Defects along the T(H)17 differentiation pathway underlie genetically distinct forms of the hyper IgE syndrome

Abstract

Background: The hyper IgE syndrome (HIES) is characterized by abscesses, eczema, recurrent infections, skeletal and connective tissue abnormalities, elevated serum IgE, and diminished inflammatory responses. It exists as autosomal-dominant and autosomal-recessive forms that manifest common and distinguishing clinical features. A majority of those with autosomal-dominant HIES have heterozygous mutations in signal transducer and activator of transcription (STAT)-3 and impaired T(H)17 differentiation.

Objective: To elucidate mechanisms underlying different forms of HIES.

Methods: A cohort of 25 Turkish children diagnosed with HIES were examined for STAT3 mutations by DNA sequencing. Activation of STAT3 by IL-6 and IL-21 and STAT1 by IFN-alpha was assessed by intracellular staining with anti-phospho (p)STAT3 and -pSTAT1 antibodies. T(H)17 and T(H)1 cell differentiation was assessed by measuring the production of IL-17 and IFN-gamma, respectively.

Results: Six subjects had STAT3 mutations affecting the DNA binding, Src homology 2, and transactivation domains, including 3 novel ones. Mutation-positive but not mutation-negative subjects with HIES exhibited reduced phosphorylation of STAT3 in response to cytokine stimulation, whereas pSTAT1 activation was unaffected. Both patient groups exhibited impaired T(H)17 responses, but whereas STAT3 mutations abrogated early steps in T(H)17 differentiation, the defects in patients with HIES with normal STAT3 affected more distal steps.

Conclusion: In this cohort of Turkish children with HIES, a majority had normal STAT3, implicating other targets in disease pathogenesis. Impaired T(H)17 responses were evident irrespective of the STAT3 mutation status, indicating that different genetic forms of HIES share a common functional outcome.

Figure 1

Characteristics of the HIES cohort for Age (A), HIES score (B), serum IgE levels (C) and blood eosinophilia (D). Disease complications in HIES-STAT3wt versus HIES-STAT3mut group. Abscesses include those in the skin and viscera; skeletal involvement includes fractures, retained primary teeth and hyper-extensibility; severe infections are those requiring hospitalization; CNS involvement includes infections, infarcts, vessel occlusion and ischemic injury. *p=0.02 for eosinophil counts (Student's two tailed t test) and ^†p=0.005 for pneumatocele formation (Fisher exact test) in HIES-STAT3wt versus HIES-STAT3mut group.

Figure 2

Defective IL-6-induced STAT3 phosphorylation in T cells of HIES subjects with, but not without, STAT3 mutations. A. Representative intracellular staining profile of pY705STAT3 in T cells of a control subject, an HIES subject with normal STAT3 sequence (HIES-STAT3wt) and HIES subjects with DNA binding and SH2 domain mutations (HIES-STAT3R382Q and HIES-STAT3Y668F, respectively), following stimulation with IL-6 for 15 min. B. *p<0.01 for HIES patients with STAT3 mutations (HIES-STAT3mut) versus HIES-STAT3wt or unrelated controls (n=4 for each group).

Figure 3

Defective IL-21-induced STAT3 phosphorylation in T cells of HIES patients with STAT3 mutations. A. Representative intracellular staining profile of pY705STAT3 in T cells of a control subject, an HIES subject with normal STAT3 sequence (HIES-STAT3wt), and HIES subjects with DNA binding and SH2 domain mutations (HIES-STAT3R382Q and HIES-STAT3Y657C, respectively), following stimulation with IL-21 for 15 min. B. *p<0.001 for unrelated and parent controls (n=6 and 17, respectively) versus HIES patients with STAT3 mutations (HIES-STAT3mut; n=5), and for HIES-STAT3wt (n=15) versus HIES-STAT3mut.

Figure 4

Impaired Th17 differentiation is a common attribute of different forms of HIES. A. Real time PCR analysis of RORγτ mRNA levels in peripheral blood T cell blasts of Parent control subjects (n= 7), HIES without STAT3 mutations (HIES-STAT3wt, n=7) and HIES subjects with STAT3 mutations (HIES-STAT3mut; n=3); p= *0.01 and **0.001 for parent controls versus HIES-STAT3mut and HIES-STAT3wt subjects, respectively. B. IL-17 production by peripheral blood T cells of HIES and control subjects following Th17 differentiation; p=***0.001 for controls (n=17) versus HIES-STAT3mut (n=6) and HIES-STAT3wt (n=12), respectively. C. IFNγ production following Th1 differentiation is not impaired in HIES groups. D, E. RORγτ mRNA expression (D) and IL-17 production (E) in Th0 and Th17 cells derived from naïve T cells of control, HIES-STAT3mut and HIES-STAT3wt subjects; *p<0.05 for Th17 HIES-STAT3wt versus Th17 HIES-STAT3mut (D), and **p<0.01 for Th17 HIES-STAT3wt versus Th17 control or Th17 HIES-STAT3mut (E).