Formula diet alters small intestine morphology, microbial abundance and reduces VE-cadherin and IL-10 expression in neonatal porcine model

Abstract

Background: Breastfeeding is associated with a variety of positive health outcomes in children and is recommended exclusively for the first 6 months of life; however, 50-70 % of infants in the US are formula-fed. To test the hypothesis that immune system development and function in neonates and infants are significantly influenced by diet, 2-day old piglets were fed soy or milk formula (n = 6/group/gender) until day 21 and compared to a sow-fed group (n = 6/gender).

Methods: Histomorphometric analyses of ileum, jejunum and Peyer's patches were carried out, to determine the inflammation status, mRNA and protein expression of pro-inflammatory, anti-inflammatory and growth-related chemokines and cytokines.

Results: In formula-fed animals, increases in ileum and jejunum villus height and crypt depth were observed in comparison to sow-fed animals (jejunum, p < 0.01 villus height, p < 0.04 crypt depth; ileum p < 0.001 villus height, p < 0.002 crypt depth). In formula-fed the lymphoid follicle size (p < 0.01) and germinal centers (p < 0.01) with in the Peyer's patch were significantly decreased in comparison to sow-fed, indicating less immune education. In ileum, formula diet induced significant up-regulation of AMCFII, IL-8, IL-15, VEGFA, LIF, FASL, CXCL11, CCL4, CCL25 and down-regulation of IL-6, IL-9, IL-10, IL-27, IFNA4, CSF3, LOC100152038, and LOC100736831 at the transcript level. We have confirmed some of the mRNA data by measuring protein, and significant down-regulation of anti-inflammatory molecule IL-10 in comparison to sow-fed piglets was observed. To further determine the membrane protein expression in the ileum, VE-cadherin, occludin, and claudin-3, Western blot analyses were conducted. Sow fed piglets showed significantly more VE-Cadherin, which associated with levels of calcium, and putrescine measured. It is possible that differences in GI tract and immune development are related to shifts in the microbiome; notably, there were 5-fold higher amounts of Lactobacillaceae spp and 3 fold higher Clostridia spp in the sow fed group in comparison to milk formula-fed piglets, whereas in milk formula-fed pigs Enterobacteriaceae spp was 5-fold higher.

Conclusion: In conclusion, formula diet alters GI morphology, microbial abundance, intestinal barrier protein VE-cadherin and anti-inflammatory molecule IL-10 expression. Further characterization of formula effects could lead to modification of infant formula to improve immune function, reduce inflammation and prevent conditions such as allergies and infections.

Keywords: GALT; Ileum; Jejunum; Nutrition; Peyer’s patches.

Fig. 1

Data are a pool of 6 males and 6 female piglets for each diet group and mean ± SEM from 12 animals are plotted for each diet group. a Small intestine length from piglets fed three different infant diets (sow milk, soy or cow’s milk formula) is reported as cm/kg body weight. Non parametric 2-factor ANOVA analyses of data indicated a group difference but no interaction or gender differences (p < 0.01 diet, p < 0.58 sex, p < 0.51 interaction). Milk fed animals showed significant differences in comparison to sow-fed (KW ^† p < 0.01). b Perimeter of the mucosa was calculated as explained in the methods section. One-factor ANOVA analyses of data showed significant differences between sow and soy (P < 0.0001) and sow and milk (P < 0.1, at tenth percentile). c Histomorphometric analyses of jejunum and ileum vacuoles from piglets fed three different infant diets (sow milk, soy or cow’s milk formula). Negative binomial regression analyses indicated statistical significance among different diet groups (p < 0.01) in jejunum

