Porcine intestinal epithelial responses to Campylobacter infection

Campylobacter infection is estimated to cause diarrhoea in 1% of the population of developed countries every year, but our understanding of this disease has been hampered by a lack of a suitable animal model. Colostrum-deprived piglets have been suggested as models since they produce similar clinical signs to humans when infected but little information currently exists regarding the response of this species to Campylobacter at cellular and molecular level. This study shows that intestinal epithelial cells from both species respond in a similar manner to Campylobacter infection regarding invasion, induction of innate immune response and effect on barrier function.     

肠炎沙门菌SEF14菌毛与肠上皮细胞IPEC-J2和Caco-2的体外黏附作用研究

为研究肠炎沙门菌SEF14菌毛对肠上皮细胞的黏附作用,本试验利用肠炎沙门菌50336株、突变株50336△sefA、50336△sefD以及互补株50336△sefA (pBRA)、50336△sefD (pACYCD)与肠上皮细胞系细胞(IPEC-J2和Caco-2)进行了黏附作用.结果显示:上述菌株均能与IPEC-J2、Caco-2细胞进行有效黏附,并且随时间延长黏附数量有所增多,相同时间各菌株与IPEC-J2细胞的黏附数量明显多于Caco-2细胞;但细菌和细胞共感染1和4h后,肠炎沙门菌野生株、相应的突变株和互补株与IPEC-J2和Caco-2细胞黏附的数量差别很小,未到达显著差异水平(P＞0.05).结果表明:SEF14菌毛并不特异性介导肠炎沙门菌与肠上皮细胞系IPEC-J2和Caco-2的黏附作用,或者不是介导黏附作用的主要因子.     

Gene expression study of two widely used pig intestinal epithelial cell lines: IPEC-J2 and IPI-2I

The intestinal epithelial cells (IEC) play an important role in the immune system of swine, protecting against infectious and non-infectious environmental insults. The IEC participate in the innate immune response of the intestine through different mechanisms such as barrier function, mucus secretion, antibacterial peptide synthesis and participation in the cytokine/chemokine networks.Most of the current knowledge of intestinal cell functions has come from studies conducted on cell cultures generated from human cancers or from classical animal models. However, because the molecular and cellular elements of the immune system have been selected over evolutionary time in response to the species-specific environment, models of immune function based on mouse and human need to be applied cautiously in pig. Few models of swine small intestine epithelium exist and these are poorly characterised. In the present study we characterised the basal expression of epithelial and immune-related genes of two pig small intestine cell lines, IPEC-J2 and IPI-2I, under different culture conditions. These data represent essential background information for future studies on pig-intestinal pathogen interactions.     

Porcine circovirus 2 inclusion bodies in pulmonary and renal epithelial cells

Porcine circovirus 2 (PCV2) is the cause of postweaning multisystemic wasting syndrome (PMWS). The most common lesions of PMWS are lymphohistiocytic to granulomatous lymphadenitis, interstitial pneumonia and interstitial nephritis, with intracytoplasmic amphophilic botryoid inclusion bodies in macrophages. In addition to these typical changes, intracytoplasmic botryoid inclusion bodies were observed in bronchial, bronchial glandular, and renal tubular epithelium of several pigs from 4 different farms in Western and Eastern Canada. PCV2 inclusion bodies were demonstrated to be located in the cytoplasm of epithelial cells by immunohistochemical staining for PCV2 and cytokeratin antigens and by ultrastructural demonstration of viral particles in the inclusion bodies within renal tubular epithelium.     

Innate immune activation of swine intestinal epithelial cell lines (IPEC-J2 and IPI-2I) in response to LPS from Salmonella typhimurium

The innate immune system has the basic function of identifying and eradicating microbial invaders and alerting the adaptative immune system to their presence. In this study, the porcine intestinal innate immune response was evaluated by analysing the expression of TLRs, cytokines and chemokines in two porcine epithelial cell lines from different regions: IPEC-J2 (jejunum) and IPI-2I (ileum). Both cells lines were stimulated with 1microg of LPS from Salmonella typhimurium. RNA was collected at 30min, 1, 2, 3 and 4h after treatment. Expression of TLR-1, -2, -3, -4, -6, -8, -9, -10, TNF-alpha, IL-1beta, -8 and MCP-1 was quantified relative to the quantity of Cyclophilin-A mRNA using real-time quantitative PCR (RTQ-PCR). The results obtained show up differences in the gene expression between both cell lines IPEC-J2 and IPI-2I as response to LPS from S. typhimurium during the activation time, which may suggest an in vivo variability in the innate immune response against pathogens in different regions of the host's gut.     

