MOLECULAR STRUCTURE AND FUNCTION OF THE TIGHT JUNCTION

Enteral versus Parenteral Nutrition: Effecton Intestinal Barrier Function

Hua Yang, M.D., Ph.D.,a,b Yongjia Feng, M.D., Ph.D.,a

Xiaoyi Sun, M.D., Ph.D.,a and Daniel H. Teitelbaum, M.D.a

aDepartment of Surgery, the University of Michigan Medical School, Ann Arbor, MichiganbDepartment of General Surgery, Xingqiao Hospital, Chongqing, China

Total parenteral nutrition (TPN), or the complete absence of enteral nutrients, is com-monly used in a clinical setting. However, a major consequence of TPN administration isthe development of mucosal atrophy and a loss of epithelial barrier function (EBF); andthis loss may lead to an increase in clinical infections and septicemia. Our laboratory hasinvestigated the mechanism of this TPN-associated loss of EBF using a mouse model.We have demonstrated that the mucosal lymphoid population significantly changeswith TPN, and leads to a rise in interferon gamma (IFN-γ) and decline in interleukin-10(IL-10) expression—both of which contribute to the loss of EBF. Associated with thesecytokine changes is a dramatic decline in the expression of tight junction and adherensjunction proteins. This article discusses the potential mechanisms responsible for thesechanges, and potential strategies to alleviate this loss in EBF.

Key words: epithelial barrier function; tight junction; intraepithelial lymphocyte;interleukin-7 (IL-7); interferon gamma (IFN-γ); IL-10; mouse, epithelial cells

Introduction

The intestinal epithelium undergoes bothmorphological and functional changes dur-ing total parenteral nutrition (TPN) admin-istration. Many patients, because of eithergastrointestinal dysfunction or a lack of intesti-nal length, are unable to tolerate enteral feed-ings. These patients are confined to a prolongedcourse of TPN. It is estimated that over 250,000patients receive TPN in the United States aloneon a yearly basis,1 resulting in over 11.5 millionpatient days of care per year. Increasingly, inves-tigators have found that the intestine undergoessignificant changes during the administrationof TPN with an absence of enteral nutrition.In several TPN animal models, the intestinalepithelium shows a loss of villus height and adecline in epithelial growth.2,3 Further analysis

Address for correspondence: Daniel H. Teitelbaum M.D., Section ofPediatric Surgery, University of Michigan Hospitals, Mott F3970, Box0245, Ann Arbor, Michigan 48109. Voice: +734-936-8464 (office); fax:+734-936-9784. dttlbm@umich.edu

of the mucosa shows a loss of epithelial bar-rier function (EBF) during the administrationof TPN.4 This loss of barrier function may re-sult in endotoxins and even bacteria enteringthe systemic circulation.

Clinical Consequencesof a Loss of Epithelial Barrier

Function with TPN

Loss of EBF with TPN may well be a poten-tial mechanism for the observed higher ratesof septicemia and other clinical infectious pro-cesses. Significantly higher rates of sepsis andinfection have been strongly associated withTPN in the clinical settings of surgery andtrauma.5,6 The etiology of these increased ratesof infection is controversial. Systemic immuno-logic changes are quite common during theadministration of TPN, and include both adecrease in absolute lymphocyte counts and adepression in lymphocyte function.7–9

Molecular Structure and Function of the Tight Junction: Ann. N.Y. Acad. Sci. 1165: 338–346 (2009).doi: 10.1111/j.1749-6632.2009.04026.x c© 2009 New York Academy of Sciences

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Yang et al.: Parenteral Nutrition and Barrier Function 339

Use of a TPN Mouse Model toStudy Mucosal Atrophy andEpithelial Barrier Function

The mouse model of TPN has yielded valu-able insights and may actually more closely re-semble clinical TPN administration than otheranimal models. Previous studies with TPN inrat models showed a rapid progression of vil-lus atrophy within a matter of 3 days.10 Suchchanges do not occur as rapidly in humans re-ceiving TPN, and thus must be carefully inter-preted. Mouse models of TPN show the devel-opment of villus atrophy and formation of anepithelial barrier leak in a delayed period ofonset (twice as long as rats), and more similar(although still more rapid) to humans.

