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

Abstract

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 probiotic Lactobacillus rhamnosus GR-1 on ameliorating E. 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 following E. coli challenge, but this increase was attenuated by L. rhamnosus GR-1 pretreatment. Immunofluorescence and Western blot analyses revealed that L. rhamnosus GR-1 pretreatment decreased the E. 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 the Pycard gene) was decreased during E. coli infection, even with L. rhamnosus GR-1 pretreatment. Pretreatment with L. rhamnosus GR-1 counteracted the E. 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 that L. rhamnosus GR-1 reduces the adhesion of E. coli to BMECs, subsequently ameliorating E. 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 of L. 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.

FIG 1

Pretreatment with live and UV-irradiated L. rhamnosus GR-1 reduced the adhesion of E. coli to BMEC monolayers. BMECs were collected from the indicated BMEC cultures at 6 h after E. coli challenge. An adhesion assay using E. coli alone served as a reference. The adhesion rate was defined as the adhered E. coli population on the BMECs pretreated with different conditions relative to the adhered E. coli population in the reference experiment. Data are presented as the means ± the standard deviations (SD) of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 2

Effect of L. rhamnosus GR-1 on E. coli-induced disruption of BMEC subcellular structure. The subcellular structure of BMECs collected from the indicated BMEC cultures at 6 h after E. coli challenge was observed using TEM. Black arrows indicate microvilli, white arrows indicate mitochondria, black arrowheads indicate tight-junction structures, white arrowheads indicate endoplasmic reticulum structures, and double white arrows indicate myelin-like figures. The data are representative of three independent experiments.

FIG 3

Changes in TLR and NLR mRNA expression during E. coli infection in BMECs preincubated with L. rhamnosus GR-1. The relative expression of mRNAs for the TLR2 (A), TLR4 (B), NOD1 (C), and NOD2 (D) genes in BMECs collected from the indicated BMEC cultures at 2, 4, 6, 12, and 24 h after E. coli challenge was analyzed using quantitative real-time PCR. Data are presented as the means ± the standard errors of the mean (SEM) of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 4

Pretreatment with L. rhamnosus GR-1 attenuated NLRP3 activation. Immunofluorescence staining for NLRP3 (red) in BMECs collected from the indicated BMEC cultures at 6 h after E. coli challenge. DAPI was used for nuclear staining (blue). Representative confocal immunofluorescence images show expression of NLRP3. Scale bars, 50 μm. Data are representative of three independent experiments.

FIG 5

Western blot detection of NLRP3, ASC, and caspase-1. Representative panels showing expression of NLRP3 (A, left panels), ASC (B, left panels), and caspase-1 (C, left panels) by BMECs collected from the indicated BMEC cultures at 2, 4, and 6 h after E. coli challenge. The intensities of NLRP3, ASC, and caspase-1 bands were determined using Quantity One software. Expression of GAPDH was measured as an internal control. Results are presented as the ratio of NLRP3, ASC, or caspase-1 band intensity to that of GAPDH (right panels). Data are presented as the means ± the SEM of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 6

Effect of L. rhamnosus GR-1 pretreatment on cytokine mRNA expression in BMECs following E. coli challenge. The relative expression of mRNAs for the IL1β (A), IL18 (B), IL8 (C), TNFα (D), IL6 (E), and IL10 (F) genes in BMECs collected from the indicated BMEC cultures at 2, 4, 6, 12, and 24 h after E. coli challenge was analyzed by quantitative real-time PCR. Data are presented as the means ± the SEM of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.