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Enteral Feeding of a Chemically Defined Diet Preserves Pulmonary Immunity but Not Intestinal Immunity: The Role of Lymphotoxin β Receptor

Kenneth A. Kudsk, MD^*,, F. Enrique Gomez, PhD, Woodae Kang, MD, PhD and Chikara Ueno, MD

From the ^* Veterans Administration Surgical Services, William S. Middleton Memorial Veterans Hospital, Madison, Wisconsin; and the Department of Surgery, University of Wisconsin–Madison College of Medicine and Public Health, Madison, Wisconsin

Correspondence: Kenneth A. Kudsk, MD, 600 Highland Avenue, H4/736 CSC, Madison, WI 53792-7375. Electronic mail may be sent to kudsk{at}surgery.wisc.edu.

Background: Compared with chow or a complex enteral diet (CED), IV administration of a parenteral nutrition solution (IV-PN) impairs intestinal and respiratory mucosal immunity, resulting in cellular and immunoglobulin A (IgA) defects in the intestine and impaired respiratory antiviral and antibacterial defenses. PN given intragastrically (IG-PN) impairs intestinal immunity similar to IV-PN but preserves antiviral defences and partially preserves antibacterial defenses. Lymphotoxin β receptor (LTβR) is a molecule essential for development and organization of lymphoid tissue. It controls many molecules important in mucosal immune integrity. This study examines effects of route (IV or enteral) and type (PN, CED, or chow) on murine intestine and lung LTβR expression. Methods: Forty-three mice randomly received IV-PN (n = 12), IG-PN (n = 11), IV saline + chow (chow; n = 11), or a CED (n = 9). After 5 days of feeding, intestinal and lung samples were obtained and processed for levels of LTβR by Western blot. Results: IV-PN significantly reduced intestinal and lung LTβR compared with CED and chow. IG-PN reduced LTβR levels only in the intestine but preserved lung levels. Conclusions: Route and type of nutrition differentially influence molecular events in the intestinal and respiratory mucosal immune systems. Enteral feeding with any diet (complex or chemically defined) maintains lung LTβR expression, whereas intestinal LTβR levels are maintained only with CEDs (chow and CED). We hypothesize that LTβR is responsible for the observed preservation of respiratory tract immunity with administration of a noncomplex, chemically defined enteral diet, whereas intestinal immunity is compromised with this diet.

Route and type of nutrition affect the histology and function of the mucosal-associated lymphoid tissue (MALT). As enteral stimulation increases, mucosal immunity retains more integrity. Our extensive animal work in mice demonstrates that enteral feeding (ENT) with either chow or a complex enteral diet (CED) maintains normal cellularity and function within the gut-associated lymphoid tissue (GALT).^1–6

Under normal conditions,⁷ naïve T and B cells destined for mucosal immunity enter Peyer's patches (PP) through interaction between ₄β₇ and L-selectin expressed on their cell surfaces and mucosal addresin adhesion molecule-1 (MAdCAM-1) expressed on the high endothelial venules of the PP. After sensitization to luminal antigens processed in the PP by antigen presenting cells, the sensitized T and B cells migrate to the mesenteric lymph nodes and the thoracic ducts. They are then distributed by the circulatory system to intestinal (GALT) and nonintestinal sites such as the upper and lower respiratory tracts.

Both chow and CED diets preserve normal total T- and B-cell numbers within the PP, and lamina propria (LP) maintain normal numbers of LP CD4⁺ cells and preserve the normal CD4/CD8 ratio of 2:1 in the LP¹ (Table I). Parenteral nutrition (PN; IV-PN) with lack of enteral stimulation exerts deleterious effects on almost all aspects of mucosal immunity. IV-PN with no enteral stimulation significantly reduces total T and B cells within the PP and LP, decreases CD4⁺ cells in the LP, and drops the CD4/CD8 ratio from 2:1 to 1:1 in LP.¹ In addition, whereas chow and CED maintain normal levels of 2 important Th-2-type immunoglobulin A (IgA) cytokines, interleukin (IL)-4 and IL-10, in the intestine, their levels plummet in parenterally fed mice.⁵ The functional effect of these changes is significant. Due to the crosstalk between the gastrointestinal (GI) and respiratory tracts, both intestinal and respiratory IgA levels drop.^1,2,5 As a result, IgA-mediated antiviral² and antibacterial defenses⁶ become severely impaired in IV-PN-fed animals. These IV-PN (with no enteral stimulation)-induced deficits readily reverse with just a few days of enteral stimulation with chow.⁸

