The in vitro host cell immune response to bovine-adapted Staphylococcus aureus varies according to bacterial lineage

Abstract

Mastitis is the most economically important disease affecting dairy cattle worldwide. Staphylococcus aureus is a highly prevalent cause of mastitis, causing infections ranging from sub-clinical to gangrenous. However, the interaction between the genotype of the infecting strain of S. aureus and the host response remains largely uncharacterised. To better understand the variation in presentation and outcomes of S. aureus-mediated bovine mastitis, we studied the interaction of a panel of mastitis isolates from several prominent bovine-associated lineages with bovine mammary epithelial cells (bMEC) and neutrophils. Significant differences in immune gene expression by infected primary or immortalised bMEC, or their elaboration of neutrophil chemoattractants, were observed and were dependent on the lineage of the infecting strain. Differences were also apparent in the invasiveness of S. aureus strains and their ability to survive killing by neutrophils. Our results demonstrate that a range of immune responses occur, suggesting the importance of S. aureus strain in dictating mastitis disease course. S. aureus lineages may therefore have adopted differing strategies for exploitation of the intramammary niche. Consequently, improved diagnosis of infecting lineage may enable better prognosis for S. aureus mastitis and reduce morbidity and economic loss.

Figure 1

Gene expression in the MAC-T bovine mammary epithelial cell line in response to infection with S. aureus from bovine-adapted lineages. Expression of (A) CCL20, (B) IL1β, (C) IL6, (D) IL8 and (E) TNFα over time in MAC-T cells infected with strains from S. aureus lineages CC71 (black), CC97 (red), ST136 (green) and CC151 (blue). Expression was determined relative to uninfected cells. Data represents mean log₂-transformed, normalised expression of 3 individual strains ± SEM. Means which share a superscript are not significantly different.

Figure 2

Gene expression in primary bovine mammary epithelial cells in response to infection with S. aureus from bovine-adapted lineages. Expression of (A) CCL20, (B) IL1β, (C) IL6, (D) IL8 and (E) TNFα over time in primary bMECs infected with strains from S. aureus lineages CC71 (black), CC97 (red), ST136 (green) and CC151 (blue). Expression was determined relative to uninfected cells. Data represents mean log₂-transformed, normalised expression of 3 individual strains ± SEM. Means which share a superscript are not significantly different.

Figure 3

Secretion of IL-6 and IL-8 by bovine mammary epithelial cells in response to infection with S. aureus from bovine-adapted lineages. Secretion of IL-6 (A,C) and IL-8 (B,D) from MAC-T cells (A,B), or primary bMEC (C,D) over time in response to infection with strains from S. aureus lineages CC71 (black), CC97 (red), ST136 (green) and CC151 (blue). Data represent mean ± SEM of three strains per lineage. Means which share a superscript are not significantly different.

Figure 4

Ability of conditioned media from MAC-T cells infected for 24 h with each strain of S. aureus to attract neutrophils. Chemotaxis was measured in real time in a two-chamber cell migration plate by cell impedance on an xCELLigence system (Roche). Positive control included 150 ng/ml IL-8 in the lower chamber; negative control included no IL-8; chemokinesis control included 150 ng/ml IL-8 in both upper and lower chambers; fugetaxis control included 150 ng/ml IL-8 in the upper chamber while uninfected controls included conditioned media from uninfected MAC-T cells in the lower chamber. Data represent the mean rate of migration ± SEM, from triplicate experiments.

Figure 5

Survival of S. aureus incubated with bovine granulocytes. Percentage of each strain of S. aureus that survived 2 hours of co-culture with bovine granulocytes compared to cells incubated without granulocytes. Data represent the mean ± SD from triplicate experiments.

Figure 6

Real-time measurement of adhesion, morphology and detachment of MAC-T cells pre- and post-infection with each of 12 strains of S. aureus. Cells were cultured to confluence in wells of an E-plate (Roche) over 20 h before addition of each S. aureus strain at an MOI of 10 (arrow indicates time of infection). Cell index, the relative change in electrical impedence and hence a measure of the strength of cell adhesion, was monitored in real-time for the following 24 hours post-infection using an xCELLigence system (Roche). Data represent mean cell index ± SD over time (N = 3 per strain).

Figure 7

Percentage of each strain of S. aureus that internalised into MAC-T cells after 2 hours of co-incubation. Percentage internalisation was determined relative to bacterial proliferation in the absence of cells. Data represent mean ± SD, N = 4.