numbers of CD68\u003csup\u003e+\u003c/sup\u003e cells in WT\u0026rarr;WT and WT\u0026rarr;\u003cem\u003eTP\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were higher than those in \u003cem\u003eTP\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e\u0026rarr;WT and \u003cem\u003eTP\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e\u0026rarr;\u003cem\u003eTP\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. Together, these results suggest that TP signaling in BM cells plays a critical role in the resolution of liver inflammation and repair in APAP-induced liver injury by accumulating macrophages in injured regions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTaken together, these results indicated that the inhibition of TP signaling aggravated APAP-induced liver injury and that TP deficiency in BM cells enhanced liver injury and delayed liver repair after APAP treatment. Additionally, the impaired accumulation of macrophages in injured regions was associated with delayed liver repair following APAP-induced liver injury.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eInhibition of TXS aggravated APAP-induced liver injury\u003c/h2\u003e \u003cp\u003eWe further confirmed that TP signaling is involved in liver repair during APAP hepatotoxicity. The TXS inhibitor Ozagrel or vehicle (PBS) was administered 2 h after APAP treatment, and liver injury and repair were assessed 48 h after APAP treatment. Ozagrel treatment sensitized mice to APAP-induced liver injury, as indicated by increased levels of ALT and hepatic necrotic area (Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eMacrophage-specific TP deficiency exacerbated APAP-induced liver injury\u003c/h2\u003e \u003cp\u003eTo confirm that TP signaling in macrophages plays a role in liver repair after APAP treatment, APAP was administered to macrophage-specific TP deficient (\u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e) or control (Controls) mice. Compared with controls, \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice exhibited sustained liver injury as indicated by increased levels of ALT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) and necrotic area (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), suggesting that liver repair was delayed in \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice. In addition, the expression of PCNA in \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice was decreased at 48 h after APAP treatment as compared with controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, changes in hepatic GSH levels, which detoxifies NAPQI via conjugation, were similar in the liver sections of both \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice and controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Both mouse models displayed rapid GSH depletion 1 h after APAP treatment, followed by gradual replenishment and recovery to pre-APAP levels within 24 h of APAP treatment. The same was true for the hepatic GSSG levels, which is a marker of oxidative stress. The similar rate of GSH depletion in the early phase of APAP hepatotoxicity between the two genotypes suggested that the difference in hepatotoxicity did not result from APAP metabolic activation.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eDeficiency of TP signaling in macrophages suppressed accumulation of macrophages in the injured regions\u003c/h2\u003e \u003cp\u003eTo understand the contribution of TP signaling in macrophages to liver injury and repair following APAP treatment, we determined the number of macrophages. The numbers of CD68\u003csup\u003e+\u003c/sup\u003e cells in controls was larger than that in \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice at 48 h after APAP treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Immunofluorescence revealed that CD68\u003csup\u003e+\u003c/sup\u003e cells accumulated extensively into the necrotic regions of livers from controls, whereas CD68\u003csup\u003e+\u003c/sup\u003e cells diffusely distributed to the liver lobules from \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). These were accompanied by higher mRNA expression levels of \u003cem\u003eCcl2, Ccl7\u003c/em\u003e, and \u003cem\u003eCcl9\u003c/em\u003e in controls than those in \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eLoss of TP signaling in macrophages polarized macrophages toward a pro-inflammatory phenotype\u003c/h2\u003e \u003cp\u003eWe further evaluated the phenotypes of infiltrated macrophages during APAP hepatotoxicity using flow cytometry. Flow cytometry analysis demonstrated that the populations of Ly6C\u003csup\u003ehigh\u003c/sup\u003e macrophages (pro-inflammatory macrophages) in \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice 48 h after APAP treatment were larger than those in control mice, while the populations of Ly6C\u003csup\u003elow\u003c/sup\u003e macrophages (reparative