Visualizing Lung Cellular Adaptations during Combined Ozone and LPS Induced Murine Acute Lung Injury

Summary

Combined ozone and bacterial endotoxin exposed mice show wide-spread cell death, including that of neutrophils. We observed cellular adaptations such as disruption of cytoskeletal lamellipodia, increased cellular expression of complex V ATP synthase subunit β and angiostatin in broncho-alveolar lavage, suppression of the lung immune response and delayed neutrophil recruitment.

Abstract

Lungs are continually faced with direct and indirect insults in the form of sterile (particles or reactive toxins) and infectious (bacterial, viral or fungal) inflammatory conditions. An overwhelming host response may result in compromised respiration and acute lung injury, which is characterized by lung neutrophil recruitment as a result of the patho-logical host immune, coagulative and tissue remodeling response. Sensitive microscopic methods to visualize and quantify murine lung cellular adaptations, in response to low-dose (0.05 ppm) ozone, a potent environmental pollutant in combination with bacterial lipopolysaccharide, a TLR4 agonist, are crucial in order to understand the host inflammatory and repair mechanisms. We describe a comprehensive fluorescent microscopic analysis of various lung and systemic body compartments, namely the broncho-alveolar lavage fluid, lung vascular perfusate, left lung cryosections, and sternal bone marrow perfusate. We show damage of alveolar macrophages, neutrophils, lung parenchymal tissue, as well as bone marrow cells in correlation with a delayed (up to 36-72 h) immune response that is marked by discrete chemokine gradients in the analyzed compartments. In addition, we present lung extracellular matrix and cellular cytoskeletal interactions (actin, tubulin), mitochondrial and reactive oxygen species, anti-coagulative plasminogen, its anti-angiogenic peptide fragment angiostatin, the mitochondrial ATP synthase complex V subunits, α and β. These surrogate markers, when supplemented with adequate in vitro cell-based assays and in vivo animal imaging techniques such as intravital microscopy, can provide vital information towards understanding the lung response to novel immunomodulatory agents.

Introduction

Acute lung injury (ALI) is a crucial pathologic response of lungs to infectious or other harmful stimuli which is marked by simultaneous activation of coagulative, fibrinolytic and innate immune systems¹. Neutrophils promptly sense microbial as well as intracellular damage patterns through the Toll-like receptor (TLR) family^2,3,4. Neutrophils release preformed cytokines and cytotoxic granule contents, which can then cause collateral tissue damage. The ensuing alveolar damage is marred with secondary cell death leading to release of molecules such as ad....

Protocol

The study design was approved by the University of Saskatchewan's Animal Research Ethics Board and adhered to the Canadian Council on Animal Care guidelines for humane animal use. Six-eight week old male C57BL/6J mice were procured. NOTE: Euthanize any animals which develop severe lethargy, respiratory distress or other signs of severe distress before scheduled end point.

NOTE: Prepare the following: 27-18 G needle-blunted (will depend on the mouse tracheal diameter), appropriately sized PE tubing to fit the blunt needle (make a PE cannula for every mouse), cannula, 2 sharp scissors, 2 blunt forceps (small), 1 sharp forceps (small), 3 1....

Results

Combined O₃ and LPS exposure leads to systemic inflammation and bone marrow mobilization at 72 h: Cell counts in different compartments revealed significant changes in peripheral blood and the femur bone marrow total cell counts upon combined O₃ and LPS exposures. Although combined O₃ and LPS exposures did not induce any changes in the total BAL (Figure 1A) or LVP (Figure 1B

Discussion

The methods presented in the current study highlight the usefulness of multiple compartment analysis to study multiple cellular events during lung inflammation. We have summarized the findings in Table 2. We and many labs have extensively studied the murine response to intranasal LPS instillation, which is marked by rapid recruitment of lung neutrophils, which peaks between 6-24 h following which, resolution kicks in. And recently, we have shown that sub-clinical O₃ (at 0.05 ppm for 2 h) alone.......

Materials

Name	Company	Catalog Number	Comments
33-plex Bioplex chemokine panel	Biorad	12002231
63X oil (NA 1.4-0.6) Microscope objectives	Leica	HCX PL APO CS (11506188)
Alexa 350 conjugated goat anti-mouse IgG (H+L)	Invitrogen	A11045
Alexa 488 conjugated goat anti-mouse IgG (H+L)	Invitrogen	A11002
Alexa 488 conjugated phalloidin	Invitrogen	A12370
Alexa 555 conjugated mouse anti-α tubulin clone DM1A	Millipore	05-829X-555
Alexa 568 conjugated goat anti-hamster IgG (H+L)	Invitrogen	A21112
Alexa 568 conjugated goat anti-rat IgG (H+L)	Invitrogen	A11077
Alexa 633 conjugated goat anti-rabbit IgG (H+L)	Invitrogen	A21070
Armenian hamster anti-CD61 (clone 2C9.G2) IgG1 kappa	BD Pharmingen	553343
C57BL/6 J Mice	Jackson Laboratories	64
Confocal laser scanning microscope	Leica	Leica TCS SP5
DAPI (4′,6-diamidino-2-phenylindole)	Invitrogen	D1306	aliquot in 2 µl stocks and store at -20°C
Inverted fluorescent wide field microscope	Olympus	Olympus IX83
Ketamine (Narketan)	Vetoquinol	100 mg/ml	Dilute 10 times to make a 10 mg/ml stock
Live (calcein)/Dead (Ethidium homodimer-1) cytotoxicity kit	Invitrogen	L3224
Mouse anti-ATP5A1 IgG2b (clone 7H10BD4F9)	Invitrogen	459240
Mouse anti-ATP5β IgG2b (clone 3D5AB1)	Invitrogen	A-21351
Mouse anti-NK1.1 IgG2a kappa (clone PK136)	Invitrogen	16-5941-82
Pierce 660 nm protein assay	Thermoscientific	22660
Rabbit anti-angiostatin (mouse aa 98-116) IgG	Abcam	ab2904
Rabbit anti-CX3CR1 IgG (RRID 467880)	Invitrogen	14-6093-81
Rat anti-Ki-67 (clone SolA15) IgG2a kappa	Invitrogen	14-5698-82
Rat anti-Ly6G IgG2a kappa (clone 1A8)	Invitrogen	16-9668-82
Rat anti-Ly6G/Ly6C (Gr1) IgG2b kappa (clone RB6-8C5)	Invitrogen	53-5931-82
Rat anti-mouse CD16/CD32 Fc block (clone 2.4G2)	BD Pharmingen	553142
Reduced mitotracker orange	Invitrogen	M7511
Xylazine (Rompun)	Bayer	20 mg/ml	Dilute 2 times to make a 10 mg/ml stock