Human TH17 cells express a functional IL-13 receptor and IL-13 attenuates IL-17A production

Abstract

Background: IL-13 is a central mediator of airway responsiveness and mucus expression in patients with allergic airway inflammation, and IL-13 is currently a therapeutic target for asthma. However, little is known about how IL-13 regulates human CD4(+) T-cell lineages because IL-13 receptor (IL-13R) α1, a subunit of IL-13R, has not previously been reported to exist on human T cells.

Objective: We sought to determine whether human CD4(+) T(H)17 cells express IL-13Rα1 and whether IL-13 regulates T(H)17 cytokine production.

Methods: Naive human CD4(+) cells were isolated from whole blood, activated with anti-CD3 and anti-CD28, and polarized to T(H)1, T(H)2, T(H)17, or induced regulatory T cells in the presence of IL-13 (0-10 ng/mL). Cell supernatants, total RNA, or total protein was examined 4 days after T(H)17 polarization.

Results: T(H)17 cells, but not T(H)0, T(H)1, T(H)2, or induced regulatory T cells, expressed IL-13Rα1. IL-13 attenuated IL-17A production, as well as expression of retinoic acid-related orphan receptor, runt-related transcription factor-1, and interferon regulatory factor 4 in T(H)17-polarized cells. IL-13 neither inhibited IFN-γ production from T(H)1 cells nor inhibited IL-4 production from T(H)2 cells. Furthermore, attenuation of IL-17A production only occurred when IL-13 was present within 24 hours of T-cell activation or at the time of restimulation.

Conclusions: IL-13Rα1 is expressed on human CD4(+) T(H)17 cells, and IL-13 attenuates IL-17A production at polarization and restimulation. Although IL-13 is an attractive therapeutic target for decreasing symptoms associated with asthma, these results suggest that therapies inhibiting IL-13 production could have adverse side effects by increasing IL-17A production.

FIG. 1

IL-13Rα1 expression on human Th17 cells. CD4+ T cells were polarized to Th1, Th2, Th17 or iTregs. (A), IL-13Rα1 relative expression normalized to GAPDH and compared to Th0 cells. (B), Dot plot of IL-13Rα1 surface expression on CD4+, CD3+ cells (C), total CD4+, CD3+ IL-13Rα1+ cells. n=5–6 from 3 experiments, *p<0.05 compared to Th0, ANOVA.

FIG. 2

IL-13 increases STAT6 phosphorylation in Th17 cells. Serum-starved Th0 and Th17 cells were stimulated with IL-13 (10ng/ml) for 1 h and examined for phospho-STAT6 by flow cytometry. (A). STAT6 phosphorylation in CD3+, CD4+ cells compared to isotype control. (B). Fold increase of phospho-STAT6+, CD3+, CD4+ cells compared to 0ng/ml IL-13 in respective cell lineage. n = 4; *p<0.05, ANOVA

FIG. 3

TGF-β, IL-1β, and IL-23 upregulate IL-13Rα1 expression. Naïve T cells were polarized using combinations of Th17 polarizating cytokines in the presence of anti-IL-4 and anti-IFN-γ. IL-13Rα1 expression levels determined by real-time PCR, normalized to GAPDH, and compared to Th0 cells. Data is representative from 3 different experiments, * p<0.05 compared to Th0, ANOVA.

FIG. 4

IL-13 attenuates Th17 cytokine production. (A–C), Naive T cells were polarized to become Th17 cells in the presence of IL-13 and IL-17A, IL-21 and IL-22 was measured. (D), Th17 cells were polarized in the presence of isotype control, anti-IL4 or anti-IL13 (all 10μg/ml) and IL-17A was measured. Data is from 3 experiments, * p<0.05 compared to isotype control, t-test (A–C) or ANOVA (D).

FIG. 5

IL-13 attenuates IL-17A production in human Th17 polarized cells. CD4+ T cells were polarized to become Th0, Th1, Th2, Th17 or iTreg 4 days in the presence of IL-13 (10ng/ml). (A–C), Supernatants were examined by ELISA for cytokines. n= 5–6 replicates from 3 experiments, * p<0.05 compared to 0ng/ml of IL-13 for respective T cell lineage, ANOVA.

FIG. 6

IL-13 attenuates expression of Th17 transcription factors. (A), Serum starved Th17 cells were stimulated with IL-1β and IL-6 for 15 min and phospho-STAT3 levels were examined. (B–D), Nuclear protein from Th17 cells were examined for RORC2, Runx1, and IRF-4. Densitometry was normalized to total STAT3 (A) or nucleolin (B–D) and then normalized to Th17 cells. * p<0.05 compared to 0ng/ml of IL-13, t-test.

FIG. 7

IL-13 decreases IL-17A within 24 h of polarization or at restimulation. (A). IL-13 was added to Th17 cells at day 0, 1, or 2 and IL-17A levels were measured 4 d after polarization. (B). Th17 cells were restimulated with anti-CD3 and anti-CD28 in the presence or absence of IL-13 and IL-17A was measured 24 h later. * p<0.05 compared to Th17, ANOVA (A) or t-test (B).