Fetal Intestinal Epithelial Cell Line Research Articles

Hydrocortisone-induced anti-inflammatory effects in immature human enterocytes depend on the timing of exposure.

The immature human gut has a propensity to exaggerated inflammatory responses that are thought to play a role in the pathogenesis of necrotizing enterocolitis (NEC). Prenatal exposure to corticosteroids has been reported to reduce the risk of NEC, while postnatal dexamethasone treatment is associated with adverse neurodevelopmental outcomes in preterm infants. The aim of this study was to investigate the direct role of hydrocortisone in gene expression patterns and inflammatory responses in immature human enterocytes. Time-dependent hydrocortisone effects in nontransformed primary human fetal intestinal epithelial cell line H4 were investigated by cDNA microarray. Fetal intestinal organ culture and cell culture experiments were conducted. Inflammatory responses were induced by stimulation with IL-1β and TNF-α with and without hydrocortisone. IL-8 and IL-6 expression and secretion were measured as functional readout. Here we report time-dependent hydrocortisone-induced changes in gene expression patterns detected by cDNA microarray. Hydrocortisone significantly attenuated IL-1β-induced inflammatory responses in the immature human gut when administered at the time of the proinflammatory insult: IL-1β-induced IL-8 and IL-6 secretion in the fetal ileum as well as H4 cells were significantly reduced. Hydrocortisone also inhibited IL-8 secretion in response to TNF-α. In contrast, TNF-α-induced IL-8 secretion was not reduced in cells treated with hydrocortisone for 48 h before stimulation. Our observations provide a physiological basis for understanding the differential clinical effects of corticosteroids in the immature human gut depending on the timing of treatment.

Lactobacillus rhamnosus GG and its SpaC pilus adhesin modulate inflammatory responsiveness and TLR-related gene expression in the fetal human gut.

Bacterial contact in utero modulates fetal and neonatal immune responses. Maternal probiotic supplementation reduces the risk of immune-mediated disease in the infant. We investigated the immunomodulatory properties of live Lactobacillus rhamnosus GG and its SpaC pilus adhesin in human fetal intestinal models. Tumor necrosis factor (TNF)-α mRNA expression was measured by qPCR in a human fetal intestinal organ culture model exposed to live L. rhamnosus GG and proinflammatory stimuli. Binding of recombinant SpaC pilus protein to intestinal epithelial cells (IECs) was assessed in human fetal intestinal organ culture and the human fetal intestinal epithelial cell line H4 by immunohistochemistry and immunofluorescence, respectively. TLR-related gene expression in fetal ileal organ culture after exposure to recombinant SpaC was assessed by qPCR. Live L. rhamnosus GG significantly attenuates pathogen-induced TNF-α mRNA expression in the human fetal gut. Recombinant SpaC protein was found to adhere to the fetal gut and to modulate varying levels of TLR-related gene expression. The human fetal gut is responsive to luminal microbes. L. rhamnosus GG significantly attenuates fetal intestinal inflammatory responses to pathogenic bacteria. The L. rhamnosus GG pilus adhesin SpaC binds to immature human IECs and directly modulates IEC innate immune gene expression.

Oral Administration of Transforming Growth Factor-β1 (TGF-β1) Protects the Immature Gut from Injury via Smad Protein-dependent Suppression of Epithelial Nuclear Factor κB (NF-κB) Signaling and Proinflammatory Cytokine Production

