Modulation of Toll-like receptor 4-mediated innate immunity in bovine intestinal epithelial cells by lactic acid bacteria isolated from feedlot cattle

MANSILLA, F.; ISLAM, A.; VIGNOLO, G.; GARCIA-CASTILLO, V.; NADER-MACIAS, M.E.; VILLENA, J.; TAKAGI, M.; ASO, H.; KITAZAWA, H.

Praga

Conferencia; XIII Conferencia Científica Internacional sobre Probióticos, Prebióticos, Microbiota Intestinal y Salud.; 2019

IPC

Lactobacillus acidophilus CRL2074, L. mucosae CRL2069, L. fermentum CRL2085, and L. rhamnosus CRL2084 isolated from feedlot cattle environment, were previously selected by their beneficial-probiotic characteristics including their ability to inhibit gastrointestinal pathogen´s growth. In this work, we aimed to go further in the evaluation of their ability to differentially modulate the innate immune response triggered by Toll-like receptor (TLR)-4 activation in bovine intestinal epithelial cells. For this purpose, a bovine intestinal epithelial cell line (BIE cells) was stimulated with CRL2074, CRL2069, CRL2085 and CRL2084 (5×108 cells/ml) strains for 48 h. Then, BIE cells were challenged with heat-stable pathogen molecular associated patterns (PAMPs) from enterotoxigenic Escherichia coli (ETEC) (O9:H- :987P+: STa+) to induce the activation of TLR4. Twelve hours after the challenge, quantitative expression of cytokines (interleukin (IL)-1α, IL-, IL-6, tumor necrosis factor (TNF)-α), and chemokines (monocyte chemoattractant protein 1 (MCP-1), IL-8) were evaluated by qPCR. In addition, the expressions of the following negative regulators of TLR4 signaling were studied: A20-binding inhibitor of nuclear factor kappa B activation 3 (ABIN-3), B cell lymphoma 3-encoded protein (Bcl-3), interleukin-1 receptor-associated kinase M (IRAK-M), mitogen-activated protein kinase (MKP-1), single immunoglobulin IL-1-related receptor (SIGIRR), and Toll interacting protein (Tollip). The challenge of BIE cells with heat-stable ETEC PAMPs significantly increased the expression of all the pro-inflammatory cytokines and chemokines evaluated. No significant differences were observed in the expression of these pro-inflammatory factors when control BIE cells were compared to the cells previously stimulated with L. acidophilus CRL2074 or L. fermentum CRL2085. On the contrary, L. mucosae CRL2069 significantly reduced the expression of TNF-α, IL-, MCP-1, and IL-8 in BIE cells after the challenge with heat-stable ETEC PAMPs. In addition, diminished expressions of IL-6, MCP-1, and IL-8 were found in BIE cells stimulated with L. rhamnosus CRL2084, although its effect was significantly lower than that observed for CRL2069 strain. The reduced levels of pro-inflammatory factors in BIE cells induced by the CRL2069 and CRL2085 strains was related to their ability of increasing the expression of TLR4 negative regulators. L. mucosae CRL2069 significantly improved the expression of ABIN-3, IRAK-M and MKP-1, while L. rhamnosus CRL2084 augmented ABIN-3 expression in heat-stable ETEC PAMPs-challenged BIE cells. The results of this work suggest that L. mucosae CRL2069 is able to regulate the innate immune response triggered by TLR-4 activation in bovine intestinal epithelial cells through the up-regulation of three TLR negative regulators: ABIN-3, IRAK-M and MKP-1, which would influence nuclear factor kB and mitogen-activated protein kinases signaling pathways, while reducing the expression of pro-inflammatory cytokines and chemokines. Therefore, L. mucosae CRL2069 is an interesting probiotic candidate for the protection of the bovine host against TLR4-mediated intestinal inflammatory damage.