Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Imaging of innate immunity activation in vivo with a redox-tuned PET reporter

Nature Biotechnology volume 40, pages 965–973 (2022)Cite this article

Subjects

Abstract

High-redox-potential reactive oxygen species and reactive nitrogen species (ROS/RNS), generated by NADPH oxidase-2 (NOX2), myeloperoxidase (MPO) and related enzymes, are key effector molecules of innate immunity. High-redox-potential radicals are difficult to distinguish by imaging from less potent ROS/RNS functioning as background biological signaling molecules. Here we present 4-[18F]fluoro-1-naphthol ([18F]4FN), a redox-tuned radiopharmaceutical that selectively binds proteins and cells when oxidized by products of human MPO plus H2O2, but not H2O2 alone, and can be detected using positron emission tomography (PET). Activating HL-60 neutrophil-like human cells with phorbol ester (PMA) caused [18F]4FN retention five-fold over unstimulated cells. An MPO-specific inhibitor (4-ABAH) blocked cellular retention by more than 95%. [18F]4FN PET/CT imaging discriminated inflammatory foci in vivo in three murine models of activated innate immunity: endotoxin-induced toxic shock, PMA-induced contact dermatitis and lipopolysaccharide-induced ankle arthritis. 4-ABAH and Cybb−/− (Nox2−/−) gene deletion strongly abrogated [18F]4FN retention in vivo. Thus, [18F]4FN shows promise as a robust reporter of innate immunity activation by PET/CT.

Access through your institution
Buy or subscribe

This is a preview of subscription content, access via your institution

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Synthesis of [18F]4FN.
Fig. 2: [18F]4FN oxidation in vitro and in cellulo.
Fig. 3: Imaging endotoxin-induced toxic shock with [18F]4FN PET.
Fig. 4: Imaging LPS-induced arthritis with [18F]4FN PET.
Fig. 5: Imaging PMA-induced contact dermatitis with [18F]4FN PET.

Similar content being viewed by others

Data availability

Source data are provided with this paper. Any remaining raw data will be available from the corresponding author upon reasonable request. Source data are provided with this paper.

References

  1. Zindel, J. & Kubes, P. DAMPs, PAMPs, and LAMPs in immunity and sterile inflammation. Annu. Rev. Pathol. 15, 493–518 (2020).

    Article  CAS  PubMed  Google Scholar 

  2. Schroder, K. & Tschopp, J. The inflammasomes. Cell 140, 821–832 (2010).

    Article  CAS  PubMed  Google Scholar 

  3. Marquez, L. A. & Dunford, H. B. Kinetics of oxidation of tyrosine and dityrosine by myeloperoxidase compounds I and II. Implications for lipoprotein peroxidation studies. J. Biol. Chem. 270, 30434–30440 (1995).

    Article  CAS  PubMed  Google Scholar 

  4. Nishinaka, Y., Arai, T., Adachi, S., Takaori-Kondo, A. & Yamashita, K. Singlet oxygen is essential for neutrophil extracellular trap formation. Biochem. Biophys. Res. Commun. 413, 75–79 (2011).

    Article  CAS  PubMed  Google Scholar 

  5. Spiller, K. L. et al. Differential gene expression in human, murine, and cell line-derived macrophages upon polarization. Exp. Cell. Res. 347, 1–13 (2016).

    Article  CAS  PubMed  Google Scholar 

  6. Montezano, A. C. & Touyz, R. M. Reactive oxygen species and endothelial function–role of nitric oxide synthase uncoupling and Nox family nicotinamide adenine dinucleotide phosphate oxidases. Basic Clin. Pharmacol. Toxicol. 110, 87–94 (2012).

    Article  CAS  PubMed  Google Scholar 

  7. Arasimowicz-Jelonek, M., Floryszak-Wieczorek, J., Abramowski, D. & Izbiańska, K. In: Nitric Oxide in Plants: Metabolism and Role in Stress Physiology (eds Khan, M. N., Mobin, M., Mohammad, F., & Corpas, F. J.) 165–184 (Springer International Publishing, 2014).

