TGF-β1 Drives Inflammatory Th Cell But Not Treg Cell Compartment Upon Allergen Exposure

Abstract

TGF-β1 is known to have a pro-inflammatory impact by inducing Th9 and Th17 cells, while it also induces anti-inflammatory Treg cells (Tregs). In the context of allergic airway inflammation (AAI) its dual role can be of critical importance in influencing the outcome of the disease. Here we demonstrate that TGF-β is a major player in AAI by driving effector T cells, while Tregs differentiate independently. Induction of experimental AAI and airway hyperreactivity in a mouse model with inducible genetic ablation of the gene encoding for TGFβ-receptor 2 (Tgfbr2) on CD4⁺T cells significantly reduced the disease phenotype. Further, it blocked the induction of pro-inflammatory T cell frequencies (Th2, Th9, Th17), but increased Treg cells. To translate these findings into a human clinically relevant context, Th2, Th9 and Treg cells were quantified both locally in induced sputum and systemically in blood of allergic rhinitis and asthma patients with or without allergen-specific immunotherapy (AIT). Natural allergen exposure induced local and systemic Th2, Th9, and reduced Tregs cells, while therapeutic allergen exposure by AIT suppressed Th2 and Th9 cell frequencies along with TGF-β and IL-9 secretion. Altogether, these findings support that neutralization of TGF-β represents a viable therapeutic option in allergy and asthma, not posing the risk of immune dysregulation by impacting Tregs cells.

Keywords: TGF-beta; Th17; Th2; Th9; allergen-specific immunotherapy; asthma; induced sputum.

Figure 1

Impact of Tgfbr2 ablation on lung inflammatory infiltrate and AHR. (A) Scheme of AAI induction, challenge and tamoxifen treatment. (B) Airway hyperreactivity measured in intubated, mechanically ventilated animals following methacholine provocation. n= 5 - 9/group; **p < 0.01; ***p < 0.001 vs WT+PBS; ^##p < 0.01 vs WT+OVA at same methacholine concentrations (two-way analysis of variance (ANOVA) with Bonferroni’s post-hoc test). (C) BAL total cell number and (D) relative population size of mice from the four experimental groups. (E) PAS-staining of lung sections from the four experimental groups. Arrows: inflammatory infiltrate; arrowheads: mucus hypersecretion; scale bar: 50 µm. (F, G) Levels of total and OVA-specific Immunoglobulin E (tIgE: n=11-16/group; OVA-sIgE: n=5/group) measured in plasma samples (two-tailed Mann-Whitney U test). (H, I) Histological scores of (H) Inflammatory cell infiltrates and (I) mucus production (mean ± SD; n=5/group). (C, I) Representative of three independent experiments each with 5 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, nd, not detected.

Figure 2

Impact of Tgfbr2 ablation on lung cytokine production and T cell subsets. (A) BAL fluid of the indicated experimental groups was analyzed using electrochemiluminescence assay. Each data point represents an individual mouse. Data is compiled from one or two independent experiments (n=5-15/group). *p<0.05, **p<0.01, n.s. not significant (t-test). (B) Flow cytometric analysis of lungs focusing on distinct T cell subsets, performed 24h after the last OVA-challenge. Total Th2 (GATA3⁺), Th9 (IRF4⁺; IL-9⁺), Th17 (RORγt⁺) and Treg (FoxP3⁺) cells within the CD3⁺CD4⁺T cell population in non-allergic WT+PBS and iCD4TGFBR2+PBS mice and allergic WT+OVA and iCD4TGFBR2+OVA mice were assessed. Each data point represents an individual mouse. Data are representative of two independent experiments (n=6-13/group). *p < 0.05, **p < 0.01, ****p < 0.0001; n.s, not significant (two-tailed Mann-Whitney U test).

Figure 3

Impact of Tgfbr2 ablation on Th-subset transcription factors and signal molecules. (A) Illustration of the complex interaction of genes in the regulation of T cell differentiation in allergy. The network indicates known molecular interactions or dependencies (either direct binding or gene regulation) and was inspired by the ImmunoNet (immunet.princeton.edu) (45) and extended manually by systematic literature research. The current knowledge was compiled in a gene regulation network of T_H2, T_H9 and T_H17 networks. The references for the network were manually added and are as follows: [a: (46), b: (47), c: (48), d: (49), e: (50), f:(51), g: (52), h (53), i: (54), j: (55), k: (56), l: (57), m: (58), n: (59), o: (60), p: (61), q: (62), r: (63), (64), s: (65)]. (B) CD4⁺ T cells were isolated from digested lungs of non-allergic and allergic WT and iCD4TGFBR2 mice and analyzed for IL-9 expression as well as T_H9 and T_H2 relevant transcription factors and signal molecules using quantitative RT-PCR. Expression levels of Il9, Pu.1, Irf4, Sgk1, Foxo1, Foxo3, Batf and Nfat5 were normalized to Gapdh house-keeping gene and relative changes were represented as 2^−ΔΔCT (ΔΔC_T=ΔC_T−ΔC_Control). Data is compiled from two independent experiments (n= 5-13/group). *p < 0.05, **p < 0.01, ***p < 0.001, n.s. not significant (t-test). (C) Comparisons of gene regulation network of T cells. Blue indicates reduced gene expression, while red indicates increased and gray unchanged gene expression of the respective factor for the respective comparison.

Figure 4

Local TGF-β is decreased along with pro-inflammatory cytokines upon AIT. Levels of selected cytokines in sputum of treated and untreated AR and AA patients and healthy controls. (A) Secreted TGF-β cytokine levels in induced sputum of allergic rhinitis and allergic asthma patients receiving (blue colored) or not (red colored) AIT as well as of control individuals were analyzed by LEGENDplex. Data presented by individual values and mean. (B) Levels of secreted cytokines IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13 and IFN-γ detected in sputum supernatants of the groups as in (A) assessed by LEGENDplex. Data presented by individual values and mean. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; statistical significance was determined by Kruskal-Wallis tests and only when medians across patient groups varied significantly, multiple single comparisons were performed using two-tailed Mann-Whitney U tests.

Figure 5

Treg cells increase and Th9 as well as Th2 cells decrease upon AIT. Th2, Th9, and Treg cell frequencies in sputum of treated and untreated AR and AA patients and healthy controls. (A) Gating strategy for Th2 and Th9 and (B) Treg cells in induced sputum. (C, D) Flow cytometric analysis of sputum derived T cell subpopulation Th2, Th9 and Tregs in healthy subjects, AR and AA patients as described in in season (C) and out of season (D). (E, F) Flow cytometric analysis of peripheral blood derived T cell subpopulation Th2, Th9, and Treg cells in healthy subjects, AR and AA patients as described in in season (E) and out of season (F). Data presented by single patient values and mean. Statistical significance was determined by Kruskal-Wallis tests and only when medians across patient groups varied significantly, multiple single comparisons were performed using two-tailed Mann-Whitney U tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (G, H) Correlation of sputum T_H2 and T_H9 cell frequencies with total serum IgE (G) and symptom score mRQLQ (H). (I, J) Correlation of peripheral blood derived T_H2 and T_H9 cell frequencies with total serum IgE (I) and symptom score mRQLQ (J). Two-sided Spearman test was used to calculate the correlations.