macrophages) in controls were larger than those in \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). These results indicated that accelerated liver repair in controls was associated with accumulation of reparative macrophages and that delayed liver repair in \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice was associated with accumulation of pro-inflammatory macrophages. We also determined the levels of pro- and anti-inflammatory mediators in the liver. The expressions of mRNA encoding pro-inflammatory mediators including \u003cem\u003eIl1b\u003c/em\u003e and \u003cem\u003eIl6\u003c/em\u003e in \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice 48 h after APAP treatment were higher than those in controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). In addition, the expressions of mRNA encoding anti-inflammatory mediators including \u003cem\u003eMr\u003c/em\u003e and \u003cem\u003eFizz1\u003c/em\u003e in \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice were lower than those in controls.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further examine whether TP signaling in macrophages polarizes macrophages from a pro-inflammatory macrophage phenotype toward a reparative macrophage phenotype, BM-derived macrophages were stimulated using LPS/IFN-γ or IL-4. The administration of LPS/IFN-γ polarizes BM-macrophages toward a pro-inflammatory phenotype, whereas IL-4 administration polarizes BM-macrophages toward a reparative phenotype [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The levels of mRNA encoding \u003cem\u003eIl1b\u003c/em\u003e and \u003cem\u003eIl6\u003c/em\u003e in macrophages from \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice stimulated with LPS/IFN-γ were enhanced as compared with those from controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). On the other hand, the mRNA expression levels of \u003cem\u003eMr\u003c/em\u003e and \u003cem\u003eFizz1\u003c/em\u003e were enhanced in macrophages from controls stimulated with IL-4 as compared with those from \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice. These results suggest that inhibition of TP signaling polarizes macrophages toward a pro-inflammatory phenotype.\u003c/p\u003e \u003cp\u003eWith respect to other immune cells, including Kuffer cells (KCs), the number of KCs in both genotypes decreased after APAP treatment (Supplementary Fig.\u0026nbsp;3). By contrast, neutrophils in controls transiently increased at 24 h, and the number of neutrophils at 24 h in controls was higher than that in \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice. At 48 h, the number of neutrophils in controls declined, whereas that in \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice was elevated. The number of neutrophils at 48 h in \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice was higher than that in controls.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eHGF treatment attenuated APAP-induced liver injury and enhanced liver repair\u003c/h2\u003e \u003cp\u003eTo gain insight into the mechanisms by which TP signaling in macrophages facilitates liver repair after APAP-induced liver injury, we determined the hepatotrophic factor, HGF. mRNA levels of \u003cem\u003eHgf\u003c/em\u003e in \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice were lower than those in controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). We also examined the localization of HGF. Immunofluorescence revealed that HGF colocalized with CD68\u003csup\u003e+\u003c/sup\u003e cells in the injured centrilobular regions of controls 48 h after APAP treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). To examine the contribution of HGF to promote liver repair, \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice were given HGF 2 h and 24 h after APAP treatment. Treatment of \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice with HGF attenuated APAP-induced liver injury as indicated by reduced levels of ALT and hepatic necrotic area (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) and enhanced liver repair as indicated by increased PCNA\u003csup\u003e+\u003c/sup\u003e hepatocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The number of CD68\u003csup\u003e+\u003c/sup\u003e cells in HGF-treated \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice was higher than that in vehicle-treated \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). The levels of mRNA for \u003cem\u003eIl1b\u003c/em\u003e and \u003cem\u003eIl6\u003c/em\u003e in HGF-treated \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice were lower than those in vehicle-treated \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice, while the levels of mRNA for \u003cem\u003eMr\u003c/em\u003e in HGF-treated \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice were higher than those in vehicle-treated \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). The mRNA levels of \u003cem\u003eCcl2\u003c/em\u003e, \u003cem\u003eCcl7\u003c/em\u003e, and \u003cem\u003eCcl9\u003c/em\u003e in HGF-treated \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice were higher than those in vehicle-treated \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). These results suggest that HGF, probably from macrophages, reduced liver inflammation and facilitated liver repair after APAP treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, we examined the role of endogenous TP signaling in liver repair following APAP-induced liver injury in mice. Our data showed that the inhibition of TP signaling or TXS aggravated APAP-induced liver inflammation and delayed liver repair following APAP-induced liver injury, which was related to the suppressed accumulation of macrophages in the injured regions. The deficiency of TP signaling in BM-derived macrophages suppresses the accumulation of macrophages in damaged areas during repair. In addition, the inhibition of TP signaling in macrophages prolonged liver inflammation and impaired liver repair, which was accompanied by reduced accumulation of reparative macrophages in the damaged area and downregulated expression of the hepatotrophic factor HGF. Treatment of mice deficient in TP signaling in macrophages with HGF mitigated liver inflammation and facilitated liver repair. These results suggest that the inhibition of TP signaling in macrophages delays liver repair after APAP hepatotoxicity by suppressing the accumulation of reparative macrophages and producing HGF.\u003c/p\u003e \u003cp\u003eThe processes of liver repair and regeneration after acute liver injury determine the outcomes of patients with APAP hepatotoxicity [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Although the mechanisms underlying liver repair appear to be complex [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], macrophages are key players in liver repair after acute liver injury, including APAP hepatotoxicity [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Macrophage recruitment is a critical event in the resolution of liver inflammation and restoration of liver function. Our data revealed an extensive accumulation of macrophages in the hepatic necrotic area during the repair phase. The recruited macrophages were derived from the BM to remove necrotic debris and injured hepatocytes. The accumulation of macrophages in injured regions is a prerequisite for proper liver repair [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. By contrast, macrophages were evenly distributed throughout the livers from \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice and were not accumulated and concentrated in the damaged regions. These results indicate that the scattered distribution of macrophages is associated with impaired liver repair following APAP-induced acute liver injury. Furthermore, control mice had more infiltrated macrophages than \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice, suggesting that accumulation of macrophages in the necrotic area would be required for adequate removal of necrotic tissues and consequently for liver recovery and regeneration [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe importance of macrophages in liver repair after acute liver injury has been highlighted by the deletion of hepatic macrophages using clodronate liposomes [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Pre-elimination of KCs with clodronate liposomes exacerbated APAP-induced liver injury, which is consistent with the present results, demonstrating increased ALT, necrosis and reduced PCNA expression.\u003c/p\u003e \u003cp\u003eMacrophage recruitment to injured areas is driven by chemokine production CCL2 is important for the recruitment of MoMFs to promote liver repair following APAP toxicity [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Consistent with this, our data showed that higher levels of CCL2 were associated with macrophage accumulation and recovery from APAP-induced liver injury. Upregulation of CCL2 expression in macrophages is dependent on TP signaling CCL2 is produced by injured hepatocytes in the centrilobular area and recruited by macrophages during APAP hepatotoxicity [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In addition, deletion of CCR2, a receptor for CCL2, inhibits the accumulation of macrophages and clearance of necrotic cell debris in APAP-treated livers [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], suggesting that downregulated CCR2 expression might lead to the suppression of phagocytosis of necrotic tissues in APAP-treated livers. Furthermore, a recent study reported that other chemokines, including CCL7 and CCL9, were elevated during APAP-induced liver injury [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], which is consistent with our results.