Staphylococcus aureus Phenol-Soluble Modulins Impair Interleukin Expression in Bovine Mammary Epithelial Cells

Abstract

The role of the recently described interleukin-32 (IL-32) in Staphylococcus aureus-induced mastitis, an inflammation of the mammary gland, is unclear. We determined expression of IL-32, IL-6, and IL-8 in S. aureus- and Escherichia coli-infected bovine mammary gland epithelial cells. Using live bacteria, we found that in S. aureus-infected cells, induction of IL-6 and IL-8 expression was less pronounced than in E. coli-infected cells. Notably, IL-32 expression was decreased in S. aureus-infected cells, while it was increased in E. coli-infected cells. We identified the staphylococcal phenol-soluble modulin (PSM) peptides as key contributors to these effects, as IL-32, IL-6, and IL-8 expression by epithelial cells exposed to psm mutant strains was significantly increased compared to that in cells exposed to the isogenic S. aureus wild-type strain, indicating that PSMs inhibit the production of these interleukins. The use of genetically complemented strains confirmed this observation. Inasmuch as the decreased expression of IL-32, which is involved in dendritic cell maturation, impairs immune responses, our results support a PSM-dependent mechanism that allows for the development of chronic S. aureus-related mastitis.

FIG 1

Upregulation of IL-32, IL-6, and IL-8 expression in E. coli-infected cells. BME-UV or PS cells (2 × 10⁵) were grown in six-well plates for 24 h. The cells were then exposed to the E. coli K-12 strain (MOI, 30:1) for 8 h, 24 h, and 48 h. Isolation of total RNA, synthesis of cDNA, and qRT-PCR were performed as described in Materials and Methods. After normalization using PPIA and RPL19 genes, interleukin expression at 8 h, 24 h, and 48 h p.i. was calculated relative to the values obtained from mock cells, arbitrarily set to 1. Data were calculated from three different experiments performed in triplicate. P values of <0.05 (*) and <0.01 (**) were considered significant.

FIG 2

Analysis of IL-32, IL-6, and IL-8 expression in S. aureus-infected cells. PS cells (2 × 10⁵) were grown for 24 h. The cells were then exposed to S. aureus NB305 or RF122 strains (MOI, 80:1) for 8 h, 24 h, and 48 h. Isolation of total RNA, synthesis of cDNA, and qRT-PCR were performed as described in Materials and Methods. After normalization using PPIA and RPL19 genes, interleukin expression at 8 h, 24 h, and 48 h p.i. was calculated relative to the values obtained from mock cells, arbitrarily set to 1. When the expression was decreased compared to that in unstimulated mock cells, data were presented as negative values. Data were calculated from three different experiments performed in triplicate. P values of <0.05 (*) and <0.01 (**) were considered significant.

FIG 3

Increased IL-32 expression during experimentally E. coli-induced mastitis. Cows were infected either with E. coli or S. aureus as described in Materials and Methods. IL-32-specific qRT-PCRs were performed with RNA extracted from tissue samples of udder quarters from uninfected or bacteria-infected cows. Data presented are values from five animals; a statistical significance (P < 0.05 [*]) of E. coli-infected versus uninfected cows was observed using exact permutation tests after global comparison using a Kruskal-Wallis test.

FIG 4

PSM production in bovine mastitis isolates. PSMs were analyzed in culture filtrates of cultures inoculated from precultures and grown for 8 h in tryptic soy broth. HPLC-MS detection was performed essentially as described previously (23). Intensity values are based on the integration of the extracted ion chromatogram using the two most abundant m/z peaks of every single PSM. Intensities can thus be directly compared between strains for a specific PSM, but only in a limited way for different PSMs. The assay was performed in triplicate. Error bars show standard deviations.

FIG 5

Analysis of the expression of IL-32, IL-6, and IL-8 in cells exposed to S. aureus LAC wt and its mutants. PS cells (2 × 10⁵) were grown for 24 h. The cells were then exposed to LAC wt and to the deletion mutants LAC Δpsmα and LAC Δpsmαβhld at an MOI of 80:1 for 8 h, 24 h, and 48 h. An mRNA expression was measured in total RNA preparation by qRT-PCR. After normalization using PPIA and RPL19 genes, interleukin expression was calculated relative to the values obtained from mock cells, arbitrarily set to 1. According to ANOVA with post hoc Tukey's HSD test, there was a statistically significant difference between interleukin expression levels in cells exposed either to LAC wt or deletion mutants and mock cells, except for IL-32 expression in LAC wt-treated cells for 8 h. There were also statistically significant differences between interleukin expression levels in cells exposed to LAC wt and corresponding deletion mutants as indicated with asterisks. Data were calculated from three different experiments performed in triplicate. P values of <0.05 (*) and <0.01 (**) were considered significant.

FIG 6

Analysis of the expression of IL-32, IL-6, and IL-8 in cells exposed to S. aureus LAC WT pTXD16, corresponding deletion mutants, and complemented strains. PS cells (2 × 10⁵) were grown for 24 h. The cells were then exposed to LAC (WT) pTX_Δ16, to the deletion PSMα-deficient mutant LAC Δpsmα pTX_Δ16, and to the complemented LAC Δpsmα pTX_Δα1-4 strain, as well as to the PSM-deficient mutant LAC Δpsmαβhld pTX_Δ16 and complemented strains LAC Δpsmαβhld pTX_Δα1-4, LAC Δpsmαβhld pTX_Δβ1-2, and LAC Δpsmαβhld pTX_Δηhld at an MOI of 80:1 for 24 h. mRNA expression was measured in total RNA preparations by qRT-PCR. After normalization using PPIA and RPL19 genes, interleukin expression was calculated relative to the values obtained from mock cells, arbitrarily set to 1. According to ANOVA with post hoc Tukey's HSD test, there was a statistically significant difference between interleukin expression levels in cells exposed to either LAC (WT) pTX_Δ16, the deletion mutants, or complemented strains and mock cells. Statistical analysis demonstrated that interleukin expression levels were significantly higher in cells infected with deletion mutants than in those from cells infected either with LAC (WT) pTX_Δ16 or complemented strains. Statistically significant differences between interleukin expression levels in cells exposed to deletion mutants and corresponding complemented strains are indicated with asterisks. Data were calculated from three different experiments performed in triplicate. P values of <0.05 (*) and <0.01 (**) were considered significant.

FIG 7

Analysis of the expression of IL-32, IL-6, and IL-8 in cells treated with synthetic PSM peptides. PS cells (2 × 10⁵) were grown for 24 h and then exposed to synthetic PSMα3 ranging from 0.01 to 2 μg/ml for 8 h, 24 h, or 48 h. mRNA expression was measured in total RNA preparation by qRT-PCR. After normalization using PPIA and RPL19 genes, interleukin expression was calculated relative to the values obtained from mock cells, arbitrarily set to 1. According to ANOVA with post hoc Tukey's HSD test, there were statistically significant differences between interleukin expression levels in cells treated with 2 μg/ml of PSMα3 for 8 h compared to those in mock cells. Data were calculated from three different experiments performed in triplicate. P values of <0.05 (*) were considered significant.