FOXP3+ Tregs require WASP to restrain Th2-mediated food allergy

Abstract

In addition to the infectious consequences of immunodeficiency, patients with Wiskott-Aldrich syndrome (WAS) often suffer from poorly understood exaggerated immune responses that result in autoimmunity and elevated levels of serum IgE. Here, we have shown that WAS patients and mice deficient in WAS protein (WASP) frequently develop IgE-mediated reactions to common food allergens. WASP-deficient animals displayed an adjuvant-free IgE-sensitization to chow antigens that was most pronounced for wheat and soy and occurred under specific pathogen-free as well as germ-free housing conditions. Conditional deletion of Was in FOXP3+ Tregs resulted in more severe Th2-type intestinal inflammation than that observed in mice with global WASP deficiency, indicating that allergic responses to food allergens are dependent upon loss of WASP expression in this immune compartment. While WASP-deficient Tregs efficiently contained Th1- and Th17-type effector differentiation in vivo, they failed to restrain Th2 effector responses that drive allergic intestinal inflammation. Loss of WASP was phenotypically associated with increased GATA3 expression in effector memory FOXP3+ Tregs, but not in naive-like FOXP3+ Tregs, an effect that occurred independently of increased IL-4 signaling. Our results reveal a Treg-specific role for WASP that is required for prevention of Th2 effector cell differentiation and allergic sensitization to dietary antigens.

Trial registration: ClinicalTrials.gov NCT01164241.

Figures

Figure 1. Increased sensitization and prevalence of food allergy among patients with WAS mutations.

(A) Schematic of WAS gene with mutations identified among cohort (n = 25). Bold indicates WAS diagnosis; unbolded indicates XLT; italicized indicates mutations associated with food allergy in childhood. (B) Total serum IgE (sIgE) levels and food allergen–specific sIgE levels among cohort; limit of detection for sIgE was 0.1 IU/ml. Light gray fill indicates the normal range; boxes indicate median and interquartile ranges for 4 foods reported in NHANES (ref. 23). White circles indicate food-allergic individuals. (C) Among patients with sera obtained prior to transplantation (n = 22), percent of WAS (n = 12) or XLT (n = 10) patients positive for sIgE against foods (minimum cutoff 0.35 IU/ml) compared with the general population as reported in NHANES analysis (left panel). Prevalence of food allergy during childhood among all WAS (n = 15) and XLT (n = 10) patients compared with those reported in the general population (NHANES) and in patients with moderate to severe atopic dermatitis (AD) (right panel) (refs. and 25). (D) Concordance between sIgE measurement and SPT for foods in individuals who underwent both modalities (n = 14) (left panel). Results of SPT and sIgE testing among patients with persistent clinical food allergy (n = 4); 2 individuals with food allergy in childhood without current evidence of sensitization were excluded (right panel). (E) Percentage of positive wheal responses to SPT with morphine titration among patients with WAS mutations (n = 13) compared with sex-matched controls (n = 15), *P = 0.03 by Fisher’s exact test. UD, undefined; ND, none detected; WH1, WASP Homology domain 1; GBD, GTPase binding domain; PPP, polyproline domain; VCA, verprolin homology, cofilin homology, and acidic region domain.

Figure 2. Spontaneous sensitization to food antigens and food allergy in Was–/– mice.

