Innate Mucosal Immune System Response of BALB/c vs C57BL/6 Mice to Injury in the Setting of Enteral and Parenteral Feeding.

BACKGROUND: Outbred mice exhibit increased airway and intestinal immunoglobulin A (IgA) following injury when fed normal chow, consistent with humans. Parenteral nutrition (PN) eliminates IgA increases at both sites. Inbred mice are needed for detailed immunological studies; however, specific strains have not been evaluated for this purpose. BALB/c and C57BL/6 are common inbred mouse strains but demonstrate divergent immune responses to analogous stress. This study addressed which inbred mouse strain best replicates the outbred mouse and human immune response to injury. METHODS: Intravenously cannulated mice received chow or PN for 5 days and then underwent sacrifice at 0 or 8 hours following controlled surgical injury (BALB/c: n = 16-21/group; C57BL/6: n = 12-15/group). Bronchoalveolar lavage (BAL) was analyzed by enzyme-linked immunosorbent assay for IgA, tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6, while small intestinal wash fluid (SIWF) was analyzed for IgA. RESULTS: No significant increase in BAL IgA occurred following injury in chow- or PN-fed BALB/c mice (chow: P = .1; PN: P = .7) despite significant increases in BAL TNF-α and SIWF IgA (chow: 264 ± 28 vs 548 ± 37, P < .0001; PN: 150 ± 12 vs 301 ± 17, P < .0001). Injury significantly increased mucosal IgA in chow-fed C57BL/6 mice (BAL: 149 ± 33 vs 342 ± 87, P = .01; SIWF: 236 ± 28 vs 335 ± 32, P = .006) and BAL cytokines. After injury, PN-fed C57BL/6 mice exhibited no difference in BAL IgA (P = .9), BAL cytokines, or SIWF IgA (P = .1). CONCLUSIONS: C57BL/6 mice exhibit similar airway responses to injury as outbred mice and humans, providing an appropriate model for studying mucosal responses to injury. The BALB/c mucosal immune system responds differently to injury and does not replicate the human injury response.

Bombesin Preserves Goblet Cell Resistin-Like Molecule ß During Parenteral Nutrition but Not Other Goblet Cell Products.

INTRODUCTION: Parenteral nutrition (PN) increases the risk of infection in critically ill patients and is associated with defects in gastrointestinal innate immunity. Goblet cells produce mucosal defense compounds, including mucin (principally MUC2), trefoil factor 3 (TFF3), and resistin-like molecule ß (RELMß). Bombesin (BBS), a gastrin-releasing peptide analogue, experimentally reverses PN-induced defects in Paneth cell innate immunity. We hypothesized that PN reduces goblet cell product expression and PN+BBS would reverse these PN-induced defects. METHODS: Two days after intravenous cannulation, male Institute of Cancer Research mice were randomized to chow (n = 15), PN (n = 13), or PN+BBS (15 µg tid) (n = 12) diets for 5 days. Defined segments of ileum and luminal fluid were analyzed for MUC2, TFF3, and RELMß by quantitative reverse transcriptase polymerase chain reaction and Western blot. Th2 cytokines interleukin (IL)-4 and IL-13 were measured by enzyme-linked immunosorbent assay. RESULTS: Compared with chow, PN significantly reduced MUC2 in ileum (P < .01) and luminal fluid (P = .01). BBS supplementation did not improve ileal or luminal MUC2 compared with PN (P > .3). Compared with chow, PN significantly reduced TFF3 in ileum (P < .02) and luminal fluid (P < .01). BBS addition did not improve ileal or luminal TFF3 compared with PN (P > .3). Compared with chow, PN significantly reduced ileal RELMß (P < .01). BBS supplementation significantly increased ileal RELMß to levels similar to chow (P < .03 vs PN; P > .6 vs chow). Th2 cytokines were decreased with PN and returned to chow levels with BBS. CONCLUSION: PN significantly impairs the goblet cell component of innate mucosal immunity. BBS only preserves goblet cell RELMß during PN but not other goblet cell products measured.

The gastrin-releasing peptide analog bombesin preserves exocrine and endocrine pancreas morphology and function during parenteral nutrition.

