Murine Intestinal Epithelial Cell Line Research Articles

Application of In vitro transcytosis models to brain targeted biologics.

The blood brain barrier (BBB) efficiently limits the penetration of biologics drugs from blood to brain. Establishment of an in vitro BBB model can facilitate screening of central nervous system (CNS) drug candidates and accelerate CNS drug development. Despite many established in vitro models, their application to biologics drug selection has been limited. Here, we report the evaluation of in vitro transcytosis of anti-human transferrin receptor (TfR) antibodies across human, cynomolgus and mouse species. We first evaluated human models including human cerebral microvascular endothelial cell line hCMEC/D3 and human colon epithelial cell line Caco-2 models. hCMEC/D3 model displayed low trans-epithelial electrical resistance (TEER), strong paracellular transport, and similar transcytosis of anti-TfR and control antibodies. In contrast, the Caco-2 model displayed high TEER value and low paracellular transport. Anti-hTfR antibodies demonstrated up to 70-fold better transcytosis compared to control IgG. Transcytosis of anti-hTfR.B1 antibody in Caco-2 model was dose-dependent and saturated at 3 μg/mL. Enhanced transcytosis of anti-hTfR.B1 was also observed in a monkey brain endothelial cell based (MBT) model. Importantly, anti-hTfR.B1 showed relatively high brain radioactivity concentration in a non-human primate positron emission tomography study indicating that the in vitro transcytosis from both Caco-2 and MBT models aligns with in vivo brain exposure. Typically, brain exposure of CNS targeted biologics is evaluated in mice. However, antibodies, such as the anti-human TfR antibodies, do not cross-react with the mouse target. Therefore, validation of a mouse in vitro transcytosis model is needed to better understand the in vitro in vivo correlation. Here, we performed transcytosis of anti-mouse TfR antibodies in mouse brain endothelial cell-based models, bEnd3 and the murine intestinal epithelial cell line mIEC. There is a good correlation between in vitro transcytosis of anti-mTfR antibodies and bispecifics in mIEC model and their mouse brain uptake. These data strengthen our confidence in the predictive power of the in vitro transcytosis models. Both mouse and human in vitro models will serve as important screening assays for brain targeted biologics selection in CNS drug development.

Oral Exposure to Polystyrene Microplastics of Mice on a Normal or High-Fat Diet and Intestinal and Metabolic Outcomes.

Microplastics (MPs) are small particles of plastic ( in diameter). In recent years, oral exposure to MPs in living organisms has been a cause of concern. Leaky gut syndrome (LGS), associated with a high-fat diet (HFD) in mice, can increase the entry of foreign substances into the body through the intestinal mucosa. We aimed to evaluate the pathophysiology of intestinal outcomes associated with consuming a high-fat diet and simultaneous intake of MPs, focusing on endocrine and metabolic systems. C57BL6/J mice were fed a normal diet (ND) or HFD with or without polystyrene MP for 4 wk to investigate differences in glucose tolerance, intestinal permeability, gut microbiota, as well as metabolites in serum, feces, and liver. In comparison with HFD mice, mice fed the HFD with MPs had higher blood glucose, serum lipid concentrations, and nonalcoholic fatty liver disease (NAFLD) activity scores. Permeability and goblet cell count of the small intestine (SI) in HFD-fed mice were higher and lower, respectively, than in ND-fed mice. There was no obvious difference in the number of inflammatory cells in the SI lamina propria between mice fed the ND and mice fed the ND with MP, but there were more inflammatory cells and fewer anti-inflammatory cells in mice fed the HFD with MPs in comparison with mice fed the HFD without MPs. The expression of genes related to inflammation, long-chain fatty acid transporter, and cotransporter was significantly higher in mice fed the HFD with MPs than in mice fed the HFD without MPs. Furthermore, the genus Desulfovibrio was significantly more abundant in the intestines of mice fed the HFD with MPs in comparison with mice fed the HFD without MPs. Muc2 gene expression was decreased when palmitic acid and microplastics were added to the murine intestinal epithelial cell line MODE-K cells, and Muc2 gene expression was increased when IL-22 was added. Our findings suggest that in this study, MP induced metabolic disturbances, such as diabetes and NAFLD, only in mice fed a high-fat diet. These findings suggest that LGS might have been triggered by HFD, causing MPs to be deposited in the intestinal mucosa, resulting in inflammation of the intestinal mucosal intrinsic layer and thereby altering nutrient absorption. These results highlight the need for reducing oral exposure to MPs through remedial environmental measures to improve metabolic disturbance under high-fat diet conditions. https://doi.org/10.1289/EHP11072.

