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. 2014 Feb 12;45(1):16.
doi: 10.1186/1297-9716-45-16.

Contribution of mammary epithelial cells to the immune response during early stages of a bacterial infection to Staphylococcus aureus

Pauline Brenaut, Lucas Lefèvre, Andrea Rau, Denis Laloë, Giuliano Pisoni, Paolo Moroni, Claudia Bevilacqua, Patrice Martin  1
Affiliations

Contribution of mammary epithelial cells to the immune response during early stages of a bacterial infection to Staphylococcus aureus

Pauline Brenaut et al. Vet Res. .

Abstract

To differentiate between the contribution of mammary epithelial cells (MEC) and infiltrating immune cells to gene expression profiles of mammary tissue during early stage mastitis, we investigated in goats the in vivo transcriptional response of MEC to an experimental intra mammary infection (IMI) with Staphylococcus aureus, using a non-invasive RNA sampling method from milk fat globules (MFG). Microarrays were used to record gene expression patterns during the first 24 hours post-infection (hpi). This approach was combined with laser capture microdissection of MEC from frozen slides of mammary tissue to analyze some relevant genes at 30 hpi. During the early stages post-inoculation, MEC play an important role in the recruitment and activation of inflammatory cells through the IL-8 signalling pathway and initiate a sharp induction of innate immune genes predominantly associated with the pro-inflammatory response. At 30 hpi, MEC express genes encoding different acute phase proteins, including SAA3, SERPINA1 and PTX3 and factors, such as S100A12, that contribute directly to fighting the infection. No significant change in the expression of genes encoding caseins was observed until 24 hpi, thus validating our experimental model to study early stages of infection before the occurrence of tissue damage, since the milk synthesis function is still operative. This is to our knowledge the first report showing in vivo, in goats, how MEC orchestrate the innate immune response to an IMI challenge with S. aureus. Moreover, the non-invasive sampling method of mammary representative RNA from MFG provides a valuable tool to easily follow the dynamics of gene expression in MEC to search for sensitive biomarkers in milk for early detection of mastitis and therefore, to successfully improve the treatment and thus animal welfare.

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Figures

Figure 1
Figure 1
Experimental infection workflow. The left udder was challenged by S. aureus whereas the right udder remained uninfected as the control. Then every six hours, milk was sampled and centrifuged to extract RNA from Milk Fat Globule (MFG). At 30 hpi, goats (n = 5) were slaughtered and mammary tissue samples were taken for MEC capture experiments using LCM (total RNA extraction from micro-dissected MEC) and total RNA extraction from deep alveolar mammary parenchyma (referred to as MG). Finally, these different sources of RNA were analyzed using microarrays (only MFG) and qPCR.
Figure 2
Figure 2
Kinetics of infection (A) and somatic cell recruitment (B). A: Staphylococcus aureus colony forming unit (CFU/mL) evolution during the 24 first hpi. B: Somatic cell counts (SCC/mL) in the left (infected) udder of the 5 goats challenged by S. aureus. The control PBS-infused udders remained free from detectable infection throughout the study for the five goats.
Figure 3
Figure 3
Determination of specific markers by qPCR to assess the level of contamination of RNA extracted from milk fat globules by RNA from immune cells. Relative expression (± SEM) is given for specific gene markers for MEC CSN3 (κ-casein, green), CSN1S2 (αs2-casein, blue), macrophages (CD68, red) and macrophages + neutrophils (CD18, orange) in the left half-udder (infected) or the right half-udder (uninfected), sampled at 30 hpi on slaughtered goats (mammary gland, left), and in milk fat globules (right), before infection (uninfected) and after IMI challenge with S. aureus (12 hpi, 18 hpi and 24 hpi). *Significance is relative to fold change in expression of uninfected MG (adjusted p-value < 0.05).
Figure 4
Figure 4
Selectivity of MEC capture by Microdissection from infected mammary tissue, assessed by real-time qPCR. A) Section of infected mammary tissue with the zone selected for laser capture microdissection (pink). B) Relative expression (± SEM) of specific marker for MEC (CSN3, green bar) and for putative contaminating myoepithelial cells (Krt14, red bar), macrophages and neutrophils (CD18, orange bar) and lymphocytes (CD3e, yellow bar) in infected mammary gland (left) and in microdissected infected MEC (right), at 30 hpi. *Significance is relative to fold change in expression of infected MG (adjusted p-value < 0.05).
Figure 5
Figure 5
Ingenuity Pathway Analysis (IPA) of the 39 differentially expressed genes in MFG transcriptome at 18 hpi vs. before infection (T0). The IPA legend defining the symbols depicted in IPA networks is given in the inset. Direct interaction is in solid line whereas indirect interaction is indicated by a dotted line.
Figure 6
Figure 6
Interaction network analysis of the differentially expressed genes at the early stages of infection. This consensus network represents the relationships identified by the analysis with the EBDBN approach (see Materials and methods).
Figure 7
Figure 7
Heat map of up- and down-regulated genes related to innate defense, selected from MFG transcriptome analysis of the IMI time course challenge with S. aureus. Gene expression was assessed using qPCR; genes shown in red are up-regulated and those shown in green are down-regulated in MFG infected at 12 hpi and 18 hpi, relative to MFG from the uninfected half-udder. Data are expressed in log10 ratios with respect to the reference genes (RPS24 and PPIA), at each time point and represent the five biological replicates at each time point.
Figure 8
Figure 8
Expression of IL-8 in MFG, at different time points of infection (12 hpi and 18 hpi) induced by an S. aureus challenge. Abundance of mRNA arising from the gene encoding the pro-inflammatory cytokine IL-8 (yellow bar) is expressed relative (± SEM) to transcripts from CSN3, a specific MEC marker (green bar) and from CD18, a specific marker for macrophages and neutrophils (red bar). *Significance is relative to the fold change in expression taking MFG from milk of the right uninfected half-udder as reference (adjusted p-value < 0.05).
Figure 9
Figure 9
Individual time-courses expression of selected genes assessed with RNA from MFG using microarrays, during the course of the IMI challenge by S. aureus. Selected genes are the following: S100A12, Il-1β, CD14, NFκB, IRAK4 and MyD88. Gene expression reflecting abundance of mRNA is given as fluorescence intensity (normalized signal).
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