产肠毒素大肠杆菌感染IPEC-J2细胞后IL-17细胞因子表达变化及其功能初探
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摘要
产肠毒素大肠杆菌(enterotoxigenic E.coli, ETEC)是引起新生和断奶仔猪腹泻(post-weaning diarrhea, PWD)的最重要的病原之一。ETEC进入肠道后与肠上皮细胞的相互作用既是该细菌致病的前提条件,也是激发宿主免疫反应的基础。本研究旨在分析ETEC感染猪肠上皮细胞后IL-17细胞因子表达变化,同时探索细胞因子在抵抗ETEC感染中的免疫防御机制。本研究采用猪肠上皮细胞系IPEC-J2和产肠毒素大肠杆菌标准株(C83901)为主要研究材料,通过RT-PCR、ELISA、免疫荧光及免疫印迹的方法分析了IL-17细胞因子及肠黏膜免疫防御相关基因在感染前后的变化。同时,进一步研究了IPEC-J2细胞中相关IL-17细胞因子刺激对肠黏膜防御相关基因的调控作用。结果表明,除IL-17D未检测到外,C83901能促进所有IL-17细胞因子mRNA的转录,尤其能显著上调IL-17A、IL-17C在基因和蛋白水平的表达。ETEC感染或者体外添加IL-17A/IL-17C有利于IPEC-J2细胞中黏蛋白(mucin-2)、紧密连接蛋白(claudin-1、 claudin-2)及防御素pBD-2的表达。结果提示,ETEC感染后,肠上皮细胞通过调节IL-17细胞因子的表达进而增强肠黏膜屏障功能以抵抗该细菌的入侵。
Enterotoxigenic E. coli(ETEC) is one of the most important cause of neonatal and post-weaning diarrhea(PWD) in piglets. The interaction between ETEC and intestinal epithelial cells is the prerequisite of bacterial pathogenesis, as well as the first step of triggering host immune response. The aim of this study was to determine the expression profile and host defense mechanisms of IL-17 cytokine in porcine intestinal epithelial cells against ETEC infection. Firstly, we determined the expression of IL-17 cytokines and several mucosal defense genes by the application of RT-PCR, ELISA, immunohistochemistry(IHC) assays and Western Blotting in the porcine intestinal epithelial cell line IPEC-J2 infected with a F4~+ETEC reference strain C83901. Later on, the regulating effect of IL-17 cytokine on the expression of above mucosal defense genes in IPEC-J2 cells was explored. The results showed that C83901 induced the mRNA expression of all IL-17 cytokines(IL-17 D was not detected), particularly upregulated the expression of IL-17 A and IL-17 C both at the transcriptional and protein level. The upregulation of IL-17 A and IL-17 C in the protein level were confirmed by IHC and ELISA. The existence of IL-17 A and/or IL-17 C either induced by ETEC infection or supplemented artificially in IPEC-J2 cells can elicit the expression of host defense gene expression of mucin-2, claudin-1, claudin-2 and beta defensins-2. These results indicated that IL-17 induction in intestinal epithelial cells upon ETEC infection are beneficial for the mucosal host defense by promoting the intestinal barrier integrity.
Enterotoxigenic E. coli(ETEC) is one of the most important cause of neonatal and post-weaning diarrhea(PWD) in piglets. The interaction between ETEC and intestinal epithelial cells is the prerequisite of bacterial pathogenesis, as well as the first step of triggering host immune response. The aim of this study was to determine the expression profile and host defense mechanisms of IL-17 cytokine in porcine intestinal epithelial cells against ETEC infection. Firstly, we determined the expression of IL-17 cytokines and several mucosal defense genes by the application of RT-PCR, ELISA, immunohistochemistry(IHC) assays and Western Blotting in the porcine intestinal epithelial cell line IPEC-J2 infected with a F4~+ETEC reference strain C83901. Later on, the regulating effect of IL-17 cytokine on the expression of above mucosal defense genes in IPEC-J2 cells was explored. The results showed that C83901 induced the mRNA expression of all IL-17 cytokines(IL-17 D was not detected), particularly upregulated the expression of IL-17 A and IL-17 C both at the transcriptional and protein level. The upregulation of IL-17 A and IL-17 C in the protein level were confirmed by IHC and ELISA. The existence of IL-17 A and/or IL-17 C either induced by ETEC infection or supplemented artificially in IPEC-J2 cells can elicit the expression of host defense gene expression of mucin-2, claudin-1, claudin-2 and beta defensins-2. These results indicated that IL-17 induction in intestinal epithelial cells upon ETEC infection are beneficial for the mucosal host defense by promoting the intestinal barrier integrity.
