Fig 2: Strongyloides venezuelensis-infection induces highly reactive ILC2s in the lungs. (A) S. venezuelensis-infected (Sv) or uninfected control (cont) mice (n = 3) were inoculated with 500 N. brasiliensis L3 (Nb). Cell populations of lung cells at the indicated days were analyzed by flow cytometry (SP6800) (Figure S6). (B) Eight weeks after S. venezuelensis infection, lung cells were stimulated with PMA and ionomycin for 3 h in the presence of brefeldin A. Intracellular IL-5 and IL-13 in ILC2s were analyzed by flow cytometry (SP6800). ILC2s were gated on Fixable Viability Dye−CD45+Lin(CD3, CD4, CD8, CD11b, B220, NK1.1, IgE, Gr-1)−ST2+Sca-1+Thy1.2+ cells. (C) The proportions of IL-5+IL-13−, IL-5−IL-13+, and IL-5+IL-13+ ILC2s in the population of CD45+ cells (n = 4–5). (D) Lung ILC2s were sorted from S. venezuelensis-infected or uninfected mice (n = 4–5) before or 2 days after infection with N. brasiliensis L3. The expression levels of Il5 and Il13 mRNA were analyzed by Q-PCR. Data are representative of two independent experiments that had similar results (Mean ± SD). (E) The expression levels of transcription factors and TSLPR (Crlf2) in lung ILC2s were analyzed by Q-PCR. Statistical analyses were performed using Student's t-tests. Data are representative of two independent experiments (Mean ± SD).

Fig 3: ILC2s are critical for the enhanced inflammation and the protection against Nippostrongylus brasiliensis infection. (A) Experimental workflow for sequential nematode infection in bone marrow chimera mice. BMT; bone marrow transplantation, sac; sacrificed. S. venezuelensis-infected (Sv) or uninfected (cont) mice (Rorasg/+, n = 6–7; Rorasg/sg, n = 7) were infected with N. brasiliensis (Nb). Five days after N. brasiliensis infection, (B) ILC2s and eosinophils among the BALF cells were analyzed by flow cytometry (LSRFortessa). Cell populations were defined as follows: ILC2s, FSCloSSCloCD45+CD4−Lin(CD3, CD8, CD19, NK1.1, IgE, siglec F, Gr-1)−Sca-1+ST2+ and Eosinophils, CD45+CD11cloCD3−B220−CCR3+. (C) Il33, Il5, and Il13 mRNA expression levels in the lungs were examined. (D) The numbers of worms in the intestine were counted at day 5 post-N. brasiliensis infection. Data are representative of two independent experiments. (E) S. venezuelensis-infected or uninfected WT mice were infected with N. brasiliensis after anti-CD4 Ab treatment (αCD4) as in Figure S5. The numbers of worms in the intestine were counted at day 5 after N. brasiliensis infection (n = 10–11). Pooled data from two independent experiments are shown (Mean ± SD).

Fig 4: 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, αIL-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; ΔdblGATA, 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 5: Plpp6-/- mice have reduced lung inflammatory responses to allergen(A–D) Plpp6-/- and WT mice were airway sensitized and challenged with HDM and their immunophenotype was determined on protocol day 16—a time of increased allergic lung inflammation. (A) Representative images of lung histology after staining with hematoxylin and eosin (H&E), arrowheads indicate leukocyte infiltration (left panels), or with periodic acid schiff (PAS), asterisks indicate mucous cells metaplasia (right panels); scale bars correspond to 100µm in 10x images (H&E) and 20x images (PAS) and 50µm in 40x images. (B) Lung differential cell numbers of eosinophils (Eos) and neutrophils (PMN) were determined by FACS (representative contour plots on the left; numbers in red represent the percentage of leukocytes); experiments were performed 3 times for a total of n = 9. (C) Lung differential cell numbers of alveolar macrophages (aMacs) and T cells determined by FACS (representative contour plots on the left; numbers in red represent the percentage of leukocytes); experiments were performed 3 times for a total of n = 9 (see Figure S3A for gating strategy). (D) Gene expression of il4, il5, and il13 by RT-qPCR in lung tissue harvested on protocol day 16; data represent fold change in gene expression with HDM relative to naive within the same genotype; experiments were performed 3 times for a total of n = 4 to 5.(E) Total serum IgE was measured by ELISA on days 4 and 16 of the HDM protocol; experiments were performed 2 times for a total of n = 3 to 5.(F) Resistance of the respiratory system (Rrs) was determined by invasive measurement in anesthetized animals by Flexivent in response to increasing doses of methacholine (MCh) (1, 3, 10, 30, and 100 mg/mL); experiments were performed 2 times for a total of n = 4 to 9; data represent percent change from baseline. *p < 0.05, **p < 0.01 and*** p < 0.001 comparing WT and Plpp6-/-, and #p < 0.05 comparing HDM and naive within same mouse genotype by unpaired nonparametric Mann-Whitney test. Bars represent median with interquartile range in (B–E) and mean ± SE in (F). See also Figure S3.