Human germline heterozygous gain-of-function STAT6 variants cause severe allergic disease

STAT6 (signal transducer and activator of transcription 6) is a transcription factor that plays a central role in the pathophysiology of allergic inflammation. We have identified 16 patients from 10 families spanning three continents with a profound phenotype of early-life onset allergic immune dysregulation, widespread treatment-resistant atopic dermatitis, hypereosinophilia with esosinophilic gastrointestinal disease, asthma, elevated serum IgE, IgE-mediated food allergies, and anaphylaxis. The cases were either sporadic (seven kindreds) or followed an autosomal dominant inheritance pattern (three kindreds). All patients carried monoallelic rare variants in STAT6 and functional studies established their gain-of-function (GOF) phenotype with sustained STAT6 phosphorylation, increased STAT6 target gene expression, and TH2 skewing. Precision treatment with the anti-IL-4Rα antibody, dupilumab, was highly effective improving both clinical manifestations and immunological biomarkers. This study identifies heterozygous GOF variants in STAT6 as a novel autosomal dominant allergic disorder. We anticipate that our discovery of multiple kindreds with germline STAT6 GOF variants will facilitate the recognition of more affected individuals and the full definition of this new primary atopic disorder

Epidermal growth factor receptor (EGFR) mutations and phosphorylation pattern in non-small cell lung cancer (NSCLC)

published_or_final_versionPathologyDoctoralDoctor of Philosoph

Immobilized Osteopontin Enhances Adhesion but Suppresses Cytokine Release of Anti-IgE Activated Human Mast Cells

Osteopontin (OPN) is an Arg-Gly-Asp (RGD)-containing extracellular matrix protein which is upregulated in inflamed tissues and has been reported to modulate mast cell activities in mice. Due to the known heterogeneity among mast cells of different species and the important roles of mast cells in allergic reactions, we investigated the effects of human OPN (hOPN) on human mast cell activities. Mature primary human cultured mast cells (HCMC) were derived from peripheral blood CD34+ progenitors and the modulation of their activation by soluble and plate-bound immobilized hOPN were examined by studying their release of inflammatory mediators (histamine, IL-5, IL-8, TNF-α, and VEGF) and matrix adhesion following stimulation by anti-IgE. Immobilized hOPN enhanced the adhesion, but suppressed the release of IL-5, IL-8, and TNF-α of anti-IgE-activated HCMC while soluble hOPN failed to demonstrate any significant effects. By employing cyclic RGD peptide and neutralizing antibodies against different classes of integrin and CD44, we demonstrated that the interaction of immobilized hOPN and HCMC was mediated by the RGD domain of hOPN and integrin but not CD44 on HCMC. Our results suggest that immobilized hOPN anchored to extracellular matrix can regulate adaptive immunity in humans by retaining mast cells at the site of inflammation and suppressing anti-IgE-induced cytokine release from HCMC

Differential Effects of the Toll-Like Receptor 2 Agonists, PGN and Pam3CSK4 on Anti-IgE Induced Human Mast Cell Activation

Mast cells are pivotal in the pathogenesis of allergy and inflammation. In addition to the classical IgE-dependent mechanism involving crosslinking of the high-affinity receptor for IgE (Fc epsilon RI), mast cells are also activated by Toll-like receptors (TLRs) which are at the center of innate immunity. In this study, we demonstrated that the response of LAD2 cells (a human mast cell line) to anti-IgE was altered in the presence of the TLR2 agonists peptidoglycan (PGN) and tripalmitoyl-S-glycero-Cys(Lys) 4 (Pam3CSK4). Pretreatment of PGN and Pam3CSK4 inhibited anti-IgE induced calcium mobilization and degranulation without down-regulation of FceRI expression. Pam3CSK4 but not PGN acted in synergy with anti-IgE for IL-8 release when the TLR2 agonist was added simultaneously with anti-IgE. Studies with inhibitors of key enzymes implicated in mast cell signaling revealed that the synergistic release of IL-8 induced by Pam3CSK4 and anti-IgE involved ERK and calcineurin signaling cascades. The differential modulations of anti-IgE induced mast cell activation by PGN and Pam3CSK4 suggest that dimerization of TLR2 with TLR1 or TLR6 produced different modulating actions on Fc epsilon RI mediated human mast cell activation

Effects of TLR2 ligands on anti-IgE induced calcium mobilization in LAD2 cells.

<p>(A, B) LAD2 cells were incubated with anti-IgE (○), PGN/Pam3CSK4 (◊), anti-IgE with PGN/Pam3CSK4 (▪), and calcium mobilization was measured at the same time. (C, D) Cells were incubated with PGN or Pam3CSK4 for 24 h prior to being challenged with anti-IgE (▪). Changes in [Ca²⁺]_i were compared in the presence or absent of PGN or Pam3CSK4. Error bars were omitted for the clarity of the graph. The peak levels of calcium influx were compared with area under the curves analysis (E, F, G, H). Significant differences following student <i>t</i>-test were indicated by asterisks: *p<0.05, **p<0.01 (n = 4–6).</p

Effects of PGN and Pam3CSK4 on the expression of FcεRI on LAD2 cells.

<p>LAD2 cells (without IgE sensitization) were incubated with PGN (50 µg/ml) (A) and Pam3CSK4 (20 µg/ml) (B) for 24 h and FcεRI surface expression was analyzed by flow cytometry after cells were incubated with FITC-conjugated anti-human FcεRI antibody, FITC-conjugated mouse IgG2b isotype control or FACS buffer for specific labelling of FcεRI, isotype and blank control respectively. The FcεRI expression of cells that were not treated (grey curve) or treated (blank curve) with the TLR2 ligands was not different as shown. Results were representative of four independent experiments.</p

Effects of inhibitors of MAPKs on TLR2 ligands and anti-IgE induced IL-8 release from LAD2 cells.

<p>(A) ERK inhibitor, PD98059 (10 µM), (B) JNK inhibitor, SP600125 (20 µM) or (C) p38 inhibitor, SB2023580 (10 µM) was incubated with LAD2 cells for 30 min before the addition of anti-IgE (2 µg/ml), PGN (50 µg/ml), Pam3CSK4 (20 µg/ml), anti-IgE with PGN or Pam3CSK4 for 24 h to induce the release of IL-8. The amounts of IL-8 release from activated LAD2 cells pre-incubated with an inhibitor and the corresponding control pre-incubated in culture medium were compared with student <i>t</i>-test. *p<0.05, **p<0.01, ***p<0.01 (n = 4–6).</p

Effects of inhibitors of calcineurin, NF-κB and PI3K on TLR2 ligands and anti-IgE induced IL-8 release from LAD2 cells.

<p>(A) ciclosporin (2 µg/ml), (B) Bay 11-7821 (10 µM) or (C) wortmannin (0.5 µM) was incubated with LAD2 cells for 30 min before the addition of anti-IgE (2 µg/ml), PGN (50 µg/ml), Pam3CSK4 (20 µg/ml), anti-IgE with PGN or Pam3CSK4 for 24 h to induce the release of IL-8. The amounts of IL-8 release from activated LAD2 cells pre-incubated with an inhibitor and the corresponding control pre-incubated in culture medium were compared with student <i>t</i>-test. *p<0.05, **p<0.01, ***p<0.001 (n = 4–5).</p