Decreased Enteral Stimulation Alters Mucosal Immune Chemokines

By Hermsen, Joshua L Gomez, F Enrique; Maeshima, Yoshinori; Sano, Yoshifumi; Kang, Woodae; Kudsk, Kenneth A

ABSTRACT. Background: Migration of lymphocytes into and through the mucosal immune system depends upon adhesion molecules to attract circulating cells and chemokines to stimulate diapedesis into tissues. Decreased enteral stimulation significantly reduces mucosal addressin cellular adhesion molecule-1 (MAdCAM-1) levels, an adhesion molecule critical for homing of T and B cells to Peyer’s patches (PP), which reduces PP and intestinal T and B cells. We studied the effect of type and route of nutrition on tissue specific chemokines in PP (CXCL-12, -13 and CCL-19, -20 and -21), small intestine (SI; CCL-20, -25 and -28) and lung (CXCL-12, CCL-28). Methods: Intravenously cannulated male Institute of Cancer Research (ICR) mice were randomized to chow or parenteral nutrition (PN) for 5 days. PP, SI, and lung chemokine mRNA levels were measured using real-time qRT-polymerase chain reaction, and analyzed semiquantitatively by the DeltaDeltaCt method. Protein levels were quantified using enzyme-linked immunosorbent assay (ELISA) techniques, and groups compared using Student’s t-test. Results: PP CXCL13 protein significantly decreased, whereas CCL21 protein increased significantly in the parenterally fed group. Parenteral feeding significantly decreased SI CCL20 and CCL 25 protein levels. CCL28 decreased significantly in the SI and lung of intravenously fed animals. mRNA levels changed in the opposite direction (compared with protein) for all chemokines except CCL28. Conclusions: Decreased enteral stimulation significantly alters key mucosal immune chemokine protein levels at multiple sites. In general, PN (and concomitant lack of enteral stimulation) results in decreased levels of chemokines that control lymphocyte migration within the mucosal immune system. (Journal of Parenteral and Enteral Nutrition 32:36-44, 2008) Several prospective randomized clinical trials and a meta-analysis demonstrate that severely injured trauma patients fed parenterally experience increased infectious morbidity compared with enterally fed or starved patients.1-4 Specifically, rates of pneumonia increase with starvation or parenteral feeding compared with enteral feeding. One cogent hypothesis for this finding proposes that decreased enteral stimulation (DES), experienced while patients receive parenteral or no nutrition, leads to phenotypic change and dysfunction of the mucosal immune system. These changes increase susceptibility of patients to infection.

The mucosal immune system consists of a bodywide network of specialized tissues and cells that function to provide antigen- specific antibody-mediated immune defense at all internal body/ environment interfaces. Collectively, these cells and tissues are referred to as mucosa-associated lymphoid tissue (MALT). Vaccination experiments in which vaccine delivered at 1 mucosal surface causes a vaccine-specific immune response detectable at separate, nonvaccinated mucosal sites provide functional evidence of linkage and communication between mucosal sites.5-8

Previous work in our laboratory and others demonstrates specific mucosal immune deficits in experimental models of DES during parenteral nutrition (PN).9-17 We use these terms interchangeably as evidence suggests that the lack of enteral stimulation, not the PN solution itself, is responsible for inducing change.18 Gut- associated lymphoid tissue (GALT) T-and B-cell mass decreases and the phenotype of these cell populations change with DES/PN.19 Simultaneously, specific lymphocyte cellular adhesion molecules in Peyer’s patches (PP)13,14 and gut epithelial immunoglobulin transport proteins20 decrease with DES/PN. As a result, levels of secretory immunoglobulin A (SIgA), the principal specific immune defense at mucosal surfaces, decrease19 and animals display impairment in established antiviral and antibacterial defenses.21,22 They also lose the ability to generate effective new responses against infectious challenges at mucosal sites.23 These observations are consistent with the increased infections observed in clinical trials.

