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

Figure 1:. Correlation between OD₆₀₀ and CFU counts for ALC2906.

3–4 ALC2906 SH1000 colonies were picked from an agar plate and transferred into TSB with 10 μg/mL chloramphenicol for overnight culture. The next day, the suspension was split 1:50 into TSB with 10 μg/mL chloramphenicol and cultured. Optical density at 600 nm (OD₆₀₀) was measured in regular intervals after 2 hours using a spectrophotometer.At each measurement the bacteria was diluted 1:100,000 in ice cold PBS and aliquoted onto an agar plate for overnight incubation. CFUs were counted the following day to calculate the initial concentration and correlated to OD₆₀₀. N = 3 with 4 different OD₆₀₀ measurements per experiment.

Figure 2:. Representative region of interests (ROIs) for data analysis.

To analyze in vivo BLI and in vivo FLI signals, large, equivalent sized ROIs were centered over the wound and total flux (photons per second) was measured. To measure wound closure, an ROI was fit to the wound edge and area (cm²) was measured.

Figure 3:. LysM-EGFP×MyD88^−/− mice are more susceptible to S. aureus infection compared to LysM-EGFP mice.

LysM-EGFP and LysM- EGFP×MyD88^−/− mice were administered a 6 mm wound on the dorsum and infected with 1 × 10⁷ CFU of bioluminescent S. aureus or sterile saline. Animals were monitored daily and (A) survival and (B) weight were recorded. Animals were imaged daily to measure (C) wound size and (D) bacterial luminescence. (E) Representative bioluminescent images are depicted from infected LysM-EGFP and LysM-EGFP×MyD88^−/− mice.Scale bar = 3 mm. Data represent 7–16 mice per group for A, C, and D and 3 mice per group for B and are expressed as mean ±SEM. * p = 0.05, ** p = 0.01, ***p = 0.001,^****p < 0.0001 between infected (A, B, and D) or uninfected (C) groups. Statistical significance was determined using Mantel-Cox test (A) and the Holm-Sidak method (B-D), with alpha = 0.05, and each time point was analyzed individually, without assuming a consistent SD.

Figure 4:. LysM-EGFP×MyD88^−/− mice have defective neutrophil recruitment to infected wounds.

LysM-EGFP and LysM-EGFP×MyD88^−/−mice were administered a 6mm wound on the dorsum and infected with 1 × 10⁷ CFU of bioluminescent S. aureus or sterile saline. Animals were imaged daily to measure (A,B) neutrophil content. (C) Representative fluorescent images are depicted from infected and non-infected LysM-EGFP and LysM-EGFP×MyD88^−/− mice. Scale bar = 5 mm. Data represent 8–16 mice per group and are expressed as mean ±SEM. *p < 0.05, ** p < 0.01, and *** p < 0.001 between infected groups and^## p < 0.01 between uninfected groups (A) or as depicted on the graph (B). Statistical significance was determined using the Holm-Sidak method (A), with alpha = 0.05, and each time point was analyzed individually, without assuming a consistent SD, and one-way ANOVA (B), with the Tukey multiple-comparisons post-test.