The nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide dampens lipopolysaccharide-induced transcriptomic changes in macrophages

Abstract

Objective

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.

Results

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.

Conclusions

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.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11

References

  1. 1.

    Liu YC, Zou XB, Chai YF, Yao YM. Macrophage polarization in inflammatory diseases. Int J Biol Sci. 2014;10(5):520–9.

    Article  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Mosser DM, Edwards JP. Exploring the full spectrum of macrophage activation. Nat Rev Immunol. 2008;8(12):958–69.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Jin MS, Lee JO. Structures of the toll-like receptor family and its ligand complexes. Immunity. 2008;29(2):182–91.

    CAS  Article  PubMed  Google Scholar 

  4. 4.

    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.

    CAS  Article  PubMed  Google Scholar 

  5. 5.

    May MJ, Ghosh S. Signal transduction through NF-kappa B. Immunol Today. 1998;19(2):80–8.

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    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.

    CAS  Article  PubMed  Google Scholar 

  7. 7.

    Finkel T. Signal transduction by reactive oxygen species. J Cell Biol. 2011;194(1):7–15.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Halliwell B. Antioxidant characterization. Methodology and mechanism. Biochem Pharmacol. 1995;49(10):1341–8.

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Domazou AS, Koppenol WH, Gebicki JM. Efficient repair of protein radicals by ascorbate. Free Radic Biol Med. 2009;46(8):1049–57.

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Janzen EG. Spin trapping. Methods Enzymol. 1984;105:188–98.

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    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.

    CAS  Article  Google Scholar 

  12. 12.

    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.

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    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.

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    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.

    CAS  Article  PubMed  Google Scholar 

  15. 15.

    Floyd RA, Kopke RD, Choi CH, Foster SB, Doblas S, Towner RA. Nitrones as therapeutics. Free Radic Biol Med. 2008;45(10):1361–74.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Hamburger SA, McCay PB. Endotoxin-induced mortality in rats is reduced by nitrones. Circ Shock. 1989;29(4):329–34.

    CAS  PubMed  Google Scholar 

  17. 17.

    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.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Ramirez DC, Mejiba SE, Mason RP. Immuno-spin trapping of DNA radicals. Nat Methods. 2006;3(2):123–7.

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    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.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. 20.

    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.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. 21.

    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.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Finkelstein E, Rosen GM, Rauckman EJ, Paxton J. Spin trapping of superoxide. Mol Pharmacol. 1979;16(2):676–85.

    CAS  PubMed  Google Scholar 

  23. 23.

    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.

    CAS  Google Scholar 

  24. 24.

    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.

    Google Scholar 

  25. 25.

    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.

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9(7):671–5.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. 27.

    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.

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    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.

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    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.

    CAS  Article  PubMed  Google Scholar 

  30. 30.

    Davies MJ, Hawkins CL. EPR spin trapping of protein radicals. Free Radic Biol Med. 2004;36(9):1072–86.

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgements

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.

Author information

Affiliations

Authors

Corresponding authors

Correspondence to S. E. Gomez Mejiba or D. C. Ramirez.

Ethics declarations

Conflict of interest

Authors declare no competing conflict of interest.

Additional information

Responsible Editor: John Di Battista.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PPTX 1474 KB)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Keywords

  • Macrophage
  • Lipopolysaccharide
  • Inflammation
  • Transcriptomics
  • DMPO