Investigating the contribution of IL-17A and IL-17F to the host response during Escherichia coli mastitis

Abstract

Mastitis remains a major disease of cattle with a strong impact on the dairy industry. There is a growing interest in understanding how cell mediated immunity contributes to the defence of the mammary gland against invading mastitis causing bacteria. Cytokines belonging to the IL-17 family, and the cells that produce them, have been described as important modulators of the innate immunity, in particular that of epithelial cells. We report here that expression of IL-17A and IL-17F genes, encoding two members of the IL-17 family, are induced in udder tissues of cows experimentally infected with Escherichia coli. The impact of IL-17A on the innate response of bovine mammary epithelial cells was investigated using a newly isolated cell line, the PS cell line. We first showed that PS cells, similar to primary bovine mammary epithelial cells, were able to respond to agonists of TLR2 and to LPS, provided CD14 was added to the culture medium. We then showed that secretion of CXCL8 and transcription of innate immunity related-genes by PS cells were increased by IL-17A, in particular when these cells were stimulated with live E. coli bacteria. Together with data from the literature, these results support the hypothesis that IL-17A and IL-17 F could play an important role in mediating of host-pathogen interactions during mastitis.

Figure 1

Analysis of gene expression during experimentally induced mastitis. RT-qPCR specific for CCL20 (A), CXCL8 (B), IL-17A (C) and IL-17F (D) were performed on RNA extracted from tissue samples of udder quarters from uninfected cows or E. coli infected cows. Data presented are values from five animals with medians and interquartile ranges indicated as vertical bars. * statistical significance (P < 0.05) of infected versus uninfected cows calculated using exact permutation tests after global comparison using a Kruskal and Wallis test.

Figure 2

Characterization of PS cells properties by flow cytometry and RT-PCR. PS cells, at passage 14, were labelled with antibodies against CD45 (A), cytokeratin 14 (B), cytokeratin 18 (C), TLR2 (D) and CD14 (E). Controls were performed either by omitting the primary antibody (A, B, C) or using unlabelled cells (D, E). As a control for CD45 labelling, bovine PBMC were labelled with the anti-CD45 antibody (F). Labelled cells are depicted as red curves while controls are represented as blue curves. (G-H) RNA was extracted from unstimulated PS cells and converted to cDNA. cDNAs for each of the indicated receptors were amplified by PCR and products were separated by agarose gel electrophoresis. MW: 50 bp molecular weight ladder.

Figure 3

Innate immune response of PS cells to different purified bacterial agonists. PS cells were incubated for 5 h with the indicated concentrations of different purified agonists. Response was analyzed in terms of expression of CCL20 (A), CXCL8 (B), TAP (C), LAP (D) genes by RT-qPCR or CXCL8 secretion by ELISA (E,F). Data presented are mean values and SD obtained from 3 independent experiments. Experiments were performed at passages 11, 12 and 24. PS cells were incubated for 5 hours in GM medium with different agonists at the indicated concentrations. (F) PS cells were incubated with ultrapure LPS (LPS) at 10 ng/mL in the presence of either 10% fetal calf serum, 5 μg/mL CD14 or 5 μg/mL LBP. Stimulation was also performed in whole milk instead of GM medium. * statistical significance (P < 0.05) of stimulated versus unstimulated PS cells using exact permutation tests after global comparison using a Kruskal and Wallis test.

Figure 4

IL-17A effect on the response of PS cells to Pam3-CSK4. PS cells were incubated for 5 h with 100 ng/mL of Pam3-CSK4 in the presence or not of 100 ng/mL IL-17A. Response was analyzed in terms CXCL8 secretion (A) by ELISA or expression of CCL20 (B) and CXCL8 (C) by RT-qPCR. Data presented are mean values and SD obtained from 3 independent experiments. Experiments were performed at passages 15, 16 and 24. * statistical significance (P < 0.05) calculated using exact permutation tests after global comparison using a Kruskal and Wallis test.

Figure 5

IL-17A effect on the response of PS cells to LPS. PS cells were incubated for 5 h with 10 ng/mL of LPS in the presence or not of 100 ng/mL IL-17A and 5 μg/mL CD14. Response was analyzed in terms CXCL8 secretion (A) by ELISA or expression of CCL20 (B) and CXCL8 (C) by RT-qPCR. Data presented are mean values and SD obtained from 3 independent experiments. Experiments were performed at passages 15, 16 and 24. * statistical significance (P < 0.05) calculated using exact permutation tests after global comparison using a Kruskal and Wallis test.

Figure 6

IL-17A increases the secretion of CXCL8 by PS cells upon stimulation with E. coli strains 1303 and P4. A-D: PS cells were incubated for 3 hours with strain E. coli 1303 or P4 at an MOI of 1. Cells were then washed twice with HBSS and medium with gentamycin was added with or without 100 ng/mL IL-17A. Response was analysed in terms of gene expression (CCL20: A, CXCL8: B, TAP: C) or CXCL8 secretion by ELISA (D) after beginning of the experiment. Data presented are mean values and SD obtained from 3 independent experiments. Experiments were performed at passages 8, 26 and 30. E-F: primary mammary epithelial cells from two cows were incubated for 3 hours with strains E. coli 1303 and P4 at an MOI of 1. Cells were then washed twice with HBSS and medium with gentamycin was added with or without 100 ng/mL IL-17A. CXCL8 secretion was measured by ELISA 24 hpi. Data presented are mean values obtained with cells stimulated in triplicates. * statistical significance (P < 0.05) calculated using exact permutation tests after global comparison using a Kruskal and Wallis test.