Microbes and Infection 16 (2014) 954e961 www.elsevier.com/locate/micinf

Original article

Mouse intestinal innate immune responses altered by enterotoxigenic Escherichia coli (ETEC) infection Wenkai Ren a,1, Jie Yin a,1, Jielin Duan a, Gang Liu a,*, Xiaoping Zhu b, Shuai Chen a, Tiejun Li a, Shengping Wang a, Yulong Tang a, Philip R. Hardwidge c a

Laboratory of Animal Nutrition and Health and Key Laboratory of Agro-Ecology, Institute of Subtropical Agriculture, The Chinese Academy of Sciences, Changsha, Hunan 410125, PR China b Division of Immunology, Virginia-Maryland Regional College of Veterinary Medicine, Maryland Pathogen Research Institute, University of Maryland, College Park, MD 20742, USA c Department of Diagnostic Medicine/Pathobiology, Kansas State University, Manhattan, KS 66506, USA Received 17 July 2014; accepted 18 September 2014 Available online 1 October 2014

Abstract Enterotoxigenic Escherichia coli (ETEC) is an important cause of human and porcine morbidity and mortality. The current study was conducted to identify intestinal immunity that is altered in a mouse model of ETEC infection. Innate immune responses and inflammation were analyzed. The activation of signal transduction pathways, including toll like receptor 4 (TLR-4)-nuclear factor kappa B (NF-kB) and mitogenactivated protein kinases (MAPK), was analyzed using immunoblotting and PCR array analyses. We found that ETEC infection promoted the expression of pro-inflammatory cytokines through the activation of the NF-kB and MAPK pathways. Meanwhile, ETEC infection affected sIgA transportation and Paneth cell function. These data improve our understanding of how ETEC causes disease in animals. © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Keywords: ETEC; Paneth cell; Innate immunity; NF-kB; MAPK

1. Introduction Enterotoxigenic Escherichia coli (ETEC) causes travelers' diarrhea [1] and is a leading cause of infectious diarrhea in children from developing nations [2]. Porcine ETEC strains also cause severe secretory diarrhea in pigs. ETEC produces several virulence factors, including colonization factors (adhesins), and/or the heat-labile (LT) and heat-stable (ST) toxins [3]. Colonization factors are responsible for bacterial adhesion to the intestinal mucosa. LT and ST induce the excessive production of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), respectively. Elevated cAMP and cGMP levels activate protein * Corresponding author. E-mail address: [email protected] (G. Liu). 1 These authors contributed equally.

kinase A (PKA)- and cGMP-dependent protein kinase II regulated pathways, ultimately leading to phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR), chloride secretion, and diarrhea [3]. Although these mechanisms were characterized long ago, other aspects of the ETEC-host interaction have been elucidated only recently. Enteric pathogens and their enterotoxins disrupt various intestinal functions, such as tight junctions (TJ) [4,5], aquaporins [6], and the inflammatory response [7], which can indirectly contribute to diarrhea [8]. While several studies have characterized these aspects of ETEC infection [9e11], the details remain elusive. It is widely known that ETEC infection induces pro-inflammatory responses in porcine intestinal epithelial IPEC-1 cells and IPEC J2 cells, associating with mitogen-activated protein kinases (MAPK) and NF-kB pathway [12,13]. Considering numerous genes involved in MAPK or NF-kB pathway, however, it remains to

http://dx.doi.org/10.1016/j.micinf.2014.09.005 1286-4579/© 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

