A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Summary

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

Abstract

Staphylococcus aureus (S. aureus) infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP×MyD88^-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

Introduction

Staphylococcus aureus (S. aureus) accounts for the majority of skin and soft tissue infections (SSTIs) in the United States¹. The incidence of methicillin-resistant S. aureus (MRSA) infections has increased steadily over the past two decades², motivating the study of the mechanisms of persistence and the discovery of new treatment strategies. The standard of care for MRSA infections is systemic antibiotic therapy, but MRSA has become increasingly resistant to antibiotics over time³ and these drugs can diminish the host's beneficial microbiome, causing negative health....

Protocol

All mouse studies were reviewed and approved by the Institutional Animal Care and Use Committee at UC Davis and were performed according to the guidelines of the Animal Welfare Act and the Health Research Extension Act. Be sure to use sterile gloves when working with animals.

1. Mouse Source and Housing

1. Derive LysM-eGFP mice on a C57BL/6J genetic background as described previously¹². Derive LysM-EGFP×MyD88^-/- mice by crossing LysM-EGFP mice with MyD88^-/- mice on a C57BL/6J background.
2. House mice in a vivarium. For these studies, animals were housed at the University of....

Results

LysM-EGFP×MyD88^-/- mice are more susceptible to S. aureus infection than LysM-EGFP mice

The strain of S. aureus used in this study, ALC2906¹⁸, was constructed with a plasmid that contains the lux construct that produces bioluminescent signals from live and actively metabolizing bacteria. When inoculated into mice, in vivo bioluminescence imaging (BLI) techniques.......

Discussion

S. aureus infection models that utilize bioluminescent S. aureus infection in a fluorescent neutrophil reporter mouse in conjunction with advanced techniques of whole animal in vivo optical imaging have advanced our knowledge of the innate immune response to infection. Previous studies using the LysM-EGFP mouse have shown that up to 1 x 10⁷ neutrophils recruit to an S. aureus infected wound over the first 24 h of infection¹⁴, and wound-recruited neutrophils ex.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
14 mL Polypropylene Round-Bottom Tube	Falcon	352059
6 mm Disposable Biopsy Punch	Integra Miltex	33-36
Bioluminescent S. aureus	Lloyd Miller, Johns Hopkins	ALC 2906 SH1000
Bovine Blood Agar, 5%, Hardy Diagnostics	VWR	10118-938
Buprenoprhine hydrochloride injectable	Western Medical Supply	7292	0.3 mg/mL
C57BL/6J Mice	Jackson Labratory	000664
Chloramphenicol (crystalline powder)	Fisher BioReagents	BP904-100
DPBS (1x)	Gibco	14190-144
Insulin Syringes	Becton, Dickson and Company	329461	0.35 mm (28 G) x 12.7 mm (1/2'')
IVIS Spectrum In Vivo Imaging System	Perkin Elmer	124262
Living Image Software – IVIS Spectrum Series	Perkin Elmer	128113
LysM-eGFP Mice	Thomas Graff Albert Einstein College of New York	NA
Microvolume Spectrophotometer	ThermoFisher Scientific	ND-2000
MyD88 KO Mice	Jackson Labratory	009088
Non-woven sponges	AMD- Ritmed Inc	A2101-CH	5 cm x 5 cm
Povidone Iodine 10% Solution	Aplicare	697731
Prism 7.0	GraphPad Software	License
Tryptic Soy Broth	Becton, Dickson and Company	211825