PMC9169891_01
The clinical manifestations of the patient. (A) Generalised eczematous skin rashes in the patient. (B) Chest computed tomographic scan of the patient shows interstitial lung disease with diffuse ground-glass opacity. (C) Histology of lung biopsy from the patient. The alveolar membranes were markedly thickened (arrow) with macrophages, lymphocytes, and granulocytes in the alveolar cavity.
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Eleven of 20 (55%) ILD patients had mutations in the DBD ( Figure 1C ).
A novel de novo heterozygous mutation is identified in STAT3.
(A) Pedigree of the affected family. Circles, females; squares, males; filled symbol, affected subject; open symbols, unaffected family members. ND, not determined. (B) Sequence analysis of the patient and his parents. The heterozygous single base substitution (c.989C>G, p.P330R) in STAT3 is shown. (C) The schematic diagram shows the location of ILD-related GOF mutations on the STAT3 protein reported before. The position of the novel mutation identified in this study is marked in red. N-terminal domain (N-term), coiled-coil domain (CCD), DNA-binding domain (DBD), Linker domain (Linker), SH2-domain (SH2), and transactivation domain (TAD). (D) WebLogo demonstrates conservation of STAT3 amino acid sequence in 35 vertebrate species. (E) Crystal structure of the STAT3 (left) is shown in cartoon representation and colored as follows: CCD (magenta), DBD (cyan), Linker (grey), and SH2 (green). Magnified pictures displayed the sticks of Met329, Pro330 (top-right), Arg330 (bottom-right), and Met331 residue. Pro330 and Arg330 are colored in orange. (F) The predicted hydrophobicity (circled, yellow) of STAT3WT and STAT3P330R. Gradient colors from red to white indicate a hydrophobicity scale from high to low. (G) The root-mean-square deviations (RMSDs) of the STAT3WT and STAT3P330R during 50 ns of molecular dynamics simulations. (H) The calculated electrostatic potentials of the STAT3WT and STAT3P330R are mapped on the surfaces and colored in a gradient from red (negative) to blue (positive).
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Instead, a de novo heterozygous missense mutation of the STAT3 gene (c.989C>G, p.P330R) was identified in the patient ( Figures 2A, B ). This mutation was located within the DNA-binding domain (DBD), resulting in a proline to arginine substitution at position 330 ( Figure 2C ). The arginine residue was not observed at this position in homologous sequences ( Figure 2D ), suggesting highly conserved among different species. The mutant residue lay on the surface of the STAT3 protein ( Figure 2E ) and decreased the hydrophobic interactions in this region ( Figure 2F ). The root-mean-square deviation (RMSD) for the P330R system showed higher fluctuations than the WT system ( Figure 2G ), suggesting an unstable protein structure. Beyond that, according to the electrostatic potential calculation, the charge changed from neutral (P) to positive (R) ( Figure 2H ), which may increase the interaction with negatively charged nucleic acids.
The mutation leads to increased phosphorylation and transcriptional activity. (A) Flow cytometric analysis of phosphorylated STAT3 in PBMCs from the patient and healthy controls with or without IL-6 stimulation. (B, C). Western blot shows the phosphorylation (B) and dephosphorylation (C) of STAT3 in the human embryonic kidney 293 (HEK293) cells transfected with STAT3 constructs. The total STAT3 and GAPPDH were used as the loading controls. EV, empty vector. The results are representative of three independent experiments, respectively. (D, E) The STAT3 relative luciferase activity in the absence (D) or presence (E) of IL-6 stimulation. The representative result from three independent experiments is shown, respectively. Relative STAT3 protein expression level was confirmed by western blotting. Data are mean  +- SEM (n = 3). *p < 0.05. **p < 0.01.
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It was demonstrated that, upon IL-6 stimulation, the patient cells have much higher phosphorylation of STAT3 compared to normal controls. In addition, the mutation also activated STAT3 under the steady-state ( Figure 3A ). The STAT3P330R mutant also displayed high phosphorylation in the basal condition ( Figure 3B ), suggesting constitutive activation. Besides, the P330R mutation also impaired the dephosphorylation of phosphorylated STAT3 on IL-6 deprivation ( Figure 3C ). We observed enhanced transcriptional activity of STAT3P330R under the steady-state ( Figure 3D ), further suggesting constitutive activation of mutant STAT3. The mutant also showed a severalfold increase in the luciferase activity compared with STAT3WT after being stimulated with IL-6 ( Figure 3E ).
The Th17 cells and immune-phenotype in the patient. (A) The black and grey bars indicate the percentage of Th17 cells, as determined by flow cytometry, in phorbol myristate acetate (PMA)/ionomycin-activated PBMCs of the patient and healthy controls, respectively. Data are expressed as mean +- SEM. *p < 0.05. (B) The IL-17A production in T cells of the patient and two healthy controls after PMA/ionomycin treatment. (C) The representative dot plot shows the Treg population in the patient and two healthy controls, respectively. (D) Flow cytometry plots show the naive, central memory (CM), effector memory (EM), and terminally differentiated effector memory (TEMRA) CD4 or CD8 subpopulations in the patient and two age-matched healthy controls.
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P330R mutation would cause Th17 and Treg cells dysregulation. PMA/ionomycin-activated PBMCs of the patient showed increased IL-17A levels in the T cells compared to the normal controls ( Figures 4A, B ), suggesting the over-activation of the Th17 pathway. Treg cells was within a normal range in the patient ( Figure 4C ). The levels of immunoglobulin G, A and M were not reduced. Table 1 ). The proportions of effector memory (EM) CD4+ T, EM CD8+ T, naive CD4+ T and naive CD8+ T cells were also normal ( Figure 4D ). Taken together, these data suggested that the STAT3P330R gain-of-function mutation would contribute to activating the Th17 pathway.
The suppression of STAT3 phosphorylation by JAK and STAT3 inhibitors. (A) The transfected HEK293 cells were treated with dimethylsulphoxide (DMSO), ruxolitinib (Rux), or tofacitinib (Tof), respectively, and then activated with IL-6 (20ng/ml). The phospho-STAT3 level was examined. (B, C) The transfected HEK293 cells were treated with ruxolitinib, tofacitinib or stattic at the indicated concentration and then activated without (B) or with (C) IL-6 (20ng/ml). The phosphorylation of STAT3 was examined by western blot. The total STAT3 and GAPPDH were used as the loading controls.
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It was found that both ruxolitinib and tofacitinib significantly reduced the phosphorylation of mutant STAT3 induced by IL-6 stimulation ( Figure 5A ). IL-6-stimulated condition ( Figures 5B, C ). In addition, the inhibitory effect was dose-dependent.
PMC9169891_02
The clinical manifestations of the patient. (A) Generalised eczematous skin rashes in the patient. (B) Chest computed tomographic scan of the patient shows interstitial lung disease with diffuse ground-glass opacity. (C) Histology of lung biopsy from the patient. The alveolar membranes were markedly thickened (arrow) with macrophages, lymphocytes, and granulocytes in the alveolar cavity.
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Eleven of 20 (55%) ILD patients had mutations in the DBD ( Figure 1C ).