Innate immune responses induced by lipopolysaccharide and lipoteichoic acid in primary goat mammary epithelial cells

Omar Bulgari,Alfred L Roca,Juan J Loor,Xianwen Dong,Anna M Caroli

doi:10.1186/s40104-017-0162-8

Abstract

BackgroundInnate immune responses induced by in vitro stimulation of primary mammary epithelial cells (MEC) using Gram-negative lipopolysaccharide (LPS) and Gram-positive lipoteichoic acid (LTA) bacterial cell wall components are well- characterized in bovine species. The objective of the current study was to characterize the downstream regulation of the inflammatory response induced by Toll-like receptors in primary goat MEC (pgMEC). We performed quantitative real-time RT-PCR (qPCR) to measure mRNA levels of 9 genes involved in transcriptional regulation or antibacterial activity: Toll-like receptor 2 (TLR2), Toll-like receptor 4 (TLR4), prostaglandin-endoperoxide synthase 2 (PTGS2), interferon induced protein with tetratricopeptide repeats 3 (IFIT3), interferon regulatory factor 3 (IRF3), myeloid differentiation primary response 88 (MYD88), nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NFKB1), Toll interacting protein (TOLLIP), and lactoferrin (LTF). Furthermore, we analyzed 7 cytokines involved in Toll-like receptor signaling pathways: C-C motif chemokine ligand 2 (CCL2), C-C motif chemokine ligand 5 (CCL5), C-X-C motif chemokine ligand 6 (CXCL6), interleukin 8 (CXCL8), interleukin 1 beta (IL1B), interleukin 6 (IL6), and tumor necrosis factor alpha (TNF).ResultsStimulation of pgMEC with LPS for 3 h led to an increase in expression of CCL2, CXCL6, IL6, CXCL8, PTGS2, IFIT3, MYD88, NFKB1, and TLR4 (P < 0.05). Except for IL6, and PTGS2, the same genes had greater expression than controls at 6 h post-LPS (P < 0.05). Expression of CCL5, PTGS2, IFIT3, NFKB1, TLR4, and TOLLIP was greater than controls after 3 h of incubation with LTA (P < 0.05). Compared to controls, stimulation with LTA for 6 h led to greater expression of PTGS2, IFIT3, NFKB1, and TOLLIP (P < 0.05) whereas the expression of CXCL6, CXCL8, and TLR4 was lower (P < 0.05). At 3 h incubation with both toxins compared to controls a greater expression (P < 0.05) of CCL2, CCL5, CXCL6, CXCL8, IL6, PTGS2, IFIT3, IRF3, MYD88, and NFKB1 was detected. After 6 h of incubation with both toxins, the expression of CCL2, CXCL6, IFIT3, MYD88, NFKB1, and TLR4 was higher than the controls (P < 0.05).ConclusionsData indicate that in the goat MEC, LTA induces a weaker inflammatory response than LPS. This may be related to the observation that gram-positive bacteria cause chronic mastitis more often than gram-negative infections.

Highlights

Innate immune responses induced by in vitro stimulation of primary mammary epithelial cells (MEC) using Gram-negative lipopolysaccharide (LPS) and Gram-positive lipoteichoic acid (LTA) bacterial cell wall components are well- characterized in bovine species
Severe clinical mastitis with systemic signs produced by S. aureus and E. coli may be due to the action of various cytotoxins and endotoxins leading to extensive tissue damage and systemic reactions in the animal [2, 3]
The greater expression of C motif chemokine ligand 2 (CCL2) with LPS than LTA was consistent with data from a study with bovine primary mammary epithelial cells (pMEC) incubated with LPS purified from E. coli strain O55:B5 [19, 20] or heat-inactivated E. coli [36], and the lack of effect of LTA isolated from Streptococcus pyogenes [19], S. aureus [20] or heat-inactivated S. aureus [36]

Summary

Introduction

Innate immune responses induced by in vitro stimulation of primary mammary epithelial cells (MEC) using Gram-negative lipopolysaccharide (LPS) and Gram-positive lipoteichoic acid (LTA) bacterial cell wall components are well- characterized in bovine species. The objective of the current study was to characterize the downstream regulation of the inflammatory response induced by Toll-like receptors in primary goat MEC (pgMEC). The most common causal agent of mastitis in goats is Staphylococcus aureus followed by Pasteurella haemolytica, Escherichia coli, Clostridium perfrigens, Streptococcus sp., Pseudomonas sp., and Nocardia sp. Toll-like receptors (TLR) play a central role in the innate immune system, and form a first line of defense against infections by recognizing pathogen associated molecular patterns [8]. TLR2 recognizes lipoteichoic acid (LTA), a major constituent of Grampositive bacteria, and TLR4 recognizes lipopolysaccharide (LPS) that is common to Gram-negative bacteria [8]

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