Characterization of Butyrate Uptake by Nontransformed Intestinal Epithelial Cell Lines

Butyrate (BT) is one of the main end products of anaerobic bacterial fermentation of dietary fiber within the human colon. Among its recognized effects, BT inhibits colon carcinogenesis. Our aim was to characterize uptake of BT by two nontransformed intestinal epithelial cell lines: rat small intestinal epithelial (IEC-6) and fetal human colonic epithelial (FHC) cells. Uptake of ¹⁴C-BT by IEC-6 cells was (1) time- and concentration-dependent; (2) pH-dependent; (3) Na⁺-, Cl⁻- and energy-dependent; (4) inhibited by BT structural analogues; (5) sensitive to monocarboxylate transporter 1 (MCT1) inhibitors; and (6) insensitive to DIDS and amiloride. IEC-6 cells express both MCT1 and Na⁺-coupled monocarboxylate transporter 1 (SMCT1) mRNA. We conclude that ¹⁴C-BT uptake by IEC-6 cells mainly involves MCT1, with a small contribution of SMCT1. Acute exposure to ethanol, acetaldehyde, indomethacin, resveratrol and quercetin reduced ¹⁴C-BT uptake. Chronic exposure to resveratrol and quercetin reduced ¹⁴C-BT uptake but had no effect on either MCT1 or SMCT1 mRNA levels. Uptake of ¹⁴C-BT by FHC cells was time- and concentration-dependent but pH-, Na⁺-, Cl⁻- and energy-independent and insensitive to BT structural analogues and MCT1 inhibitors. Although MCT1 (but not SMCT1) mRNA expression was found in FHC cells, the characteristics of ¹⁴C-BT uptake by FHC cells did not support either MCT1 or SMCT1 involvement. In conclusion, uptake characteristics of ¹⁴C-BT differ between IEC-6 and FHC cells. IEC-6 cells demonstrate MCT1- and SMCT1-mediated transport, while FHC cells do not.     

Gap Junctional Communication between Murine Macrophages and Intestinal Epithelial Cell Lines

In intestinal inflammation, inflammatory cells infiltrate the submucosa and are found juxtaposed to intestinal epithelial cell (IEC) basolateral membranes and may directly regulate IEC function. In this study we determined whether macrophage (M), P388D1 and J774A.1, are coupled by gap junctions to IEC lines, Mode-K and IEC6. Using flow cytometric analysis, we show bi-directional transfer of the fluorescent dye, calcein (700 Da) between IEC and M resulting in a 3.5–20-fold increase in recipient cell fluorescence. Homocellular and heterocellular dye transfer between M and/or IEC was detected in cocultures of P388D1, J774A.1, Mode-K, IEC6 and CMT93. However, transfer between P388D1 and Mode-K was asymmetrical in that transfer from P388D1 to Mode-K was always more efficient than transfer from Mode-K to P388D1. Dye transfer was strictly dependent on IEC-M? adhesion which in turn was dependent on the polarity of IEC adhesion molecule expression. Both calcein dye transfer and adhesion were inhibited by the addition of heptanol to cocultures. Furthermore we demonstrate both IEC homocellular, and M?-IEC heterocellular propagation of calcium waves in response to mechanical stimulation, typical of gap junctional communication. Finally, areas of close membrane apposition were seen in electron micrographs of IEC-M? cocultures, suggestive of gap junction formation. These data indicate that IEC and MM? are coupled by gap junctions suggesting that gap junctional communication may provide a means by which inflammatory cells might regulate IEC function.     