There are a number of differences between the chow-, CED-, and IV-PN-fed groups. Although the CED and IV-PN diets are isocaloric and isonitrogenous, they are by no means equivalent in complexity (eg, the CED contains fat, complex proteins, and complex carbohydrates that are not present in the IV-PN). To control for these differences, we used a control group receiving the same PN solution administered intragastrically (IG-PN). This allows testing of the individual effects of route of nutrition (IG-PN vs IV-PN) and complexity of the diet (IG-PN vs CED). Chow represents the normal conditions of a complex diet and intermittent feeding to maintain normal host integrity. In essence, IG-PN is a chemically defined elemental diet that presumably is absorbed in the very proximal intestine. Because it contains no fat or complex proteins or carbohydrates, it produces less enteral stimulation than the CED or chow. Consistently, our results show that this degree of stimulation (chow > CED > IG-PN > IV-PN) is reflected in histologic and functional parameters of the GALT and MALT (Table I). Interestingly, although IG-PN fails to maintain normal T- and B-cell numbers in the PP and LP, the CD4/CD8 ratio in the LP, intestinal IL-4 levels, or intestinal IgA levels, respiratory IgA levels are maintained with IG-PN. This exerts a functional effect in that there is complete maintenance of IgA-mediated antiviral respiratory immunity and partial maintenance of antibacterial respiratory immunity. This disconnect between intestinal mucosal immunity and respiratory immunity is the focus of this paper.

Lymphotoxin-β receptor (LTβR) is a critical signaling molecule for the development, organization, and differentiation of lymphoid tissue.^9,10 The interaction between lymphotoxin (LT₁β₂) on activated T and B lymphocytes with LTβR activates intracellular NFB, which contributes to cell survival, cell migration, and other events. LTβR activation results in products that include IL-4 and adhesion molecules such as MAdCAM-1, which are important in cell entry into the GALT and subsequent distribution to peripheral tissues.¹¹ This work examines the effect of route and type of nutrition on LTβR expression in the gut and lung tissues in an attempt to examine why IG-PN maintains respiratory immunity but fails to maintain intestinal mucosal immunity.

	MATERIALS AND METHODS

Top MATERIALS AND METHODS RESULTS DISCUSSION

 
The Animal Care and Use Committee of the University of Wisconsin–Madison and the research committee of the Middleton Veteran's Administration Hospital approved all experimental protocols. Animals were housed in an American Association for Accreditation Laboratory Animal Care–accredited conventional facility. The environment was controlled for temperature and humidity, with a daily 12-hour light-dark cycle.

Animals, Cannulation and Feeding Regimens
Outbred male ICR mice 6 weeks of age, purchased from Harlan (Madison, WI), were fed ad libitum chow (Lab Diet 5001; PMI International, Brentwood, MO) and water for 2 weeks prior to protocol entry. Chow and IV-PN mice received catheters for IV infusion after intraperitoneal injection of a ketamine (100 mg/kg body weight) and acepromazine maleate (5 mg/kg body weight) mixture. A silicone rubber catheter (0.012-inch ID by 0.025-inch OD; Helix Medical, Inc, Carpinteria, CA) was inserted into the vena cava through the right jugular vein. The distal end of the catheter was tunneled subcutaneously and exited the midpoint of the tail. For IG feeding of IG-PN or the CED, mice received a gastrostomy catheter (0.020-inch ID and 0.037-inch OD; Helix Medical, Inc) fixed with a pursestring suture and tunneled as the IV catheter. Mice were partially immobilized by tail restraint to protect the catheter during infusion. Catheterized mice were immediately connected to an infusion apparatus, and 0.9% saline solution was infused at an initial rate of 4 mL/d with ad libitum access to chow and water. This technique of infusion in the mouse has proven to be an acceptable method of nutrition support and does not produce physical or biochemical evidence of stress.¹²

