Investigating the Effects of Particulate Matter on House Dust Mite and Ovalbumin Allergic Airway Inflammation in Mice

Alejandro R. Castañeda1, Kent E. Pinkerton2

1 Center for Health and the Environment, University of California, Davis, California, 2 Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California, Davis, California
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 18.18
DOI:  10.1002/cptx.5
Online Posting Date:  May, 2016
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Particulate matter (PM), a component of air pollution, has been shown to enhance allergen‐mediated airway hypersensitivity and inflammation. Surprisingly, exposure to PM during the sensitization to allergen is sufficient to produce immunological changes that result in heightened inflammatory effects upon future allergen exposures (challenge) in the absence of PM. This suggests that PM has the ability to modulate the allergic immune response, thereby acting as an adjuvant by enhancing the immunological memory formed during the adaptive immune response; however, the mechanisms through which this occurs remain elusive. Establishing a reproducible animal model to study the PM‐mediated immunotoxicological effects that enhance allergy, may provide insights to understand how air pollution activates the immune system and thereby modulates the pathophysiology of asthma. The basic protocol can be used to study various characteristics of air pollution, such as PM size, source, or chemical composition, to help elucidate how such features may affect the allergic response in a mouse model of asthma. Using a BALB/c model of acute exposure (14 days), mice are first sensitized with allergen and PM, and then subsequently challenged with allergen only. The endpoints of this basic protocol include the assessment of inflammation via cells recovered from broncho‐alveolar lavage (BAL), histopathological analysis, gene expression profiles, and protein quantification of inflammatory markers. © 2016 by John Wiley & Sons, Inc.

Keywords: allergic mouse model; particulate matter (PM); air pollution; allergen; ovalbumin (OVA); house dust mite (HDM); airway inflammation

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Table of Contents

  • Introduction
  • Basic Protocol 1: Intranasal Sensitization (Allergen and Particulate Matter) and Challenge (Allergen Alone)
  • Support Protocol 1: Assessment of Broncho‐Alveolar Lavage Fluid for Pulmonary Inflammatory Cellular Profiles
  • Support Protocol 2: Assessment of Plasma IgE
  • Support Protocol 3: Assessment of Pulmonary Inflammation via Histopathology
  • Support Protocol 4: Assessment of Pulmonary Inflammation via Gene Expression
  • Support Protocol 5: Assessment of Pulmonary Inflammation via Protein Levels
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Intranasal Sensitization (Allergen and Particulate Matter) and Challenge (Allergen Alone)

  • Allergen (Ovalbumin, Sigma or House Dust Mite Allergen, Greer Laboratories)
  • Delivery vehicle: saline, phosphate‐buffered saline (PBS), or Hank's balanced salt solution (HBSS); use the same delivery vehicle for all further support protocols
  • Particulate matter (PM)
  • Mice (we recommend 8‐week‐old male BALB/c mice)
  • Isoflurane
  • Pentobarbital sodium diluted in sterile saline to 65 mg/ml
  • Sterile saline, PBS, or HBSS (same as delivery vehicle; for broncho‐alveolar lavage)
  • Ice
  • Reservoir with liquid nitrogen
  • RNAlater Solution (optional; Ambion)
  • 4% paraformaldehyde made in PBS
  • Vortex mixer
  • 1.5‐ml microcentrifuge tubes
  • Ultrasonic cleaner water bath
  • Veterinary anesthesia machine with anesthesia induction chamber
  • Timer
  • 20‐, 200‐, and 1000‐μl pipets with appropriate tips
  • Balance for weighing mice
  • Data sheets for recording animal weights and dosing schedule
  • 1‐ml syringe equipped with a 25‐G hypodermic needle
  • Absorbent paper (inscribe with animal ID numbers)
  • Surgical dissecting scissors (blunt)
  • Forceps (serrated, curved tip, and blunt)
  • 1‐ml syringe (optional for cardiac puncture) with 21‐G hypodermic needle
  • EDTA‐coated tubes (optional for cardiac puncture; BD Microtainer)
  • Surgical hemostats (serrated and non‐serrated)
  • Braided silk suture (USP sizes 1 and 2‐0)
  • Razor blade
  • Cannula (21‐G, 1‐in., blunt end for tracheal cannulation)
  • 1‐ml syringe (for broncho‐alveolar lavage)
  • 5‐ml polystyrene round‐bottom tubes (BD Falcon, Fisher Scientific)
  • Cryogenic tubes (Nunc, Thermo Scientific)
  • Tissue perfusion system
  • 20‐ml glass scintillation vials for storing fixed lungs (Sigma‐Aldrich)
  • Data sheets for project date, treatment, animal ID numbers, anesthetic amounts, BAL volume recovery, and lung fixation time
  • Biohazard disposal bag for animals
  • Gauze
  • Cotton swabs
  • Weight boats