The mucosal immune system (Figure 1) is composed of inductive and effector sites. PP represent inductive sites, where naive lymphocytes first encounter and are sensitized to antigens. Those lymphocytes then migrate through mesenteric lymph nodes and the thoracic duct into the systemic circulation. The antigen-specific lymphocytes home to, and populate effector sites in the lamina propria underlying mucosal sites where their main function is production of antigen-specific IgA.24

FIGURE 1. Schematic overview of mucosal immune system cellular trafficking highlighting the major known roles of chemokines. IgA, immunoglobulin A; SIgA, secretory IgA.

Multiple mechanisms control mucosal immune lymphocyte homing and trafficking into and through inductive and effector sites. Naive T and B cells enter the MALT through the PP.25 Initial interaction between L-selectin and alpha4beta7 integrin on naive lymphocytes and mucosal addressin cellular adhesion molecule-1 (MAdCAM-1) expressed on the high endothelial venules in PP tether the cells, causing them to “roll” along the endothelium.26 After tethering, chemokine receptor activation causes firm arrest and diapedesis of cells into the tissue.27 Chemokines are small (generally < 12 kDa), soluble, tissue-specific proteins that form a complex with their respective receptor(s) to activate cell signaling systems,28,29 increase integrin-cellular adhesion molecule binding avidity,30 induce changes in lymphocyte polarity,31 and stimulate chemotaxis.29,32 In addition, some chemokines, CCL28 for example, also have direct antimicrobial properties.33

Because chemokines direct lymphocyte movement from the blood into the mucosal immune tissues and because DES and PN affect many other mucosal immune variables, we studied the effect of DES/PN on mRNA and protein levels of key mucosal immune chemokines (Table I) within the PP, small intestine (SI), and lung.

MATERIALS AND METHODS

Animals

The Animal Care and Use Committee of the University of Wisconsin- Madison, and the Middleton Veterans Administration Hospital in Madison approved all protocols. Male Institute of Cancer Research (ICR) mice (Harlan, Indianapolis, IN) were housed in an American Association for Accreditation of Laboratory Animal Care-accredited facility. Mice were housed in an environment controlled for temperature, humidity and light (12-hour light:dark) and fed ad libitum chow (LabDiet; PMI Nutrition International, St. Louis, MO) and water for 1 week before initiation of study protocol. After entry into study protocol, mice were housed individually in metal metabolism cages with wire grid floors to prevent coprophagia.

TABLE I

Chemokines assayed

Experimental Protocol

One hundred forty-three male ICR mice aged 6-8 weeks were randomized to chow (n = 54) or PN (n = 89). Per experimental protocol, mice were anesthetized and centrally cannulated via the external jugular vein (catheter dimensions = 0.012-inch ID and 0.025- inch OD; Helix Medical, Inc, Carpinteria, CA). Catheters were tunneled subcutaneously over the back and exited midtail. Mice were restrained by the tail, which has been shown not to induce significant physical or biochemical stress.34

After catheterization, mice were connected to infusion pumps and recovered for 48 hours while receiving 4 mL/d of 0.9% saline to maintain open lines; mice had free access to food and water during the recovery period. Study diets were initiated after the recovery period. Chow mice received 0.9% saline at 4 mL/d and had free access to chow and water throughout the study. PN mice received solution at 4 mL/d (day 1), 7 mL/d (day 2), and 10 mL/d (days 3-5). 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 nitrogen. PN mice had free access to water only during the study period.

After 5 days of feeding, mice were anesthetized and exsanguinated by cardiac puncture. The SI was removed by dissection from mesenteric fat, lymph nodes, and vasculature. Twenty milliliters of Hanks’ balanced salt solution (HBSS; Bio Whittaker, Walkersville, MD) was irrigated through the intestinal lumen to obtain intestinal washing specimens. After washing, PP were sharply dissected from the intestine. The SI was sectioned and 2-cm portions of midjejunum were snap frozen in liquid nitrogen. Protein extracts from these full- thickness 2-cm-long jejunal sections were used for small-intestinal analysis. The chest was opened and lung tissue sharply removed and washed in HBSS. All samples were snap frozen in liquid nitrogen immediately after sectioning and stored at -80[degrees]C until assayed. Samples for this experiment were randomly selected from the available banked specimens.

