M1-like inflammatory phenotype of macrophages plays a critical role in tissue damage in chronic inflammatory diseases. Previously, we found that the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) dampens lipopolysaccharide (LPS)-triggered inflammatory priming of RAW 264.7 cells. Herein, we tested whether DMPO by itself can induce changes in macrophage transcriptome, and that these effects may prevent LPS-induced activation of macrophages.
Materials and methods
To test our hypothesis, we performed a transcriptomic and bioinformatics analysis in RAW 264.7 cells incubated with or without LPS, in the presence or in the absence of DMPO.
Functional data analysis showed 79 differentially expressed genes (DEGs) when comparing DMPO vs Control. We used DAVID databases for identifying enriched gene ontology terms and Ingenuity Pathway Analysis for functional analysis. Our data showed that DMPO vs Control comparison of DEGs is related to downregulation immune-system processes among others. Functional analysis indicated that interferon-response factor 7 and toll-like receptor were related (predicted inhibitions) to the observed transcriptomic effects of DMPO. Functional data analyses of the DMPO + LPS vs LPS DEGs were consistent with DMPO-dampening LPS-induced inflammatory transcriptomic profile in RAW 264.7. These changes were confirmed using Nanostring technology.
Taking together our data, surprisingly, indicate that DMPO by itself affects gene expression related to regulation of immune system and that DMPO dampens LPS-triggered MyD88- and TRIF-dependent signaling pathways. Our research provides critical data for further studies on the possible use of DMPO as a structural platform for the design of novel mechanism-based anti-inflammatory drugs.
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Liu YC, Zou XB, Chai YF, Yao YM. Macrophage polarization in inflammatory diseases. Int J Biol Sci. 2014;10(5):520–9.
Mosser DM, Edwards JP. Exploring the full spectrum of macrophage activation. Nat Rev Immunol. 2008;8(12):958–69.
Jin MS, Lee JO. Structures of the toll-like receptor family and its ligand complexes. Immunity. 2008;29(2):182–91.
Kawai T, Akira S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol. 2010;11(5):373–84.
May MJ, Ghosh S. Signal transduction through NF-kappa B. Immunol Today. 1998;19(2):80–8.
Ullah MO, Sweet MJ, Mansell A, Kellie S, Kobe B. TRIF-dependent TLR signaling, its functions in host defense and inflammation, and its potential as a therapeutic target. J Leukoc Biol. 2016;100(1):27–45.
Finkel T. Signal transduction by reactive oxygen species. J Cell Biol. 2011;194(1):7–15.
Halliwell B. Antioxidant characterization. Methodology and mechanism. Biochem Pharmacol. 1995;49(10):1341–8.
Domazou AS, Koppenol WH, Gebicki JM. Efficient repair of protein radicals by ascorbate. Free Radic Biol Med. 2009;46(8):1049–57.
Janzen EG. Spin trapping. Methods Enzymol. 1984;105:188–98.
Janzen EG, Jandrisits LT, Shetty RV, Haire DL, Hilborn JW. Synthesis and purification of 5,5-dimethyl-1-pyrroline-N-oxide for biological applications. Chem Biol Inter. 1989;70(1–2):167–72.
Janzen EG, Poyer JL, Schaefer CF, Downs PE, DuBose CM. Biological spin trapping. II. Toxicity of nitrone spin traps: dose-ranging in the rat. J Biochem Biophys Methods. 1995;30(4):239–47.
Janzen EG, West MS, Kotake Y, DuBose CM. Biological spin trapping methodology. III. Octanol-water partition coefficients of spin-trapping compounds. J Biochem Biophys Methods. 1996;32(3):183–90.
Lees KR, Zivin JA, Ashwood T, Davalos A, Davis SM, Diener HC, Grotta J, Lyden P, Shuaib A, Hardemark HG, Wasiewski WW. NXY-059 for acute ischemic stroke. N Engl J Med. 2006;354(6):588–600.
Floyd RA, Kopke RD, Choi CH, Foster SB, Doblas S, Towner RA. Nitrones as therapeutics. Free Radic Biol Med. 2008;45(10):1361–74.
Hamburger SA, McCay PB. Endotoxin-induced mortality in rats is reduced by nitrones. Circ Shock. 1989;29(4):329–34.
