Abstract
Inflammation is the hallmark of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli-induced bovine mastitis. Organic selenium can activate pivotal proteins in immune responses and regulate the immune system. The present study aimed to investigate whether selenomethionine (SeMet) attenuates ESBL E. coli-induced inflammation in bovine mammary epithelial cells (bMECs) and macrophages. Cells were treated with 0, 5/10, 10/20, 20/40, or 40/60 μM SeMet for 12 h and/or inoculated with ESBL-E. coli [multiplicity of infection (MOI) = 5] for 4/6 h, respectively. We assessed inflammatory responses, including selenoprotein S (SeS), Toll-like receptor 4 (TLR4), Ikappa-B (IκB), phospho-NF-κB p65 (Ser536), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and lactate dehydrogenase (LDH) activities. Treatment with 40/60 μM SeMet promoted cell viability and inhibited LDH activities in both bMECs and macrophages. Inoculation with ESBL-E. coli reduced cell viability, which was attenuated by SeMet treatment in bMECs and macrophages. SeMet increased ESBL E. coli-induced downregulation of SeS and decreased LDH activities, TLR4, IκB, phospho-NF-κB p65 (Ser536), IL-1β, and TNF-α protein expressions in bMECs and macrophages. In addition, knockdown of SeS promoted protein expression of TLR4-mediated nuclear factor-kappa (NF-κB) pathway and BAY 11-708 inhibited TNF-α and IL-1β protein levels in bMECs and macrophages after ESBL-E. coli treatment. Moreover, ESBL-E. coli inoculation increased monocyte chemoattractant protein 1 (MCP-1), C–C motif ligand 3 (CCL-3), and CCL-5 mRNA expressions in bMECs. In conclusion, ESBL-E. coli induced expression of MCP-1, CCL-3, and CCL-5 in bMECs and then recruited and activated macrophages, whereas SeMet attenuated ESBL E. coli-induced inflammation through activated SeS-mediated TLR4/NF-κB signaling pathway in bMECs and macrophages.
Highlights
Escherichia coli is an opportunistic environmental pathogen affecting bovine mammary glands, causing clinical mastitis, during early lactation in dairy cows (Olde Riekerink et al, 2008; Gao et al, 2017)
Primary antibodies to Toll-like receptor 4 (TLR4), NF-κB, Selenoprotein S (SeS), IKB, tumor necrosis factor-α (TNF-α), IL-1β, GAPDH, and PCNA were obtained from Proteintech (Proteintech, Wuhan, China), and primary antibodies to the phospho-NF-κB p65
To assess cytotoxic effects of selenium (Figure 1), bovine mammary epithelial cells (bMECs) and macrophages were cultured for 12 h with SeMet at various concentrations and cell viability and lactate dehydrogenase (LDH) activity were measured
Summary
Escherichia coli is an opportunistic environmental pathogen affecting bovine mammary glands, causing clinical mastitis, during early lactation in dairy cows (Olde Riekerink et al, 2008; Gao et al, 2017). Escherichia coli penetrates the lumen of the mammary gland, promoted by lactate dehydrogenase (LDH) release (Vangroenweghe et al, 2005) and incites distinct inflammatory and innate immune responses (Gilbert et al, 2013). Bovine mammary epithelial cells (bMECs), and macrophage lines are an ideal model to evaluate in vitro inflammation induced by E. coli. They are well characterized, readily available, and moderately priced. Mammary epithelial cells and macrophages each have an array of germline-encoded pattern recognition receptors/sensors that recognize pathogen-associated molecular patterns and which activate the nuclear factor-kappa B (NFκB) signaling pathway to mediate inflammatory and innate immune responses. The P65 subunit of NF-κB contains the transcriptional activation domain, which regulates transcription of inflammatory proteins during infection, e.g., tumor necrosis factor-α (TNF-α), and interleukin1β (IL-1β; Lecoq et al, 2017)
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