Yongjia Feng

Loss of enteral nutrition in a mouse model results in intestinal epithelial barrier dysfunction

Total parenteral nutrition (TPN) administration in a mouse model leads to a local mucosal inflammatory response, resulting in a loss of epithelial barrier function (EBF). Although, the underlying mechanisms are unknown, a major contributing factor is a loss of growth factors and subsequent critical downstream signaling. An important component of these is the p‐Akt pathway. An additional contributing factor to the loss of EBF with TPN is an increase in proinflammatory cytokine abundance within the mucosal epithelium, including TNF‐α and IFN‐γ. Loss of critical nutrients, including glutamine and glutamate, may affect EBF, contributing to the loss of tight junction proteins. Finding protective modalities for the small intestine during TPN administration may have important clinical applications. Supplemental glutamine and glutamate may be examples of such agents.

Intestinal epithelial cell apoptosis and loss of barrier function in the setting of altered microbiota with enteral nutrient deprivation

Total parenteral nutrition (TPN), a commonly used treatment for patients who cannot receive enteral nutrition, is associated with significant septic complications due in part to a loss of epithelial barrier function (EBF). While the underlying mechanisms of TPN-related epithelial changes are poorly understood, a mouse model of TPN-dependence has helped identify several contributing factors. Enteral deprivation leads to a shift in intestinal microbiota to predominantly Gram-negative Proteobacteria. This is associated with an increase in expression of proinflammatory cytokines within the mucosa, including interferon-γ and tumor necrosis factor-α. A concomitant loss of epithelial growth factors leads to a decrease in epithelial cell proliferation and increased apoptosis. The resulting loss of epithelial tight junction proteins contributes to EBF dysfunction. These mechanisms identify potential strategies of protecting against TPN-related complications, such as modification of luminal bacteria, blockade of proinflammatory cytokines, or growth factor replacement.

Glutamine Prevents Total Parenteral Nutrition-Associated Changes to Intraepithelial Lymphocyte Phenotype and Function: A Potential Mechanism for the Preservation of Epithelial Barrier Function

Total parenteral nutrition (TPN) results in a number of derangements to the intestinal epithelium, including a loss of epithelial barrier function (EBF). As TPN supplemented with glutamine has been thought to prevent this loss, this article further defined the impact of glutamine on EBF, and investigated potential mechanisms that contributed to the preservation of EBF. C57BL/6J male mice were randomized to enteral nutrition (control), TPN, or TPN supplemented with glutamine (TPN+GLN). Changes in intraepithelial lymphocyte (IEL)-derived cytokine expression were measured, and EBF was assessed with electrophysiologic methods and assessment of junctional protein expression. TPN resulted in a significant decline in EBF, and this loss of EBF was significantly prevented in the TPN+GLN group. Coincident with these changes was a loss of intraepithelial lymphocyte (IEL, mucosal lymphocyte)-derived IL-10 and increase in interferon-gamma (IFN-gamma) expression, and a decline in IEL numbers in the TPN group. A prevention in the increase in IFN-gamma and decline in IL-10 expression was seen in the TPN+GLN group. To determine the mechanism responsible for these glutamine-associated cytokine changes, we tested whether blockade of the IL-7 signaling pathway between epithelial cells (EC) and IEL would prevent these changes; however, blockade failed to influence IEL-derived cytokine changes. Glutamine-supplemented TPN leads to a specific IEL-derived cytokine profile, which may account for the preservation of EBF; and such action may be due to a direct action of glutamine on the IEL.

Epidermal growth factor/TNF-α transactivation modulates epithelial cell proliferation and apoptosis in a mouse model of parenteral nutrition.

Epidermal growth factor (EGF) and tumor necrosis factor-α (TNF-α) signaling are critical for effective proliferative and apoptotic actions; however, little is known about the codependency of these signaling pathways in the intestinal epithelium. Because total parenteral nutrition (TPN) is associated with loss of intestinal epithelial cell (IEC) proliferation and increased apoptosis, we utilized a mouse model to explore these transactivation pathways in small bowel epithelium. Mice underwent intravenous cannulation and were given enteral nutrition or TPN for 7 days. Outcomes included IEC proliferation, apoptosis, and survival. To address transactivation or dependence of EGF and TNF on IEC physiology, TNF-α receptor knockout (KO) mice, TNFR1-KO, R2-KO, or R1R2-double KO, were used. Exogenous EGF and pharmacological blockade of ErbB1 were performed in other groups to examine the relevance of the ErB1 pathway. TPN increased IEC TNFR1 and decreased EGF and ErbB1 abundance. Loss of IEC proliferation was prevented by exogenous EGF or blockade of TNFR1. However, EGF action was prevented without effective TNFR2 signaling. Also, blockade of TNFR1 could not prevent loss of IEC proliferation without effective ErbB1 signaling. TPN increased IEC apoptosis and was due to increased TNFR1 signaling. Exogenous EGF or blockade of TNFR1 could prevent increased apoptosis, and both pathways were dependent on effective ErbB1 signaling. Exogenous EGF prevented increased apoptosis in mice lacking TNFR2 signaling. TPN mice had significantly decreased survival vs. controls, and this was associated with the TNFR1 signaling pathway. We concluded that these findings identify critical mechanisms that contribute to TPN-associated mucosal atrophy via altered TNF-α/EGF signaling. It emphasizes the importance of both TNFR1 and TNFR2 pathways, as well as the strong interdependence on an intact EGF/ErbB1 pathway.