RNA Extraction

Tissues were homogenized in 1 mL Trizol Reagent (Invitrogen, Carlsbad, CA), followed by chloroform extraction and precipitation with isopropanol. The resultant RNA pellet was dissolved in diethyl pyrocarbonate-treated water (DEPC-H2O). All samples were assayed for RNA integrity by electrophoresis. Samples were reverse transcribed only if they showed clean and distinct bands of 28s and 18s RNA and minimal smearing within each lane. Total RNA in the extracted samples was quantified by ultraviolet (UV) spectrophotometry at 260 nm. Reverse Transcription

Two micrograms of total RNA (dissolved in DEPC-H2O to a volume of 9 [mu]L) was combined with 1 [mu]L of oligo (dT)^sub 15^. The other reagents totaling a volume of 15 [mu]L (for a total reaction volume of 25 [mu]L) were as follows: DEPC-H2O (4.5 [mu]L), 5 x avian myeloblastoma virus (AMV) buffer (5 [mu]L), dNTP’s (2.5 [mu]L), RNAsin (1 [mu]L), and AMV-reverse transcriptase (AMV-RT) enzyme (2 [mu]L). All reagents listed were obtained from Promega, Madison, WI. The reaction was carried out at 42[degrees]C for 1 hour and stopped by heating at 70[degrees]C for 10 minutes. The resultant cDNA samples were diluted 1:20 in DEPC-H2O and stored at -20[degrees]C until assayed.

Real-Time Polymerase Chain Reaction (RT-PCR)

RT-PCR was performed using a Rotor-Gene 3000 thermal cycling system (Corbett Research, Mortlake, New South Wales, Australia) with SYBR-Green I (Molecular Probes, Eugene, OR) as fluorescent probe. Two microliters of cDNA template was combined with 8 [mu]L of the following reaction mixture: 423.4 [mu]L 2 x super mix (Invitrogen) + 136.1 [mu]L DEPC-H2O + 22.7 [mu]L SybrGreen (diluted 1:1000 in DMSO) + 22.7 [mu]L 50 mmol/L MgCl^sub 2^ + 43 [mu]L of forward and reverse primer mix. Primers were developed using the SDSC Biology Workbench (http://workbench.sdsc.edu/) for each target gene. Primer sequences were cross-referenced against PubMed sequencing data using BLAST to assess for possible nonspecific amplification. Each primer matched only its intended transcript sequence and produced amplicons of the calculated size. beta-Actin was used as housekeeping gene. Primers were constructed by Integrated DNA Technologies, Coralville, LA. Table II shows all primer sequences and sizes of amplified products. Optimum primer concentrations were titrated for each gene in each tissue. Primer specificity was inferred from both melting curve analyses homogeneity and product size on 1% agarose gel electrophoresis. The efficiency of the system was tested by performing reactions on 10-fold serial dilutions (up to 1:200,000) of samples and assessing the change in cycle threshold (Ct; change of ~3.3 between neighboring dilutions correlates to 10-fold difference in starting cDNA amount). Reactions were highly efficient throughout the range of dilutions tested.

Protein Quantification

Protein extracts were prepared from tissue samples by homogenization of the samples with a RIPA lysis buffer (Upstate Cell Signaling Solutions, Lake Placid, NY) plus 1% proteases-inhibitor cocktail (Sigma-Aldrich, St. Louis, MO), incubation for 30 minutes at 4[degrees]C, followed by centrifugation for 10 minutes to remove cellular debris. The clear supernatant was transferred to a clean tube and the protein content was quantified with the dye-binding Bradford reagent (BioRad, Hercules, CA).

TABLE II

Primer sequences

Commercially available sandwich enzyme-linked immunosorbent assay (ELISA) kits were purchased for CXCL13, CCL20, CCL21 and CCL25 (R&D Systems, Minneapolis, MN). The sandwich ELISA for CCL28 was developed in our laboratory and consisted of the following components: standard; mouse recombinant CCL28 (E. coli derived), capture Ab; rat antimouse CCL28 (monoclonal), detection Ab; goat antimouse CCL28 (polyclonal, all from R&D Systems), enzyme detection; rabbit antigoat IgG-HRP conjugate (Santa Cruz Biotechnology, Santa Cruz, CA).

