Fig 1: Extracellular signals activate the IRE1-XBP1s cascade and control TH17 function.(A) Western blot of p-IRE1 and XBP1s expression in TH17 cells treated with or without anti-CD3/CD28, IL-23, or IL-6. p-IRE1 and XBP1s abundances were relative to IRE1 and Tubulin, respectively. (B) Western blot XBP1s in TH17 cells treated with 10 µM IRE1 inhibitor 4µ8C or a vehicle in the presence or absence of IL-23. Abundances of XBP1s was normalized to Tubulin. (C) RT-qPCR of mRNA expression of indicated genes in TH17 cells treated as (B). Actb was used as a loading control. (D) ELISA of cytokines, IL-17 and IL-22. Data (means ± SD) were pooled from 3 independent experiments. Student’s t test, *p < 0.05, **p < 0.005.
Fig 2: IRE1 is induced in neutrophilic asthma and intensifies TH17 differentiation and function.(A) RT-qPCR of Ern1 mRNA in lungs from asthmatic and healthy mice. Asthma was elicited by using Candida albican extract. n = 6 per group. (B) RT-qPCR of Ern1 mRNA in TH17 cells and naive CD4+ T cells. (C-F) Characterization of TH17 cells polarized from Ern1fl/fl and Ern1fl/fl;Cd4Cre naïve CD4+ T cells. (C) Flow cytometry of IL-17+ CD4+ T cells. (D) Statistical analysis of IL-17+ CD4+ T cell frequencies (left) and ELISA of IL-17 expression (rigt) in (C). (E) RT-qPCR of mRNA expression of TH17 signature genes, Il17, Il17f, Rorc(gt), Il6, and Il21. (F) RT-qPCR of mRNA expression of IRE1 downstream genes, Xbp1u, Xbp1s, Hspa5, Becn1, and Ddit3. mRNA values were normalized to the internal reference gene Actb (A-B, E-F). Data (mean ± SD) shown are a representative (A, C) or a combination (B, D-F) of 3 experiments. Student’s t-test, *p < 0.05; ** p < 0.005.
Fig 3: 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 4: 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 5: T cell-specific Ern1-deficiency alleviates neutrophilic airway inflammation.Neutrophilic asthma was induced in Ern1fl/fl and Ern1fl/fl;Cd4Cre mice by i.n. challenges with C. albicans extract and OVA. (A) Intracellular stain of XBP1s in asthmatic lung infiltrating TH17 cells on a CD3+ CD4+ IL-23R+ gate. Iso, isotype control antibody. (B) Intracellular stain of CD4+ IL-17+ cells in LLNs on a CD3+ gate. (C-D) ELISA of cytokines in BALF (C) and culture supernatant of LLNs after ex vivo recall with OVA (D). (E) Profiles of macrophages and neutrophils in BALF. (F) H&E stain of lung sections. Scale bar, 50µm. Statistical analysis of neutrophils (average counts per 20x field per sample). Data (mean ± SD) are a representative of two experiments. n = 5–6 per group. Student’s t test, *p < 0.05.
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