Fig. 2

Histomorphometric analyses of jejunum and ileum from piglets fed three different infant diets (sow milk, soy or cow’s milk formula). Data are a pool of 6 males and 6 female piglets for each diet group and mean ± SEM from 12 animals are plotted for each diet group. Villus height and crypt depth are indicated in micrometers on Y-axis. Non parametric 2-factor ANOVA analyses of data indicated a group difference but no interaction or gender differences. a Representative pictures of ileum villus height and crypt depth of sow, soy and milk fed piglets. Villus height and crypt depth are indicated with arrows. b Jejunum statistical analyses for villus height (p < 0.01 diet, p < 0.26 gender, and p < 0.71 interaction) and crypt depth (*p < 0.05 diet, p < 0.67 gender, and p < 0.14 interaction) indicates increased villus height and crypt depth in formula groups in comparison to sow fed (KW ^† p < 0.01 villus height, *p < 0.05 crypt depth). c Ileum statistical analyses for villus height (p < 0.0008 diet, p < 0.33 gender, and p < 0.43 interaction), crypt depth (p < 0.0005 diet, p < 0.12 gender, and p < 0.95 interaction) showed increased villus height and crypt depth in formula groups in comparison to sow fed (KW #p < 0.001 villus height, ^‡ p < 0.005 crypt depth)

Fig. 3

Histomorphometric analyses of ileum and jejunum lymphoid follicle. Data are a pool of 6 males and 6 female piglets for each diet group and mean ± SEM from 12 animals are plotted for each diet group. Non parametric 2-factor ANOVA analyses of data indicated a group difference but no interaction or gender differences. The entire area of the lymphoid follicle from ileum and jejunum area were measured which is indicated as size. The follicle size is 10,000 times to the Y-axis (μm). a Representative pictures of ileum lymphoid follicle of sow, soy and milk fed piglets. Sow fed piglets show bigger follicle than soy or milk fed piglets. b No significant differences were observed in size of the follicle within the Ileum with different diets (p < 0.14 diet, p < 0.54 gender and p < 0.18 interaction). However significant differences were observed in the diameter of germinal centers (GC) measured (p < 0.0005 diet, p < 0.06 gender and p < 0.3751 interaction) in the ileum. c Similarly to ileum the entire area of the lymphoid follicle of jejunal PP was measured which is indicated as size and significant differences were observed in size of the follicle in jejunal PP with different infant diets (p < 0.009 diet, p < 0.70 gender and p < 0.37 interaction). In addition, significant differences were observed in the diameter of germinal centers (GC) measured (p < 0.005 diet, p < 0.7 gender and p < 0.54 interaction). KW p values are indicated on the graphs of ileum and PP in soy and milk formula groups with respect to sow fed (^† p < 0.01, ^‡ p < 0.005) on the graphs

Fig. 4

Data are a pool of 12 piglets (6/gender) for each diet group for calcium and protein data. Data are presented as mean ± SEM and are analyzed by 2-tailed t –test. a Western blot image of E-cadherin. b Relative expression of VE-Cadherin showed significantly lower levels in milk group in comparison to sow (^† p < 0.01) c Calcium d Ileum protein expression of milk group showed significant difference with IL-10 (#P < 0.001), IL-8 (P < 0.1, at tenth percentile) and IL-6 (*P < 0.05,) in comparison to sow

Fig. 5

Data are a pool of 12 piglets (6/gender) for each diet group for polyamine data. Microbiome data for milk group N is 11 (5 males and 6 females) and for sow group N is 6 (3 males and 3 females). Data are presented as mean ± SEM and are analyzed by 2-tailed t –test. a Ileum tissue polyamine measurements showed significantly higher Putrescine (*p < 0.05), spermidine (*p < 0.07, at tenth percentile) and no significant differences with spermine in sow fed piglets in comparison to milk formula fed piglets. b PCoA plot of ileum microbiome c Ileum contents microbiome analyses showed significant differences between sow and milk fed piglets with Lactobacillaceae spp ($P < 0.000001), Clostridiaceae spp (*p < 0.05), Enterobacteriaceae spp (^† p < 0.01), and Streptococcaceae spp (*p < 0.05)