Mice were randomized to saline + chow (chow; n = 11), PN (IV-PN; n = 12), IG-PN (IG-PN; n = 11), or a CED (n = 9). Chow mice received 4 mL/d of saline, with free access to food and water throughout the length of the study. IV-PN mice initially received 4 mL/d of PN and advanced to a goal rate of 10 mL/d by the third day. The PN solution contains 6.0% amino acids, 34.9% dextrose (6002 kJ/L), electrolytes, and multivitamins, with a nonprotein calorie/nitrogen ratio of 535.8 kJ/g N. The IG-PN mice received 4 mL/d of the PN formula and advanced to a rate of 10 mL/d by the third day. CED mice received 4 mL/d of Nutren (Nestlé, Chicago, IL) via gastrostomy, with the rate increased to a goal of 14 mL/d by the third day. Nutren contains 12.7% carbohydrate, 3.8% fat, and 4% protein (4186 kJ/L), in addition to electrolyte and vitamins. The nonprotein calorie/nitrogen ratio of the CED is 549.4 kJ/g N, and therefore, PN and CED diets were almost isocaloric and isonitrogenous but not iso-osmolar. These feedings meet the calculated nutrient requirements of mice weighing 25–30 g. All animals had free access to water at all times.

Tissue Collection and Processing
After 5 days of dietary treatments, animals were anesthetized and exsanguinated by cardiac puncture. The small intestine was removed, and the mesenteric fat and external vasculature were dissected away. The intestine was washed with 20 mL cold calcium and magnesium-free Hanks' balanced salt solution (HBSS; BioWhittaker, Walkersville, MD), and the PP were removed from the serosal side. A 3-cm-long section of the middle small intestine (jejunum) was cut and flash frozen in liquid N₂. Next, the thoracic cavity was opened and the lungs were removed by cutting each one at the junction with the main stem bronchus and frozen immediately in liquid N₂. The intestine and lung samples were stored at –80°C until the levels of LTβR expression were measured by Western blot.

Quantitation of LTβR by Western Blot Analysis
The small intestine and lung samples were homogenized in 1 mL of RIPA lysis buffer (Upstate, Lake Placid, NY) with 1% proteases inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). The crude homogenate was kept on ice for 30 minutes and spun at 16,100 x g for 10 minutes at 4°C in a bench-top Eppendorf 5415 refrigerated centrifuge (Eppendorf, Hamburg, Germany). The supernatant was transferred to a clean tube, and the protein content was quantified with the dye-binding Bradford method.¹³ Twenty micrograms of protein was separated on a 10% PAGE under denaturing conditions (Tris 25 mmol/L, glycine 192 mmol/L, SDS 0.1%) at 150 volts (V) for 1 hour. After electrophoresis, the proteins were transferred to a 0.45-µm PVDF membrane (Immobilon-P; Millipore Corporation, Bedford, MA) at 4°C, 80 V for 45 minutes in transfer buffer (Tris 48 mmol/L, glycine 39 mmol/L, methanol 20%). The membrane was washed 3 times, for 5 minutes each wash, with TBS-Tween (Tris-buffered saline plus 0.05% Tween-20) and was blocked for 3 hours with blocking solution (5% non-fat dry milk in TBS-Tween) with constant agitation at room temperature. The membrane was incubated with the primary antibody, goat antimouse LTβR (R&D Systems, Minneapolis, MN) diluted 1:100 in blocking solution, overnight at 4°C with constant agitation. The membrane was washed as before and was incubated 1 hour at room temperature with the secondary antibody: rabbit antigoat IgG-HRP conjugate (Santa Cruz Biotechnology, Inc, Santa Cruz, CA) diluted 1:40,000 in blocking solution.

