Adrianus C. J. M. de Bruijn

Mucin Muc2 Deficiency and Weaning Influences the Expression of the Innate Defense Genes Reg3β, Reg3γ and Angiogenin-4

Background Mucin Muc2 is the structural component of the intestinal mucus layer. Absence of Muc2 leads to loss of this layer allowing direct bacterial-epithelial interactions. We hypothesized that absence of the mucus layer leads to increased expression of innate defense peptides. Specifically, we aimed to study the consequence of Muc2 deficiency (Muc2−/−) on the expression of regenerating islet-derived protein 3 beta (Reg3β), regenerating islet-derived protein 3 gamma (Reg3γ), and angiogenin-4 (Ang4) in the intestine shortly before and after weaning. Methods Intestinal tissues of Muc2−/− and wild-type (WT) mice were collected at postnatal day 14 (P14, i.e. pre-weaning) and P28 (i.e. post-weaning). Reg3β, Reg3γ, and Ang4 expression was studied by quantitative real-time PCR, Western-blot, in situ hybridization, and immunohistochemistry. Results Reg3β and Reg3γ were expressed by diverging epithelial cell types; namely enterocytes, Paneth cells, and goblet cells. Additionally, Ang4 expression was confined to Paneth cells and goblet cells. Expression of Reg3β, Reg3γ, and Ang4 differed between WT and Muc2−/− mice before and after weaning. Interestingly, absence of Muc2 strongly increased Reg3β and Reg3γ expression in the small intestine and colon. Finally, morphological signs of colitis were only observed in the distal colon of Muc2−/− mice at P28, where and when expression levels of Reg3β, Reg3γ, and Ang4 were the lowest. Conclusions Expression of Reg3 proteins and Ang4 by goblet cells point to an important role for goblet cells in innate defense. Absence of Muc2 results in up-regulation of Reg3β and Reg3γ expression, suggesting altered bacterial-epithelial signaling and an innate defense response in Muc2−/− mice. The inverse correlation between colitis development and Reg3β, Reg3γ, and Ang4 expression levels might point toward a role for these innate defense peptides in regulating intestinal inflammation.

Epithelial functions of the residual bowel after surgery for necrotising enterocolitis in human infants.

Objectives: Information on epithelial functions of the residual small or colonic bowel after resection for necrotising enterocolitis (NEC) in human infants is scarce. Our aim is to evaluate epithelial functions in the intestinal resection margins of tissue obtained at bowel resection for acute NEC and consecutive stoma closure. Materials and Methods: Epithelial morphology, proliferation, and protein expression were (immuno)histochemically studied. Results: Acute NEC was associated with severe and mild epithelial damage varying from epithelial loss to fairly unaffected epithelium. Epithelial proliferation was increased both at acute NEC and at stoma closure. In acute NEC, lactase, glucose transporter-2 and -5 expression was down-regulated in severely affected epithelium, whereas sucrase-isomaltase and intestinal fatty acid binding protein expression was maintained. Goblet cells continued to express mucin 2 and trefoil factor 3, however, their numbers were decreased. Moreover, in acute NEC, Paneth cells were weakly lysozyme positive and were reduced in number. At stoma closure, expression of the above cell type–specific markers had completely been re-established. Conclusions: Residual bowel after resection for acute NEC shows a disturbed epithelial proliferation/differentiation balance. Acute NEC was associated with downregulation of distinct enterocyte-specific proteins. Because of goblet cell and Paneth cell loss in acute NEC, mucosal barrier, and defense functions may be impaired.

Paneth cell hyperplasia and metaplasia in necrotizing enterocolitis.

Paneth cell dysfunction has been suggested in necrotizing enterocolitis (NEC). The aim of this study was to i) study Paneth cell presence, protein expression, and developmental changes in preterm infants with NEC and ii) determine Paneth cell products and antimicrobial capacity in ileostomy outflow fluid. Intestinal tissue from NEC patients (n = 55), preterm control infants (n = 22), and term controls (n = 7) was obtained during surgical resection and at stoma closure after recovery. Paneth cell abundance and protein expression were analyzed by immunohistochemistry. RNA levels of Paneth cell proteins were determined by real-time quantitative RT-PCR. In ileostomy outflow fluid, Paneth cell products were quantified, and antimicrobial activity was measured in vitro. In acute NEC, Paneth cell abundance in small intestinal tissue was not significantly different from preterm controls. After recovery from NEC, Paneth cell hyperplasia was observed in the small intestine concomitant with elevated human alpha-defensin 5 mRNA levels. In the colon, metaplastic Paneth cells were observed. Ileostomy fluid contained Paneth cell proteins and inhibited bacterial growth. In conjunction, these data suggest an important role of Paneth cells and their products in various phases of NEC.