Inflammatory immune responses play an important role in mucosal homeostasis and gut diseases. Nuclear factor κB (NF-κB), central to the proinflammatory cascade, is activated in necrotizing enterocolitis (NEC), a devastating condition of intestinal injury with extensive inflammation in premature infants. TGF-β is a strong immune suppressor and a factor in breast milk, which has been shown to be protective against NEC. In an NEC animal model, oral administration of the isoform TGF-β1 activated the downstream effector Smad2 in intestine and significantly reduced NEC incidence. In addition, TGF-β1 suppressed NF-κB activation, maintained levels of the NF-κB inhibitor IκBα in the intestinal epithelium, and systemically decreased serum levels of IL-6 and IFN-γ. The immature human fetal intestinal epithelial cell line H4 was used as a reductionistic model of the immature enterocyte to investigate mechanism. TGF-β1 pretreatment inhibited the TNF-α-induced IκBα phosphorylation that targets the IκBα protein for degradation and inhibited NF-κB activation. Chromatin immunoprecipitation (ChIP) assays demonstrated decreased NF-κB binding to the promoters of IL-6, IL-8, and IκBα in response to TNF-α with TGF-β1 pretreatment. These TGF-β1 effects appear to be mediated through the canonical Smad pathway as silencing of the TGF-β central mediator Smad4 resulted in loss of the TGF-β1 effects. Thus, TGF-β1 is capable of eliciting anti-inflammatory effects by inhibiting NF-κB specifically in the intestinal epithelium as well as by decreasing systemic IL-6 and IFN-γ levels. Oral administration of TGF-β1 therefore can potentially be used to protect against gastrointestinal diseases.

Lactoferrin (Lf) modulates microbe‐associated molecular pattern (MAMP)‐ and cytokine‐induced inflammation in human fetal intestinal epithelial cells

Immune dysregulation of postnatal enteropathies includes aberrant exposure to microbe‐associated molecular patterns (MAMP) that potentiate an exaggerated inflammatory cytokine profile. The antimicrobial protein lactoferrin (Lf) associates with its cognate receptor (LfR) on intestinal epithelial cells (IEC) of breast‐fed infants where it may exert immunomodulatory activity. Lf binds lipopolysaccharide (LPS) with high affinity possibly mitigating overt pattern‐recognition receptor signaling; however, little is know regarding the capacity of Lf to interrupt alternative MAMPs and their downstream cytokine profiles. Using a human fetal intestinal epithelial cell line (FHs74), we investigated effects of commercial bovine Lf (C‐bLf) on MAMP‐ (LPS: 1 μg/ml; DNA: 10 μg/ml, and flagellin: 1 μg/ml) and cytokine‐ (IL1β, TNFα, and IFNγ) induced inflammation. Analysis of protein expression showed that C‐bLf attenuates IL1β‐ and TNFα‐mediated up‐regulation of IL6 across time. Compared to unstimulated cells, LfR protein expression was up‐regulated in IL1β‐ (10 ng/ml) and TNFα‐ (50 ng/ml) stimulated cells. C‐bLf (400 μg/ml) inhibited MAMP‐induced IL6 and IL8 gene expression in unstimulated and cytokine‐stimulated cells at 4, 12, and 24 h. These findings suggest that Lf modulates cytokine‐ and MAMP‐induced inflammation in IECs suggesting a role for Lf in limiting the pathogenesis of postnatal enteropathies.Grant Funding Source: N/A

Vascular Endothelial Growth Factor in Human Milk

After completing this article, readers should be able to: 1. Delineate the fundamental advantages conferred by dietary regulation of genes expressed in the intestinal epithelium. 2. Describe the relationship between vascular endothelial growth factor in human milk and gestational age and duration of lactation. As discussed in the article on the physicochemical environment of the neonatal intestine in this issue, the intestine interacts with the external environment to the benefit of the host. The intestine changes the environment (albeit minimally), and the removal of nutrients provides the energy and building materials for life. Thus, the interaction of the intestine and the environment is a basic function. Dietary regulation of genes expressed in the epithelium confers three fundamental advantages for mammals. First, it enables the epithelium to adapt to the luminal environment to digest absorbed food better. Second, it provides the means for human milk to influence the development of the gastrointestinal tract. Third, epithelial expression of proteins that act on the immune system constitutes a signaling mechanism from the intestinal lumen to host defenses. This third mechanism is described in the article on dietary regulation of immune genes …

Rapid protection of gnotobiotic pigs against experimental salmonellosis following induction of polymorphonuclear leukocytes by avirulent Salmonella enterica.

