Trends in Immunology
Volume 41, Issue 11, November 2020, Pages 994-1005
Journal home page for Trends in Immunology

Review
Illuminating Epigenetics and Inheritance in the Immune System with Bioluminescence

https://doi.org/10.1016/j.it.2020.09.001Get rights and content

Highlights

  • BLI is a powerful technique for non-invasive visualization of in vivo processes, with key advantages being its low background and high sensitivity.

  • BLI can be used to study dynamic gene expression and epigenetic changes in vivo, with increasing opportunities for applying this imaging approach more widely in immunology.

  • Technological advances are expanding the toolkit of luciferases and substrates available for BLI, such as by enabling deeper visualization and dual-reporter imaging.

  • Newly characterized bioluminescent systems are opening the possibility of engineering autonomously bioluminescent organisms, potentially revolutionizing the field of in vivo BLI.

  • Recent advances in genome-editing tools are rendering the insertion of reporters into endogenous loci to generate new and better transgenic models for BLI widely feasible.

The remarkable process of light emission by living organisms has fascinated mankind for thousands of years. A recent expansion in the repertoire of catalytic luciferase enzymes, coupled with the discovery of the genes and pathways that encode different luciferin substrates, means that bioluminescence imaging (BLI) is set to revolutionize longitudinal and dynamic studies of gene control within biomedicine, including the regulation of immune responses. In this review article, we summarize recent advances in bioluminescence-based imaging approaches that promise to enlighten our understanding of in vivo gene and epigenetic control within the immune system.

Section snippets

Bioluminescence and Luciferase-Based Assays

Bioluminescence is a process in which light is emitted from a living organism during oxygenation of a substrate (a luciferin, see Glossary) by an enzyme (a luciferase) [1]. Descriptions of this extraordinary phenomenon date back thousands of years, with bioluminescence occurring extensively in marine life and in a range of terrestrial organisms, including fungi and soft-bodied beetles, such as fireflies. Bioluminescent systems encompass a huge variety of enzymes and substrates, each of which

Using Bioluminescence for In Vivo Imaging

A variety of technologies now exist for in vivo reporter imaging [6,7] (Box 1); however, BLI offers a relatively low-cost and accessible approach with several distinct advantages over other modalities, including over other optical techniques, such as fluorescence imaging (FLI) [1,3,4]. In particular, BLI offers an exceptionally high signal-to-noise ratio, since no external illumination is required, and background light emission is essentially zero. This contrasts with FLI, which relies on

Using BLI to Monitor Gene Expression In Vivo

Advances in BLI technology are now allowing researchers to expand applications beyond cell tracking and revisit luciferase as a reporter for transcriptional dynamics, in an in vivo setting. Although BLI can be used to report in vivo gene expression in a variety of different contexts, as exemplified later, imaging requires three basic components: a luciferase enzyme (encoded genetically), a luciferin substrate (often administered by injection), and a sensitive imaging system for light detection,

Applying BLI Approaches to the Immune System

In the body, immune cells do not operate in isolation, but as part of a spatially and temporally complex and dynamic system, influenced by multiple cellular interactions and environmental factors. However, much of our current molecular understanding of the immune system is derived from ex vivo studies of individual cells and factors [43]. BLI offers a complementary and non-invasive approach to study immune responses in a physiological setting in vivo.

Seeing More with Bioluminescence

BLI provides a powerful tool for studying biology in vivo and new technological advances are on the horizon. There has been significant progress in the discovery and synthetic development of new luciferase enzymes and improved substrates for in vivo imaging [1., 2., 3., 4.] (Table 1). Despite the sensitivity of BLI, one of the biggest challenges remains imaging deeper internal sites, due to absorption and scattering of light by host tissue, especially for shorter wavelengths below 600 nm [69].

Concluding Remarks

As researchers strive to reach a more complete understanding of the immune system upon challenge and across organism lifespans, non-invasive gene reporters promise to make increasingly important contributions. The intrinsic advantages of BLI, in particular its low background and high sensitivity, make this approach ideal for studying complex, spatially and temporally dynamic processes that are hallmarks of immunology. The BLI toolkit continues to grow and further innovations for imaging deep

Acknowledgments

This work was funded by core support from the Medical Research Council UK to the London Institute of Medical Sciences.

Glossary

2A self-cleaving peptide sequences
(e.g., T2A, P2A) allow production of separate proteins from a single open reading frame.
Autonomous bioluminescence
light production from cells or an organism that does not require external addition of luciferin or other components, as is the case in naturally occurring bioluminescence. This requires that both the luciferase and the pathways for substrate synthesis be genetically encoded, which is currently rare for artificial reporter systems.
Bacterial

References (105)

  • V.H. Nguyen

    In vivo dynamics of regulatory T-cell trafficking and survival predict effective strategies to control graft-versus-host disease following allogeneic transplantation

    Blood

    (2007)
  • X. Yang

    Tracing the dynamic expression of the Nfκb2 gene during inflammatory processes by in vivo bioluminescence imaging in transgenic mice

    Biochem. Biophys. Res. Commun.

    (2018)
  • M. Yasunaga

    Highly sensitive luciferase reporter assay using a potent destabilization sequence of calpain 3

    J. Biotechnol.

    (2015)
  • B.R. Branchini

    Thermostable red and green light-producing firefly luciferase mutants for bioluminescent reporter applications

    Anal. Biochem.

    (2007)
  • B.R. Branchini

    Red-emitting luciferases for bioluminescence reporter and imaging applications

    Anal. Biochem.

