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Intestinal Epithelial Chemokine (C-C Motif) Ligand 7 Overexpression Enhances Acetaminophen-Induced Hepatotoxicity in Mice

  • Mengwei Niu
    Affiliations
    Department of Pathophysiology, School of Basic Medical Sciences, Guangdong Provincial Key Laboratory of Proteomics, Southern Medical University, Guangzhou, China

    State Key Laboratory of Organ Failure Research, Southern Medical University, Guangzhou, China
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  • Zhihong Luo
    Affiliations
    Department of Pathophysiology, School of Basic Medical Sciences, Guangdong Provincial Key Laboratory of Proteomics, Southern Medical University, Guangzhou, China

    State Key Laboratory of Organ Failure Research, Southern Medical University, Guangzhou, China
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  • Shenhai Gong
    Affiliations
    Department of Pathophysiology, School of Basic Medical Sciences, Guangdong Provincial Key Laboratory of Proteomics, Southern Medical University, Guangzhou, China

    State Key Laboratory of Organ Failure Research, Southern Medical University, Guangzhou, China
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  • Sanda Win
    Affiliations
    University of Southern California Research Center for Liver Diseases, Keck School of Medicine, University of Southern California, Los Angeles
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  • Neil Kaplowitz
    Affiliations
    University of Southern California Research Center for Liver Diseases, Keck School of Medicine, University of Southern California, Los Angeles
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  • Yong Jiang
    Correspondence
    Address correspondence to Peng Chen, Ph.D., or Yong Jiang, Ph.D., Department of Pathophysiology, Southern Medical University, N. No.1838, Guangzhou Ave., Guangzhou, Guangdong, 510515, China.
    Affiliations
    Department of Pathophysiology, School of Basic Medical Sciences, Guangdong Provincial Key Laboratory of Proteomics, Southern Medical University, Guangzhou, China

    State Key Laboratory of Organ Failure Research, Southern Medical University, Guangzhou, China
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  • Peng Chen
    Correspondence
    Address correspondence to Peng Chen, Ph.D., or Yong Jiang, Ph.D., Department of Pathophysiology, Southern Medical University, N. No.1838, Guangzhou Ave., Guangzhou, Guangdong, 510515, China.
    Affiliations
    Department of Pathophysiology, School of Basic Medical Sciences, Guangdong Provincial Key Laboratory of Proteomics, Southern Medical University, Guangzhou, China

    State Key Laboratory of Organ Failure Research, Southern Medical University, Guangzhou, China

