Fig 2: SNHht Signals through TLR2 and MyD88 to Induce IL-10(A) Induction of IL-10 production in different knockout C57BL/6 BMDMs stimulated for 3 hr with SNHht normalized to WT BMDMs (three independent experiments). Two-way ANOVA and Bonferroni’s multiple comparisons test, p < 0.05. Ifnar, interferon-a/b receptor; Mr, mannose receptor; Sra, scavenger receptor A.(B) Il10 mRNA and protein induction in different knockout BMDMs stimulated for 3 hr with SNHht (one of three independent experiments). One-way ANOVA and Tukey’s multiple comparisons test, p < 0.05.(C) Induction of IL-10 production in WT and Tlr2-/- BMDMs stimulated for 3 hr with TSBt, SNHht, CPHht (SNHht crude polysaccharide fraction), LPS UP (TLR4 ligand), or Pam3 (TLR2/1) (one of three independent experiments). Two-way ANOVA and Sidak’s multiple comparisons test, p < 0.05.(D) Induction of IL-10 production in WT BMDMs pre-treated for 2 hr with a blocking antibody specifically inhibiting TLR2 signaling (monoclonal a-mTLR2-IgG) or its isotype control before stimulation for 3 hr with TSBt, SNHht, Pam2 (TLR2/6), Pam3, or LPS UP (three independent experiments). Two-way ANOVA and Sidak’s multiple comparisons test, p < 0.05.(E) Western blot showing the phosphorylation of ERK1/2 and p38 and the total ERK1/2 and p38 protein amounts in WT and Tlr2-/- BMDMs after stimulation with TSBt, SNHht, Pam3, or LPS UP for 15 min, 30 min, and 3 hr. Mean ± SD. See also Figure S4.

Fig 3: The effect of a bicyclic peptide (16) on ERK and p38 pathways. (A) Serum-starved HEKT cells were pre-treated with 0, 25, 50, 100 µM peptide 16 for 2 h, and then stimulated by 50 ng/ml EGF for 15 min or 200 ng/ml EGF for 10 min. Samples were subjected to Western-blot analysis; activity of the ERK pathway was followed by anti-pp-ERK and anti-ERK antibody; anti-tubulin antibody was used as load control and demonstrates equal sample load in addition to the anti-ERK Western-blot signal. Bar charts show the ppERK2/ERK2 intensity ratio of each sample. (B) Serum-starved HEKT cells were pre-treated with the peptide 16 at 0.1, 1, 3, 10, 25, 100 µM for 1 h and then stimulated by 0.4 M Sorbitol for 10 min (upper panel) or treated only with the medium (lower panel; Mock). p38 activation was confirmed by using anti-pp-p38 antibody, and the total p38 amount of the samples by using the anti-p38 antibody. The graph shows the pp-p38/p38 intensity ratio of each band obtained from the densitometry of Western-blots in the case of Sorbitol (in grey) or medium (Mock; in black) treatment (C) Cell viability test with peptide 16. HEKT cells were incubated with the peptide at 10 and 100 µM for 24 h, then the viability of cells was determined by PrestoBlue™ reagent according to the manufacturer’s instructions (ThermoFisher Scientific, #A13261). Note that peptide incubation times before stimulation was only up to 2 h at maximun on Panel A and B. Error bars show SD of 3 independent measurements. *Indicates statistical significance between peptide-treated and control (p < 0.05 with two-sided independent Student’s t-test).

Fig 4: ER stress augments inflammatory response in the isolated intestinal cells of large yellow croaker in vitro. (A) Relative mRNA expression of inflammation-related genes in the isolated intestinal cells of large yellow croaker after TM treatment for 12 h (n = 3). (B) Western blot analysis of inflammation-related protein levels in the isolated intestinal cells of large yellow croaker after TM treatment for 12 h (normalized to GAPDH, n = 3). il-1ß, interleukin-1 beta; il-6, interleukin-6; il-8, interleukin-8; cox2, cyclooxygenase 2; tnfa, tumor necrosis factor alpha; ERK1/2, extracellular signal-regulated protein kinases 1 and 2; p38, p38 mitogen-activated protein kinase; p65, NF-kappaB p65; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Results were analyzed using independent t-test (*P < 0.05, **P < 0.01), and they were presented as mean ± standard deviation.

Fig 5: Inflammatory pathways of PBECs activated by M. haemolytica and LPS. Passage 1 (P1) of PBECs were incubated with LPS (10 µg/ml) and M. haemolytica (1 × 105 CFU/ml) for 24 h in the SCC system and (A) interleukin (IL)-6, (B) tumor necrosis factor (TNF)-a and (C) IL-8 levels in the supernatants of control and LPS/M. haemolytica-treated PBECs were assessed by ELISA. (D) Expression of TLR4/5 and phosphorylation of p38, ERK1/2, JNK1/2 and NF-?B p65 were determined by western blot in control and LPS/M. haemolytica-treated PBECs (original blots are shown in Supplementary Figure S5A. *P<0.05; **P<0.01; ***P <0.001; (A–C) one-way ANOVA; and (D) two-way ANOVA. Data are presented as means ± SE (n = 5–6).