Cytotoxic effects of manganese oxide nanoparticles in combination with microbial components on intestinal epithelial cells

Background: Manganese oxide has been shown to cause toxicity and is associated with occupational-related disease (e.g., welders). With the goal to improve several biomedical areas, manganese oxide nanoparticles (MnO NP) are being considered for use in drug delivery and magnetic resonance imaging (MRI) to obtain high resolution anatomical images of tumors and gastrointestinal (GI) inflammation. Regardless of whether it is intentional or unintentional ingestion, the GI tract has been shown to be the primary route of entry for metal nanoparticles including MnO NP. However, studies assessing toxicity of MnO NP for intestinal epithelial cells (IECs) are virtually nonexistent. Methods: Given the proximity to the GI lumen, assessing the effects of nanoparticles on IECs in the presence of bacterial components presents a more holistic model of exposure. Therefore, we examined the effects of MnO NP alone and MnO NP in combination with Escherichia coli LF82 bacterial lysate on selected functions of MODE-K cells, a murine intestinal epithelial cell line. Data were analyzed using one-way ANOVA. Differences with p < 0.05 were considered significant. Results: Results showed MnO NP plus E. coli LF82 lysate added to MODE-K cells severely inhibited monolayer scratch wound healing, enhanced the secretion of interleukin 6 (IL-6), and induced mitochondrial dysfunction. Conclusions: Overall, our findings show that toxicity of MnO NP deleteriously affected MODE-K cells and demonstrated the necessity to integrate other environmental factors, such as microbial components and/or inflammatory cytokines, into studies assessing effects of nanoparticles on mucosal epithelia.

Regulation of Intestinal Epithelial Thymic Stromal Lymphopoietin Expression by Retinoic Acid Receptor Alpha

We have previously targeted the retinoic acid receptor alpha (RARα) isoform in murine intestinal epithelial cells (IECs) to explore the role of epithelial intrinsic retinoid signaling in intestinal immune homeostasis. We noted a significant decrease in the CD11c+ and CD103+ dendritic cell (DC) subpopulations in the intestines of intestinal‐epithelial specific RARα‐deficient (RARαvillin) mice. In the present study, we identified Thymic Stromal Lymphopoietin (TSLP) as a contributing factor that influences intrinsic RARα signaling within IECs as this epithelial‐derived cytokine preferentially stimulates CD103+ DCs to a more tolerogenic phenotype in the GI tract, induces Tregs and drives TH2 polarization. TSLP also influences immune homeostasis by modulating the activation of myeloid cells, inhibition of proinflammatory DCs, and production of IL‐12. Notably, TSLP levels are decreased in Crohn’s disease (CD) where inflammation is driven by IL‐12.We hypothesize that RA intrinsic signaling within IECs modulates TSLP expression via activation of RARα. We sought to generate a RARα−/− murine intestinal epithelial cell line utilizing the CRISPR/CAS9 system to examine the effects of RARα ablation. Second, we aim to establish RARα’s role in regulating intestinal epithelial TSLP expression in both the RARαvillin mice and the CRISPR‐generated RARα−/− cell line.Exon 5 in the open reading frame of the RARα gene in Mode‐K murine IECs was targeted using CRISPR/CAS9. CRISPR cleavage and knockdown of RARα activity was confirmed via surveyor/luciferase assays. The cell lines were phenotyped with regards to their morphology, proliferative ability, and viability. Next, we examined TSLP mRNA expression in the colons of RARαvillin mice and the CRISPR RARα−/− knockout cell line via qRT‐PCR under baseline and stimulated conditions. Wild‐type (WT) and RARα−/− cells were stimulated with RARα‐selective agonist, BMS753, and muramyl dipeptide (MDP), a NOD2‐selective agonist.TSLP expression in surface colonic epithelium was elevated 2.3‐fold in RARαvillin mice compared to WT litter mates. TSLP expression was elevated 10‐fold in the Mode‐K RARα−/− cells versus parent cells at baseline. BMS753 stimulation resulted in a 15‐fold increase in TSLP expression in the RARα−/− cells compared to baseline. When stimulated with MDP, a NOD2‐selective agonist and known stimulus of TSLP production, TSLP expression was elevated 60‐fold in the RARα−/− cells, but significantly impaired compared to the WT cells (400‐fold), suggesting a different regulatory mechanism under stimulated conditions.Identifying a link between dietary factors, such as RA, its receptors and the TSLP molecular pathway can facilitate our understanding of how environmental components contribute to the pathogenesis of inflammatory bowel disease. TSLP expression is controlled by RARα in colonic IECs where it may act as a repressor of TSLP promoter transactivation under baseline conditions. This suggests an important role for RA on myeloid and T cell function via effects on intestinal epithelial TSLP expression.Support or Funding InformationUniversity of Calgary, Department of Medicine and Crohn's Colitis Canada