    Microbiome Medicine Center, Zhujiang Hospital, Southern Medical University, Guangzhou, China
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Open ArchivePublished:October 11, 2019DOI:https://doi.org/10.1016/j.ajpath.2019.09.009
      Acetaminophen (APAP) overdose–induced hepatotoxicity is the leading cause of drug-induced liver injury worldwide. The related injury pathogenesis is mainly focused on the liver. Here, the authors report that gut barrier disruption may also be involved in APAP hepatotoxicity. APAP administration led to gut leakiness and colonic epithelial chemokine (C-C motif) ligand 7 (CCL7) up-regulation. Intestinal epithelial cell (IEC)-specific CCL7 transgenic mice (CCL7tgIEC mice) showed markedly increased myosin light chain kinase phosphorylation, and elevated gut permeability and bacterial translocation into the liver compared to wild-type mice. Global transcriptome analysis revealed that the expression of hepatic proinflammatory genes was enhanced in CCL7tgIEC mice compared with wild-type animals. Moreover, CCL7 overexpression in intestinal epithelial cells significantly augmented APAP-induced acute liver injury. These data provide new evidence that dysfunction of CCL7-mediated gut barrier integrity may be an important contributor to APAP-induced hepatotoxicity.
      Acetaminophen (APAP) is a common over-the-counter drug and is generally safe at recommended therapeutic doses. However, APAP overdose is the predominant cause of acute liver failure in the Western world.
      • Lee W.M.
      Acetaminophen (APAP) hepatotoxicity-isn't it time for APAP to go away?.
      ,
      • Bernal W.
      • Auzinger G.
      • Dhawan A.
      • Wendon J.
      Acute liver failure.
      The pathogenesis of APAP hepatotoxicity is complex. Briefly, APAP is metabolized into N-acetyl-p-benzoquinone-imine (NAPQI) by CYP2E1 in the liver and is then conjugated and detoxified with glutathione (GSH). Upon exhausting hepatic GSH, excess NAPQI ultimately leads to mitochondrial damage and hepatocyte death.
      • Lancaster E.M.
      • Hiatt J.R.
      • Zarrinpar A.
      Acetaminophen hepatotoxicity: an updated review.
      ,
      • Chun L.J.
      • Tong M.J.
      • Busuttil R.W.
      • Hiatt J.R.
      Acetaminophen hepatotoxicity and acute liver failure.
      It is believed that APAP mainly targets the liver and has low side effects on the intestine.
      • Marin T.M.
      • de Carvalho Indolfo N.
      • Rocco S.A.
      • Basei F.L.
      • de Carvalho M.
      • de Almeida Goncalves K.
      • Pagani E.
      Acetaminophen absorption and metabolism in an intestine/liver microphysiological system.
      However, recent reports have shown that APAP was able to change the Caco-2 cell membrane properties and influence intestinal functional gene expression in vitro,
      • Schafer C.
      • Schroder K.R.
      • Hoglinger O.
      • Tollabimazraehno S.
      • Lornejad-Schafer M.R.
      Acetaminophen changes intestinal epithelial cell membrane properties, subsequently affecting absorption processes.
      ,
      • Ghanem C.I.
      • Arias A.
      • Novak A.
      • Carpini G.D.
      • Villanueva S.
      • Blazquez A.G.
      • Marin J.J.
      • Mottino A.D.
      • Rubio M.C.
      Acetaminophen-induced stimulation of MDR1 expression and activity in rat intestine and in LS 174T human intestinal cell line.
      thus indicating that gut barrier function may be involved in APAP toxicity. However, the details of the functional link between APAP and intestinal barrier integrity are poorly understood.
      The gut barrier is predominately formed by intestinal epithelial cell (IEC) connections, and it effectively provides a biochemical and physical barrier that limits bacterial toxins and antigens from passing through the mucosa.
      • Marchiando A.M.
      • Graham W.V.
      • Turner J.R.
      Epithelial barriers in homeostasis and disease.
      ,
      • Peterson L.W.
      • Artis D.
      Intestinal epithelial cells: regulators of barrier function and immune homeostasis.
      The gut barrier integrity is mainly regulated by the intestinal inflammatory microenvironment.
      • Munoz L.
      • Borrero M.J.
      • Ubeda M.
      • Conde E.
      • Del Campo R.
      • Rodriguez-Serrano M.
      • Lario M.
      • Sanchez-Diaz A.M.
      • Pastor O.
      • Diaz D.
      • Garcia-Bermejo L.
      • Monserrat J.
      • Alvarez-Mon M.
      • Albillos A.
      Intestinal immune dysregulation driven by dysbiosis promotes barrier disruption and bacterial translocation in rats with cirrhosis.
      Specifically, inflammatory factors released either from immune cells or epithelial cells trigger key intracellular kinase activation, such as myosin light chain kinase (MLCK); in turn, MLCK disrupts tight junction expression and finally causes elevated gut permeability.
      • Cunningham K.E.
      • Turner J.R.
      Myosin light chain kinase: pulling the strings of epithelial tight junction function.
      Bacteria or their products could then penetrate into the circulatory system and access remote organs through the leaky gut and then activate immune cells to induce low-grade inflammation that sensitizes organs to toxin-induced damage, which mainly influences the liver.
      • Giannelli V.
      • Di Gregorio V.
      • Iebba V.
      • Giusto M.
      • Schippa S.
      • Merli M.
      • Thalheimer U.
      Microbiota and the gut-liver axis: bacterial translocation, inflammation and infection in cirrhosis.
      In the present study, the authors explored the detailed association between APAP toxicity and gut leakiness in an animal model, and determined the key intestinal molecule that mediates APAP hepatotoxicity.

      Materials and Methods

      Animal Model

      Six– to 8–week-old male specific-pathogen–free C57BL/6J mice were used. Intestinal epithelial CCL7–specific transgenic mice (carrying the human CCL7 gene, CCL7tgIEC mice, C57BL/6J background) and their littermates used in this study were generated and purchased from GemPharmatech Co. (Nanjing, China). The mice (nonfasted) were injected intraperitoneally with 300 mg/kg APAP dissolved in phosphate-buffered saline (PBS) and were sacrificed 3 or 24 hours after APAP treatment. Mice were fed ad libitum and were housed in a temperature-controlled room on a 12 hour/12 hour light/dark cycle. All animal experimental protocols were in agreement with the NIH guidelines and were approved by the Animal Care and Use Committee of the Southern Medical University.

      Histologic Analysis

      Ten percent buffered formalin was used to fix the harvested tissues. The sample was embedded in paraffin and sliced, and then hematoxylin and eosin staining was performed. Immunofluorescence and immunohistochemistry were performed using the primary antibodies for occludin (Invitrogen, Carlsbad, CA), claudin-4 (Invitrogen), and CD11b (Abcam, Cambridge, UK). For necrosis area quantification, 6 to 8 random fields per slide were examined with microscopy and analyzed by ImageJ software version 1.8.0 (NIH, Bethesda, MD; https://imagej.nih.gov/ij).

      Western Blot Analysis

      Total protein was isolated with lysis buffer (Thermo Fisher Scientific, Waltham, MA). Protein levels were analyzed by Western blot through primary antibodies targeting CYP2E1 (Proteintech, Wuhan, China), p-MLCK (Thermo Fisher Scientific), occludin (Invitrogen), claudin-4 (Invitrogen), Actin (Cell Signaling Technology, Danvers, MA), and GAPDH (Cell Signaling Technology).