Support Protocol 1: Assessment of Broncho‐Alveolar Lavage Fluid for Pulmonary Inflammatory Cellular Profiles

  • 5‐ml round‐bottom tubes containing BAL (see Basic Protocol)
  • Sterile saline, PBS, or HBSS
  • Trypan blue solution (0.4%)
  • 100% methanol
  • Hematoxylin and eosin stain
  • ClearMount mounting medium
  • Centrifuge (refrigerated)
  • 1.5‐ and 0.5‐ml microcentrifuge tubes
  • Vortex mixer
  • 20‐, 200‐, and 1000‐μl pipets with appropriate tips
  • Hemacytometer
  • Light microscope
  • Cytospin funnel
  • Aqua color frost glass slides (Fisher Scientific)
  • Absorbent paper
  • Shandon Cytospin 4 centrifuge
  • Coverslips
  • Microscope cover glasses
  • Data sheet to count/calculate cells
  • Shandon filter cards (for samples with <0.4 ml volume)

Support Protocol 2: Assessment of Plasma IgE

  • Blood collected in EDTA‐coated tubes (see Basic Protocol)
  • Mouse IgE ELISA kit (various manufacturers)
  • Centrifuge (refrigerated)
  • 1.5‐ml microcentrifuge tubes
  • 1000‐μl pipets with appropriate tips

Support Protocol 3: Assessment of Pulmonary Inflammation via Histopathology

  • Lung lobes (see Basic Protocol)
  • Ice
  • TRI Reagent (Sigma‐Aldrich)
  • Chloroform
  • RNA extraction kit (various manufacturers)
  • Reverse transcription PCR kit (various manufacturers)
  • Nuclease‐free water
  • SYBR Green Master Mix (various manufacturers)
  • Gene specific primers (species‐specific)
  • 20‐, 200‐, and 1000‐μl pipets with appropriate tips
  • 2‐ml round‐bottom microcentrifuge tubes
  • Stainless steel beads, 5 mm (Qiagen)
  • Fume hood
  • TissueLyser (Qiagen)
  • 1.5‐ml microcentrifuge tubes
  • Centrifuge (refrigerated)
  • Ultraviolet‐visible light spectrophotometer (Nanodrop, Thermo Scientific)
  • Real‐time PCR instrument (various manufacturers)

Support Protocol 4: Assessment of Pulmonary Inflammation via Gene Expression

  • Right lung lobes (see Basic Protocol)
  • Ice
  • Tissue/cell lysis kit (various manufacturers)
  • Lowry protein assay kit (various manufacturers)
  • Mouse enzyme‐linked immunoabsorbant assay (ELISA) kit (various manufacturers)
  • 2‐ml round‐bottom microcentrifuge tubes
  • Spectrophotometer (to measure protein content)
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Literature Cited

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Internet Resources
  The National Institute of Standards and Technology Standard Reference Materials provide a variety of commercially available PM sources that can be used for this protocol.
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