Ninety-six-well plates were coated with capture antibody diluted to working dilution in phosphate-buffered saline (PBS) and incubated overnight at room temperature (RT) in a humid chamber. The following morning, plates were washed 3 times with Tris-buffered saline + 0.05% Tween 20 (TBS-Tween), and nonspecific binding was blocked by blotto (5% nonfat dry milk in TBS-Tween) for 1 hour. Plates were washed 3 times before addition of samples. Samples (100 [mu]L/well) were loaded at no dilution and incubated for 2 hours at RT. Plates were washed 3 times with TBS-Tween before addition of the detection antibody (100 [mu]L/well) diluted 1:500 in blotto and incubated at RT for 2 hours. Plates were washed 3 times before addition (100 [mu]L/well) of the IgG-HRP conjugate at a dilution of 1:20,000 in blotto. The conjugate was incubated at RT for 1 hour. Tetramethyl benzidine (TMB; BD Biosciences, San Diego, CA; 100 [mu]L/well) substrate was added and incubated at RT for 15 minutes before the reaction was stopped with 50 [mu]L/well of 2-N H^sub 2^SO^sub 4^. Similar protocols were followed for the commercial kits according to the manufacturer’s recommendations. We used a sequential ELISA method for PP CCL21 and CXCL13 (in that order) owing to small sample volume.35

Statistical Analysis

The amount of target gene mRNA was compared between chow and IV- PN groups for a total of 7 genes in 3 tissues, using the Delta- Delta cycle36 threshold (DeltaDeltaCt) method. Briefly, this involves calculating a DeltaCt between the average target gene Ct and average housekeeping gene Ct for a given target in each treatment group. The DeltaDeltaCt is the difference between ACts (DeltaCt^sub IV-PN^ – DeltaCt^sub chow^) and is used to calculate the “n-fold” change in gene expression between groups by insertion into the following operation: 2^sup -(-DeltaDeltaCt)^. The range of this fold change with 95% confidence is found as follows: The standard deviation of Ct values for the target gene in each treatment group is squared, these values are summed, and the square root of this value is taken. This value is then both added to and subtracted from the DeltaDeltaCt and placed in the 2^sup -(- DeltaDeltaCt)^ operation. If this range crosses the value of 1.0, the n-fold change was not statistically significant at an alpha = .05. The value of 1.0 is derived by 2^sup -(DeltaCt chow-DeltaCt chow)^ or 2^sup (0)^, which equals 1. Protein assay results were compared with an unpaired Student’s t-test, with alpha = .05.

RESULTS

Messenger RNA Levels

mRNA levels measured for all chemokines are shown in Table I.

PP (Figure 2)

Levels of 2 chemokine transcripts significantly changed with DES. CCL21 (which regulates T-cell entry) mRNA decreased (n-fold change, 0.39; range, 0.17-0.87) and CXCL13 (which regulates B-cell entry) mRNA increased significantly (n-fold change, 1.7; range, 1.09-2.65) compared with chow. The other chemokines studied showed no statistically significant differences between groups.

Figure 2. Chow, n = 11; PN, n = 10; *indicates statistically significant (p < .05) change vs chow by DeltaDeltaCt analysis. n- Fold change: CCL 21 = 0.39, CXCL13 = 1.7. PP, Peyer's patches.

FIGURE 3. Chow, n = 11; PN, n = 10; *indicates statistically significant (p < .05) change vs chow by DeltaDeltaCt analysis. n- Fold change: CCL20 = 1.95. SI, small intestine.

SI (Figure 3)

DES/PN significantly increased CCL20 (a dendritic cell [DC] attractant) mRNA (n-fold change, 1.95; range, 1.04-3.6). CCL25 (which recruits antibody-secreting cells [ASCs] showed a strong trend toward increase, just missing statistical significance (n- fold change, 1.37; range, 0.97-1.93). CCL28, which also recruits ASCs, showed a trend to decrease also just missing statistical significance (n-fold change, 0.74; range, 0.53-1.04).

