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Ceramides and Sphingosine-1-Phosphate Mediate the Distinct Effects of M1/M2-Macrophage Infusion on Liver Recovery After Hepatect

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Ceramides and Sphingosine-1-Phosphate Mediate the Distinct Effects of M1/M2-Macrophage Infusion on Liver Recovery After Hepatect Sun et al. Cell Death and Disease (2021) 12:324 https://doi.org/10.1038/s41419-021-03616-9 Cell Death & Disease ARTICLE Open Access Ceramides and sphingosine-1-phosphate mediate the distinct effects of M1/M2-macrophage infusion on liver recovery after hepatectomy Hang Sun1,ShiboSun1, Gang Chen1,HaorongXie1,2, Sheng Yu1, Xinxin Lin3, Jianping Qian1,CunguiMao4, Hongxian Peng1,HaoChen1, Xuefang Chen1,2,YiyiLi5,CuitingLiu6,JunminShi6, Bili Zhu7,LinghongGuo1,8, Qingping Li1, Pengxiang Huang1,YiranWei3, Xixin Huang3,MeiqiLiu3, Zhonglin Cui1, Qifan Zhang 1, Jie Zhou 1, Chuanjiang Li 1 and Kai Wang 1 Abstract Post-hepatectomy liver dysfunction is a life-threatening morbidity that lacks efficient therapy. Bioactive lipids involved in macrophage polarization crucially regulate tissue injury and regeneration. Herein, we investigate the key bioactive lipids that mediate the cytotherapeutic potential of polarized-macrophage for post-hepatectomy liver dysfunction. Untargeted lipidomics identified elevation of ceramide (CER) metabolites as signature lipid species relevant to M1/M2 polarization in mouse bone-marrow-derived-macrophages (BMDMs). M1 BMDMs expressed a CER-generation- metabolic pattern, leading to elevation of CER; M2 BMDMs expressed a CER-breakdown-metabolic pattern, resulting in upregulation of sphingosine-1-phosphate (S1P). After infusing M1- or M2-polarized BMDMs into the mouse liver after hepatectomy, we found that M1-BMDM infusion increased M1 polarization and CER accumulation, resulting in exaggeration of hepatocyte apoptosis and liver dysfunction. Conversely, M2-BMDM infusion enhanced M2 polarization 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; and S1P generation, leading to alleviation of liver dysfunction with improved hepatocyte proliferation. Treatment of exogenous CER and S1P or inhibition CER and S1P synthesis by siRNA targeting relevant enzymes further revealed that CER induced apoptosis while S1P promoted proliferation in post-hepatectomy primary hepatocytes. In conclusion, CER and S1P are uncovered as critical lipid mediators for M1- and M2-polarized BMDMs to promote injury and regeneration in the liver after hepatectomy, respectively. Notably, the upregulation of hepatic S1P induced by M2-BMDM infusion may have therapeutic potential for post-hepatectomy liver dysfunction. Introduction Hepatectomy is frequently performed in the manage- ment of a variety of benign and malignant liver diseases. Post-hepatectomy liver dysfunction caused by impaired Correspondence: Qifan Zhang ([email protected])or liver recovery is still a life-threatening morbidity1,2. Due Jie Zhou ([email protected]) or Chuanjiang Li ([email protected])or to lack of efficient therapy, the mortality of post- Kai Wang ([email protected]) 2 1Division of Hepatobiliopancreatic Surgery, Department of General Surgery, hepatectomy liver failure is reported at 50% . Clinical Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, and experimental hepatectomy elicits a centric role of China 2 fi macrophages in regulating injury and regeneration in the Department of Hepatopancreatobiliary Surgery, The Second Af liated 3 Hospital of Guangzhou University of Chinese Medicine, Guangzhou, liver . Currently, the functional plasticity of macrophages Guangdong, China is characterized by tissue-damaging or repairing proper- Full list of author information is available at the end of the article ties4. Classical (M1) polarization driven by lipopoly- These authors contributed equally: Hang Sun, Shibo Sun, Gang Chen, Haorong Xie saccharide (LPS) and alternative polarization (M2) driven Edited by A. Finazzi-Agrò © The Author(s) 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association Sun et al. Cell Death and Disease (2021) 12:324 Page 2 of 16 by interleukin-4 (IL-4) represent the extreme states Willenbring21. The liver regeneration index was demon- associated with tissue injury and regeneration5. Elchani- strated using the ratio of the remnant liver weight and nov et al. reported that the increase of M2-like macro- body weight. Liver tissues for immunohistochemistry phages was likely correlated with liver regeneration after were fixed in 4% paraformaldehyde (PFA) (Biosharp, hepatectomy6. The study from Melgar-Lesmes et al. also Hefei, China) then embedded in paraffin or Tissue-Tek demonstrated that the increased infiltration of M2 mac- OCT compound (Sakura Finetek, Chuo-ku, Tokyo, rophages in the liver was linked to the enhancement of Japan). Liver tissues for RNA isolation, protein extraction, liver regeneration7. Notably, cytotherapy with polarized or sphingolipid extraction were snap-frozen in liquid macrophages was found to alleviate certain hepatopathy nitrogen then stored at −80 °C. Blood samples were by altering immune response in the liver, such as fibrosis8. allowed to clot at room temperature, and the serum was These results altogether lead to a hypothesis that hepa- collected by centrifugution and then stored at −80 °C toprotective macrophages which improve liver repair may until analyzed. Serum total bilirubin (TBIL), alanine have cytotherapeutic potential for post-hepatectomy liver aminotransferase activity (ALT), and albumin (ALB) were dysfunction. determined using Bilirubin Assay Kit (Sigma, St. Louis, Emerging studies have demonstrated that bioactive MO, USA), Alanine Transaminase Colorimetric Activity lipids are critical in mediating the distinct functions of Assay Kit (Cayman Chemical, Ann Arbor, MI, USA), and – polarized macrophages in tissue damage and repair9 12. Albumin Assay Kit (Sigma, St. Louis, MO, USA), Giannakis et al. reported that dynamic changes in fatty respectively, following the manufacturer’s instructions. acids support the phenotype transition of macrophages in All animal experiments were carried out following the muscle regeneration, providing substantial evidence for protocols approved by the Institutional Animal Care and exploring lipid mediators in subtypes of macrophages Use Committee of Nanfang Hospital Southern Medical which distinctly regulates tissue injury and regeneration9. University (NFYY-2017-03). Ceramides (CERs) and sphingosine-1-phosphate (S1P) in the sphingolipid metabolism are particularly highlighted Bone-marrow-derived macrophages culture, small to distinctly regulate hepatocyte injury and repair13. interfering RNA transfection, and BMDM infusion Accumulation of CER in hepatocytes has been reported to Isolation, culture, and polarization of bone-marrow- cause cell death and eventually lead to liver dysfunction, derived macrophages (BMDMs) were performed accord- – liver atrophy, and liver cancer14 16. In contrast to CER, ing to the protocol published by Ying et al.22. BMDMs S1P is a pro-survival lipid that is implicated in promoting were incubated with phosphate buffer saline (PBS) (Gibco, tissue regeneration through enhancing cell proliferation Waltham, MA, USA) as vehicle control, 100 ng/ml LPS and angiogenesis17,18. Lentsch and Hla’s studies demon- (Sigma, St. Louis, MO, USA), and 10 ng/ml IL-4 (Pepro- strated that extracellular S1P is important in promoting tech, Rocky Hill, NJ, USA) for 72 h to induce M1 and M2 liver regeneration by enhancing angiogenesis and polarization, respectively. For in vivo tracking after hepatocyte proliferation19,20. However, much remains BMDM infusion, BMDM were transfected with green unknown about the bioactive lipids in mediating the fluorescent protein (GFP) plasmid (Jikai, Shanghai, China) functions of polarized macrophages in post-hepatectomy with Lipofectamine 2000 (Invitrogen, Waltham, MA, liver recovery. USA) before inducing polarization. For disturbing CER In this study, we investigated the role of bioactive lipids metabolism in polarized BMDMs, M1 and M2 BMDMs that mediated the effects of polarized-macrophages were transfected with small interfering RNA (siRNA) infusion on post-hepatectomy liver recovery. Our study (Jikai, Shanghai, China) targeting Cers2 (5ʹ-CCUACA uncovers CER and S1P as critical lipids that mediate the CUGCACGAUGAUAUATT-3ʹ,5ʹ-UAUAUCAUCGUGC hepatotoxic and hepatoprotective effects of M1- and M2- AGUGUAGGTT-3ʹ) or Sphk1 (5ʹ-ACCUUCUUUCGCC macrophage infusion on liver recovery after hepatectomy, UAGCAATT-3ʹ,5ʹ-UUGCUAGGCGAAAGAAGGUTT- highlighting a cytotherapeutic capacity of M2 macro- 3ʹ) using X-treme GENE siRNA Transfection Reagent phage via generation of hepatoprotective S1P for post- (Roche Diagnostics, Mannheim, Germany). Transfection hepatectomy liver dysfunction. was performed at 24 h before induction of polarization. Polarized BMDM infusion was performed as described8,23 Materials and methods with slight modification. Non-polarized (M0), M1, and Mouse model M2 BMDMs were trypsinized
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