Zuo L, Chen YR, Reyes LA, Lee HL, Chen CL, Villamena FA, Zweier JL. The radical trap 5,5-dimethyl-1-pyrroline N-oxide exerts dose-dependent protection against myocardial ischemia-reperfusion injury through preservation of mitochondrial electron transport. J Pharmacol Exp Ther. 2009;329(2):515–23.
Ramirez DC, Mejiba SE, Mason RP. Immuno-spin trapping of DNA radicals. Nat Methods. 2006;3(2):123–7.
Gomez-Mejiba SE, Zhai Z, Akram H, Deterding LJ, Hensley K, Smith N, Towner RA, Tomer KB, Mason RP, Ramirez DC. Immuno-spin trapping of protein and DNA radicals: “tagging” free radicals to locate and understand the redox process. Free Radic Biol Med. 2009;46(7):853–65.
Zhai Z, Gomez-Mejiba SE, Gimenez MS, Deterding LJ, Tomer KB, Mason RP, Ashby MT, Ramirez DC. Free radical-operated proteotoxic stress in macrophages primed with lipopolysaccharide. Free Radic Biol Med. 2012;53(1):172–81.
Zhai Z, Gomez-Mejiba SE, Zhu H, Lupu F, Ramirez DC. The spin trap 5,5-dimethyl-1-pyrroline N-oxide inhibits lipopolysaccharide-induced inflammatory response in RAW 264.7 cells. Life Sci. 2012;90(11–12):432–9.
Finkelstein E, Rosen GM, Rauckman EJ, Paxton J. Spin trapping of superoxide. Mol Pharmacol. 1979;16(2):676–85.
Makino KH, Murakami AT. A mini review: fundamental aspects of spin trapping with DMPO. Int J Radiat Appl Instrum Part C Radiat Phys Chem. 1991;37(5):657–65.
Hochberg YBY. Controlling the false discovery rate: a practical and powerfull approach to multiple testing. J Roy Stat Soc Ser B (Methodol). 1995;57(1):289–300.
Gomez-Mejiba SE, Zhai Z, Della-Vedova MC, Munoz MD, Chatterjee S, Towner RA, Hensley K, Floyd RA, Mason RP, Ramirez DC. Immuno-spin trapping from biochemistry to medicine: advances, challenges, and pitfalls. Focus on protein-centered radicals. Biochim Biophys Acta. 2014;1840(2):722–9.
Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9(7):671–5.
Huang H, Park CK, Ryu JY, Chang EJ, Lee Y, Kang SS, Kim HH. Expression profiling of lipopolysaccharide target genes in RAW 264.7 cells by oligonucleotide microarray analyses. Arch Pharm Res. 2006;29(10):890–7.
Kotake Y, Sang H, Miyajima T, Wallis GL. Inhibition of NF-kappaB, iNOS mRNA, COX2 mRNA, and COX catalytic activity by phenyl-N-tert-butylnitrone (PBN). Biochim Biophys Acta. 1998;1448(1):77–84.
Tabatabaie T, Vasquez AM, Moore DR, Floyd RA, Kotake Y. Direct administration of interleukin-1 and interferon-gamma to rat pancreas leads to the in vivo production of nitric oxide and expression of inducible nitric oxide synthase and inducible cyclooxygenase. Pancreas. 2001;23(3):316–22.
Davies MJ, Hawkins CL. EPR spin trapping of protein radicals. Free Radic Biol Med. 2004;36(9):1072–86.
Authors want to express their gratitude to Dr Bart Frank (OMRF) for early processing of raw microarray data used in this study and to Dr Paula Di Sciullo for excellent technical assistance. This study was supported in part by the following agencies: PICT-3369 (FONCyT, AGENCIA, Argentina), PIP 916 (CONICET) and PROICO 2332 and PROICO 100414 (National University of San Luis). In addition, this research was also supported in part by the National Institute of Environmental Health Sciences.
Conflict of interest
Authors declare no competing conflict of interest.
Responsible Editor: John Di Battista.
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Muñoz, M.D., Della Vedova, M.C., Bushel, P.R. et al. The nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide dampens lipopolysaccharide-induced transcriptomic changes in macrophages. Inflamm. Res. 67, 515–530 (2018). https://doi.org/10.1007/s00011-018-1141-z