Lactobacillus reuteri strains reduce incidence and severity of experimental necrotizing enterocolitis via modulation of TLR4 and NF-κB signaling in the intestine

Abstract

Necrotizing enterocolitis (NEC) is the leading gastrointestinal cause of mortality and morbidity in the premature infant. Premature infants have a delay in intestinal colonization by commensal bacteria and colonization with potentially pathogenic organisms. Lactobacillus reuteri is a probiotic that inhibits enteric infections, modulates the immune system, and may be beneficial to prevent NEC. In previous studies, L. reuteri strains DSM 17938 and ATCC PTA 4659 differentially modulated inflammation in vitro; however, the strains had equivalent anti-inflammatory responses in LPS feeding-induced ileitis in neonatal rats in vivo. The impact of these two strains in the prevention of NEC has not been previously investigated. NEC was induced in newborn rats by orogastric formula feeding and exposure to hypoxia. L. reuteri was added to the formula to prevent NEC. NEC score, Toll-like receptor (TLR)-signaling genes, phospho-IκB activity, and cytokine levels in the intestine were examined. Both strains significantly increased survival rate and decreased the incidence and severity of NEC, with optimal effects from DSM 17938. In response to probiotic, mRNA expression of IL-6, TNF-α, TLR4, and NF-κB was significantly downregulated, while mRNA levels of anti-inflammatory cytokine IL-10 were significantly upregulated. In parallel, L. reuteri treatment led to decrease intestinal protein levels of TLR4 and cytokine levels of TNF-α and IL-1β in newborn rats with NEC. Both strains significantly inhibited not only intestinal LPS-induced phospho-IκB activity in an ex vivo study but also decreased the levels of phospho-IκB in the intestines of NEC rat model. Cow milk formula feeding produced a similar but milder proinflammatory profile in the intestine that was also ameliorated by 17938. Our studies demonstrate that each of the two L. reuteri strains has potential therapeutic value in our NEC model and in enteritis associated with cow milk feeding. These results support the concept that L. reuteri may represent a valuable treatment to prevent NEC.

Fig. 1.

Survival rate of rat pups with necrotizing enterocolitis (NEC) compared with NEC pups that were supplemented with Lactobacillus reuteri strains. Newborn rats were separated from their mothers and housed in an incubator. NEC was induced by formula feeding plus exposure to hypoxia (5% oxygen-95% nitrogen for 10 min, 3 times per day) from days 1 to 3 (indicated as arrow). Rat pups were euthanized on day 4 (indicated by “X”), and tissues were collected. L. reuteri strain DSM 17938 or ATCC PTA 4659 (10⁶ CFU·g body wt⁻¹·day⁻¹, where CFU is colony-forming unit) was added in formula for treatment. Groups studied were as follows: 1): NEC + 17938 (n = 38); 2) NEC + 4659 (n = 36); and 3) NEC + no probiotic (n = 46). Both strains reduced mortality rate on days 3 and 4 compared with NEC without probiotics. *P < 0.05, **P < 0.01, ***P < 0.001, NEC + 17938 or NEC + 4659 vs. NEC without probiotic; ^#P < 0.05, NEC + 17938 vs. NEC + 4659.

Fig. 2.

Survival rate of rat pups with cow milk-based formula feeding compared with formula plus L. reuteri. Newborn rats were separated from their mothers and fed with formula (without hypoxia exposure) beginning on day 1 for 3 days (indicated with arrow). Rat pups were euthanized on day 4 (indicated by “X”). L. reuteri strain DSM 17938 or strain ATCC PTA 4659 (10⁶ CFU·g body wt⁻¹·day⁻¹) was added in formula for treatment without hypoxia. Groups studied were as follows: 1) formula + 17938 (n = 22); 2) formula + 4659 (n = 17); and 3) formula alone (n = 31). *P < 0.05.

Fig. 3.