Lung (Figure 4)

DES/PN feeding significantly lowered lung CCL28 (which attracts ASCs) mRNA (n-fold change, 0.16; range, 0.09-0.29). DES/PN exerted no effect on CXCL12 mRNA compared with chow.

Protein Levels

Chemokine protein levels were assayed for all chemokines in which the mRNA levels were significantly different between diet groups. SI CCL25 and CCL28 protein levels were also measured because mRNA analysis displayed strong trends that just missed statistical significance. Numerical values given are pg/mg protein +- standard error.

Figure 4. Chow, n = 9; PN, n = 10; *indicates statistically significant (p < .05) change vs chow by DeltaDeltaCt analysis. n- Fold change: CCL28 = 0.16.

Figure 5. Chow, n = 10; PN, n = 10; *indicates statistically insignificant (p = .06) change vs chow by Student’s t-test. #Indicates statistically significant (p < .05) change vs PN by Student's t-test. PP, Peyer's patches.

PP (Figure 5)

In contrast to mRNA results, DES/PN significantly increased CCL21 protein levels compared with chow (PN: 1008 +- 168 vs chow: 582 +- 91; p = .04) and displayed a strong trend toward decreased CXCL13 protein levels (PN: 458 +- 88 vs chow: 948 +- 209; p = .06).

SI (Figures 6, 7)

DES/PN significantly lowered levels of CCL20 (PN: 8.6 +- 1.9 vs chow: 40.6 +- 8.6; p = .0012) and CCL25 protein (PN: 9.1 +- 1.8 vs chow: 24.0 +- 5.5; p = .017) in contrast to the mRNA results. Consistent with the mRNA findings, DES/PN significantly decreased CCL28 protein levels (PN: 0.9 +- 0.2 vs chow: 1.6 +- 0.24; p = .03).

FIGURE 6. Chow, n = 14; PN, n = 14; *indicates statistically significant (p < .05) change vs chow by Student's t-test. SI, small intestine.

FIGURE 7. Chow, n = 14; PN, n = 14; *indicates statistically significant (p < .05) change vs chow by Student's t-test. SI, small intestine.

Lung (Figure 8)

Consistent with the mRNA findings, DES/PN significantly lowered CCL28 protein levels (PN: 3.4 +- 0.76 vs chow: 10.6 +- 2.2; p = .003).

DISCUSSION

The mucosal immune system provides protection against infectious pathogens at all epithelial-lined body/external environmental interfaces. The gastrointestinal (GI) tract contains the majority of cells (lymphocytes) and produces the largest amount of the critical end product (SIgA) in this system.37 Interestingly, clinical and experimental evidence to date suggests that stimulation of the GI tract (by feeding), or lack thereof, affects mucosal immune function and susceptibility to infection in critically ill patients after traumatic injury.1-4

FIGURE 8. Chow, n = 10; PN, n = 12; *indicates statistically significant (p < .05) change vs chow by Student's t-test.

TABLE III

Results of mRNA and protein analyses

Previous experimental work demonstrated changes in multiple mucosal immune variables when the GI tract is not stimulated/fed. These changes include decreases in GALT lymphocyte populations and changes in cell phenotype,19 reductions in expression of mucosal immune cell adhesion molecules,14 decreases in Th2-type IgA stimulating cytokines (specifically IL-4 and IL-10),12 reduced levels of the IgA transport protein polymeric immunoglobulin receptor (pIgR),20 and most important clinically, lower SIgA at mucosal surfaces.19-38-40 This work addresses the effects of enteral stimulation (or lack of stimulation) on mucosal immune chemokine mRNA and protein levels in the PP, SI, and lung. We hypothesized that chemokine levels would decrease in animals receiving DES/PN as mRNA levels dropped for these proteins. Surprisingly, we found that intestinal and PP mRNA and protein levels moved in opposite directions in all cases except for intestinal CCL28 (Table III) where levels of protein and mRNA decreased together. CCL 28 mRNA and protein levels also moved in concert (both decreased) in the lung.

