Immunobiotic Lactobacillus jensenii elicits anti-inflammatory activity in porcine intestinal epithelial cells by modulating negative regulators of the Toll-like receptor signaling pathway

Abstract

The effect of Lactobacillus jensenii TL2937 on the inflammatory immune response triggered by enterotoxigenic Escherichia coli (ETEC) and lipopolysaccharide (LPS) in a porcine intestinal epitheliocyte cell line (PIE cells) was evaluated. Challenges with ETEC or LPS elicited Toll-like receptor 4 (TLR4)-mediated inflammatory responses in cultured PIE cells, indicating that our cell line may be useful for studying inflammation in the guts of weaning piglets. In addition, we demonstrated that L. jensenii TL2937 attenuated the expression of proinflammatory cytokines and chemokines caused by ETEC or LPS challenge by downregulating TLR4-dependent nuclear factorκB (NF-κB) and mitogen-activated protein kinase (MAPK) activation. Furthermore, we demonstrated that L. jensenii TL2937 stimulation of PIE cells upregulated three negative regulators of TLRs: A20, Bcl-3, and MKP-1, deepening the understanding of an immunobiotic mechanism of action. L. jensenii TL2937-mediated induction of negative regulators of TLRs would have a substantial physiological impact on homeostasis in PIE cells, because excessive TLR inflammatory signaling would be downregulated. These results indicated that PIE cells can be used to study the mechanisms involved in the protective activity of immunobiotics against intestinal inflammatory damage and may provide useful information for the development of new immunologically functional feeds that help to prevent inflammatory intestinal disorders, including weaning-associated intestinal inflammation.

Fig 1

Expression of IL-1α, IL-6, IL-8, and MCP-1 mRNA in porcine intestinal epithelial (PIE) cells after inflammatory stimulation. (A) PIE cells were challenged with ETEC (5 × 10⁷ cells/ml) (solid line) or LPS (1 μg/ml) (broken line), and the levels of IL-1α, IL-6, IL-8, and MCP-1 mRNAs were evaluated at various time points after the inflammatory challenge. Unchallenged PIE cells at the indicated time were used as controls, and statistical analyses were conducted at each indicated time point. (B) PIE cells were stimulated with ETEC or LPS at various concentrations as indicated, and the levels of IL-1α, IL-6, IL-8, and MCP-1 mRNAs were evaluated 12 h after the inflammatory challenge. Unchallenged, untreated PIE cells were used as controls. Values with different superscript letters are significantly different (P < 0.05). (C) PIE cells were stimulated with ETEC (5 × 10⁷ cells/ml) pretreated with the LPS inhibitor polymyxin B at the indicated concentrations (in micrograms per milliliter). The levels of IL-1α, IL-6, IL-8, and MCP-1 mRNAs were evaluated 12 h after the inflammatory challenge. Unchallenged PIE cells were used as controls. The results represent data from three independent experiments. Values with different letters are significantly different (P < 0.05).

Fig 2

Expression of IL-1α, IL-6, IL-8, and MCP-1 mRNAs in porcine intestinal epithelial (PIE) cells after treatment with different lactobacillus strains. PIE cells were pretreated with lactobacilli for 48 h, and subsequently the levels of IL-1α, IL-6, IL-8, and MCP-1 mRNAs were evaluated. The results represent three independent experiments. Values with different letters are significantly different (P < 0.05).

Fig 3

Expression of IL-1α, IL-6, IL-8, and MCP-1 mRNAs in porcine intestinal epithelial (PIE) cells after lactobacillus treatment and inflammatory challenge. (A) PIE cells were pretreated with L. jensenii TL2937, L. reuteri MEP221102, or L. rhamnosus MEP221111 for 3 or 12 h and then challenged with ETEC (5 × 10⁷ cells/ml). The levels of IL-1α, IL-6, IL-8, and MCP-1 mRNAs were evaluated 12 h after the inflammatory challenge. ETEC-challenged PIE cells that were not pretreated with lactobacilli were used as the ETEC controls. (B) PIE cells were pretreated with L. jensenii TL2937, L. reuteri MEP221102, or L. rhamnosus MEP221111 for 48 h and then challenged with ETEC (5 × 10⁷ cells/ml). The levels of IL-1α, IL-6, IL-8, and MCP-1 mRNAs were evaluated 24 h after the inflammatory challenge. ETEC-challenged PIE cells that were not pretreated with lactobacilli were used as the ETEC controls. (C) PIE cells were pretreated with L. jensenii TL2937, L. reuteri MEP221102, or L. rhamnosus MEP221111 for 48 h and then challenged with ETEC (5 × 10⁷ cells/ml). The concentration of IL-6 and IL-8 protein in culture supernatants was determined 48 h after the inflammatory challenge. Unchallenged, untreated PIE cells were used as controls. The results represent data from two independent experiments. Values with different superscript letters were significantly different (P < 0.05).

