Molecular Mechanism of Lactobacillus plantarum Inhibiting Inflammatory Response Induced by Enterotoxigenic Escherichia coli in Porcine Intestinal Epithelial Cells

LI Haihua; LIANG Dongmei; QIAO Jiayun

doi:10.3969/j.issn.1006-267x.2021.07.043

2021 , Vol. 33 >Issue 7: 4018 - 4029

DOI: https://doi.org/10.3969/j.issn.1006-267x.2021.07.043

MOLECULAR AND CELLULAR NUTRITION

Molecular Mechanism of Lactobacillus plantarum Inhibiting Inflammatory Response Induced by Enterotoxigenic Escherichia coli in Porcine Intestinal Epithelial Cells

LI Haihua ,
LIANG Dongmei ,
QIAO Jiayun

Expand

1. Tianjin Key Laboratory of Agricultural Animal Breeding and Healthy Husbandry, College of Animal Science and Animal Medicine, Tianjin Agricultural University, Tianjin 300384, China;
2. Tianjin Key Laboratory of Conservation and Utilization of Animal Diversity, College of Life Sciences, Tianjin Normal University, Tianjin 300387, China

Received date: 2020-12-30

Online published: 2021-07-06

Supported by

Fold

Abstract

The aim of this study was to investigate the molecular mechanism of Lactobacillus plantarum (LP) inhibiting inflammatory response induced by enterotoxigenic Escherichia coli (ETEC) in porcine intestinal epithelial cells. In this experiment, LP was used to incubate porcine intestinal epithelial cells IPEC-J2 for 3 h, then ETEC was used to stimulate cells for 1, 3, 6, 12 and 24 h. The culture supernatants and cells were collected. The contents of interleukin (IL)-1β, IL-8 and tumor necrosis factor-α (TNF-α) in the supernatant were detected by enzyme linked immunosorbent assay (ELISA), the expressions of Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) and NOD like receptor 3 (NLRP3) and NOD like receptor 6 (NLRP6) in cells were analyzed by real-time fluorescent quantitative PCR, and the phosphorylation levels of mitogen activated protein kinase (MAPK), including p38 and extracellular signal-regulated kinase (ERK), and nuclear factor-κB (NF-κB) as well as the expression of zona occludens-1 (ZO-1) and occludin in cells were examined by Western blot. IPEC-J2 cells were treated with p38 MAPK inhibitor, ERK-MAPK inhibitor and NF-κB p65 inhibitor for 1 h, respectively. The inhibitor treated-cells were incubated with LP for 3 h, and then were treated with ETEC for 24 h. The culture supernatant and cells were collected for detecting the contents of IL-1β, IL-8 and TNF-α and lactate dehydrogenase (LDH) activity by enzyme-linked immunosorbent assay (ELISA). The results showed as follows:1) compared with the ETEC treatment, the LP pretreatment extremely significantly decreased the contents of IL-1β, IL-8 and TNF-α produced by cells infected with ETEC for 12 to 24 h (P<0.01), significantly or extremely significantly increased the mRNA relative expression levels of TLR2, TLR4, NLRP3 and NLRP6 in cells infected with ETEC for 3 to 12 h (P<0.05 or P<0.01), extremely significantly decreased the mRNA relative expression levels of TLR2 and NLRP3 in cells infected with ETEC for 24 h (P<0.01), extremely significantly decreased the phosphorylation levels of p38 MAPK and NF-κB p65 in cells infected with ETEC for 3 to 6 h (P<0.01), and significantly or extremely significantly increased the expression of ZO-1 and occludin in cells infected with ETEC for 6 to 24 h (P<0.05 or P<0.01). 2) Compared with the ETEC treatment, the combination of inhibitor and LP extremely significantly reduced the contents of IL-1β, IL-8 and TNF-α and LDH activity produced by ETEC infected cells (P<0.01), the LP alone also extremely significantly reduced the LDH activity produced by ETEC infected cells (P<0.01), and the signaling pathway inhibitors had synergistic effects on the production of some proinflammatory cytokines regulated by LP. In conclusion, LP can reduce the production of inflammation-related factors by weakening p38 MAPK phosphorylation and blocking the activation of NF-κB signaling pathway, and displays the potential to improve the integrity of porcine intestinal epithelial cells.

Key words： Lactobacillus plantarum; porcine intestinal epithelial cells; enterotoxigenic Escherichia coli; inflammatory response; molecular mechanism

Cite this article

LI Haihua , LIANG Dongmei , QIAO Jiayun . Molecular Mechanism of Lactobacillus plantarum Inhibiting Inflammatory Response Induced by Enterotoxigenic Escherichia coli in Porcine Intestinal Epithelial Cells[J]. Chinese Journal of Animal Nutrition, 2021 , 33(7) : 4018 -4029 . DOI: 10.3969/j.issn.1006-267x.2021.07.043

References

[1] 何再平.微生物内分泌学研究引起断奶仔猪腹泻的机理[J].养猪,2020(3):116-118. HE Z P.Study on the mechanism of weaned piglets diarrhea by microbial endocrinology[J].Swine Production,2020(3):116-118.(in Chinese)
[2] HANKE D,POHLMANN A,SAUTER-LOUIS C,et al.Porcine epidemic diarrhea in Europe:in-detail analyses of disease dynamics and molecular epidemiology[J].Viruses,2017,9(7):177.
[3] LÓPEZ-HABER C,LEVIN-KONIGSBERG R,ZHU Y Y,et al.Phosphatidylinositol-4-kinase Ⅱα licenses phagosomes for TLR4 signaling and MHC-Ⅱ presentation in dendritic cells[J].Proceedings of the National Academy of Sciences of the United States of America,2020,117(45):28251-28262.

