Fig 1.
Neutrophilic and eosinophilic asthma can successfully be modeled in mice.
(A) Experimental design and immunization timeline. C57BL/6 mice were either immunized three times weekly intraperitoneal (i.p.) with Alum (1 mg/mouse) for eosinophilic asthma or once subcutaneous (s.c.) with CFA (0.5 mg/mL) for neutrophilic asthma along with the model antigen OVA (20 μg/mouse). Three weeks after the first administration, the experimental groups were challenged with OVA (50 μg/mouse) daily for two days. Samples were collected 16–18 hours post-challenge. Cells from bronchoalveolar lavage fluid (BALF) and lung homogenates were stained for (B) eosinophils (live CD45+ CD19- CD11c- CD11b- Ly6G- Siglec-F+) and (C) neutrophils (live CD45+ CD19- CD11b+/lo Ly6G+). The graphs showing the absolute cell counts of eosinophils and neutrophils in the BALF are shown in S1A and S1B Fig, respectively. (D) H&E (upper panel) and Alcian Blue/PAS (lower panel) immunohistochemistry analysis of inflamed lungs collected from indicated groups (10X magnification, scale 127 μm). (E) ELISA values of IFNɣ, IL-5, and IL-13 from indicated BALF supernatants. (F) OVA-specific IgE, total IgG, and IgG1 ELISA results in serum indicated groups. For B, C, and F, combined data from four independent experiments are shown with 6 (F) or 12 (B, C) mice in the naïve group and 16 (F) or 18–19 (B, C) mice in the experimental asthma groups in total. For E, combined data from three independent experiments are shown with n = 9 mice in the naïve and n = 14–15 mice in experimental groups. The gating strategy for the myeloid cells is detailed in the S2 Fig.
Fig 2.
DC subsets, mast cells, and basophils are differentially recruited to the inflamed lungs in neutrophilic and eosinophilic asthma.
C57BL/6 mice were immunized as outlined in Fig 1A to induce neutrophilic (CFA/OVA) or eosinophilic (Alum/OVA) asthma. Cells from bronchoalveolar lavage fluid (BALF) and lung homogenates were analyzed for indicated cell populations. (A) Total cell counts of all leukocytes (live CD45+ cells), macrophages (live CD19- CD45+ Siglec-F- Ly6G- F4/80+ CD64+), dendritic cells (live CD19- CD45+ Siglec-F- Ly6G- F4/80- CD64- CD24+ CD11c+ MHC class II+/-), and monocytes (live CD19- CD45+ Siglec-F- Ly6G- F4/80- CD64- CD24- CD11c- MHC class II+/- Ly6C+) in the BALF. Total cell counts and relative cell frequencies of (B) basophils (live CD45+ CD19- Siglec-F- Ly6G- CD11b+ FcεRIα+ CD117- cells); (C) mast cells (live CD45+ CD19- Siglec-F- Ly6G- CD11b+ FcεRIα+ CD117+ cells); (D) conventional DCs (cDC, live CD45+ CD19- Siglec-F- Ly6G- CD24+ CD11c+ MHC class II+ CD103- CD11b+ cells); (E) CD103+ DCs (live CD45+ CD19- Siglec-F- Ly6G- CD24+ CD11c+ MHC class II+ CD103+ cells); and (F) plasmacytoid dendritic cells (pDC, live CD19- CD45+ Siglec-F- Ly6G- CD11b- CD45R+ Ly6C+) in indicated organs. Combined data from four (A, D-F) or three (B, C) independent experiments are shown with 3 mice per naïve group and 4–5 mice per experimental asthma group in each experiment.
Fig 3.
The distribution of lung macrophage subsets among asthma endotypes.
