A Common IL-4 Receptor Variant Promotes Asthma Severity via a T_reg Cell GRB2-IL-6-Notch4 Circuit

Association of the IL4R⁵⁷⁶ allele with asthma diagnosis and disease severity.

The IL4R⁵⁷⁶ allele has previously been implicated in asthma severity and is highly prevalent in African Americans (12, 15, 16, 23). To investigate the relationship between genotype and asthma severity we studied a group of 184 children and young adults, including 132 subjects with asthma and 52 non-atopic controls. Age ranged from 1 to 21 years (mean 8.6 ± 3.8 years), while gender distribution showed a preponderance of males (n=105, 57.1%) vs females (n=79, 42.9%)(Table S1). Participants were recruited in the Allergy clinic at Boston Children’s Hospital and from inner city cohort populations (23, 24). Informed consent was obtained from adult participants and from legal guardians of minor subjects. Asthma severity was assigned based on EPR3 criteria and medication use. Asthmatic patients were grouped as having either intermittent/mild persistent asthma (n=78) or moderate/severe persistent asthma (n=54). Based on genotyping at the IL4R 576 codon, subjects were categorized as being either homozygous for the dominant allele (Q/Q, n=67, 36.4%), heterozygous (R/Q, n= 69, 37.5%) or homozygous variant allele (R/R, n= 48, 26.1%). The demographic and clinical characteristics of asthmatic and control subjects and the ethnicity distribution among each individual genotype are summarized in Table S1 and Figure S1. Among asthmatic individuals, allergic rhinitis with evidence of sensitization to aeroallergens (including tree pollen, grass pollen, weed pollen, animal dander, dust mites, mold) by either skin prick testing and/or specific IgE was present in 75.2% (98/125) individuals with available data (missing n=7), while 31.8% (42/132) of asthmatic subjects carried a diagnosis of food allergy based on history of clinical reaction and positive IgE testing to the culprit food. Distribution of individual allergies is shown in Table S1, with most children suffering from multiple food and/or environmental allergies. Total IgE and circulating eosinophil data were available for 80 and 92 asthmatic children, respectively. Mean total IgE level was 825.2 kU/L (range 2–7491), with 65% (52/80) of children manifesting elevated IgE levels for age. Mean circulating eosinophil count was 334.5/mcL (range 0–1250), with age-adjusted peripheral eosinophilia detected in 47.8% (44/92) of subjects (Table S1). Taken altogether, these data suggest a high prevalence of allergic, Th2-driven asthma among our study subjects, consistent with previous reports of asthma in pediatric populations. In our population, individuals carrying either one (IL4R^Q576/R576) or two copies of the variant allele (IL4R^R576/R576) were more likely to have a diagnosis of asthma (OR 1.98, 95% C.I. 1.04–3.82) (Table S2). Furthermore, among Hispanic individuals with asthma, those who were either heterozygous or homozygous for the variant allele were more likely to present with moderate/severe asthma as compared to individuals who were homozygous for the WT allele (OR 5.76, 95% C.I. 1.30–25.51) (Table S3). No significant association was detected between genotype and asthma severity among Caucasian or African American asthmatics, suggesting that a possible contribution of the genotype at the IL4R⁵⁷⁶ allele to asthma severity may be at least in part dependent on the individual’s ethnic background.

IL4R^R576 Asthmatics exhibit increased T_H2 and T_H17 cytokine expression in their T_eff and T_reg cells.

We analyzed the cytokine expression profiles of peripheral blood T_eff and T_reg cells as a function of IL4R^Q576/Q576 (IL4R^Q576) versus IL4R^Q576/R576 and IL4R^R576/R576 (IL4R^R576) genotypes and disease severity in the asthmatic and control subjects. Results revealed that patients either heterozygous or homozygous for IL4R^R576 allele expressed higher levels of Th2 cytokines and markers (CRTH2) in their circulating T_reg and T_eff cells that progressively increased in relation with asthma severity (Fig. 1, A–B). They also present a higher levels of Th17 cytokines and markers (CCR6) (Fig. 1, C–D).