Intestinal morphology of neonatal rat ileum. Ileal tissues of rat pups were collected after 3 days of induction of NEC, with or without L. reuteri treatment. Tissue sections were stained with hematoxylin and eosin with magnification = ×100. Groups studied were as follows: 1) dam fed (A); 2) formula fed (B1–B2); 3) NEC (C1–C3); 4) NEC + 17938 (D1–D3); and 5) NEC + 4659 (E1–E3).

Fig. 4.

Expression of Toll-like receptor-4 (TLR4) protein in the ileum. Tissue lysates were prepared for Western blot analysis, and 50 μg of total protein were loaded and immunoblotted with anti-TLR4 antibody with β-actin as a loading control. A: typical blot. Numbers represent number of animals examined in each group. B: relative densitometric values, means ± SE. Groups studied were as follows: 1) dam fed (n = 6); 2) formula fed (n = 6); 3) NEC (n = 12); 4) NEC + 17938 (n = 12); and 5) NEC + 4659 (n = 12). *P < 0.05, NEC vs. dam fed or formula fed; ^##P < 0.01, ^###P < 0.001, NEC + 17938 or NEC + 4659 vs. NEC.

Fig. 5.

Phospho-IκB activation in the intestines of newborn rats in an ex vivo experiment (A) and in the NEC model (B). A: ileal tissues from each newborn rat (day of life 0) were collected in an antibiotic-free DMEM complete medium and immediately treated with probiotics (strain17938 or strain 4659) or control bacteria [Lactobacillus acidophilus DDS (La DDS) or Escherichia coli DH5α; 3–4 × 10⁷ CFU] for 4 h, washed, and subsequently treated with LPS (E. coli subtype 0111:B4), 100 μg/ml for 30 min. Tissues were washed with PBS and homogenized in a lysis buffer containing protease inhibitors. Tissue lysates were used to determine phospho-IκB activity by ELISA. Activity of phospho-IκB (Ser32) was expressed as units/ mg total protein in tissue lysates; n = 6 rats for each group. ^aGroups compared with no treatment; ^b17938-LPS or 4659-LPS compared with LPS. Strains 17938 and 4659 both inhibited IκB phosphorylation by > 90%: ***P < 0.001. B: phospho-IκB analysis in the ileum of rat pup with NEC as examined by Western blot analysis. Fifty micrograms of total protein were loaded and immunoblotted with anti-phospho-IκB and IκB antibodies, with β-actin as a loading control. Top: a typical blot; numbers represent number of animals examined in each group. Bottom: relative densitometric values are means ± SE; n = 6 for each groups. **P < 0.01, NEC vs. dam fed; ^##P < 0.01, NEC + 17938 vs. NEC.

Fig. 6.

TNF-α (A) and IL-1β (B) levels in the intestines of rats with NEC compared with rats with NEC fed with supplemental L. reuteri. Tissue lysates were examined cytokine levels by using MSD multiplex cytokine assay; n = 6–7 rats per group. ^aGroups were compared with dam fed; ^bNEC group was compared with formula-fed group; ^cgroups NEC + 17938 or NEC + 4659 were compared with NEC without probiotic. Increased cytokine levels in NEC were both decreased by strains 17938 and 4659. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 7.

Cytokine levels of TNF-α, IL-1β, and IFN-γ in the intestines of rats with formula ± 17938 feeding (no hypoxia). Newborn rats were left with their mothers (dam fed) or separated from their mothers and housed in an incubator for formula feeding only without hypoxic exposure. L. reuteri strain DSM 17938 (10⁶ CFU·g body wt⁻¹·day⁻¹) was added to formula. Ileal tissues were homogenized in a lysis buffer containing protease inhibitors. Tissue lysates were examined cytokine levels by using MSD multiplex cytokine assay; n = 6–9 rats per group. Comparisons were as follows: ^a,c,eformula fed vs. dam fed; ^b,d,fL. reuteri strain 17938-supplemented vs. formula without 17938. Strain 17938 significantly decreased formula-induced cytokine levels in the intestines of newborn rats. *P < 0.05, **P < 0.01, ***P < 0.001.