  8. Cronin, S. J. F., Woolf, C. J., Weiss, G. & Penninger, J. M. The role of iron regulation in immunometabolism and immune-related disease. Front. Mol. Biosci. 6, 116 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Sharrock, J. & Sun, J. C. Innate immunological memory: from plants to animals. Curr. Opin. Immunol. 62, 69–78 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Zámocký, M. et al. Independent evolution of four heme peroxidase superfamilies. Arch. Biochem. Biophys. 574, 108–119 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. Davies, M. J. Myeloperoxidase-derived oxidation: mechanisms of biological damage and its prevention. J. Clin. Biochem. Nutr. 48, 8–19 (2011).

    Article  CAS  PubMed  Google Scholar 

  12. Jelcic, M., Enyedi, B., Xavier, J. B. & Niethammer, P. Image-based measurement of H2O2 reaction-diffusion in wounded zebrafish larvae. Biophys. J. 112, 2011–2018 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Elias, J. M. A rapid, sensitive myeloperoxidase stain using 4-chloro-1-naphthol. Am. J. Clin. Pathol. 73, 797–799 (1980).

    Article  CAS  PubMed  Google Scholar 

  14. Eastmond, D. A., French, R. C., Ross, D. & Smith, M. T. Metabolic activation of 1-naphthol and phenol by a simple superoxide-generating system and human leukocytes. Chem. Biol. Interact. 63, 47–62 (1987).

    Article  CAS  PubMed  Google Scholar 

  15. Sreekanth, R., Prasanthkumar, K. P., Sunil Paul, M. M., Aravind, U. K. & Aravindakumar, C. T. Oxidation reactions of 1- and 2-naphthols: an experimental and theoretical study. J. Phys. Chem. A 117, 11261–11270 (2013).

    Article  CAS  PubMed  Google Scholar 

  16. Islam, A., Lee, S.-E. & Jang-Eok, K. Enhanced enzymatic transformation of 1-naphthol in the presence of catechol by peroxidase. J. Korean Soc. Appl. Biol. Chem. 57, 209–215 (2014).

    Article  CAS  Google Scholar 

  17. Tredwell, M. et al. A general copper-mediated nucleophilic 18F fluorination of arenes. Angew. Chem. Int. Ed. Engl. 53, 7751–7755 (2014).

    Article  CAS  PubMed  Google Scholar 

  18. Daiber, A. et al. Measurement of NAD(P)H oxidase-derived superoxide with the luminol analogue L-012. Free Radic. Biol. Med. 36, 101–111 (2004).

    Article  CAS  PubMed  Google Scholar 

  19. Xu, F., Koch, D. E., Kong, I. C., Hunter, R. P. & Bhandari, A. Peroxidase-mediated oxidative coupling of 1-naphthol: characterization of polymerization products. Water Res. 39, 2358–2368 (2005).

    Article  CAS  PubMed  Google Scholar 

  20. Seitz, P. M. et al. Development of a high-throughput cell-based assay for superoxide production in HL-60 cells. J. Biomol. Screen. 15, 388–397 (2010).

    Article  CAS  PubMed  Google Scholar 

  21. Augsburger, F. et al. Pharmacological characterization of the seven human NOX isoforms and their inhibitors. Redox Biol. 26, 101272 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Doi, K., Leelahavanichkul, A., Yuen, P. S. & Star, R. A. Animal models of sepsis and sepsis-induced kidney injury. J. Clin. Invest. 119, 2868–2878 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Cunningham, P. N., Wang, Y., Guo, R., He, G. & Quigg, R. J. Role of Toll-like receptor 4 in endotoxin-induced acute renal failure. J. Immunol. 172, 2629–2635 (2004).

    Article  CAS  PubMed  Google Scholar 

  24. Gross, S. et al. Bioluminescence imaging of myeloperoxidase activity in vivo. Nat. Med. 15, 455–461 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Hayeri, M. R., Ziai, P., Shehata, M. L., Teytelboym, O. M. & Huang, B. K. Soft-tissue infections and their imaging mimics: from cellulitis to necrotizing fasciitis. Radiographics 36, 1888–1910 (2016).

    Article  PubMed  Google Scholar 

  26. Galban, C. J. et al. Applications of molecular imaging. Prog. Mol. Biol. Transl. Sci. 95, 237–298 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Jones, H. A. et al. Dissociation between respiratory burst activity and deoxyglucose uptake in human neutrophil granulocytes: implications for interpretation of 18F-FDG PET images. J. Nucl. Med. 43, 652–657 (2002).

    CAS  PubMed  Google Scholar 

  28. Bruno, J. G. & Kiel, J. L. Luminol and diazoluminomelanin as indicators of HL-60 cell differentiation. Vitr. Cell Dev. Biol. Anim. 29A, 737–741 (1993).

