The Utilization of Oropharyngeal Intratracheal PAMP Administration and Bronchoalveolar Lavage to Evaluate the Host Immune Response in Mice

Summary

The host immune response to pathogen infection is a tightly regulated process. Utilizing a lipopolysaccharide lung exposure model in mice, it is possible to conduct high resolution evaluations of the complex mechanisms associated with disease pathogenesis.

Abstract

The host immune response to pathogens is a complex biological process. The majority of in vivo studies classically employed to characterize host-pathogen interactions take advantage of intraperitoneal injections of select bacteria or pathogen associated molecular patterns (PAMPs) in mice. While these techniques have yielded tremendous data associated with infectious disease pathobiology, intraperitoneal injection models are not always appropriate for host-pathogen interaction studies in the lung. Utilizing an acute lung inflammation model in mice, it is possible to conduct a high resolution analysis of the host innate immune response utilizing lipopolysaccharide (LPS). Here, we describe the methods to administer LPS using nonsurgical oropharyngeal intratracheal administration, monitor clinical parameters associated with disease pathogenesis, and utilize bronchoalveolar lavage fluid to evaluate the host immune response. The techniques that are described are widely applicable for studying the host innate immune response to a diverse range of PAMPs and pathogens. Likewise, with minor modifications, these techniques can also be applied in studies evaluating allergic airway inflammation and in pharmacological applications.

Introduction

Pulmonary infections associated with pathogenic bacteria species are a common cause of global morbidity and mortality. Determining the mechanisms that drive the host immune response to these pathogens will promote the development of novel prevention strategies and therapeutic agents that will attenuate the impact of these infections. The overall goal of the protocol described here is to provide the user with a flexible method to evaluate the host innate immune response to pathogen infection using a pathogen associated molecular pattern (PAMP) as a surrogate for live bacteria. The majority of previous studies evaluating the host innate immune response to bacteria have ....

Protocol

All studies were conducted under the approval of the Institutional Care and Use Committee (IACUC) for Virginia Tech and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

1. Intratracheal (i.t.) Inoculation of LPS Using Oropharyngeal Administration

1. Ensure that each animal is uniquely identified using either an ear punch, ear tag, or other institutionally approved method.
2. Record the baseline body weight and body temperature for each animal.
3. Prepare the working stock of LPS. Each mouse will receive a 50 µl dose of 1 mg/kg LPS in 1x phosphate buffere....

Results

The cell walls of gram-negative bacteria are composed of LPS, which is highly abundant in the environment. Inhalation of LPS in sensitive human populations exacerbates airway reactivity and is capable of triggering a robust immune response11. LPS is also a common PAMP used in mouse models to elicit a robust innate immune response. In the protocol described here, the mice received an i.t. dose of LPS isolated from E. coli (serotype 0111:B4) using oropharyngeal i.t. administration. In models of LPS exposure, both .......

Discussion

The most critical steps for successfully evaluating the host immune response in mouse lungs is as follows: 1) choose the appropriate mouse strain and sex for the model being evaluated; 2) optimize PAMP delivery to the lungs; 3) correctly collect and process the BALF; and 4) properly fix and prepare of the lungs for histopathological assessments.

The choice of mouse strain is an important factor in evaluating the host immune response. C57Bl/6 mice are typically considered the optimal mouse back.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
C57Bl/6J	The Jackson Laboratory	Stock 000664
Compact Scale	Ohaus Scale Corporation	71142845
Small Animal Rectal Thermometer	Braintree Scientific	TH 5
Rectal Probe for Rodents	Braintree Scientific	RET 3
Ear Punch	Braintree Scientific	EP-S 901
Lipopolysaccharide from E. coli 0111:B4	InvivoGen	LPS-EB
1x Phosphate Buffered Saline	Life Technologies	10010-023
Isoflurane	Baxter	40032609
Intratrachael Administration and Lung Inflation Stand	ICAP Manufacturing	n/a
Rodent Intubation Stand	Braintree Scientific	RIS 100
Scissors (blunt/sharp)	Fisher Scientific	13-806-2
forceps (straight)	Fisher Scientific	22-327-379
forceps (45º, curved)	Fisher Scientific	10-275
Scissors (blunt/blunt)	Fisher Scientific	08-940
Pipette (200 µl Capacity)	Gilson	F123601
Ethanol	Sigma	459844
1 ml Syringe	BD Medical	301025
10 ml Syringe	BD Medical	301604
27 G x 0.5 in Needle	BD Medical	305109
Refrigerated Microcentrifuge	Fisher Scientific	13-100-676
1.2 mm Tracheal Cannulae with Luer-adapter	Harvard Apparatus	732836
Hank's Balanced Salt Solution	Life Technologies	14025-076
4-0 Silk Braided Surgical Suture	Ethicon	A183
Luer to Tube Connector Kits	Harvard Apparatus	721406
Luer Stopcock Kit	Harvard Apparatus	721664
Tygon formula E-3603 Laboratory Tubing	Sigma	R-3603
Formalin Solution, neutral buffered, 10%	Sigma	HT501128-4L
Mouse IL-1β OptEIA ELISA Kit	BD Biosciences	559603
Mouse IL-6 OptEIA ELISA Kit	BD Biosciences	550950
Mouse TNF-α OptEIA ELISA Kit	BD Biosciences	560478
Hemocytometer	Hausser Scientific	3520
Hemocytometer Cover Glasses	Thermo Scientific	22-021-801
Trypan Blue	Thermo Scientific	SV3008401
Cytology Funnel Clips	Fisher Scientific	10-357
Cytology Funnels	Fisher Scientific	10-354
Filter Cards	Fisher Scientific	22-030-410
Microscope Slides	Fisher Scientific	12-544-1
Cover Glasses	Fisher Scientific	12-540A
Cytospin Cytocentrifuge	Thermo Scientific	A78300003
Diff Quick Staining Kit	Fisher Scientific	47733150
Permount Mounting Medium	Fisher Scientific	SP15-500