Diminished allergic disease in patients with STAT3 mutations reveals a role for STAT3 signaling in mast cell degranulation Andrea M. Siegel, PhD, Kelly.

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Diminished allergic disease in patients with STAT3 mutations reveals a role for STAT3 signaling in mast cell degranulation Andrea M. Siegel, PhD, Kelly D. Stone, MD, PhD, Glenn Cruse, PhD, Monica G. Lawrence, MD, Ana Olivera, PhD, Mi-yeon Jung, PhD, John S. Barber, BA, Alexandra F. Freeman, MD, Steven M. Holland, MD, Michelle O'Brien, RN, Nina Jones, RN, Laura B. Wisch, RN, Heidi H. Kong, MD, MHSc, Avanti Desai, MA, Orly Farber, Alasdair M. Gilfillan, PhD, Juan Rivera, PhD, Joshua D. Milner, MD Journal of Allergy and Clinical Immunology Volume 132, Issue 6, Pages 1388-1396.e3 (December 2013) DOI: 10.1016/j.jaci.2013.08.045 Copyright © 2013 Terms and Conditions

Fig 1 STAT3-mutant (AD-HIES) patients have reduced incidence of food allergies compared with patients with similar degrees of atopic dermatitis, high IgE, and no STAT3 mutation. A, Physician-diagnosed eczema in healthy volunteer control, patients with AD-HIES (STAT3-mutant), and atopic (no STAT3 mutation) control cohorts. B, Serum IgE levels in healthy volunteer controls (N = 25), patients with AD-HIES (N = 72), and atopic controls (N = 60). Each symbol represents a single patient. Line represents the median concentration of serum IgE. C, Physician-diagnosed food allergies in healthy volunteers, patients with AD-HIES, and atopic control patients were determined by interview. D, Incidence of physician-diagnosed anaphylaxis in patents with AD-HIES and control patients with atopy. Significance for panels C and D as determined by a 2-tailed χ2 test. E-G, Allergen-specific IgE in serum or plasma was measured by ImmunoCAP assay for 3 common food allergens: egg, milk, and peanut. Dotted line represents the 95% positive predictive value of each allergen as previously reported.5 P values between healthy volunteers and atopic controls are P = .0013 for milk, P = .0001 for egg, and P = .0001 for peanut, respectively. Significance was determined by 2-tailed Mann-Whitney test. Journal of Allergy and Clinical Immunology 2013 132, 1388-1396.e3DOI: (10.1016/j.jaci.2013.08.045) Copyright © 2013 Terms and Conditions

Fig 2 Reduced mast cell degranulation in STAT3-mutant humans and mice. A, Healthy volunteers (N = 26) and patients with AD-HIES (N = 31) were skin prick tested for morphine reactivity with increasing doses of morphine. Scores of 0 or 1 were considered nonreactive. Error was determined by 1-tailed Mann-Whitney test. B, Histamine reactivity in healthy volunteers (N = 26) and patients with AD-HIES (N = 31). C, A basophil activation test was performed on healthy volunteers (N = 5) and patients with AD-HIES (N = 5) with increasing concentrations of anti-IgE. Activated basophils were gated on Lin1– CD123+ CD203c+ CD63high. Error was determined by 1- tailed Mann-Whitney test (*P = .0278, **P = .0476). D, Immunohistochemical staining of tryptase (magnification, ×20) from skin biopsies of human flank in 2 controls (top) and 2 patients with AD-HIES (bottom). Journal of Allergy and Clinical Immunology 2013 132, 1388-1396.e3DOI: (10.1016/j.jaci.2013.08.045) Copyright © 2013 Terms and Conditions

Fig 3 Reduced mast cell degranulation in STAT3-mutant mice. A, IgE-mediated systemic anaphylaxis in mut-Stat3 mice compared with wild-type littermate controls. Data are representative of 2 independent experiments with 4 to 5 mice per group. B, Compound 48/80 was used to induce IgE-independent anaphylaxis in mut-Stat3 and wild-type mice. Data are representative of 4 mice per group. C, Dot plot of surface FcεRI expression versus CD117 expression. D, Percentage of Lin–IgE+FcεRI+CD117+ peritoneal mast cells. Data are cumulative of 2 independent experiments of age- and sex-matched animals with 4 to 6 mice per group. Journal of Allergy and Clinical Immunology 2013 132, 1388-1396.e3DOI: (10.1016/j.jaci.2013.08.045) Copyright © 2013 Terms and Conditions