Oral inoculation of 5-day-old gnotobiotic pigs with Salmonella enterica serovar Typhimurium strain F98 resulted in severe enteritis and invasive disease. Preinoculation 24 h earlier with an avirulent mutant of Salmonella enterica serovar Infantis (1326/28) completely prevented disease for up to 14 days (when the experiment was terminated). S. enterica serovar Infantis colonized the alimentary tract well, with high bacterial counts in the intestinal lumen but with almost no invasion into the tissues. Unprotected pigs had high S. enterica serovar Typhimurium counts in the intestines, blood, and major nonintestinal organs. Recovery of this strain from the blood and major organs in S. enterica serovar Infantis-protected pigs was substantially reduced despite the fact that intestinal counts were also very high. Protection against disease thus did not involve a colonization exclusion phenomenon. Significant (P < 0.05) infiltration of monocytes/macrophages was observed in the submucosal regions of the intestines of both S. enterica serovar Infantis-protected S. enterica serovar Typhimurium-challenged pigs and unprotected S. enterica serovar Typhimurium-challenged pigs. However, only polymorphonuclear neutrophils (PMNs) were observed throughout the villus, where significant (P < 0.05) numbers infiltrated the lamina propria and the subnuclear and supranuclear regions of the epithelia, indicating that PMN induction and positioning following S. enterica serovar Infantis inoculation was consistent with rapid protection against the challenge strain. Similarly, in vitro experiments using a human fetal intestinal epithelial cell line (INT 407) demonstrated that, although significantly (P < 0.05) fewer S. enterica serovar Infantis than S. enterica serovar Typhimurium organisms invaded the monolayers, S. enterica serovar Infantis induced an NF-kappaB response and significantly (P < 0.05) raised interleukin 8 levels and transmigration of porcine PMN. The results of this study suggest that attenuated Salmonella strains can protect the immature intestine against clinical salmonellosis by PMN induction. They also demonstrate that PMN induction is not necessarily associated with clinical symptoms and/or intestinal pathology.

Distribution of the IgG Fc receptor, FcRn, in the human fetal intestine.

The intestinal Fc receptor, FcRn, functions in the maternofetal transfer of gamma globulin (IgG) in the neonatal rodent. In humans, most of this transfer is presumed to occur in utero via the placenta. Although the fetus swallows amniotic fluid that contains immunoglobulin, it is unknown whether this transfer also occurs via the fetal intestine. A human FcRn has been identified in the syncytiotrophoblast that mediates the maternofetal transfer of antibody. It has also been identified in human fetal intestine and is postulated to function in IgG transport. We hypothesize that the human fetal intestinal FcRn may play a role in IgG transport from the amniotic fluid into the fetal circulation. The aim of this study was to characterize the distribution of the FcRn along the human fetal intestine. Lysates prepared from human fetal intestine and from a nonmalignant human fetal intestinal epithelial cell line (H4) were subjected to Western blot analysis and probed using anti-FcRn antibodies. A 42-kD band, consistent with the known molecular weight of the FcRn, was detected along the human fetal intestine and in H4 cells. Expression of the human FcRn was confirmed with immunohistochemistry. Our study demonstrates the expression of FcRn along the human fetal intestine and in a human nonmalignant fetal intestinal epithelial cell line (H4), which by location indicates that FcRn could play a role in the uptake and transport of IgG in the human fetus.

Sodium butyrate blocks interferon-gamma (IFN-gamma)-induced biosynthesis of MHC class III gene products (complement C4 and factor B) in human fetal intestinal epithelial cells.

Human intestinal epithelial cells have been established as local sites for complement biosynthesis. In this study, we investigated the effects of IFN-gamma and sodium butyrate on biosynthesis of MHC class III gene products (complement C4 and factor B) in the human fetal intestinal epithelial cell line INT-407. IFN-gamma induced a dose- and time-dependent increase in C4 and factor B secretion. However, sodium butyrate dose-dependently inhibited IFN-gamma-induced C4 and factor B secretion. These effects were also observed at the mRNA level. Immunoblotting indicated that IFN-gamma induced a rapid activation of Stat1alpha, and fluorescence immunohistochemistry detected a translocation of Stat1alpha into the nucleus within 1 h. However, the translocation of Stat1alpha was not affected by the addition of sodium butyrate. Nuclear run-on assay indicated that IFN-gamma induced a weak increase in the transcription rate of factor B gene, and sodium butyrate did not affect this response. IFN-gamma and sodium butyrate induced a counter-regulatory effect on C4 and factor B secretion: IFN-gamma acted as a potent inducer, but sodium butyrate potently abrogated these responses. These are mainly regulated through the post-transcriptional mechanism.