      Determination of APAP Adducts

      APAP-protein adducts (APAP-AD) were determined as previously described.
      • Win S.
      • Than T.A.
      • Min R.W.
      • Aghajan M.
      • Kaplowitz N.
      c-Jun N-terminal kinase mediates mouse liver injury through a novel Sab (SH3BP5)-dependent pathway leading to inactivation of intramitochondrial Src.
      Liver samples were collected 2 hours after APAP treatment and homogenized in radioimmunoprecipitation assay buffer with protease and phosphatase inhibitor cocktails. An equal protein amount was used for SDS-PAGE electrophoresis and transferred to nitrocellulose membrane. Membrane was incubated on a shaker with 5% milk in tris-buffered saline (TBS) with 0.05% Tween-20 (TBS-T) for 1 hour at room temperature. The blocking buffer was discarded, and the membrane was incubated on a shaker with polyclonal anti-serum for APAP-AD (1:2000 diluted in 5% bovine serum albumin–TBS) overnight at 8°C. Next, the membrane was transferred to a washing buffer (TBS-T) and kept on a shaker for 10 minutes at room temperature, and the washing step was repeated three times. The washing buffer was discarded, and the membrane was incubated on a shaker with anti-rabbit IgG–horseradish peroxidase (1:2000 diluted in 5% milk TBS-T) for 1 hour at room temperature. The washing step was repeated before developing the signal on film with enhanced chemiluminescence reagents. For a loading control, the membrane used to determine APAP-AD was stripped with stripping buffer (BioLand Scientific, Paramount, CA), blocked with 5% milk–TBS-T, and incubated with anti–GAPDH–horseradish peroxidase (Sigma Aldrich, St. Louis, MO). Multiple difference exposure confirmed the linear range of signal.
      Immunoblots were scanned and signal intensity was semiquantitated using ImageJ software. The full length of each lane in a single blot was selected to measure densitometry quantitation of APAP-AD. A single specific band of GAPDH was selected to measure densitometry. The densitometry ratio was calculated as APAP-AD/GAPDH.

      Biochemical Analysis

      The serum levels of alanine aminotransferase (ALT) activity were measured with commercial kits (Jiancheng Bioengineering, Nanjing, China) according to the manufacturer's instructions. Human and mouse CCL7 protein levels were detected by an enzyme-linked immunosorbent assay kit (CUSABIO, Wuhan, China).

      Gene Expression Analysis

      TRIzol reagent was used to isolate total RNA, and a reverse transcription enzyme (TOYOBO, Shanghai, China) was employed for reverse transcription. The real-time quantitative PCR primers are shown in Table 1. Transcriptome analysis has been described previously.
      • Zhang Q.
      • Lai M.M.
      • Lou Y.Y.
      • Guo B.H.
      • Wang H.Y.
      • Zheng X.Q.
      Transcriptome altered by latent human cytomegalovirus infection on THP-1 cells using RNA-seq.
      Briefly, 3 μg of total RNA of each sample was used to construct the Illumina sequencing libraries according to the manufacturer's instructions (Illumina, San Diego, CA). The libraries were sequenced using the Illumina NovaSeq 6000 platform to generate high-quality paired-end reads. The function and pathway enrichment for differentially expressed coding genes were referred by Gene Ontology (GO; http://geneontology.org) functional annotations and KEGG database (https://www.genome.jp/kegg/pathway.html). Raw sequencing data have been uploaded to the BioProject database (https://www.ncbi.nlm.nih.gov/bioproject; accession number PRJNA555333).
      Table 1PCR Primers Used in This Study
      GenePrimer pair
      18sLeft5′-CGATCCGAGGGCCTCACTA-3′
      Right5′-AGTCCCTGCCCTTTGTACACA-3′
      16sLeft5′-GTGSTGCAYGGYTGTCGTCA-3′
      Right5′-ACGTCRTCCMCACCTTCCTC-3′
      TNFALeft5′-CCACCACGCTCTTCTGTCTAC-3′
      Right5′-AGGGTCTGGGCCATAGAACT-3′
      IL6Left5′-TGATGCACTTGCAGAAAACA-3′
      Right5′-ACCAGAGGAAATTTTCAATAGGC-3′
      IL1BLeft5′-TGTGAAATGCCACCTTTTGA-3′
      Right5′-GGTCAAAGGTTTGGAAGCAG-3′
      CCL2Left5′-CCTGCTGTTCACAGTTGCC-3′
      Right5′-ATTGGGATCATCTTGCTGGT-3′
      CCL3Left5′-ACCATGACACTCTGCAACCA-3′
      Right5′-GTGGAATCTTCCGGCTGTAG-3
      CCL4Left5′-CATGAAGCTCTGCGTGTCTG-3′
      Right5′-GAAACAGCAGGAAGTGGGAG-3′
      CCL5Left5′-GTGCCCACGTCAAGGAGTAT-3′
      Right5′-CCACTTCTTCTCTGGGTTGG-3′
      CCL7(Mus)Left5′-CTGCTTTCAGCATCCAAGTG-3′
      Right5′-TTCCTCTTGGGGATCTTTTG-3′
      CCL7(Homo)Left5′-GAAGGACCACCAGTAGCCACTGTC-3′
      Right5′-AGTCCTGGACCCACTTCTGTGTG-3′
      CCL8Left5′-TCTTTGCCTGCTGCTCATAG-3′
      Right5′-GAAGGGGGATCTTCAGCTTT-3′
      CXCL1Left5′-CCACACTCAAGAATGGTCGC-3′
      Right5′-TCTCCGTTACTTGGGGACAC-3′
      CXCL2Left5′-CGGTCAAAAAGTTTGCCTTG-3′
      Right5′-TCCAGGTCAGTTAGCCTTGC-3′
      CXCL10Left5′-CTCATCCTGCTGGGTCTGAG-3′
      Right5′-CCTATGGCCCTCATTCTCAC-3′
      TLR2Left5′-AGGGGCTTCACTTCTCTGCT-3′
      Right5′-CCAAAGAGCTCGTAGCATCC-3′
      TLR4Left5′-GGACTCTGATCATGGCACTG-3′
      Right5′-TGTCATCAGGGACTTTGCTG-3′
      TLR9Left5′-GAATCCTCCATCTCCCAACA-3′
      Right5′-CAGCTCACAGGGTAGGAAGG-3′
      CYP2E1Left5′-GGCTGTCAAGGAGGTGCTAC-3′
      Right5′-CCTTCCATGTGGGTCCATTA-3′