Intestinal Threonine Utilization for Protein and Mucin Synthesis Is Decreased in Formula-Fed Preterm Pigs

Threonine is an essential amino acid necessary for synthesis of intestinal (glyco)proteins such as mucin MUC2 to maintain adequate gut barrier function. In premature infants, reduced barrier function may contribute to the development of necrotizing enterocolitis (NEC). Human milk protects against NEC compared with infant formula. Therefore, we hypothesized that formula feeding decreases the MUC2 synthesis rate concomitant with a decrease in intestinal first-pass threonine utilization, predisposing the preterm neonate to NEC. Preterm pigs were delivered by caesarian section and received enteral feeding with formula (FORM; n = 13) or bovine colostrum (COL; n = 6) for 2 d following 48 h of total parenteral nutrition. Pigs received a dual stable isotope tracer infusion of threonine to determine intestinal threonine kinetics. NEC developed in 38% of the FORM pigs, whereas none of the COL pigs were affected (P = 0.13). Intestinal fractional first-pass threonine utilization was lower in FORM pigs (49 ± 2%) than in COL pigs (60 ± 4%) (P = 0.02). In FORM pigs compared with COL pigs, protein synthesis (369 ± 31 mg·kg(-1)·d(-1) vs. 615 ± 54 mg·kg(-1)·d(-1); P = 0.003) and MUC2 synthesis (121 ± 17%/d vs. 184 ± 15%/d; P = 0.02) were lower in the distal small intestine (SI). Our results suggest that formula feeding compared with colostrum feeding in preterm piglets reduces mucosal growth with a concomitant decrease in first-pass splanchnic threonine utilization, protein synthesis, and MUC2 synthesis in the distal SI. Hence, decreased intestinal threonine metabolism and subsequently impaired gut barrier function may predispose the formula-fed infant to developing NEC.

Modulation of the gut microbiota with antibiotic treatment suppresses whole body urea production in neonatal pigs

We examined whether changes in the gut microbiota induced by clinically relevant interventions would impact the bioavailability of dietary amino acids in neonates. We tested the hypothesis that modulation of the gut microbiota in neonatal pigs receiving no treatment (control), intravenously administered antibiotics, or probiotics affects whole body nitrogen and amino acid turnover. We quantified whole body urea kinetics, threonine fluxes, and threonine disposal into protein, oxidation, and tissue protein synthesis with stable isotope techniques. Compared with controls, antibiotics reduced the number and diversity of bacterial species in the distal small intestine (SI) and colon. Antibiotics decreased plasma urea concentrations via decreased urea synthesis. Antibiotics elevated threonine plasma concentrations and turnover, as well as whole body protein synthesis and proteolysis. Antibiotics decreased protein synthesis rate in the proximal SI and liver but did not affect the distal SI, colon, or muscle. Probiotics induced a bifidogenic microbiota and decreased plasma urea concentrations but did not affect whole body threonine or protein metabolism. Probiotics decreased protein synthesis in the proximal SI but not in other tissues. In conclusion, modulation of the gut microbiota by antibiotics and probiotics reduced hepatic ureagenesis and intestinal protein synthesis, but neither altered whole body net threonine balance. These findings suggest that changes in amino acid and nitrogen metabolism resulting from antibiotic- or probiotic-induced shifts in the microbiota are localized to the gut and liver and have limited impact on whole body growth and anabolism in neonatal piglets.

Small intestinal MUC2 synthesis in human preterm infants

Mucin 2 (MUC2) is the structural component of the intestinal protective mucus layer, which contains high amounts of threonine in its peptide backbone. MUC2 synthesis rate might be a potential parameter for intestinal barrier function. In this study, we aimed to determine whether systemic threonine was used for small intestinal MUC2 synthesis and to calculate the MUC2 fractional synthetic rate (FSR) in human preterm infants. Seven preterm infants with an enterostomy following bowel resection for necrotizing enterocolitis received intravenous infusion of [U-(13)C]threonine to determine incorporation of systemic threonine into secreted MUC2 in intestinal outflow fluid. Small intestinal MUC2 was isolated using cesium chloride gradient ultracentrifugation and gravity gel filtration chromatography. MUC2-containing fractions were identified by SDS-PAGE/periodic acid-Schiff staining and Western blot analysis and were subsequently pooled. Isotopic enrichment of threonine, measured in MUC2 using gas chromatography isotopic ratio mass spectrometry, was used to calculate the FSR of MUC2. Systemically derived threonine was indeed incorporated into small intestinal MUC2. Median FSR of small intestinal MUC2 was 67.2 (44.3-103.9)% per day. Systemic threonine is rapidly incorporated into MUC2 in the small intestine of preterm infants, and thereby MUC2 has a very high synthesis rate.

High beta-palmitate fat controls the intestinal inflammatory response and limits intestinal damage in mucin Muc2 deficient mice.