Stimulation of digestive organs by enteric peptides is lost during total parental nutrition (PN). Here we examine the role of the enteric peptide bombesin (BBS) in stimulation of the exocrine and endocrine pancreas during PN. BBS protects against exocrine pancreas atrophy and dysfunction caused by PN. BBS also augments circulating insulin levels, suggesting an endocrine pancreas phenotype. While no significant changes in gross endocrine pancreas morphology were observed, pancreatic islets isolated from BBS-treated PN mice showed a significantly enhanced insulin secretion response to the glucagon-like peptide-1 (GLP-1) agonist exendin-4, correlating with enhanced GLP-1 receptor expression. BBS itself had no effect on islet function, as reflected in low expression of BBS receptors in islet samples. Intestinal BBS receptor expression was enhanced in PN with BBS, and circulating active GLP-1 levels were significantly enhanced in BBS-treated PN mice. We hypothesized that BBS preserved islet function indirectly, through the enteroendocrine cell-pancreas axis. We confirmed the ability of BBS to directly stimulate intestinal enteroid cells to express the GLP-1 precursor preproglucagon. In conclusion, BBS preserves the exocrine and endocrine pancreas functions during PN; however, the endocrine stimulation is likely indirect, through the enteroendocrine cell-pancreas axis.

Impaired Cellular Immunity in the Murine Neural Crest Conditional Deletion of Endothelin Receptor-B Model of Hirschsprung's Disease.

Hirschsprung's disease (HSCR) is characterized by aganglionosis from failure of neural crest cell (NCC) migration to the distal hindgut. Up to 40% of HSCR patients suffer Hirschsprung's-associated enterocolitis (HAEC), with an incidence that is unchanged from the pre-operative to the post-operative state. Recent reports indicate that signaling pathways involved in NCC migration may also be involved in the development of secondary lymphoid organs. We hypothesize that gastrointestinal (GI) mucosal immune defects occur in HSCR that may contribute to enterocolitis. EdnrB was deleted from the neural crest (EdnrBNCC-/-) resulting in mutants with defective NCC migration, distal colonic aganglionosis and the development of enterocolitis. The mucosal immune apparatus of these mice was interrogated at post-natal day (P) 21-24, prior to histological signs of enterocolitis. We found that EdnrBNCC-/- display lymphopenia of their Peyer's Patches, the major inductive site of GI mucosal immunity. EdnrBNCC-/- Peyer's Patches demonstrate decreased B-lymphocytes, specifically IgM+IgDhi (Mature) B-lymphocytes, which are normally activated and produce IgA following antigen presentation. EdnrBNCC-/- animals demonstrate decreased small intestinal secretory IgA, but unchanged nasal and bronchial airway secretory IgA, indicating a gut-specific defect in IgA production or secretion. In the spleen, which is the primary source of IgA-producing Mature B-lymphocytes, EdnrBNCC-/- animals display decreased B-lymphocytes, but an increase in Mature B-lymphocytes. EdnrBNCC-/- spleens are also small and show altered architecture, with decreased red pulp and a paucity of B-lymphocytes in the germinal centers and marginal zone. Taken together, these findings suggest impaired GI mucosal immunity in EdnrBNCC-/- animals, with the spleen as a potential site of the defect. These findings build upon the growing body of literature that suggests that intestinal defects in HSCR are not restricted to the aganglionic colon but extend proximally, even into the ganglionated small intestine and immune cells.

Effects of diurnal variation of gut microbes and high-fat feeding on host circadian clock function and metabolism.

Circadian clocks and metabolism are inextricably intertwined, where central and hepatic circadian clocks coordinate metabolic events in response to light-dark and sleep-wake cycles. We reveal an additional key element involved in maintaining host circadian rhythms, the gut microbiome. Despite persistence of light-dark signals, germ-free mice fed low or high-fat diets exhibit markedly impaired central and hepatic circadian clock gene expression and do not gain weight compared to conventionally raised counterparts. Examination of gut microbiota in conventionally raised mice showed differential diurnal variation in microbial structure and function dependent upon dietary composition. Additionally, specific microbial metabolites induced under low- or high-fat feeding, particularly short-chain fatty acids, but not hydrogen sulfide, directly modulate circadian clock gene expression within hepatocytes. These results underscore the ability of microbially derived metabolites to regulate or modify central and hepatic circadian rhythm and host metabolic function, the latter following intake of a Westernized diet.

Gut Lymphocyte Phenotype Changes After Parenteral Nutrition and Neuropeptide Administration.

