Lactobacillus Attenuates Inflammatory Damage Of Bovine Mammary Epithelial Cells Infected With Escherichia Coli

Escherichia coli(E.coli)is a major environmental pathogen causing bovine mastitis,and induces damage of mammary gland and cell death.Antibiotics are the main therapy for controlling bovine mastitis.However,excess use of antibiotics leads to several severe threats,including food safety,spread of antibiotic resistance and environment pollution.Probiotics are the potential alternatives to antibiotics and play an important role in preventing bovine mastitis.Using the in vitro bovine mammary epithelial cells(BMECs)model of E.coli infection,the present study explores the effect of probiotic Lactobacillus rhamnosus GR-1(LGR-1)on ameliorating E.coli-induced inflammatory response of BMECs and cell damage and the underlying molecular mechanisms.Primary BMECs were isolated from the mmmary gland of health bovine,using tissue explant and differential centrifugation method.Cytokeratin 18 staining showed that BMECs were characterized by epithelial cells.BMECs were preincubated with LGR-1(1 × 107 CFU/ml)for 3 h,and then exposed to E.coli(1 × 107 CFU/ml).Expression of Toll-like receptor 4(TLR4),NOD1 and NOD2 mRNA increased following E.coli infection,however,preincubation of LGR-1 attenuated this increase.Immunoflurescene and Western blot analysis showed that preincubation of LGR-1 inhibited E.coli-induced increased expression of NOD-like receptor family member pyrin domain-containing protein 3(NLRP3)and caspase-1.Decreased expression of adaptor protein apoptosis-associated speck-like protein(ASC)was observed after E.coli infection.Preincubation of LGR-1 suppressed increased expression of interleukin(IL)1?,-6,-8,-18 and tumor necrosis factor a mRNA by E.coli,but up-regulated IL-10 mRNA expression.Our data show that LGR-1 inhibites the E.coli adhesion to BMECs,subsequently attenuating E.coli-induced damage of cellular morphology and ultrastructure and ameliorating detrimental inflammatory responses,partly via promoting TLR2 and NOD1 synergism and reducing ASC-independent NLRP3 inflammasome activation.To further confirm the role of ASC in LGR-1 preventing E.coli infection,the ASC gene was knocked out in bovine mammary epithelial MAC-T cells using clustered,regularly interspaced,short palindromic repeat(CRISPR)/CRISPR-associated(Cas)-9 technology.MAC-T cells were preincubated with LGR-1(1 × 107 CFU/ml)for 3 h,and then exposed to E.coli(1 × 107 CFU/ml).Western blot analysis showed that E.coli infection increased NLRP3 and NLRC4 expression,however,preincubation of LGR-1 reduced this increase,regardless of ASC knockout.Immunoflurescene and Western blot analysis showed that LGR-1 inhibited E.coli-induced caspase-1 activation,as shown in ASC-knockout MAC-T cells.LGR-1 also inhibited E.coli-induced caspase-4 activation.ASC knockout decreased,but not abolished E.coli-induced increased production of IL-1? and IL-18,as well as cell pyroptosis.However,preincubation of LGR-1 inhibited this increase.The present study shows that L.rhamnosus GR-1 suppresses activation of ASC-dependent NLRP3 and NLRC4 inflammasomes and production of downstream IL-1? and IL-18 during E.coli infection.Lactobacillus rhamnosus GR-1 also inhibited E.coli-induced cell pyroptosis,in part through attenuation of NLRC4 and non-canonical caspase-4 activation independently of ASC.Our data enhance understanding of the mechanism of action of this candidate probiotic and provide scientific theory support for protiobics controlling pathogen infection of the bovine mammary gland.