Fig 2: Rab27A and Rab27B have distinct tissue expression and regulate gut exosome release.(A) NTA of WT and Rab27DKO colon explant EVs (n = 3). (B) Quantification of 50–200 nm particles (the gray shaded area) between genotypes of A. (C) CD63 and CD9 immunoblot of WT and Rab27DKO colon EVs. (D) Rab27A (n = 7) and (E) Rab27B expression in distal SI, distal colon tissue, and splenocytes at baseline (n = 3). (F) Rab27A expression in FACS WT CD4+, CD8+, B220+, and CD11b+ populations (n = 7). (G) Rab27A and Rab27B expression in mouse CD45+ SI cells from ref. 31. (H) Analysis of mouse CD45– SI cells from ref. 31. Clusters as previously annotated for G and H. Unpaired 2-tailed t test for all bar graphs. ***P < 0.0005. EPs, enterocyte progenitors; TA, transit amplifying cells.

Fig 3: Eosinophils are essential for resistance against Nippostrongylus brasiliensis. (A–C) S. venezuelensis-infected (Sv) or uninfected (cont) mice (n = 5) were treated with anti-IL-5 Ab (TRFK5, aIL-5) or control Ab (cont IgG1) at day 0 and day 2 post-infection with N. brasiliensis (Nb). (A) The numbers of worms in the intestine were counted at day 5 after N. brasiliensis infection. (B) ILC2s, eosinophils, and CD4+ cells among the BALF cells were analyzed by flow cytometry (SP6800). Cell populations were defined as follows: ILC2s, FSCloSSCloCD45+CD4-Lin(CD3, CD8, CD19, NK1.1, IgE, siglec F, Gr-1)-Sca-1+ST2+; Eosinophils, CD45+CD11cloCD3-B220-CCR3+; and CD4+ cells, FSCloSSCloCD45+CD4+. (C) Il33, Il13, and Epx mRNA expression levels in the lungs were examined. Data are representative of two independent experiments that had similar results. (D–F) S. venezuelensis-infected or uninfected (cont) mice (WT, n = 7; ?dblGATA, n = 5–6) were infected with N. brasiliensis. (D) The numbers of worms in the intestine were counted at day 5 after N. brasiliensis infection. (E) Eosinophils, ILC2s, and CD4+ cells among the BALF cells were analyzed as in (B). (F) Il33, Il5, and Il13 mRNA expression levels in the lungs were examined. Data are representative of two independent experiments that had similar results.

Fig 4: IL-33 is not sufficient for the induction of trained ILC2s. WT C57BL/6 mice were subcutaneously inoculated with 5000 S. venezuelensis L3 at day 0 (Sv) or were intranasally administrated 100 ng of IL-33 at days 0, 1, 2, and 3 (IL-33). cont, untreated control. (A) Cell populations in the lung cells, BALF cells, and peripheral blood leukocytes (PBLs) at indicated days were analyzed by flow cytometry (SP6800) (n = 4). Cell populations were defined as follows: Eosinophils, PI-CD45+CD11cintCD3-B220-CCR3+ and ILC2s, PI-FSCloSSCloCD45+Thy1.2+Lin-Sca-1+ST2+. (B) Four weeks after treatment, lung cells were stimulated with PMA and ionomycin for 3 h in the presence of brefeldin A. Intracellular levels of IL-5 and IL-13 in ILC2s were analyzed by flow cytometry (SP6800) as in Figure S8. (C) The proportions of IL-5+IL-13-, IL-5+IL-13+, and IL-5-IL-13+ cells within the population of ILC2s (n = 4). (D,E) Mice were infected with N. brasiliensis 4 weeks after S. venezuelensis infection or IL-33 treatment. Control mice were treated with PBS (PBS) instead of IL-33. Five days after N. brasiliensis infection, (D) the numbers of worms in the intestine were counted (n = 7). (E) The numbers of ILC2s, eosinophils, and macrophages (n = 5–7) among the BALF cells were examined. Statistical analyses were performed using a two-way ANOVA (A,C) or one-way ANOVA (D) with Bonferroni post-hoc tests. Data are representative of three independent experiments (Mean ± SD).

Fig 5: Loss of the miR-15 family results in B cell progenitor expansion in vitro. (A) Schematic illustration of the sorting strategy for the B cell progenitor in vitro culture. B220+c-KIT+NK1.1-CD19- B cell progenitors were sorted from the bone marrow of miR-15a/bfl/fl Vav-Cre and miR-15a/b+/+ Vav-Cre mice and cultured in the presence of IL-7, FLT3L and SCF. A representative FACS plot depicts the percentages of pro-B (c-KIT+CD25-), intermediate B (c-KIT-CD25-) and pre-B cells (c-KIT-CD25+) under basal conditions after 1 week. (B) Statistical analysis of the distribution of pro-, intermediate and pre-B cell progenitors derived from miR-15a/bfl/fl Vav-Cre mice and miR-15a/b+/+ Vav-Cre mice under basal conditions. (C) Representative FACS blots and statistical analysis of the progenitor B cell differentiation upon IL-7 withdrawal in vitro. (D) Progenitors from miR-15a/bfl/fl Vav-Cre mice and miR-15a/b+/+ Vav-Cre mice were labeled via stable expression of either dsRed or GFP, mixed at equal ratios at a cell density of 5 x 104 cells/ml and tracked over time by flow cytometry in the presence of all growth factors. (E) The bar graph depicts the percentages of EdU+ progenitors derived from miR-15a/bfl/fl Vav-Cre mice or miR-15a/b+/+ Vav-Cre mice after labeling with 10 ug/ml EdU for 4 hrs. (F) Quantification of the percentage of apoptotic progenitors under steady state conditions as analyzed by AnnexinV staining. Error bars depict the standard deviation of the mean. Numbers indicate the percentage of cells in the respective region. MiR-15a/bfl/fl Vav-Cre progenitors were statistically compared to miR-15a/b+/+ Vav-Cre progenitors by an unpaired two-tailed Student’s t-test. *P < 0.05.