      FD-4 Permeability Experiment

      In vivo gut leakiness was assessed by fluorescein isothiocyanate–dextran 4 KDa (FD-4) (Sigma, Shanghai, China) penetration. Mice were intragastrically administered with FD-4 (1 mg/kg) and 3 hours later, plasma FD-4 level was determined spectrofluorometrically with a 485-nm excitation wavelength and 530-nm emission wavelength in a microplate fluorescence reader.

      Fecal Albumin Measurement

      Fecal samples collected before sacrifice were immediately frozen and stored at −80°C until analysis. Fecal albumin concentration was determined by enzyme-linked immunosorbent assay (Bethyl Laboratories, Montgomery, AL) according to the manufacturer's instructions.

      Intestinal Epithelial Cell Isolation

      The mouse intestines were rinsed in ice-cold PBS, opened longitudinally, and cut into 1-cm–long pieces. These pieces were shaken in ice-cold RPMI1640 medium (Gibco; Thermo Fisher Scientific) with 5% fetal calf serum and dithiothreitol (1 mmol/L). The supernatant was discarded, and the tissue was incubated in RPMI1640 with 5% fetal calf serum and 1 mmol/L EDTA at 37°C with shaking for 20 minutes. To completely isolate the epithelial cells, this step was repeated.

      Glutathione Assay

      Glutathione (reduced) was measured in liver homogenate using reduced GSH assay kit (Jiancheng Bioengineering) according to the manufacturer's instructions. Briefly, GSH in the samples reacted with DNTB [5,5′-dithiobis(2-nitrobenzoic acid)] and generated a yellow compound. After the chromogenic reaction, the optical density at 405 nm was recorded, and the concentration was calculated.

      Statistical Analysis

      The results are expressed as means ± SEM. A two-tailed t-test was used for statistical analysis. Statistical differences between groups were analyzed; P < 0.05 was considered significant.

      Results

      APAP Induces Gut Permeability and Colonic Epithelial CCL7 Overexpression

      The mouse APAP-induced liver injury model was first established, and the effects of APAP on the intestine were examined (Figure 1A). The gut permeability was evaluated by circulatory FD-4 and fecal albumin levels. The FD-4 concentration in the plasma of the APAP group was significantly higher than that in the PBS group at both 3 hours and 24 hours after APAP (Figure 1B). Furthermore, fecal albumin was prominently increased in the APAP group at both 3 hours and 24 hours after APAP (Figure 1C). Because gut barrier dysfunction is accompanied with bacterial translocation, the 16s/18s ratio was higher in liver of APAP-treated mice (Figure 1D). Additionally, APAP treatment markedly decreased tight junction occludin and claudin-4 protein levels (Figure 1E), and immunofluorescence also showed a significantly disrupted colonic epithelium barrier (Figure 1F).
      Figure thumbnail gr1
      Figure 1Acetaminophen (APAP) both induces hepatotoxicity and develops gut barrier defects and colon epithelial inflammation. C57BL/6J mice were treated with APAP for 3 and 24 hours, and the control group was treated with phosphate-buffered saline (PBS). A: Plasma alanine aminotransferase (ALT) levels at 24 hours after APAP. B: Fluorescein isothiocyanate–dextran 4 KDa (FD-4) level in the plasma at 3 hours and 24 hours after APAP. C: Fecal albumin content at 3 hours and 24 hours after APAP. D: Relative 16s/18s ratio in the liver. E: Occludin and claudin-4 protein levels in the colon at 3 hours and 24 hours after APAP. F: Representative immunofluorescence staining for occludin and claudin-4 in the colon at 3 hours and 24 hours after APAP. G: mRNA levels of cytokines and chemokines in the colon and colonocyte at 24 hours after APAP. The Black box indicates the CCL7 mRNA expression that was focused on for the following study. Data are expressed as means ± SEM. n = 6 to 8. *P < 0.05. Original magnification, ×400.
      Because a leaky gut is predominantly induced by intestinal inflammation, the main cytokines and chemokines produced throughout the entire colonic tissue and colonic enterocytes were detected. Although there were no obvious changes in these cytokines and chemokines throughout the colon, CCL7 was observed to exhibit the greatest elevation among all inflammatory factors in the colonic epithelial cells (Figure 1G). Furthermore, CCL7 expression in colonic lamina propria was comparable between the PBS and APAP groups (Supplemental Figure S1A). These data indicated that colonic epithelial cells, but not immune cells, may be the primary cells expressing CCL7 in APAP-overdosed mice. In addition, the CCL7 relative expression is higher in liver after APAP treatment (Supplemental Figure S1B), which resulted from the toxic and proinflammatory effects of APAP. These results revealed that APAP treatment caused development of mucosal damage, including an increase in intestinal permeability and increased expression of CCL7 in the colonic epithelium.

