Host response signature to Staphylococcus aureus alpha-hemolysin implicates pulmonary Th17 response

Abstract

Staphylococcus aureus pneumonia causes significant morbidity and mortality. Alpha-hemolysin (Hla), a pore-forming cytotoxin of S. aureus, has been identified through animal models of pneumonia as a critical virulence factor that induces lung injury. In spite of considerable molecular knowledge of how this cytotoxin injures the host, the precise host response to Hla in the context of infection remains poorly understood. We employed whole-genome expression profiling of infected lungs to define the host response to wild-type S. aureus compared with the response to an Hla-deficient isogenic mutant in experimental pneumonia. These data provide a complete expression profile at 4 and at 24 h postinfection, revealing a unique response to the toxin-expressing strain. Gene ontogeny analysis revealed significant differences in the extracellular matrix and cardiomyopathy pathways, both of which govern cellular interactions in the tissue microenvironment. Evaluation of individual transcript responses to Hla-secreting staphylococci was notable for upregulation of host cytokine and chemokine genes, including the p19 subunit of interleukin-23. Consistent with this observation, the cellular immune response to infection was characterized by a prominent Th17 response to the wild-type pathogen. These findings define specific host mRNA responses to Hla-producing S. aureus, coupling the pulmonary Th17 response to the secretion of this cytotoxin. Expression profiling to define the host response to a single virulence factor proved to be a valuable tool in identifying pathways for further investigation in S. aureus pneumonia. This approach may be broadly applicable to the study of bacterial toxins, defining host pathways that can be targeted to mitigate toxin-induced disease.

Fig 1

Heat map of gene expression levels for differentially regulated genes in the mouse lung obtained by pairwise comparison between PBS control and WT infection at 24 h postinoculation. Criteria for analysis: FDR of <1% and a minimum of a 3-fold change. Red represents increased gene expression; blue represents downregulation.

Fig 2

Top 10 significant KEGG pathways enriched by the differentially regulated genes in the mouse lung in response to WT S. aureus infection at 24 h postinoculation compared to PBS control. The corresponding results for the comparison between Hla⁻ infection and PBS control are also listed. The differentially expressed genes with an FDR of <1% and a minimum of a 3-fold change were included in this analysis. The vertical dashed line indicates the cutoff of significance (P < 0.05). P values were corrected by the Benjamini-Hochberg procedure (3).

Fig 3

Sustained IL-23 p19 gene expression in S. aureus pneumonia depends on bacterial expression of Hla. qRT-PCR analysis of IL-23 p19 gene expression in the mouse lung in response to infection with WT or Hla⁻ S. aureus or PBS control at 4 h (A), 8 h (B), 12 h (C), and 24 h (D) postinoculation. Bars represent average ± SEM. P values are indicated: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Fig 4

Infection with WT S. aureus is associated with increased immune cell recovery from lung tissue. Histogram plots of total cell count (A), CD3⁺ T cells (B), and GR1-positive (GR1⁺) neutrophils (C) recovered from the lungs of mice infected with WT S. aureus, Hla⁻ S. aureus, or PBS control. Bars represent average ± SEM. Results are derived from 4 PBS-treated mice, 21 WT-infected mice, and 14 Hla⁻-infected mice. P values are indicated: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 5

Hla expression is required for induction of a pulmonary Th17 response to S. aureus infection. Flow cytometric analysis of cells stimulated with PMA and ionomycin and then stained with anti-CD3 allophycocyanin (APC) and anti-IL-17 PE. (A to C) Live cell gate from mouse lung following infection with PBS (A), WT S. aureus (B), or Hla⁻ S. aureus (C). (D) Histogram quantifying IL-17⁺ cells recovered from the lungs of mice. (E to G) CD3⁺ lymphocyte gate from mouse lung following infection with PBS (E), WT S. aureus (F), or Hla⁻ S. aureus (G). Representative plots are shown. The percentage of IL-17⁺ cells is indicated. (H) Histogram of the percentage CD3⁺ cells that are IL-17⁺. Bars represent the average ± SEM. Results are derived from 4 PBS-treated mice, 21 WT-infected mice, and 14 Hla⁻-infected mice. P values are indicated: *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.