Fig 4 STAT3 contributes to human mast cell proximal and distal FcεRI signaling events. A, STAT3 phosphorylation on Serine727 and Tyrosine705 after 2 minutes of IgE cross-linking in LAD2 and primary human mast cells (HuMCs). The change in mean fluorescence intensity between stimulated (black line) and unstimulated (gray solid) cells is given in the upper left corner. As a positive control, ERK phosphorylation at 0 (solid gray), 2 (black line), and 15 (dashed red line) minutes after cross-linking was measured by flow cytometry. Data are representative of 2 independent experiments with LAD2 cells and 1 experiment in primary human mast cells. B, Western blot of lysates from the human mast cell line LAD2 transduced with 5 different shRNAs directed against STAT3. C, Mast cell degranulation was measured by FcεRI cross-linking and subsequent β-hexosaminidase (β-hex) release in LAD2 cells transduced with 5 different shRNAs against STAT3. Data are representative of 2 independent experiments. LAD2, unstimulated control; LAD+, FcεRI cross-linking. D, Mast cell degranulation was measured by FcεRI cross-linking and subsequent β-hexosaminidase release in primary human mast cells transduced with 2 different shRNAs against STAT3. HuMC, unstimulated control; HuMC+, FcεRI cross-linking. E, Silencing of STAT3 reduced proximal and distal FcεRI-induced mast cell signaling. Phosphorylation of STAT3 (S727) was evident with antigen stimulation that was much less marked with shSTAT3. Early and proximal FcεRI signaling occurs through Lyn and Syk kinases and is evident at 2 minutes (arrows) in the scramble control cells. However, silencing of STAT3 reduces phosphorylation at 2 minutes. FcεRI signaling defects were also evident with phospholipase C, γ 1 (PLCγ1) phosphorylation at 2 minutes. Defects in distal FcεRI signaling (AKT and ERK), which occur later, were also evident with STAT3 silencing. Ag, Antigen. Journal of Allergy and Clinical Immunology 2013 132, 1388-1396.e3DOI: (10.1016/j.jaci.2013.08.045) Copyright © 2013 Terms and Conditions

Fig E1 A, IgE-mediated basophil activation in atopic controls is not significantly different than basophil activation in healthy controls. Data are representative of 5 healthy volunteers and 8 atopic controls. Basophil activation after stimulation with f-Met-Leu-Phe (fMLP) is not significantly different between patients with AD-HIES and healthy volunteers (B) or patients and healthy volunteers (C). n.s., Not significant. Journal of Allergy and Clinical Immunology 2013 132, 1388-1396.e3DOI: (10.1016/j.jaci.2013.08.045) Copyright © 2013 Terms and Conditions

Fig E2 STAT3 signaling and silencing in LAD2 mast cells. A, Phosphoflow measurement of pSTAT3 S727. Light gray, isotype control; dark gray, unstimulated; solid line, 2 minutes after surface IgE crosslink. B, Densitometry of STAT3 protein expression after shRNA knockdown in LAD2 cells. C, Correlation between STAT3 protein expression and inhibition of mast cell degranulation after STAT3 knockdown (r2 = 0.95). Journal of Allergy and Clinical Immunology 2013 132, 1388-1396.e3DOI: (10.1016/j.jaci.2013.08.045) Copyright © 2013 Terms and Conditions

Fig E3 Signaling in LAD2 mast cells after STAT3 knockdown. A, Effect of STAT3 shRNA silencing on IgE-dependent LAD-2 human mast cell degranulation with the use of 2 different shSTAT3 constructs. B, IgE-independent degranulation induced by the G-protein coupled receptor (GPCR) agonist C3a. C, Degranulation induced by the mast cell activator, compound 48/80. D, Receptor-independent activation with thapsigargin. E, Surface FcεRI expression as measured by bound IgE to the cell surface. F, C3a induced total and phosphorylated phospholipase C, γ 1 (pPLCγ1), AKT, and ERK, as measured by Western blot analysis. *P < .05 compared to scramble shRNA. Journal of Allergy and Clinical Immunology 2013 132, 1388-1396.e3DOI: (10.1016/j.jaci.2013.08.045) Copyright © 2013 Terms and Conditions