引文
[1] 张秀媛,袁万哲,孙继国.产肠毒素性大肠杆菌主要毒力因子及其疫苗的研究进展[J].畜牧与兽医,2014,46(5):112-116.ZHANG X Y,YUAN W Z,SUN J G.Research progress on main virulence factors and vaccines of enterotoxigenic Escherichia coli[J].Animal Husbandry & Veterinary Medicine,2014,46(5):112-116.(in Chinese)
[2] GYLES C L,PRESCOTT J F,SONGER G,et al.Pathogenesis of bacterial infections in animals[M].4th ed.Ames,Iowa:Wiley-Blackwell,2010.
[3] PETERSON L W,ARTIS D.Review.Intestinal epithelial cells:regulators of barrier function and immune homeostasis[J].Nat Rev Immunol,2014,14(3):141-153.
[4] BROSNAHAN A J,BROWN D R.Porcine IPEC-J2 intestinal epithelial cells in microbiological investigations[J].Vet Microbiol,2012,156(3-4):229-237.
[5] ZHOU C L,LIU Z Z,JIANG J C,et al.Differential gene expression profiling of porcine epithelial cells infected with three enterotoxigenic Escherichia coli strains[J].BMC Genomics,2012,13:330
[6] GAO Y,HAN F,HUANG X,et al.Changes in gut microbial populations,intestinal morphology,expression of tight junction proteins,and cytokine production between two pig breeds after challenge with Escherichia coli K88:a comparative study[J].J Anim Sci,2013,91(12):5614-5625.
[7] NGENDAHAYO MUKIZA C,DUBREUIL J D.Escherichia coli heat-stable toxin b impairs intestinal epithelial barrier function by altering tight junction proteins[J].Infect Immun,2013,81(8):2819-2827.
[8] NASSOUR H,DUBREUIL J D.Escherichia coli STb enterotoxin dislodges claudin-1 from epithelial tight junctions[J].PLoS One,2014,9(11):e113273.
[9] KUMAR P,LUO Q W,VICKERS T J,et al.EatA,an immunogenic protective antigen of enterotoxigenic Escherichia coli,degrades intestinal mucin[J].Infect Immun,2014,82(2):500-508.
[10] LUO Q W,KUMAR P,VICKERS T J,et al.Enterotoxigenic Escherichia coli secretes a highly conserved mucin-degrading metalloprotease to effectively engage intestinal epithelial cells[J].Infect Immun,2014,82(2):509-521.
[11] ZHOU M,ZHU J,YU H,et al.Investigation into in vitro and in vivo models using intestinal epithelial IPEC-J2 cells and Caenorhabditis elegans for selecting probiotic candidates to control porcine enterotoxigenic Escherichia coli[J].J Appl Microbiol,2014,117(1):217-226.
[12] SPITZER F,SPEISER S,VAHJEN W,et al.Effect of different feed ingredients and additives on IPEC-J2 cells challenged with an enterotoxigenic Escherichia coli strain[J].Cytotechnology,2016,68(4):1463-1471.
[13] KARIMI S,JONSSON H,LUNDH T,et al.Lactobacillus reuteri strains protect epithelial barrier integrity of IPEC-J2 monolayers from the detrimental effect of enterotoxigenic Escherichia coli[J].Physiol Rep,2018,6(2):e13514.
[14] KERN M,GüNZEL D,ASCHENBACH J R,et al.Altered cytokine expression and barrier properties after in vitro infection of porcine epithelial cells with enterotoxigenic Escherichia coli and probiotic Enterococcus faecium[J].Mediators Inflamm,2017,2017:2748192.
[15] SONG X Y,HE X,LI X X,et al.The roles and functional mechanisms of interleukin-17 family cytokines in mucosal immunity[J].Cell Mol Immunol,2016,13(4):418-431.
[16] LEE J S,TATO C M,JOYCE-SHAIKH B,et al.Interleukin-23-independent IL-17 production regulates intestinal epithelial permeability[J].Immunity,2015,43(4):727-738.
