STAT3 mutation-associated airway epithelial defects in Job syndrome

Abstract

Background: Job syndrome is a disease of autosomal dominant hyper-IgE syndrome (AD-HIES). Patients harboring STAT3 mutation are particularly prone to airway remodeling and airway infections.

Objectives: Airway epithelial cells play a central role as the first line of defense against pathogenic infection and express high levels of STAT3. This study thus interrogates how AD-HIES STAT3 mutations impact the physiological functions of airway epithelial cells.

Methods: This study created human airway basal cells expressing 4 common AD-HIES STAT3 mutants (R382W, V463del, V637M, and Y657S). In addition, primary airway epithelial cells were isolated from a patient with Job syndrome who was harboring a STAT3-S560del mutation and from mice harboring a STAT3-V463del mutation. Cell proliferation, differentiation, barrier function, bacterial elimination, and innate immune responses to pathogenic infection were quantitatively analyzed.

Results: STAT3 mutations reduce STAT3 protein phosphorylation, nuclear translocation, transcription activity, and protein stability in airway basal cells. As a consequence, STAT3-mutated airway basal cells give rise to airway epithelial cells with abnormal cellular composition and loss of coordinated mucociliary clearance. Notably, AD-HIES STAT3 airway epithelial cells are defective in bacterial killing and fail to initiate vigorous proinflammatory responses and neutrophil transepithelial migration in response to an experimental model of Pseudomonas aeruginosa infection.

Conclusions: AD-HIES STAT3 mutations confer numerous abnormalities to airway epithelial cells in cell differentiation and host innate immunity, emphasizing their involvement in the pathogenesis of lung complications in Job syndrome. Therefore, therapies must address the epithelial defects as well as the previously noted immune cell defects to alleviate chronic infections in patients with Job syndrome.

Keywords: AD-HIES STAT3 mutation; cell differentiation; innate immunity; mucociliary clearance; neutrophil chemotaxis.

Figure 1.. AD-HIES STAT3 mutations result in the loss of function of STAT3 signaling activity in human airway basal cells.

A. Schematic model of STAT3 delineating the domain location for four AD-HIES single-amino-acid missense mutation or in-frame deletion. B. Schematic diagram of the generation of airway basal cell lines expressing WT-STAT3 or AD-HIES STAT3. C-D. Western blotting (C) and protein signal quantification (D) of total STAT3 and phosphorylated STAT3 in engineered airway basal cell lines before and after treatment of IL-6 (10 ng/ml) for 1 h. The stain-free imaging technology was used to indicate the total protein loaded. E-F. STAT3 immunostaining (E) and the ratio of nuclear versus cytoplasmic fluorescent intensity (F) in indicated airway basal cells before and after treatment of IL-6 (10 ng/ml) for 1 h. Scale bar: 10 μm. G. Indicated airway basal cells were transfected with the STAT3-dependent luciferase reporter (m67-luciferase). At 24 h post-transfection, the cells were treated with IL-6 (10 ng/ml) for 6 h and then analyzed for luciferase activity. H. Representative images of STAT3 protein determined by western blotting in the presence of 2 μg/mL CHX for the indicated times. I. Half-lives of indicated STAT3 mutants were determined using monoexponential decay curve fitting of STAT3/β-actin ratios at various time post CHX treatment. The data are expressed as the mean ± SEM of three independent experiments. ***P < 0.001, using one-way ANOVA followed by Tukey’s multiple comparisons test (D, F and G).

Figure 2.. AD-HIES STAT3 airway basal cells generate airway epithelia with altered cellular composition.

A. Representative staining of ALI cultures for ciliated cells (Ac-Tub, green), club cells (CCSP, red), and goblet cells (MUC5AC, red). Scale bar: 100 μm. B. Quantification of various cell types based on whole-mount staining (A). C. Representative staining of Ac-Tub⁺ ciliated cells and MUC5AC⁺ goblet cells generated from normal human airway basal cells treated with 10 ng/ml IL-6 or 5 μM Stattic during ALI differentiation. Scale bar: 100 μm. D-E. Representative staining (D) and quantification (E) of Ac-Tub⁺ ciliated cells and MUC5AC⁺ goblet cells generated from control airway basal cells (shRNA-Control) and from STAT3 knockdown airway basal cells (shRNA-STAT3). The data are expressed as the mean ± SEM of three or four independent experiments. ***P < 0.001, using one-way ANOVA followed by Dunnett’s multiple comparisons test (B) or unpaired two-tailed Student’s t-test (E).

Figure 3.. AD-HIES STAT3 airway epithelial cells exhibit impaired mucociliary clearance and decreased bacterial killing ability.

A-B. Mucociliary transport (MCT) was imaged (A) and quantified (B) by using μOCT technology in ALI culture from WT and AD-HIES STAT3 airway basal cells. C-D. Measurement of PAO1 number adhered to the apical surface of WT-STAT3 and AD-HIES STAT3 airway epithelial cells (C) or to normal airway epithelial cells generated in the presence of 5 μM Stattic or 10 ng/ml IL-6 (D) after 1h incubation. E. Measurement of total survived PAO1 number after 4 h incubation with indicated airway epithelial cells. F-G. Western blotting (F) and quantification (G) of BPIFA1 apical secretion from indicated airway epithelial cells. The total loaded protein is detected by Stain-Free technology (F). The data are expressed as the mean ± SEM of three or four independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001, using one-way ANOVA followed by Dunnett’s multiple comparisons test (B-E and G).