A58 REGULATION OF INTESTINAL EPITHELIAL THYMIC STROMAL LYMPHOPOIETIN GENE EXPRESSION BY RETINOIC ACID RECEPTOR ALPHA

Abstract Background We have targeted the retinoic acid receptor alpha (RARα) isoform in murine intestinal epithelial cells (IECs) to explore the role of epithelial intrinsic retinoid signaling in intestinal immune homeostasis. We noted a significant decrease in the CD11c+ and CD103+ dendritic cell (DC) subpopulations in the intestines of intestinal-epithelial specific RARα-deficient (RARα villin) mice. In the present study, we identified Thymic Stromal Lymphopoietin (TSLP) as a contributing factor that influences intrinsic RARα signaling within IECs. This cytokine preferentially stimulates CD103+ DCs to a more tolerogenic phenotype in the GI tract, induces Tregs and drives TH2 polarization. TSLP also influences immune homeostasis by modulating the activation of myeloid cells, inhibition of proinflamamtory DCs, and production of IL-12. Notably, TSLP levels are decreased in Crohn’s disease (CD) where inflammation is driven by IL-12. Aims We hypothesize that RA intrinsic signaling within IECs modulates TSLP expression via activation of RARα. We sought to generate a RARα -/- murine intestinal epithelial cell line utilizing the CRISPR/CAS9 system to examine the effects of RARa ablation. Second, we aim to establish RARα’s role in regulating intestinal epithelial TSLP expression in both the RARα villin mice and the CRISPR-generated RARα -/- cell line. Methods Exon 5 in the open reading frame of the RARα gene in Mode-K murine IECs was targeted using CRISPR/CAS9. CRISPR cleavage and knockdown of RARα activity was confirmed via surveyor/luciferase assays. The cell lines were phenotyped with regards to their morphology, proliferative ability, and viability. Next, we examined TSLP mRNA expression in the colons of RARα villin mice and the CRISPR RARα -/- knockout cell line via qRT-PCR under baseline and stimulated conditions. Wild-type (WT) and RARα-/- cells were stimulated with RARα-selective agonist, BMS753, and muramyl dipeptide (MDP), a NOD2-selective agonist. Results TSLP expression in surface colonic epithelium was elevated 2.3-fold in RARα villin mice compared to WT litter mates. TSLP expression was elevated 10-fold in the Mode-K RARα-/- cells versus parent cells at baseline. BMS753 stimulation resulted in a 15-fold increase in TSLP expression in the RARα-/- cells compared to baseline. When stimulated with MDP, a NOD2-selective agonist known to stimulate TSLP expression, TSLP expression was elevated 60-fold in the RARα-/- cells, but significantly impaired compared to the WT cells (400-fold). Conclusions TSLP expression is controlled by RARα in colonic IECs where it may act as a repressor of TSLP promoter transactivation under baseline conditions. This suggests an important role for RA on myeloid and T cell function via effects on intestinal epithelial TSLP expression. Funding Agencies CCCUniversity of Calgary, Department of Medicine

Inhibition of Pathological Mitochondrial Fission Restores DSS Associated Respiratory Impairments in an Intestinal Epithelial Cell Line