[4] MAISONNEUVE C,BERTHOLET S,PHILPOTT D J,et al.Unleashing the potential of NOD- and Toll-like agonists as vaccine adjuvants[J].Proceedings of the National Academy of Sciences of the United States of America,2014,111(34):12294-12299.

[5] MANSILLA F,TAKAGI M,GARCIA-CASTILLO V,et al.Modulation of Toll-like receptor-mediated innate immunity in bovine intestinal epithelial cells by lactic acid bacteria isolated from feedlot cattle[J].Beneficial Microbes,2020,11(3):269-282.

[6] QIAO J Y,SUN Z Y,LIANG D M,et al.Lactobacillus salivarius alleviates inflammation via NF-κB signaling in ETEC K88-induced IPEC-J2 cells[J].Journal of Animal Science and Biotechnology,2020,11:76.
[7] CHON H,CHOI B,JEONG G,et al.Suppression of proinflammatory cytokine production by specific metabolites of Lactobacillus plantarum 10hk2 via inhibiting NF-κB and p38 MAPK expressions[J].Comparative Immunology,Microbiology and Infectious Diseases,2010,33(6):e41-e49.
[8] SANTECCHIA I,FERRER M F,VIEIRA M L,et al.Phagocyte escape of Leptospira:the role of TLRs and NLRs[J].Frontiers in Immunology,2020,11:571816.
[9] BRUBAKER S W,BONHAM K S,ZANONI I,et al.Innate immune pattern recognition:a cell biological perspective[J].Annual Review of Immunology,2015,33:257-290.
[10] WU Q,LIU M C,YANG J,et al.Lactobacillus rhamnosus GR-1 ameliorates Escherichia coli-induced inflammation and cell damage via attenuation of ASC-independent NLRP3 inflammasome activation[J].Applied Environmental Microbiology,2015,82(4):1173-1182.
[11] JIANG Y J,LI L,SUN H X,et al.Induction of cytokines via NF-κB and p38 MAP kinase signalling pathways associated with the immunomodulation by Lactobacillus plantarum NDC 75017in vitro and in vivo[J].Journal of Functional Foods,2016,20:215-225.
[12] LI H H,ZHANG L,CHEN L B,et al.Lactobacillus acidophilus alleviates the inflammatory response to enterotoxigenic Escherichia coli K88 via inhibition of the NF-κB and p38 mitogen-activated protein kinase signaling pathways in piglets[J].BMC Microbiology,2016,16:273.
[13] BAEUERLE P A,HENKEL T.Function and activation of NF-κB in the immune system[J].Annual Review of Immunology,1994,12:141-179.
[14] WANG Z L,WANG L,CHEN Z,et al.In vitro evaluation of swine-derived Lactobacillus reuteri:probiotic properties and effects on intestinal porcine epithelial cells challenged with enterotoxigenic Escherichia coli K88[J].Journal of Microbiology Biotechnology,2016,26(6):1018-1025.

[15] RATHINAM V A K,VANAJA S K,FITZGERALD K A.Regulation of inflammasome signaling[J].Nature Immunology,2012,13(4):333-342.

[16] SHIMAZU T,VILLENA J,TOHNO M,et al.Immunobiotic Lactobacillus jensenii elicits anti-inflammatory activity in porcine intestinal epithelial cells by modulating negative regulators of the Toll-like receptor signaling pathway[J].Infection and Immunity,2012,80(1):276-288.

[17] FINAMORE A,ROSELLI M,IMBINTO A,et al.Lactobacillus amylovorus inhibits the TLR4 inflammatory signaling triggered by enterotoxigenic Escherichia coli via modulation of the negative regulators and involvement of TLR2 in intestinal Caco-2 cells and pig explants[J].PLoS One,2014,9(4):e94891.
[18] SUN K Y,XU D H,XIE C,et al.Lactobacillus paracasei modulates LPS-induced inflammatory cytokine release by monocyte-macrophages via the up-regulation of negative regulators of NF-kappa B signaling in a TLR2-dependent manner[J].Cytokine,2017,92:1-11.
[19] LI R Z,ZHU S.NLRP6 inflammasome[J].Molecular Aspects of Medicine,2020,76:100859.
[20] LEVY M,SHAPIRO H,THAISS C A,et al.NLRP6:A multifaceted innate immune sensor[J].Trends in Immunology,2017,38(4):248-260.

[21] YAP A S,MULLIN J M,STEVENSON B R.Molecular analyses of tight junction physiology:insights and paradoxes[J].The Journal of Membrane Biology,1998,163(3):159-167.

[22] TSUKITA S,FURUSE M,ITOH M.Structural and signalling molecules come together at tight junctions[J].Current Opinion in Cell Biology,1999,11(5):628-633.

[23] YU H T,DING X L,SHANG L J,et al.Protective ability of biogenic antimicrobial peptide microcin J25 against enterotoxigenic Escherichia coli-induced intestinal epithelial dysfunction and inflammatory responses IPEC-J2 cells[J].Frontiers in Cellular Infection Microbiology,2018,8:242.
[24] MCLAMB B L,GIBSON A J,OVERMAN E L,et al.Early weaning stress in pigs impairs innate mucosal immune responses to enterotoxigenic E. coli challenge and exacerbates intestinal injury and clinical disease[J].PLoS One,2013,8(4):e59838.
[25] KOH J Y,CHOI D W.Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase efflux assay[J].Journal of Neuroscience Methods,1987,20(1):83-90.

Options

Outlines

模态框（Modal）标题

Abstract

Cite this article

References