Fig 1: TLR-5 mediated IL-17C induce pBD-2, claudin-1 and -2 expression in IPEC-J2 cells. IPEC-J2 monolayers were pre-incubated with 0.5 µM TH1020 or the equivalent amount of its solvent DMSO for 2 h. Then, the monolayers were inoculated with HB101 or C83901 at MOI 100 for 1 h, washed and further incubated for 23 h. Non-treated cells were supplemented with PBS or IL-17C (20 or 40 ng/mL). A pBD-2, claudin-1 and -2 mRNA expression in the IPEC-J2 monolayers was assessed by qPCR. The mRNA expression level was normalized to the reference genes and then to the control group. Different letters indicate significant differences (p < 0.05). Data are presented as the mean ± SD (n = 5 per group). B pBD-2, claudin-1 and -2 protein were detected by Western blotting. IPEC-J2 monolayers were pre-incubated with TH1020 (0.5 µM) or the equivalent amount of its solvent DMSO for 2 h. Then, the monolayers were inoculated with HB101 and C83901 at MOI 100 for 1 h, washed and further incubated for 23 h. Non-treated cells were supplemented 20 ng/mL IL-17C. C Cellular localization of claudin-1 and -2 in IPEC-J2 cells 24 h post PBS (left panel) and C83901 (right pane, MOI 100) stimulation. The cells were stained with anti-claudin-1 (Texas-Red, Red), anti-claudin-2 (FITC, green). The nuclei were counterstained with Hoechst and representative confocal images are shown (n = 3). Scale bar = 10 µm.
Fig 2: Induction of IL-17C and TLR-5, -8 in the IPEC-J2 cells after F4+ETEC infection. IPEC-J2 monolayers were inoculated with F4+ ETEC (C83901), non-pathogenic E. coli strain HB101 at MOI 100 or PBS for 1 h and further incubated for another 1 h, 3 h or 23 h, respectively. The mRNA expression of IL-17C (A) and TLR5 and TLR8 (C) in the IPEC-J2 monolayers was assessed by qPCR. The mRNA expression level was normalized to the reference genes and then to the control group of 2 h treatment. Data are presented as the mean ± SD (n = 3 per group), different letters indicate significant differences between groups (p < 0.05). B IPEC-J2 monolayers were inoculated with the different bacterial strains at MOI 100 for 1 h and then further incubated for 23 h. The cells were stained with anti-IL-17C (FITC, green). The nuclei were counterstained with Hoechst and representative confocal images are shown (n = 3). Scale bar = 25 µm.
Fig 3: F4+ETEC infection elicits an IL-17C response under the control of TLR5 in IPEC-J2 cells. IPEC-J2 monolayers were first pre-cultured with 0.5 µM TH1020 (0.5 µM), 0.5 µM oligodeoxyribonucleotide (ODN) 2088 or the equivalent amount of their solvent DMSO and TE or PBS for 2 h. Then, the monolayers were inoculated with the C83901 at MOI 100 for 1 h, washed and further incubated for 23 h. A IL-17C cytokine production was analysed in the cell culture supernatant by ELISA. Data are presented as the mean ± SD (n = 5 per group). Different letters indicate significant differences between groups (a:b, p < 0.05; a:c, p < 0.01). B IL-17C protein was detected by Western blotting. C IPEC-J2 monolayers were first pre-cultured with TH1020 (0.5 µM, green bars), DMSO (red bars) or PBS (blue bars) for 2 h. Then, the monolayers were stimulated with flagellin (FLA, 100 ng/mL) and further incubated for 4 h and 24 h for mRNA and protein expression of IL-17C, respectively. Control samples only treated with PBS were marked with open bars. Different letters indicate significant differences between groups (a:b, p < 0.05; a:c, p < 0.01). Data are presented as the mean ± SD (n = 5 per group).
Fig 4: F4+ ETEC induces IL-17C and TLR2, 5, 8 and -10 mRNA expression in small intestinal tissues. Jejunal segments of three piglets were perfused with F4+ ETEC (C83901), non-pathogenic E. coli strain HB101 or PBS for 4 h. A The mRNA expression of IL-17A, IL-17C and IL-17RE in the intestinal segments was analyzed by qPCR. The mRNA expression level was normalized to the reference genes and then to the control group. Data are presented as the mean ± standard deviation (SD) (n = 3 per group). * p < 0.05. B The mRNA expression of different IL-17 receptors in jejunal segments in homeostatic conditions. The mRNA expression level was normalized to the reference genes and then to IL-17RC. Data are presented as the mean ± SD (n = 6), different letters indicate significant differences between groups (p < 0.01). C TLR mRNA expression profile in jejunal segments after F4+ETEC perfusion. Jejunal segments of three piglets were perfused with F4+ ETEC (C83901), non-pathogenic E. coli strain HB101 or PBS for 4 h. The mRNA expression of TLR-1 to 10 in the intestinal segments was analyzed by qPCR. The mRNA expression level was normalized to the reference genes and then to the control group. Data are presented as the mean ± SD (n = 3 per group). *p < 0.05, **p < 0.01.
Fig 5: Gene expression of tight junctions and proinflammatory cytokines and epithelial permeability changes after bacterial infection. IPEC-J2 cells were cultured and differentiated on transwell inserts and then incubated with HB101 and C83901 at MOI 100 or PBS and IL-17C (20 ng/mL) for 1 h, washed and further incubated for another 23 h. A Occludin, ZO-1, TNF-a and IL-8 mRNA expression was assessed by qPCR in the IPEC-J2 monolayers at 2 and 4 h post-infection/stimulation. The mRNA expression level was normalized to the reference genes and then to the control group. B Apical and basolateral IL-8 secretion by differentiated IPEC-J2 cells 24 h post-infection/stimulation. IPEC-J2 monolayers were pre-incubated with TH1020 (0.5 µM) for 2 h. Then, the monolayers were inoculated with HB101 and C83901 at MOI 100 or PBS and IL-17C (20 ng/mL) for 1 h, washed and further incubated for 23 h. C Transepithelial electrical resistance (TEER) was measured in IPEC-J2 cells after bacterial infection or IL-17C stimulation. Different letters indicate significant differences (p < 0.05). Data are presented as the mean ± SD (n = 5 per group).
Supplier Page from Abcam for Anti-IL-17C antibody