      IEC Overexpression of CCL7 Exacerbates APAP Hepatotoxicity

      To further define the role of CCL7 from enterocytes in mediating the susceptibility of APAP hepatotoxicity in mice, transgenic mice that express constitutively active CCL7 specifically within the intestinal epithelia were chosen (CCL7tgIEC mice). Vector construction is shown in Figure 2, A and B . It was confirmed that CCL7 expression is much higher in CCL7tgIEC mice than wild-type (WT) mice at both mRNA and protein levels in isolated intestinal epithelial cells (Figure 2, C and D). The CCL7 protein expression was not altered in the liver of CCL7tgIEC mice (Supplemental Figure S2A). CCL7tgIEC mice displayed more severe liver damage upon APAP treatment, as revealed by significantly increased plasma ALT (Figure 2E), markedly increased necrotic area compared with WT mice (Figure 2F), and more hepatic CD11b-positive cells after APAP in CCL7tgIEC mice (Supplemental Figure S2B). Taken together, APAP-induced hepatotoxicity is more prominent in mice with CCL7 overexpressed in intestinal epithelium cells.
      Figure thumbnail gr2
      Figure 2Chemokine (C-C motif) ligand (CCL7) overexpressed in intestinal epitheliums enhances acetaminophen (APAP)-induced hepatotoxicity. A and B: Vector construction for CCL7tgIEC mice generation. C: CCL7 mRNA levels in isolated intestinal epithelium cells. D: CCL7 protein levels in isolated intestinal epithelium cells. E: Plasma alanine aminotransferase (ALT) levels after 24 hours APAP treatment. F: Histopathological examination of hepatic hematoxylin and eosin staining (left panel, green dashed lines indicate necrotic areas) and quantification of the necrotic area after 24 hours of APAP treatment (right panel). Data are expressed as means ± SEM. n = 8 to 10. *P < 0.05. Original magnification, ×200. DSI, distal small intestine; PSI, proximal small intestine; WT, wild-type.

      Hepatic APAP Metabolism Is Not Altered in CCL7tgIEC Mice

      GSH levels and APAP protein adduct (APAP-AD) formation are key modulators in the development of APAP hepatotoxicity.
      • Jaeschke H.
      • McGill M.R.
      • Ramachandran A.
      Oxidant stress, mitochondria, and cell death mechanisms in drug-induced liver injury: lessons learned from acetaminophen hepatotoxicity.
      ,
      • Hinson J.A.
      • Roberts D.W.
      • James L.P.
      Mechanisms of acetaminophen-induced liver necrosis.
      To preclude the possibility that CCL7 overexpressed on IECs increased APAP-induced hepatotoxicity as a result of altered GSH and CYP2E1 levels, the depletion of GSH and APAP-AD formation 2 hours after APAP administration was examined in the liver. No differences were observed between CCL7tgIEC mice and WT mice (Figure 3, A and B ). Additionally, the gene expression levels and protein levels of CYP2E1, the main metabolic enzyme of APAP, were measured. No changes were observed in the baseline characteristics (Figure 3C). These data indicated that APAP-induced augmented hepatotoxicity in CCL7tgIEC mice was not dependent on the alteration of metabolism process.
      Figure thumbnail gr3
      Figure 3Hepatic acetaminophen (APAP) metabolism is not affected in CCL7tgIEC mice. A: Glutathione (GSH) levels in the liver homogenates after APAP treatment for 2 hours. B: APAP adducts levels in the livers after APAP treatment for 2 hours (the numbers inside the bottom box are the relative density of APAP adducts to GAPDH). C: CYP2E1 mRNA and protein levels in the liver. Data are expressed as means ± SEM. n = 4 to 6. *P < 0.05. WT, wild-type.

      CCL7 Overexpression in IECs Induces Gut Barrier Defects and Bacterial Translocation

