Figures and data
Recombinant Ym1 and Ym2 crystals are structurally similar to in vivo Ym1 and Ym2 crystals and to each other.
A, B) Light microscopy pictures of recombinant Ym1 (A) and Ym2 (B) crystals. C-E) Structural superposition of the X-ray structures determined from a native in vivo grown Ym1 crystal isolated from the BAL fluid of mev/mev mice (C+E) (yellow), a native in vivo grown Ym2 crystal isolated from the BAL fluid of a C57BL/6J-Tg(CAG-Gal10)Bla mouse (D+E) (blue) and a recombinant Ym1 (C) and Ym2 (D) crystal (grey). The low root-mean-square deviations (RMSDs) establish structural similarity. F, G) Crystal packing of ex vivo Ym1 (F) and Ym2 (G) crystals revealing their crystallographic equivalence.
Ym1 crystals activate innate immunity.
A) Scheme representing the innate immune response experiment. B) Flow cytometric analysis of the immune cell influx in the lungs of PBS, Ym1 crystal and soluble Ym1 treated mice measured 6 and 24 hours after i.t. injection. Data are pooled from 2 (for 6h time point) or 3 (for 24h time point) independent experiments with n = 13, 13, 13, 14, 15 and 15, respectively. C) Concentration of IL-6 and TNFα in the BAL fluid measured by ELISA. D, E) Concentration of IL-1β, IL-33 (D), CCL2 and CCL24 (E) per milligram of protein of dispersed lung tissue measured by ELISA. Data are pooled from 2 independent experiments with n = 13 per group. Data are shown as mean ± SEM. ns P ≥ 0.05; *P < 0.05; **P<0,01; ***P <0.001; ****P < 0.0001. F) ImageStream pictures of the BAL fluid of Ym1 crystal and soluble Ym1 treated mice 6 and 24 hours after i.t. injection. Samples were stained with an antibody recognizing Ym1/2 (red).
Ym1 crystals activate DCs in the lymph nodes.
A) Gating strategy to distinguish different subsets of DCs and recruited monocytes. B) Scheme representing the DC activation experiment. C, D) Flow cytometric analysis of the DC and monocyte influx in the mLNs of PBS, OVA-AF488 alone, OVA-AF488 plus Ym1 crystals and OVA-AF488 plus soluble Ym1 treated mice measured 24 hours after i.t. injection. E) Median Fluorescence Intensity (MFI) of activation marker CD86 on OVA-AF488+ cDC1s and cDC2s of PBS, OVA-AF488 alone, OVA-AF488 plus Ym1 crystals and OVA-AF488 plus soluble Ym1 treated mice measured 24 hours after i.t. injection. Data are pooled from 2 independent experiments with n = 10 mice per group. Data are shown as mean ± SEM. ns P ≥ 0.05; *P < 0.05; **P<0,01; ***P <0.001; ****P < 0.0001.
Ym1 crystals boost adaptive cellular immunity.
A) Scheme representing the used adoptive transfer model. B-E) Proliferation and KN2 expression of OT-II T cells in the mLNs and lungs 4 and 7 days after i.t. administration of PBS, OVA alone, OVA plus Ym1 crystals or OVA plus soluble Ym1. (B) Representative histograms of the division profile of the adoptively transferred OT-II T cells (CD3e+ CD4+ Vα2+ CD45.1.2+) in the mLNs on day 4. (C, D) The numbers of OT-II T cells and KN2+ OT-II T cells in the mLNs on day 4 (C) and day 7 (D). (E) The numbers of OT-II T cells and KN2+ OT-II T cells in the lungs on day 7. Data are pooled from 2 independent experiments with n = 10 mice per group. Data are shown as mean ± SEM. ns P ≥ 0.05; *P < 0.05; **P<0,01; ***P <0.001; ****P < 0.0001.
Ym1 crystals can act as type 2 adjuvant in OVA-induced asthma model and activate adaptive humoral immunity.
A) Scheme representing the used OVA-induced asthma model. B) Flow cytometric analysis of the immune cell influx in the BAL fluid of mice sensitized i.t. with PBS, OVA alone, OVA plus Ym1 crystals or OVA plus soluble Ym1 and challenged i.n. with OVA. Data are pooled from 2 independent experiments with n = 18 per group. C) Levels of IL-5, IL-10 and IL-13 in the supernatant of mLN cells after restimulation with OVA for 20 hours. Data are from 1 experiment with n = 5, 7, 9 and 9, respectively. D) Serum OVA-specific IgG1 antibodies were measured by ELISA. E) Serum Ym1-specific IgM antibodies were measured by ELISA. Data are pooled from 2 independent experiments with n = 18 per group. F) Scheme representing the adaptive humoral immune response experiment. G) Serum Ym1-specific IgM antibodies were measured by ELISA. Data are pooled from 3 independent experiments with n = 13, 13, 9, 9, 12 and 8, respectively. Data are shown as mean ± SEM. ns P ≥ 0.05; *P < 0.05; **P<0,01; ****P < 0.0001. If pairwise comparison is not shown, the result is not significant.