OBJECTIVE: To define gut-associated lymphoid tissue (GALT) phenotype changes with parenteral nutrition (PN) and PN with bombesin (BBS). BACKGROUND: PN reduces respiratory tract (RT) and GALT Peyer patch and lamina propria lymphocytes, lowers gut and RT immunoglobulin A (IgA) levels, and destroys established RT antiviral and antibacterial immunity. BBS, an enteric nervous system neuropeptide, reverses PN-induced IgA and RT immune defects. METHODS: Experiment 1: Intravenously cannulated ICR mice received chow, PN, or PN + BBS injections for 5 days. LSR-II flow cytometer analyzed Peyer patches and lamina propria isolated lymphocytes for homing phenotypes (L-selectin and LPAM-1) and state of activation (CD25, CD44) in T (CD3)-cell subsets (CD4 and CD8) along with homing phenotype (L-selectin and LPAM-1) in naive B (IgD) and antigen-activated (IgD or IgM) B (CD45R/B220) cells. Experiment 2: Following the initial experiment 1 protocol, lamina propria T regulatory cell phenotype was evaluated by Foxp3 expression. RESULTS: Experiment 1: PN significantly reduced lamina propria (1) CD4CD25 (activated) and (2) CD4CD25LPAM-1 (activated cells homed to the lamina propria) T cells, whereas PN-BBS assimilated chow levels. PN significantly reduced lamina propria (1) IgD (naive), (2) IgDLPAM (antigen-activated homed to the lamina propria) and CD44 memory B cells, whereas PN-BBS assimilated chow levels. Experiment 2: PN significantly reduced lamina propria CD4CD25Foxp3 T regulatory cells compared with chow-fed mice, whereas PN + BBS assimilated chow levels. CONCLUSIONS: PN reduces lamina propria activated and T regulatory cells and also naive and memory B cells. BBS addition to PN maintains these cell phenotypes, demonstrating the intimate involvement of the enteric nervous system in mucosal immunity.

Bombesin improves adaptive immunity of the salivary gland during parenteral nutrition.

BACKGROUND: The parotid and submandibular salivary glands are gut-associated lymphoid tissues (GALTs) that secrete immune compounds into the oral cavity. Parenteral nutrition (PN) without enteral stimulation decreases GALT function, including intestinal lymphocyte counts and secretory immunoglobulin A (sIgA) levels. Since the neuropeptide bombesin (BBS), a gastrin-releasing peptide analogue, stimulates intestinal function and restores GALT parameters, we hypothesized that PN + BBS would stimulate parotid and salivary gland IgA levels, T lymphocytes, and IgA plasma cell counts compared with PN alone. METHODS: Male (Institute of Cancer Research) ICR mice received intravenous catheters and were randomized to chow with saline, PN, or PN + BBS (15 µg/tid/mouse) for 5 days (8/group), 2 days after cannulation. Salivary glands were weighed and either frozen for IgA and amylase analysis or fixed for histological analysis of acinar cells, IgA+ plasma cells, and T lymphocytes. Small intestinal wash fluid was collected for IgA regression analysis with salivary glands. RESULTS: PN reduced organ weight, acinar cell size, and amylase activity compared with chow; BBS had no significant effects on these parameters. Compared with chow, PN significantly reduced salivary gland IgA levels, IgA+ plasma cells, and T lymphocytes. PN + BBS significantly elevated IgA and restored cellularity compared with PN. Salivary gland tissue homogenate IgA levels significantly correlated with intestinal fluid IgA levels. CONCLUSIONS: Compared with chow, PN results in atrophy of the salivary glands characterized by reduced amylase, IgA, and immune cellularity. BBS has no effect on acinar cells or amylase activity compared with PN but maintains tissue IgA and plasma cells and T-lymphocyte numbers compared with chow.

Glutamine Improves Innate Immunity and Prevents Bacterial Enteroinvasion During Parenteral Nutrition.

BACKGROUND: Patients receiving parenteral nutrition (PN) are at increased risk of infectious complications compared with enteral feeding, which is in part explained by impaired mucosal immune function during PN. Adding glutamine (GLN) to PN has improved outcome in some clinical patient groups. Although GLN improves acquired mucosal immunity, its effect on innate mucosal immunity (defensins, mucus, lysozymes) has not been investigated. METHODS: Forty-eight hours following venous cannulation, male Institute of Cancer Research mice were randomized to chow (n = 10), PN (n = 12), or PN + GLN (n = 13) for 5 days. Small intestine tissue and luminal fluid were collected for mucin 2 (MUC2), lysozyme, cryptdin 4 analysis, and luminal interleukin (IL)-4, IL-10, and IL-13 level measurement. Tissue was also harvested for ex vivo intestinal segment culture to assess tissue susceptibility to enteroinvasive Escherichia coli. RESULTS: In both luminal and tissue samples, PN reduced MUC2 and lysozyme (P < .0001, respectively) compared with chow, whereas GLN addition increased MUC2 and lysozyme (luminal, P < .05; tissue, P < .0001, respectively) compared with PN alone. PN significantly suppressed cryptdin 4 expression, while GLN supplementation significantly enhanced expression. IL-4, IL-10, and IL-13 decreased significantly with PN compared with chow, whereas GLN significantly increased these cytokines compared with PN. Functionally, bacterial invasion increased with PN compared with chow (P < .05), while GLN significantly decreased enteroinvasion to chow levels (P < .05). CONCLUSIONS: GLN-supplemented PN improves innate immunity and resistance to bacterial mucosal invasion lost with PN alone. This work confirms a clinical rationale for providing glutamine for the protection of the intestinal mucosa.