SignificanceRecent evidence has shown that mitochondrial dynamics and more specifically, mitochondrial fission, play an important role in numerous disease of energy intensive tissues. In particular, mitochondrial fission mediated by the enzyme dynamin related protein‐1 (DRP1) and the receptor fission protein‐1 (Fis1) is associated with reduced ATP production, ROS generation and inflammation. Examination of this interaction and corresponding pathological mitochondrial fission has yet to be examined in the colon in the context of inflammatory bowel disease.ObjectiveTo identify if a common murine colitis agent, dextran sodium sulfate (DSS), induces mitochondrial respiration dysfunction and if the novel peptide (P110), can restore these deficiencies by inhibiting DRP1 and Fis1 mediated fission.DesignThe murine intestinal epithelial cell line, IEC4.1, was separated into control, P110, DSS and DSS + P110. Cell were seeded and allowed to incubate for 24 hrs before receiving their respective treatments and further incubation for another 24hrs.MethodsOptimization of both DSS (1–2w./v.%) and P110 (0.5–1.5 uM) dosage and exposure time was performed with the AlamarBlue cell proliferation assay. Basal, mitochondrial complex and maximal respiratory activity were assessed using the Oxygraph‐2k (OROBOROS Instruments, Austria). Respiratory function of cells under various substrate pathways was also examined.ResultsDSS (2%) significantly reduced cell proliferation compared to control and P110 groups (p<0.05). Administration of P110 in 2% DSS cells dose dependently increased cell proliferation, with 1.5 uM P110 exhibiting cell proliferation similar to control. Analysis of mitochondrial complexes revealed that DSS reduced basal, complex I, II, IV and maximal activity (p<0.05). P110 was found to restore DSS associated respiratory deficiencies in complex I, II and IV (p<0.05).ConclusionThis study highlights novel findings regarding the role of mitochondrial fission in a model system of epithelial dysfunction. Importantly, we show i) evidence that DRP1 and Fis1 mediated fission hold potential; pathological consequences in energetically demanding tissues like the colon and ii) P110 treatment reduces DSS induced damage and its associated respiratory deficiencies. These results suggest mitochondrial dysfunction could be intricately involved in gut barrier deterioration seen in inflammatory bowel disease.Support or Funding InformationCrohn's and Colitis Canada. The authors wish to thank Daria Mochly‐Rosen for donating P110.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Representational difference analysis identifies specific genes in the interaction of Giardia duodenalis with the murine intestinal epithelial cell line, IEC-6

Giardia duodenalis is a re-emerging protozoan parasite that causes diarrhoea in humans, significantly affecting the health of many people globally. To date, little is known about the genetic events underpinning the establishment of infection in host cells; however, the parasite’s ventral disc, proteases and variable surface proteins (VSPs) are recognised as important pathogenic factors. In this study, representational difference analysis (RDA) was used to identify differentially expressed genes in four different Giardia isolates (WB, P-1, NF and GS/M) during the first 2h of in vitro interaction with the rat intestinal epithelial cell line, IEC-6. RDA showed that more than 40 genes were differentially expressed in each of the four Giardia isolates upon IEC-6 cells infection. Most of the up-regulated genes were common to the four isolates except for those encoding proteins possibly involved in immune evasion such as VSPs, high cysteine membrane proteins (HCMp), hypothetical proteins, and oxygen defence proteins (e.g., thioredoxin, peroxiredoxin 1). Differences in the expressed VSPs and HCMp may account for the variation in symptoms during giardiasis. Interestingly, the NF isolate solely expressed genes involved in encystation during interaction with IEC-6 (e.g., glucosamine 6-phosphate isomerase, dynamin, acid sphingomyelinase-like phosphodiesterase) suggesting that encystation signals could be different for this isolate. Common to the four isolates, transcripts for genes involved in glycolysis (e.g., glucose-6-phosphate dehydrogenase, fructose bisphosphate aldolase, enolase), attachment (γ and α1 giardins) and cysteine proteases were frequently detected. Genes involved in transcription, translation, signalling and cell cycle control were also up-regulated. This study shows that the RDA technique has selectively isolated genes involved in host–parasite interactions and complements previous microarray data. Some of the detected genes are also discussed as potential virulence factors and treatment targets in giardiasis.