We have previously established that the IL-4RαR576 variant promotes asthma severity and dysregulates Th2 and Th17 responses by destabilizing lung tissue induced T_reg cells in an IL-6-dependent manner. Moreover, IL-6 promotes NOTCH4 expression on lung tissue induced T_reg cell to increased asthma severity through a T_reg cells destabilization. To establish the relationship between the IL-4RαR576 variant and NOTCH4, we analyzed by flow cytometry the expression of NOTCH4 on circulating T_reg cells of human subjects with asthma as a function of IL4R^R576 allele carriage and disease severity. NOTCH4 expression was increased on circulating T_reg cells of human asthmatics as a function of both disease severity and IL4R^R576 allele carriage (Fig. 1E; see Fig. S2 for gating strategy). The IL4R^R576 allele was associated with increased NOTCH4 expression on T_reg cells of asthmatic subjects in a gene dose-dependent manner (Fig. 1F). Other Notch receptors are not upregulated in T_reg or T_eff as a function of either disease severity and IL4R^R576 allele carriage (Fig. S3). Consistent with the promotion by the IL4RαR576 variant of IL-6 production and the dependency of NOTCH4 expression in T_reg cells on IL-6 signaling^16,22, we found a positive correlation between serum IL-6 levels and Notch4 expression in asthmatics that segregated as a function of IL4R^R576 allele carriage and disease severity (Fig. 1, G and H). These results established the upregulation of the IL-6-Notch4 axis in IL4R^R576+ asthmatics in direct correlation with disease severity.

Increased Notch4 expression on T_reg cells underlies heightened airway inflammation in Il4ra^R576 mice.

To further decipher the functional impact of Notch4⁺T_reg cells in the exacerbated asthmatic inflammation imparted by the IL4R^R576 allele, we employed an allergic airway inflammation mouse model using the previously described Il4ra^R576 mice, whose interleukin 4 receptor alpha chain gene (Il4ra) encodes the R576 substitution at the same position as the human protein variant^15,16. We further examined the impact of T_reg cell-specific deletion of Notch4 in these mice on their allergic airway inflammatory response using a floxed Notch4 allele and a Cre recombinase driven by the Foxp3 gene (Foxp3^YFPCre). Mice were sensitized with chicken egg ovalbumin (OVA) and then challenged with aerosolized OVA either without or together with intranasal treatment with Traffic-related ultrafine pollutant particles (UFP), as described^17,21,22. In agreement with our previous results, the Il4ra^R576Foxp3^YFPCre mice showed increased airway hyperresponsiveness (AHR) and exacerbated tissue inflammation scores upon OVA sensitization and challenge as compared to Foxp3^YFPCre control mice. Both responses were further augmented by UFP co-treatment during the challenge phase (Fig. 2, A – C, Fig.S4). T_reg cell-specific deletion of Notch4 profoundly inhibited both AHR and tissue inflammation down to similar levels in both set of mice (Il4ra^R576Foxp3^YFPCreNotch4^Δ/Δ and control Foxp3^YFPCreNotch4^Δ/Δ), indicating that the augmentation of AHR and allergic lung inflammation by the Il4ra^R576 allele was Notch4-dependent (Fig. 2, A – C). Consistent with these results, flow cytometric analysis confirmed that allergic airway inflammation was associated with upregulation of Notch4 expression on T_reg cell as a function of disease severity, which was completely abrogated upon Cre-mediated deletion of Notch4. (Fig. 2D, E). In addition to suppressing AHR and tissue inflammation, Notch4 deletion in T_reg cells also suppressed the OVA-specific IgE response, lung tissue eosinophilia and CD4⁺ T cell lymphocytosis, and Th2 and Th17 cell cytokine expression (Fig. 2, D–H).

The above results were reproduced in a separate set of studies that employed house dust mite (HDM) as an inciting allergen in airway inflammation. Similar to the case with OVA, HDM-induced airway hyperresponsiveness and tissue inflammation, eosinophilia and Th2 and Th17 cell cytokine production were all upregulated in the Il4ra^R576Foxp3^YFPCre mice in a T_reg cell Notch4-dependent manner (Fig. S5). Together, these findings confirmed the crucial role played by Notch4 expression on T_reg cells in the augmented allergic airway inflammation induced by the IL-4RαR576 variant.

To determine whether acute Notch4 inhibition would rescue disease phenotype, we examined the capacity of therapy with a blocking Notch4 antibody to inhibit OVA-induced allergic airway inflammation in Il4ra^R576Foxp3^YFPCre mice. Results showed that treatment with the anti-Notch4 antibody at the time of allergen sensitization and challenge suppressed both OVA and OVA+UFP induced allergic airway inflammatory responses to levels approaching those achieved with the T_reg cell specific Notch4 deletion. Parameters inhibited by the antibody therapy included AHR (Fig. 3, A and B), OVA-specific IgE (Fig. 3C) CD4⁺ T cell lymphocytosis, tissue eosinophilia and neutrophilia (Fig. 3, D – F) and tissue Th cell infiltration (Fig.3, G and H).