\u003c/p\u003e \u003cp\u003eMacrophages are instrumental in the repair and regeneration processes after acute liver damage [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Pro-inflammatory macrophages peak at 24 h after APAP treatment, and reparative macrophages peak at 48 h [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], suggesting \u003cem\u003ein situ\u003c/em\u003e reprogramming of the macrophage phenotype [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The current study showed that increased numbers of reparative macrophages were associated with the promotion of liver repair, whereas increased numbers of pro-inflammatory macrophages were associated with the impairment of liver repair. These changes were associated with increased expression of genes related to the pro-inflammatory macrophage phenotype and decreased expression of genes related to the reparative macrophage phenotype in the livers of mice deficient in TP signaling. These results suggest that TP signaling in macrophages is involved in macrophage differentiation to repair damaged liver tissues exposed to APAP overdoses. Additionally, TP signaling in macrophages appears to be involved in macrophage polarization [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]; however, further studies are required to elucidate the mechanism underlying macrophage polarization via TP signaling.\u003c/p\u003e \u003cp\u003eCells other than MoMFs are likely involved in liver repair following APAP hepatotoxicity [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The current study demonstrated that APAP treatment reduced the number of liver-resident macrophages and KCs in both genotypes. Loss of KCs is a common feature of liver diseases [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] including APAP toxicity [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. These results suggest that KCs contribute little to liver repair via TP signaling in macrophages. Neutrophils are another type of immune cells that accumulate in APAP-treated livers. However, the role of neutrophils in the APAP-induced liver injury and repair remains controversial [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The present study demonstrated that control mice had more hepatic neutrophils at 24 h and a smaller hepatic necrotic area at 48 h, whereas mice lacking TP in macrophages had more hepatic neutrophils and more robust hepatic necrosis at 48 h. These results suggest that the accumulation of neutrophils prior to the accumulation of reparative macrophages may induce liver repair, and excessive accumulation of neutrophils in sustained inflammatory livers impairs the resolution of inflammation and delays liver repair [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Thus, the role of neutrophil accumulation in APAP hepatotoxicity appears to be context-dependent. Additionally, it remains unclear why the deletion of TP signaling in macrophages prevented the accumulation of neutrophils in the injured regions induced by APAP treatment. Further studies are necessary to elucidate the underlying mechanisms that regulate the role of neutrophils in APAP hepatotoxicity and their involvement in TP signaling.\u003c/p\u003e \u003cp\u003eHepatic accumulation of platelets is also involved in the progression of APAP-induced liver injury and delays liver repair after APAP treatment [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Because platelets aggregate through TP signaling, TP signaling in platelets would participate in APAP hepatotoxicity; however, in the present study, the area of platelet did not differ between WT and \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, suggesting that TP signaling-induced platelet accumulation did not contribute to APAP-induced liver injury and repair. These results indicate that platelet accumulation and aggregation in the liver are independent of TP signaling.\u003c/p\u003e \u003cp\u003eVarious trophic factors facilitate liver regeneration and repair after APAP toxicity [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. We found that hepatic HGF levels in control mice were higher than those in \u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e mice 48 h after APAP treatment, which was consistent with our previous results [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Furthermore, the current study demonstrated that HGF administration attenuated APAP-induced liver injury and facilitated liver repair from the injury. HGF is characterized by pro-proliferative properties. HGF deletion impairs liver regeneration in mice [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In contrast, recombinant HGF appears to confer protection against liver failure by boosting liver regeneration in mice [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Consistent with this, HGF overexpression in BM-derived mesenchymal stem cells facilitates liver repair following liver