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Laser capture microdissection-seq analysis of local and systemic responses of mammary epithelial cells in cows locally treated with lipopolysaccharide.

R. K. Choudhary

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06-22-2020

Abstract:

79
Laser capture microdissection-seq analysis of local and systemic responses of mammary epithelial cells in cows locally treated with lipopolysaccharide.
R. K. Choudhary*1, A. Spitzer1, T. B. McFadden2, E. M. Shangraw2, R. O. Rodrigues2, F.-Q. Zhao1. 1Department of Animal and Veterinary Sciences, University of Vermont Burlington, VT, 2Division of Animal Sciences, University of Missouri Columbia, MO.

We aimed to analyze transcriptional changes of mammary epithelial cells (MEC) isolated from bovine mammary glands after intramammary challenge with lipopolysaccharide (LPS). Ten multiparous cows were used in the study. Five treatment (T) and 5 control (C) cows were paired based on days in milk, milk yield and parity. For T cows, both mammary glands on one side of the udder were infused with LPS (50 �g in 10 mL saline); these glands were designated (TL). The contralateral glands received 10 mL saline and were designated (TS). Likewise, for C cows 2 ipsilateral glands received saline (CS) and the contralateral glands remained uninfused (CU). Mammary tissues were biopsied before (0 h) and at 3 and 12 h post-infusion and processed for laser capture microdissection (LCM). MEC were collected using LCM, total RNA was isolated and subjected to low-put RNA sequencing. Among various comparisons, we found 3167 (TL3 vs. TL0), 670 (TL12 vs. TL0), 2555 (TL3 vs. TS3), and 3823 (TL3 vs. CS3) differentially expressed genes [DEGs; FDR < 0.05, Log2 (fold change) ≥ 1]. The major local responses of MEC in TL glands at 3h included upregulation of ribosome biogenesis, innate immunity and KEGG pathways of TNF, NOD-like receptor and NFKB signaling. Ingenuity pathway analysis showed activation of TNFR2, PI3/AKT, iNOS and acute phase protein response. Upstream regulators of these pathways predicted invasion of cell, chemotaxis and cell migration, and showed activated HIF1A network. Downregulated genes included network of carbohydrate metabolism, PPAR fatty acid biosynthesis, and several ionic transporters. Major systemic responses of MEC in TS glands showed weak cell mediated immune response, lymphocyte activation, and cytokine production responses. Ingenuity pathway analysis of systemic response genes at 3 h showed p53 senescence pathway with activated upstream regulators as NFKB and TNFA. These results of comprehensive transcriptome profiling of MEC may explain gene regulation of local and systemic responses of MEC during E. coli mastitis.

Keywords: endotoxin, mastitis, RNA-sequencing.