Intestinal dysbiosis and bacterial enteroinvasion in a murine model of Hirschsprung's disease.

BACKGROUND/PURPOSE: Hirschsprung's disease (HSCR), characterized by the absence of ganglia in the distal colon, results in functional obstruction. Despite surgical resection of the aganglionic segment, around 40% of patients suffer recurrent life threatening Hirschsprung's-associated enterocolitis (HAEC). The aim of this study was to investigate whether gut microbiota and intestinal immunity changes contribute to the HAEC risk in an HSCR model. METHODS: Mice with neural crest conditional deletion of Endothelin receptor B (EdnrB) and their littermate controls were used (EdnrB-null and EdnrB-het). Bacterial DNA was prepared from cecal contents of P16-18 and P21-24 animals and pyrosequencing employed for microbiome analysis. Ileal tissue was isolated and secretory phospholipase A2 (sPLA2) expression and activity determined. Enteroinvasion of Escherichia coli into ileal explants was measured using an ex vivo organ culture system. RESULTS: EdnrB-het and EdnrB-nulls displayed similar flora, sPLA2 expression and activity at P16-18. However, by P21-24, EdnrB-hets demonstrated increased Lactobacillus and decreased Bacteroides and Clostridium, while EdnrB-nulls exhibited reciprocal changes. EdnrB-nulls also showed reduced sPLA2 expression and luminal activity at this stage. Functionally, EdnrB-nulls were more susceptible to enteroinvasion with E. coli ex vivo and released less sPLA2 than EdnrB-hets. CONCLUSIONS: Initially, EdnrB-het and EdnrB-nulls contain similar cecal flora but then undergo reciprocal changes. EdnrB-nulls display dysbiosis, demonstrate impaired mucosal defense, decreased luminal sPLA2 and increased enteroinvasion of E. coli just prior to robust colonic inflammation and death. These findings suggest a role for the intestinal microbiome in the development of HAEC.

The enteric nervous system neuropeptide, bombesin, reverses innate immune impairments during parenteral nutrition.

BACKGROUND: Lack of enteral stimulation during parenteral nutrition (PN) impairs mucosal immunity. Bombesin (BBS), a gastrin-releasing peptide analogue, reverses PN-induced defects in acquired immunity. Paneth cells produce antimicrobial peptides (AMPs) of innate immunity for release after cholinergic stimulation. OBJECTIVE: Determine if BBS restores AMPs and bactericidal function during PN. METHODS: Intravenously cannulated male ICR mice were randomized to Chow, PN, or PN+BBS (15 µg 3 times daily, n = 7 per group) for 5 days. Ileum was analyzed for AMPs (Protein: sPLA2 by fluorescence, lysozyme and RegIII-γ by western andcryptdin-4 by ELISA; mRNA: all by RT-PCR). Cholinergic stimulated (100 µM bethanechol) ileal specimens assessed Pseudomonas bactericidal activity. Ileum (Chow: n = 7; PN: n = 9; PN+BBS: n = 8) was assessed for Escherichia coli invasion in ex-vivo culture. RESULTS: PN significantly decreased most AMPs versus Chow while BBS maintained Chow levels (sPLA2: Chow: 107 + 14*, PN: 44.6 + 7.2, PN+BBS: 78.7 + 13.4* Fl/min/µL/total protein; Lysozyme: Chow: 63.9 + 11.9*, PN: 26.8 + 6.2; PN+BBS: 64.9 + 13.8* lysozyme/total protein; RegIII-γ: Chow: 51.5 + 10.0*, PN: 20.4 + 4.3, PN+BBS: 31.0 + 8.4 RegIII-γ/total protein; Cryptdin-4: Chow: 18.4 + 1.5*, PN: 12.7 + 1.6, PN+BBS: 26.1 + 2.4*† pg/mg [all *P < 0.05 vs PN and †P < 0.05 vs Chow]). Functionally, BBS prevented PN loss of bactericidal activity after cholinergic stimulation (Chow: 25.3 + 3.6*, PN: 13.0 + 3.2; PN+BBS: 27.0 + 4.7* percent bacterial killing, *P < 0.05 vs PN). BBS reduced bacterial invasion in unstimulated tissue barely missing significance (P = 0.06). CONCLUSIONS: The enteric nervous system (ENS) controls AMP levels in Paneth cells during PN but mucosal protection by innate immunity requires both ENS and parasympathetic stimulation.

