Aging results in accumulation of M1 and M2 hepatic macrophages and a differential response to gadolinium chloride
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Macrophages have vital roles in innate immunity by modulating the inflammatory response via their ability to alter their phenotype from pro-inflammatory (M1) to anti-inflammatory (M2). Aging increases activation of the innate immune system, and macrophage numbers increase in the aged liver. Since macrophages also produce free radical molecules, they are a potential source of age-related oxidative injury in the liver. This study evaluated macrophage phenotype in the aged liver and whether the increase in the number of macrophages with aging is associated with enhanced hepatic oxidative stress. Hepatic macrophage phenotype and oxidative stress were evaluated 2 days after a single intraperitoneal injection of saline or gadolinium chloride (GdCl3, 10 mg/kg) in young (6 months) and aged (24 months) Fischer 344 rats. GdCl3 has been shown to decrease the expression of macrophage-specific markers and impair macrophage phagocytosis in the liver. Saline-treated aged rats demonstrated greater numbers of both M1 (HO-1+/iNOS+) and M2 (HO-1+/CD163+) macrophages, without evidence of a phenotypic shift. GdCl3 did not alter levels of dihydroethidium fluorescence or malondialdehyde, suggesting that macrophages are not a major contributor to steady-state levels of oxidative stress. However, GdCl3 decreased M1 and M2 macrophage markers in both age groups, an effect that was attenuated in aged rats. In old animals, GdCl3 decreased iNOS expression to a greater extent than HO-1 or CD163. These results suggest a novel effect of aging on macrophage biology and that GdCl3 shifts hepatic macrophage polarization to the M2 phenotype in aged animals.
KeywordsFluorescence microscopy Immunolabelling iNOS Kupffer cells Oxidative stress Inflammation
We thank Tom Moninger at the University of Iowa Central Microscopy Research Facility for assistance with confocal microscopy.
SAB was supported by a Faculty Development Grant from Penn State Abington. EDM was supported by research funds from the Schreyer Honors College of the Pennsylvania State University, and an Erickson Summer Discovery Grant from the Office of Undergraduate Research at Penn State. This work was also supported by NIH grant AG12350 to KCK. No funding source had a role in study design, data collection, analysis or interpretation of the data, in the writing of the final report, or the decision to submit the article for publication.
Compliance with ethical standards
Conflicts of interest
The authors declare that they have no competing interests.
All applicable national and institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the University of Iowa (University of Iowa Institutional Animal Care and Use Committee, ACURF #0606117).
- Amanzada A, Moriconi F, Mansuroglu T, Cameron S, Ramadori G, Malik I (2013) Induction of chemokines and cytokines before neutrophils and macrophage recruitment in different regions of rat liver after TAA administration. Lab Invest 94:235–247. https://doi.org/10.1038/labinvest.2013.134 CrossRefPubMedGoogle Scholar
- Elmore MRP, Hohsfield LA, Kramar EA, Soreq L, Lee RJ, Pham ST, Najafi AR, Spanenberg EE, Wood MA, West BL, Green KN (2018) Replacement of microglia in the aged brain reverses cognitive, synaptic, and neuronal deficits in aged mice. Aging Cell 17(6):e12832. https://doi.org/10.1111/acel.12832 CrossRefPubMedPubMedCentralGoogle Scholar
- Fukuda M, Yokoyama H, Mizukami T, Ohgo H, Okamura Y, Kamegaya Y, Horie Y, Kato S, Ishii H (2004) Kupffer cell depletion attenuates superoxide anion release into the hepatic sinusoids after lipopolysaccharide treatment. J Gastroenterol Hepatol 19(10):1155–1162. https://doi.org/10.1111/j.1440-1746.2004.03408.x CrossRefPubMedGoogle Scholar
- Goda N, Suzuki K, Naito M, Takeoka S, Tsuchida E, Ishimura Y, Tamatani T, Suematsu M (1998) Distribution of heme oxygenase isoforms in rat liver topographic basis for carbon monoxide-mediated microvascular relaxation. J Clin Invest 101(3):604–612. https://doi.org/10.1172/jci1324 CrossRefPubMedPubMedCentralGoogle Scholar
- Harrison-Findik DD, Klein E, Evans J, Gollan J (2009) Regulation of liver hepcidin expression by alcohol in vivo does not involve kupffer cell activation or TNF-α signaling. Am J Physiol-Gastrointest Liver Physiol 296(1):G112–G118. https://doi.org/10.1152/ajpgi.90550.2008 CrossRefPubMedPubMedCentralGoogle Scholar
