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, 299 (5), G1087-96

Human-derived Probiotic Lactobacillus Reuteri Strains Differentially Reduce Intestinal Inflammation

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Human-derived Probiotic Lactobacillus Reuteri Strains Differentially Reduce Intestinal Inflammation

Yuying Liu et al. Am J Physiol Gastrointest Liver Physiol.

Abstract

Lactobacillus reuteri (L. reuteri) is a probiotic that inhibits the severity of enteric infections and modulates the immune system. Human-derived L. reuteri strains DSM17938, ATCC PTA4659, ATCC PTA 5289, and ATCC PTA 6475 have demonstrated strain-specific immunomodulation in cultured monocytoid cells, but information about how these strains affect inflammation in intestinal epithelium is limited. We determined the effects of the four different L. reuteri strains on lipopolysaccharide (LPS)-induced inflammation in small intestinal epithelial cells and in the ileum of newborn rats. IPEC-J2 cells (derived from the jejunal epithelium of a neonatal piglet) and IEC-6 cells (derived from the rat crypt) were treated with L. reuteri. Newborn rat pups were gavaged cow milk formula supplemented with L. reuteri strains in the presence or absence of LPS. Protein and mRNA levels of cytokines and histological changes were measured. We demonstrate that even though one L. reuteri strain (DSM 17938) did not inhibit LPS-induced IL-8 production in cultured intestinal cells, all strains significantly reduced intestinal mucosal levels of KC/GRO (∼IL-8) and IFN-γ when newborn rat pups were fed formula containing LPS ± L. reuteri. Intestinal histological damage produced by LPS plus cow milk formula was also significantly reduced by all four strains. Cow milk formula feeding (without LPS) produced mild gut inflammation, evidenced by elevated mucosal IFN-γ and IL-13 levels, a process that could be suppressed by strain 17938. Other cytokines and chemokines were variably affected by the different strains, and there was no toxic effect of L. reuteri on intestinal cells or mucosa. In conclusion, L. reuteri strains differentially modulate LPS-induced inflammation. Probiotic interactions with both epithelial and nonepithelial cells in vivo must be instrumental in modulating intrinsic anti-inflammatory effects in the intestine. We suggest that the terms anti- and proinflammatory be used only to describe the effects of a probiotic in the living host.