TPN Administration Results in a Loss ofMucosal Barrier Function

Loss of EBF has been identified both in ex-perimental models of TPN3,10,11 and in hu-mans.12 The mechanism by which TPN leadsto a loss of epithelial integrity has only re-cently been approached. In vitro cultures of in-testinal epithelial cells (EC) have given criticalinsight into these mechanisms. A number ofcytokines influence EBF. This was first shownin human intestinal EC (T84 cell line) incu-bated with interferon gamma (IFN-γ).13 Usinga similar in vitro model, others have shown thatpretreatment with transforming growth factorbeta 1 (TGF-β1) can prevent the effects of IFN-γ.14 A number of other cytokines have alsobeen shown to affect EBF. Similar to TGF-β1, interleukin-10 (IL-10) can attenuate the lossof epithelial barrier in T84 monolayers causedby IFN-γ.15 Interleukin-4 (IL-4), however, canlead to a loss of EBF in a similar in vitro model.16

Other cytokines which have been associatedwith a loss of EBF include interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α).17

Loss of barrier function with TPN appears tobe closely associated with the increased expres-sion of IFN-γ by intraepithelial lymphocytes(IEL).11,18

Gut-Associated Lymphoid Tissue

The gastrointestinal tract is exposed to anumber of foreign antigens on a daily ba-sis, including toxins, bacteria, viruses and anumber of diverse enteral nutrients. A criticalpart of protecting the host from these factorsis the gut-associated lymphoid tissue (GALT).The GALT is a complex immunologic organthat contains several layers within the intesti-nal tract.19–21 The layer of the GALT closest tothe lumen is the IEL. The IEL has a uniqueT-cell phenotype and plays a critical role inboth proliferative and cytotoxic T-lymphocyteactions.22,23 The IEL phenotype predomi-nately consists of CD4−,CD8+ (70–85%, formice, single positive), with other less frequentsubpopulations: CD4−,CD8− (5–10%, doublenegative); CD4+,CD8− (5–12%, single posi-tive); and CD4+,CD8+ (4–10%, double posi-tive).24,25 The IEL also has a large number ofγδ-TCR+ cells (30–70%, compared to less than2% in peripheral blood lymphocytes of mice).26

The IEL is thought to be derived from both athymic-dependent and a thymic-independentsource. Although still controversial,27 studiessuggest that the IEL originate from both theintestinal tract and the thymus.27,28 RAG2−/−

mice injected with bone marrow of nude micefailed to develop peripheral T-cells but did de-velop a nearly normal IEL population, whichcontained CD8+ T-cells with both γδ- or αβ-TCR+ phenotype.29 Although still controver-sial, the thymic-independent portion of the IELis best defined by the CD8αα (homodimeric)T-cell population. In contrast, CD8αβ (het-erodimeric) cells are believed to be of thymicorigin. The function of the IEL has not beencompletely determined. Using a redirected cy-totoxic functional assay without in vitro activa-tion, the IEL shows considerable activity.30 Theactual significance of these actions has not beencompletely defined but suggests a role in pro-tecting the organism from harmful intralumi-nal pathogens. The IEL is also a rich sourceof cytokines, including: IFN-γ,31 IL-2,32 IL-4,33 IL-6,34 TNF-α,35 and TGF-β1.36 TGF-β1

340 Annals of the New York Academy of Sciences

has been detected in both αβ-TCR+ and γδ-TCR+ IEL subsets.37 IL-10 has recently beenshown to be expressed in the IEL, and mayclosely relate to the modulation and functionof T-regulatory cells.38 Cytokine production inthe αβ-TCR+ fraction of the IEL has been ex-tensively studied, and the CD4+,CD8− pop-ulation expresses IFN-γ, IL-4 and IL-5.37,39

The CD4+,CD8+ population expresses greateramounts of IFN-γ and IL-5. The CD4−,CD8+

population produces IFN-γ, IL-5 and someIL-6. It appears that a major aspect of IELregulatory function is achieved by specific cy-tokine production. It is hypothesized that IELcytokine production leads to modulation in ep-ithelial cell function,40 as well as to a downreg-ulation of immunologic sensitization to foreignantigen.33