New Model for Studying the Migration of Immune Cells into Intestinal Epithelial Cell Monolayers

A novel cell culture system was constructed to analyze the direct interaction between intestinal epithelial cells and immune cells. Human intestinal epithelial Caco-2 cells were monolayer-cultured on the under side of a permeable membrane (12 μm pore size) in a Millicell insert. Integrated monolayers of Caco-2 cells had formed after 12 days of culture. Human monocyte/macrophage-like THP-1 cells were then added to the upper chamber of the insert, and their migration into the Caco-2 cell monolayers was observed by confocal laser scanning microscopy, after staining the cells with specific antibodies. When MCP-1, a β-chemokine, was added to the apical side of the monolayer, a greater number of THP-1 cells migrated into the Caco-2 cell monolayers. This cell culture system will be useful for studying the behavior of macrophages in the intestinal epithelial cell monolayers at the initial stage of an intestinal immune reaction.     

Evidence For Cell Generation Controlled Proliferation In the Small Intestinal Crypt

Abstract. In this paper we discuss the hypothesis that cell proliferation is controlled by the number of generations after leaving an 'eternal' stem cell. the theory is based on a simulation of the kinetic behaviour of cells in the intestinal crypts. There is evidence of three, four and five generations of cells which are allowed to enter mitosis in the lower and upper part of the normal intestinal tract, and in some disease states, respectively. We suggest an internal proliferation control: some kind of knowledge that cells carry from generation to generation. It is an open question what sets and changes the generation counter: internal genetic information or external influences such as growth factors or chalones.
The geometric shape of the epithelial tissue in the intestinal tract can be understood as the steady state of a highly dynamic process. Age and death are determined from the beginning; cell-cell interaction or communication is not necessary and can be neglected. Our theory will be illustrated using the intestinal crypts as they are easily accessible, of a simple structure and completely described in the literature.     

Mycoplasma hyorhinis-Contaminated Cell Lines Activate Primary Innate Immune Cells via a Protease-Sensitive Factor

Mycoplasma are a frequent and occult contaminant of cell cultures, whereby these prokaryotic organisms can modify many aspects of cell physiology, rendering experiments that are conducted with such contaminated cells problematic. Chronic Mycoplasma contamination in human monocytic cells lines has been associated with suppressed Toll-like receptor (TLR) function. In contrast, we show here that components derived from a Mycoplasma hyorhinis-infected cell line can activate innate immunity in non-infected primary immune cells. Release of pro-inflammatory cytokines such as IL-6 by dendritic cells in response to Mycoplasma hyorhinis-infected cell components was critically dependent on the adapter protein MyD88 but only partially on TLR2. Unlike canonical TLR2 signaling that is triggered in response to the detection of Mycoplasma infection, innate immune activation by components of Mycoplasma-infected cells was inhibited by chloroquine treatment and sensitive to protease treatment. We further show that in plasmacytoid dendritic cells, soluble factors from Mycoplasma hyorhinis-infected cells induce the production of large amounts of IFN-α. We conclude that Mycoplasma hyorhinis-infected cell lines release protein factors that can potently activate co-cultured innate immune cells via a previously unrecognized mechanism, thus limiting the validity of such co-culture experiments.     

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.     

肝脏固有免疫系统对肠源性感染的免疫应答与免疫耐受机制

肝特殊的解剖结构及生理特征使其成为暴露肠源性抗原的主要器官。由于肝具有独特的固有免疫系统,在正常情况下,肝分布多种致耐受的抗原提呈细胞,对持续性表达或递呈于肝的肠源性抗原物质,诱发针对该抗原的系统性免疫耐受,避免肝受到不必要的免疫损伤。当炎症发生及肝脏固有免疫系统活化时,则通过免疫效应细胞及免疫效应因子对肠源性病原体发挥强烈地免疫应答以控制感染。该过程形成机制的研究对肝功能的理解及肝性疾病的预防与治疗至关重要。本文就肝固有免疫系统对肠源性感染的免疫应答与免疫耐受形成机制作一综述。     

Innate Immune Responses of Pulmonary Epithelial Cells to Burkholderia pseudomallei Infection

Background

Burkholderia pseudomallei, a facultative intracellular pathogen, causes systemic infection in humans with high mortality especially when infection occurs through an infectious aerosol. Previous studies indicated that the epithelial cells in the lung are an active participant in host immunity. In this study, we aimed to investigate the innate immune responses of lung epithelial cells against B. pseudomallei.