Cytotoxicity and metabolic stress induced by deoxynivalenol in the porcine intestinal IPEC-J2 cell line

The digestive tract is a target for the Fusarium toxin deoxynivalenol (DON), a major cereal grain contaminant of animal and public health concern. Toxic effects of DON range from diarrhoea, vomiting and gastrointestinal inflammation to necrosis of several tissues. Following ingestion of contaminated food or feed, intestinal epithelial cells are exposed to a high concentration of ingested DON, potentially affecting intestinal functions. Pigs are considered to be the species most sensitive to DON toxicity. However, only few studies directly evaluated DON effects on porcine intestinal epithelial cells. Therefore, we used the porcine intestinal cell line (IPEC-J2) to assess short-term effects of DON on functional characteristics of the intestinal epithelial cells. The cytotoxic effect of DON on IPEC-J2 cells was evaluated by measuring the count of living cells and the activity of lactate dehydrogenase (LDH) released in the culture media at a DON concentration range from 0, 0.5, 2.5 and 10 μm. We demonstrated that DON at concentrations of 2.5 and 10 μm decreased significantly (p < 0.001) the cell count in a dose-dependent manner. At a concentration of 10 μm, DON caused cell damage, including rounding of cells, autolysis and cell loss from the monolayer. The mycotoxin, DON, increased LDH release into the culture medium compared with the control value. The alterations of LDH showed a good agreement with the decrease in cell count. Deoxynivalenol decreased the l-lactate concentration in the fluid supernatant of IPEC-J2 cells at 2.5 μm (p < 0.05) with a maximal effect at 10 μm of DON. To determine whether the altered lactate production may be linked to alterations of energy balance, we measured cellular ATP levels in IPEC-J2 cells. A significant decrease in ATP levels was seen at 48 h in a dose-dependent manner. It could be demonstrated that DON has a distinct cytotoxic effect on IPEC-J2 cells.     

Campylobacter-induced interleukin-8 responses in human intestinal epithelial cells and primary intestinal chick cells

Campylobacter (C.) jejuni and C. coli can cause gastrointestinal disorders in humans characterized by acute inflammation. Inflammatory signals are initiated during interaction between these pathogens and human intestinal cells, but nothing is known about the stimulation of avian intestinal cells by Campylobacter. Interleukin-8 (IL-8) as a proinflammatory chemokine plays an important role in mobilizing cellular defence mechanism. IL-8 mRNA expression in both human intestinal cells (INT 407) and primary intestinal chick cells (PIC) was determined by quantitative real-time RT-PCR. The secretion of IL-8 protein by INT407 was measured using ELISA. Although C. jejuni and C. coli are considered to be harmless commensals in the gut of birds, the avian Campylobacter isolates investigated were able to induce the proinflammatory IL-8 in PIC as well as in INT407. In an in vitro system, C. jejuni as well as C. coli were able to induce IL-8 mRNA in PIC. Relation between the virulence properties like toxin production, the ability to invade and to survive in Caco-2 cells and the level of IL-8 mRNA produced by INT 407 and PIC after infection with Campylobacter strains was also investigated.     

乳酸锌对猪空肠上皮细胞IPEC-J2增殖及相关调控基因mRNA表达的影响

旨在探讨乳酸锌对猪空肠上皮细胞增殖及相关调控基因ZnT2、DMT1、IREG-1、MT1和ZIP4 mRNA表达的影响.用乳酸锌的锌浓度分别为50、100、150、200 mg·L-1的培养基培养IPEC-J2细胞,采用比色法测定分析细胞增殖变化;用实时荧光定量RT-PCR方法检测Zn T2、DMT1、IREG-1、MT1及ZIP4 mRNA表达,以TBPmRNA的表达水平作为内参对照.在细胞培养前36 h,乳酸锌对IPEC-J2细胞基本没有影响,随锌浓度递增细胞增殖幅度升高;乳酸锌处理IPEC-J2细胞后,Zn T2、DMT1、IREG-1及MT1 mRNA表达随锌浓度增高而升高,ZIP4 mRNA表达则随锌浓度增高而降低.添加乳酸锌可以促进IPEC-J2细胞增殖,上调ZnT2、DMT1、IREG-1、 MT1 mRNA表达,下调ZIP4 mRNA表达.     

