An asthma-associated IL4R variant exacerbates airway inflammation by promoting conversion of regulatory T cells to TH17-like cells

Amir Hossein Massoud, Louis-Marie Charbonnier, David Lopez, Matteo Pellegrini, Wanda Phipatanakul, Talal A Chatila, Amir Hossein Massoud, Louis-Marie Charbonnier, David Lopez, Matteo Pellegrini, Wanda Phipatanakul, Talal A Chatila

Abstract

Mechanisms by which regulatory T (Treg) cells fail to control inflammation in asthma remain poorly understood. We show that a severe asthma-associated polymorphism in the gene encoding the interleukin (IL)-4 receptor alpha chain (Il4ra(R576)) promotes conversion of induced Treg (iTreg) cells toward a T helper 17 (TH17) cell fate. This skewing is mediated by the recruitment by IL-4Rα(R576) of the growth-factor-receptor-bound protein 2 (GRB2) adaptor protein, which drives IL-17 expression by activating a pathway that involves extracellular-signal-regulated kinase, IL-6 and the transcription factor STAT3. Treg cell-specific deletion of genes that regulate TH17 cell differentiation, including Il6ra and RAR-related orphan receptor gamma (Rorc), but not of Il4 or Il13, prevented exacerbated airway inflammation in mice expressing Il4ra(R576) (hereafter referred to as Il4ra(R576) mice). Furthermore, treatment of Il4ra(R576) mice with a neutralizing IL-6-specific antibody prevented iTreg cell reprogramming into TH17-like cells and protected against severe airway inflammation. These findings identify a previously unknown mechanism for the development of mixed TH2-TH17 cell inflammation in genetically prone individuals and point to interventions that stabilize iTreg cells as potentially effective therapeutic strategies.