Tumour necrosis factor‐α‐induced loss of intestinal barrier function requires TNFR1 and TNFR2 signalling in a mouse model of total parenteral nutrition

•  Total parenteral nutrition (TPN) is critical for patients who cannot tolerate enteral nutrition. However, TPN‐associated loss of barrier function leads to an increase in enterically derived pathogens that may harm the patient. •  Tumour necrosis factor‐α (TNF‐α) is involved in the dysregulation of intestinal barrier function in many animal models. •  The mouse model of TPN provides an excellent, non‐destructive approach to examine epithelial barrier dysfunction. •  Tumour necrosis factor‐α is shown to be a major mediator of epithelial barrier dysfunction using this TPN model. •  Tumour necrosis factor‐α signalling is reliant on both the TNFR1 and TNFR2 pathways to effect epithelial barrier dysfunction. •  Anti‐TNF treatment protected against TPN‐associated epithelial barrier dysfunction and might prove to be a viable future clinical approach.

Total Parenteral Nutrition–Associated Lamina Propria Inflammation in Mice Is Mediated by a MyD88-Dependent Mechanism

Enteral nutrient deprivation via total parenteral nutrition (TPN) administration leads to local mucosal inflammatory responses, but the underlying mechanisms are unknown. Wild-type (WT) and MyD88−/− mice underwent jugular vein cannulation. One group received TPN without chow, and controls received standard chow. After 7 d, we harvested intestinal mucosally associated bacteria and isolated small-bowel lamina propria (LP) cells. Bacterial populations were analyzed using 454 pyrosequencing. LP cells were analyzed using quantitative PCR and multicolor flow cytometry. WT, control mucosally associated microbiota were Firmicutes-dominant, whereas WT TPN mice were Proteobacteria-domiant. Similar changes were observed in MyD88−/− mice with TPN administration. UniFrac analysis showed divergent small bowel and colonic bacterial communities in controls, merging toward similar microbiota (but distinct from controls) with TPN. The percentage of LP T regulatory cells significantly decreased with TPN in WT mice. F4/80+CD11b+CD11cdull/− macrophage–derived proinflammatory cytokines significantly increased with TPN. These proinflammatory immunologic changes were significantly abrogated in MyD88−/− TPN mice. Thus, TPN administration is associated with significant expansion of Proteobacteria within the intestinal microbiota and increased proinflammatory LP cytokines. Additionally, MyD88 signaling blockade abrogated decline in epithelial cell proliferation and epithelial barrier function loss.

Decreased phospho-Akt signaling in a mouse model of total parenteral nutrition: a potential mechanism for the development of intestinal mucosal atrophy.

Total parenteral nutrition (TPN) leads to a decline in phosphatidylinositol 3-kinase (PI3K)/phospho-Akt (p-Akt) activity, affecting downstream signaling, reducing epithelial cell (EC) proliferation, and contributing to intestinal mucosal atrophy. We hypothesized that promoting Akt activity would prevent these changes. We used a novel Akt-activating peptide, TCL1 (a head-to-tail dimer of the Akt-binding domain of T-cell lymphoma-1), or an inactive mutant sequence TCL1G conjugated to a transactivator of transcription peptide sequence to promote intracellular uptake. Four groups of mice were studied, enteral nutrition group (control), control mice given a functioning TCL1 (control + TCL1), TPN mice given TCL1G (control peptide, TPN + TCL1G); and TPN mice given TCL1. TPN mice given TCL1G showed a significant decrease in jejunal EC p-Akt (Ser473 and Thr308) abundance, whereas TPN + TCL1 mice showed increased p-Akt (Ser473) abundance. Phosphorylation of beta-catenin and glycogen synthase kinase-3beta (downstream targets of Akt signaling) were also decreased in the TPN + TCL1G group and completely prevented in the TPN + TCL1 group. Use of TCL1 nearly completely prevented the decline in EC proliferation seen in the TPN + TCL1G group, as well as partly returned EC apoptosis levels close to controls. The mammalian target of rapamycin pathway demonstrated a similar reduction in activity in the TPN + TCL1G group that was significantly prevented in the TPN + TCL1 group. These results support a significant loss of PI3K/p-Akt signaling upon replacing enteral nutrition with TPN, and prevention of this loss demonstrates the key importance of PI3K/p-Akt signaling in maintaining gut integrity including EC proliferation and reduction in apoptosis.