Intraepithelial LymphocytesClosely Interact with Mucosal

Epithelial Cells

Because of the close physical association ofthe IEL with mucosal EC, interactions betweenthe two populations have been examined. ECexpress essential ligands which are needed forIEL to adhere and reside within the mucosalepithelium.41 Interestingly, these ligands are ex-pressed along the basolateral surface of the EC,in juxtaposition to IEL. Perhaps the best char-acterized of these is the integrin αEβ7 on theIEL and E-cadherin on the surface of the ep-ithelial cells.42 Another important manner inwhich the IEL and EC intercommunicate isvia the large number of cytokines, growth fac-tors and chemokines which are expressed byEC. One such example is that EC-derived IL-7 strongly influences the development, growthand phenotype of the adjacent IEL.43 IL-7−/−

or IL-7R−/− mice show distinct declines in ab-solute numbers of thymocytes and IEL, with avirtual absence of the γδ-TCR+ population,and fewer αβ-TCR+ T cells than wild-typemice.44

Changes in the IntraepithelialLymphocytes with Total Parenteral

Nutrition Administration

Phenotypic Changes

A number of IEL phenotypic alterations oc-cur with TPN, and include a marked decline inthe CD4+, CD44+, and CD8αβ+ populations(Table 1). The CD4+ population is known to bevery responsive to exogenous stimulation, andits loss may explain an observed loss of IELproliferative responsiveness with TPN.31 Theloss of the CD8αβ+ population with TPN mayrepresent a loss of the thymic-dependent pop-ulation of the IEL,29 and the loss of CD44+

cells suggests a shift to a less mature IEL.45 In-terestingly, the alteration in IEL phenotype didnot correlate with enteral anaerobic or aerobicbacterial cultures, in that the number and strainof these bacteria did not significantly changewith TPN.31 An additional group is shown inTable 1—that of TPN mice allowed smallamounts of enteral food (TPN + Food group;comprising 25% of caloric needs). Interestingly,IEL phenotype changes were significantly in-fluenced by the loss of enteral nutrition; mostof the observed IEL changes with TPN werereversed with the addition of this small amountof enteral nutrition. The mechanism by whichenteral food prevents the observed IEL changeshas not been determined.

Cytokine Expression

Table 2 shows changes in cytokine expressionwith TPN.31,36,46 This panel of cytokines waschosen because each of these has been shownto alter either barrier function or apoptotic sig-naling in the intestine.15,16,47–51 Our labora-tory reported a rise in IFN-γ expression withTPN, and this could well impact both the ob-served increase of EC apoptosis52 and a loss ofEBF (see below). In addition to IFN-γ, othercytokines may also have relevance to EC physi-ology, including IL-4, IL-6 and IL-10 (Table 2).As mentioned above, each of these cytokines

Yang et al.: Parenteral Nutrition and Barrier Function 341

TABLE 1. Small Bowel IEL Phenotype in Control and TPN Mice

IEL Phenotypes Description Control (range) TPN (range) TPN + Food (range)

CD4 and CD8α

CD4+CD8− single positive 4.7 (3.9–6.1) 0.6 (0.2–0.7)∗

2.5 (1.9–2.8)CD4+CD8+ double positive 2.8 (2.5–4.3) 0.6 (0.1–1)

∗5.4 (3.8–12.1)

CD4−CD8− double negative 28 (23–34) 35 (19–64) 16 (12–28)CD4−CD8+ single positive 65 (58–70) 64 (37–79) 72 (67–77)

CD8αα and CD8αβ

CD8αα+ thymic-independent 64 (59–71) 52 (40–63) 72 (66–78)CD8αβ+ thymic-dependent 4.8 (3.0–6.5) 0.4 (0.1–0.8)

∗7.4 (5.6–8.6)

TCR-αβ vs. TCR-γδ

TCR-αβ+ T-cell receptor 39 (33–43) 22 (21–31) 24 (18–28)TCR-γδ+ T-cell receptor 62 (53–68) 70 (57–76) 57 (51–62)CD44 T-cell maturity

CD8+,CD44+ 29 (28–37) 10 (1.0–18)∗

40 (38–46)

Column on right are mice maintained on TPN and allowed 25% of their nutritional intake via the enteral route.Results are from flow cytometry studies after gating on the IEL population. CD4,CD44 results not shown because ofthe marked loss of the CD4+ population with TPN. Results are expressed as mean% gated IEL (ranges) and representsan N = 6 mice for each group.

