Advanced Imaging of Lung Homing Human Lymphocytes in an Experimental In Vivo Model of Allergic Inflammation Based on Light-sheet Microscopy

Summary

The here introduced protocol allows characterization of the lung homing capacity of primary human lymphocytes under in vivo inflammatory conditions. Pulmonary infiltration of adoptively transferred human immune cells in a mouse model of allergic inflammation can be imaged and quantified by light-sheet fluorescence microscopy of chemically cleared lung tissue.

Abstract

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an attractive therapeutic target in the clinical management of affected patients. In order to further optimize strategies aiming at therapeutic regulation of pathologically imbalanced tissue infiltration of pro-inflammatory immune cells, it will be of particular importance to achieve improved insights into disease- and organ-specific homing properties of peripheral lymphocytes. The here described experimental protocol allows to monitor lung accumulation of fluorescently labeled and adoptively transferred human lymphocytes in the context of papain-induced pulmonary inflammation. In contrast to standard in vitro assays frequently used for the analysis of immune cell migration and chemotaxis, the now introduced in vivo setting takes into account lung-specific aspects of tissue organization and the influence of the complex inflammatory scenario taking place in the living murine organism. Moreover, three-dimensional cross-sectional light-sheet fluorescence microscopic imaging does not only provide quantitative data on infiltrating immune cells, but also depicts the pattern of immune cell localization within the inflamed lung. Overall, we are able to introduce an innovative technique of high value for immunological research in the field of chronic inflammatory lung diseases, which can be easily applied by following the provided step-by-step protocol.

Introduction

Classic inflammatory disorders of the lung, such as allergic asthma and chronic obstructive pulmonary disease (COPD), are well known to be driven by an increased recruitment of activated lymphocytes into the pulmonary tissue^1,2. Lymphocyte-released cytokines (e.g., IL-4, IL-5, IL-9, IL-13, IFN-γ and TNF-α) further promote chemotaxis of innate and adaptive immune cells, induce fibrotic airway remodeling or directly damage the lung parenchyma². So far, the underlying mechanisms responsible for the pathological accumulation of lymphocytes within lung tissue are not yet fully unde....

Protocol

Experiments involving animals were performed in accordance with protocols approved by the relevant local authorities in Erlangen (Regierung von Unterfranken, Würzburg, Germany). Mice were housed under specific pathogen-free conditions. The collection of human blood was approved by the local ethical committee and the institutional review board of the University of Erlangen-Nuremberg. Each patient gave written informed consent.

1. Induce Allergic Lung Inflammation in Mice

NOTE: As described in earlier studies²⁷, the following experimental procedure allows inducing allergi....

Results

The presented protocol describes an experimental mouse model, which allows monitoring and quantifying the accumulation of adoptively transferred human T lymphocytes in the lung via light-sheet fluorescence microscopy. Figure 1A provides a schematic overview of the in vivo steps of the experimental schedule. In order to guarantee reliable results, it is of substantial importance to ensure a good quality of the isolated and fluorescently labeled human CD4⁺ T cells, .......

Discussion

The here described experimental setting provides the opportunity to monitor the lung homing capacity of primary human immune cells under in vivo inflammatory conditions and thereby relevantly complements classically performed in vitro adhesion and chemotaxis assays. To take into account specific anatomic organ characteristics of the lung, important aspects of immune cell homing (including chemotaxis and cell distribution within the target organ) as well as the clinical relevance and transfera.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agarose NEEO Ultra	Carl Roth GmbH + Co. KG, Karlsruhe, Germany	2267.4
AlexaFlour594 anti-human CD45 antibody	BioLegend, San Diego, USA	304060
Ammonium chloride	Carl Roth GmbH + Co. KG, Karlsruhe, Germany	K2981
Cannula 21 G	Becton, Dickinson and Company, Franklin Lakes, USA	301300
Cell proliferation dye eflour670	eBioscience Inc., San Diego, USA	65-0840-85
CD4 MicroBeads, human	Miltenyi Biotech GmbH, Bergisch-Gladbach, Germany	130-045-101
EDTA (ethylenediaminetetraacetic acid)	Carl Roth GmbH + Co. KG, Karlsruhe, Germany	8043.1
Potassium-EDTA blood collection tube, 9 mL	Sarstedt AG & Co., Nümbrecht, Germany	21066001
Ethly cinnamate (ECi)	Sigma-Aldrich, Steinheim, Germany	112372-100G
Ethanol ≥ 99.5% (EtOH)	Carl Roth GmbH + Co. KG, Karlsruhe, Germany	5054.3
FBS (fetal bovine serum) Good Forte	PAN-Biotech GmbH, Aidenbach, Germany	P40-47500
Filter 100 µm	VWR International Germany GmbH, Darmstadt, Germany	732-2758
Imaris Image Analysis Software 9.0.2	Bitplane AG, Zurich, Switzerland	n.a.
ImspectorPro software	Abberior Instruments GmbH, Göttingen, Germany	n.a.
Ketamin	Inresa Arzneimittel GmbH, Freiburg, Germany	3617KET-V
LaVision UltraMicroscope II	LaVision BioTec GmbH, Bielefeld, Germany	n.a.
MACS MultiStand	Miltenyi Biotech GmbH, Bergisch-Gladbach, Germany	130-042-303
Multifly cannula 20 G	Sarstedt AG & Co., Nümbrecht, Germany	851638035
30 G needle	B. Braun Melsungen AG, Melsungen, Hessen, Germany	9161502
Neubauer counting chamber	neoLab Migge GmbH, Heidelberg, Germany	C-1003
Pattex Glue	Henkel AG & Co, Düsseldorf, Germany	PSK1C
LS column	Miltenyi Biotech GmbH, Bergisch-Gladbach, Germany	130-042-401
Lymphocyte Separation Media (Density 1,077 g/mL)	anprotec	AC-AF-0018
Papain	Merck	1,071,440,025
PBS Dulbecco (phosphate buffered saline)	Biochrom GmbH, Berlin, Germany	L182-10
PerCP/Cy5.5 anti-human CD4	BioLegend, San Diego, USA	317428
PerCP/Cy5.5 mouse IgG2b, κ isotype Ctrl	BioLegend, San Diego, USA	400337
PFA (paraformaldehyde)	Carl Roth GmbH + Co. KG, Karlsruhe, Germany	0335.1
Potassium hydrogen carbonate	Carl Roth GmbH + Co. KG, Karlsruhe, Germany	P7481
Serological pipette 10 mL	Sarstedt AG & Co., Nümbrecht, Germany	86.1254.001
Syringe 1 mL	B. Braun Melsungen AG, Melsungen, Hessen, Germany	9166017V
Syringe 5 mL	Becton, Dickinson and Company, Franklin Lakes, USA	260067
Syringe 20 mL	Becton, Dickinson and Company, Franklin Lakes, USA	260069
Tube 1.5 mL	Sarstedt AG & Co., Nümbrecht, Germany	72,706,400
Tube 2 mL	Sarstedt AG & Co., Nümbrecht, Germany	72.695.400
Tube 2 mL, brown	Sarstedt AG & Co., Nümbrecht, Germany	72,695,001
Tube 15 mL	Sarstedt AG & Co., Nümbrecht, Germany	62.554.502
Tube 50 mL	Sarstedt AG & Co., Nümbrecht, Germany	62.547.254
QuadroMACS Separator	Miltenyi Biotech GmbH, Bergisch-Gladbach, Germany	130-090-976
Xylazin (Rompun 2%)	Bayer Vital GmbH, Leverkusen, Germany	KPOBD32