Tissue-specific patterning of host innate immune responses by Staphylococcus aureus α-toxin

Abstract

Immunomodulatory cytotoxins are prominent virulence factors produced by Staphylococcus aureus, a leading cause of bacterial sepsis, skin infection, and pneumonia. S. aureus α-toxin is a pore-forming toxin that utilizes a widely expressed receptor, ADAM10, to injure the host epithelium, endothelium, and immune cells. As each host tissue is characterized by a unique composition of resident cells and recruited immune cells, the outcome of α-toxin-mediated injury may depend on the infected tissue environment. Utilizing myeloid lineage-specific Adam10 knockout mice, we show that α-toxin exerts tissue-specific effects on innate immunity to staphylococcal infection. Loss of ADAM10 expression exacerbates skin infection, yet affords protection against lethal pneumonia. These diverse outcomes are not related to altered immune cell recruitment, but rather correlate with a defect in toxin-induced IL-1β production. Extension of these studies through analysis of ADAM10 double-knockout mice affecting both the myeloid lineage and either the skin or lung epithelium highlight the prominence of toxin-induced injury to the epithelium in governing the outcome of infection. Together, these studies provide evidence of tissue specificity of pore-forming cytotoxin action in the modulation of host immunity, and illustrate that the outcome of infection is a collective manifestation of all effects of the toxin within the tissue microenvironment.

Fig. 1

Myeloid lineage knockout of Adam10 worsens clinical outcome in S. aureus skin infection. Abscess (a) and dermonecrosis (b) area in control and Adam10^−/− mice following subcutaneous infection with 1 × 10⁷ CFU S. aureus USA300/LAC. One representative experiment of 10 using 6-10 individual mice per group is shown. Lesion sizes were recorded by noninvasive measurements and the lesion area calculated according to the formula A = ([π/2] × l × w). * p < 0.05, Student's t test. c, d Mice were infected as in a and skin lesions were harvested 72 h (c) or 7 days (d) after infection with an 8-mm punch biopsy tool, then homogenized in PBS for quantification of tissue bacterial burden by serial dilution analysis. e Hla protein content in lesion homogenates 24 h after infection, measured by sandwich ELISA. f Gross pathologic image of control and Adam10^−/− skin lesions 4 days after infection, where the abscess area (delineated by a dotted circle) surrounds the central dermonecrosis lesion. g HE-stained tissues of day 4 lesions in infected control and Adam10^−/− mice. Black scale bar (low magnification images) = 500 μm; yellow scale bar (high magnification images) = 200 μm.

Fig. 2

Myeloid lineage knockout of Adam10 reduces lethality from S. aureus pneumonia. a Survival of control and Adam10^−/− mice following intranasal infection with 2-3 × 10⁸ CFU S. aureus USA300/LAC, scored over 5 days. One representative experiment of 6 using 7-13 mice per group is shown. * p = 0.01, Gehan-Breslow-Wilcoxon test. b BAL fluid protein concentration in mice infected as in a, harvested for analysis 24 h after infection. c S. aureus recovery from left lung tissue harvested 24 h after infection and homogenized for bacterial CFU quantification. d Lung histopathology in control and Adam10^−/− mice infected as in a, harvested 24 h after infection and stained with HE. Black scale bar (low magnification images) = 1 mm; yellow scale bar (high magnification images) = 50 μm.

Fig. 3

Hla-mediated production of IL-1β by myeloid lineage cells is impaired in Adam10^−/− mice. Tissue IL-1β levels in control and Adam10^−/− mice were assessed by ELISA in skin homogenates (a) and BAL fluid (b) from mice infected with S. aureus. To standardize against slight variations in the degree of tissue homogenization, tissue IL-1β was standardized to total protein in each sample. Results are 1 representative experiment of 4 from skin and 5 from BAL fluid. *** p < 0.001, Student's t test. c Recruitment of neutrophils and macrophages to S. aureus-infected skin tissue, examined by immunofluorescence microscopy performed on frozen skin sections. Control and Adam10^−/− mice were examined 48 h after infection, demonstrating similar neutrophil recruitment by Ly-6G staining and macrophage infiltration by MoMa-2 staining. The imaged area immediately adjacent to and encompassing the abscess is highlighted with an orange box, while lesions from the overlying epidermis are highlighted in blue boxes. Images are representative of results obtained from analysis of 9 independent mice from 3 experiments. d Flow cytometric analysis of neutrophil and macrophage populations in lung homogenates prepared 8 h following intranasal infection with S. aureus. Cell populations were examined in 9 independent mice from 3 experiments as a function of all CD45+ cells, where the data are a representative experiment. e Ex vivo chemotaxis assay performed on primary neutrophils harvested from control and Adam10^−/− mice where chemotaxis in response to media alone or 1 × 10⁶ CFU S. aureus was scored by counting neutrophils that migrated across a Transwell filter toward the chemotactic stimulus. Migration was recorded as a percentage of total neutrophil input into the assay. ** p < 0.01, two-way ANOVA. NS = Not significant. f, g IL-1β production in response to 6-hour culture of primary neutrophils (f) or macrophages (g) from control or Adam10^−/− mice with purified, endotoxin-free Hla (25 μg/ml), HKSA (equivalent to 2 × 10⁷ staphylococci), or Hla + HKSA. ** p < 0.01, *** p < 0.001, Student's t test.

Fig. 4

Epithelial and myeloid lineage expression of ADAM10 co-modulate the host response to tissue infection. a, b Mice harboring a double knockout of Adam10 in the epidermis and myeloid lineage (LysM/K14 Adam10^−/−) are more resistant to S. aureus-induced abscess formation (a) and development of dermonecrotic lesions (b) following skin infection relative to mice harboring myeloid lineage-specific Adam10 deletion alone (LysM Adam10^−/−). One representative experiment of 3 using 7-15 individual mice per group is shown, where statistical significance was calculated by repeated-measures two-way ANOVA with Bonferroni post hoc tests to compare each group to the wild-type control. c, d Mice harboring a double knockout of Adam10 in the alveolar epithelium and myeloid lineage (LysM/SpcTet Adam10^−/−) are more resistant to lethal S. aureus pneumonia than mice harboring myeloid lineage-specific Adam10 deletion alone (LysM Adam10^−/−). c One representative experiment of 3 using 8-16 mice per group is shown. NS = Not significant. d Three independent experiments in which results from 25-38 mice per group were pooled to assess for significant differences in mortality between LysM Adam10^−/− and LysM/SpcTetAdam10^−/− mice. Statistical significance in these experiments was calculated by pairwise comparisons using the Gehan-Breslow-Wilcoxon test. * p < 0.05, ** p < 0.01, *** p < 0.001.