Cranberry proanthocyanidins improve intestinal sIgA during elemental enteral nutrition.

BACKGROUND: Elemental enteral nutrition (EEN) decreases gut-associated lymphoid tissue (GALT) function, including fewer Peyer's patch lymphocytes and lower levels of the tissue T helper 2 (Th2) cytokines and mucosal transport protein polymeric immunoglobulin receptor (pIgR), leading to lower luminal secretory immunoglobulin A (sIgA) levels. Since we recently demonstrated that cranberry proanthocyanidins (PACs) maintain the Th2 cytokine interleukin (IL)-4 when added to EEN, we hypothesized the addition of PACs to EEN would normalize other GALT parameters and maintain luminal levels of sIgA. METHODS: Institute of Cancer Research mice were randomized (12/group) to receive chow, EEN, or EEN + PACs (100 mg/kg body weight) for 5 days, starting 2 days after intragastric cannulation. Ileum tissue was collected to measure IL-4 by enzyme-linked immunosorbent assay, pIgR by Western blot, and phosphorylated STAT-6 by microarray. Intestinal wash fluid was collected to measure sIgA by Western blot. RESULTS: Compared with chow, EEN significantly decreased tissue IL-4, phosphorylated STAT-6, and pIgR. The addition of PACs to EEN prevented these alterations. Compared with chow, EEN resulted in significantly lower levels of luminal sIgA. The addition of PACs to EEN increased luminal sIgA levels compared with EEN alone. CONCLUSIONS: This study suggests the addition of PACs to EEN may support GALT function and maintain intestinal sIgA levels compared with EEN administration alone.

IL-25 improves luminal innate immunity and barrier function during parenteral nutrition.

BACKGROUND: Parenteral nutrition (PN) increases risks of infections in critically injured patients. Recently, PN was shown to reduce intestine luminal levels of the Paneth cell antimicrobial molecule secretory phospholipase A2 (sPLA2) and the goblet cell glycoprotein mucin2 (MUC2). These molecules are critical factors for innate mucosal immunity and provide barrier protection. Interleukin-4 (IL-4) and IL-13 regulate sPLA2 and MUC2 production through the IL-13 receptor. Because IL-25 stimulates IL-4 and IL-13 release and PN reduces luminal sPLA2 and MUC2, we hypothesized that adding IL-25 to PN would restore these innate immune factors and maintain barrier function. METHODS: Two days after venous cannulation, male ICR (Institute of Cancer Research) mice were randomized to receive chow (n = 12), PN (n = 9), or PN + 0.7 µg of exogenous IL-25 (n = 11) daily for 5 days. Small-intestine wash fluid (SIWF) was collected for analysis of sPLA2 activity, MUC2 density, and luminal levels of IL-4 and IL-13. Small-intestinal tissue was harvested for analysis of tissue sPLA2 activity or immediate use in an ex-vivo intestinal segment culture (EVISC) to assess susceptibility of the tissue segments to enteroinvasive Escherichia coli. RESULTS: PN reduced luminal sPLA2 (P < 0.0001) and MUC2 (P <0.002) compared with chow, whereas the addition of IL-25 to PN increased luminal sPLA2 (P < 0.0001) and MUC2 (P < 0.02) compared with PN. Tissue IL-4 and IL-13 decreased with PN compared with chow (IL-4: P < 0.0001, IL-13: P < 0.002), whereas IL-25 increased both cytokines compared with PN (IL-4: P < 0.03, IL-13: P < 0.02). Tissue levels of sPLA2 were significantly decreased with PN compared with chow, whereas IL-25 significantly increased tissue sPLA2 levels compared with PN alone. Functionally, more bacteria invaded the PN-treated tissue compared with chow (P < 0.01), and the addition of IL-25 to PN decreased enteroinvasion to chow levels (P < 0.01). CONCLUSIONS: PN impairs innate mucosal immunity by suppressing luminal sPLA2 activity and MUC2 density compared with chow. PN also increases bacterial invasion in ex-vivo tissue. Administration of exogenous IL-25 reverses this dysfunction and increases luminal sPLA2 and MUC2. PN tissue treated with IL-25 was significantly more resistant to bacterial invasion than with PN alone, suggesting that IL-25-induced effects augment the barrier defense mechanisms.

