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-1␤ (IL-1␤), -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. Mastitis affects cows in all regions of the world and can cause a decrease in milk production and quality, resulting in a major economic burden to the dairy industry (1). Escherichia coli is by far the most common cause of mastitis, since it is isolated in more than 80% of cases of coliform mastitis (2). Antibiotic therapy frequently leaves residues in milk, potentially facilitating the development of antibiotic resistance. Probiotics represent a novel alternative to antibiotics for controlling pathogen infections.Lactobacillus rhamnosus GR-1 is a probiotic bacterium isolated from the female urethra, and oral ingestion of L. rhamnosus GR-1 and L. fermentum RC-14 reduces vaginal colonization by pathogenic bacteria and yeasts and maintains urogenital health in women (3). Pretreatment of pregnant CD-1 mice with L. rhamnosus GR-1 culture supernatant decreases lipopolysaccharide (LPS)-induced production of various cytokines and chemokines (4). Lactobacillus rhamnosus GR-1 suppresses expression of nuclear factor (NF)-B-related inflammatory genes and activates alternate mitogen-activated protein kinase (MAPK) and activator protein 1 pathways to recruit host defense factors...

Dietary supplementation with Clostridium butyricum helps to improve the intestinal barrier function of weaned piglets challenged with enterotoxigenic Escherichia coli K88

“…However, the probiotic L. rhamnosus GR‐1 has been reported to alleviate ETEC‐induced increases in NLRP3 protein, as well as increased IL‐1β and IL‐18 mRNA expression in primary bovine mammary epithelial cells (Wu et al . ). In addition, in the DSS‐ and TNBS‐models, NLRP3 −/− mice are more susceptible to experimental colitis and exhibit reduced expression of pro‐inflammatory cytokine IL‐1β and the anti‐inflammatory cytokine IL‐10 (Hirota et al .…”

Vitamin D/VDR, Probiotics, and Gastrointestinal Diseases

“…They may stimulate the immune system, balance commensal and pathogenic bacteria to decrease the incidence of infections, reduce symptoms, restore homeostasis, and modify toxic compounds and host products. Recent trials using Lactobacillus rhamnosus GR-1 pretreatment demonstrated counteraction in the E. coli -induced production of various cytokines and chemokines in ameliorating inflammation and cell damage [33] . Zhang et al [34] demonstrated that pre-treatment with L. rhamnosus suppressed apoptosis of intestinal epithelia cells, which is consistent with the study in which Lactobacillus rhamnosus GG prevents cytokine-induced apoptosis in intestinal epithelial cell models [35] .…”

“…Another aspect with regard to immunomodulation is that pretreatment with probiotics may decrease various cytokines and chemokines production and induce anti-inflammatory molecules, mainly through Th17/Treg, TLRs, or NF-κB signaling pathways [89, 90] . Probiotic Lactobacillus rhamnosus GR-1 ameliorated E. coli induced disruption of cellular structure and inflammation, partly by promoting TLR2 and NOD1 synergism and decreasing NLRP3 inflammasome activation [33] .…”

Evaluation of Immunomodulatory Activities of the Heat-Killed Probiotic Strain Lactobacillus casei IMAU60214 on Macrophages In Vitro

“…Macrophages also participate in the activation and regulation of the immune response through antitumor activities, antigen presentation, and secretion of both pro-inflammatory cytokines (e.g., TNF-α, IL-1, and IL-6) and anti-inflammatory cytokines (IL-10 and transforming growth factor (TGF)-β) that act to regulate immune homeostasis [20][21][22]. In addition, probiotics act as mediators of inflammation and play an important role in the control of infection of several pathogens [23,24]. Several recent reports have examined the regulation of the macrophage immune response to probiotic strains of Lactobacillus casei [25].…”