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 effects, especially in children⁴. Preclinical studies have examined alternative strategies to treat MRSA infections⁵, but translating these approaches to the clinic has proved challenging due to emergence of virulence factors that thwart host immune responses⁶. To dissect the host-pathogen dynamics that drive S. aureus SSTIs, we combine noninvasive and longitudinal readouts of the number of neutrophils recruited to the wound bed with kinetic measures of bacterial abundance and wound area.

Neutrophils are the most abundant circulating leukocyte in humans and the first responders to a bacterial infection⁷. Neutrophils are a necessary component for an effective host response against S. aureus infections due to their bactericidal mechanisms, including production of reactive oxygen species, proteases, antimicrobial peptides and functional responses including phagocytosis and neutrophil extracellular trap production^8,9. Human patients with genetic defects in neutrophil function, such as chronic granulomatous disease and Chediak-Higashi syndrome, show an increased susceptibility to S. aureus infection. In addition, patients with genetic (such as congenital neutropenia) and acquired (such as neutropenia seen in chemotherapy patients) defects in neutrophil numbers are also highly susceptible to S. aureus infection¹⁰. Given the importance of neutrophils in clearing S. aureus infections, enhancing their immune capacity or tuning their numbers within a S. aureus lesion may prove an effective strategy in resolving infection.

Over the past decade, transgenic mice with fluorescence neutrophil reporters have been developed to study their trafficking^11,12. Combining neutrophil reporter mice with whole animal imaging techniques permits spatiotemporal analysis of neutrophils in tissues and organs. When combined with bioluminescent strains of S. aureus, it is possible to track the accumulation of neutrophils in response to S. aureus abundance and persistence in the context of bacterial virulence factors that directly and indirectly perturb neutrophil numbers in affected tissue^13,14,15,16.

Mice are less susceptible to S. aureus virulence and immune evasion mechanisms than humans. As such, wild-type mice may not be an ideal animal model to investigate the efficacy of a given therapeutic to treat chronic S. aureus infection. MyD88-deficient mice (i.e., MyD88^-/- mice), an immunocompromised mouse strain that lacks functional interleukin-1 receptor (IL-1R) and Toll-like receptor (TLR) signaling, show greater susceptibility to S. aureus infection compared to wild-type mice¹⁷ and an impairment in neutrophil trafficking to a site of S. aureus infection in the skin¹⁸. Development of a mouse strain that possesses a fluorescent neutrophil reporter in MyD88^-/- mice has provided an alternative model for investigating the efficacy of therapies to treat S. aureus infection compared to current neutrophil reporter mice.

In this protocol, we characterize S. aureus infection in the immunocompromised LysM-EGFP×MyD88^-/- mice, and compare the time course and resolution of infection with the LysM-EGFP mice. LysM-EGFP×MyD88^-/- mice develop a chronic infection that does not resolve, and 75% succumb to infection after 8 days. A significant defect in initial neutrophil recruitment occurs over 72 h of the inflammatory phase of infection, and 50% fewer neutrophils recruit during the latter stage of infection. The increased susceptibility of the LysM-EGFP×MyD88^-/- mice makes this particular strain a rigorous preclinical model to evaluate the efficacy of new therapeutic techniques targeting S. aureus infection compared to current models that utilize wild-type mice, especially techniques aiming to boost the innate immune response against infection.

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 California, Davis in groups prior to surgery and single housed following surgery. Use mice between the ages of 10-16 weeks.

2. Bacterial Preparation and Quantification

1. Remove the bioluminescent S. aureus strain of interest from -80 °C storage to thaw on ice. Streak on a 5% bovine blood agar plate. Incubate the streaked plate in a humidified incubator at 37 °C overnight (16 h).
   NOTE: In this protocol, the ALC2906 SH1000 strain was used. This strain contains the shuttle plasmid pSK236 with the penicillin-binding protein 2 promoter fused to the luxABCDE reporter cassette from Photohabdus luminescens¹⁸.
2. Prepare tryptic soy broth (TSB) by mixing 0.03 g of TSB powder per mL of pure water, and autoclave TSB to sterilize. When cooled, add any necessary antibiotics using sterile technique. In this protocol, add 10 µg/mL of chloramphenicol¹⁸ to the TSB to select for expression of the pSK236 shuttle plasmid, which contains the bioluminescence luxABCDE cassette.
3. Pick 3-4 separate colonies from the S. aureus plate into TSB with 10 µg/mL chloramphenicol to start an overnight culture. Incubate bacteria on an incubating shaker at 37 °C overnight (16 h).
4. Start a new bacterial culture from the overnight culture by diluting a sample 1:50 into TSB with 10 µg/mL chloramphenicol. Culture in an incubating shaker at 200 rpm and 37 °C.
5. Two hours after splitting the S. aureus, monitor the optical density at 600 nm (OD₆₀₀) on a spectrophotometer. Observe the OD₆₀₀ vs. time to find mid-logarithmic phase growth. For the ALC2906 SH1000 strain, an OD₆₀₀ of 0.5 is mid-logarithmic and corresponds to a concentration of 1 x 10⁸ CFU/mL (Figure 1).
6. When OD₆₀₀ is 0.5, wash bacteria 1:1 with ice-cold DPBS. Centrifuge the bacteria for 10 min at 3,000 x g and 4 °C. Carefully decant the supernatant and add additional chilled DPBS and vortex thoroughly. Centrifuge once more for 10 min at 3,000 x g and 4 °C.
7. Carefully decant the supernatant. Resuspend the bacterial pellet at a desired concentration. For these studies, collect 3 mL of ALC2906 SH1000 and resuspend in 1.5 mL of PBS, correlating to a bacteria concentration of about 2 x 10⁸ CFU/mL. Keep bacteria on ice until use.
8. To verify bacteria concentration, dilute 100 µL of the bacterial sample 1:10,000 and 1:100,000 in PBS. Plate 20 µL aliquots on an agar plate. Incubate at 37 °C in a humidified incubator for 16 h. Count CFUs by gross examination and calculate a bacterial concentration the following day.