W. Ren et al. / Microbes and Infection 16 (2014) 954e961

know which gene is targeted by ETEC infection to modulate MAPK or NF-kB pathway. Secretory IgA (SIgA), mucus layer produced from goblet cell, and antimicrobial peptides released from Paneth cells play indispensable functions in mucosal defense and homeostasis [14]. However, we are lacking of the data about the effects of ETEC on the intestinal mucosal defense, especially on Paneth cell. Thus, this study was conducted to explore the effects of ETEC infection on MAPK or NF-kB pathway with PCR array, which can detect 84 genes in each pathway at same time. Meanwhile, this study was conducted to test our hypothesis that ETEC infection affects the intestinal mucosal defense using a mouse model of ETEC infection. 2. Materials and methods 2.1. Bacterial strains and antibodies This study used ETEC strain SEC470 (serotype O4; F18; STa, STb, LT, SLT-IIe), which was isolated from a 39-day-old piglet with diarrhea in Jingxi Province, China [15,16]. Antibodies against Jnk (Sc-571), p-Jnk (Sc-12882), neonatal Fc receptor (FcRn) (Sc-66892) and TLR4 (Sc-10741) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, Texas, USA). Antibodies against ERK1/2 (CST 4695), p-ERK1/2 (CST 4370), p38 (CST 8690) and p-p38 (CST 4511) were purchased from Cell Signaling Technology (Danvers, MA, USA). 2.2. Animal model This study was performed according to the guidelines of the Laboratory Animal Ethical Commission of the Chinese Academy of Sciences. The ETEC infection model was established according to the method from Allen et al. [17]. Female ICR (Institute for Cancer Research) mice were purchased from SLAC Laboratory Animal Central (Changsha, China). At six weeks of age, mice were randomly assigned into either ETEC infection or control groups and inoculated by oral gavage with either 1  109 CFUs SEC470 or with sterile phosphate-buffered saline (PBS). Before the oral infection, all mice were treated according to the description from Allen et al. [17]. Briefly, all mice received streptomycin (5 g/liter) in their drinking water from 48 to 12 h prior to ETEC inoculation to eradicate normal resident bacterial flora in the intestinal tract. The streptomycin-treated water also contained fructose (6.7%) (Shanghai Kayon Biological Technology Co. Ltd, Shanghai, China) to encourage water consumption prior to ETEC inoculation. Food was withdrawn for 12 h prior to inoculation, and fructose-treated water was replaced with unsweetened, sterile water 12 h prior to ETEC inoculation. Cimetidine (50 mg/kg; SigmaeAldrich) was administered intraperitoneally to all mice 3 h prior to inoculation with ETEC to reduce the effect of stomach acidity on the bacterial organisms. At 24 h post infection, all active mice were sacrificed to collect the jejunum and ileum.

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2.3. Morphological analyses For light microscopic observation, jejunum tissues were fixed with 10% formalin PBS at 4  C, dehydrated in a graded series of ethanol, and then embedded in paraffin wax. Five mm sections of tissues were mounted on slides, dewaxed, hydrated, and then stained with Hematoxylin & Eosin (H&E). 2.4. Immunoblotting Equal amounts of proteins obtained from jejunum were separated by SDS-PAGE, transferred to PVDF membranes (Millipore, MA, USA), and blocked with 5% non-fat milk in Tris-Tween buffered saline buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween-20) for 3 h. Primary antibodies were incubated overnight at 4  C and HRP-conjugated secondary antibodies were incubated for 1 h at room temperature before development and analysis using Alpha Imager 2200 software (Alpha Innotech Corporation, CA, USA). Signal intensity was digitally quantified and normalized to actin protein abundance. 2.5. Quantitative real-time RT-PCR Total RNA was isolated from jejunum or ileum samples with TRIZOL regent (Invitrogen, USA) and then treated with DNase I (Invitrogen, USA) according to the manufacturer's instructions. Primers were presented in our previous studies [18,19]. b-actin was used as an internal control to normalize target gene transcript levels. Real-time PCR was performed according to our previous study [20]. Briefly, 1.0 ml cDNA template was added to a total volume of 10.0 ml containing 5.0 ml SYBR Green mix (Takara, Dalian, China), 0.2 ml Rox Reference Dye (Takara, Dalian, China), 3.0 ml ddH2O, and 0.4 ml each of forward and reverse primers. We used the following protocol: (1) pre-denaturation (10 s at 95  C); (2) amplification and quantification (40 cycles of 5 s at 95  C, 20 s at 60  C); (3) melting (60e99  C with a heating rate of 0.1  C/ s and fluorescence measurement). The relative expression was expressed as a ratio of the target gene to the control gene using the formula 2-(DDCt), where DDCt ¼ (CtTarget-Ct®-actin)E® TEC-(CtTarget-Ct -actin)control. Relative expression was normalized and expressed as a ratio to the expression in the control group. 2.6. PCR array analysis RNA was prepared using the RNeasy Mini Kit and reverse transcribed using the RT First Strand kit (Qiagen, Germany). cDNA was analyzed using RT Profiler PCR Arrays (Qiagen, Germany) according to the manufacturer's instructions. 2.7. Statistical analyses Data were expressed as means ± the standard error of the mean (SEM). All statistical analyses were performed using SPSS 16.0 software (Chicago, IL, USA). Data were analyzed

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W. Ren et al. / Microbes and Infection 16 (2014) 954e961

using the student's t-test with a p-value

Mouse intestinal innate immune responses altered by enterotoxigenic Escherichia coli (ETEC) infection.

Enterotoxigenic Escherichia coli (ETEC) is an important cause of human and porcine morbidity and mortality. The current study was conducted to identif...
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