- Isidro RA, Isidro AA, Cruz ML, Hernandez S, Appleyard CB (2015) Double immunofluorescent staining of rat macrophages in formalin-fixed paraffin-embedded tissue using two monoclonal mouse antibodies. Histochem Cell Biol 144(6):613–621. https://doi.org/10.1007/s00418-015-1364-9 CrossRefPubMedPubMedCentralGoogle Scholar
- Jenkins SJ, Ruckerl D, Cook PC, Jones LH, Finkelman FD, van Rooijen N, MacDonald AS, Allen JE (2011) Local macrophage proliferation, rather than recruitment from the blood, is a signature of Th2 inflammation. Science 332(6035):1284–1288. https://doi.org/10.1126/science.1204351 CrossRefPubMedPubMedCentralGoogle Scholar
- Khan HA, Ibrahim KE, Khan A, Alrokayan SH, Alhomida AS, Lee YK (2016) Comparative evaluation of immunohistochemistry and real-time PCR for measuring proinflammatory cytokines gene expression in livers of rats treated with gold nanoparticles. Exp Toxicol Pathol 68(7):381–390. https://doi.org/10.1016/j.etp.2016.05.006 CrossRefPubMedGoogle Scholar
- Maeso-Díaz R, Ortega-Ribera M, Fernández-Iglesias A, Hide D, Muñoz L, Hessheimer AJ, Vila S, Francés R, Fondevila C, Albillos A, Peralta C, Bosch J, Tacke F, Cogger VC, Gracia-Sancho J (2018) Effects of aging on liver microcirculatory function and sinusoidal phenotype. Aging Cell 17(6):e12829. https://doi.org/10.1111/acel.12829 CrossRefPubMedPubMedCentralGoogle Scholar
- Murray PJ, Allen JE, Biswas SK, Fisher EA, Gilroy DW, Goerdt S, Gordon S, Hamilton JA, Ivashkiv LB, Lawrence T, Locati M, Mantovani A, Martinez FO, Mege JL, Mosser DM, Natoli G, Saeij JP, Schultze JL, Shirey KA, Sica A, Suttles J, Udalova I, van Ginderachter JA, Vogel SN, Wynn TA (2014) Macrophage activation and polarization: nomenclature and experimental guidelines. Immunity 41(1):14–20. https://doi.org/10.1016/j.immuni.2014.06.008 CrossRefPubMedPubMedCentralGoogle Scholar
- Pilkington SM, Barron MJ, Watson REB, Griffiths CEM, Bulfone-Paus S (2019) Aged human skin accumulates mast cells with altered functionality that localize to macrophages and vasoactive intestinal peptide-positive nerve fibres. Bri J Dermatol 180(4):849–858. https://doi.org/10.1111/bjd.17268 CrossRefGoogle Scholar
- Rana B, Mischoulon D, Xie Y, Bucher NL, Farmer SR (1994) Cell-extracellular matrix interactions can regulate the switch between growth and differentiation in rat hepatocytes: reciprocal expression of C/EBP alpha and immediate-early growth response transcription factors. Moll Cell Biol 14(9):5858–5869CrossRefGoogle Scholar
- Smallwood HS, López-Ferrer D, Squier TC (2011) Aging enhances the production of reactive oxygen species and bactericidal activity in peritoneal macrophages by upregulating classical activation pathways. Biochemistry 50(45):9911–9922. https://doi.org/10.1021/bi2011866 CrossRefPubMedPubMedCentralGoogle Scholar
- Uhlén M, Fagerberg L, Hallström BM, Lindskog C, Oksvold P, Mardinoglu A, Sivertsson Å, Kampf C, Sjöstedt E, Asplund A, Olsson I, Edlund K, Lundberg E, Navani S, Szigyarto CAK, Odeberg J, Djureinovic D, Takanen JO, Hober S, Alm T, Edqvist PH, Berling H, Tegel H, Mulder J, Rockberg J, Nilsson P, Schwenk JM, Hamsten M, von Feilitzen K, Forsberg M, Persson L, Johansson F, Zwahlen M, von Heijne G, Nielsen J, Pontén F (2015) Tissue-based map of the human proteome. Science 347(6220):1260419. https://doi.org/10.1126/science.1260419 CrossRefPubMedGoogle Scholar
- Wan J, Benkdane M, Teixeira-Clerc F, Bonnafous S, Louvet A, Lafdil F, Pecker F, Tran A, Gual P, Mallat A, Lotersztajn S, Pavoine C (2014) M2 kupffer cells promote M1 kupffer cell apoptosis: a protective mechanism against alcoholic and nonalcoholic fatty liver disease. Hepatology 59(1):130–142. https://doi.org/10.1002/hep.26607 CrossRefPubMedGoogle Scholar
- Yan BC, Gong C, Song J, Krausz T, Tretiakova M, Hyjek E, Al-Ahmadie H, Alves V, Xiao SY, Anders RA, Hart JA (2010) Arginase-1: a new immunohistochemical marker of hepatocytes and hepatocellular neoplasms. Am J Surg Pathol 34(8):1147–1154. https://doi.org/10.1097/PAS.0b013e3181e5dffa CrossRefPubMedPubMedCentralGoogle Scholar
- Zhong S, Xu J, Li P, Tsukamoto H (2012) Caveosomal oxidative stress causes Src-p21ras activation and lysine 63 TRAF6 protein polyubiquitination in iron-induced M1 hepatic macrophage activation. J Biol Chem 287(38):32078–32084. https://doi.org/10.1074/jbc.M112.377358 CrossRefPubMedPubMedCentralGoogle Scholar
- Zhu F, Li X, Jiang Y, Zhu H, Zhang H, Zhang C, Zhao Y, Luo F (2015) GdCl3 suppresses the malignant potential of hepatocellular carcinoma by inhibiting the expression of CD206 in tumor-associated macrophages. Oncol Rep 34(5):2643–2655. https://doi.org/10.3892/or.2015.4268 CrossRefPubMedGoogle Scholar