Parenteral nutrition decreases paneth cell function and intestinal bactericidal activity while increasing susceptibility to bacterial enteroinvasion.

INTRODUCTION: Parenteral nutrition (PN) increases the risk of infection in patients with contraindication to enteral feeding. Paneth cells produce and secrete antimicrobial products that protect the mucosa from pathogens. Their loss is associated with increased host-pathogen interactions, mucosal inflammation, and altered microbiome composition. HYPOTHESIS: We hypothesized that PN reduces Paneth cell product expression, and these changes would reduce bactericidal properties of tissue secretions following cholinergic stimulation, increase mucosal enteroinvasion, and shift the intestinal microbiome. METHOD: Experiment 1: Male ICR mice were randomized to Chow (n = 8) or PN (n = 8). Ileum tissue was collected for Paneth cell antimicrobial expression using RT-PCR, stimulated with a cholinergic agonist degranulate Paneth cells bactericidal activity, or used to assess bacterial enteroinvasion in EVISC. Experiment 2: Mice were randomized to Chow (n = 11) or PN (n = 8) and ileum washing was collected for 16s pyrosequencing analysis. RESULTS: Compared to Chow, PN decreased tissue expression of REGIII-g (p < 0.002), lysozyme (p < 0.002), and cryptdin-4 (p < 0.03). At the phylum level, PN decreased total Firmicutes but increased total Bacteroidetes, and Proteobacteria. Functionally, secretions from PN tissue was less bactericidal (p < 0.03) and demonstrated increased susceptibility to enteroinvasion by E coli (p < 0.02). CONCLUSION: PN without enteral nutrition impairs innate mucosal immune function. Tissue expression of Paneth cell antimicrobial proteins decreases associated with compositional shifts to the microbiome, decreased bactericidal activity of mucosal secretions and greater susceptibility of to enteroinvasion by E coli. These changes may explain in-part the increased risk of infection in parenterally fed patients.

Pharmaconutrition review: physiological mechanisms.

The search to improve outcomes in critically ill patients through nutrition support has steadily progressed over the past 4 decades. One current approach to this problem is the addition of specific nutrients as primary therapy to improve host defenses and improve the outcome of critically ill patients. The field is referred to as "pharmaconutrition," with the hope of focusing investigations on each nutrient to understand its pharmacological effects on immune and clinical outcomes. The purpose of this review is to describe some of the known physiological mechanisms of pharmaconutrients such as glutamine, arginine, ω-3 fatty acids, and selenium.

Colonic enteric nervous system analysis during parenteral nutrition.

INTRODUCTION: Parenteral nutrition (PN) is a necessary therapy used to feed patients with gastrointestinal dysfunction. Unfortunately, PN results in intestinal atrophy and changes to host immune function. PN may also induce additional effects on gut motility that we hypothesized would result from changes in the enteric nervous system. METHODS: Mice received an intravenous (i.v.) catheter and were randomized to chow (n = 5), i.v. PN (n = 6), or i.v. PN + bombesin (BBS, 15 µg/kg, 3×/d) (n = 6) for 5 d. Colons were removed and dissected to measure the length and circumference. Enteric neuronal density and neurotransmitter expression were determined by co-immunostaining whole-mount tissue with Hu and neuronal nitric oxide synthase (nNOS). RESULTS: The number of myenteric neurons expressing Hu and nNOS increased per unit length in the mid-colon during PN treatment compared with chow. This increase was abrogated by the addition of BBS to the PN regimen. However, the percentage of nNOS-expressing neurons was not significantly altered by PN. Morphometric analysis revealed a decrease in the length and circumference of the colon during PN administration that was partially normalized by supplementation of PN with BBS. A significant reduction in total fecal output was observed in PN animals compared with chow and was increased by mice receiving BBS in addition to PN. CONCLUSIONS: PN causes a constriction of the bowel wall, reducing not only the length but also the circumference of the colon. These changes cause a condensation of enteric neurons but no difference in neurotransmitter expression. BBS supplementation partially restores the constriction and increases the fecal output during PN treatment compared with PN treatment alone.

Parenteral nutrition increases susceptibility of ileum to invasion by E coli.

