Multicolor Two-photon Imaging of in Vivo Cellular Pathophysiology Upon Influenza Virus Infection Using the Two-photon IMPRESS

Overview

Journal Nat Protoc

Specialties Biology
Pathology
Science

Date 2020 Jan 31

PMID 31996843

Citations 20

Authors

Hiroshi Ueki

I-Hsuan Wang

Dongming Zhao

Matthias Gunzer

Yoshihiro Kawaoka

Affiliations

Soon will be listed here.

Abstract

In vivo two-photon imaging is a valuable technique for studies of viral pathogenesis and host responses to infection in vivo. In this protocol, we describe a methodology for analyzing influenza virus-infected lung in vivo by two-photon imaging microscopy. We describe the surgical procedure, how to stabilize the lung, and an approach to analyzing the data. Further, we provide a database of fluorescent dyes, antibodies, and reporter mouse lines that can be used in combination with a reporter influenza virus (Color-flu) for multicolor analysis. Setup of this model typically takes ~30 min and enables the observation of influenza virus-infected lungs for >4 h during the acute phase of the inflammation and at least 1 h in the lethal phase. This imaging system, which we termed two-photon IMPRESS (imaging pathophysiology research system), is broadly applicable to analyses of other respiratory pathogens and reveals disease progression at the cellular level in vivo.

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References

Wang J, Bair A, King S, Shnayder R, Huang Y, Shieh C . Ly6G ligation blocks recruitment of neutrophils via a β2-integrin-dependent mechanism. Blood. 2012; 120(7):1489-98. PMC: 3423786. DOI: 10.1182/blood-2012-01-404046. View

Choi M, Kwok S, Yun S . In vivo fluorescence microscopy: lessons from observing cell behavior in their native environment. Physiology (Bethesda). 2015; 30(1):40-9. PMC: 4285577. DOI: 10.1152/physiol.00019.2014. View

Tinevez J, Perry N, Schindelin J, Hoopes G, Reynolds G, Laplantine E . TrackMate: An open and extensible platform for single-particle tracking. Methods. 2016; 115:80-90. DOI: 10.1016/j.ymeth.2016.09.016. View

Katsura H, Fukuyama S, Watanabe S, Ozawa M, Neumann G, Kawaoka Y . Amino acid changes in PB2 and HA affect the growth of a recombinant influenza virus expressing a fluorescent reporter protein. Sci Rep. 2016; 6:19933. PMC: 4742795. DOI: 10.1038/srep19933. View

Neher R, Neher E . Optimizing imaging parameters for the separation of multiple labels in a fluorescence image. J Microsc. 2003; 213(1):46-62. DOI: 10.1111/j.1365-2818.2004.01262.x. View

Hanna R, Cekic C, Sag D, Tacke R, Thomas G, Nowyhed H . Patrolling monocytes control tumor metastasis to the lung. Science. 2015; 350(6263):985-90. PMC: 4869713. DOI: 10.1126/science.aac9407. View

Mertens M, Tabuchi A, Meissner S, Krueger A, Schirrmann K, Kertzscher U . Alveolar dynamics in acute lung injury: heterogeneous distension rather than cyclic opening and collapse. Crit Care Med. 2009; 37(9):2604-11. DOI: 10.1097/CCM.0b013e3181a5544d. View

Matheu M, Teijaro J, Walsh K, Greenberg M, Marsolais D, Parker I . Three phases of CD8 T cell response in the lung following H1N1 influenza infection and sphingosine 1 phosphate agonist therapy. PLoS One. 2013; 8(3):e58033. PMC: 3606384. DOI: 10.1371/journal.pone.0058033. View

Surewaard B, Thanabalasuriar A, Zeng Z, Tkaczyk C, Cohen T, Bardoel B . α-Toxin Induces Platelet Aggregation and Liver Injury during Staphylococcus aureus Sepsis. Cell Host Microbe. 2018; 24(2):271-284.e3. PMC: 6295203. DOI: 10.1016/j.chom.2018.06.017. View

10.

Yipp B, Kim J, Lima R, Zbytnuik L, Petri B, Swanlund N . The Lung is a Host Defense Niche for Immediate Neutrophil-Mediated Vascular Protection. Sci Immunol. 2017; 2(10). PMC: 5472445. DOI: 10.1126/sciimmunol.aam8929. View

11.

Narasaraju T, Yang E, Perumal Samy R, Ng H, Poh W, Liew A . Excessive neutrophils and neutrophil extracellular traps contribute to acute lung injury of influenza pneumonitis. Am J Pathol. 2011; 179(1):199-210. PMC: 3123873. DOI: 10.1016/j.ajpath.2011.03.013. View

12.

Sakai K, Miyazaki J . A transgenic mouse line that retains Cre recombinase activity in mature oocytes irrespective of the cre transgene transmission. Biochem Biophys Res Commun. 1997; 237(2):318-24. DOI: 10.1006/bbrc.1997.7111. View

13.

MacGregor R . Examination of the pulmonary circulation with the microscope. J Physiol. 1933; 80(1):65-77. PMC: 1394355. DOI: 10.1113/jphysiol.1933.sp003071. View

14.

Denk W, Holt J, Shepherd G, Corey D . Calcium imaging of single stereocilia in hair cells: localization of transduction channels at both ends of tip links. Neuron. 1995; 15(6):1311-21. DOI: 10.1016/0896-6273(95)90010-1. View

15.

Zheng Z, Chiu S, Akbarpour M, Sun H, Reyfman P, Anekalla K . Donor pulmonary intravascular nonclassical monocytes recruit recipient neutrophils and mediate primary lung allograft dysfunction. Sci Transl Med. 2017; 9(394). PMC: 5568853. DOI: 10.1126/scitranslmed.aal4508. View

16.

Chojnacki A, Wojcik K, Petri B, Aulakh G, Jacobsen E, LeSuer W . Intravital imaging allows real-time characterization of tissue resident eosinophils. Commun Biol. 2019; 2:181. PMC: 6513871. DOI: 10.1038/s42003-019-0425-3. View

17.

Funakoshi N, Onizuka M, Yanagi K, Ohshima N, Tomoyasu M, Sato Y . A new model of lung metastasis for intravital studies. Microvasc Res. 2000; 59(3):361-7. DOI: 10.1006/mvre.2000.2238. View

18.

Schmidt E, Yang Y, Janssen W, Gandjeva A, Perez M, Barthel L . The pulmonary endothelial glycocalyx regulates neutrophil adhesion and lung injury during experimental sepsis. Nat Med. 2012; 18(8):1217-23. PMC: 3723751. DOI: 10.1038/nm.2843. View

19.

WAGNER Jr W, Latham L, Capen R . Capillary recruitment during airway hypoxia: role of pulmonary artery pressure. J Appl Physiol Respir Environ Exerc Physiol. 1979; 47(2):383-7. DOI: 10.1152/jappl.1979.47.2.383. View

20.

Looney M, Headley M . Live imaging of the pulmonary immune environment. Cell Immunol. 2018; 350:103862. PMC: 6742573. DOI: 10.1016/j.cellimm.2018.09.007. View