      The proinflammatory mediators during inflammatory response have been shown to increase gut permeability,
      • Suzuki T.
      • Yoshinaga N.
      • Tanabe S.
      Interleukin-6 (IL-6) regulates claudin-2 expression and tight junction permeability in intestinal epithelium.
      • Al-Sadi R.
      • Ye D.
      • Dokladny K.
      • Ma T.Y.
      Mechanism of IL-1beta-induced increase in intestinal epithelial tight junction permeability.
      • Capaldo C.T.
      • Nusrat A.
      Cytokine regulation of tight junctions.
      and gut barrier dysfunction may sensitize liver to toxin-induced injury responses. To decipher the potential mechanism by which intestinal epithelial CCL7 promotes APAP-induced liver damage, the integrity of the intestinal mucosa was studied by measuring the plasma FD-4 and fecal albumin levels in CCL7tgIEC mice. As expected, the penetration of FD-4 and the levels of fecal albumin were higher in mice with the selective activation of CCL7 on intestinal epithelial cells than in controls (Figure 4, A and B ). The expression of the key tight-junction proteins occludin and claudin-4 was assessed in the colon. CCL7tgIEC mice exhibited significantly decreased levels of these proteins compared with WT mice (Figure 4, C and D). Electron microscopy also confirmed the disrupted colonic tight junctions (Figure 4E). Phosphorylation of MLCK, a key regulator of tight junction expression and gut barrier integrity,
      • Chelakkot C.
      • Ghim J.
      • Ryu S.H.
      Mechanisms regulating intestinal barrier integrity and its pathological implications.
      was markedly enhanced in the colon of CCL7tgIEC mice compared with WT animals (Figure 4F). These data clearly demonstrate that MLCK activation maybe involved in the CCL7-mediated alteration of tight junctions and gut barrier dysfunction.
      Figure thumbnail gr4
      Figure 4CCL7 overexpression in intestinal epithelial cells (IECs) leads to gut barrier dysfunction and promotes bacterial translocation into the liver. A: Fluorescein isothiocyanate–dextran 4 KDa (FD-4) level in the plasma. B: Fecal albumin (Alb) content. C: Occludin and claudin-4 protein levels in the colon. D: Representative immunofluorescence staining for occludin and claudin-4 in the colon. E: Electron microscopy of mice colon. Arrows indicate the connection between two epithelia cells. F: Western blotting for phosphorylated myosin light chain kinase (p-MLCK) in the colon. G: Plasma endotoxin level and relative 16s/18s ratio in the liver. H: mRNA levels of toll-like receptors (TLRs) in the liver. I: mRNA levels of key cytokines and chemokines in the liver. Data are expressed as means ± SEM. n = 4 to 6. *P < 0.05. Original magnification: ×400 (D); ×40,000 (E). WT, wild-type.
      The onset of increased intestinal permeability could lead to bacterial product translocation into the systemic circulation and remote organs.
      • Wiest R.
      • Lawson M.
      • Geuking M.
      Pathological bacterial translocation in liver cirrhosis.
      ,
      • Hu J.
      • Luo H.
      • Wang J.
      • Tang W.
      • Lu J.
      • Wu S.
      • Xiong Z.
      • Yang G.
      • Chen Z.
      • Lan T.
      • Zhou H.
      • Nie J.
      • Jiang Y.
      • Chen P.
      Enteric dysbiosis-linked gut barrier disruption triggers early renal injury induced by chronic high salt feeding in mice.
      Plasma endotoxin levels and 16s bacterial DNA were measured to recognize bacterial translocation. The plasma endotoxin levels and hepatic 16s/18s ratio were higher in CCL7tgIEC mice than WT, which is consistent with the increased gut permeability observed in transgenic mice (Figure 4G). In addition, increased hepatic toll-like receptor mRNA levels, particularly TLR2, were detected in CCL7tgIEC mice (Figure 4H), which may induce a low-grade inflammation. Consequently, intrahepatic cytokines such as tumor necrosis factor-α (TNF-α), IL-1β, and IL-6 or other key chemokines were all increased in CCL7tgIEC mice compared with WT mice (Figure 4I). These data indicate that IECs with CCL7 overexpression exhibited disruption of the integrity of the intestinal barrier, leading to bacterial translocation and the initiation of an inflammatory response in the liver.

      Hepatic Transcriptome Analysis Confirms Inflammatory Signaling Activation in CCL7tgIEC Mice

      Because bacterial translocation promotes liver inflammation, the hepatic transcriptomes of CCL7tgIEC mice and WT littermates were profiled. The expression profile of the two groups could clearly be separated by principal component analysis (Figure 5A). The heat map shows the up-regulated and down-regulated genes in CCL7tgIEC mice (Figure 5B). These results indicate that CCL7 overexpression in IECs induces prominent transcriptome alterations. Further, Gene Ontology (GO) enrichment analysis was employed on differentially expressed genes. The top 20 enrichment map highlighted that IEC CCL7 overexpression up-regulated several inflammatory pathways such as T-cell aggregation and activation, response to cytokines, and regulation of the immune system process (Figure 5C). Similar to GO analysis, the KEGG pathways indicate that hepatic inflammatory pathways including the TNF signaling pathway, cytokine–cytokine receptor interaction, and toll-like receptor signaling pathway were all altered between the two strains (Figure 5D). Importantly, among the genes associated with inflammation pathway, the up-regulated genes were much more abundant than down-regulated genes in CCL7tgIEC mice compared with controls (Figure 5E). These data clearly reveal that the immune system was activated in the liver of CCL7tgIEC mice, which indicates that the livers of these mice had low-grade inflammation at baseline.
      Figure thumbnail gr5
      Figure 5Liver transcriptome analysis. A: Principal component analysis displaying the overall gene expression profiles across all samples between WT and CCL7tgIEC mice. B: The heat map depicting the gene expression alteration of the differentially expressed genes. C: The top 20 enrichment GO pathways of differentially expressed genes. D: The key KEGG pathways with P < 0.05 related to inflammation activation. E: Up-regulated and down-regulated gene counts for the inflammation pathways in CCL7tgIEC mice. n = 5. WT, wild-type.

