Fig 1: Treatment with β5 inhibitor prevents asthma in HDM-treated mice. (A) Unsupervised analysis of the lung from control, HDM (10 μg/mL), or HDM (10 μg/mL)+IPI-β5-1 (300 μg/kg) using t-SNE and mapping the following populations: eosinophils (CD45+F4/80+SiglecF+CD11c−), ILC2s (CD45+Lin−CD127+CD90.2+ST2+), tissue-resident alveolar macrophages (TR-AM, CD45+F4/80+SiglecF+ CD11c+), monocyte-derived alveolar macrophages (Mo-AM, CD45+F4/80+SiglecF−CD11c−), CD4 (CD45+CD3+CD4+), CD8 (CD45+CD3+CD8+), cDC1 (CD45+CD11b-CD11c+), and cDC2 CD45+CD11b+CD11c+). (B and C) Percentage of lung mILC2s and eosinophils in CD45+ from the lung of control, HDM, and HDM+IPI-β5-1 mice measured by flow cytometry (n = 14). (D and E) IL13 and IL5 expression was quantified by PCR in total lung from control mice, HDM-injected mice, and HDM-injected mice treated with IPI-β5-1 (n = 9). (F) IL-13, IL-4, IL-5, IL-6, IL-10, and TNF were measured by flow cytometry in BAL from WT mice, WT mice injected with HDM and WT mice injected with HDM and treated with IPI-β5-1 (n = 6). (G–I) Intracellular staining of IL-13 (G), IL-4 (H), and IL-5 (I) was quantified in lung mILC2s of WT mice, WT mice injected with HDM and WT mice injected with HDM and treated with IPI-β5-1 by flow cytometry (n = 6). (J) Representative images of H&E staining from lung sections at a ×10 magnification. (K and L) Epithelium thickness (K) and inflammation score (L) in WT mice, WT mice injected with HDM and WT mice injected with HDM and treated with IPI-β5-1 (n = 8). All results are represented as means ± SEM. Data are cumulative of three independent experiments and statistical significance was evaluated using a Mann–Whitney test. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 2: Treatment with β5 inhibitor reduces ILC2 activation during airway inflammation by affecting their metabolism. (A) Model of IL-33 induced airway inflammation with four injections of PBS or IL-33 (500 ng) either alone or with IPI-β5-1 inhibitor (30 μg/kg). (B) Percentage of lung mILC2s (CD45+Lin−CD127+CD90.2+ST2+) in CD45+ from the lung of control, IL-33, and IL-33+IPI-β5-1 mice measured by flow cytometry (n = 12). (C) Percentage of eosinophils (Eos, CD45+CD11c−SiglecF+), macrophages (Mac, CD45+Ly6G−SiglecF+CD11c+F4/80+), neutrophils (PMN, CD45+Ly6G+SiglecF−CD11b+), and T cells (CD45+CD3+) in BAL of IL-33 injected mice and IL-33 injected mice treated with IPI-β5-1 (n = 6 or 8). (D and E) IL13 and IL5 expression was quantified by PCR in total lung from control mice, IL-33 injected mice, and IL-33 injected mice treated with IPI-β5-1 (n = 9). (F) IL-13, IL-4, IL-5, IL-6, IL-10, and TNF were measured by flow cytometry in BAL from WT mice, WT mice injected with IL-33 and WT mice injected with IL-33 and treated with IPI-β5-1 (n = 6). (G–I) Intracellular staining of IL-13 (G), IL-5 (H), and IL-4 (I) was quantified in lung mILC2s by flow cytometry (n = 6). (J–L) ROS production was measured in lung mILC2s by flow cytometry (n = 6). (M–O) TMRM and mitotracker were measured in lung mILC2s by flow cytometry (n = 6). All results are represented as means ± SEM. Data are cumulative of two to four independent experiments and statistical significance was evaluated using a Mann–Whitney test. *P < 0.05, **P < 0.01, ***P < 0.001. ns = not significant.
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