Levels of LTβR protein were detected by electrochemiluminescence (ECL) with the Supersignal West-Femto Maximum Sensitivity Substrate (Pierce Biotechnology, Inc, Rockford, IL), and the pixel intensities were quantitated using the TotalLab gel imaging analysis software (Nonlinear Dynamics, Durham, NC).

Statistical Analysis
Levels of LTβR protein are presented as mean ± SEM. Statistical analysis was performed with Statview 5.0 (SAS Institute, Cary, NC). Groups were compared with ANOVA followed, in case of significance, by Fisher's protected least significant difference post hoc test. Significance was set at p < .05.

	RESULTS

Top MATERIALS AND METHODS RESULTS DISCUSSION

 
Body Weight
There were no significant differences in initial body weight. Mice fed CED, IV-PN, or IG-PN lost significantly more weight than chow-fed mice (Table II), but there were no differences between those 3 groups. Chow mice have approximately 1.5 g of residual feces, whereas the GI tracts of IV-PN and IG-PN mice are empty. CED animals hold approximately 0.5 g of feces. Therefore, body weight differences are exaggerated in the chow group. No enterally fed mice developed diarrhea at any time.

Effect of Type and Route of Nutrition on LTβR Levels
Route and type of nutrition affected levels of LTβRin the lung and intestine differently. Pixel level intensities of LTβR protein levels in the intestine of IV-PN and IG-PN animals (111 ± 40 and 112 ± 12 units, respectively) were significantly lower than those in animals fed CED (168 ± 23 units) or the chow group (168 ± 14 units; p < .05; Figure 1). Interestingly, pixel level intensity of levels of lung LTβR were significantly reduced only in the IV-PN group (127 ± 17; p < .05). Normal levels were maintained in the IG-PN, CED, and chow-fed mice (160 ± 41,153 ± 29, and 157 ± 20, respectively).

View larger version (15K): [in this window] [in a new window]   FIGURE 1. Effect of route and type of nutrition on lymphotoxin-β receptor levels in the intestine and the lung (mean ± SEM).

	DISCUSSION

Top MATERIALS AND METHODS RESULTS DISCUSSION

This system of mucosal immune cell sensitization and movement is directly influenced by LTβR, a molecule expressed in the stroma of PP and LP.¹⁵ Expression of both MAdCAM-1 and tissue specific chemokines (which augment diapedesis of the attached T and B cells into the PP) is controlled by LTβR stimulated by lymphotoxin expressed on activated T and B cells.⁹ LTβR activation stimulates differentiation of dendritic cells, which process antigen for presentation to and sensitization of the naïve T and B cells in the PP.⁹ LTβR activation also augments IL-4 levels, an important Th-2-type IgA-stimulating cytokine, which drops within the intestine with either IG- or IV-PN. Within the LP, LTβR is important for IgA production.¹⁶

Our previous work focused on the effects of route and type of nutrition on intestinal mechanisms of mucosal integrity, in particular on adhesion molecules and Th-2-type cytokines which modulate mucosal immunity within the intestine.^3–5,14 PP MAdCAM-1 expression is proportional to the degree of ENT stimulation (chow > CED > IG-PN > IV-PN). As PP MAdCAM-1 expression decreases, total T- and B-cells lymphocytes simultaneously decrease within the PP, LP, and intraepithelial spaces of the intestine. We know that MAdCAM-1 is clearly important in cell entry and migration because blockade of this molecule with anti-MAdCAM-1 antibody reproduces the same cellular changes in GALT as IV-PN (with lack of enteral stimulation) despite chow feeding.³ Intestinal IL-4, a Th-2-type cytokine that drops with lack of enteral stimulation, plays 2 roles in the intestinal tract. First, IL-4 is a costimulant (with LTβR) of MAdCAM-1 expression in the PP.¹⁷ Second, IL-4 is an important Th-2 IgA- stimulating cytokine for production of IgA by the B cells (which transform into plasma cells) in the LP to maintain immunologic integrity.⁵