    Article  CAS  Google Scholar 

  29. Locksley, R. M., Wilson, C. B. & Klebanoff, S. J. Increased respiratory burst in myeloperoxidase-deficient monocytes. Blood 62, 902–909 (1983).

    Article  CAS  PubMed  Google Scholar 

  30. Werry, E. L. et al. Recent developments in TSPO PET imaging as a biomarker of neuroinflammation in neurodegenerative disorders. Int. J. Mol. Sci. 20, 3161 (2019).

  31. Shao, X. et al. Imaging of carrageenan-induced local inflammation and adjuvant-induced systemic arthritis with [11C]PBR28 PET. Nucl. Med. Biol. 40, 906–911 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Lee, Y., Park, Y., Nam, H., Lee, J. W. & Yu, S. W. Translocator protein (TSPO): the new story of the old protein in neuroinflammation. BMB Rep. 53, 20–27 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Zhang, L. et al. Recent developments on PET radiotracers for TSPO and their applications in neuroimaging. Acta Pharm. Sin. B 11, 373–393 (2021).

    Article  CAS  PubMed  Google Scholar 

  34. Colovic, M. et al. Non-invasive use of positron emission tomography to monitor diethyl maleate and radiation-induced changes in system xC− activity in breast cancer. Mol. Imaging Biol. 21, 1107–1116 (2019).

    Article  CAS  PubMed  Google Scholar 

  35. Yang, H. et al. 18F-5-fluoroaminosuberic acid as a potential tracer to gauge oxidative stress in breast cancer models. J. Nucl. Med. 58, 367–373 (2017).

  36. Sivapackiam, J. et al. Galuminox: preclinical validation of a novel PET tracer for non-invasive imaging of oxidative stress in vivo. Redox Biol. 37, 101690 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Wu, J. et al. Feasibility study of PET dynamic imaging of [18F]DHMT for quantification of reactive oxygen species in the myocardium of large animals. J. Nucl. Cardiol. https://doi.org/10.1007/s12350-020-02184-3 (2020).

  38. Wang, S. T. et al. Sensitivity of activatable reactive oxygen species probes by fluorescence spectroelectrochemistry. Analyst 138, 4363–4369 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Hou, C. et al. Development of a positron emission tomography radiotracer for imaging elevated levels of superoxide in neuroinflammation. ACS Chem. Neurosci. 9, 578–586 (2018).

    Article  CAS  PubMed  Google Scholar 

  40. Egami, H. et al. [18F]-Labeled dihydromethidine: positron emission tomography radiotracer for imaging of reactive oxygen species in intact brain. Org. Biomol. Chem. 18, 2387–2391 (2020).

    Article  CAS  PubMed  Google Scholar 

  41. Carroll, V. N. et al. [11C]Ascorbic and [11C]dehydroascorbic acid, an endogenous redox pair for sensing reactive oxygen species using positron emission tomography. Chem. Commun. (Camb.) 52, 4888–4890 (2016).

    Article  CAS  Google Scholar 

  42. Wang, C. et al. An activatable PET imaging radioprobe is a dynamic reporter of myeloperoxidase activity in vivo. Proc. Natl Acad. Sci. USA 116, 11966–11971 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Halliwell, B. & Gutteridge, J. M. Biologically relevant metal ion-dependent hydroxyl radical generation. An update. FEBS Lett. 307, 108–112 (1992).

    Article  CAS  PubMed  Google Scholar 

  44. Bhattacharjee, S., Chatterjee, S., Jiang, J., Sinha, B. K. & Mason, R. P. Detection and imaging of the free radical DNA in cells–site-specific radical formation induced by Fenton chemistry and its repair in cellular DNA as seen by electron spin resonance, immuno-spin trapping and confocal microscopy. Nucleic Acids Res. 40, 5477–5486 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Leto, T. L. & Geiszt, M. Role of Nox family NADPH oxidases in host defense. Antioxid. Redox Signal. 8, 1549–1561 (2006).

    Article  CAS  PubMed  Google Scholar 

  46. Kamada, N. & Rogler, G. The innate immune system: a trigger for many chronic inflammatory intestinal diseases. Inflamm. Intest. Dis. 1, 70–77 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Tay, M. Z., Poh, C. M., Renia, L., MacAry, P. A. & Ng, L. F. P. The trinity of COVID-19: immunity, inflammation and intervention. Nat. Rev. Immunol. 20, 363–374 (2020).