Several possibilities exist to account for the discordant mRNA and protein findings for all of the chemokines except CCL28. Dissociation of transcription and translation alterations due to local physical4 or chemical42 factors have been described in vitro. DES/PN could affect either mRNA binding proteins43 that inhibit translation of transcribed message or translation initiation proteins.44 These chemokines may also be autoregulated through a negative feedback type mechanism. For example, if the chemokine protein levels are decreased, more message might be produced to compensate for low protein expression. Conversely, if protein levels increase there might be a lack of stimulus for mRNA transcription and a decrease in message levels. Interleukin-10, a key mucosal immune cytokine, has been shown to be autoregulated by negative feedback in response to LPS stimulation. DES/PN-induced alterations in posttranscriptional maturation and processing may also affect our results because we only detected (through reverse transcription) mature mRNA containing a poly-A tail.

To further investigate mRNA/protein level discrepancies we would first perform a time-course experiment and kill groups of animals at varying time points throughout the 5-day feeding protocol. This would allow us to temporally track mRNA and protein levels of these molecules vs the current study design, which yields a “snapshot” of mRNA and protein levels after 5 days of feeding. Additionally we could assay such samples for the known mRNA inhibitory binding protein YB-1, the translation initiation factor eIF4G, or its inhibitor Pdcd4.

The reasons for alteration in lung chemokine levels are intuitively unclear. The intestinal mucosa is extremely sensitive to DES/PN. Elimination of luminal gut stimulation (ie, “gut starvation”) alters both functional and local environmental factors (digestion, peristalsis, absorption, villous structure, mucosal thickness, the neuropeptide milieu, etc). Lack of feeding clearly affects proteins important in mucosal immune integrity, as well as many histologic and digestive processes. We have documented significant decreases in a number of important proteins, including lymphotoxin beta receptor15 (which controls MAdCAM-1 and IL-4 production), MAdCAM-113,14 (which controls lymphocyte entry into PP), and pIgR20 (which binds and transports dimeric IgA produced by plasma cells in the lamina propria). The lungs, however, never come in direct contact with the nutrient stream and remain normally ventilated and perfused in their respiratory role. We hypothesize that neuropeptides and possibly other gut hormones released by the GI tract during feeding up-regulate lung protein expression including the chemokines. Our previous work demonstrated that bombesin, a neuropeptide analogous to gastrin-releasing peptide, maintained normal levels of respiratory IgA and preserved established respiratory antiviral and antibacterial immunologic defenses in IV-fed mice, whereas IV feeding alone did not preserve this defense.46 We will soon test this hypothesis using exogenously administered neuropeptides in IV-fed mice.

The functional implications of our work relate to actual protein expression and not mRNA results, and our results clearly demonstrate the importance of confirming mRNA changes with actual protein expression. DES/PN decreased protein levels of CXCL13, which exerts major effects on B lymphocyte entry to pp.47,48 We hypothesize that decreases in CXCL13 contribute to the decrease in PP lymphocyte mass previously observed.19,38 The highest expression of CXCL13 occurs on areas of the high endothelial venules that also express MAdCAM-1, an important mucosal immune adhesion molecule. We previously showed that MAdCAM-1 decreases with DES/PN and that MAdCAM-1 blockade with a specific monoclonal antibody reproduces changes in GALT cell populations that are similar to those created by DES/PN.12,14 We know of no evidence linking production and expression of CXCL13 with MAdCAM-1, however.

CXCL12 is an ubiquitous chemokine with substantial mRNA expression in most tissues. It is a “constitutive” chemokine that is essential for life, probably in part because it regulates bone marrow hematopoietic stem cell traffic.49-52 Given its vast distribution and relative importance, we included it as a type of negative control and were not surprised that type and route of nutrition did not alter its level.