Cooperation of interleukin-17 and interferon-gamma on chemokine secretion in human fetal intestinal epithelial cells.

Interleukin (IL)-17 is a newly identified T cell-derived cytokine that can regulate the functions of a variety of cell types. In this study, we investigated the effects of IL-17 and interferon (IFN)-gamma on chemokine secretion in human fetal intestinal epithelial cells. IL-8 and monocyte chemoattractant protein (MCP)-1 secretion by the human fetal intestinal epithelial cell line, intestine-407, was evaluated by ELISA and Northern blot. The expression of IL-17 receptor (R) was analysed by a binding assay using [(125)I]-labelled IL-17. The activation of nuclear factor-kappa B (NF-kappa B), NF-IL6 and AP-1 was assessed by an electrophoretic gel mobility shift assay (EMSA). IL-17 induced a dose-dependent increase in IL-8 and MCP-1 secretion. The inducing effects of IL-17 on IL-8 and MCP-1 mRNA abundance reached a maximum as early as 3 h, and then gradually decreased. IL-17 and IFN-gamma synergistically increased IL-8 and MCP-1 secretion and mRNA abundance. IFN-gamma induced a weak increase in IL-17 R mRNA abundance, and incubation with IFN-gamma for 24 h enhanced [(125)I]-labelled IL-17-binding by 2.4-fold. IL-17 rapidly induced the phosphorylation and degradation of I kappa B alpha molecules, and the combination of IL-17 and IFN-gamma induced a marked increase in NF-kappa B DNA-binding activity as early as 1.5 h after the stimulation. Furthermore, this combination induced an increase in NF-IL-6 and AP-1 DNA-binding activity. In conclusion, it becomes clear that IL-17 is an inducer of IL-8 and MCP-1 secretion by human fetal intestinal epithelial cells. The combination of IL-17 with IFN-gamma synergistically enhanced chemokine secretion. These effects of IL-17 and IFN-gamma might play an important role in the inflammatory responses in the intestinal mucosa.

The neonatal IgG receptor FcRn, is expressed in the human fetal intestine and the non-malignant fetal intestinal epithelial cell line

The neonatal Fc receptor FcRn functions in rodents to transfer maternal gammaglobulins (IgG) from mother to young via the neonatal intestine. In humans most of this transfer is presumed to occur in utero via the placenta. The fetus swallows amniotic fluid that contains immunogiobulins but it is unknown whether this transfer also occurs via the fetal intestine. Human FcRn has been identified in the human syncitiotrophoblast and plays a role in the materno-fetal transfer of antibody. It is also expressed in adult intestine and may be important in IgG catabolism. Objective: We hypothesize that the FcRn receptor is expressed in the human fetal intestine and may play a role in IgG transport from the amniotic fluid/colostrum into the fetal circulation. We wish to demonstrate expression of the FcRn 1) along different segments of the human fetal intestine and 2) in a human non-malignant fetal intestinal epithelial cell line (H4). Methods: Lysates were prepared from human fetal stomach, small intestine and colon from a 22 week gestation fetus and from H4 cells. Caco-2 cells and T-84 cells were used as positive controls. Prepared lysates were run on Western blots and were probed with anti-FcRn antibodies. Bound antibody was detected by HRP-conjugated secondary antibody and enhanced chemiluminescence. FcRn expression was normalized against GAPDH and quantitated by densitometry. Immunostaining of human fetal intestine, neonatal mouse intestine (+ contro[), H4 cells and T-B4 ceils (+ control), was done with a rabbit anti-human FcRn antibody and detected with the appropriate conjugated secondary antibody for microscopy. Results: The FcRn heavy chain was detected from the fetal intestine and H4 cells on Western blot analysis. On immunostaining, a subapical staining pattern for FcRn was seen on human fetal intestine. The H4 cells also stained positive for FcRn. Conclusion: FcRn expression is demonstrated in the human fetal intestine and in the human non-malignant fetal intestinal epithelial cell line (H4) suggesting that FcRn may play a role in the uptake and transport of IgG in the fetus.