Methodology and Principal Findings

Using a murine lung epithelial cell line, primary lung epithelial cells and an inhalational murine infection model, we characterized the types of innate immunity proteins and peptides produced upon B. pseudomallei infection. Among a wide panel of immune components studied, increased levels of major pro-inflammatory cytokines IL-6 and TNFα, chemokine MCP-1, and up-regulation of secretory leukocyte protease inhibitor (SLPI) and chemokine (C-C motif) ligand 20 (CCL20) were observed. Inhibition assays using specific inhibitors suggested that NF-κB and p38 MAPK pathways were responsible for these B. pseudomallei-induced antimicrobial peptides.

Conclusions

Our findings indicate that the respiratory epithelial cells, which form the majority of the cells lining the epithelial tract and the lung, have important roles in the innate immune response against B. pseudomallei infection.     

Segmented Filamentous Bacteria Are Indigenous Intestinal Bacteria That Activate Intraepithelial Lymphocytes and Induce MHC Class II Molecules and Fucosyl Asialo GM1 Glycolipids on the Small Intestinal Epithelial Cells in the Ex-Germ-Free Mouse

In ex-germ-free mice conventionalized by association with fecal microorganisms, the induction of major histocompatibility complex class II molecules and fucosylation of asialo GM1 glycolipid occur in the small intestinal epithelial cells (IEC). The intestinal intraepithelial lymphocytes (IEL), especially αβ T-cell receptor-bearing ones, also remarkably expand and show cytolytic activity. In this study, we investigated the immunological and physiological characteristics of the small intestine induced by a kind of indigenous bacteria of the small intestine, segmented filamentous bacteria (SFB), among chloroform-resistant intestinal bacteria. Monoassociation of SFB with germ-free mice was confirmed by the determination of the base sequences of polymerase chain reaction products of 16S rRNA genes of the fecal bacteria of these mice and in situ hybridization using fluorescein-labeled probes based on them. SFB increased the number of αβTCR-bearing IEL and induced Thy-1 expression and cytolytic activity of IEL. The induction of MHC class II molecules and fucosyl asialo GM1 glycolipids and the increases in the mitotic activity and the ratio of the number of columnar cells to those of goblet cells also occurred in the small intestinal epithelial cells on monoassociation of these bacteria. SFB are important indigenous bacteria for the development of the mucosal architecture and immune system in the small intestine, at least in mice.     

Use of A Mouse Chimaeric Model to Study Cell Migration Patterns In the Small Intestinal Epithelium

The pattern of cells migration in the small intestinal epithelia of a RIII/?ro C57BL/6J mouse aggregation chimaera is demonstrated using Dolichos biflorus agglutinin-peroxidase (DBA) conjugate as a strain-specific marker. Using serial tangential sections of heterogeneously stained villi and plotting the distribution of labelled/unlabelled cells with a drawing tube, and by three-dimensional reconstruction with the aid of computer graphics, we show the migration pathway to be in tight cohorts of similar DBA-peroxidase staining type, which move upwards in straight lines. There is little cell mixing either on the villus or along the crypt-villus junctions. Our observations also show for the first time that a single crypt can feed cells to more than one villus. They also suggest that either cell loss is not confined to the villus tips but can take place from the villus sides, or that there is marked asynchrony of cell production between crypts.     