Only CCL21 in the PP increased with DES/PN. CCL21 exerts a major influence on T cell entry into PP53 across the high endothelial value. The increase observed may be a temporally late or delayed attempt to compensate for a lowered PP total lymphocyte mass associated with DES/PN. Conversely, this response may direct antigenically stimulated T cells out of PP. Such a mechanism affects T-cell expulsion from the thymus.54,55

Intestinal CCL20 and CCL25 protein expression decreased with DES/ PN. CCL20 activates DC expressing CC-chemokine receptor 6, which causes chemotaxis and enables appropriate DC:lymphocyte interaction.56 We postulate decreases in CCL20 might yield decreases in DC activation that hinder proper induction of mucosal immune responses. CCL20 is also made by follicle-associated epithelial cells in the intestine,57 and we examined washing fluid from the SI for CCL20 but found none (data not shown). Because CCL25 recruits ASCs,58 this finding remains consistent with reductions in intestinal GALT cell mass with DES/PN. We postulate that decreased levels of this chemokine, together with reduced MAdCAM-1, reduce ASC in intestinal lamina propria and subsequent IgA production after DES/ PN.

DES/PN decreased levels of CCL28 in both the lung and SI. CCL28 is also an ASC chemoattractant,58-60 and such decreases could contribute to decreased lymphocyte recruitment and subsequent lower mucosal IgA levels seen with DES/PN. We observed decreases in lung lymphocyte mass (similar to PP and SI) with DES/PN in an inbred (Balb/c) mouse model (unpublished data).

In future work, the impact and importance of the changes in chemokine ligands documented in this experiment could be tested by administering exogenous chemokine(s) to animals undergoing DES/PN or by blocking specific chemokines with exogenous binding antibodies as we have previously done with MAd-CAM-1 blockade.14 The individual effects could be ascertained by examining cell populations and IgA levels in PP, SI, and lung of such animals compared with both chow and DES/PN-alone animals. If lymphocyte populations and/or mucosal IgA levels in DES/PN animals were restored to chow levels with exogenous chemokine treatment (or reduced in the chow animals with chemokine blockade), our hypotheses concerning the importance of these molecules to mucosal immune function would be validated. Such findings would also suggest that chemokine pathways represent therapeutic targets. In addition, the issues could be addressed by subjecting mice that overexpress chemokine(s) to our diet protocol, although this is less desirable as we prefer to avoid knockout or otherwise genetically engineered, “non-wild-type” animals. Future experiments may also investigate the effect of DES/PN with regard to chemokine receptor expression on circulating lymphocytes.

CONCLUSIONS

This work demonstrates that mucosal immune chemokines, like a multitude of other mucosal immune variables, are subject to change under conditions of DES. In general, a decrease in enteral stimulation results in decreasing protein levels of these important molecules, which do not necessarily correlate with alterations in their mRNA. The majority of these observations fit theoretically with previously observed mucosal immune changes under conditions of DES/PN. Further work will characterize these changes, explore the interaction between the gut and lung immune defenses, and define their subsequent effects on mucosal immunity and infectious morbidity.

ACKNOWLEDGMENTS

Supported by NIH grant R01 6M53439 and also based upon work supported in part by the Office of Research and Development Biomedical Laboratory R&D Service, Department of Veterans Affairs.

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Joshua L. Hermsen, MD*; F. Enrique Gomez, PhD*; Yoshinori Maeshima, MD*; Yoshifumi Sano, MD*; Woodae Kang, MD, PhD*; and Kenneth A. Kudsk, MD*[dagger]

From the * Department of Surgery, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin; and [dagger] William S. Middleton Veterans Administration Hospital, Madison, Wisconsin

Received for publication June 12, 2007.

Accepted for publication August 20, 2007.

Presented at Nutrition Week, January 2007, in Phoenix, Arizona.

Correspondence: Kenneth A. Kudsk, MD, Department of Surgery, School of Medicine and Public Health, University of Wisconsin- Madison, H4/736 Clinical Science Center, Madison, WI 53792-7375. Electronic mail may be sent to kudsk@surgery.wisc.edu.

Copyright American Society for Parenteral and Enteral Nutrition Jan/ Feb 2008

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