Figure 4.. AD-HIES STAT3 airway epithelial cells fail to mount robust inflammatory responses to pathogenic infection.

A-B. Western blotting (A) and quantification (B) of STAT3 and p-STAT3 in normal airway epithelial cells after 1 h infection with PAO1. The total loaded protein is detected by Stain-Free technology. C. Volcano plot of RNA-seq data showing statistical significance (−log10 of p value, y axis) versus magnitude of gene expression change (log2 fold-change, x axis) of normal airway epithelial cells before and after 1 h of PAO1 infection. The red dots indicate the gene expression having p < 0.05 and absolute log2 (fold-change) >1. D-G. The expression of IL6, IL17C, and CXCL2 was measured by qRT-PCR in indicated airway epithelial cells after 1 h of PAO1 infection. The data are expressed as the mean ± SEM of three independent experiments. ns, not significant; ***P < 0.001, using unpaired two-tailed Student’s t-test (B) or one-way ANOVA followed by Tukey’s multiple comparisons test (D-G).

Figure 5.. Airway epithelial cells isolated from a Job Syndrome patient exhibit similar defects in cell differentiation, mucociliary clearance, and innate immunity.

A. Schematic of airway basal cell isolation from healthy donors and a Job Syndrome patient harboring STAT3-S560del mutation. B-C. Western blotting (B) and quantification (C) of total STAT3 and p-STAT3 in healthy and Job Syndrome patient airway basal cells before and after 1 h of 10 ng/ml IL-6 treatment. Total protein loaded is detected by Stain-Free technology. D. Healthy and Job Syndrome patient airway basal cells were transfected with the STAT3-dependent luciferase reporter. At 24 h post-transfection, the cells were treated with IL-6 (10 ng/ml) for 6 h and then analyzed for luciferase activity. E. Representative staining of Ac-Tub⁺ ciliated cells and MUC5AC⁺ goblet cells of ALI culture generated by healthy and Job Syndrome patient’s airway basal cells. Scale bar: 100 μm. F. Measurement of PAO1 number adhered to the apical surface of healthy and Job Syndrome patient’s airway epithelial cells after 1 h incubation. G. Measurement of total survived PAO1 number after 4 h incubation with healthy and Job Syndrome patient’s airway epithelial cells. H. PCA for the global transcriptional profiles of healthy and Job Syndrome patient’s airway epithelial cells before and after 1 h of PAO1 infection. I. Heatmap depicting selected cytokines and chemokines that are differentially expressed in healthy and Job Syndrome patient’s airway epithelial cells after 1 h of PAO1 infection. J-K. The measurement of indicated cytokines and chemokines by qPCR in healthy and Job Syndrome patient’s airway epithelial cells before and after 1 h of PAO1 infection. L. The strategic diagram of transepithelial migration assay using inverted ALI culture and MPO measurement as a readout. M. The quantification of neutrophil migration across healthy and Job Syndrome patient’s airway epithelial cells assessed by MPO activity. The data are expressed as the mean ± SEM of three or four independent experiments. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001, using unpaired two-tailed Student’s t-test (F and G) or one-way ANOVA followed by Tukey’s multiple comparisons test (C, D, J, K and M).

Figure 6.. Job Syndrome mouse tracheal epithelial cells exhibit abnormal cell differentiation, mucociliary clearance, and innate immune response.

A. Schematic of tracheal basal cell isolation from WT C57-B6 mouse and the Job Syndrome mouse harboring STAT3-V463del mutation. B-C. Western blotting (B) and quantification (C) of total STAT3 and p-STAT3 in WT and STAT3-V463del mouse tracheal basal cells before and after 1 h of 10 ng/ml IL-6 treatment. Total protein loaded is detected by Stain-Free technology. D. Western blotting and quantification of nuclear and cytoplasmic STAT3 distribution in WT and STAT3-V463del mouse tracheal basal cells before and after 1 h of IL-6 (10 ng/ml) treatment. E. WT and STAT3-V463del mouse tracheal basal cells were transfected with the STAT3-dependent luciferase reporter (m67-luciferase). At 24 h post-transfection, the cells were treated with IL-6 (10 ng/ml) for 6 h and then analyzed for luciferase activity. F. Western blotting of STAT3 in WT and STAT3-V463del mouse tracheal basal cells after treatment with 2 μg/mL CHX for the indicated times. G-H. Representative staining (G) and quantification (H) of Ac-Tub⁺ ciliated cells and SSEA1⁺ club cells generated from WT and STAT3-V463del mouse tracheal basal cells after ALI culture. Scale bar: 100 μm. I. Measurement of PAO1 number adhered to the apical surface of WT and STAT3-V463del mouse tracheal epithelial cells after 1 h incubation. J. Measurement of total survived PAO1 number after 4 h incubation with WT and STAT3-V463del mouse tracheal epithelial cells. K. qPCR quantification of indicated cytokines and chemokines in WT and STAT3-V463del mouse tracheal epithelial cells before and after 1 h of PAO1 infection. L. Immunofluorescence of Ac-Tub⁺ ciliated cells and SSEA1⁺ club cells grown on 3 μm inverted transwells. Scale bar: 25 μm. M. The quantification of neutrophil migration across WT and STAT3-V463del mouse tracheal epithelial cells assessed by MPO activity. The data are expressed as the mean ± SEM of three or four independent experiments. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001, using unpaired two-tailed Student’s t-test (H-J) or one-way ANOVA followed by Tukey’s multiple comparisons test (C-E, K and M).