Characterization of Immune Cells and Proinflammatory Mediators in the Pulmonary Environment

Summary

This protocol describes the use of flow cytometry to identify the changes in immune cell composition, cytokine profile, and chemokine profile in the pulmonary environment following transient middle cerebral artery occlusion, a murine model of ischemic stroke.

Abstract

Immune cell expansion, activation, and trafficking to the lungs, which are controlled by the expression of multiple cytokines and chemokines, may be altered by severe brain injury. This is evidenced by the fact that pneumonia is a major cause of mortality in patients who have suffered from ischemic stroke. The goal of this protocol is to describe the use of multicolor flow cytometric analysis to identify 13 types of immune cells in the lungs of mice, including alveolar macrophages, interstitial macrophages, CD103+ or CD11b+ dendritic cells (DCs), plasmacytoid DCs, eosinophils, monocytes/monocyte-derived cells, neutrophils, lymphoid-derived  T and B cells, NK cells, and NKT cells, following ischemic stroke induction by transient middle cerebral artery occlusion. Moreover, we describe the preparation of lung homogenates using a bead homogenization method, to determine the expression levels of 13 different cytokines or chemokines simultaneously by multiplex bead arrays coupled with flow cytometric analysis. This protocol can also be used to investigate the pulmonary immune response in other disease settings, such as infectious lung disease or allergic disease.

Introduction

The lungs are a barrier organ, exposed to the external environment and, therefore, are constantly receiving immunological challenges such as pathogens and allergens¹. The activation of lung-resident immune cells and the infiltration of immune cells from the periphery are required to clear pathogens from the pulmonary environment. Additionally, lung-resident immune cells maintain tolerance to commensal bacteria, suggesting that these cells play a role in pathogen clearance and maintaining homeostasis¹. Alveolar and interstitial macrophages are among the lung-resident sentinel immune cells that sense pathogens via pattern ....

Protocol

All protocols and procedures performed were approved by the Institutional Animal Care and Use Committee (IACUC) of West Virginia University. The mice were housed under specific-pathogen-free conditions in the vivarium at West Virginia University.  

1. Preparation of solutions

1. Prepare perfusion buffer (phosphate buffered saline, PBS). Use approximately two 10 mL aliquots of ice-cold PBS per mouse.
2. Prepare lung cell medium/FACS buffer. FACS buffer contains PBS supplemented with 1% fetal bovine serum (FBS). Keep the medium cold for the entire process of lung excision and transfer. Prepare approximately 8 mL per lung ....

Results

We recently reported that ischemic stroke induction in mice alters the immune cell composition of the lungs¹¹. Specifically, transient cerebral ischemia increased percentages of alveolar macrophages, neutrophils, and CD11b+ DCs, while diminishing percentages of CD4+ T cells, CD8+ T cells, B cells, NK cells, and eosinophils in the pulmonary compartment. Moreover, cellular alteration corresponded to significantly diminished levels of multiple chemokines in the lungs. Described here is a method for t.......

Discussion

The protocols described here allow for the identification of lung immune cell types and the expression of chemokines or cytokines in the same mouse. If a histopathology study is desired, an individual lobe can be removed and fixed for that purpose prior to proceeding to the single cell isolation steps. One limitation of this method is that this approach may not be suitable in some disease settings if the change in the immune cell composition and the expression of chemokines and/or cytokines are anticipated to be unequall.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
B220-APC, clone RA3-6B2	Biolegend	103212	1:200 dilution
Beadbug 3 position bead homogenizer	Benchmark Scientific	D1030	Tissue homogenizer
CCR2-BV421, clone SA203G11	Biolegend	150605	1:200 dilution
CD103-BV421, clone 2E7	Biolegend	121422	1:200 dilution
CD11b-PE/Cy7, clone M1/70	Biolegend	101216	1:400 dilution
CD11c-PE/Cy7, clone N418	Biolegend	117318	1:200 dilution
CD11c-Percp/Cy5.5, clone N418	Biolegend	117328	1:200 dilution
CD4-BV421, clone GK1.5	Biolegend	100443	1:200 dilution
CD45-FITC, clone 30-F11	Biolegend	103108	1:200 dilution
CD64-APC, clone X54-5/7.1	Biolegend	139306	1:200 dilution
CD8-PE, clone 53-6.7	Biolegend	100708	1:800 dilution
Collagenase D	Sigma Aldrich	11088882001	Component in the dissociation buffer
Conical screw cap tube	ThermoFisher	02-681-344	Tube for tissue homogenization
DNase I	Sigma Aldrich	10104159001	Component in the dissociation buffer
Fc block CD16/32 antibody	Biolegend	101320	1:100 dilution
genlteMACS dissociator	Miltenyi Biotec	130-093-235	Comparsion of lung digestion with or without mechanical dissociator
gentleMACS C tubes	Miltenyi Biotec	130-093-237	Tube for tissue disscoiation with genlteMACS dissociator
Halt protease and phosphatase inhibitor cocktial	ThermoFisher	78442	Component in the homogenization buffer
Laser doppler monitor	Moor	MOORVMS-LDF	Blood flow monitoring during tMCAO
LEGENDplex proinflammatory chemokine panel	Biolegend	740451	Multiplex bead array
LIVE/DEAD fixable near-IR stain	ThermoFisher	L34976	Use for dead cell exclusion during flow cytometric analysis
Ly6C-PE, clone HK1.4	Biolegend	128008	1:800 dilution
Ly6G-BV510, clone 1A8	Biolegend	127633	1:200 dilution
MCAO suture L56 reusable 6-0 medium	Doccol	602356PK10Re	tMCAO
MHC II-BV510, clone M5/114.15.2	Biolegend	107636	1:800 dilution
NK1.1-Percp/Cy5.5, clone PK136	Biolegend	108728	1:200 dilution
Siglec F-PE, clone E50-2440	BD Biosciences	552126	1:200 dilution
Silk suture thread, size 6/0	Fine Science Tools	18020-60	tMCAO
SomnoSuite anesthesia system	Kent Scientific	SS-01	Mouse anaesthetization for tMCAO
TCRb-BV510, clone H57-897	Biolegend	109234	1:200 dilution
Zirconia/silica beads, 2.3 mm	Biospec	11079125z	Beads for tissue homogenization