Fig 4

Expression of IL-1α, IL-6, IL-8, and MCP-1 mRNAs in porcine intestinal epithelial (PIE) cells after lactobacillus treatment and inflammatory challenge. (A) PIE cells were pretreated with L. jensenii TL2937, L. reuteri MEP221102, or L. rhamnosus MEP221111 for 48 h and then stimulated with LPS (1 μg/ml). The levels of IL-1α, IL-6, IL-8, and MCP-1 mRNAs were evaluated 12 h after the inflammatory challenge. LPS-challenged PIE cells that were not pretreated with lactobacilli were used as the LPS controls. (B) PIE cells were pretreated with the supernatant from cocultures of PIE cells and L. jensenii TL2937 and then challenged with ETEC (5 × 10⁷ cells/ml). The levels of IL-1α, IL-6, IL-8, and MCP-1 mRNAs were evaluated 12 h after the inflammatory challenge. (C) Expression of the TLR4 protein was determined by flow cytometry after 48 h of pretreatment with L. jensenii TL2937. PIE cells without any treatments were used as controls. Histograms show flow cytometric analysis as follow: PIE cells stained with anti-TLR4 antibody (black line) and isotype-matched controls (gray line). Values of mean fluorescence intensity (MFI) are also shown. The results represent data from three independent experiments. Values with different letters are significantly different (P < 0.05).

Fig 5

Western blot analysis of MAPK and NF-κB activation in PIE cells following inflammatory challenge. PIE cells were pretreated with lactobacilli for 48 h and then challenged with ETEC (A) or LPS (B). Samples were collected at the indicated time points after ETEC or LPS challenge. (C) The intensities of the IκBα, p-p38, and p38 bands were determined using image analysis software (Image J; Research Services Branch, NIMH, NIH, Bethesda, MD). The densitogram of each band was determined, and the relative density was expressed as the area of the peak in the densitogram. (D) Expression levels of IL-1α, IL-6, IL-8, and MCP-1 mRNA in PIE cells treated with p38 inhibitor PD169316 (10 nM) for 1 h and then stimulated with ETEC for 12 h. Values with different letters are significantly different (P < 0.05).

Fig 6

Expression of IL-6 and IL-8 proteins on porcine intestinal epithelial (PIE) cells following inflammatory challenge. PIE cells were cocultured with Peyer's patch immunocompetent cells, pretreated with L. jensenii TL2937 for 48 h, and then challenged with ETEC (5 × 10⁷ cells/ml). The levels of IL-6 and IL-8 proteins were determined by flow cytometry 12 h after the inflammatory challenge. PIE cells without any treatments were used as controls. (A) Histograms show flow cytometric analyses as follows: PIE cells stained with anti-IL-8 or anti-IL-6 antibodies (black lines) and isotype-matched controls (gray lines). (B) Values of mean fluorescence intensity (MFI) are shown for each group. The results represent data from three independent experiments. Values with different letters are significantly different (P < 0.05).

Fig 7

Expression of three negative regulators of Toll-like receptors (TLRs): A20, Bcl-3, and MKP-1 mRNAs in porcine intestinal epithelial (PIE) cells. (A) PIE cells were pretreated with L. jensenii TL2937, L. reuteri MEP221102, or L. rhamnosus MEP221111 for 36 h, and then the levels of A20, Bcl-3, and MKP-1 mRNA were evaluated. The results represent data from three independent experiments. Values with different letters are significantly different (P < 0.05). (B) PIE cells were stimulated with Pam3CSK4 (200 ng/ml), and then the levels of A20, Bcl-3, and MKP-1 mRNAs were evaluated at the indicated time points. A20 and Bcl-3 mRNA expression in PIE cells stimulated with Pam3CSK4 was significantly higher at 48 h than at 0 h (P < 0.05).