(A) Comparative analysis of total sIgE and IgG1 levels in 3-month-old WT BALB/c (open circles, n = 7) and Was–/– mice (gray circles, n = 9) of mixed genders. (B) IgE and IgG1 reactivity against the 5 main (% w/w) chow components as determined by ELISA in 1:30 (IgE) or 1:1,000 (IgG1) diluted serum samples. (C) Loading of WT bone marrow–derived mast cells with serum of food-allergic (FA sens) or non–food-allergic (FA non-sens) Was–/– mice compared with no-serum control (left panel). Appearance of surface LAMP-1 as a marker of mast cell degranulation after stimulation with antigen extracts from conventional chow (CCh), elemental chow (ECh), or PBS (–). (D) Intestinal mast cell expansion as determined by chloroacetate esterase staining of jejunal cross-sections (×20, with digital magnification to ×50 shown in window) and quantification per 4 high-power fields (4hpf) in WT (n = 7) and Was–/– mice (n = 8). (E) Serum levels of mast cell protease 1 (MCPT1) determined by ELISA. (F) Effect of 7-day treatment with elemental diet on serum MCPT1 in Was–/– mice (n = 11). Spearman’s rank correlation between cumulative anti-food IgE titers of mice and response to allergen elimination defined as ΔMCPT1. (G) Effect of oral rechallenge with 12.5 mg soy protein extract on body temperature and serum MCPT1 after 4 hours. Symbols represent individual mice and error bars depict SEM. *P < 0.05, **P < 0.01, ***P < 0.001 as determined by 2-tailed Student’s t test. NS, not significant; ND, not detectable. Results in C are representative of 2 independent experiments. Equivalent results were obtained in a cohort of WT and Was–/– mice on the 129SvEv background.

Figure 3. Commensals are dispensable for spontaneous sensitization to food in Was–/– mice but shape the isotype composition of the humoral anti-food response.

(A) Comparison of total IgE and total IgG1 serum levels in 4- to 6-month-old WT (open circles, n = 5) or Was–/– (gray circles) on the 129SvEv background that were housed under either SPF (n = 10) or GF (n = 14) conditions. (B) Food-specific IgE and IgG1 for the 5 main chow constituents in IgE (serum dilution 1:100) or IgG1 (1:5,000) from SPF (n = 8) and GF (n = 7) Was–/– mice. (C) Comparison of serum MCPT1 levels. (D) Comparison of cumulative anti-food titers of IgE, IgG1, IgG2a (1:1,000), IgG2b (1:1,000), IgG3 (1:200), and IgA (1:5,000) in SPF (n = 8) and GF (n = 7) Was–/– animals. Symbols represent individual mice and error bars depict SEM. *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant as determined by 2-tailed Student’s t test. Results are shown from sera obtained from mice from ≥ 3 independent cohorts.

Figure 4. WASP deficiency in Tregs is sufficient for the development of spontaneous food allergy and results in more severe disease.

(A) Comparison of MCPT1 levels in mice with cell type–specific WASP deletions. Mice with conditional deletion of Wasfl/fl alleles in B cells (Wasfl/fl Mb1-Cre), CD11c+ dendritic cells (Wasfl/fl Itgax-Cre) or Tregs (Wasfl/fl Foxp3-Cre) of ≥ 2 months of age, n ≥ 5 per group. (B) Representative H&E (×10 magnification) and chloroacetate esterase (CAE) staining (×20 magnification) of intestinal cross-sections in Wasfl/fl Foxp3-Cre or WasWT Foxp3-Cre littermates on the C57BL/6 background. (C) Comparison of total and soy-specific IgE and IgG1 at 2 months in cohoused WT (open circles, n = 9), Was–/– (gray circles, n = 12), and Wasfl/fl Foxp3-Cre (black circles, n = 9) mice of mixed genders on the C57BL/6 background. (D) Comparison of serum protein and jejunal mRNA expression levels of mucosal mast cell marker MCPT1. Symbols represent individual mice and error bars depict SEM. *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant as determined by 2-tailed Student’s t test (A) or 1-way ANOVA with Tukey’s multiple comparisons test (C and D). BDL, below detection limit. Data in C and D are representative of 2 independent cohorts.

Figure 5. WASP deficiency in Tregs results in Th2-type small intestinal inflammation.

