Fig 1: IL-23 activates the JAK2–IRE1–XBP1s pathway in vivo and enhances Candida-caused asthma.B6 mice were sensitized with C. albicans extract plus OVA followed by rechallenges with OVA in the presence IL-23 or a vehicle. (A) Intracellular stain of XBP1s, pJAK2 and pIRE1 in lung infiltrating TH17 cells on a CD3+ CD4+ IL-23R+ gate. (B-C) ELISA of cytokine expression in BALF (B) and culture supernatant of LLNs after ex vivo recall with OVA (C). (D) Intracellular stain of IL-17-expressing cells in LLNs on a CD3+ CD4+ gate. (E) Profiles of CD4+ T cells and TH17 cells in BALF. (F) Profiles of macrophages and neutrophils in BALF. (G) H&E stain of lung sections. Scale bar, 50μm. Right, neutrophil counts were average numbers per field (x20). Data (mean ± SD) are a representative of two experiments. n = 5 per group. Student’s t test, *p< 0.05.
Fig 2: p-JAK2 interacts with and activates IRE1.(A) Immunofluorescence stain and colocalization of p-JAK2 (red) and p-IRE (green). DAPI marked cell nuclei (blue). Arrow heads indicate overlaps of p-JAK2 and p-IRE1. Scale bars, 10 μm. (B-C) Co-immunoprecipitation of p-JAK2 with p-IRE1 by anti-p-JAK2 (B) or anti-p-IRE1 (C). Iso, isotype control antibody. Tubulin and IRE1 or JAK2 were used as a loading control. (D) p-JAK2 phosphorylates recombinant IRE1 (rIRE1) in vitro. p-JAK2 was isolated by IP from IL-23 activated TH17 cells and incubated with rIRE1 (shorter than endogenous IRE1). p-IRE1 and inputs p-JAK2 and rIRE1 were detected by western blot. Tubulin was used as a loading control for IP of p-JAK2. Data shown are a representative of 2 (D) or 3 (A-C) experiments. (E) Schematic of extracellular signal-driven activation of the IRE1-XBP1s axis.
Fig 3: JAK2 is required for IL-23 to induce the IRE1–XBP1s axis in TH17 cells.(A) Western blot of p-JAK2 in TH17 cells treated with or without IL-23. p-JAK2 abundances were relative to JAK2. (B) Western blot of p-JAK2, p-IRE1, and Xbp1s in TH17 cells following JAK2 inhibition (inh) with Fedratinib or a vehicle in the presence or absence of IL-23. Abundances of p-JAK2, p-IRE1, and XBP1s were normalized to JAK2, IRE1 and Tubulin, respectively. (C) RT-qPCR of mRNA expression of indicated genes in TH17 cells treated as (B). mRNA abundances were normalized to Actb. (D) ELISA of indicated cytokines in TH17 cell culture supernatant treated as (B). Data (means ± SD) were pooled from 3 (A-C) or 5 (D) independent experiments. Student’s t test, *p < 0.05, **p < 0.005.
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