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. 2006 Mar;74(3):1907-15.
doi: 10.1128/IAI.74.3.1907-1915.2006.

Genome-wide expression analysis of lipopolysaccharide-induced mastitis in a mouse model

Jiamao Zheng  1 , Anjanette D Watson, David E Kerr
Affiliations

Genome-wide expression analysis of lipopolysaccharide-induced mastitis in a mouse model

Jiamao Zheng et al. Infect Immun. 2006 Mar.

Abstract

To better understand the acute host response to Escherichia coli mastitis, we analyzed gene expression patterns of approximately 23,000 transcripts 4 h after an intramammary infusion of lipopolysaccharide (LPS) in a mouse model. A total of 489 genes were significantly affected, of which 391 were induced and 98 were repressed. Gene ontology analysis demonstrated that most of the induced genes were associated with the innate immune response, apoptosis, and cell proliferation. Substantial induction of the chemokines CXCL1, CXCL2, and S100A8; the acute-phase protein SAA3; and the LPS binding protein CD14 were confirmed by Northern blot analysis. A subsequent time course experiment revealed CXCL1 induction prior to that of CD14 and SAA3. Mammary epithelial cell cultures also showed marked expression of these factors in response to LPS. The expression of immune-related genes in mammary epithelial cells indicates the importance of this cell type in initiating the inflammatory responses. Repressed genes include several carbohydrate and fatty acid metabolic enzymes and potassium transporters, which may contribute to milk composition changes during mastitis. Therefore, the overall transcription profile, in conjunction with gene ontology analysis, provides a detailed picture of the molecular mechanisms underlying the complex biological processes that occur during LPS-induced mastitis.

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Figures

FIG. 1.
FIG. 1.
Microarray analysis of selected mammary gland gene expression changes 4 h after intramammary infusion of saline or LPS was verified by Northern blot and Western blot analyses. (A) Northern blot analysis. A total of nine representative genes were selected for this assay, as indicated. Samples of the same RNA used for microarray analysis were separated by agarose gel electrophoresis, and the resulting blots were hybridized with appropriate mouse probes. Changes (n-fold) in gene expression detected by Northern analysis were calculated by dividing the average value of LPS treatment groups (T1 to T3) by that of saline control groups (C1 to C3). The expression level of mouse GAPDH was analyzed as an internal control. (B) Western blot analysis for CD14 and S100A8. Protein (80 μg) extracted from saline-control- and LPS-treated mammary tissues (4 h post-LPS infusion) were fractionated on an SDS-12% polyacrylamide gel and then transferred to the nitrocellulose membranes. Anti-CD14 and anti-S100A8 antibodies were used.
FIG. 2.
FIG. 2.
Infiltration of immune cells into the mammary gland tissues in response to LPS infection. (A) HE staining of mouse mammary tissues at 4 h after intramammary infusion of saline and LPS (1 μg). The infiltration of inflammatory cells (dark dots) into the mammary gland tissue is evident in LPS-treated glands. Shown are representative photographs of mammary tissue sections from either three saline-treated mice or three LPS-treated mice. (B) Graphical representation of induced chemotactic factors. Shown are expression levels of induced genes that are categorized by chemokines, chemokine receptors, adhesion molecules, and chemotactants, respectively. The mean and standard error for each gene are calculated from three independent normalized hybridization intensities from the microarray data set. The number on the top of the bar of each gene represents changes (n-fold) derived from microarray analysis.
FIG. 3.
FIG. 3.
Expression of immune-related genes in mammary epithelial cells. (A) Immunohistochemistry analysis of CD14 protein in mouse mammary tissues at 4 h after intramammary infusion of saline and LPS (1 μg). One saline control tissue section and one LPS-treated tissue section that were stained with an anti-CD14 antibody are shown. (B) Dose-dependent induction of CD14, CXCL1, and SAA3 genes in murine mammary epithelial cells (HC11) by LPS. Total RNA (5 μg) was extracted from HC11 cells that were stimulated with various doses of LPS for 24 h and was fractioned in the agarose gel. CD14, CXCL1, and SAA3 probes were randomly hybridized with three blots produced from three independent experiments. The mouse β-actin bands serve as a control for RNA integrity and loading amount.
FIG. 4.
FIG. 4.
Time course of CD14, CXCL1, and SAA3 gene expression during LPS-induced mastitis. Mice were killed at 1, 2, and 6 h after intramammary infusion of either saline (−) or LPS (+). Total RNA (20 μg) was isolated from mammary glands and subjected to Northern blot analysis for expression of CD14, CXCL1, and SAA3. The GAPDH band serves as a control for RNA integrity and loading amount.
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