T_reg cell-specific Grb2 deletion abrogates the augmentation of allergic airway inflammation by Il4ra^R576.

Our previous studies have demonstrated that the IL-4RαR576 variant uniquely recruits the intracellular signaling intermediate Grb2 to mediate microtubule associated protein kinase (MAPK) activation and IL-6 production¹⁶. This process was incriminated in corrupting lung tissue allergen-specific induced T_reg cell formation towards a Th17 fate. Accordingly, we examined the capacity of T_reg cell specific deletion of Grb2 to reverse the augmentation in allergic airway inflammation induced in Il4ra^R576 mice. Analysis revealed that Il4ra^R576 mice whose T_reg cells lacked GRB2 (Il4ra^R576Foxp3^YFPCreGrb2^Δ/Δ) failed to exhibit the expected augmentation in attributes of airway inflammatory normally associated with the Il4ra^R576 allele upon sensitization and challenge with HDM. Measures including AHR, total IgE, and tissue eosinophilia and neutrophilia were all normalized to levels achieved in similarly treated Grb2-sufficient control mice bearing the wild-type Il4ra^Q576 allele (Foxp3^YFPCre) (Fig. 4, A–F). Importantly, deletion of Grb2 in control mice bearing the wild-type Il4ra^Q576 allele (Foxp3^YFPCreGrb2^Δ/Δ) did not affect the allergic airway inflammatory response induced by HDM as compared to Grb2-sufficient Foxp3^YFPCre mice. Further analysis revealed that Grb2 deletion completely abrogated the augmentation in Th17, cell responses associated with the Il4ra^R576 allele (Fig. 4G–H). Grb2 deletion suppressed the increased in IgE production and eosinophilia associated with the Il4ra^R576 allele but had a more modest impact on the Th2 response, suggesting additional mechanisms mobilized by IL-4RαR576 variant in promoting Th2 skewing.

The upregulation of Notch4 signaling in Il4ra^R576 T_reg cells is Grb2-dependent.

To determine the role of the IL-4RαR576-coupled Grb2 pathway in the upregulation of Notch4 signaling in Il4ra^R576 T_reg cells, we analyzed Notch4 expression in Grb2-sufficient and deficient lung T_reg cells of mice bearing the WT Il4ra allele (Foxp3^YFPCre and Foxp3^YFPCreGrb2^Δ/Δ, respectively), compared to those of Il4ra^R576 mice (Il4ra^R576Foxp3^YFPCre and Il4ra^R576Foxp3^YFPCreGrb2^Δ/Δ, respectively). The respective mouse groups were subjected to HDM-induced allergic airway inflammation, as shown in Fig. 4, and their lung T_reg cells were analyzed for Notch4 and IL-6 expression. The T_reg cells were also analyzed for the expression of the Notch4 downstream mediators Yap1 and β-catenin, effector proteins of the Hippo and Wnt pathways respectively, which control the lung tissue Th17 and Th2 cell responses²². Results showed that the increased Notch4 expression in the lung T_reg cells of Il4ra^R576Foxp3^YFPCre mice was completely abrogated upon T_reg cell-specific deletion of Grb2, both at baseline and especially after HDM sensitization and challenge (Fig. 5A; see Fig. S6 for gating strategy).

In view of our previous demonstration that Notch4 induction on lung T_reg cells proceeds by an IL-6-dependent mechanism²², we analyzed IL-6 production in Grb2-sufficient and deficient T_reg cells of sham and HDM-treated mice bearing the WT and Il4ra^R576 allele. T_reg cell-specific Grb2 deletion abrogated the increase in IL-6 production mediated by the IL-4RαR576 variant¹⁶, consistent with a mechanistic autocrine loop involving Grb2-dependent IL-6 production mediating the super-induction of Notch4 on the T_reg cells of Il4ra^R576 mice (Fig. 5B). The role of IL-6 in the upreglation of Notch4 by IL-4RαR576 variant was further established using mice with T_reg cell-specific deletion of Il6ra, encoding the IL-6 receptor alpha chain. Il6ra deletion in T_reg cells inhibited Notch4 expression in both control and Il4ra^R576 mice (Foxp3^YFPCreIl6ra^Δ/Δ and Il4ra^R576Foxp3^YFPCreIl6ra^Δ/Δ, respectively) and abrogated differential disease severity in the latter mice (Fig. S7A–H). T_reg cell-specific Grb2 deletion also abrogated the super-induction of the Notch4 downstream mediators Yap1 and β-catenin in the T_reg cells of HDM-treated Il4ra^R576Foxp3^YFPCre mice (Fig. 5, C and D). These results are in agreement with the abrogation in HDM-treated Il4ra^R576Foxp3^YFPCreGrb2^Δ/Δ mice of the augmented Th2 and Th17 cells responses typically associated with the Il4ra^R576 allele. Together, these results indicated that the destabilization of lung T_reg cells upon signaling via IL-4RαR576 variant in allergic airway inflammation proceeded by a Grb2-IL-6-Notch4 circuit.