Impact of a probiotic Enterococcus faecalis in a gnotobiotic mouse model of experimental colitis

IL-10-deficient (IL-10(-/-) ) mice are susceptible to the development of chronic intestinal inflammation in response to the colonization with commensal Enterococcus faecalis isolates. The aim of this study was to characterize the impact of a probiotic E. faecalis strain in germ-free, wild-type (WT), and disease-susceptible IL-10(-/-) mice. The probiotic E. faecalis and the colitogenic control strain OG1RF induced IL-6 and IFN-γ inducible protein-10 secretion in the murine intestinal epithelial cell line Mode K. Epithelial cell activation involved nuclear factor κ B, p38 and extracellular signal-regulated kinase 1/2-dependent pathways. Mouse embryonic fibroblasts from WT and toll-like receptor-2-deficient (TLR-2(-/-) ) mice confirmed that both E. faecalis strains trigger pro-inflammatory responses via the pattern recognition receptor TLR-2. Monoassociation of germ-free IL-10(-/-) mice with the probiotic E. faecalis strain revealed pro-inflammatory epithelial cell activation and colonic tissue pathology. The non-pathogenic nature of E. faecalis was confirmed in monoassociated WT mice. 2-DE and MALDI-TOF MS identified the ER stress chaperone Hspa5 (glucose-regulated protein 78) and 3-mercaptopyruvate sulfurtransferase as key targets in the epithelium from IL-10(-/-) and TLR-2(-/-) mice. This study shows the potential of probiotic bacteria to initiate pro-inflammatory responses in the disease-susceptible but not the normal host.

A Commensal Helicobacter sp. of the Rodent Intestinal Flora Activates TLR2 and NOD1 Responses in Epithelial Cells

Helicobacter spp. represent a proportionately small but significant component of the normal intestinal microflora of animal hosts. Several of these intestinal Helicobacter spp. are known to induce colitis in mouse models, yet the mechanisms by which these bacteria induce intestinal inflammation are poorly understood. To address this question, we performed in vitro co-culture experiments with mouse and human epithelial cell lines stimulated with a selection of Helicobacter spp., including known pathogenic species as well as ones for which the pathogenic potential is less clear. Strikingly, a member of the normal microflora of rodents, Helicobacter muridarum, was found to be a particularly strong inducer of CXC chemokine (Cxcl1/KC, Cxcl2/MIP-2) responses in a murine intestinal epithelial cell line. Time-course studies revealed a biphasic pattern of chemokine responses in these cells, with H. muridarum lipopolysaccharide (LPS) mediating early (24–48 h) responses and live bacteria seeming to provoke later (48–72 h) responses. H. muridarum LPS per se was shown to induce CXC chemokine production in HEK293 cells stably expressing Toll-like receptor 2 (TLR2), but not in those expressing TLR4. In contrast, live H. muridarum bacteria were able to induce NF-κB reporter activity and CXC chemokine responses in TLR2–deficient HEK293 and in AGS epithelial cells. These responses were attenuated by transient transfection with a dominant negative construct to NOD1, and by stable expression of NOD1 siRNA, respectively. Thus, the data suggest that both TLR2 and NOD1 may be involved in innate immune sensing of H. muridarum by epithelial cells. This work identifies H. muridarum as a commensal bacterium with pathogenic potential and underscores the potential roles of ill-defined members of the normal flora in the initiation of inflammation in animal hosts. We suggest that H. muridarum may act as a confounding factor in colitis model studies in rodents.

Differentiation of a murine intestinal epithelial cell line (MIE) toward the M cell lineage

M cells are a kind of intestinal epithelial cell in the follicle-associated epithelium of Peyer's patches. These cells can transport antigens and microorganisms into underlying lymphoid tissues. Despite the important role of M cells in mucosal immune responses, the origin and mechanisms of differentiation as well as cell death of M cells remain unclear. To clarify the mechanism of M cell differentiation, we established a novel murine intestinal epithelial cell line (MIE) from the C57BL/6 mouse. MIE cells grow rapidly and have a cobblestone morphology, which is a typical feature of intestinal epithelial cells. Additionally, they express cytokeratin, villin, cell-cell junctional proteins, and alkaline phosphatase activity and can form microvilli. Their expression of Musashi-1 antigen indicates that they may be close to intestinal stem cells or transit-amplifying cells. MIE cells are able to differentiate into the M cell lineage following coculture with intestinal lymphocytes, but not with Peyer's patch lymphocytes (PPL). However, PPL costimulated with anti-CD3/CD28 MAbs caused MIE cells to display typical features of M cells, such as transcytosis activity, the disorganization of microvilli, and the expression of M cell markers. This transcytosis activity of MIE cells was not induced by T cells isolated from PPL costimulated with the same MAbs and was reduced by the depletion of the T cell population from PPL. A mixture of T cells treated with MAbs and B cells both from PPL led MIE cells to differentiate into M cells. We report here that MIE cells have the potential ability to differentiate into M cells and that this differentiation required activated T cells and B cells.