BACKGROUND: Parenteral nutrition (PN), with the lack of enteral feeding, compromises mucosal immune function and increases the risk of infections. We developed an ex vivo intestinal segment culture (EVISC) model to study the ex vivo effects of PN on susceptibility of the ileum to invasion by extra-intestinal pathogenic Escherichia coli (ExPEC) and on ileal secretion of antimicrobial secretory phospholipase A2 (sPLA2) in response to the pathogen. MATERIALS AND METHODS: Study 1: Using mouse (n = 7) ileal tissue, we examined the effects of ileal region (proximal versus distal) and varying ExPEC inoculum concentrations on ex vivo susceptibility to ExPEC invasion and sPLA2 secretion. Study 2: Ten mice were randomized to oral chow or intravenous PN feeding for 5 d (n = 5/group). Using the EVISC model, we compared the susceptibility of ileal tissue to invasion by ExPEC and sPLA2 secretion in response to the pathogen. RESULTS: Study 1: The proximal ileum was more susceptible to invasion (P < 0.0001) and secreted lower amounts of sPLA2 (P = 0.0002) than the distal ileum. Study 2: Ileal tissue from PN-fed animals was more susceptible (approximately 4-fold, P = 0.018) to invasion than those from chow-fed animals. Ileal tissue from PN-fed animals secreted less sPLA2 (P < 0.02) than those from chow-fed animals. CONCLUSIONS: The data illustrate EVISC as a reproducible model for studying host-pathogen interactions and the effects of diet on susceptibility to infections. Specifically, the findings support our hypothesis that PN with the lack of enteral feeding decreases mucosal responsiveness to pathogen exposure and provides a plausible mechanism by which PN is associated with increased risk of infectious complication.

Parenteral nutrition suppresses the bactericidal response of the small intestine.

BACKGROUND: Parenteral nutrition (PN) increases infectious risk in critically ill patients compared with enteral feeding. Previously, we demonstrated that PN feeding suppresses the concentration of the Paneth cell antimicrobial protein secretory phospholipase A2 (sPLA2) in the gut lumen. sPLA2 and other Paneth cell proteins are released in response to bacterial components, such as lipopolysaccharide (LPS), and they modulate the intestinal microbiome. Because the Paneth cell protein sPLA2 was suppressed with PN feeding, we hypothesized PN would diminish the responsiveness of the small bowel to LPS through reduced secretions and as a result exhibit less bactericidal activity. METHODS: The distal ileum was harvested from Institute of Cancer Research mice, washed, and randomized for incubation with LPS (0, 1, or 10 µg/mL). Culture supernatant was collected and sPLA2 activity was measured. Bactericidal activity of the ileum segment secretions was assessed against Pseudomonas aeruginosa with and without an sPLA2 inhibitor at 2 concentrations, 100 nmol/L and 1 µmol/L. Institute of Cancer Research mice were randomized to chow or PN for 5 days. Tissue was collected for immunohistochemistry (IHC) and ileal segments were incubated with LPS (0 or 10 µg/mL). sPLA2 activity and bactericidal activity were measured in secretions from ileal segments. RESULTS: Ileal segments responded to 10 µg/mL LPS with significantly greater sPLA2 activity and bactericidal activity. The bactericidal activity of secretions from LPS stimulated tissue was suppressed 50% and 70%, respectively, with the addition of the sPLA2-inhibitor. Chow displayed greater sPLA2 in the Paneth cell granules and secreted higher levels of sPLA2 than PN before and after LPS. Accordingly, media collected from chow was more bactericidal than PN. IHC confirmed a reduction in Paneth cell granules after PN. CONCLUSION: This work demonstrates that ileal segments secrete bactericidal secretions after LPS exposure and the inhibition of the Paneth cell antimicrobial protein sPLA2 significantly diminishes this. PN feeding resulted in suppressed secretion of the sPLA2 and resulted in increased bacterial survival. This demonstrates that PN significantly impairs the innate immune response by suppressing Paneth cell function.

Cranberry proanthocyanidins improve the gut mucous layer morphology and function in mice receiving elemental enteral nutrition.