      Discussion

      The present study demonstrates that APAP overdose increases gut permeability and induces CCL7 expression in colonic epithelial cells, and therefore provides novel insights into the role of CCL7 in mediating gut barrier dysfunction and determining susceptibility to APAP-induced liver damage. In vitro, APAP changes the topography of Caco-2 cells; even subtoxic doses of APAP can induce gene transcription alteration in the intestinal epithelium.
      • Schafer C.
      • Schroder K.R.
      • Hoglinger O.
      • Tollabimazraehno S.
      • Lornejad-Schafer M.R.
      Acetaminophen changes intestinal epithelial cell membrane properties, subsequently affecting absorption processes.
      ,
      • Ghanem C.I.
      • Arias A.
      • Novak A.
      • Carpini G.D.
      • Villanueva S.
      • Blazquez A.G.
      • Marin J.J.
      • Mottino A.D.
      • Rubio M.C.
      Acetaminophen-induced stimulation of MDR1 expression and activity in rat intestine and in LS 174T human intestinal cell line.
      In this study, APAP overdose strongly increased the CCL7 mRNA level in colonocytes and remarkably increased gut permeability. Although there were no obvious differences in CCL7 protein levels in colon epithelium between the PBS and APAP groups (Supplemental Figure S1C), it cannot be excluded that CCL7 could be transiently secreted from colonic epithelial cells upon APAP overdose. Once the gut barrier is disrupted, bacteria translocation into liver occurs and finally induces inflammatory responses. Although the inflammation may contribute to hepatocyte necrosis, it is also possible that the factors released from permeabilized gut could directly target the hepatocytes and sensitize the early events in APAP-induced necrosis.
      Intestinal homeostasis is maintained through the interplay of the intestinal mucosa, immune activation, and microbiota.
      • Hill D.A.
      • Artis D.
      Intestinal bacteria and the regulation of immune cell homeostasis.
      Variations in intestinal microbiota could influence APAP hepatotoxicity.
      • Possamai L.A.
      • McPhail M.J.
      • Khamri W.
      • Wu B.
      • Concas D.
      • Harrison M.
      • Williams R.
      • Cox R.D.
      • Cox I.J.
      • Anstee Q.M.
      • Thursz M.R.
      The role of intestinal microbiota in murine models of acetaminophen-induced hepatotoxicity.
      ,
      • Gong S.
      • Lan T.
      • Zeng L.
      • Luo H.
      • Yang X.
      • Li N.
      • Chen X.
      • Liu Z.
      • Li R.
      • Win S.
      • Liu S.
      • Zhou H.
      • Schnabl B.
      • Jiang Y.
      • Kaplowitz N.
      • Chen P.
      Gut microbiota mediates diurnal variation of acetaminophen induced acute liver injury in mice.
      Within the gastrointestinal mucosa, chemokines, cytokines, and other small molecules play key roles in lymphocyte and leukocyte recirculation, and maintain the balance of mucosal immunity.
      • Ahmad R.
      • Sorrell M.F.
      • Batra S.K.
      • Dhawan P.
      • Singh A.B.
      Gut permeability and mucosal inflammation: bad, good or context dependent.
      Chemokines can lead to increased immune cell infiltration into the lamina propia and to subsequent inflammation and tissue damage. Consistent with this, CD11b-positive immune cells were recruited into the colon lamina propia in CCL7tgIEC during the early phase of APAP challenge (Supplemental Figure S2C). Cytokine-mediated barrier dysfunction contributes to the pathology of intestinal diseases, during which MLCK is activated and plays a key role in cytokine-induced barrier loss.
      • Chen P.
      • Starkel P.
      • Turner J.R.
      • Ho S.B.
      • Schnabl B.
      Dysbiosis-induced intestinal inflammation activates tumor necrosis factor receptor I and mediates alcoholic liver disease in mice.
      MLCK phosphorylation was associated with CCL7-induced tight junction dysregulation. However, it is unclear whether MLCK phosphorylation alone is sufficient to regulate CCL7-mediated tight junction permeability. A more detailed signaling pathway should be studied in the future.
      Gut barrier dysfunction and bacterial translocation are common in liver diseases such as non-alcoholic fatty liver disease and cirrhosis.
      • Fouts D.E.
      • Torralba M.
      • Nelson K.E.
      • Brenner D.A.
      • Schnabl B.
      Bacterial translocation and changes in the intestinal microbiome in mouse models of liver disease.
      ,
      • Hackstein C.P.
      • Assmus L.M.
      • Welz M.
      • Klein S.
      • Schwandt T.
      • Schultze J.
      • Forster I.
      • Gondorf F.
      • Beyer M.
      • Kroy D.
      • Kurts C.
      • Trebicka J.
      • Kastenmuller W.
      • Knolle P.A.
      • Abdullah Z.
      Gut microbial translocation corrupts myeloid cell function to control bacterial infection during liver cirrhosis.
      Gut-derived bacterial products or components such as microbe-associated molecular patterns induce a proinflammatory intrahepatic milieu.
      • Chu H.
      • Duan Y.
      • Yang L.
      • Schnabl B.