Differential Regulation of Glucocorticoid Synthesis in Murine Intestinal EpithelialVersusAdrenocortical Cell Lines

Glucocorticoids are steroid hormones with important functions in development, immune regulation, and glucose metabolism. The adrenal glands are the predominant source of glucocorticoids; however, there is increasing evidence for extraadrenal glucocorticoid synthesis in thymus, brain, skin, and vascular endothelium. We recently identified intestinal epithelial cells as an important source of glucocorticoids, which regulate the activation of local intestinal immune cells. The molecular regulation of intestinal glucocorticoid synthesis is currently unexplored. In this study we investigated the transcriptional regulation of the steroidogenic enzymes P450 side-chain cleavage enzyme and 11beta-hydroxylase, and the production of corticosterone in the murine intestinal epithelial cell line mICcl2 and compared it with that in the adrenocortical cell line Y1. Surprisingly, we observed a reciprocal stimulation pattern in these two cell lines. Elevation of intracellular cAMP induced the expression of steroidogenic enzymes in Y1 cells, whereas it inhibited steroidogenesis in mICcl2 cells. In contrast, phorbol ester induced steroidogenic enzymes in intestinal epithelial cells, which was synergistically enhanced upon transfection of cells with the nuclear receptors steroidogenic factor-1 (NR5A1) and liver receptor homolog-1 (NR5A2). Finally, we observed that basal and liver receptor homolog-1/phorbol ester-induced expression of steroidogenic enzymes in mICcl2 cells was inhibited by the antagonistic nuclear receptor small heterodimer partner. We conclude that the molecular basis of glucocorticoid synthesis in intestinal epithelial cells is distinct from that in adrenal cells, most likely representing an adaptation to the local environment and different requirements.

Role of gamma interferon in chemokine expression in the ileum of mice and in a murine intestinal epithelial cell line after Cryptosporidium parvum infection.

Cryptosporidium parvum is a protozoan parasite that infects intestinal epithelial cells and induces inflammation of the intestine. To better understand the inflammatory process occurring during cryptosporidiosis, we investigated in this study the kinetics of chemokine expression in the mucosa of mice by quantitative reverse transcription-PCR. Our results demonstrate that among the chemokine mRNAs studied, gamma interferon (IFN-gamma)-inducible protein 10 (IP-10), monokine induced by IFN-gamma (MIG), i-TAC, lymphotactin, macrophage inflammatory protein 1 beta (MIP-1 beta), and RANTES mRNAs were strongly up-regulated in infected neonate mice, which correlated with the immunofluorescence staining results showing T-cell and macrophage infiltration in the mucosa. Our in vitro data showed that intestinal epithelial cells infected by C. parvum or stimulated by the proinflammatory cytokines (IFN-gamma, interleukin-1 beta, and tumor necrosis factor alpha) produce a pattern of chemokine secretion similar to that observed in vivo, suggesting that these cells may take part in the initial production of chemokines. In order to identify the chemokines responsible for the recruitment of the inflammatory cells leading to a protective immune response, we compared the patterns of chemokine expression in a healing neonate mouse model and a nonhealing IFN-gamma knockout (GKO) mouse model of cryptosporidiosis. In the absence of IFN-gamma, the chemokine response was altered for IP-10, MIG, i-TAC, RANTES, and MIP-1 beta mRNAs, while the three ELR C-X-C chemokine mRNAs studied (lipopolysaccharide-induced C-X-C chemokine, MIP-2 alpha, and KC mRNAs) were strongly overexpressed. These results are consistent with the neutrophil recruitment observed in the lamina propria of GKO mice at day 9 postinfection but are not consistent with the hypothesis that these cells play an important role in the resolution of the infection. On the contrary, the altered response of chemokines responsible for the recruitment of macrophages and T cells in GKO mice suggests that these two populations may be critical in the development of a protective immune response.