Both IL-4 and MAdCAM-1 levels are products of LTβR activation.¹¹ We confirmed that altered intestinal LTβR levels are associated with, and under experimental conditions of exogenous stimulation and blockade replicate, these nutrition-related alterations. We previously showed that (1) IV-PN with lack of enteral stimulation is associated with reduced LTβR protein and mRNA in PP and the intestine compared with chow,¹³ (2) agonistic antibody stimulation of LTβR in IV-PN-fed mice results in normal PP cell numbers and intestinal IgA levels,¹³ and (3) blockade of LTβR with a fusion protein reduces MAdCAM-1 mRNA levels and cells within the PP of chow-fed mice receiving maximal enteral stimulation.¹⁸

The current work expands our knowledge of LTβR's role in the respiratory tract. It at least partially explains the discrepancy between the ability of an enterally administered, chemically defined PN diet to maintain respiratory (but not intestinal) mucosal immune defenses while there is complete loss of both respiratory and intestinal immunity with a parenteral administration of that same diet. Our work concludes that IG-PN preserves LTβR expression within the lung but not within the gastrointestinal tract. As a result, IG-PN preserves mucosal immunity within the respiratory tract, without preserving intestinal mucosal immunity.

There is a very important limitation in the interpretation of these data. It is unclear whether the reduced lung LTβR is due to direct pulmonary toxicity of the PN solution when administered intravenously or due to the stimulation of pulmonary LTβR expression with IG-PN. PN administered intragastrically does not stimulate normal GALT histology and function. Because the nutrients are processed via the splanchnic system, it eliminates any potential pulmonary toxicity. However, the lung is directly perfused with this solution when administered intravenously. Although it is not surprising that the gastrointestinal tract mucosal immunity deteriorates with the lack of ENT during parenteral feeding, one cannot dismiss the concept that the PN solution itself has some direct toxic effect on mechanisms that maintain or stimulate LTβR levels within this bed. Our inclination is to believe that it is not a direct toxic effect, because the supplementation of IV-PN with 2% glutamine or IV or subcutaneous administration of the intestinal neuropeptide bombesin (an analog of gastrin releasing peptide) either partially (glutamine) or completely (bombesin) abrogates deleterious effects of the solution in both the gastrointestinal and respiratory tracts. Thus, it is unlikely that either the dextrose or the amino acid component of PN is toxic to the respiratory tract, but rather that there is loss of some intestinal product of enteral stimulation that maintains respiratory immunity. This is consistent with our general observations that as the complexity of diet increases (chow > CED > IG-PN > IV-PN), both intestinal and respiratory mucosal immunity improve toward the normal state (chow feeding). Further work is necessary to unravel this confounding variable.

This work shows that the route and type of nutrition influence different molecular events in the intestinal and respiratory mucosal immune system. ENT of chow, CED, or the less complex IG-PN maintains pulmonary LTβR expression, whereas IV-PN does not. Conversely, intestinal LTβR levels are maintained only with a CED. We conclude that expression of LTβR is at least partly responsible for the preservation of respiratory tract immunity when intestinal immunity is severely compromised with IG-PN.

This article was supported by National Institutes of Health grant R01 GM53439.

Received for publication February 25, 2007. Accepted for publication May 4, 2007.

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Journal of Parenteral and Enteral Nutrition, Vol. 31, No. 6, 477-481 (2007)
DOI: 10.1177/0148607107031006477


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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via Google Scholar Citing Articles via Scopus Google Scholar Articles by Kudsk, K. A. Articles by Ueno, C. Search for Related Content PubMed PubMed Citation Articles by Kudsk, K. A. Articles by Ueno, C. Social Bookmarking                   What's this?