    Article  CAS  PubMed  Google Scholar 

  48. Wood, P. M. The potential diagram for oxygen at pH 7. Biochem. J. 253, 287–289 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Kucera, J. Biofouling of polyamide membranes: fouling mechanisms, current mitigation and cleaning strategies, and future prospects. Membranes (Basel) 9, 111 (2019).

  50. Koppenol, W. H., Moreno, J. J., Pryor, W. A., Ischiropoulos, H. & Beckman, J. S. Peroxynitrite, a cloaked oxidant formed by nitric oxide and superoxide. Chem. Res. Toxicol. 5, 834–842 (1992).

    Article  CAS  PubMed  Google Scholar 

  51. Arnhold, J., Furtmuller, P. G. & Obinger, C. Redox properties of myeloperoxidase. Redox Rep. 8, 179–186 (2003).

    Article  CAS  PubMed  Google Scholar 

  52. Koppenol, W. H., Stanbury, D. M. & Bounds, P. L. Electrode potentials of partially reduced oxygen species, from dioxygen to water. Free Radic. Biol. Med. 49, 317–322 (2010).

    Article  CAS  PubMed  Google Scholar 

  53. Das, T. N. & Neta, P. Reduction potentials of naphthoxyl and pyridoxyl radicals in aqueous solutions. J. Phys. Chem. A 102, 7081–7085 (1998).

    Article  CAS  Google Scholar 

  54. Harvey, D. Standard reduction potentials by element. https://chem.libretexts.org/ (2021).

  55. Koppenol, W. H. Thermodynamic considerations on the formation of reactive species from hypochlorite, superoxide and nitrogen monoxide. Could nitrosyl chloride be produced by neutrophils and macrophages? FEBS Lett. 347, 5–8 (1994).

    Article  CAS  PubMed  Google Scholar 

  56. Bartberger, M. D. et al. The reduction potential of nitric oxide (NO) and its importance to NO biochemistry. Proc. Natl Acad. Sci. USA 99, 10958–10963 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Halliwell, B., Clement, M. V. & Long, L. H. Hydrogen peroxide in the human body. FEBS Lett. 486, 10–13 (2000).

    Article  CAS  PubMed  Google Scholar 

  58. Trujillo, M., Alvarez, B. & Radi, R. One- and two-electron oxidation of thiols: mechanisms, kinetics and biological fates. Free Radic. Res. 50, 150–171 (2016).

    Article  CAS  PubMed  Google Scholar 

  59. Jensen, S. B., Nielsen, K. M., Mewis, D. & Kaufmann, J. Fast and simple one-step preparation of 68Ga citrate for routine clinical PET. Nucl. Med. Commun. 34, 806–812 (2013).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We acknowledge support from the Gerald Dewey Dodd, Jr. Endowed Distinguished Chair at The University of Texas MD Anderson Cancer Center and a Faculty UT STARs Award. We would like to thank the Small Animal Imaging Facility and the Science Park Research Histology, Pathology and Imaging Core at MD Anderson Cancer Center for animal imaging and histology, respectively, especially C. Kingsley and J. DeLacerda. Both core facilities obtain support from the National Cancer Institute Cancer Center Support Grant (P30 CA016672).

Author information

Author notes

  1. These authors contributed equally: Federica Pisaneschi, Seth T. Gammon.

Authors and Affiliations

  1. Department of Cancer Systems Imaging, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Federica Pisaneschi, Seth T. Gammon, Sarah A. Qureshy & David Piwnica-Worms

  2. Center for Advanced Biomedical Imaging, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

    Vincenzo Paolillo

Authors
  1. Federica Pisaneschi
    View author publications

    Search author on:PubMed Google Scholar

  2. Seth T. Gammon
    View author publications

    Search author on:PubMed Google Scholar

  3. Vincenzo Paolillo
    View author publications

    Search author on:PubMed Google Scholar

  4. Sarah A. Qureshy
    View author publications

    Search author on:PubMed Google Scholar

  5. David Piwnica-Worms
    View author publications

    Search author on:PubMed Google Scholar

Contributions

Conceptualization: F.P., S.T.G. and D.P.W.; Methodology: F.P. and S.T.G.; Experimental performance: F.P., S.T.G., S.Q. and V.P.; Formal analysis: F.P., S.T.G., S.Q., V.P. and D.P.W.; Writing—original draft preparation: F.P., S.T.G. and D.P.W.; Writing—review and editing: F.P., S.T.G., S.Q., V.P. and D.P.W.; Funding acquisition: D.P.W. All authors have read and agreed to the published version of the manuscript.