Effects of daidzein on antioxidant capacity in weaned pigs and IPEC-J2cells

Our previous study found that soybean isoflavones in soybean meal play an important role in improving growth performance and antioxidant capacity in pigs. However, it is still unknown whether long-term supplementation with daidzein, an active molecule deglycosylated from daidzin, in a corn-soybean meal diet can enhance growth performance in pigs. Thus, in the present study, an animal trial was carried out to investigate the effects of dietary supplementation with daidzein on the growth performan...     

猪METTL3基因表达水平与DON诱导IPEC-J2细胞损伤的关系

旨在初步探究猪m6A甲基化酶METTL3基因表达水平与脱氧雪腐镰刀菌烯醇(deoxynivalenol,DON)诱导猪小肠上皮细胞(IPEC-J2)损伤的关系.本研究构建了稳定干扰METTL3基因表达水平的IPEC-J2细胞系,用1 μg·mL-1 DON诱导METTL3干扰组和对照组猪肠上皮细胞48 h,通过实时荧光...     

Reduced differentiation of intestinal epithelial cells in wasting marmoset syndrome

Wasting marmoset syndrome (WMS) is a serious disease in captive common marmoset (Callithrix jacchus) colonies. Because of the high mortality rates, elucidation of the underlying mechanisms is essential. In this study, we compared the histopathology, the number of each epithelial cell in the jejunum and colon, and the expression patterns of some molecular markers between healthy and WMS-affected marmosets. Atrophy of villi in the jejunum and mononuclear cell infiltration in the lamina propria were observed in the intestinal tract of WMS-affected marmosets. Although the numbers of transient amplifying cells and tuft cells were increased, the number of goblet cells was obviously decreased in the jejunum and colon of WMS-affected marmosets compared to healthy marmosets. In addition, the number of enterocytes in the jejunum was decreased in WMS animals. There was no apparent difference in the numbers of stem cells, enteroendocrine cells, or Paneth cells. The expression of β-catenin and Tcf7l2 was increased in WMS, and the co-existence of β-catenin and Tcf7l2/Cyclin D1 was observed around the crypts in WMS-affected marmosets. These findings suggest that cell proliferation continues, but cell differentiation is halted in the intestinal tract due to the enhanced β-catenin/Tcf7l2/Cyclin D1signaling pathway in WMS, which results in malfunction of the villus and mucosa.     

大肠杆菌侵染猪肠上皮细胞IPEC-J2后TAP1基因表达量的变化分析

为了在细胞水平上探究抗原处理相关转运体1(TAP1)基因与仔猪大肠杆菌性腹泻间的关系以及在机体抗大肠杆菌侵染过程中是否发挥了作用,选用3种主要产肠毒素大肠杆菌(F18ab,F18ac和K88ac)刺激离体培养的猪肠上皮细胞(IPEC-J2),用实时荧光定量PCR检测3种大肠杆菌刺激后IPEC-J2细胞中TAP1基因表达量的变化。结果显示:IPEC-J2细胞经过3种大肠杆菌刺激后,TAP1基因在细胞中的表达量均显著高于对照组(P0.05),差异倍数分别是对照组的1.7,1.8和1.5倍;3种不同的大肠杆菌刺激,TAP1基因在IPEC-J2细胞中的表达量不存在显著差异(P0.05)。通过分析3种大肠杆菌侵染离体培养的猪肠上皮细胞后TAP1基因表达量的变化,在细胞水平上验证了TAP1基因的表达对仔猪抗细菌性腹泻具有一定的作用,为TAP1基因作为机体免疫调控的相关基因提供了一定的理论依据。     

Stanniocalcin-1 protects bovine intestinal epithelial cells from oxidative stress-induced damage

Chronic enteritis can produce an excess of reactive oxygen species resulting in cellular damage. Stanniocalcin-1(STC-1) reportedly possesses anti-oxidative activity, the aim of this study was to define more clearly the direct contribution of STC-1 to anti-oxidative stress in cattle. In this study, primary intestinal epithelial cells (IECs) were exposed to hydrogen peroxide (H₂O₂) for different time intervals to mimic chronic enteritis-induced cellular damage. Prior to treatment with 200 µM H₂O₂, the cells were transfected with a recombinant plasmid for 48 h to over-express STC-1. Acridine orange/ethidium bromide (AO/EB) double staining and trypan blue exclusion assays were then performed to measure cell viability and apoptosis of the cells, respectively. The expression of STC-1 and apoptosis-related proteins in the cells was monitored by real-time PCR and Western blotting. The results indicated that both STC-1 mRNA and protein expression levels positively correlated with the duration of H₂O₂ treatment. H₂O₂ damaged the bovine IECs in a time-dependent manner, and this effect was attenuated by STC-1 over-expression. Furthermore, over-expression of STC-1 up-regulated Bcl-2 protein expression and slightly down-regulated caspase-3 production in the damaged cells. Findings from this study suggested that STC-1 plays a protective role in intestinal cells through an antioxidant mechanism.     

