Name: Advanced Imaging of Lung Homing Human Lymphocytes in an Experimental In Vivo Model of Allergic Inflammation Based on Light-sheet Microscopy (Video) | JoVE Journal
Uploaded: 2019-04-16T13:30:00.000+00:00
Duration: 10 min 39 s
Description: Learn light-sheet microscopy for imaging lung-homing human lymphocytes in a mouse model of allergic inflammation. This protocol enables precise quantification of immune cell accumulation to advance translational immunology research.

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The authors gratefully acknowledge funding by the DFG Collaborative Research Centers SFB 1181 and TRR 241. The Optical Imaging Centre Erlangen (OICE) and in particular Ralf Palmisano, Philipp Tripal and Tina Fraa&#223; (Project Z2 of the DFG CRC 1181) are acknowledged for expert technical support for light-sheet fluorescence microscopic imaging.

M.F. Neurath has served as an advisor for Pentax, Giuliani, MSD, Abbvie, Janssen, Takeda and Boehringer. The remaining authors do not disclose any conflict.

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...

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 tissue1,2. Lymphocyte-released cytokines (e.g., IL-4, IL-5, IL-9, IL-13, IFN-&#947; and TNF-&#945;) 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 understood. In analogy to tissue-selective T cell imprinting described for gut and skin homing, pulmonary dendritic cells (DCs) are obviously able to prime peripheral T cells for preferential lung infiltration, at least partly via the induction of CCR4 expression on the surface of lymphocytes3. Besides CCR4, airway-infiltrating T cells are also characterized by a particularly increased expression of the chemokine receptors CCR5 and CXCR3 compared to T cells within the peripheral blood1,4,5. Overall, existing data are consistent with the concept that lung homing of T lymphocytes under physiological or inflammatory conditions involves a number of different chemokine receptors and their respective ligands and thus crucially depends on a closely controlled collaboration between innate and adaptive immune cells1. Especially, during the initial phase of pathogen or allergen exposure, cells of the innate immune system respond to TLR stimulation or to IgE-mediated cross-linking by the immediate release of different chemoattractants, like LTB4, CCL1, CCL17, CCL22, CCL20, CXCL10 and PGD21,6,7. As a prime example, the interaction between PGD2 and the chemoattractant receptor CRTh2 is known to be of particular importance for chemotaxis of Th2 cells and thus appeared as promising therapeutic target in the clinical management of asthma. Indeed, patients with moderate asthma showed an improvement of symptoms and a significant increase of the forced expiratory volume in one second (FEV1) after treatment with a selective CRTh2 antagonist compared to the placebo group8,9. In a more progressed state of the inflammatory response, already recruited T cells are able to further amplify pulmonary lymphocyte accumulation via the release of IL-4 and IL-13 as potent stimuli for pulmonary DCs. Subsequently, these myeloid-derived innate cells up-regulate the expression of CCL17 and CCL22 in a STAT6-dependent manner1,10,11.&#160; Although the complexity of the described scenario still hinders a complete understanding of T cell lung homing, it offers a plethora of molecular targets for a potentially optimized therapeutic control of inflammatory or allergic pulmonary diseases. Therefore, there is an urgent need of innovative experimental techniques, which are able to further deepen and complement our knowledge in the field of T cell chemotaxis and lung homing.
Due to the fact that lung homing of lymphocytes within the human body is influenced by multiple cellular, humoral and physical parameters1, most of the existing experimental methods are not able to model the whole complexity of this immunological process. Instead, many standard protocols for the analysis of lung homing selectively focus on a specific aspect involved in the cascade of lymphocyte attraction, adhesion, migration and retention. Besides a purely descriptive determination of the mRNA or protein expression pattern of integrins and chemokine receptors on peripheral or lung-infiltrating lymphocytes and the complementary measurement of respective chemokine levels in blood, bronchoalveolar lavage (BAL) or pulmonary tissue12,13,14,15, well-established in vitro cell culture assays allow a functional characterization of lymphocyte adhesion or chemotaxis upon defined experimental conditions16,17,18. In principle, static in vitro adhesion assays monitor the binding capacity of cultured lymphocytes to an endothelial monolayer or to glass slides coated with recombinant endothelial adhesion molecules (e.g., MAdCAM-1, VCAM-1), while standard in vitro chemotaxis assays