Type I Interferon Signaling Accelerates Liver Regeneration by Metabolic Modulation in Noninfectious Conditions

Type I interferon (IFN-I) has a well-known function in controlling viral infections, but its contribution hepatocyte proliferation and hepatocellular carcinoma (HCC) formation remains unclear. Mice deficient IFN-α receptor expression whole mice or only hepatocytes (Ifnar−/− IfnarΔliver) were used to investigate the role of IFN-I signaling cell cancer liver. Ifnar−/− resistant chemical-induced HCC absence infection. The results show that low grade interferon-stimulated gene expressed substantially naïve mouse level activation is constantly present liver after weaning negatively modulates forkhead box O hepatic expression. can be partially blocked by clearance lipopolysaccharide. lacking have lower basal activity delayed regeneration processes two-thirds partial hepatectomy. on controls glucose homeostasis lipid metabolism support potency long-term tumorigenesis. Our reveal positive low-grade singling modulating metabolism. both metabolic immunoregulatory functions. 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French A.R. Piwnica-Worms D. Virgin H.W. Lenschow D.J. Stappenbeck T.S. interferons link enhanced repair.Cell Host Microbe. 2015; 17: 85-97Abstract (58) effect fate context dependent. In study, knockout (Ifnar−/−) examine development, proliferation, regeneration. A was found positively regulate metabolism, contribute development. Wild-type (WT) C57BL/6 male (C57BL/6 background)8Yu Scholar,13Muller Steinhoff Reis L.F. Hemmi Pavlovic Zinkernagel R.M. Aguet Functional II defense.Science. 1994; 264: 1918-1921Crossref (1931) maintained at National Health Research Institutes. IfnarF/F obtained from Jackson Laboratory (Bar Harbor, ME), albumin-Cre kindly provided Dr. Michael Karin. crossed obtain IfnarΔliver line. Unless specified, weaned 4 weeks age fed diet normal chow. All mouse-related experiments conducted compliance guidelines Animal Center Institutes approved animal protocols (National Institutes–Institutional Care Use Committee 100013, 102126, 103104, 104006, 106051). 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Antibodies studying (FoxO) 1, FoxO3, epidermal growth factor Met, cyclin D1, A2, B1, p27, p21, p15 purchased Cell Signaling Technology (Danvers, MA). Cyclin E1 Santa Cruz Biotechnology (Dallas, TX). Tubulin antibodies Sigma-Aldrich (St. MO). Adipose differentiation-related protein (ADRP) antibody PROGEN Biotechnik Gmbh (Heidelberg, Germany). anti-mouse APOL9 raised rabbits (Animal Institute, Taiwan) immunized purified His-tagged protein. Insulin intralipid emulsion Sigma-Aldrich. Paraffin-embedded sections antigen retrieval Target Retrieval Solution (S1700; DakoCytomation, Carpinteria, CA) 95°C 45 minutes stained anti–Ki-67 (GeneTX, Hsinchu, methods described previously.18Wu C.H. Tseng F.C. Yang H.J. Lo Y.C. Kuo Y.P. Interleukin-17F elevated patients fibrosis carcinoma.Infect Agent Cancer. 