The level of protein in milk formula modifies ileal sensitivity to LPS later in life in a piglet model

Abstract

Background: Milk formulas have higher protein contents than human milk. This high protein level could modify the development of intestinal microbiota, epithelial barrier and immune functions and have long-term consequences.

Methodology/principal findings: We investigated the effect of a high protein formula on ileal microbiota and physiology during the neonatal period and later in life. Piglets were fed from 2 to 28 days of age either a normoprotein (NP, equivalent to sow milk) or a high protein formula (HP, +40% protein). Then, they received the same solid diet until 160 days. During the formula feeding period ileal microbiota implantation was accelerated in HP piglets with greater concentrations of ileal bacteria at d7 in HP than NP piglets. Epithelial barrier function was altered with a higher permeability to small and large probes in Ussing chambers in HP compared to NP piglets without difference in bacterial translocation. Infiltration of T cells was increased in HP piglets at d28. IL-1β and NF-κB sub-units mRNA levels were reduced in HP piglets at d7 and d28 respectively; plasma haptoglobin also tended to be reduced at d7. Later in life, pro-inflammatory cytokines secretion in response to high doses of LPS in explants culture was reduced in HP compared to NP piglets. Levels of mRNA coding the NF-κB pathway sub-units were increased by the challenge with LPS in NP piglets, but not HP ones.

Conclusions/significance: A high protein level in formula affects the postnatal development of ileal microbiota, epithelial barrier and immune function in piglets and alters ileal response to inflammatory mediators later in life.

Figure 1. Ileal ex vivo permeability of NP and HP piglets.

Permeability to FD-4 (A) and HRP (B) in the ileum of 2-d old piglets (grey bars) and of NP (open bars) and HP (solid bars) piglets at 7 and 28 days of age was determined in Ussing chambers. Values are means ± SE, n = 5–6. *, P<0.05. The HP formula induced an increase of FD-4 permeability at d 7 and HRP permeability at both d 7 and d 28 in the piglet ileum.

Figure 2. IgG and haptoglobin concentrations in the plasma of NP and HP piglets.

IgG (A) and haptoglobin (B) concentrations were determined in the plasma of 2-d old piglets (grey bars) and of NP (open bars) and HP (solid bars) piglets at 7 and 28 days of age. Values are means ± SE; n = 5–6. *, P<0.05 ; #, P≤0.06. HP piglets exhibited lower plasma concentrations of IgG and haptoglobin than NP piglets at d 7.

Figure 3. Ileal explant responses to LPS of NP and HP piglets at d 160 of life.

TNFα (A) and IL-8 (B) release by ileal explants of 160 day-old piglets fed either the NP (open squares) or the HP (solid squares) formula from d 2 to d 28 of life was measured in response to different doses of LPS. NF-κB subunits mRNA levels were measured in explants stimulated with 200 µg/mL of LPS and compared to that of non-stimulated explants (C). Values are means ± SE; n = 8–10. *, P<0.05., FC fold-change. Ileal explants from HP pigs exhibited less cytokine release in response to high doses of LPS. Moreover, while NF-κB1 and 2 mRNA levels were increased after challenge with 200 µg/mL LPS in explants from NP pigs, this increase did not occur in explants from HP ones.