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Escherichia coli is a major environmental pathogen causing bovine mastitis, which leads to mammary tissue damage and cell death. We explored the effects of the probioticLactobacillus rhamnosus GR-1 on amelioratingE. coli -induced inflammation and cell damage in primary bovine mammary epithelial cells (BMECs). Increased Toll-like receptor 4 (TLR4), NOD1, and NOD2 mRNA expression was observed followingE. coli challenge, but this increase was attenuated byL. rhamnosus GR-1 pretreatment. Immunofluorescence and Western blot analyses revealed thatL. rhamnosus GR-1 pretreatment decreased theE. coli -induced increases in the expression of the NOD-like receptor family member pyrin domain-containing protein 3 (NLRP3) and the serine protease caspase 1. However, expression of the adaptor protein apoptosis-associated speck-like protein (ASC, encoded by thePycard gene) was decreased duringE. coli infection, even withL. rhamnosus GR-1 pretreatment. Pretreatment withL. rhamnosus GR-1 counteracted theE. coli -induced increases in interleukin-1β (IL-1β), -6, -8, and -18 and tumor necrosis factor alpha mRNA expression but upregulated IL-10 mRNA expression. Our data indicate thatL. rhamnosus GR-1 reduces the adhesion ofE. coli to BMECs, subsequently amelioratingE. coli -induced disruption of cellular morphology and ultrastructure and limiting detrimental inflammatory responses, partly via promoting TLR2 and NOD1 synergism and attenuating ASC-independent NLRP3 inflammasome activation. Although the residual pathogenic activity ofL. rhamnosus , the dosage regimen, and the means of probiotic supplementation in cattle remain undefined, our data enhance our understanding of the mechanism of action of this candidate probiotic, allowing for development of specific probiotic-based therapies and strategies for preventing pathogenic infection of the bovine mammary gland.

applied and environmental microbiology

Lactobacillus rhamnosus GR-1 Ameliorates Escherichia coli-Induced Inflammation and Cell Damage via Attenuation of ASC-Independent NLRP3 Inflammasome Activation