2017; 42Crossref (5) Ki-67 staining images taken under microscope quantified ImageJ version 1.50i software (NIH, Bethesda, MD; https://imagej.nih.gov/ij, last accessed March 26, 2016). For FOXO1 staining, performed citrate buffer, pH 6.0, 0.1% Tween-20 80°C 6 hours anti-FOXO1 (GeneTX). hematoxylin eosin pathology core laboratory cDNA synthesis quantitative real-time PCR primer sequences this study listed Table 1.Table 1Quantitative Real-Time Primer SequencesGeneForward sequenceReverse sequenceCph5′-ATGGTCAACCCCACCGTGT-3′5′-TTCTTGCTGTCTTTGGAACTTTGTC-3′Ifna15′-ACCCAGCAGATCCTGAACAT-3′5′-AATGAGTCTAGGAGGGTGTATTCC-3′Ifna25′-GTCCTGGCACAGATGAGGA-3′5′-ACCTTCTCCAGGGGGAATC-3′Ifna45′-TCAAGCCATCCTTGTGCTAA-3′5′-GTCTTTTGATGTGAAGAGGTTCAA-3′Ifna55′-GCCTTAACCCTCCTGGTAAAA-3′5′-TCCTGTGGGAATCCAAAGTC-3′Ifna65′-AGGACTTTGGATTCCCCTTG-3′5′-CTCAGTCAGGACAGGGATGG-3′Ifna75′-CTGCTGCTTGGGATGGAT-3′5′-ACAGCCTTGCAGGTCATTG-3′Ifna85′-GGAGGTGGTCAGAGCAGAAG-3′5′-TTGGTCTCAGGACTCACTCGT-3′Ifna95′-TTCCTGATGGTCTTGGTGGT-3′5′-TGTGCCAGGAGTGTCAAGAT-3′Ifna115′-AGGACTTTGGATTCCCCTTG-3′5′-AGGGATGGATTGAGCCTTCT-3′Ifna125′-TGACTCTCCTGGCACAAATG-3′5′-CCTTCTCCTGGGGGAATCTA-3′Ifna135′-GGTGGTCAGAGCAGAAGTCC-3′5′-TCCTCCTTGCTCAGTCTTGC-3′Ifna145′-GGAGGTGGTCAGAGCAGAAAT-3′5′-CTTTCAGGCTGGTCAGCAAC-3′Ifna155′-GCCATCCCTGTCCTACAAGA-3′5′-GCAGCAGATGAGTCCTTTGA-3′Ifna165′-GCCATCCCTGTCCTAAGTGA-3′5′-ATCCCAAGCAGCAGATGAAT-3′Ifnb5′-AGCTCCAAGAAAGGACGAACAT-3′5′-GCCCTGTAGGTGAGGTTGATCT-3′Ifng5′-ATCTGGAGGAACTGGCAAAA-3′5′-TTCAAGACTTCAAAGAGTCTGAGGTA-3′Apol95′-AGGCCATCTGGGTCCTGT-3′5′-TTGCCATGTTCTGAGATACCC-3′Ccnd15′-TTTCTTTCCAGAGTCATCAAGTGT-3′5′-TGACTCCAGAAGGGCTTCAA-3′Ccne15′-TTTCTGCAGCGTCATCCTC-3′5′-TGGAGCTTATAGACTTCGCACA-3′Ccna25′-CTTGGCTGCACCAACAGTAA-3′5′-CAAACTCAGTTCTCCCAAAAACA-3′Ccnb15′-GCGCTGAAAATTCTTGACAAC-3′5′-TTCTTAGCCAGGTGCTGCAT-3′E2f15′-GCCCTTGACTATCACTTTGGTCTC-3′5′-CCTTCCCATTTTGGTCTGCTC-3′E2f75′-GATGCGTTCGTGAACTCCCTG-3′5′-AGAAACTTCTGGCACAGCAGCC-3′E2f85′-GAGAAATCCCAGCCGAGTC-3′5′-CATAAATCCGCCGACGTT-3′G6pc5′-TGGCTGGAGTCTTGTCAGG-3′5′-GTAGAATCCAAGCGCGAAAC-3′Fasn5′-GTAAGTTCTGTGGCTCCAGAG-3′5′-GCCCTCCCGTACACTCACTC-3′Acly5′-CAGCCAAGGCAATTTCAGAGC-3′5′-CTCGACGTTTGATTAACTGGTCT-3′Acc5′-ACAGTGAAGGCTTACGTCTG-3′5′-AGGATCCTTACAACCTCTGC-3′Apob5′-GGAACAACTCTTGTTGGTCCTCT-3′5′-ATCCTGAACATCAAGAGGGG-3′Mtp5′-CCCAACGTACTTCTAATTTATGGTC-3′5′-TGCTGGCCAACACGTCTA-3′Cd365′-GATGACGTGGCAAAGAACAG-3′5′-TCCTCGGGGTCCTGAGTTAT-3′Ppara5′-TCGGCGAACTATTCGGCTG-3′5′-GCACTTGTGAAAACGGCAGT-3′ Open table tab Frozen fixed formalin 10 washed tap water. being rinsed 60% isopropanol water, 1.5% oil red (Sigma) water mixture (3:2) 20 further counterstained hematoxylin. Lipid droplet fractionation previously,19Brasaemle D.L. Wolins N.E. Isolation droplets density gradient centrifugation.Curr Protoc Biol. 72: 3.15.1-3.15.13Crossref (35) minor modification. brief, 300 mg livers lysed HLM buffer (20 mmol/L Tris-HCl, 7.4, EDTA, sodium fluoride, 1× Roche protease inhibitor) grinding glass Dounce homogenizer centrifuged 1000 × g, 4°C. post-nuclear supernatant mixed one-third volume ice-cold containing sucrose layered bottom ultracentrifuge tube SW 41 Ti rotor. total 5 mL 5% 0% top sequentially. Flotation 30 160,000 Fractions (1 mL/fraction) immunoblotting. 100 μg lysates, prepared radioimmunoprecipitation assay (10 8.0, 140 NaCl, 1% Triton X-100, deoxycholate, SDS), SDS-PAGE gel electrophoresis immunoblotting specific primary horseradish peroxidase–conjugated secondary (Jackson ImmunoResearch, West Grove, PA). blot developed SuperSignal Pico PLUS Chemiluminescent Substrate (Thermo Scientific, Rockford, IL). quantification within linear range. As closely associated repetitive processes, whether examined. Carcinogen DEN induce young injection.14Maeda Acute causes mild injury, followed regeneration.14Maeda To test two-week–old WT (25 mg/kg) months (Figure 1A). 