Background Palmitic-acid esterified to the sn-1,3 positions of the glycerol backbone (alpha, alpha’-palmitate), the predominant palmitate conformation in regular infant formula fat, is poorly absorbed and might cause abdominal discomfort. In contrast, palmitic-acid esterified to the sn-2 position (beta-palmitate), the main palmitate conformation in human milk fat, is well absorbed. The aim of the present study was to examine the influence of high alpha, alpha’-palmitate fat (HAPF) diet and high beta-palmitate fat (HBPF) diet on colitis development in Muc2 deficient (Muc2−/−) mice, a well-described animal model for spontaneous enterocolitis due to the lack of a protective mucus layer. Methods Muc2−/− mice received AIN-93G reference diet, HAPF diet or HBPF diet for 5 weeks after weaning. Clinical symptoms, intestinal morphology and inflammation in the distal colon were analyzed. Results Both HBPF diet and AIN-93G diet limited the extent of intestinal erosions and morphological damage in Muc2−/− mice compared with HAPF diet. In addition, the immunosuppressive regulatory T (Treg) cell response as demonstrated by the up-regulation of Foxp3, Tgfb1 and Ebi3 gene expression levels was enhanced by HBPF diet compared with AIN-93G and HAPF diets. HBPF diet also increased the gene expression of Pparg and enzymatic antioxidants (Sod1, Sod3 and Gpx1), genes all reported to be involved in promoting an immunosuppressive Treg cell response and to protect against colitis. Conclusions This study shows for the first time that HBPF diet limits the intestinal mucosal damage and controls the inflammatory response in Muc2−/− mice by inducing an immunosuppressive Treg cell response.

Dietary protein absorption of the small intestine in human neonates

BACKGROUND The intestine plays a key role in the absorption of dietary proteins, which determines growth of human neonates. Bowel resection in the neonatal period brings loss of absorptive and protective surface and may consequently lead to malabsorption of dietary nutrients. However, there are no data on net dietary protein absorption of the small intestine in the period after intestinal surgery in human neonates. We therefore evaluated dietary feeding tolerance and quantified net dietary protein absorption capacity of the small intestine in human neonates in whom a temporary jejunostomy or ileostomy was created. METHODS Seventeen patients were included in the study. We collected small intestinal outflow fluid at the level of the enterostomy weekly for 24-48 hours during weeks 3 through 6 postoperatively. Protein levels in the intestinal outflow fluid were determined by bicinchoninic acid (BCA) assay. RESULTS In 14 patients, an enteral intake of >100 mL/kg/d was reached at a median of 17 days (range, 8-32 days) postoperatively. Three patients did not reach this level within the study period. Overall, the net dietary protein absorption capacity was 70%-90% of the total enteral protein intake. CONCLUSIONS This study demonstrates that the dietary protein absorption capacity of the small intestine is intact in most human neonates after intestinal surgery in a very critical period of their lives. Furthermore, our results do not support the use of hydrolyzed or elemental formula in newborns with an enterostomy to improve amino acid uptake.

Endoplasmic Reticulum Stress, Unfolded Protein Response and Altered T Cell Differentiation in Necrotizing Enterocolitis

Background Endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) play important roles in chronic intestinal inflammation. Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in preterm infants and is characterized by acute intestinal inflammation and necrosis. The objective of the study is to investigate the role of ER stress and the UPR in NEC patients. Methods Ileal tissues from NEC and control patients were obtained during surgical resection and/or at stoma closure. Splicing of XBP1 was detected using PCR, and gene expression was quantified using qPCR and Western blot. Results Splicing of XBP1 was only detected in a subset of acute NEC (A-NEC) patients, and not in NEC patients who had undergone reanastomosis (R-NEC). The other ER stress and the UPR pathways, PERK and ATF6, were not activated in NEC patients. A-NEC patients showing XBP1 splicing (A-NEC-XBP1s) had increased mucosal expression of GRP78, CHOP, IL6 and IL8. Similar results were obtained by inducing ER stress and the UPR in vitro. A-NEC-XBP1s patients showed altered T cell differentiation indicated by decreased mucosal expression of RORC, IL17A and FOXP3. A-NEC-XBP1s patients additionally showed more severe morphological damage and a worse surgical outcome. Compared with A-NEC patients, R-NEC patients showed lower mucosal IL6 and IL8 expression and higher mucosal FOXP3 expression. Conclusions XBP1 splicing, ER stress and the UPR in NEC are associated with increased IL6 and IL8 expression levels, altered T cell differentiation and severe epithelial injury.

The Role of B Cells in Carriage and Clearance of Mycoplasma pneumoniae From the Respiratory Tract of Mice

Background Carriage of Mycoplasma pneumoniae (Mp) in the nasopharynx is considered a prerequisite for pulmonary infection. It is interesting to note that Mp carriage is also detected after infection. Although B cells are known to be involved in pulmonary Mp clearance, their role in Mp carriage is unknown. Methods In this study, we show in a mouse model that Mp persists in the nose after pulmonary infection, similar to humans. Results Infection of mice enhanced Mp-specific immunoglobulin (Ig) M and IgG levels in serum and bronchoalveolar lavage fluid. However, nasal washes only contained elevated Mp-specific IgA. These differences in Ig compartmentalization correlated with differences in Mp-specific B cell responses between nose- and lung-draining lymphoid tissues. Moreover, transferred Mp-specific serum Igs had no effect on nasal carriage in B cell-deficient μMT mice, whereas this enabled μMT mice to clear pulmonary Mp infection. Conclusions We report the first evidence that humoral immunity is limited in clearing Mp from the upper respiratory tract.