[17] MAXWELL J R,ZHANG Y,BROWN W A,et al.Differential roles for interleukin-23 and interleukin-17 in intestinal immunoregulation[J].Immunity,2015,43(4):739-750.
[18] IWAKURA Y,ISHIGAME H,SAIJO S,et al.Functional specialization of interleukin-17 family members[J].Immunity,2011,34(2):149-162.
[19] RAMIREZ-CARROZZI V,SAMBANDAM A,LUIS E,et al.IL-17C regulates the innate immune function of epithelial cells in an autocrine manner[J].Nat Immunol,2011,12(12):1159-1166.
[20] SONG X Y,ZHU S,SHI P Q,et al.IL-17RE is the functional receptor for IL-17C and mediates mucosal immunity to infection with intestinal pathogens[J].Nat Immunol,2011,12(12):1151-1158.
[21] HUANG J L,MENG S H,HONG S J,et al.IL-17C is required for lethal inflammation during systemic fungal infection[J].Cell Mol Immunol,2016,13(4):474-483.
[22] LOOS M,GEENS M,SCHAUVLIEGE S,et al.Role of heat-stable enterotoxins in the induction of early immune responses in piglets after infection with enterotoxigenic Escherichia coli[J].PLoS One,2012,7(7):e41041.
[23] LUO Y,VAN NGUYEN U,DE LA FE RODRIGUEZ P Y,et al.F4+ ETEC infection and oral immunization with F4 fimbriae elicits an IL-17-dominated immune response[J].Vet Res,2015,46:121.
[24] JIAO W,MA Q,LV X,et al.Gene expression and tissue distribution of β-defensins in Chinese Min pigs and Landrace pigs[J].Czech J Anim Sci,2017,62(4):178-183.
[25] PASTERNAK J A,AIYER V I A,HAMONIC G,et al.Molecular and physiological effects on the small intestine of weaner pigs following feeding with deoxynivalenol-contaminated feed[J].Toxins (Basel),2018,10(1):40.
[26] BRUEL T,GUIBON R,MELO S,et al.Epithelial induction of porcine suppressor of cytokine signaling 2 (SOCS2) gene expression in response to Entamoeba histolytica[J].Dev Comp Immunol,2010,34(5):562-571.
[27] VON DER HARDT K,KANDLER M A,FINK L,et al.High frequency oscillatory ventilation suppresses inflammatory response in lung tissue andmicrodissected alveolar macrophages in surfactant depleted piglets[J].Pediatr Res,2004,55(2):339-346.
[28] MELKEBEEK V,SONCK E,VERDONCK F,et al.Optimized FaeG expression and a thermolabile enterotoxin DNA adjuvant enhance priming of an intestinal immune response by an FaeG DNA vaccine in pigs[J].Clin Vaccine Immunol,2007,14(1):28-35.
[29] STARNES T,BROXMEYER H E,ROBERTSON M J,et al.Cutting edge:IL-17D,a novel member of the IL-17 family,stimulates cytokine production and inhibits hemopoiesis[J].J Immunol,2002,169(2):642-646.
[30] FRIEDRICH M,DIEGELMANN J,SCHAUBER J,et al.Intestinal neuroendocrine cells and goblet cells are mediators of IL-17A-amplified epithelial IL-17C production in human inflammatory bowel disease[J].Mucosal Immunol,2015,8(4):943-958.
[31] IM E,JUNG J,RHEE S H.Toll-like receptor 5 engagement induces interleukin-17C expression in intestinal epithelial cells[J].J Interf Cytok Res,2012,32(12):583-591.
[32] PFEIFER P,VOSS M,WONNENBERG B,et al.IL-17C is a mediator of respiratory epithelial innate immune response[J].Am J Respir Cell Mol Biol,2013,48(4):415-421.
[33] KUSAGAYA H,FUJISAWA T,YAMANAKA K,et al.Toll-like receptor-mediated airway IL-17C enhances epithelial host defense in an autocrine/paracrine manner[J].Am J Respir Cell Mol Biol,2014,50(1):30-39.
[34] REYNOLDS J M,ANGKASEKWINAI P,DONG C.IL-17 family member cytokines:regulation and function in innate immunity[J].Cytok Growth Factor Rev,2010,21(6):413-423.