    (2010)
  • B.A. Tannous

    Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo

    Mol. Ther.

    (2005)
  • T. Abe

    Pronuclear microinjection during S-phase increases the efficiency of CRISPR-Cas9-assisted knockin of large DNA donors in mouse zygotes

    Cell Rep.

    (2020)
  • H. Yang

    One-step generation of mice carrying reporter and conditional alleles by CRISPR/cas-mediated genome engineering

    Cell

    (2013)
  • D. Xiao

    A novel luciferase knock-in reporter system for studying transcriptional regulation of the human Sox2 gene

    J. Biotechnol.

    (2016)
  • H.W. Yeh et al.

    Development and applications of bioluminescent and chemiluminescent reporters and biosensors

    Annu. Rev. Anal. Chem.

    (2019)
  • T. Xu

    The expanding toolbox of in vivo bioluminescent imaging

    Front. Oncol.

    (2016)
  • S.T.M. Allard et al.

    Luciferase reporter assays: powerful, adaptable tools for cell biology research

    Cell Notes

    (2008)
  • D.T. Lauber

    State of the art in vivo imaging techniques for laboratory animals

    Lab. Anim.

    (2017)
  • M. Li

    Multimodality reporter gene imaging: construction strategies and application

    Theranostics

    (2018)
  • X. Yan

    A transgenic tri-modality reporter mouse

    PLoS One

    (2013)
  • R.A. Levin

    An optimized triple modality reporter for quantitative in vivo tumor imaging and therapy evaluation

    PLoS One

    (2014)
  • C. Genevois

    In vivo follow-up of brain tumor growth via bioluminescence imaging and fluorescence tomography

    Int. J. Mol. Sci.

    (2016)
  • J.E. Kim

    In vivo cell tracking with bioluminescence imaging

    Nucl. Med. Mol. Imaging

    (2015)
  • J.H. Chewning

    Bioluminescence-based visualization of CD4 T cell dynamics using a T lineage-specific luciferase transgenic model

    BMC Immunol.

    (2009)
  • S. Iwano

    Single-cell bioluminescence imaging of deep tissue in freely moving animals

    Science

    (2018)
  • J. Charo

    Visualizing the dynamic of adoptively transferred T cells during the rejection of large established tumors

    Eur. J. Immunol.

    (2011)
  • J.B. Kim

    Non-invasive detection of a small number of bioluminescent cancer cells in vivo

    PLoS One

    (2010)
  • L. Mezzanotte

    Sensitive dual color in vivo bioluminescence imaging using a new red codon optimized firefly luciferase and a green click beetle luciferase

    PLoS One

    (2011)
  • M. Aswendt

    Quantitative in vivo dual-color bioluminescence imaging in the mouse brain

    Neurophotonics

    (2019)
  • C.L. Stowe

    Near-infrared dual bioluminescence imaging in mouse models of cancer using infraluciferin

    Elife

    (2019)
  • L. Mezzanotte

    Evaluating reporter genes of different luciferases for optimized in vivo bioluminescence imaging of transplanted neural stem cells in the brain

    Contrast Media Mol. Imaging

    (2013)
  • E.B. Santos

    Sensitive in vivo imaging of T cells using a membrane-bound Gaussia princeps luciferase

    Nat. Med.

    (2009)
  • Y. Su

    Novel NanoLuc substrates enable bright two-population bioluminescence imaging in animals

    Nat. Methods

    (2020)
  • C. Daniel

    Dual-color bioluminescence imaging for simultaneous monitoring of the intestinal persistence of Lactobacillus plantarum and Lactococcus lactis in living mice

    Appl. Environ. Microbiol.

    (2015)
  • M. Chang

    Real-time bioluminescence imaging of mixed mycobacterial infections

    PLoS One

    (2014)
  • G. Vande Velde

    Towards non-invasive monitoring of pathogen-host interactions during Candida albicans biofilm formation using in vivo bioluminescence

    Cell. Microbiol.

    (2014)
  • T. Kuchimaru

    A luciferin analogue generating near-infrared bioluminescence achieves highly sensitive deep-tissue imaging

    Nat. Commun.

    (2016)
  • A.M. Zagozdzon

    Generation of a new bioluminescent model for visualisation of mammary tumour development in transgenic mice

    BMC Cancer

    (2012)
  • N. Zhang

    A spontaneous acinar cell carcinoma model for monitoring progression of pancreatic lesions and response to treatment through noninvasive bioluminescence imaging

    Clin. Cancer Res.

    (2009)
  • J. Wang

    Molecular imaging of mesenchymal stem cell mechanistic insight into cardiac repair after experimental myocardial infarction

    Circ. Cardiovasc. Imaging

    (2012)
  • A.C. Stacer

    NanoLuc reporter for dual luciferase imaging in living animals

    Mol. Imaging

    (2013)
  • N. Gueven

    Regulation of the Atm promoter in vivo

    Genes Chromosom. Cancer

    (2006)
  • T. Hamada

    In vivo imaging of clock gene expression in multiple tissues of freely moving mice

    Nat. Commun.

    (2016)
  • K.L. Tinkum

    Bioluminescence imaging captures the expression and dynamics of endogenous p21 promoter activity in living mice and intact cells

    Mol. Cell. Biol.

    (2011)
  • D. Olmeda

    Whole-body imaging of lymphovascular niches identifies pre-metastatic roles of midkine

    Nature

    (2017)
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