Human subjects.

Asthmatic children and young adults and age matched control subjects were recruited in the Allergy clinic at Boston Children’s Hospital and from previously described inner city cohort populations^23,24. Demographics, including age, self identified ethnicity and gender, and clinical information were obtained from the subjects’ clinical electronic records. Informed consent was obtained from adult participants and from legal guardians of minor subjects.

Mice.

The following mouse strains were obtained from the JAX Laboratories: Foxp3^YFPCre (B6.129(Cg)-Foxp3^{tm4(YFP/icre)Ayr}/J)³⁴, Il4ra^R576 (C.129X1-Il4ra^tm2Tch/J)¹⁵, floxed Il6ra (Il6ra^fl/fl; B6;SJL-Il6ratm1.1Drew/J) floxed Grb2 (Grb2^fl/fl; B6.C(Cg)-Grb2^tm1.1Lnit/J)³⁵ and floxed Notch4 (Notch4^fl/fl; Notch4^{tm1c(NCOM)Mfgc}) were obtained from the Canadian mutant mouse repository. Foxp3^YFPCreIl6ra^Δ/Δ, Il4ra^R576Foxp3^YFPCreIl6ra^Δ/Δ, Foxp3^YFPCreNotch4^Δ/Δ, Il4ra^R576Foxp3^YFPCreNotch4^Δ/Δ, Foxp3^YFPCreGrb2^Δ/Δ and Il4ra^R576Foxp3^YFPCreGrb2^Δ/Δ were generated by crossing the respective component strains. Mice were maintained in at Boston Children’s Hospital Animal facility under breeding and research protocols approved by the institutional animal care and use committee (IACUC). All experiments were approved by Boston Children’s Hospital IACUC

Particles.

UFP (≤0.18 μm) were collected in an urban area of downtown Los Angeles, as previously reported²¹. The respective particles were suspended in an aqueous solution, with the hydrophilic components becoming part of the solution, while the solid non-soluble UFP cores are left in suspension. The entire mixture was administered intranasally, as indicated below.

Isolation of Human peripheral blood mononuclear cells (PBMCs).

Human PBMCs were isolated from full blood from either healthy control, mild asthmatics, moderate asthmatics or severe asthmatics probands via density gradient using Ficoll (GE Healthcare). PBMCs were then stored frozen in Fetal Calf Serum (FCS) (Sigma Aldrich) and 15% Dimethyl sulfoxide (DMSO) (Sigma Aldrich). The cells were later thawed for flow cytometry analysis. For intracellular cytokine staining, cells were stimulated with PMA (100 ng/ml) and ionomycin (1 μg/ml) for 4 hours in the presence of GolgiPlug monensin (1 μg/ml, BD Biosciences) at 37°C in a humidified, 5% CO₂ atmosphere.

IL-6 ELISA.

EDTA Plasma from 88 probands (Healthy controls, mild, moderate and severe persistent asthma patients) were used to measure IL-6 using enzyme-linked immunosorbent assay (ELISA) (Invitrogen) according to manufacturer’s protocol.

Allergic sensitization and challenge.

Mice were sensitized to OVA by intraperitoneal (i.p.) injection of 100 μg OVA in 100 μl PBS, then boosted two weeks later with a second i.p. injection of OVA in PBS. Control mice were sham sensitized and boosted with PBS alone. Starting on day 29, both OVA and sham-sensitized mice were challenged with aerosolized OVA at 1%, for 30 minutes daily for 3 days. Two hours before each OVA aerosol exposure, subgroups of mice were given intranasally (i.n.) either PBS or UFP at 10 μg/100μl PBS/instillation. Mice were euthanized on day 32 post sensitization and analyzed. For dust mite-induced allergic airway inflammation, mice received 5 μg of lyophilized D. Pteronyssinus extract (Greer) in 100 μl PBS intranasally for 3 days at the start of the protocol then challenged with the same dose of D. Pteronyssinus extract on days 15–17 with or without UFP at the same concentration as before. Mice were euthanized on day 18 and analyzed for measures of airway inflammation. Bronchoalveolar lavage (BAL) fluid and lung tissues were obtained and analyzed for cellular components and T cell cytokine expression as described¹⁶

Measurement of airway functional responses.

