Fig 2: Experimental design and protein levels of markers for M1/M2 polarization and TLR4–MyD88 signaling in mouse hippocampi across time points in the acute phase after SE. (A) Schematic illustration of the experimental procedure. CP or MIP was injected intrahippocampally 6 h prior to an intraperitoneal injection of lithium chloride. Twenty-four hours after the intrahippocampal injection, pilocarpine was administered intraperitoneally. SE was halted by diazepam administration. The mice were sacrificed at different time points after SE, and their brains were collected for analysis. (B) Site of stereotactic unilateral hippocampal injection. (C1) Representative immunoblots illustrate that the abundance of TLR4 and MyD88 in mouse hippocampi increased over time in the acute phase after SE. (C2–C3) Group comparison of TLR4 and MyD88 immunoblots (calibrated relative to β-actin). (D1) Representative immunoblots showing increases in TNF-α and IFN-γ in mouse hippocampi over time up to 72 h after SE. (D2–D3) Group comparison of TNF-α and IFN-γ levels. (E1) Representative immunoblots of MR, iNOS, and ARG-1 for each group; note the relatively rapid increase in iNOS levels over time. (E2–E4) Comparison of MR, iNOS, and ARG-1 levels among the above groups. N = 6 per group in the Western blots; *p < 0.05, **p < 0.01, ***p < 0.001 between groups; ANOVA followed by Tukey’s test

Fig 3: Representative pictures of MyD88-immunoreactive MG/MΦ and their distribution in the DG, CA1, and CA3 of the hippocampus. (A1–A3) Sections from mice in the control group showed neither activated MG/MΦ nor MyD88-positive cells in these regions. Sections from mice at 1 day (B1–B3) and 3 days (C1–C3) after SE showed increased expression of MyD88 specifically in MG/MΦ (identified by iba-1 immunoreactivity). (D1–D3) Higher magnification of the boxes in (C1–C3). Arrows show cells with strong MyD88 and iba-1 immunoreactivity. The insets of (D1, D2, and D3) show additional high-magnification images of MyD88-positive MG/MΦ. (E) Quantitative analysis of iba-1/MyD88 double-labeled cells in hippocampi of control group mice 1 or 3 days after SE (means ± SEM, n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 between groups. One-way ANOVA followed by Tukey’s test. ND = not detectable. Scale bars: (A1–C3) 100 μm; (D1–D3) 50 μm; (D3) (inset) 12.5 μm

Fig 4: Hippocampal GLT-1 and NR1 expression 3 days after SE with MyD88 inhibition. Sections from the hippocampi of mice in the CP group (A1–A3) and MIP group (B1–B3) 3 days after SE with GLT-1 immunoreactivity in astrocytes and neuronal processes. (A4, B4) Higher magnification of the boxes in (A3) and (B3). (C) Comparison of the numbers of GFAP/GLT-1 double-labeled cells in the DG, CA1, and CA3 between the CP and MIP groups (means ± SEM, n = 3). *p < 0.05 versus the CP group; **p < 0.01 versus the CP group. Independent samples t tests were performed. (D1) Immunoblots of NR1, NR2a, and NR2b for the control, CP, and MIP groups. (D2–D4) Comparison of NR1, NR2a, and NR2b levels among the above groups (calibrated to β-actin). *p < 0.05; ***p < 0.001 between groups. One-way ANOVA followed by Tukey’s test. Scale bars: (A1–A3, B1–B3) 100 μm; (A4, B4) 50 μm

Fig 5: DC apoptosis induced by C. krusei mannan was dependent on MyD88 signaling pathway. (A) BMDCs were pre-treated with vehicle control or Syk inhibitor, and the cells were incubated with 25 μg/ml of β-glucan or 25 μg/ml of C. krusei mannan for 48 h. (B) BMDCs were pre-treated with control peptide or MyD88 inhibitor, and the cells were incubated with 0.5 μg/ml LPS or 25 μg/ml of C. krusei mannans for 48 h. The BMDC viability was determined by MTT assay. The percentage of cell viability was expressed as a percentage relative to the unstimulated BMDCs. (C) and (D) BMDCs were pre-treated with control peptide or MyD88 inhibitor, and the cells were incubated with 0.5 μg/ml of LPS or 25 μg/ml of C. krusei mannan for 48 h. Then, the cells were stained with CD11c, Annexin V and 7AAD. CD11c+ cells were gated and the Annexin V+, and Annexin V+7AAD+ were identified as apoptotic fraction. The number in the dot plot indicated the percentage of apoptotic DCs. (D) The percentage of apoptotic CD11c+ cells was shown as the bar graph. n = 3; Data are representatives of two independent experiments. *p < 0.05. (−), unstimulated BMDCs; Ca, C. albicans; Ct, C. tropicalis; Cg, C. glabrata; Ck, C. krusei.