are usually applied in order to quantify the ability of lymphocytes to migrate along a chemokine gradient in a transwell system19. Both in vitro settings enable a controlled adjustment and modulation of experimental conditions, but on the other hand lack important variables known to critically impact on in vivo chemotaxis and adhesion of lymphocytes. Predominantly, static cell culture assays disregard the influence of shear forces caused by the permanent blood flow19 and potentially neglect the involvement of the surrounding immunological milieu and interacting non-lymphocyte immune cells, both present in a living organism. In order to overcome these limitations, the interpretation of results acquired in static in vitro chemotaxis or adherence assays need further validation in dynamic adhesion experiments under flow conditions20,21 and in in vivo models of inflammatory organ pathology19. Indeed, important conclusions on the regulation of T cell lung homing under inflammatory or allergic conditions could be drawn from animal studies analyzing genetically modified mice in defined models of different pulmonary diseases3,22,23. The quantitative comparison of lung infiltrating lymphocytes between wildtype mice and mice with a deficiency for a specific gene of interest represents a well-established and broadly used tool for defining the impact of particular cellular pathways or receptors on the disease-driven pattern of T cell distribution. However, in contrast to before discussed in vitro cell culture assays, a study design based on classical animal models lacks the ability to analyze and monitor primary human T cells directly derived from the blood or BAL of patients suffering from an inflammatory lung disease. Thus, it still remains challenging to functionally validate whether a diagnostically specified lung disease is able to imprint human lymphocytes for preferential lung tropism and how far clinical parameters might impact on this scenario. Recently, a very elegant in vivo approach was introduced in the context of inflammatory bowel diseases (IBD), which was able to overcome most of these limitations and opened new avenues for advanced translational studies on intestinal lymphocyte homing24. Taking advantage of protocols for solvent-based tissue clearing followed by cross-sectional light-sheet fluorescence microscopy as a powerful imaging tool, it was possible to visualize the infiltration and distribution of adoptively transferred human T cells in the intestine of colitic immunodeficient mice24. In particular, this experimental setting implemented two main innovations: (1) Primary human immune cells can be analyzed under experimentally defined in vivo conditions; (2) a rather large area of the diseased organ (about 1.5&#160;cm&#160;x&#160;1.5&#160;cm) can be imaged in high resolution quality, followed by 3D-reconstruction. Moreover, several recent studies successfully established the use of solvent-based tissue clearing and light-sheet fluorescence microscopy as important tools for advanced lung imaging25,26. In order to benefit from this technological progress in the field of pulmonary immunology, we now adopted the system for analysis of lung homing.
The here presented protocol provides a step-by-step introduction how to purify and fluorescently label primary human T cells for transfer into mice with induced pulmonary inflammation and, moreover, describes in detail the subsequent process of light-sheet fluorescence microscopic imaging, including organ preparation and image processing. Overall, we hope to support future translational studies in the field of inflammatory or allergic lung diseases by introducing a sophisticated, but nevertheless feasible, experimental model for monitoring human lymphocyte lung homing upon in vivo conditions.

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

advanced imaging of lung homing human lymphocytes in an experimental in vivo model of allergic inflammation based on light-sheet microscopy

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

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.

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, ...

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.

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an ...

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Learn light-sheet microscopy for imaging lung-homing human lymphocytes in a mouse model of allergic inflammation. This protocol enables precise quantification of immune cell accumulation to advance translational immunology research.

Advanced Imaging of Lung Homing Human Lymphocytes in an Experimental In Vivo Model of Allergic Inflammation Based on Light-sheet Microscopy (Video) | JoVE Journal

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

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