      Small metabolites, possible big changes: a microbiota-centered view of non-alcoholic fatty liver disease.
      ,
      • Shi H.
      • Lv L.
      • Cao H.
      • Lu H.
      • Zhou N.
      • Yang J.
      • Jiang H.
      • Dong H.
      • Hu X.
      • Yu W.
      • Jiang X.
      • Zheng B.
      • Li L.
      Bacterial translocation aggravates CCl4-induced liver cirrhosis by regulating CD4(+) T cells in rats.
      Blocking bacterially derived products such as lipopolysaccharide has been shown to ameliorate liver injury.
      • Su G.L.
      • Hoesel L.M.
      • Bayliss J.
      • Hemmila M.R.
      • Wang S.C.
      Lipopolysaccharide binding protein inhibitory peptide protects against acetaminophen-induced hepatotoxicity.
      Inflammatory mediators and toll-like receptors that were activated, particularly TLR2 in CCL7tgIEC mice, were detected and showed a proinflammatory pattern of immune dysregulation associated with accelerated progression of APAP-induced liver injury (Figure 4). Thus, the disturbed intestinal barrier could be defined as inducing a proinflammatory response in the liver. The analysis of hepatic transcriptomes for CCL7tgIEC mice and WT littermates at baseline suggests that the related gene alterations belong to several inflammatory pathways, thus the liver of the CCL7tgIEC mice had low-grade inflammation at baseline. Upon APAP challenge, the immune reaction was further activated in the liver, and more injury occurred. However, the roles of the mucosal immune system in the effects of epithelial CCL7 overexpression on the gut barrier need further study. Furthermore, although baseline inflammation in the liver in CCL7tgIEC mice was enhanced, it is unclear whether this hepatic inflammation or its enhancement after gut permeabilization directly influences the APAP-induced liver damage. Alternatively, the bacterial products or metabolites released from the gut may directly sensitize APAP toxicity. Greater permeabilization in the transgenic mice may then enhance the exposure of the liver to gut products after APAP.
      Reportedly, the blockade of HMGB1 may present a novel therapy to prevent organ failure from gut bacterial translocation in APAP overdose.
      • Yang R.
      • Zou X.
      • Tenhunen J.
      • Zhu S.
      • Kajander H.
      • Koskinen M.L.
      • Tonnessen T.I.
      HMGB1 neutralization is associated with bacterial translocation during acetaminophen hepatotoxicity.
      However, the detailed mechanism of how bacterial pathogen–associated molecular patterns or other microbial products trigger APAP-induced liver injury requires further study. Pretreatment with d-glucose protects the liver against APAP hepatotoxicity via the activation of the transporter SGLT-1 expressed by intestinal epithelial cells.
      • Zanobbio L.
      • Palazzo M.
      • Gariboldi S.
      • Dusio G.F.
      • Cardani D.
      • Mauro V.
      • Marcucci F.
      • Balsari A.
      • Rumio C.
      Intestinal glucose uptake protects liver from lipopolysaccharide and D-galactosamine, acetaminophen, and alpha-amanitin in mice.
      Therefore, targeting the intestine may provide a novel strategy to prohibit APAP hepatotoxicity.
      In conclusion, using tissue-specific genetically modified mice, this study is the first to reveal that IEC CCL7 disrupts intestinal the epithelial barrier function and to highlight the necessity of a fully operational mucosal barrier against APAP-induced liver failure. This study also provides new evidence that the disruption of colonic integrity accompanies APAP hepatotoxicity. Maintaining intestinal integrity may serve as a novel strategy for combating APAP-induced liver damage. Further studies are required to understand the mechanism by which hepatic inflammation at baseline in CCL7tgIEC mice sensitizes the liver to APAP toxicity.

      Author Contributions

      M.N., Z.L., S.G., and S.W. acquired and analyzed data; N.K., Y.J., and P.C. conceived and designed the study, wrote the manuscript, and supervised the study.

      Supplemental Data

      • Supplemental Figure 1

        CCL7 expression of colon and liver in acetaminophen (APAP) overdosed mice. C57BL/6J mice were treated with APAP for 24 hours and the control group was treated with phosphate-buffered saline (PBS). A and B: CCL7 mRNA levels in colonic lamina propria (A) and liver (B). C: CCL7 protein levels in isolated intestinal epithelium cells. Data are expressed as means ± SEM. n = 4 to 6. *P < 0.05.

      • Supplemental Figure 2

        CCL7 expression and immune cell infiltration in CCL7tgIEC mice. A: CCL7 protein levels in liver of wild-type (WT) and CCL7tgIEC mice. B: WT and CCL7tgIEC mice were treated with acetaminophen (APAP) for 24 hours. Representative images of CD11b immunohistochemistry of liver. C: WT and CCL7tgIEC mice were treated with APAP for 2 hours. Representative images of CD11b immunohistochemistry of colon. Data are expressed as means ± SEM. n = 4 to 6 (A). Original magnification, ×200 (B and C).

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