Effects of Small Intestinal Submucosa (SIS) on the Murine Innate Immune Microenvironment Induced by Heat-Killed Staphylococcus aureus

The use of biological scaffold materials for wound healing and tissue remodeling has profoundly impacted regenerative medicine and tissue engineering. The porcine-derived small intestinal submucosa (SIS) is a licensed bioscaffold material regularly used in wound and tissue repair, often in contaminated surgical fields. Complications and failures due to infection of this biomaterial have therefore been a major concern and challenge. SIS can be colonized and infected by wound-associated bacteria, particularly Staphylococcus aureus. In order to address this concern and develop novel intervention strategies, the immune microenvironment orchestrated by the combined action of S. aureus and SIS should be critically evaluated. Since the outcome of tissue remodeling is largely controlled by the local immune microenvironment, we assessed the innate immune profile in terms of cytokine/chemokine microenvironment and inflammasome-responsive genes. BALB/c mice were injected intra-peritoneally with heat-killed S. aureus in the presence or absence of SIS. Analyses of cytokines, chemokines and microarray profiling of inflammasome-related genes were done using peritoneal lavages collected 24 hours after injection. Results showed that unlike SIS, the S. aureus-SIS interactome was characterized by a Th1-biased immune profile with increased expressions of IFN-γ, IL-12 and decreased expressions of IL-4, IL-13, IL-33 and IL-6. Such modulation of the Th1/Th2 axis can greatly facilitate graft rejections. The S. aureus-SIS exposure also augmented the expressions of pro-inflammatory cytokines like IL-1β, Tnf-α, CD30L, Eotaxin and Fractalkine. This heightened inflammatory response caused by S. aureus contamination could enormously affect the biocompatibility of SIS. However, the mRNA expressions of many inflammasome-related genes like Nlrp3, Aim2, Card6 and Pycard were down-regulated by heat-killed S. aureus with or without SIS. In summary, our study explored the innate immune microenvironment induced by the combined exposure of SIS and S. aureus. These results have practical implications in developing strategies to contain infection and promote successful tissue repair.     

Glycosylation of Candida albicans Cell Wall Proteins Is Critical for Induction of Innate Immune Responses and Apoptosis of Epithelial Cells

C. albicans is one of the most common fungal pathogen of humans, causing local and superficial mucosal infections in immunocompromised individuals. Given that the key structure mediating host-C. albicans interactions is the fungal cell wall, we aimed to identify features of the cell wall inducing epithelial responses and be associated with fungal pathogenesis. We demonstrate here the importance of cell wall protein glycosylation in epithelial immune activation with a predominant role for the highly branched N-glycosylation residues. Moreover, these glycan moieties induce growth arrest and apoptosis of epithelial cells. Using an in vitro model of oral candidosis we demonstrate, that apoptosis induction by C. albicans wild-type occurs in early stage of infection and strongly depends on intact cell wall protein glycosylation. These novel findings demonstrate that glycosylation of the C. albicans cell wall proteins appears essential for modulation of epithelial immunity and apoptosis induction, both of which may promote fungal pathogenesis in vivo.     

Dose Effects of Apical versus Basolateral Zinc Supplementation on Epithelial Resistance,Viability, and Metallothionein Expression in Two Intestinal Epithelial Cell Lines

Zinc supplementation is used to reduce diarrhea incidence in piglets and it has been shown in vitro that the antisecretory effects are maximal after basolateral zinc application. To examine whether the application site and dose of zinc also influence passive ion permeability and viability, porcine (IPEC‐J2) and human (Caco‐2) intestinal epithelial cells were treated with increasing zinc concentrations (0–200 μM) at either the apical or basolateral side. Transepithelial electrical resistance and viability decreased and expression of metallothionein and the efflux zinc transporter 1 increased most prominently when zinc was added in high concentrations at the basolateral side of IPEC‐J2 cells. Zinc transporter 4, a zinc importer, was not affected. Heat shock protein 70 mRNA expression increased only after basolateral addition of 200 μM zinc in IPEC‐J2 cells. Thus, zinc can elicit toxic effects especially when added at the basolateral side, with IPEC‐J2 cells being more susceptible than Caco‐2 cells.     

