Fig 2: 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 3: 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 4: Airway mycosis induces eosinophilic, TH2-high inflammation in the upper and lower airways. Mice were challenged with 4 × 105 A. niger (AN) or PBS daily for 2 months. Representative flow plots of lung single cell suspensions from (A) PBS or (B) fungal challenged (AN) wild-type C57BL/6 mice, with gates for eosinophils (Eos), macrophages (Mac), neutrophils (Neut), and TCRß+CD4+ T cells in the negative population (NP), and sub-gates for Eos/Mac. (C) Differential cell counts from lungs. (D) Representative flow plots of naïve sinus, showing gating scheme for differential cell analysis and (E) sorting of cells for direct (F) imaging of H&E-stained cells (border colors match sorted population). (G) Overlay of fungal and PBS-treated mice and (H) differential cell counts of sinus inflammatory cells. (I) Representative flow plots of ex vivo stimulated TCRß+CD4+ T cell cytokine production from sinuses was analyzed, with (J) total T cells, (K) IFN-?+ and IL-17+, and (L) IL-13+ and IL-5+ cells quantified. (M) Fluorescent immunohistochemical staining of mouse sinus after PBS and A. niger challenge showing distribution of major basic protein, yellow box and arrow highlighting an eosinophil. Results presented as mean ± s.e.m. from at least two independent experiments with n = 3-5. *p < 0.05, n = 3; by two-way ANOVA for differential counts and one-tailed t-test for cytokine production.

Fig 5: Differentiation of icCD3e+ cells in the small intestine(A) Lin-CD127+ cells from WT small intestine were first divided into CD45lo and CD45hi. icCD3e levels were determined for each subset. Transcription factor levels in CD45hi KLRG1- cells were further analyzed and the correlation between the percentages of icCD3e+ cells and the levels of GATA3 or ROR?t are plotted on the right.(B) MLN cells from mice with or without Nb infection for 6 days were analyzed. Lin-CD127+ cells were separated into Thy1+icCD3e- and Thy1+icCD3e+ subsets, which were then scored for IL-5 and IL-13 expression. Numbers of cytokine+ cells were calculated. One-way ANOVA was used for determining the statistical significance. *p<0.05. Data are represented as mean ± SD.