Expression of the Neutrophil Chemokine KC in the Colon of Mice with Enterocolitis and by Intestinal Epithelial Cell Lines: Effects of Flora and Proinflammatory Cytokines

IL-10 plays an important role in preventing excessive inflammation to the normal flora in the intestinal lumen. The purpose of this study was to compare the effect of normal flora on inflammation in mice in which the IL-10 gene was disrupted. IL-10 knock-out mice housed in germfree conditions remained healthy while those housed in conventional conditions developed colitis after weaning, suggesting that IL-10 inhibits the adverse responses to luminal Ag. Crypt abscesses were present in virtually all of the diseased animals as evidenced by flattening of the epithelial cells and a large number of neutrophils in the lumen of the crypt. Since KC is a chemokine that is capable of recruiting neutrophils in mice, mRNA and protein for KC was measured. Increased levels of both KC mRNA and protein were detected in the colon of diseased mice. To determine whether the epithelial cells were capable of synthesizing KC and contributing to neutrophil accumulation in the crypts, a murine intestinal epithelial cell line (Mode-K) was shown to express mRNA and protein for KC. Two cytokines induced in association with colitis in these mice, TNF-alpha and IFN-gamma, increased the expression of KC mRNA and protein in murine epithelial cells. However, IL-10 was incapable of decreasing the induction of KC, even though the cells expressed the IL-10 receptor. These results suggest that the neutrophil chemokine KC is produced by gastrointestinal epithelial cells in response to inflammatory mediators that are expressed following exposure to normal flora in animals lacking IL-10.

Cytokine-stimulated nitric oxide production by Caco-2 human colonic epithelial cells is not inhibited by the corticosteroid, dexamethasone

NO production by inducible NOS (iNOS) may be involved in the pathogenesis of inflammatory bowel disease (IBD). In many cell types, iNOS expression is regulated transcriptionally and post-transcriptionally by corticosteroids, one of the main treatments for IBD. Aim: To study iNOS expression and iNOS sensitivity to corticosteroids in the human enterocytic differentiated colonic cell line, Caco-2. Methods: Caco-2 cells were grown under standard culture conditions (DMEM, FCS 20%) and used 10 days after cell confluence to permit differentiation. NOS activity was assessed by using nitrate reduction and Griess reaction, which measures the NO stable oxidative products nitrite and nitrate (NOx). Cytokine stimulation was IFN'/ (200U/ml), IL1-13 (5 ng/ml), and TNFa (100 ng/ml) (cytomix). All results are for a 24 hours incubation. Results: Combined cytokine stimulation induced NOx production in Caco-2 cells (unstimulated ceils: 1.2 ± 0.4 pM; cytomix-treated ceils: 8.5 -+ 0.7 pM, P<0.0001, Student t test). The cytokine-induced NOx production was dependent on de novo transcription as shown by its complete inhibition with actinomycin D (5 pg/ml). This production was calcium-independent since EGTA (0.1-1 mM) had no effect on cytokine-induced NOx release. This production was inhibited by the NOS inhibitor L-nitro-arginine and by the novel selective iNOS inhibitor 1400W (EC50 12±4 pM). Moreover, the cytokine-induced NOx production was inhibited by pre-treatment with pyrrolidine dithiocarbamate and dichloroisocoumarine, specific inhibitors of NF-lcB (EC50 150±38 pM and 50 pM, respectively). Conversely, dexamethasone (0.1-10 pM) had no effect on NOx production even with 6 hours pre-treatment before induction. Under the same conditions dexamethasone inhibited cytokine-induced nitrite production in the wellcharacterised murine macrophage cell line J774 (53 ±4 % with dexamethasone 10 }aM) and the murine intestinal epithelial cell line IEC-6 (60 ± 2% with 0.1 }aM dexamethasone). Conclusions: Thus, the differentiated human intestinal cell line Caco-2 produced NO after cytokine stimulation via the transcriptional induction of a calcium-independent NOS, and NF4cB was involved in this pathway. In contrast to many other cell lines, the corticosteroid, dexamethasone did not inhibit NOS induction. This could be due to a defect at the steroid receptor level or in the transduction pathway involving this receptor. M.Cavicchi received a research grant from Institut de Recherche des Maladies de rAppareil Digestif, France.