Ghrelin对猪小肠上皮细胞增殖的影响

以体外分离培养的猪小肠上皮细胞为试验材料,通过分别添加1×10-5、1×10-6、1×10-7、1×10-8、1×10-9和1×10-10mol/L的Ghrelin液,来测定Ghrelin对猪小肠上皮细胞增殖的影响。试验结果表明,Ghrelin的浓度在1×10-7-1×10-6mol/L时能够显著促进体外培养的猪小肠上皮细胞的增殖(P<0.05),且在刺激后第5天达到增殖高峰。     

乳酸杆菌黏附鸡消化道上皮细胞超微结构的观察

用扫描电镜观察健康鸡嗉囊黏膜表面正常菌群与黏膜细胞结合的状态,结果发现,嗉囊黏膜表面覆盖着大量的乳酸杆菌,将乳酸杆菌放大可见,其表面以伪足样丝状物与嗉囊黏膜细胞紧密结合。用透射电镜观察乳酸杆菌黏附消化道上皮细胞(CaCo-2cell)的状态发现,上皮细胞与乳酸杆菌相结合后其结构没有变化,菌体结构也完好无损。另外,用透射电镜对乳酸杆菌表面黏附物质被提取前后的形态变化进行了观察,结果表明,细菌被提取蛋白后,细菌细胞壁变薄、透明、凹凸不平,而未提取蛋白的细菌表面结构正常,表明乳酸杆菌表面存在着某种蛋白物质,这种物质就是上皮细胞发生结合的黏附素蛋白。     

Porcine intestinal epithelial cell lines as a new in vitro model for studying adherence and pathogenesis of enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) infections result in large economic losses in the swine industry worldwide. The organism causes diarrhea by adhering to and colonizing enterocytes in the small intestines. While much progress has been made in understanding the pathogenesis of ETEC, no homologous intestinal epithelial cultures suitable for studying porcine ETEC pathogenesis have been described prior to this report. In the current study, we investigated the adherence of various porcine ETEC strains to two porcine (IPEC-1 and IPEC-J2) and one human (INT-407) small intestinal epithelial cell lines. Each cell line was assessed for its ability to support the adherence of E. coli expressing fimbrial adhesins K88ab, K88ac, K88ad, K99, F41, 987P, and F18. Wild-type ETEC expressing K88ab, K88ac, and K88ad efficiently bound to both IPEC-1 and IPEC-J2 cells. An ETEC strain expressing both K99 and F41 bound heavily to both porcine cell lines but an E. coli strain expressing only K99 bound very poorly to these cells. E. coli expressing F18 adhesin strongly bound to IPEC-1 cells but did not adhere to IPEC-J2 cells. The E. coli strains G58-1 and 711 which express no fimbrial adhesins and those that express 987P fimbriae failed to bind to either porcine cell line. Only strains B41 and K12:K99 bound in abundance to INT-407 cells. The binding of porcine ETEC to IPEC-J2, IPEC-1 and INT-407 with varying affinities, together with lack of binding of 987P ETEC and non-fimbriated E. coli strains, suggests strain-specific E. coli binding to these cell lines. These findings suggest the potential usefulness of porcine intestinal cell lines for studying ETEC pathogenesis.     

鸡小肠上皮细胞分离培养及NLRP3在该细胞中的表达

为了探明NLRP3在鸡小肠上皮细胞中表达情况,为鸡的NLRP3基因在小肠细胞中的功能研究奠定基础。采用18胚龄的鸡胚,分离培养鸡的小肠上皮细胞(intestinal epithelial cells,IEC);采用免疫细胞化学(immunocytochemistry,ICC)和反转录PCR(RT-PCR)方法,检测NLRP3基因在鸡小肠上皮细胞中的表达。结果表明:分离培养出活性较强的IEC;ICC检测显示获得的小肠上皮细胞表面NLRP3抗原阳性,RT-PCR法可扩增出520 bp的目的片段。表明成功分离培养出鸡小肠上皮细胞,并证明鸡NLRP3在鸡小肠上皮细胞中表达。