The neonatal IgG receptor FcRn, is expressed in the human fetal intestine and the non-malignant fetal intestinal epithelial cell line

The neonatal IgG receptor FcRn, is expressed in the human fetal intestine and the non-malignant fetal intestinal epithelial cell line

Medium- and Long-Chain Fatty Acids Differentially Modulate Interleukin-8 Secretion in Human Fetal Intestinal Epithelial Cells

The primary therapeutic effects of enteral nutrition in patients with Crohn's disease have been reported previously. Although the quantity and type of fat in enteral nutrition are considered to be important, it is unclear how fat modulates mucosal inflammatory responses in the intestine. In the present study, we evaluated the effects of medium-chain and long-chain fatty acids (MCFA and LCFA) on interleukin (IL)-8 secretion in a fetal intestinal epithelial cell line, intestine-407 cells. IL-8 expression was evaluated at the protein and mRNA levels. The activation of nuclear factor-kappaB was assessed with an electrophoretic gel mobility shift assay. The addition of oleic acid (LCFA) micelles, but not octanoic acid (MCFA) micelles, weakly but significantly enhanced basal IL-8 secretion in the intestine-407 cells. The addition of MCFA (5 mmol/L) induced a 40% increase in IL-1beta-induced IL-8 secretion and a 35% increase in tumor necrosis factor (TNF)-alpha-induced IL-8 secretion, respectively. The addition of LCFA (5 mmol/L) induced a 140% increase in IL-1beta-induced IL-8 secretion and a 110% increase in TNF-alpha-induced IL-8 secretion, respectively. These responses were also observed at the mRNA levels. The electrophoretic gel mobility shift assay indicated that both MCFA and LCFA enhanced IL-1beta- and TNF-alpha-induced nuclear factor-kappaB activation. We demonstrated the proinflammatory activities of MCFA and especially LCFA. It is likely that medium-chain triglycerides may be more suitable than long-chain triglycerides as an energy source in enteral diets in the treatment of patients with Crohn's disease.

Counter-regulatory effects of interleukin-1β and transforming growth factor-β on complement C3 synthesis in human fetal intestinal epithelial cells

In the intestinal tract, local synthesis of complement C3 has previously been demonstrated in vivo. However, the cellular source of C3 in the intestine has not been identified. In this study, we attempted to demonstrate complement C3 synthesis in the human fetal intestinal epithelial cell line, FHs74Int. Unstimulated FHs74Int cells secreted a low level of C3 protein. The addition of interleukin (IL)-1β induced a dose- and time-dependent increase in C3 synthesis. These effects of IL-1β were observed at a concentration as low as 0.01 ng/ml and reached a plateau at a concentration of 10 ng/ml. Transforming growth factor (TGF)-β, by itself, had no effect on C3 synthesis, but TGF-β dose-dependently suppressed IL-1/β-induced C3 synthesis. TGF-β-mediated suppression of C3 synthesis was observed at a concentration of as low as 0.1 ng/ml. These effects of IL-1β and TGF-β were also observed at the mRNA level. The findings presented in this study indicate that human intestinal epithelial cells are indeed capable of secreting complement C3 in response to IL-1β and TGF-β.

Adherence of Lactobacillus Species to Human Fetal Intestinal Cells

Thirty-two lactobacilli were tested for ability to adhere to a human fetal intestinal epithelial cell line. By an in vitro system, two adherence mechanisms were found. One mechanism, requiring calcium in the adherence reaction, was nonspecific and allowed all lactobacilli tested to adhere. The other system, not requiring calcium, was found in four strains, all human Lactobacillus acidophilus isolates. Colonial morphology, serial broth passage, and exposure of cell crops to freezing or lyophilization did not affect adherence of Lactobacillus acidophilus. In vitro adherence, combined with subsequent in vivo studies, may provide a basis for screening candidate organisms for use in microbiotic supplements.