injury [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In addition, transplantation of HGF-knockout mesenchymal stem cells reduced the survival rate of APAP-treated mice, and HGF administration increased the survival rate during APAP hepatotoxicity, indicating that HGF improved mouse liver failure induced by APAP administration. Macrophages are one of the main sources of HGF during liver regeneration. In liver regeneration induced by partial hepatectomy in mice, accumulated reparative macrophages generate HGF, which contributes to hepatocyte proliferation [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The other possible origins of HGF secretion during APAP hepatotoxicity are likely located in hepatic stellate cells (HSCs) and liver sinusoidal endothelial cells (LSECs) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn conclusion, our study demonstrates that TP signaling in macrophages facilitates liver repair after APAP overdose. Accumulation of TP-expressing macrophages in injured regions induced by APAP is a critical step in the resolution of liver inflammation and recovery. The activation of TP signaling in macrophages may be a therapeutic target for APAP-induced liver injury and liver recovery. Specific activation of TP signaling in macrophages may be a therapeutic target for liver repair and regeneration after APAP hepatotoxicity.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eALT, alanine transaminase; APAP, N-acetyl-p-aminophenol; BM, bone marrow; CCR2, C-C motif chemokine receptor 2; CL, clodronate liposomes; DAPI, 4\u0026apos;-6-diamidino-2-phenylindole; HGF, hepatocyte growth factor; GFP, green fluorescent protein; GSH, glutathione; GSSG, glutathione disulfide; H\u0026amp;E, hematoxylin and eosin; HGF, hepatocyte growth factor; IFN-\u0026gamma;, interferon-gamma; IL, interleukin; KC, Kupffer cell; LPS, lipopolysaccharide; MoMF, monocyte-derived macrophages; NAPQI, N-acetyl-p-benzoquinone imine; PBS, phosphate-buffered saline; PCNA, proliferating cell nuclear antigen; TP, thromboxane prostanoid; TXA\u003csub\u003e2\u003c/sub\u003e, Thromboxane A\u003csub\u003e2\u003c/sub\u003e; TP, thromboxane prostanoid; TXS, Thromboxane synthase ; WT, wild type.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Ms. Michiko Ogino and Ms. Kyoko Yoshikawa for technical assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.T. designed and performed experiments, analyzed the data, and wrote the article. K.H., A.Y., and Y.I. performed experiments and analyzed the data. M.M. provided the TP-flox mice. S.N. provided the TP-deficient mice. C.K. supervised this study. H.A. .interpreted the data, supervised this study, and edited the article.. All authors have reviewed and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (22K08856 to YI) and the Takeda Science Foundation (to KH). This study was also supported by the Integrative Research Program of the Graduate School of Medical Science at Kitasato University and the Parents’ Association Grant of Kitasato University School of Medicine.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included within the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the Kitasato University School of Medicine (Approval no. 2023-062). All experimental studies were performed in accordance with the institutional guidelines for animal experimentation, based on the Guidelines for Proper Conduct of Animal Experiments published by the Science Council of Japan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eStravitz RT, Fontana RJ, Karvellas C, Durkalski V, McGuire B, Rule JA, et al. Future directions in acute liver failure. Hepatology. 2023;78(4):1266-89. https://doi.org/10.1097/HEP.0000000000000458.\u003c/li\u003e\n \u003cli\u003eRamachandran A, Jaeschke H. A mitochondrial journey through acetaminophen hepatotoxicity. 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Hepatology. 1999;30(1):151-9. https://doi.org/10.1002/hep.510300102.\u003c/li\u003e\n \u003cli\u003eZhang Y, Li R, Rong W, Han M, Cui C, Feng Z, et al. Therapeutic effect of hepatocyte growth factor-overexpressing bone marrow-derived mesenchymal stem cells on CCl(4)-induced hepatocirrhosis. Cell Death Dis. 2018;9(12):1186. https://doi.org/10.1038/s41419-018-1239-9.\u003c/li\u003e\n \u003cli\u003eHuang M, Jiao J, Cai H, Zhang Y, Xia Y, Lin J, et al. C-C motif chemokine ligand 5 confines liver regeneration by down-regulating reparative macrophage-derived hepatocyte growth factor in a forkhead box O 3a-dependent manner. Hepatology. 2022;76(6):1706-22. https://doi.org/10.1002/hep.32458.