Fig 2: 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 3: Blocking 6PGD induces an effector T cell phenotype(A and B) CD8+ T cells were isolated from C57BL/6 mice and were stimulated for 4 days with aCD3 + aCD28 mAbs and IL-2 in the presence of either 6-aminonicotinamide (6-AN) (10 µM), DHEA (20 µM), or vehicle control (DMSO) (A). IFN-? expression was assessed by intracellular staining and flow cytometry, and (B) viability was calculated as percentage of total. The plot shown is representative of three independent experiments with n = 4 per experiment.(C and D) Thymic T cells were collected from 6PGD-/- and 6PGDfl/fl mice, and fractions of CD4+ and CD8+ T cells as well as expression of CD44 and CD25 on gated double-negative (DN) T cells were assessed by flow cytometry. Results are representative of three independent experiments.(E and F) Percentage of CD4+ and CD8+ T cells in spleen (E) and lymph nodes (F) of 6PGD-/- and 6PGDfl/fl mice was examined by flow cytometry.(G and H) Absolute numbers of CD4+ (G) and CD8+ (H) T cells in thymus, lymph nodes, and spleen of 6PGD-/- and 6PGDfl/fl mice were calculated from three replicates.(I–K) Splenocytes from 6PGD-/- and 6PGDfl/fl mice were analyzed for expression of CD44 and CD62L (I), KLRG1 and CD127 (J), and CD69 (K) by flow cytometry. Results are representative of five independent experiments with n = 4 mice per group. Error bars represent ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 4: HpBARI binds to murine ST2, blocking IL-33 ligation.(A) HpBARI or HpARI (1 µg/ml each) were coated onto wells of an ELISA plate, followed by addition of mouse ST2-Fc (ST2) (top panel) or mouse IL-33 TRAP (bottom panel) or IgG controls, followed by detection using anti-human IgG-HRP. Representative of 3 repeat experiments. Two-way ANOVA comparing HpBARI-mST2 to HpARI-mST2. Error bars show SEM. (B) Mouse ST2-Fc, mouse IL-33 TRAP or IgG were bound to protein G-coated beads, and used to immunoprecipitate HpBARI. Anti-myc western blots shown. Representative of 2 repeat experiments. (C–D) Bone marrow cells of wild type (C–D) or ST2-deficient mice (C) were cultured for 5 days with IL-2, IL-7 and IL-25 (all 10 ng/ml), then incubated with biotinylated HpARI or HpBARI (0.1 µg/ml) for 20 min at 4°C, followed by detection of biotin with avidin-PE. Representative plots gated on ICOS+lineage–CD45+ ILC2. In (D), where indicated samples were treated with HES (10 µg/ml) or unlabelled HpBARI (10 µg/ml) for 20 min prior to HpBARI staining (D). Representative of at least two repeat experiments. (E) Surface plasmon resonance of HpBARI binding to chip-coated mouse ST2 (left panel) or mouse IL-33 TRAP (right panel). (F) Anti-IL-33 western blot, after immunoprecipitation of mouse IL-33 with mouse ST2-Fc or mouse IL-33 TRAP-Fc on protein G dynalbeads, in the presence of HpBARI or heat-treated HpBARI (HT). Representative of 2 repeat experiments.

Fig 5: 6PGD blockade induce mitochondrial ROS and expression of antioxidant enzymes(A) Naive CD8+ T cells from 6PGD-/- and 6PGDfl/fl mice were stimulated for 4 days with aCD3 + aCD28 mAbs and IL-2 (20 IU/mL), and expression of CD62L and CD44 was examined by flow cytometry.(B–L) Naive CD8+ stimulated for 4 days with aCD3 + aCD28 mAbs and IL-2 (20 IU/mL), mitochondrial ROS was assessed using MitoSOX Red (B), and lipid peroxidation capacity was assessed using BODIPY 581/591 C11 (C) and flow cytometry. (D–L) The indicated enzymes of ROS metabolism pathway were assessed by real-time PCR (D–K), and their quantitative changes in 6PGDfl/fl and 6PGD-/- T cells during activation were also depicted by the heatmap (L). Results are representative of three independent experiments with n = 3 per experiment. Gsr, glutathione reductase; Gpx4, glutathione peroxidase 4; Nrf2, nuclear factor erythroid 2-related factor 2; Prdx2, peroxiredoxin 2; SOD2: superoxide dismutase 2; Txn1, thioredoxin 1; Txnrd1, thioredoxin reductase 1. Error bars represent ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.