Fig 1: OVA-releasing scaffolds implanted in vaccinated mice enhance TH2 responses after ischemic injury.(A) Experimental setup with (left) BALB/c mice undergoing (right) timeline of procedures involving vaccination and boost with OVA/ALUM, followed by hindlimb ischemia induction and scaffold implantation. (B to J) In these figures, naïve + OVA denotes mice receiving no vaccination and implanted with an OVA-containing scaffold, vacc + blank denotes mice receiving vaccination with OVA/ALUM and a blank scaffold, and vacc + OVA denotes mice receiving vaccination with OVA/ALUM and an OVA-containing scaffold. (B) Number of CD4+ T cells (TCR-β+/CD4+ cells) per milligram of tissue in the ischemic upper leg at days 4 and 7 after ischemic ligation in the various treated groups. (C) Representative images of IL-5 enzyme-linked immunospot (ELISPOT) assay, where cells derived from the ischemic upper leg were cultured in the presence of splenocytes presenting either OVA or CT26-gp70 peptide as an irrelevant peptide control. (D) Quantification of OVA-specific IL-5 spot-forming cells per milligram of tissue determined by subtracting the number of IL-5 spot-forming cells in the CT26-gp70 condition from that in the OVA condition. (E to G) Concentrations of (E) IL-5, (F) IL-10, and (G) IFN-γ produced from OVA-stimulated cells isolated from the ischemic upper leg 7 days after ischemic ligation (n = 6 mice for naïve + OVA; n = 4 mice for vacc + blank; and n = 4 mice for vacc + OVA). (H to J) Number of (H) macrophages (F4/80+ cells), (I) M2a macrophages (F4/80+/CD206+ cells), and (J) eosinophils (Siglec-F+/CD11b+ cells) per milligram of tissue in the ischemic upper leg at days 4 and 7 after ischemic ligation in the various treated groups. For day 4 data in (B) to (D) and (H) to (J), n = 5 mice for naïve + OVA, n = 6 mice for vacc + blank, and n = 6 mice for vacc + OVA. For day 7 data in (B) to (D) and (H) to (J), n = 3 mice for naïve + OVA, n = 6 mice for vacc + blank, and n = 5 mice for vacc + OVA. Data are presented as means ± SD. Significance is denoted by *P ≤ 0.05, **P ≤ 0.01, or ***P ≤ 0.001 by one-way analysis of variance (ANOVA) with Bonferroni’s post hoc test.
Fig 2: Early-life viral infection results in secondary response with increased CD4+ Th2 signature.(A) Representative flow plots and quantification of intracellular GATA3 staining in CD4+ T cells isolated from the lung at day 7 after rechallenge showing increased levels in nV/aV mice. (B–D) Intracellular staining of IL-4, IL-5, and IL-13 production after ex vivo HMPV peptide stimulation in lung CD4+ T cells 7 days after rechallenge. *P < 0.05, **P < 0.01, ***P < 0.005 by 2-tailed Student’s t test.
Fig 3: Early JAK2 inhibition reduces asthma pathology.(A) UMAP visualization of Jak2 expression, showing upregulation in green circle corresponding to the Th2 cluster. (B) Mice were treated with fedratinib (120 mg/kg) once prior to rechallenge followed by BID dosing for the first 4 days after rechallenge. DMSO was used for vehicle control treatment. Created with BioRender.com. (C) Weight changes after rechallenge with HMPV. (D) Quantification of eosinophils 7 days after rechallenge. *P < 0.05 by Mann-Whitney U test. (E) N217-specific IL-4 and IFN-γ production after HMPV rechallenge, with a trend (P = 0.09, 2-tailed Student’s t test) toward reduced IL-4 production in JAK2i-treated mice. (F and G) IL-4, IL-5, IL-13, IL-2, IFN-γ, and GM-CSF protein quantity from whole lung homogenate. (H) Representative images of PAS staining demonstrating mucus production in large airways with reduction in JAK2i. Scale bar: 250 μm. (I) Quantification of PAS+ staining as percentage of total cell detections (left) and lung area (right). *P < 0.05 by 2-tailed Student’s t test.
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