Figures

Fig. 1.
Fig. 1.
IL-8 [cytokine-induced neutrophil chemoattractant-1 (CINC-1)] production from cultured intestinal epithelial cells after treatment with Lactobacillus reuteri strains in combination with LPS. Cells (1 × 105 cells/well) were treated with Lactobacillus reuteri strains in combination with indicated different concentrations of LPS for 16 h, respectively. IL-8 (or CINC-1) levels in culture supernatants were examined by ELISA assay for porcine IL-8 or rat CINC-1. A: IPEC-J2 cells. All L. reuteri strains (ATCC PTA 4659, 5289, and 6475) except DSM 17938 suppressed LPS-induced IL-8 secretion by IPEC-J2 cells. B: IEC-6 cells. Only strain 4659 inhibited LPS-induced CINC-1 production by IEC-6 cells. Data are represented as means ± SE, N = 4–6. *P < 0.05 L. reuteri strain + LPS compared with LPS.
Fig. 2.
Fig. 2.
Effects of L. reuteri strains on LPS-induced chemokine keratinocyte-derived chemokine/growth-related oncogene (KC/GRO) production by newborn rat intestine. One-day-old rat pups were dam-fed or fed with formula or formula + LPS (0.25 mg·kg body wt−1·day−1) or formula + L. reuteri (106 colony-forming units·g body wt−1·day−1) ± LPS for 3 days. KC/GRO production in ileal tissue lysates were assayed by using a MSD rat multiplex assay. Data are represented as means ± SE, N = 8–10 animals per group. Formula + LPS group compared with either dam-fed or formula-fed control: **P < 0.01. Formula + L. reuteri + LPS group compared with formula + LPS group: #P < 0.05, ##P < 0.01. LPS feeding significantly increased the level of KC/GRO in rat intestine, compared with either breast milk or formula feeding. None of the L. reuteri strains given alone increased intestinal KC/GRO production. All measured L. reuteri strains significantly inhibit LPS-induced KC/GRO production in rat intestines.
Fig. 3.
Fig. 3.
Effects of L. reuteri strains on LPS-induced IFN-γ levels in rat intestine. A: IFN-γ mRNA expression levels from the ilea of rat pups with L. reuteri strains and LPS feeding. RNA was isolated from the ileum of newborn rats. Quantitative RT-PCR (qRT-PCR) analysis was performed to determine the expression level (fold change) of IFN-γ comparing L. reuteri + LPS-fed rats with LPS-fed rats. Data are represented as means ± SE, N = 6. The dotted lines show cutoff values of fold change: > +2 indicating upregulation, < −2 downregulation. *P < 0.05 vs. LPS control. All 4 L. reuteri strains significantly downregulated mRNA expression of IFN-γ induced by LPS. B: IFN-γ protein level in rat intestine. IFN-γ protein in ileal tissue lysates were assayed by using a MSD rat multiplex assay. Data are represented as means ± SE, N = 8–10. Formula ± LPS-fed groups compared with dam-fed control: **P < 0.01. Formula + LPS compared with formula group: δP < 0.05. Formula + L. reuteri groups compared with formula-fed group: $P < 0.05. L. reuteri + LPS groups compared with LPS group: #P < 0.05. All 4 L. reuteri strains inhibited LPS-induced IFN-γ production. Only L. reuteri DSM 17938 significantly reduced formula-induced inflammation.
Fig. 4.
Fig. 4.
Effects of L. reuteri strains on LPS-induced IL-13 levels in rat intestine. A: IL-13 mRNA expression levels from the ilea of rat pups with L. reuteri strains and LPS feeding. qRT-PCR analysis was performed to determine the expression level (fold changes) of IL-13 comparing L. reuteri + LPS-fed rats with LPS-fed rats. Data are represented as means ± SE, N = 6. Dotted lines show cutoff values of fold change: > +2 indicating upregulation, < −2 downregulation. *P < 0.05 vs. LPS control. L. reuteri strains DSM 17938 and ATCC PTA 4659 significantly downregulated mRNA expression of IL-13 induced by LPS. B: IL-13 protein level in rat intestine. IL-13 protein in ileal tissue lysates were assayed by using a MSD rat multiplex assay. Data are represented as means ± SE, N = 8–10. Formula ± LPS-fed groups compared with dam-fed control: **P < 0.01. Formula + L. reuteri groups compared with formula-fed group: $P < 0.05. L. reuteri + LPS groups compared with LPS group: #P < 0.05. L. reuteri strains DSM 17938 and ATCC PTA 4659 inhibit LPS-induced IL-13 production. Only L. reuteri strain DSM 17938 significantly reduced formula-stimulated IL-13.
Fig. 5.
Fig. 5.
Effects of L. reuteri strains on intestinal morphology. Ileal morphology was observed by light microscopy. Ileal villus length and villus density were measured. A: ileal morphology. a: dam-fed (n = 8). b: formula-fed (n = 9). c: formula + LPS (n = 11). d: formula + L. reuteri DSM 17938 + LPS (n = 10). e: formula + L. reuteri ATCC PTA 4659 + LPS (n = 10). f: formula + L. reuteri ATCC PTA 5289 + LPS (n = 10). g: formula + L. reuteri ATCC PTA 6475 + LPS (n = 10). Magnification = ×200. B: comparison of villous length (in μm) among different treatment groups. C: comparison of villous density (number of villi per unit intestinal circumference) among different groups. Note: &P < 0.05 formula-fed without LPS rats compared with dam-fed rats; *P < 0.05 rat pups fed with formula containing LPS compared with formula-fed without LPS; #P < 0.05 comparing LPS without L. reuteri strains compared with LPS with L. reuteri strains. The data are expressed as means ± SE. Feeding of 4 L. reuteri strains was associated with improved villus length and density after LPS damage.

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