[35] ZAPH C,DU Y R,SAENZ S A,et al.Commensal-dependent expression of IL-25 regulates the IL-23-IL-17 axis in the intestine[J].J Exp Med,2008,205(10):2191-2198.
[36] SAWA S,LOCHNER M,SATOH-TAKAYAMA N,et al.RORγt+ innate lymphoid cells regulate intestinal homeostasis by integrating negative signals from the symbiotic microbiota[J].Nat Immunol,2011,12(4):320-326.
[2] GYLES C L,PRESCOTT J F,SONGER G,et al.Pathogenesis of bacterial infections in animals[M].4th ed.Ames,Iowa:Wiley-Blackwell,2010.
[3] PETERSON L W,ARTIS D.Review.Intestinal epithelial cells:regulators of barrier function and immune homeostasis[J].Nat Rev Immunol,2014,14(3):141-153.
[4] BROSNAHAN A J,BROWN D R.Porcine IPEC-J2 intestinal epithelial cells in microbiological investigations[J].Vet Microbiol,2012,156(3-4):229-237.
[5] ZHOU C L,LIU Z Z,JIANG J C,et al.Differential gene expression profiling of porcine epithelial cells infected with three enterotoxigenic Escherichia coli strains[J].BMC Genomics,2012,13:330
[6] GAO Y,HAN F,HUANG X,et al.Changes in gut microbial populations,intestinal morphology,expression of tight junction proteins,and cytokine production between two pig breeds after challenge with Escherichia coli K88:a comparative study[J].J Anim Sci,2013,91(12):5614-5625.
[7] NGENDAHAYO MUKIZA C,DUBREUIL J D.Escherichia coli heat-stable toxin b impairs intestinal epithelial barrier function by altering tight junction proteins[J].Infect Immun,2013,81(8):2819-2827.
[8] NASSOUR H,DUBREUIL J D.Escherichia coli STb enterotoxin dislodges claudin-1 from epithelial tight junctions[J].PLoS One,2014,9(11):e113273.
[9] KUMAR P,LUO Q W,VICKERS T J,et al.EatA,an immunogenic protective antigen of enterotoxigenic Escherichia coli,degrades intestinal mucin[J].Infect Immun,2014,82(2):500-508.
[10] LUO Q W,KUMAR P,VICKERS T J,et al.Enterotoxigenic Escherichia coli secretes a highly conserved mucin-degrading metalloprotease to effectively engage intestinal epithelial cells[J].Infect Immun,2014,82(2):509-521.
[11] ZHOU M,ZHU J,YU H,et al.Investigation into in vitro and in vivo models using intestinal epithelial IPEC-J2 cells and Caenorhabditis elegans for selecting probiotic candidates to control porcine enterotoxigenic Escherichia coli[J].J Appl Microbiol,2014,117(1):217-226.
[12] SPITZER F,SPEISER S,VAHJEN W,et al.Effect of different feed ingredients and additives on IPEC-J2 cells challenged with an enterotoxigenic Escherichia coli strain[J].Cytotechnology,2016,68(4):1463-1471.
[13] KARIMI S,JONSSON H,LUNDH T,et al.Lactobacillus reuteri strains protect epithelial barrier integrity of IPEC-J2 monolayers from the detrimental effect of enterotoxigenic Escherichia coli[J].Physiol Rep,2018,6(2):e13514.
[14] KERN M,GüNZEL D,ASCHENBACH J R,et al.Altered cytokine expression and barrier properties after in vitro infection of porcine epithelial cells with enterotoxigenic Escherichia coli and probiotic Enterococcus faecium[J].Mediators Inflamm,2017,2017:2748192.
[15] SONG X Y,HE X,LI X X,et al.The roles and functional mechanisms of interleukin-17 family cytokines in mucosal immunity[J].Cell Mol Immunol,2016,13(4):418-431.
[16] LEE J S,TATO C M,JOYCE-SHAIKH B,et al.Interleukin-23-independent IL-17 production regulates intestinal epithelial permeability[J].Immunity,2015,43(4):727-738.
[17] MAXWELL J R,ZHANG Y,BROWN W A,et al.Differential roles for interleukin-23 and interleukin-17 in intestinal immunoregulation[J].Immunity,2015,43(4):739-750.
[18] IWAKURA Y,ISHIGAME H,SAIJO S,et al.Functional specialization of interleukin-17 family members[J].Immunity,2011,34(2):149-162.
