Assessment of the immune capacity of mammary epithelial cells: comparison with mammary tissue after challenge withEscherichia coli

Abstract

We examined the repertoire and extent of inflammation dependent gene regulation in a bovine mammary epithelial cell (MEC) model, to better understand the contribution of the MEC in the immune defence of the udder. We challenged primary cultures of MEC from cows with heat inactivated Escherichia coli pathogens and used Affymetrix DNA-microarrays to profile challenge related alterations in their transcriptome. Compared to acute mastitis, the most prominently activated genes comprise those encoding chemokines, interleukins, beta-defensins, serum amyloid A and haptoglobin. Hence, the MEC exert sentinel as well as effector functions of innate immune defence. E. coli stimulated a larger fraction of genes (30%) in the MEC belonging to the functional category Inflammatory Response than we recorded with the same microarrays during acute mastitis in the udder (17%). This observation underscores the exquisite immune capacity of MEC. To more closely examine the adequacy of immunological regulation in MEC, we compared the inflammation dependent regulation of factors contributing to the complement system between the udder versus the MEC. In the MEC we observed only up regulation of several complement factor-encoding genes. Mastitis, in contrast, in the udder strongly down regulates such genes encoding factors contributing to both, the classical pathway of complement activation and the Membrane Attack Complex, while the expression of factors contributing to the alternative pathway may be enhanced. This functionally polarized regulation of the complex complement pathway is not reflected in the MEC models.

Microfluidic high-throughput RT-qPCR measurements of the immune response of primary bovine mammary epithelial cells cultured from milk to mastitis pathogens

“…Generally, they identified higher fold changes in the regulated genes, but this could be because of the microarray technique or to different cell culture conditions. In their study, SAA3 was also the most up regulated gene by E. coli (Gunther et al (2009)), followed by the chemokine CCL5, lingual antimicrobial peptide (LAP) and MX2 (Gunther et al, 2009), which were also highly upregulated in our study. Our cells proved to be able to express a similar set of inflammatory cytokines (IL6, IL10 and tumour necrosis factor (TNF)) and chemokines (chemokine CCL2, CCL5, chemokine CCL20, chemokine (C-X-C motif) ligand 5 (CXCL5), and chemokine CXCL8 ) compared with the study by Gunther et al (2009).…”

“…In their study, SAA3 was also the most up regulated gene by E. coli (Gunther et al (2009)), followed by the chemokine CCL5, lingual antimicrobial peptide (LAP) and MX2 (Gunther et al, 2009), which were also highly upregulated in our study. Our cells proved to be able to express a similar set of inflammatory cytokines (IL6, IL10 and tumour necrosis factor (TNF)) and chemokines (chemokine CCL2, CCL5, chemokine CCL20, chemokine (C-X-C motif) ligand 5 (CXCL5), and chemokine CXCL8 ) compared with the study by Gunther et al (2009). Lutzow et al (2008) measured the intra-mammary immune response of dairy cows to S. aureus in vivo and found upregulated inflammatory cytokines and chemokines, as well as defence proteins.…”

“…Heatinactivated E. coli 1303 and S. aureus 1027 (Petzl et al, 2008) were added in a multiplicity of infection (MOI) of 30 colony forming units per cultured cell to ensure that every culture received the same bacterial load per cell. This MOI was chosen on review of the literature as a typical bacterial load often used in similar experiments (Gunther et al, 2009;Danowski et al, 2012). E. coli treated (6 and 30 h), S. aureus (30 and 78 h) and control wells (6, 30 and 78 h) were sampled in duplicates by washing the wells with phosphate buffered saline (PBS) and dissolving the cell layer in lysis buffer of the AllPrep RNA/Protein Kit (Qiagen, Hilden, Germany).…”

“…To our knowledge, this is the first time that a high-throughput RT-qPCR technique was applied to study a large set of genes in pbMEC cultured from milk. So far large sets of GE data are only available from pbMEC extracted from udder tissue in microarray studies (Gunther et al, 2009). Gunther et al (2009) also reported that the immune response to E. coli was much faster and stronger than to S. aureus; however, the authors were still able to identify several significantly upregulated genes by S. aureus.…”

“…So far large sets of GE data are only available from pbMEC extracted from udder tissue in microarray studies (Gunther et al, 2009). Gunther et al (2009) also reported that the immune response to E. coli was much faster and stronger than to S. aureus; however, the authors were still able to identify several significantly upregulated genes by S. aureus. Generally, they identified higher fold changes in the regulated genes, but this could be because of the microarray technique or to different cell culture conditions.…”