(A) Hierarchical cluster analysis of differentially expressed genes within a panel of 86 inflammatory targets in tissue sections obtained from jejunum of cohoused female mice. Row-normalized, log-transformed mRNA counts are shown from 4 animals per group. (B) Bar graph representation and statistical analysis of mRNA expression of Il4, Ifng, and Il17a in jejunal sections as shown in A. Symbols represent individual mice and error bars depict SEM. **P < 0.01. NS, not significant as determined by 1-way ANOVA with Tukey’s multiple comparisons test.

Figure 6. WASP-deficient FOXP3+ Tregs fail to suppress Th2-type lymphoproliferation in vivo.

(A) Quantification by flow cytometry of FOXP3+ Tregs amongst CD4+ T cells obtained from MLNs or PPs of WT (open circles, n = 5), Was–/– (gray circles, n = 6) and Wasfl/fl Foxp3-Cre (black circles, n = 4) mice. (B) Production of IL-2 by CD4+ mesenteric T lymphocytes stimulated with anti-CD3/CD28 ex vivo. Each dot represents the average cytokine production from triplicate cell suspensions from a single mouse. (C) Total CD4+ T cell numbers obtained from MLNs and PPs. (D) Gating strategy of GATA3+ICOS+ Th2-type effector cells within the parent gate of effector memory T cells from MLNs of representative samples, with quantification and statistical testing in the right panels. (E) Fraction of T-bet+ and RORγt+ effector memory T cells. (F) Production of IL-4, IL-13, IFN-γ and IL-17a by CD4+ mesenteric T lymphocytes stimulated with anti-CD3/CD28 ex vivo. Each data point represents the average cytokine production from triplicate cell suspensions from a single mouse. (G) Serum levels of anti-soy specific IgE and IgG1, and MCPT1 in Was–/– mice on the 129SvEv background with either Il4+/+ (gray circles, n = 10 or 15) or Il4–/– alleles (gray squares, n = 8). (H) Anti-soy IgG2b titer as determined by ELISA in 1:1,000 serum dilution. Symbols represent individual mice and error bars depict SEM. *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant as determined by 2-tailed Student’s t test or 1-way ANOVA with Tukey’s multiple comparisons test. In B and F, data were log-transformed prior to statistical testing. Data from 2 pooled experiments (C, G, and H) or representative results from ≥ 2 independent experiments (A, B, and D–F) are shown.

Figure 7. WASP-deficient effector Tregs assume a Th2-like phenotype in mice and human patients.

(A) Fraction of CD44hiCD62LloCD4+FOXP3+ Tregs that coexpress GATA3 and ICOS in WT (open circles, n = 4), Was–/– (gray circles, n = 6) and Wasfl/fl Foxp3-Cre (black circles, n = 5) mice, with gating strategy in representative samples depicted on the right. (B) Intracellular GATA3 levels in CD44loCD62LhiCD4+FOXP3+ Tregs determined by mean fluorescence intensity (MFI) using flow cytometry. (C) GATA3+ICOS+ effector T cells in WT or Was–/– mice with either Il4+/+ (gray circles, n = 4) or Il4–/– alleles (gray squares, n = 3) and IL-4 production by CD4+ mesenteric lymphocytes stimulated with anti-CD3/CD28 ex vivo. Each data point represents the average cytokine production from triplicate cell suspensions from a single mouse. (D) Percentage of FOXP3+ Tregs coexpressing GATA3 and ICOS, T-bet, or RORgt in WT or Was–/– mice with either Il4+/+ or Il4–/– alleles. (E) Gating strategy and cumulative data of percentage of total effector (CD45RO+) Tregs coexpressing GATA3 in isolated PBMCs from XLT (n = 10) and WAS (n = 11) patients compared with age- and sex-matched controls (n = 10) or WAS patients following HSCT (n = 3). Data points represent individual mice or patients and error bars depict SEM. *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant as determined by 1-way ANOVA with Tukey’s multiple comparisons test. Mouse experiments representative of 2 independent experiments. Results in E are cumulative data from 1 experiment.