Identification and cytokine regulation of gap junction proteins in macrophages and intestinal epithelial cell lines

In Inflammatory Bowel Disease (IBD), inflammatory ceils, including macrophages, infiltrate the mucosa and are located along epithelial cell basolateral membranes. We have previously presented functional evidence of gap junctional communication between murine macrophages and intestinal epithelial cell (IEC) lines in an in vitro coculture system. Gap junctional channels are formed by the association of two hemichannels (one from each cell). The hemichannel is composed of six structural proteins referred to as connexins. The type of connexin expressed determines the gating and permeation characteristics of the gap junctional channel. AIM: The purpose of this study was to identify the type of connexin(s) expressed by macrophages and IEC and to determine whether inflammatory cytokines could regulate the level of connexin expression in these cells. METHODS: Identification and quantitation of connexins was done by Western Blotting using antibodies to connexin26 (Cx26), Cx32 and Cx43. Cx26 and Cx43 were detectable in lysates of whole cells and isolated plasma membranes, whereas Cx32 could only be detected in membrane lysates. RESULTS: Murine macrophage cell lines P388D1 and J774A. 1 both expressed Cx26, while only J774A.1 expressed Cx43. Cx32 was readily detected in membranes of J774A.1, while P388D1 expressed only very low levels of this connexin. Murine IEC lines Mode-K and CMT93 and the rat IEC line IEC6, expressed Cx26 and C43. Low levels of Cx32 were also detected in Mode-K membranes. One of the most significant findings was that while Cx43 was not detected in unstimulated murine resident peritoneal macrophages (RPM), Cx43 was expressed by thioglycoUateinduced, inflammatory peritoneal macrophages (IPM). This suggested that cytokines may regulate connexin expression. Murine IEC lines Mode-K and CMT93 and macrophage lines P388D1 and J774A.1 were stimulated with IL-1 or interferon gamma (IFN'/) for 18-20 hours prior to sample preparation for Western blotting. IFN~t stimulation resulted in increased expression of Cx43 by macrophage line I774A. 1 (an average of 238% n=4) but did not induce expression of Cx43 by P388D1 or IEC lines. IL-1 induced a 35-176% increase in expression Cx43 by Mode-K (n=3) but not expression by CMT93 or macrophage cell lines. Cx26 expression was unaltered by cytokine stimulation of any cell line. CONCLUSIONS: Macrophages and IEC express multiple connexins allowing for heterocellular communication via channels containing like or different connexins. In IBD, inflammatory cytokines may regulate the expression of these connexins and, by extension, may effect macrophage-IEC gap junctional communication.

A role for stem cell factor (SCF): c-kit interaction(s) in the intestinal tract response to Salmonella typhimurium infection.

Cholera toxin (CT) has been shown to induce stem cell factor (SCF) production in mouse ligated intestinal loops. Further, SCF interaction(s) with its receptor (c-kit) was shown to be important for the intestinal tract secretory response after CT exposure. In this study, we have investigated whether SCF production is induced in the intestinal tract after exposure to Salmonella typhimurium and whether this production could be an important intestinal tract response to Salmonella infection. Using a mouse ligated intestinal loop model, increased levels of SCF mRNA were detected at 2-4 h post-Salmonella challenge. Intestinal fluid obtained from Salmonella-challenged loops contained high levels of SCF by ELISA. Human and murine intestinal epithelial cell lines were also shown to have increased levels of SCF mRNA after exposure to Salmonella. Inhibition of Salmonella invasion of epithelial cells was shown to be one potentially important role for SCF:c-kit interactions in host defense to Salmonella infection. Pretreatment of human or murine intestinal cell lines with SCF resulted in a cellular state that was resistant to Salmonella invasion. Finally, mice having mutations in the white spotting (W) locus, which encodes the SCF-receptor (c-kit), were significantly more susceptible to oral Salmonella challenge than their control littermates. Taken together, the above results suggest that an important intestinal tract response to Salmonella infection is an enhanced production of SCF and its subsequent interactions with c-kit.