100% DEN-injected approximately tumors average 1B). contrast, had slightly rate (70%) multiplicity (six average) smaller size C), suggesting promoting Interestingly, transient full-length nonstructural 5B adenoviral vector8Yu could enhance DEN-induced model 4-week–old (Supplemental Figure S1). microscopic alterations tumor-harboring livers, DEN-treated old 1D). addition presence nodules, more vacuoles shown some than 1D Supplemental S2). STAT1-dependent activation, PI3K, AKT FOXO transcription proteasome-dependent degradation.20Eijkelenboom Burgering B.M. FOXOs: signalling integrators maintenance.Nat Mol 2013; 83-97Crossref (620) FOXOs genes related cycle, apoptosis.20Eijkelenboom therefore immunohistochemistry. 1E, sporadically nontumor region regions. significantly stronger nodules nodules. activated physiological state liver, mRNA adult evaluated PCR. 2A, Ifna9 main Most IFN-Is, Ifnb, a1, a2, a4, a5, a6, a7, a8, a11, a12, a13, a15, a16, not detected Ifng (type IFN) unchallenged tissues. No difference observed between spleen Ifna6, a9, a13 levels spleen. lymphocytes abundant spleen, much higher suggest high constantly. evaluate during postnatal gene, Apol9, using Apol9 increased 2B). undetectable 2C). began age. Interferon-γ (Ifngr−/−) comparable amount mRNA, S3), IFN did PI3K/AKT/FOXO pathway, 2D, FOXO3 relative fine-tune pathway negative regulation recombinant adenovirus expressing mutant, production S4).8Yu Human hepatoma Huh7 treated IFN-β showed reduction S5). These modulate Lipopolysaccharide (LPS), component Gram-negative wall, TLR4 expression.21Meredith T.C. Aggarwal P. Mamat Lindner B. Woodard R.W. Redefining requisite lipopolysaccharide structure Escherichia coli.ACS Chem 2006; 1: 33-42Crossref (107) LPS translocated trigger subnanometer gold nanocluster modified methyl motifs nanoparticles block LPS-associated inflammation22Liao F.H. T.H. Huang Y.T. Lin W.J. Su C.J. Jeng U.S. S.C. S.Y. Subnanometer clusters adhere protection against endotoxin-induced sepsis.Nano Lett. 2018; 18: 2864-2869Crossref (21) used. 2E, (75 3 days 40% level, LPS/TLR4-dependent participated marker 3, , 2-week–old contained proliferating cells, reduced mice. number Ki-67+ (aged weeks) different less weeks), 3B. Hepatocytes diploid birth differentiate into mature multiple chromosome copies (polyploidy) fulfill needs Mature hepatocytes, therefore, large nuclear size.23Gentric Desdouets Polyploidization tissue.Am J Pathol. 2014; 184: 322-331Abstract (117) Quantification 3C) indicated arbitrary units based pixel size. differentiated population ranging 3000 size, 2000 units. any group, >2000 3C), differentiation program start earlier Assessment cycle–associated cyclins ages E2f1 Ccnd1 S6). Decreased early onset confirmed challenged mutant S7). Taken together, data participates balance pathogen invasion, process monitored PHx. Two-thirds PHx surgery removes ability.17Mitchell peak occurs 36 48 resection, remaining completes original week.17Mitchell 4A, extensive few day 7 surgery. numbers still ongoing 4A), because mass reached homeostasis. sustain homeostasis, proportion body size.24Yokoyama H.O. Wilson Tsuboi K.K. Stowell R.E. Regeneration hepatectomy.Cancer Res. 1953; 13: 80-85PubMed weight/body weight ratio S8). line slow rate, amounts Ccna2 Ccnb1 4B). Reduced A2 B1 4C). control cycle progression, kinases regulated members Kip/Cip family kinase inhibitors, p27. p27 remained fewer inhibitor p21 accumulation compared wit