[19] RAMIREZ-CARROZZI V,SAMBANDAM A,LUIS E,et al.IL-17C regulates the innate immune function of epithelial cells in an autocrine manner[J].Nat Immunol,2011,12(12):1159-1166.
[20] SONG X Y,ZHU S,SHI P Q,et al.IL-17RE is the functional receptor for IL-17C and mediates mucosal immunity to infection with intestinal pathogens[J].Nat Immunol,2011,12(12):1151-1158.
[21] HUANG J L,MENG S H,HONG S J,et al.IL-17C is required for lethal inflammation during systemic fungal infection[J].Cell Mol Immunol,2016,13(4):474-483.
[22] LOOS M,GEENS M,SCHAUVLIEGE S,et al.Role of heat-stable enterotoxins in the induction of early immune responses in piglets after infection with enterotoxigenic Escherichia coli[J].PLoS One,2012,7(7):e41041.
[23] LUO Y,VAN NGUYEN U,DE LA FE RODRIGUEZ P Y,et al.F4+ ETEC infection and oral immunization with F4 fimbriae elicits an IL-17-dominated immune response[J].Vet Res,2015,46:121.
[24] JIAO W,MA Q,LV X,et al.Gene expression and tissue distribution of β-defensins in Chinese Min pigs and Landrace pigs[J].Czech J Anim Sci,2017,62(4):178-183.
[25] PASTERNAK J A,AIYER V I A,HAMONIC G,et al.Molecular and physiological effects on the small intestine of weaner pigs following feeding with deoxynivalenol-contaminated feed[J].Toxins (Basel),2018,10(1):40.
[26] BRUEL T,GUIBON R,MELO S,et al.Epithelial induction of porcine suppressor of cytokine signaling 2 (SOCS2) gene expression in response to Entamoeba histolytica[J].Dev Comp Immunol,2010,34(5):562-571.
[27] VON DER HARDT K,KANDLER M A,FINK L,et al.High frequency oscillatory ventilation suppresses inflammatory response in lung tissue andmicrodissected alveolar macrophages in surfactant depleted piglets[J].Pediatr Res,2004,55(2):339-346.
[28] MELKEBEEK V,SONCK E,VERDONCK F,et al.Optimized FaeG expression and a thermolabile enterotoxin DNA adjuvant enhance priming of an intestinal immune response by an FaeG DNA vaccine in pigs[J].Clin Vaccine Immunol,2007,14(1):28-35.
[29] STARNES T,BROXMEYER H E,ROBERTSON M J,et al.Cutting edge:IL-17D,a novel member of the IL-17 family,stimulates cytokine production and inhibits hemopoiesis[J].J Immunol,2002,169(2):642-646.
[30] FRIEDRICH M,DIEGELMANN J,SCHAUBER J,et al.Intestinal neuroendocrine cells and goblet cells are mediators of IL-17A-amplified epithelial IL-17C production in human inflammatory bowel disease[J].Mucosal Immunol,2015,8(4):943-958.
[31] IM E,JUNG J,RHEE S H.Toll-like receptor 5 engagement induces interleukin-17C expression in intestinal epithelial cells[J].J Interf Cytok Res,2012,32(12):583-591.
[32] PFEIFER P,VOSS M,WONNENBERG B,et al.IL-17C is a mediator of respiratory epithelial innate immune response[J].Am J Respir Cell Mol Biol,2013,48(4):415-421.
[33] KUSAGAYA H,FUJISAWA T,YAMANAKA K,et al.Toll-like receptor-mediated airway IL-17C enhances epithelial host defense in an autocrine/paracrine manner[J].Am J Respir Cell Mol Biol,2014,50(1):30-39.
[34] REYNOLDS J M,ANGKASEKWINAI P,DONG C.IL-17 family member cytokines:regulation and function in innate immunity[J].Cytok Growth Factor Rev,2010,21(6):413-423.
[35] ZAPH C,DU Y R,SAENZ S A,et al.Commensal-dependent expression of IL-25 regulates the IL-23-IL-17 axis in the intestine[J].J Exp Med,2008,205(10):2191-2198.
[36] SAWA S,LOCHNER M,SATOH-TAKAYAMA N,et al.RORγt+ innate lymphoid cells regulate intestinal homeostasis by integrating negative signals from the symbiotic microbiota[J].Nat Immunol,2011,12(4):320-326.