∗P < 0.01, by ANOVA.

TABLE 2. mRNA Expression of IEL Cytokines in Control, TPN and TPN + Food Groups

Groups IL-4 IL-6 IL-10 IFN-γ

Control 0.10 ± 0.1 0.02 ± 0.0 0.55 ± 0.2 0.15 ± 0.1TPN 0.73 ± 0.3

∗ ∗0.95 ± 0.3

∗ ∗0.23 ± 0.1

∗ ∗0.29 ± 0.1

∗

TPN + Food 0.11 ± 0.1 0.05 ± 0.0 0.65 ± 0.2 0.14 ± 0.0

Note the significant alterations in all measured cytokines with administration of TPN, and the prevention of thesechanges with the addition of enteral feedings to TPN mice. N = 6,

∗P < 0.05, ∗∗P < 0.01 TPN versus Control and

TPN + Food; using ANOVA. In general, a Bonferonni post hoc t test is used for remaining preliminary results. Resultsare the mean (±SD) mRNA expression of cytokine from purified IEL (via magnetic beads) samples, and expressed asthe ratio of cytokine expression to beta actin. (Ref. 55)

can contribute to changes in EBF. It is possi-ble that the decline in IL-10 mRNA expres-sion and the rise in IL-4 and IL-6 may allcontribute to the development of the TPN-associated epithelial leak and increased ratesin EC apoptosis. The administration of 25%enteral nutrition to TPN mice prevented thesecytokine changes. As mentioned earlier, EC-derived IL-7 is another important cytokinewithin the intestinal mucosa. Our laboratoryhas shown a close physical relation between ECthat express IL-7 and IEL.53 Subsequently, weshowed that the administration of TPN resultsin a significant decline in IL-7 expression.54

With the administration of exogenous IL-7,much of the IEL phenotypic changes associatedwith TPN can be prevented,54 suggesting that

IL-7 may have an important role in the media-tion of the changes to the mucosa with TPNadministration. Based on these observations,our group developed a transgenic mouse withintestinal epithelial cell overexpression of IL-7.These mice failed to undergo the loss of IELand phenotype changes observed in wild-typemice.56

Impact of IntraepithelialLymphocytes–derived Cytokinesand Epithelial Barrier Function

TPN-associated increases in IEL-derivedIFN-γ and decline in IL-10 expression haveboth been associated with a loss of EBF.11,18,57

342 Annals of the New York Academy of Sciences

Figure 1. Transepithelial resistance (TER) in micewas measured in Ussing chambers after a 30-minuteequilibration period. Results (mean ± SD) in sixmice per group. TPN significantly decreased in-testinal TER in wild-type mice. Elimination of IFN-γwas achieved using knockout mice (IFNKO). Signif-icance was found between the TPN and all otherstudy groups; the other groups were not signifi-cantly different among themselves. ANOVA was usedfor statistical analysis. ∗P < 0.05 comparing TPNgroup to other groups, with a Bonferonni post hocanalysis.

