Fig 1: Cathelicidin suppresses TH1 differentiation in the presence of TGF-β1.Splenocytes from C57BL/6 J mice were cultured in Th17-driving conditions for 48 h in the presence or absence of 2.5 μM cathelicidin. A production of IL-2 was quantified by ELISA and B–D expression of Tbet and IFN-γ were assessed by flow cytometry. E Tbet and RORγt co-expression was quantified by flow cytometry and F–H the cytokine production by each subset assessed. I Tbet expression was determined following incubation with individual cytokines. J IFN-γ production was quantified by ELISA after 48 h incubation under Th1-driving or Th2-driving conditions. Data shown are individual mice used in separate experiments. A, B, D, E, F, G, H and I were analysed by paired two-tailed t-tests with no correction, N values: A - 4, B - 6, D - 12, E - 9, F - 9, G - 9, H - 9, I - 4, J - 3. The error bars in J show standard error of the mean. Black symbols represent untreated samples and open symbols are samples treated with cathelicidin.
Fig 2: PCV2 infection induces IL-10 production to promote viral replication. Wild-type mice and il10−/− mice were infected with PCV2, and samples were collected at 7, 14, and 28 d.p.i. (A) The serum IL-10 expression was measured by ELISA n = 15. (B) The PCV2 copy numbers in lungs were detected by qPCR. The data are presented as mean ± SEM of three independent experiments n = 15. (C–H) Other groups of wild-type mice and il10−/− mice were infected with PCV2. The IL-2, IL-6, IL-12, TNF-α, IFN-α, and IFN-γ production were measured by ELISA at 0, 1, 3, and 6 h.p.i., respectively. The value of the cytokines in wild-type mice is the value of PCV2-infected wild-type mice minus the value of mock-infected wild-type mice; the value of pro-inflammatory cytokines in il10−/− mice is the value of PCV2-infected il10−/− mice minus the value of mock-infected il10−/− mice. The production of cytokines at 0 h post-infection was undetectable. The data are presented as mean ± SEM of three independent experiments n = 15 mice. (B) *p < 0.05, **p < 0.01 versus same group at 7 d.p.i.; &p < 0.05, &&p < 0.01 versus same group at 14 d.p.i.; ##p < 0.01 versus wild-type mice at same infection time. (C–H) *p < 0.05, **p < 0.01 versus same group at 0 h post-infection; &p < 0.05, &&p < 0.01 versus same group at 1 h post-infection; #p < 0.05 versus wild-type mice at same infection time.
Fig 3: Cathelicidin protects Th17 cells but not Th1 cells from death.CD4 + T cells isolated from C57BL/6 J mice were cultured in Th17-driving conditions for 24 h in the presence or absence of 2.5 μM cathelicidin. A Gene expression differences were assessed by Nanostring mouse immunology chip. Cell death was assessed in culture by B, C annexin V/propidium iodide staining and D–F binding of a fixable live/dead marker. G Annexin/PI staining was repeated at 48 h of culture following inclusion of IL-2 in culture medium. H–J T cells were labelled with CFSE and proliferation assessed following 24, 48 or 72 h culture—I and J show 72 h culture analysis. Data shown are individual mice used in separate experiments with line at median. A, E, F and I were analysed with paired two-tailed t-tests—A and I with Bonferroni post correction, C and G with a two-way ANOVA with Sidak’s post-test. N values: A - 3, C - 6, E - 20, F - 19, G - 5, I - 3, J - 3. Black symbols represent untreated samples and open symbols are samples treated with cathelicidin.
Fig 4: Effects of β-damascone on the DC-mediated activation of T cells. (A) Structure of β-damascone. (B) IL-2 concentrations in the culture media of OT-II splenocytes. A total of 1.0 × 105 cells obtained from the whole spleen of OT-II mice were maintained in the presence or absence of 500 ng of the OVA peptide and the indicated concentration of β-damascone (β-D) in 200 μl pf culture media for 72 h. (C) Division of OT-II CD4+ T cells cocultured with OVA-pulsed BMDCs. CFSE-labeled OT-II CD4+ T cells (5.0 × 104) were cocultured with or without C57BL/6 BMDCs (1.0 × 104), which were pretreated with 500 ng of the OVA peptide, in the presence or absence of the indicated concentrations of β-damascone in 200 μl of culture media for 72 h. (D,E) Effects of β-damascone on the frequencies of Th1 and Th2 cells that developed under polarizing conditions in a DC-dependent manner. OT-II naïve CD4+ T cells were cocultured with OVA-pulsed C57BL/6 BMDCs under Th1- or Th2-polarizing conditions in the presence or absence of β-damascone (30 μM) for 72 h. The percentages of (IFN-γ+/CD4+ T cells) / (total CD4+ T cells) (D) and (IL-4+/CD4+ T cells) / (total CD4+ T cells) (E) were shown, and a gating strategy of the flow cytometric analysis of CD4+ T cells and the actual number of cells are shown in Supplementary Figure 2. The Tukey-Kramer test was used (B–E). **p < 0.01, n.s.: not significant.
Fig 5: vdPVR-Fc as a ligand suppresses T-cell activity in mouse splenocyte cultures in-vitro. (A) Representative CFSE profiles showing the proliferation percentages of murine T-cells in splenocyte cultures stimulated with anti-CD3/CD28 beads in the presence or absence of vdPVR-Fc (10 µg/mL) for 3 days. (B) Graph showing the mean proliferation percentages of CD4+ and CD8+ T-cells unstimulated, stimulated with anti-CD3/CD28 coated beads, or with anti-CD3/CD28 beads in the presence of either vdPVR-Fc (10 µg/mL) or mutant vdPVR-Fc (10 µg/mL) after 3 days of culture. Proliferation was measured by flow cytometry as a measure of the percentage of CFSE signal associated with cell divisions over 3 days. The culture supernatants were harvested from the murine splenocyte culture after 3 days and the level of (C) IL-2 and (D) IFNγ were quantified by ELISA. Red line represents maximum stimulation using anti-CD3/CD28 coated beads. Each point on the graph represents a biological replicate. Error bars represent mean ± SEM, *p < 0.05, **p < 0.01 and ****p < 0.0001 evaluated by one-way ANOVA relative to anti-CD3/CD28 coated beads alone.
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