Figures

Figure 1
Figure 1
Il4raR576 polymorphism promotes enhanced lung inflammation and AHR, associated with increased IL-17, IL-6 and IL-13 expression. (a) Representative PAS staining of lung sections isolated from WT and Il4raR576 mice either sham treated with PBS or immunized and challenged with HDM and analyzed 24 hr after the last challenge (20×, scale bar 50 μm) (n = 11 counts per field-of-view). (b,c) Inflammation and PAS scores from the group of mice shown in a. (d) Methacholine–induced AHR for the same groups shown in a (n = 8 mice per group). (e) Absolute numbers of lung tissue eosinophils, neutrophils and lymphocytes, in the respective mouse groups (n = 5 mice for PBS and 7 mice for HDM groups). (f) Flow cytometric analysis, frequencies and absolute numbers of CD4+Foxp3+ Treg cells within lung tissue (n = 8 mice per group). (g–i) Flow cytometric analysis of IL-13 (g), IL-17 (h) and IL-6 (i) expression by CD4+Foxp3− Tconv or CD4+Foxp3+ Treg cells within CD90.2+ gated cells (representing all T lymphocytes) in lung tissues of WT and Il4raR576 mice treated with PBS or HDM (n = 5 mice for PBS and 7 mice for HDM groups). Results represent means ± s.e.m. from two independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 by one-way ANOVA with Bonferroni posttest analysis. For AHR analysis, *P < 0.05 and **P < 0.01 by two-way repeated measures ANOVA.
Figure 2
Figure 2
Defective formation and impaired suppressive function of Il4raR576 induced-Treg cells. (a,b) Flow cytometric plot (a) and bar graph (b) demonstrating in vitro generation of iTreg cells form DO11.10+Rag2−/−Foxp3EGFP and DO11.10+Il4raR576Rag2−/−Foxp3EGFP naïve CD4+ Tconv cells in the presence of anti-CD3 and anti–CD28 mAbs and TGF β1 in the absence or presence of IL-4 for 5 d (n = 6 replicates per group). (c,d) Flow cytometric analysis of IL-17 and IL-13 expression by converted Foxp3+ iTreg cells (c) and CD4+Foxp3− Tconv cells (d) in culture. (e,f) Bar graphs demonstrating the frequencies of converted Foxp3+ iTreg and CD4+Foxp3− Tconv cells IL-17 and RORγt (e) and IL-13 and GATA3 expression (f) (n = 6 replicates for IL-17 and IL-13 and 6 replicates for RORγt and GATA3 expression). (g) Flow cytometric analysis of dual IL-6 and IL-17 expression by converted iTreg cells. (h) Bar graph demonstrating the frequencies of double IL-6 and IL-17 expression within converted iTreg cells (n = 6 replicates per group). Each dot represents one replicate. Data represent means ± s.e.m. from two independent experiments. ***P < 0.001 by one-way ANOVA with Bonferroni posttest analysis.
Figure 3
Figure 3
CCR6+ IL-17+Il4R576 Treg cells exhibit instability and compromised suppressive activity (a) Methylation status of CpG motifs in Treg cells isolated from lung tissue of OVA–sensitized and challenged mice. Numbers on the left side indicate the position of the respective motifs. (b) Global methylation status of Foxp3 CNS2 in the respective Treg cell populations (n = 3 mice per group with 7-12 clones per mouse). (c) In vitro suppression of the proliferation of WT responder CD4+ T cells (Teff) by the respective Treg cell populations (n = 3 replicates per group) (d) Gene expression profiles (volcano plot) of EGFP+CCR6− versus EGFP+CCR6+ Treg cells isolated by FACS from lung digests of OVA-sensitized and challenged Foxp3EGFPIl4R576 mice (n = 3–4 mice). FDR: false discovery rate; Log2FC: Log2 fold change. (e) Flow cytometric analysis and frequencies of exTreg (GFP−YFP+) cells, plotted as a fraction of exTreg to total Treg cells in lung tissue. (f,g) Flow cytometric analysis and frequencies of CCR6 producing (f) and IL-17 and IL-13 producing (g) exTreg cells in lung tissues. (h) Flow cytometric analysis and frequencies of exTreg and Treg cells among CD4+IL-17+ Tconv cells in lung tissues of the respective mouse groups (n = 6 mice for PBS- and 9 mice for OVA-treated groups for e–h). Data represent means ± s.e.m. from two independent experiments. *P < 0.05, **P < 0.005 and ****P < 0.0001 by one-way ANOVA with Bonferroni posttest analysis. For suppression assay ****P < 0.0001 by repeated measures two-way ANOVA.
Figure 4
Figure 4
IL-4Rα-R576 activates GRB2–coupled MAPK. (a) Immunoprecipitates (IP) from medium and IL-4-treated WT and Il4raR576 splenocytes using IgG or anti–IL4Rα mAb were probed with anti-GRB2, -GAB2, and -IL-4Rα mAbs. Lys: lysates (b) Binding of GRB2 and STAT6 in splenocyte lysates to biotinylated murine IL-4Rα Y575Q576 (YQ), pY575Q576 (pYQ) and pY575R576 (pYR) peptides. Immunoblotted proteins (upper panel) were quantified by densitometry (lower panel). (c) Immunoblots of phospho- and total ERK1/2 and p38, and of GRB2 in medium or IL-4–treated WT and Il4raR576 BMDM transfected with scrambled or GRB2–specific siRNA. (d) RT-PCR analysis of Il6 transcripts in the same groups as c (e) Il6 transcripts in splenocytes treated with medium or IL-4 and the indicated concentrations of MEK-Inh. (f) ChIP analysis of C/EBP-β, NF-κB and AP-1 binding at the Il6 promoter in medium (Un-Stim) or IL-4–treated WT and Il4raR576 splenocytes. (g) Flow cytomeric analysis (left) and mean fluorescence intensity (MFI, right) of pSTAT3 in WT and Il4raR576 iTreg cells differentiated with anti–CD3, anti–C28 mAbs and TGF β1 without or with IL-4, anti-IL-4 or anti–IL-6 mAbs (n = 3–6 replicates per group for b–g). (h–j) Frequencies of iTreg cells (h) IL-17 and IL-13 expressing Foxp3+ iTreg cells (i) and CD4+Foxp3− Tconv cells (j) treated with the indicated combinations of cytokines, inhibitors and mAbs (n = 3–6 replicates per group). Data represent means ± s.e.m. from 2–3 independent experiments. *P < 0.05, ***P < 0.001 and ****P < 0.0001 by one-way ANOVA with Bonferroni posttest analysis.
Figure 5
Figure 5
Naïve CD4+ Tconv cells from asthmatic subjects bearing R576 mutation show defective induction of iTreg cells and their skewing towards a TH17-like phenotype. (a) Flow cytometric analysis of CD4+Foxp3+ iTreg cells induced from purified naïve CD4+ Tconv in the presence of anti-CD3 + anti-CD28 mAbs and TGF β1 + IL-4 in the absence or presence of anti-IL-6 mAb. (b,c) Cumulative frequencies of CD4+Foxp3+ iTreg cells and IL-17 and IL-13 producing Foxp3+ iTreg cells within converted iTreg cells of non-asthmatic (n = 3–13) (b) and asthmatic (n = 4–19) (c) subjects with respective genotypes differentiated in the absence or presence of TGF β1 or TGF β1 + IL-4 or TGF β1 + IL-4 + anti-IL-6 mAb. (d) IL-17 and IL-13 cytokine levels in the supernatant of differentiated iTreg cells in the treated groups mentioned in b (n = 2–5). (e-g) Frequencies of total CD4+Foxp3+ Treg cells (n = 5–14) (e) CD4+Foxp3+Helioslow and Foxp3+CCR6+Helioslow Treg cells (n = 4) (f) and frequencies of IL-17 and IL-13 producing Foxp3+ Treg and CD4+ Tconv cells in respective groups within PBMC of asthmatic subjects (n = 4–14). Each dot represents one subject. Data represent means ± s.e.m. For subjects see Supplementary Tables 2 and 3. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P <0.001 by one-way ANOVA with Bonferroni posttest analysis.
Figure 6
Figure 6
Treg cell lineage-specific deletion of Rorc reverses the aggravated airway inflammation in Il4raR576 mice. (a) PAS staining of lung sections isolated from the mouse groups shown and immunized and challenged with OVA (20×, scale bar 50 μm). (b) Inflammation and PAS scores in the mouse groups shown (n = 8–9 counts per field of view). (c) Methacholine–induced AHR in the respective mouse groups (n = 4 mice for PBS- and 6 mice for OVA–treated mouse groups). (d) Total and OVA–specific serum IgE concentrations in the mouse groups shown in b (n = 8). (e) Frequencies of lung tissue eosinophils, neutrophils and lymphocytes (n = 8 mice for PBS- and 9 mice for OVA-treated mouse groups). (f–i) Frequencies of lung tissue Foxp3+ Treg cells (f), and IL-17 (g), CCR6 (h) and IL-13 (i) producing CD4+ Tconv and Foxp3+ Treg and CD4+ Tconv cells (n = 5–9 mice per group). Each dots represents one animal. Data represent means ± s.e.m. from two to three independent experiments. *P <0.05, **P <0.01, ***P <0.001 and ****P <0.0001 by one-way ANOVA with Bonferroni posttest analysis. For AHR studies, *P < 0.05 and **P < 0.01 by repeated measures two-way ANOVA.

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