A significant decline in transepithelial resis-tance (TER) of full-thickness ileum is notedin the TPN group. In order to determine theinfluence that increased expression of IFN-γhad on this change in EBF, a series of IFN-γknockout mice underwent TPN administra-tion. In the absence of IFN-γ, the degree ofTPN-associated epithelial barrier breakdown,as measured by either the permeability coef-ficient (Papp) or by transepithelial resistance(TER) (Fig. 1), was significantly lessened.46 Thefact that permeability levels did not completelyreturn to normal suggested that other media-tors of this breakdown exist. Whether this is adirect action of IFN-γ on the EBF, or if acts toprime tumor necrosis factor receptors remainsto be determined.58 Subsequently, our groupthen determined the influence of the declinein IL-10 with TPN administration. Sun et al.

demonstrated that the additional administra-tion of exogenous IL-10 to mice receiving TPNresulted in a return of EBF toward normal.57

Similar to the IFN-γ mice, exogenous IL-10partially prevented this barrier loss; however,the effect was only about 50% effective. This

Figure 2. Immunofluorescent images of represen-tative portions of mid-small bowel mucosa in control(enterally fed) and TPN study groups. Note the loss ofjunctional and adherens proteins with TPN. Addition-ally, note the movement of some of these proteins (oc-cludens) into the cytoplasm with TPN administration.

suggests that the cytokine changes with TPNadministration account for a substantial degreeof EBF loss.

Alteration in the Tight Junctionwith Total Parenteral Nutrition

The etiology of this loss of EBF was furtherinvestigated. Our laboratory detected a sub-stantial decline in the expression of a numberof tight junction molecules,57 including ZO-1,occluden, several claudins and JAM-1, as wellas the adherens molecule E-cadherin. Figure 2shows immunofluorescent images of some ofthese factors. In addition to a loss of expres-sion, internalization (endocytosis) of some ofthese factors can be seen—a finding similarlyobserved in vitro with cytokine exposure to ep-ithelial monolayers, as well as other clinical dis-ease processes where EBF is lost, such as ininflammatory bowel disease.50,59,60

Yang et al.: Parenteral Nutrition and Barrier Function 343

Figure 3. Effect of TPN on the permeability co-efficient (Papp) of tritiated mannitol as measure inUssing chambers of full-thickness ileal specimens withTPN administration (∗P < 0.05). Note also the returnof permeability to control levels when TPN mice aregiven exogenous keratinocyte growth factor (KGF)(5 mg/kg/day).64

Therapeutic Options to PreventBarrier Loss with Total Parenteral

Nutrition

The IEL not only expresses a number ofcytokines, but the γδ-TCR subpopulation hasbeen shown to express keratinocyte growthfactor (KGF, or fibroblast growth factor-7).61

Our group has subsequently demonstrated thatwith TPN administration, there is a markeddecline in KGF expression by this IEL sub-population.62 Further, the exogenous admin-istration of KGF to mice receiving TPN wasable to significantly prevent loss of villus height,crypt depth, and restored EBF (Fig. 3).63 Thissuggests that the use of KGF may have a thera-peutic potential for patients on long-term TPNand may prevent many of the complicationsassociated with EBF loss.

Conclusions

TPN results in a loss of villus height, loss ofEC proliferation, rise in EC apoptosis, and amarked decline in EBF. Using a mouse modelof TPN, the IEL has been shown to undergosignificant changes. Such changes may partially

account for the mechanism of EBF loss withTPN, and may contribute to the increased in-cidence of septicemia with TPN administra-tion. The mechanisms by which IEL changewith TPN are not known, but data suggeststhat such changes are due to a lack of enteralnutrition and may be mediated by a declinein EC-derived IL-7. Understanding the mech-anisms that are responsible for such changesmay lead to the potential for therapeutic op-tions in patients receiving TPN. Further, un-derstanding the changes in the IEL with TPNadministration may also yield important infor-mation in how EC-IEL interactions take place.Such interactions appear to directly influenceobserved alterations in the physiology of theintestinal mucosa. Thus, these cell-cell interac-tions may have important applications in otherprocesses, such as gut adaptation after bowel re-section, or during autoimmune processes, suchas inflammatory bowel disease.

Acknowledgments

This research was supported by NIH grant2R01-AI044076-10 (to DHT), and the ASPENRhoads Research Foundation Maurice ShilsGrant (to HY), and the University of MichiganDNA Core, University of Michigan TransgenicCore. National Cancer Institute through theUniversity of Michigan’s Cancer Center Sup-port Grant (5 P03 CA46592).

Conflicts of Interest

The authors declare no conflicts of interest.

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