Additional file 2. IPEC-J2 cells pretreated with TLR2 ligand maintained the expression of MCP-1, GM-CSF and TLR2 against DON exposure. IPEC-J2 cells pretreated with or without TLR2 ligand for 24 h were exposed to DON. (A) The bar graph showed the mRNA levels of porcine mcp-1, gm-csf measured using real time-PCR at 1 and 6 h after DON exposure (n = 3). (B) The mRNA levels of porcine tlr2 were measured using real-time quantitative PCR analysis at 6 h. NT represents no treatment. Expression of each mRNA was presented relative to the expression of housekeeping gene, gapdh (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001, determined by one-way ANOVA with Tukey’s posttest

Byung-Chul Park

Cheol-Heui Yun

Hak-Kyo Lee

In Kyu Lee

Ki-Duk Song

Min Jeong Gu

Sang Yun Ji

Seongyeol Ko

Seung Eun Han

Seung Hyun Han

Suhan Bae

Sun Kwang Song

Veterinary Research

PubMed

Springer - Publisher Connector

Barrier protection via Toll-like receptor 2 signaling in porcine intestinal epithelial cells damaged by deoxynivalnol

International audienceAbstractIntestinal barrier is the first line of defense inside the body and comprises intercellular tight junction (TJ) proteins that regulate paracellular permeability. Deoxynivalenol (DON), a fungal metabolite often found in the contaminated food of domestic animals, is known to impair intestinal barrier function and may be involved in intestinal inflammation. Unlike in humans and mice, the importance of Toll-like receptor (TLR) 2 expressed in porcine intestinal epithelial cells is largely unclear. Therefore, the aim of the present study was to investigate whether TLR2 stimulation enhances intestinal barrier function and protects against DON exposure. We found that the cells treated with TLR2 ligands decreased the epithelial barrier permeability and enhanced TJ protein expression in intestinal porcine epithelial cells (IPEC-J2). In addition, pretreatment with TLR2 ligand, including Pam3CSK4 (PCSK) and lipoteichoic acid from Bacillus subtilis, prevented DON-induced barrier dysfunction by increasing the expression of TJ proteins via the PI3K-Akt-dependent pathway. It is likely that the DON-disrupted intestinal barrier caused biological changes of immune cells in the lamina propria. Thus, we conducted co-culture of differentiated IPEC-J2 cells in the upper well together with peripheral blood mononuclear cells in the bottom well and found that apical TLR2 stimulation of IPEC-J2 cells could alleviate the reduction in cell survival and proliferation of immune cells. Conclusively, TLR2 signaling on intestinal epithelial cells may enhance intestinal barrier function and prevent DON-induced barrier dysfunction of epithelial cells

Gu, Min Jeong

Song, Sun Kwang

Lee, In Kyu

Ko, Seongyeol

Han, Seung Eun

Bae, Suhan

Ji, Sang Yun

Park, Byung-Chul

Song, Ki-Duk

Lee, Hak-Kyo

Han, Seung Hyun

Yun, Cheol-Heui

HAL: Hyper Article en Ligne

Additional file 3. Co-culture of IPEC-J2 cells with PBMCs induced positive effect on immune cells. IPEC-J2 cells were cultured together with PBMCs on the transwell plate (0.4 μm pore). (A) Annexin V expression was measured at 48 h after co-culture (n = 3). * and ** indicate P < 0.05 and P < 0.01, respectively when compared to PBMCs only, determined by one-way ANOVA with Tukey’s posttest. (B) Phenotype of T lymphocyte subsets (CD3+ gated) and CD172a+ monocytes was examined by flow cytometry at 48 h after co-culture (n = 3)

Min Gu (305818)

Sun Song (3557705)

In Lee (3557690)

Seongyeol Ko (3557696)

Seung Han (3447431)

Suhan Bae (3557702)

Sang Ji (3557699)

Byung-Chul Park (3557693)

Ki-Duk Song (673013)

Hak-Kyo Lee (540982)

Cheol-Heui Yun (78927)

The Francis Crick Institute

MOESM3 of Barrier protection via Toll-like receptor 2 signaling in porcine intestinal epithelial cells damaged by deoxynivalnol

MOESM2 of Barrier protection via Toll-like receptor 2 signaling in porcine intestinal epithelial cells damaged by deoxynivalnol

Additional file 1. The primer sequences for real time-PCR. Primer sequences for real time-PCR in supplementary data (Additional files 2, 3, 4)

MOESM1 of Barrier protection via Toll-like receptor 2 signaling in porcine intestinal epithelial cells damaged by deoxynivalnol

Intestinal barrier is the first line of defense inside the body and comprises intercellular tight junction (TJ) proteins that regulate paracellular permeability. Deoxynivalenol (DON), a fungal metabolite often found in the contaminated food of domestic animals, is known to impair intestinal barrier function and may be involved in intestinal inflammation. Unlike in humans and mice, the importance of Toll-like receptor (TLR) 2 expressed in porcine intestinal epithelial cells is largely unclear. Therefore, the aim of the present study was to investigate whether TLR2 stimulation enhances intestinal barrier function and protects against DON exposure. We found that the cells treated with TLR2 ligands decreased the epithelial barrier permeability and enhanced TJ protein expression in intestinal porcine epithelial cells (IPEC-J2). In addition, pretreatment with TLR2 ligand, including Pam3CSK4 (PCSK) and lipoteichoic acid from Bacillus subtilis, prevented DON-induced barrier dysfunction by increasing the expression of TJ proteins via the PI3K-Akt-dependent pathway. It is likely that the DON-disrupted intestinal barrier caused biological changes of immune cells in the lamina propria. Thus, we conducted co-culture of differentiated IPEC-J2 cells in the upper well together with peripheral blood mononuclear cells in the bottom well and found that apical TLR2 stimulation of IPEC-J2 cells could alleviate the reduction in cell survival and proliferation of immune cells. Conclusively, TLR2 signaling on intestinal epithelial cells may enhance intestinal barrier function and prevent DON-induced barrier dysfunction of epithelial cells

SNU Open Repository and Archive

Additional file 4. Apical DON treatment down-regulated CD163 expression of monocytes co-cultured with IPEC-J2 cells. IPEC-J2 cells were cultured with PBMCs on the transwell plate (0.4 μm pore). (A) Percentages of CD163+ and CD163− among CD172a+ cells were measured 72 h after 2 μg/mL of DON treatment. NT denotes no treatment. * P < 0.05 versus NT (n = 3). (B) Supernatant in basolateral side of transwell plate was collected and TNF-α was measured by ELISA at 5 days after DON treatment (0, 0.2 and 2 μg/mL) (n = 3). *P < 0.05; **P < 0.01, determined by one-way ANOVA with Tukey’s posttest

MOESM4 of Barrier protection via Toll-like receptor 2 signaling in porcine intestinal epithelial cells damaged by deoxynivalnol

https://hal.science/hal-01341476v1/file/13567_2016_Article_309.pdf

Barrier protection via Toll-like receptor 2 signaling in porcine intestinal epithelial cells damaged by deoxynivalnol

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