3. Excisional Skin Wounding and Inoculation with S. aureus

1. Administer 100 µL of 0.03 mg/mL buprenorphine hydrochloride (~0.2 mg/kg) to each mouse via intraperitoneal injection. 
2. Twenty minutes post-injection, place 2-4 mice in a chamber with 2-3 LPM oxygen with 2-4% isoflurane. Once mice are fully anesthetized, transfer the mice one at a time to a nose cone connected to 2-3 LPM oxygen with 2-4% isoflurane. Verify mice are fully anesthetized by firmly pinching each rear paw between a thumb and forefinger. Proceed to the next step if the animal does not respond to the pinch.
3. Shave a 1 inch by 2 inch section on the back of the mouse with electric clippers and clear the area of fur clippings using a clean wipe or gauze. Avoid using depilatory lotion because it may cause excess inflammation.
4. Clean the back of the mouse first with 10% povidone-iodine soaked gauze and then with a 70% ethanol soaked gauze. Clean the area in a spiral pattern, moving outward from the center of the surgical area. Wait approximately 1 min for the surgical area to dry prior to surgery.
5. Hold the shaved back of the mouse loosely between two fingers and firmly press a sterile 6 mm punch biopsy at the center of the prepared surgical area. Do not pull the skin taut.
   1. Twist the punch biopsy to create a circular outline on the skin that fully cuts through the skin in at least one section of the outline. Be careful not to cut into the underlying fascia or tissue.
   2. Use sterile scissors and forceps to cut through the epidermis and dermis following the circular pattern imprinted by the punch biopsy.

4. S. aureus Inoculation

1. Fill a 28 G insulin syringe with the desired bioluminescent bacterial inoculant. In this study, administer a concentration of 1 x 10⁸ CFU/mL (50 µL). Do not administer more than 100 µL of volume.
2. Inject 50 µL of inoculant between the fascia and tissue in the center of the wound on the back of the mouse. Ensure that the inoculant forms a bubble at the center of the wound with minimal leakage or dispersion.
   1. Pull the dermis to the side, hold the syringe nearly parallel to the tissue, and slowly push the syringe into the tissue until a sudden decrease in resistance is felt, which indicates piercing of the fascia. Carefully lead the syringe into the center of the wound and dispense the inoculant slowly. Remove the syringe slowly from the animal.
3. Inject the same volume of sterile PBS into the wounds of uninfected animals as described above.
4. Return the animal to its cage. Place the cage under a heat lamp or on top of a heating pad, and monitor the animal until recovery from anesthesia.

5. In Vivo BLI and FLI

1. Initialize the whole animal imager through the instrument software. Anesthetize mice in a chamber receiving 2-3 LPM oxygen with 2-4% isoflurane. Deliver anesthesia to the nosecones inside the imager.
2. Place the wounded and infected mouse into the imager. Position the mouse such that the wound is as flat as possible. Use the following sequence set-up to image the mice.
   1. Select Luminescence and Photograph as the imaging mode. The exposure time is 1 min at small binning and F/stop 1 (luminescence) and F/stop 8 (photograph). The emission filter is Open. Click the Acquire button to record the image.
   2. Select Fluorescence and Photograph as the imaging mode. The exposure time is 1 s at small binning and F/stop 1 (Fluorescence) and F/stop 8 (photograph) with an excitation wavelength of 465/30 nm and an emission wavelength 520/20 nm with a high lamp intensity. Click the Acquire button to record the image.
3. Return the animal to its cage and monitor until recovery from anesthesia.
4. Image mice daily as described above.

6. Image Analysis

1. Open images to be quantified.
2. Place a large circular region of interest (ROI) over the entire wound area including surrounding skin for each mouse in the image. The neutrophil in vivo FLI EGFP signals and in vivo BLI S. aureus signals extend beyond the wound edge after several days and were included in these studies (Figure 2A and 2B). Click Measure ROI and record values for mean flux for each mouse. Plot the mean flux of each signal (p/s) versus time.
3. If absolute numbers of neutrophils or S. aureus in the wound are desired, perform the following experiments.
   1. To correlate neutrophil numbers to the in vivo FLI EGFP signals, wound C57BL/6J mice as described above, and transfer a range of bone marrow-derived neutrophils (5 x 10⁵ to 1 x 10⁷) from LysM-EGFP or LysM-EGFPxMyD88^-/- donors directly on top of different wounds. Image as described above and correlate the in vivo FLI EGFP signals to the known quantity of neutrophils.
   2. To correlate S. aureus CFU to the in vivo BLI signals, wound and infect mice as described above. On day 1 post-infection record in vivo BLI signals from the mice and immediately euthanize and chill carcasses. Excise the wound, homogenize the tissue, and plate bacterial dilutions on agar for overnight incubation. The next day, count colonies to determine CFU per wound.
4. To measure wound healing fit a circular ROI over the wound edge and measure the area of the wound (cm²) and plot the fractional change from baseline vs. time (Figure 2C).

7. Statistics

NOTE: All data are presented as mean ±SEM. p < 0.05 were considered statistically significant

1. Determine differences between two groups on a single day using the Holm-Sidak method, with alpha = 0.05, and analyze each time point individually, without assuming a consistent SD.
2. Compare differences between multiple groups on the same day by one-way ANOVA with the Tukey multiple-comparisons posttest. Survival between experimental groups was analyzed by the Mantel-Cox method.

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