Allergen-induced airway hyperreactivity (AHR) was measured, as previously described¹⁷. Anesthetized mice were exposed to doubling concentrations of aerosolized acetyl-β-methacholine (Sigma-Aldrich) by using a Buxco small-animal ventilator (Data Sciences International). The relative peak airway resistance for each methacholine dose, normalized to the saline baseline, was calculated.

Lung histopathology staining.

Paraffin-embedded lung sections were stained with hematoxylin and eosin (H&E) or Paraffin-acid-Schiff staining (PAS). The lung pathology was scored by blinded operators. Inflammation was scored separately for cellular infiltration around blood vessels and airways: 0, no infiltrates; 1, few inflammatory cells; 2, a ring of inflammatory cells 1 cell layer deep; 3, a ring of inflammatory cells 2–4 cells deep; 4, a ring of inflammatory cells of >4 cells deep¹⁵. A composite score was determined by the adding the inflammatory scores for both vessels and airways.

Anti-Notch4 antibody treatment.

The anti Notch4 neutralizing antibodies (InVivoMAb anti-mouse Notch4, HMN4-14) were introduced at the concentration of 10 ug/ml of mAb i.P. with a final volume of 100μl two hours prior to sensitizations or challenge on days 1, 14, and 26,27,28 respectively. On day 30, the mice were analyzed for airway hyperresponsiveness and then sacrificed for further analysis.

Flow cytometric analysis of mouse and human cells.

Antibodies against the following murine antigens were used for flow cytometric analyses: IL-4 (clone 11B11, 1:300 dilution), Siglec-F (E50-2440, 1:300), Foxp3 (FJK-16S, 1:300), IFN-γ (XMG1.2, 1:300), IL-13 (eBio13a, 1:300), CD11c (30-f11, 1:500), CD11b (M1/70, 1:500) (eBioscience), CD4 (RM4-5, 1:500), CD3 (145-2C11, 1:500), IL-17 (TC11-18H10.1, 1:200), GR-1 (RB6-8C5, 1:500), CD45 (30-F11, 1:300), Notch4 (HMN4-14 1:200) (Biolegend), IL-6 (MP5-20F3, 1:200, Biolegend), Yap1 (147295 1:200) and β-Catenin (196624, 1:500). Antibodies against the following human antigens were used: CD3 (HIT3a 1:300), CD4 (RPA-T4, 1:300), Foxp3 (236A/E7, 1:200), Notch1 (HMN1-519 1:100), Notch2 (HMN2-25 1:100), Notch3 (HMN3-21 1:100), Notch4 (HMN4-2 1:50), IL-4 (MP4-25D2, 1:250), IL-13 (JES10-5A2, 1:200), IL-17 (BL168, 1:200), CCR6 (G034E3, 1:250), CRTH2 (BM16, 1:300), CD127 (A019D5, 1:200) (Biolegend) The specificity and optimal dilution of each antibody was validated by testing on appropriate negative and positive controls or otherwise provided on the manufacturer’s website. Intracellular cytokine staining was performed as previously described³⁶. Cytokines were stained overnight as previously prescribed in³⁶. Dead cells were routinely excluded from the analysis based on the staining of eFluor 780 Fixable Viability Dye (1:1000 dilution) (eBioscience). Stained cells were analyzed on a BD LSR Fortessa cell analyzer (BD Biosciences) and data were processed using Flowjo (Tree Star Inc.).

Statistical analysis.

Logistic regression and Chi-square statistics were used to investigate the independent effect of race and genotype on asthma severity. Student’s two-tailed t-test, one- and two-way ANOVA and repeat measures two-way ANOVA with Sidak post-test analysis of groups were used to compare test groups, as indicated. A p-value <0.05 was considered statistically significant.

Study approval.

Recruitment of human subjects was approved by the Institutional Review Board at Boston Children’s Hospital. All animal studies were reviewed and approved by the Boston Children’s Hospital office of Animal Care Resources.