Corresponding author

Correspondence to David Piwnica-Worms.

Ethics declarations

Competing interests

The University of Texas MD Anderson Cancer Center has filed a patent application on compounds and methods described in this report (S.T.G., F.P. and D.P.W., inventors). The remaining authors have no conflicts of interest.

Peer review

Peer review information

Nature Biotechnology thanks Delphine Chen, Xiaoyuan Chen and Kenneth Krohn for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Chemical characterization.

1H NMR (compound 2), top; 13C NMR (compound 2), mid; HPLC analysis of [18F]4FN, bottom, A: QC of radioactive [18F]4FN (gamma trace). B: QC of radioactive [18F]4FN (UV trace at 254 nm). C: cold 4FN as reference compound. HPLC column: Bridge C18, 3.5 µm, 4.6×250 mm; Method: A-water (0.1% TFA), B-MeCN (0.1% TFA) B: 40% for 3 min, 40%→95% in 7 min, 95% for 1 min, flow 1 mL/min. NOTE the compressed scale of B to highlight the high purity of and molar activity of the compound.

Extended Data Fig. 2 PET image-based clearance and blood-to-brain ratios.

Individual PET image-based time-activity curves derived from heart (representing the blood pool) and brain VOIs.

Source data

Extended Data Fig. 3 [18F]4FN PET/CT in a model of mild contact dermatitis.

A) BALB/cN mice were treated with either PMA or vehicle and imaged 24 hr post treatment by PET/CT scanning at 1 hr post-injection (i.p.) of [18F]4FN. B) photograph 3 hr post [18F]4FN injection. C) BLI 10 min post luminol injection.

Extended Data Fig. 4 [18F]4FN PET/CT in a model of mild contact dermatitis.

A) In BALB/cN mice, 4-ABAH treatment mildly inhibited PMA-induced retention of [18F]4FN when compared to untreated animals. 2-way ANOVA with replication, test for interaction. B) 4-ABAH-induced inhibition appeared more evident when data were plotted as PMA/vehicle ratio showing a 50% reduction in PMA-induced retention (p = 0.1, one-tailed non-parametric t-test). C) In Mpo-/- mice (KO), any effect was below the limit of detection and appeared to be compensated by another ROS-producing mechanism. Data are presented as mean + /- SEM. Each point represents an individual animal.

Source data

Extended Data Fig. 5 Conservation of sequence across multiple species, but rodents underestimate MPO content and ROS burst broadly.

CYBB sequence of the NOX2 complex is conserved from yeast to plant to mouse to man; MPO sequence is conserved at the vertebrate level from frogs to mouse to man, but MPO content and ROS/RNS flux (as measured by PMA-induced chemiluminescence (CL activity) through these pathways) show significant interspecies variance (data from literature reports with human activity set to unity) (Surgery 126, 248-254 (1999); J. Immunol. Methods 115, 141-147 (1988); Comp Biochem Physiol B 75, 335-340 (1983); Blood 46, 913-919 (1975)).

Source data

Extended Data Fig. 6 Imaging of WT vs Nox2-/- (KO) mice confirmed that the KO mice had functionally impaired RONS production as validated by L-012 BLI imaging.

The right ears were treated with PMA as described and then imaged by [18F]4FN PET/CT. Mice were subsequently injected with L-012 i.p. (20 mg/kg) and imaged for bioluminescence (IVIS SPECTRUM, Perkin Elmer). Data are presented as mean + /- SEM. Each point represents an individual animal.

Source data

Extended Data Table 1 Radiochemical characteristics of [18F]4FN
Full size table
Extended Data Table 2 Medicinal chemistry properties
Full size table

Supplementary information

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pisaneschi, F., Gammon, S.T., Paolillo, V. et al. Imaging of innate immunity activation in vivo with a redox-tuned PET reporter. Nat Biotechnol 40, 965–973 (2022). https://doi.org/10.1038/s41587-021-01169-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue date:

  • DOI: https://doi.org/10.1038/s41587-021-01169-y

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing