Comparison of the pathogen species-specific immune response in udder derived cell types and their models

Abstract

The outcome of an udder infection (mastitis) largely depends on the species of the invading pathogen. Gram-negative pathogens, such as Escherichia coli often elicit acute clinical mastitis while Gram-positive pathogens, such as Staphylococcus aureus tend to cause milder subclinical inflammations. It is unclear which type of the immune competent cells residing in the udder governs the pathogen species-specific physiology of mastitis and which established cell lines might provide suitable models. We therefore profiled the pathogen species-specific immune response of different cell types derived from udder and blood. Primary cultures of bovine mammary epithelial cells (pbMEC), mammary derived fibroblasts (pbMFC), and bovine monocyte-derived macrophages (boMdM) were challenged with heat-killed E. coli, S. aureus and S. uberis mastitis pathogens and their immune response was scaled against the response of established models for MEC (bovine MAC-T) and macrophages (murine RAW 264.7). Only E. coli provoked a full scale immune reaction in pbMEC, fibroblasts and MAC-T cells, as indicated by induced cytokine and chemokine expression and NF-κB activation. Weak reactions were induced by S. aureus and none by S. uberis challenges. In contrast, both models for macrophages (boMdM and RAW 264.7) reacted strongly against all the three pathogens accompanied by strong activation of NF-κB factors. Hence, the established cell models MAC-T and RAW 264.7 properly reflected key aspects of the pathogen species-specific immune response of the respective parental cell type. Our data imply that the pathogen species-specific physiology of mastitis likely relates to the respective response of MEC rather to that of professional immune cells.

Figure 1

Basal expression level of immune genes and its modulation after challenging with heat-killed E. coli . A mRNA copy numbers relative to similar RNA inputs of TNF, IL6 and CCL20 as measured from the different cell types, as indicated. cDNA copy numbers were titrated against external standards and normalized according to the amount of RNA input. Note the broken ordinate in the graph of TNF. B Visualization of the data from several genes using the EXPANDER software. Each line displays the relative copy number of the respective gene as indicated over the time [h] of the challenge (0, 1, 3, 24), normalized across all cell types to the average of 0 and variance 1. Data are taken from Additional file 3. Data are mean values (error bars, ±SEM) from two replica experiments, each assayed in duplicate.

Figure 2

Pathogen species-specific immune response of different cell types. Upper panel: Changes in the level of TNF expression (ordinate) over time (abscissa) after challenging with heat-killed particles of the indicated pathogens. Lower panel: visualization of the data from several genes using the EXPANDER software. Each line displays the relative copy number of the respective gene as indicated over the time [h] of the challenge (1, 3, 24), normalized across all cell types to the average of 0 and variance 1. Data are taken from Additional file 4. Data are mean values (error bars, ±SEM) from two replica experiments, each assayed in duplicate.

Figure 3

Pathogen species-specific induction of NF-κB activity in different cells. Cells were transiently transfected with the NF-κB reporter plasmid and stimulated with 30 µg/mL of protein from preparations of the heat-killed pathogens, as indicated. The increase in NF-κB activity was measured from cell lysates sampled 24 h after the challenge. Mean values from two independent experiments, each assayed in triplicate. *, P < 0.05; ***, P < 0.001.