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"inflammation-and-regeneration","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ireg","sideBox":"Learn more about [Inflammation and Regeneration](http://inflammregen.biomedcentral.com/)","snPcode":"41232","submissionUrl":"https://www.editorialmanager.com/ireg/default2.aspx","title":"Inflammation and Regeneration","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Acetaminophen, liver repair, thromboxane, macrophage","lastPublishedDoi":"10.21203/rs.3.rs-4078778/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4078778/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAcetaminophen (APAP)-induced liver injury is the most common cause of acute liver failure. Macrophages are key players in liver restoration following APAP-induced liver injury. Thromboxane A\u003csub\u003e2\u003c/sub\u003e (TXA\u003csub\u003e2\u003c/sub\u003e) and its receptor, thromboxane prostanoid (TP) receptor, have been shown to be involved in tissue repair. However, whether TP signaling plays a role in liver repair after APAP hepatotoxicity by affecting macrophage function remains unclear.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMale TP knockout (\u003cem\u003eTP\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e) and C57BL/6 wild-type (WT) mice were treated with APAP (300 mg/kg). In addition, macrophage-specific TP-knockout (\u003cem\u003eTP\u003c/em\u003e\u003csup\u003e△mac\u003c/sup\u003e) and control WT mice were treated with APAP. We explored changes in liver inflammation, liver repair, and macrophage accumulation in mice treated with APAP.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCompared with WT mice, \u003cem\u003eTP\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice showed aggravated liver injury as indicated by increased levels of alanine transaminase (ALT) and necrotic area as well as delayed liver repair as indicated by decreased expression of proliferating cell nuclear antigen (PCNA). Macrophage deletion exacerbated APAP-induced liver injury and impaired liver repair. Transplantation of \u003cem\u003eTP\u003c/em\u003e-deficient bone marrow (BM) cells to WT or \u003cem\u003eTP\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice aggravated APAP hepatotoxicity with suppressed accumulation of macrophages, while transplantation of WT-BM cells to WT or \u003cem\u003eTP\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice attenuated APAP-induced liver injury with accumulation of macrophages in the injured regions. Macrophage-specific \u003cem\u003eTP\u003c/em\u003e \u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice exacerbated liver injury and delayed liver repair, which was associated with increased pro-inflammatory macrophages and decreased reparative macrophages and hepatocyte growth factor (HGF) expression. HGF treatment mitigated APAP-induced inflammation and promoted liver repair after APAP-induced liver injury.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusions\u003c/b\u003e\u003c/p\u003e \u003cp\u003eDeletion of TP signaling in macrophages delays liver repair following APAP-induced liver injury, which is associated with reduced accumulation of reparative macrophages and the hepatotrophic factor HGF. Specific activation of TP signaling in macrophages may be a potential therapeutic target for liver repair and regeneration after APAP hepatotoxicity.\u003c/p\u003e","manuscriptTitle":"Deletion of TP signaling in macrophages delays liver repair following APAP-induced liver injury by reducing accumulation of reparative macrophage and production of HGF","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-03 12:39:41","doi":"10.21203/rs.3.rs-4078778/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2024-05-01T03:02:09+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-04-02T23:58:24+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-29T06:34:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-12T07:43:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"Inflammation and Regeneration","date":"2024-03-11T20:14:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"inflammation-and-regeneration","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ireg","sideBox":"Learn more about [Inflammation and Regeneration](http://inflammregen.biomedcentral.com/)","snPcode":"41232","submissionUrl":"https://www.editorialmanager.com/ireg/default2.aspx","title":"Inflammation and Regeneration","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"09fee616-d906-4665-be63-07ea625f4c2e","owner":[],"postedDate":"April 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-07T16:01:21+00:00","versionOfRecord":{"articleIdentity":"rs-4078778","link":"https://doi.org/10.1186/s41232-024-00356-z","journal":{"identity":"inflammation-and-regeneration","isVorOnly":false,"title":"Inflammation and Regeneration"},"publishedOn":"2024-10-03 15:57:28","publishedOnDateReadable":"October 3rd, 2024"},"versionCreatedAt":"2024-04-03 12:39:41","video":"","vorDoi":"10.1186/s41232-024-00356-z","vorDoiUrl":"https://doi.org/10.1186/s41232-024-00356-z","workflowStages":[]},"version":"v1","identity":"rs-4078778","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4078778","identity":"rs-4078778","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}