BACKGROUND: Lamina propria Th2 cytokines, interleukin (IL)-4 and IL-13, stimulate goblet cell (GC) proliferation and MUC2 production, which protect the intestinal mucosa. Elemental enteral nutrition (EEN) reduces tissue IL-4 and impairs barrier function. Proanthocyanidins (PACs) stimulate oral mucin levels. We hypothesized that adding PAC to EEN would maintain Th2-without stimulating Th1-cytokines and preserve luminal MUC2 vs EEN alone. MATERIALS AND METHODS: Seventy mice were randomized to 5 diet groups-standard chow, intragastric EEN, or EEN with lowPAC, midPAC (50 mg), or highPAC (100 mg PAC/kg BW)-for 5 days, starting 2 days after gastric cannulation. Ileal tissue was analyzed for histomorphology and the cytokines IL-4, IL-13, IL-1ß, IL-6, and TNF-α by enzyme-linked immunosorbent assay. MUC2 was measured in intestinal washes. RESULTS: EEN lowered IL-13 (P < .05) compared with standard chow, whereas IL-4 was not significant (P < .07). LowPAC and midPAC increased IL-13 (P < .05), whereas highPAC increased both IL-4 and IL-13 (P < .05) compared with EEN. All EEN diets reduced (P < .05) crypt depth compared with the chow group. Compared with standard chow, GC numbers and luminal MUC2 were reduced with EEN (P < .05). These effects were attenuated (P < .05) with midPAC and highPAC. No changes were observed in tissue Th1 cytokines. CONCLUSIONS: Adding PACs to EEN reverses impaired intestinal barrier function following EEN by improving the gut mucous layer and function through increased GC size and number as well as levels of MUC2 and ileal IL-4 and IL-13.

IL-25 improves IgA levels during parenteral nutrition through the JAK-STAT pathway.

INTRODUCTION: Parenteral nutrition (PN) impairs mucosal immunity and increases the risk of infection in part via lower IgA levels at mucosal surfaces. Transport of immunoglobulin A (IgA) across the mucosa to the gut lumen depends on the epithelial transport protein, polymeric immunoglobulin receptor (pIgR), which is reduced during PN. In vitro, studies demonstrate that IL-4 up-regulates pIgR production via Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling. Because IL-4 stimulates IgA and is reduced during PN, we hypothesized that the suppressed pIgR is a result of decreased JAK-1 and STAT-6 phosphorylation. Because IL-4 is mediated by IL-25, we also hypothesized that PN + IL-25 would restore luminal IgA by increasing phosphorylated JAK-1 and STAT-6, resulting in increased tissue pIgR and luminal IgA. METHOD: Experiment 1: 2 days after intravenous cannulation, male Institute of Cancer Research mice were randomized to chow (n = 11) or PN (n = 9). Experiment 2: 2 days after intravenous cannulation, male Institute of Cancer Research mice were randomized to chow (n = 12), PN (n = 10), or PN + 0.7 µg of exogenous IL-25 (n = 11) per day. After 5 days, distal ileum tissue was collected, homogenized, and protein extracted for JAK-STAT expression levels using a phospho-specific antibody microarray. Tissue was homogenized to measure pIgR expression via Western blot or fixed in 4% paraformaldehyde to measure pIgR expression via immunohistochemistry. Small intestinal wash fluid was collected and IgA was quantified using enzyme-linked immunosorbent assay. RESULTS: Experiment 1: PN significantly reduced phosphorylated JAK-1 and STAT-6 compared with chow. PN also decreased the tissue levels of the Th2 cytokines, IL-4 and IL-13, as well as pIgR, and luminal IgA compared with chow. Experiment 2: Exogenous administration of PN + IL-25 increased the phosphorylated JAK-1 and STAT-6 compared with PN alone. IL-25 completely restored expression of IL-13 to chow levels. IL-4, pIgR, IgA, and phosphorylated JAK-1 were significantly increased with IL-25 treatment compared with PN but failed to reach levels measured in chow. STAT-6 was significantly increased with IL-25 treatment compared with chow and PN. CONCLUSIONS: PN significantly decreases the JAK-STAT pathway by reducing levels of phosphorylated STAT-6 and JAK-1. Consistent with our previous work, sIgA, pIgR, and IL-4 decreased with PN, whereas the addition of IL-25 to PN reversed these decreases and demonstrated the role of the JAK-STAT pathway in vivo during PN.

JAK-STAT and intestinal mucosal immunology.

The intestinal mucosal immune system is challenged with bacteria, viruses, and parasites, in addition to food and environmental antigens, that require dynamic immune responsiveness for homeostasis. One central signaling pathway is JAK-STAT, which regulates the adaptive and innate immune arms of mucosal immunity as well as epithelial repair and regeneration. Adaptive immunity includes lymphocyte mediated secretion of specific antibodies, while innate immune respones include secretion of non-antigen specific compounds. This review examines effects of specialized nutrition support on JAK-STAT in innate immune function and in lymphocyte modulation and epithelial antibody transport in gut-associated lymphoid tissue.