C57BL/6 mice were immunized as outlined in Fig 1A to induce neutrophilic (CFA/OVA) or eosinophilic (Alum/OVA) asthma. Cells from bronchoalveolar lavage fluid (BALF) and lung homogenates were analyzed for indicated cell populations: (A) alveolar macrophages (AMs, live CD45+ CD19- Siglec-F+ Ly6G- CD11c+ cells), (B) interstitial macrophages (IMs, live CD45+ CD19- Siglec-F- Ly6G- CD24- F4/80+ CD64+/- Ly6C- CD11b+ cells); and (C) exudate macrophages (ExMs, live CD45+ CD19- Siglec-F- Ly6G- CD24- F4/80+ CD64+/- Ly6C+ CD11b+ cells). (D) Representative dot plots showing IMs (Ly6C- CD11b+ cells) and ExMs (Ly6C+ CD11b+ cells) in BALF (upper panel) and lung (lower panel) from indicated groups. (E) Representative histograms showing the expression of MHC class II, CD11b, and CD11c on bronchial IMs from indicated groups. (F) Bronchial IMs (BIMs) were subdivided according to their expression of CD11c and MHC class II and the frequencies of BIM1 (CD11c+ MHC class II+, left panel), BIM2 (CD11c- MHC class II+, middle panel), and BIM3 (CD11c+ MHC class IIneg, right panel) cells are shown. Combined data from four independent experiments are shown (naïve group n = 12 mice/group, experimental asthma groups n = 17–18 mice/group in total).
Fig 4.
Frequency of CD4+ T and iNKT cell subsets in the inflamed lungs during neutrophilic and eosinophilic asthma.
C57BL/6 mice were immunized as outlined in Fig 1A to induce neutrophilic (CFA/OVA) or eosinophilic (Alum/OVA) asthma. Cells from lung homogenates were stained for indicated cell populations. (A) The relative cell frequency of lung Th1 cells (live CD19- CD3ε+ CD4+ FoxP3- Tbet+ cells), Th2 cells (live CD19- CD3ε+ CD4+ FoxP3- Gata3+ cells), Th17 cells (live CD19- CD3ε+ CD4+ FoxP3- RORɣt+ cells), and Tregs (live CD19- CD3ε+ CD4+ CD127lo/- FoxP3+ cells) is shown. (B) Production of the indicated cytokines by lung CD4+ T cells (live CD19- CD1d/PBS57-tetramer- CD3ε+ CD4+ cells) following in vitro stimulation with PMA and ionomycin. (C) Gating for iNKT cell (live CD19- CD3ε+ CD1d/PBS57-tetramer+ cells) subsets in the lung (NKT1 cells: PLZFlo RORγt- cells, NKT2 cells: PLZFint/hi RORγt- cells, NKT17 cells: PLZFint RORγt+ cells). (D) Relative frequency of NKT1 and NKT17 cells in the lung of indicated mice. Combined data from three (C, D), four (A), or two (B) independent experiments are shown with 3 mice per naïve group and 4–5 mice per experimental asthma group in each experiment. The gating strategy for the lymphoid cells is detailed in the S3 Fig.
Fig 5.
iBALT formation is more pronounced in neutrophilic than in eosinophilic asthma.
C57BL/6 mice were immunized as outlined in Fig 1A to induce neutrophilic (CFA/OVA) or eosinophilic (Alum/OVA) asthma. Cells from bronchoalveolar lavage fluid (BALF) and lung homogenates were analyzed for indicated cell populations. (A) Total cell count (left panel) and relative cell frequencies of B cells (live CD45+) in indicated organs. (B) Production of the indicated cytokines by lung B cells (live CD45+) following in vitro stimulation with PMA and ionomycin. The values are given as fold-change of the two experimental groups over the control group. (C) Relative cell frequencies of germinal center (GC) B cells (live CD3ε- CD45R+ CD95+ GL7+ cells) in lung homogenates. (D) Inflamed lungs were fixed with 4% PFA (4h, 4°C), prepared for cryostat sectioning (7–10 μm), and stained with DAPI (white), CD45R-AF647 (blue), GL7-AF488 (green), and TCRβ-AF594 (red). Scale = 50 μm. Representative data from three biological replicates are shown. Unless indicated otherwise, combined data from two independent experiments are shown (naïve group n = 5 mice/group, experimental asthma groups n = 8–9 mice/group in total).