在体分离小鼠小肠隐窝、绒毛上皮细胞的生化完整性研究

目的:检测低温条件下用螯合剂沉淀法分离的小鼠小肠上皮隐窝和绒毛细胞是否具有生化完整性.方法:使用螯合剂在低温(冰浴)条件下分离和富集小肠上皮绒毛和隐窝细胞;抽提DNA、RNA和总蛋白,用电泳的方法检测完整性;用Real-time PCR检测溶菌酶Lysozyme的表达以判断隐窝、绒毛细胞富集程度.结果:低温条件下分离的肠上皮隐窝、绒毛细胞形态完整;基因组DNA完整,未出现明显的DNA ladder现象;富集细胞的RNA完整;富集隐窝、绒毛细胞的蛋白未降解,两组总蛋白具有表达谱差异性;隐窝细胞富集物溶菌酶mRNA表达水平较绒毛细胞富集物高30倍以上.结论:小肠隐窝绒毛的生物学性状可在低温螯合剂沉底法分离过程中得到保存,提示此方法可以用来分析生理和创伤痛理条件下小肠上皮基因和蛋白表达改变.     

Endoplasmic Reticulum Aminopeptidase-1 Functions Regulate Key Aspects of the Innate Immune Response

Endoplasmic reticulum aminopeptidase-1 (ERAP1) is a multifunctional, ubiquitously expressed enzyme whose peptide-trimming role during antigen processing for presentation by MHC I molecules is well established, however, a role for ERAP1 in modulating global innate immune responses has not been described to date. Here we demonstrate that, relative to wild type mice, mice lacking ERAP1 exhibit exaggerated innate immune responses early during pathogen recognition, as characterized by increased activation of splenic and hepatic NK and NKT cells and enhanced production of pro-inflammatory cytokines such as IL12 and MCP1. Our data also revealed that ERAP1 is playing a critical role in NK cell development and function. We observed higher frequencies of terminally matured NK cells, as well as higher frequencies of licensed NK cells (expressing the Ly49C and Ly49I receptors) in ERAP1-KO mice, results that positively correlated with an enhanced NK activation and IFNγ production by ERAP1-KO mice challenged with pro-inflammatory stimuli. Furthermore, during pathogen recognition, ERAP1 regulates IL12 production by CD11c⁺ DCs specifically, with increases in IL12 production positively correlated with an increased phagocytic activity of splenic DCs and macrophages. Collectively, our results demonstrate a previously unrecognized, more central role for the ERAP1 protein in modulating several aspects of both the development of the innate immune system, and its responses during the initial stages of pathogen recognition. Such a role may explain why ERAP1 has been implicated by GWAS in the pathogenesis of autoimmune diseases that may be precipitated by aberrant responses to pathogen encounters.     

Adherence of Yersinia enterocolitica to Mammalian Epithelial Cell Lines

Yersinia enterocolitica RIMD 2501003 grown at 25 C avidly adhered to various kinds of cultured epithelial cell lines (HeLa, FL, Y-1 adrenal, human intestine, human conjunctiva) but the bacteria grown at 37 C did not adhere. This phenomenon paralleled the temperature-dependent motility of the bacteria. To clarify the adherence mechanism, we obtained two kinds of mutants, an immobile mutant and a nonadherent mutant, by treatment with A-methyl-A-nitro-A-nitrosoguanidine. The immobile mutant did not move on soft agar but retained the capacity to adhere to cultured epithelial cells when grown at 25 C. The nonadherent mutant did not adhere to cultured epithelial cells but retained the ability to move on soft agar when grown at 25 C. When the bacteria were killed by heat, ultraviolet light irradiation or formaldehyde they lost their capacity to adhere to the cultured epithelial cells. Antiserum against Y. enterocolitica RIMD 2501003 grown at 25 C was absorbed with the bacteria grown at 37 C, with the bacteria grown at 25 C, with the nonadherent mutant grown at 25 C and with the bacteria killed by various means. Only the antiserum absorbed with bacteria grown at 37 C inhibited the adherence of bacteria. These data indicate that motility does not correlate with adherence of Y. enterocolitica. It appears that the adherence factor involves both a temperature-dependent surface factor and a factor synthesized de novo during the interaction of susceptible cells with the bacteria.