Dissociation of E-cadherin and β-catenin in a mouse model of total parenteral nutrition: a mechanism for the loss of epithelial cell proliferation and villus atrophy

Total parenteral nutrition (TPN) leads a loss of epithelial barrier function, decline in epithelial cell (EC) proliferation, and decreased expression of E‐cadherin. As a large portion of intracellular β‐catenin is tightly associated with E‐cadherin, we hypothesized that the loss of E‐cadherin would result in a redistribution of intracellular β‐catenin, and could be a contributing mechanism for this TPN‐associated loss of EC proliferation. An assessment of small bowel epithelium was performed in mice given either enteral nutrition (Control) or intravenous nutrition (TPN). TPN significantly down‐regulated E‐cadherin and β‐catenin expression, and resulted in a loss of a colocalization of these factors. TPN also up‐regulated phosphorylated (p)‐β‐catenin (Ser31/33,Thr41) and down‐regulated (p)‐β‐catenin(Ser552) expression. To further address mechanisms driving this, we observed a significant decrease in the abundance of p‐AKT and p‐GSK3β expression, and an associated decline in tcf‐4 transcription factors (cyclin D1, c‐myc and Axin2), as well as a loss of EC proliferation by 7 days. To address whether the mechanism driving these changes was the absence of nutritional factors, glutamine was added to the TPN solution. This resulted in a partial restoration of β‐catenin expression and EC proliferation, suggesting that an alteration in nutrient delivery may affect many of these changes. Based on these findings, the loss of EC proliferation with TPN may well be due to a loss of total β‐catenin, as well as a concomintant change in the differential expression of β‐catenin phosphorylation sites, and a reduction in β‐catenin mediated tcf‐4 transcription. This potential pathway may well explain many of the findings of mucosal atrophy associated with TPN.

Enteral nutrient deprivation in patients leads to a loss of intestinal epithelial barrier function

OBJECTIVE To investigate the effect of nutrient withdrawal on human intestinal epithelial barrier function (EBF). We hypothesized that unfed mucosa results in decreased EBF. This was tested in a series of surgical small intestinal resection specimens. DESIGN Small bowel specifically excluding inflamed tissue, was obtained from pediatric patients (aged 2 days to 19 years) undergoing intestinal resection. EBF was assessed in Ussing chambers for transepithelial resistance (TER) and passage of fluorescein isothiocyanate (FITC)-dextran (4 kD). Tight junction and adherence junction proteins were imaged with immunofluorescence staining. Expression of Toll-like receptors (TLR) and inflammatory cytokines were measured in loop ileostomy takedowns in a second group of patients. RESULTS Because TER increased with patient age (P < .01), results were stratified into infant versus teenage groups. Fed bowel had significantly greater TER versus unfed bowel (P < .05) in both age populations. Loss of EBF was also observed by an increase in FITC-dextran permeation in enteral nutrient-deprived segments (P < .05). Immunofluorescence staining showed marked declines in intensity of ZO-1, occludin, E-cadherin, and claudin-4 in unfed intestinal segments, as well as a loss of structural formation of tight junctions. Analysis of cytokine and TLR expression showed significant increases in tumor necrosis factor (TNF)-α and TLR4 in unfed segments of bowel compared with fed segments from the same individual. CONCLUSION EBF declined in unfed segments of human small bowel. This work represents the first direct examination of EBF from small bowel derived from nutrient-deprived humans and may explain the increased incidence of infectious complications seen in patients not receiving enteral feeds.

Glutamate prevents intestinal atrophy via luminal nutrient sensing in a mouse model of total parenteral nutrition.

Small intestine luminal nutrient sensing may be crucial for modulating physiological functions. However, its mechanism of action is incompletely understood. We used a model of enteral nutrient deprivation, or total parenteral nutrition (TPN), resulting in intestinal mucosal atrophy and decreased epithelial barrier function (EBF). We examined how a single amino acid, glutamate (GLM), modulates intestinal epithelial cell (IEC) growth and EBF. Controls were chow‐fed mice, T1 receptor‐3 (T1R3)‐knockout (KO) mice, and treatment with the metabotropic glutamate receptor (mGluR)‐5 antagonist MTEP. TPN significantly changed the amount of T1Rs, GLM receptors, and transporters, and GLM prevented these changes. GLM significantly prevented TPN‐associated intestinal atrophy (2.5‐fold increase in IEC proliferation) and was dependent on up‐regulation of the protein kinase pAkt, but independent of T1R3 and mGluR5 signaling. GLM led to a loss of EBF with TPN (60% increase in FITC‐dextran permeability, 40% decline in transepithelial resistance); via T1R3, it protected EBF, whereas mGluR5 was associated with EBF loss. GLM led to a decline in circulating glucagon‐like peptide 2 (GLP‐2) during TPN. The decline was regulated by T1R3 and mGluR5, suggesting a novel negative regulator pathway for IEC proliferation not previously described. Loss of luminal nutrients with TPN administration may widely affect intestinal taste sensing. GLM has previously unrecognized actions on IEC growth and EBF. Restoring luminal sensing via GLM could be a strategy for patients on TPN.—Xiao, W., Feng, Y., Holst, J. J., Hartmann, B., Yang, H., Teitelbaum, D. H. Glutamate prevents intestinal atrophy via luminal nutrient sensing in a mouse model of total parenteral nutrition. FASEB J. 28, 2073–2087 (2014). www.fasebj.org