Fig 2: Lung cDC1s are necessary and sufficient for the induction of eosinophilia. a Lung cDC1s from WT and Batf3-/- mice. b Eosinophil counts in the lung or Balf in WT (solid circle) and Batf3-/- (empty circle) mice 1.5 days after the first OVA challenge. n = 4 or 6 mice (Saline) or n = 5–6 mice (OVA). c, d Mice were sensitized and challenged with papain, and eosinophils in the lung or Balf in WT (solid circle) and Batf3-/- (empty circle) mice were analyzed. n = 5–7 mice per group. e Lung cDC1s from DT treatment langerin-DTR or WT mice. f, g OVA/alum-sensitized (f) or papain-sensitized (g) langerin-DTR (empty circle) or WT (solid circle) mice were treated with DT on day -1, and eosinophils in the lung or Balf were assessed after challenge. n = 5–9 mice per group. h, i Eosinophils recruited into the air pouches injected with pulmonary cDC1s (SiglecF-CD11c+IA/IE+CD103+CD11b-) or CD11b+ DCs (1.5 × 104, 200 µl) sorted from C57BL/6 mice 1.5 days after the first OVA challenge (h) or 1 day after the first papain challenge (i). n = 4–6 mice per group. j Eosinophils recruited into the air pouches injected with pulmonary cDC1s, CD24+ cDC2s, CD24-CD11b+ DC2s, MCs, or IMs (1.5 × 104, 200 µl) sorted from C57BL/6 mice 1.5 days after the first OVA challenge. n = 4–6 per group. k The direct chemotactic effect of lung cDC1s, CD24+ cDC2s, or MCs (from C57BL/6 mice 1.5 days after the first OVA challenge) on eosinophils was evaluated in a transwell culture system. n = 3–4 per group. l Protocol (left) and lung eosinophil counts (right) of allergic airway induction by adoptive i.t. injection of lung cDC1s, CD24+ cDC2s, or MCs; see Methods *P < 0.05, **P < 0.01, ***P < 0.001, unpaired Student’s t test. Means ± SD are shown. Data represent two (c, d, g, i, l) and three (a, b, e, f, h, j, k) independent experiments

Fig 3: Mir223 deficiency increased autophagy and resting microglia in the brains of EAE mice. (A) Flow cytometric analysis of microglia, demonstrating PTPRC and ITGAM cells isolated from the CNS of EAE mice (n = 6 mice per group), detected on the 15th day after the induction of EAE. The data are shown in a representative plot. (B) The absolute numbers of the cell subpopulations are shown; the black column is for mir223-/- mice. Rest, ITGAM+ PTPRClow; mc, ITGAM+ PTPRChi and low. (C) Autophagy was measured in the brains of the mice. LC3 puncta were visualized by confocal imaging of microglia immunostained for LC3 and AIF1, followed by Alexa Fluor 488/555-conjugated secondary antibodies (green/red); nuclei were stained with DAPI. Representative images are shown. Scale bars: 20 µm. (D) and (E) BCL2 and BECN1 expression were visualized by fluorescence imaging of microglia. Scale bars: 50 µm.

Fig 4: Selection of lead anti-motile sperm domain-containing protein 2 (MOSPD2) monoclonal antibodies (mAbs). (a) Migration of primary CD14+ human monocytes towards stromal derived factor-1 alpha + monocyte chemotactic factor 1 (SDF-1+MCP-1) and CD3 T cells towards SDF-1 + regulated upon activation, normal T cell expressed and secreted (RANTES) in the presence of 10 µg/ml anti-MOSPD2 mAb Clone-s1 or -3 compared with matched isotype controls. One of three experiments is shown. P-values were calculated by Student’s t-test. Data are of triplicates and shown as mean ± standard error (s.e.). *P < 0·005; **P < 0·001. (b) Linear epitope mapping of clones-1 and -3 against the extracellular region of human MOSPD2. (c) Alignment of human and mouse MOSPD2. Differences are indicated by vertical lines. (d) Western blots of human embryonic kidney 293 (HEK) 293 cells transfected with human and mouse MOSPD2 and detected with anti-human MOSPD2 mAbs clones-1 or -3. One of two experiments is shown. (e) Fluorescence activated cell sorter (FACS) analysis for the recognition of anti-human MOSPD2 mAbs clones-1 and -3 of surface-expressed mouse MOSPD2. One of two experiments is shown. (f) Migration of mouse splenic CD11b cells towards SDF-1 + MCP-1 in the presence of 10 µg/ml anti-MOSPD2 mAb clone-1 or clone-3 compared with matched isotype controls. One of three experiments is shown. P-values were calculated by Student’s t-test. Data are of triplicates and shown as mean ± s.e. *P < 0·005; **P < 0·001.

Fig 5: L-DOPA treatment in EAU Gnat1rd17 mice decreases retinal infiltration of immune cells. Absolute numbers of neuroretina-infiltrating CD4+, CD8+, and CD11b+ cells in EAU Gnat1rd17 mice treated with vehicle or L-DOPA given (a) i.p. or as (b) eye drops (day 14 after immunization) were measured by flow cytometry. The data shown are the mean ± SEM of 6–8 neuroretinas from 6–8 mice per group and are representative of two experiments. * p 0.05 and *** p 0.001 by unpaired t-test for comparisons between groups receiving i.p. injections of vehicle and L-DOPA, respectively, and paired t-test for comparisons between groups of vehicle-treated contralateral eyes and L-DOPA-treated ipsilateral eyes in the same animals. (c) Absolute numbers of CD3+ T cells, naïve CD4+ T cells (CD4+CD44low), activated CD4+ T cells (CD4+CD44high), CD11b+ (CD11bhighCD11clow, macrophages, and neutrophils), and CD11c+ (CD11chighMHC-IIhigh, dendritic cells) in spleens of EAU Gnat1rd17 mice treated with i.p. injections of vehicle or L-DOPA. Flow cytometry was used to analyze the data on day 14 after immunization, and the results are shown as the means ± SEM of 4 mice in each group (unpair t-test). Shown is one representative experiment of two independently performed experiments. (d) qRT-PCR analysis of Crp mRNA levels in the liver of EAU Gnat1rd17 mice treated with i.p. injections of vehicle or L-DOPA. Data were collected on day 14 following immunization and are presented as the mean ± SEM of 4 mice in each group (unpair t-test).