Interleukin 4 is inactivated via selective disulfide-bond reduction by extracellular thioredoxin

Abstract

Thioredoxin 1 (TRX), an essential intracellular redox regulator, is also secreted by mammalian cells. Recently, we showed that TRX activates extracellular transglutaminase 2 via reduction of an allosteric disulfide bond. In an effort to identify other extracellular substrates of TRX, macrophages derived from THP-1 cells were treated with NP161, a small-molecule inhibitor of secreted TRX. NP161 enhanced cytokine outputs of alternatively activated macrophages, suggesting that extracellular TRX regulated the activity of interleukin 4 (IL-4) and/or interleukin 13 (IL-13). To test this hypothesis, the C35S mutant of human TRX was shown to form a mixed disulfide bond with recombinant IL-4 but not IL-13. Kinetic analysis revealed a k_cat/K_M value of 8.1 μM^-1⋅min^-1 for TRX-mediated recognition of IL-4, which established this cytokine as the most selective partner of extracellular TRX to date. Mass spectrometry identified the C46-C99 bond of IL-4 as the target of TRX, consistent with the essential role of this disulfide bond in IL-4 activity. To demonstrate the physiological relevance of our biochemical findings, recombinant TRX was shown to attenuate IL-4-dependent proliferation of cultured TF-1 erythroleukemia cells and also to inhibit the progression of chronic pancreatitis in an IL-4-driven mouse model of this disease. By establishing that IL-4 is posttranslationally regulated by TRX-promoted reduction of a disulfide bond, our findings highlight a novel regulatory mechanism of the type 2 immune response that is specific to IL-4 over IL-13.

Keywords: M2; disulfide bond; interleukin 4; macrophages; thioredoxin.

Fig. 1.

Molecular tools to investigate the biology of extracellular TRX. (A) NP161 inactivates TRX by oxidizing its C₃₂XXC₃₅ active site via disulfide-bond formation. Whereas oxidized TRX in the cytosol is rapidly regenerated by thioredoxin reductase in an NADPH-dependent manner, extracellular TRX has no known mechanism of regeneration; therefore, this mechanism of inactivation is selective for extracellular TRX (12, 13). (B) The C35S mutant of human TRX enables covalent trapping of its extracellular substrates. A mixed disulfide intermediate is formed between C32 and one of the two Cys residues comprising a disulfide bond in a target protein substrate. Whereas the corresponding mixed disulfide bond with wild-type TRX is highly transient, the complex involving the C35S mutant is more stable (13).

Fig. 2.

TRX inhibitor NP161 stimulates cytokine secretion in M2 cells while inhibiting secretion in M1 cells. THP-1 cells were differentiated into M0 macrophages with PMA for 48 h, followed by polarization into M1 macrophages with IFN-γ (20 ng/mL) and LPS (1 ng/mL) or into M2 macrophages with IL-4 (20 ng/mL) for 36 h. Then, M1 or M2 cells were exposed to vehicle or NP161 (33 µM). Fold changes in secreted cytokine concentrations (pg/mL) are plotted in binary-logarithmic scale in a heatmap. Cytokines that are changed by fivefold or more by NP161 are shown for either M1 or M2 macrophages. For a full list of cytokine measurements, see SI Appendix, Table S1. Cytokine and chemokine levels were determined by a multiplexed Luminex assay. Data are the means from three biological replicates.

Fig. 3.

Structure of IL-4 and IL-13. Interleukin 4 (A) [Protein Data Bank (PDB) ID code 1HIK] and interleukin 13 (B) (PDB ID code 3LB6) are four-helix bundles that each possess three disulfide bonds (shown in yellow).

Fig. 4.

TRX selectively recognizes and reduces IL-4. (A) The C35S mutant of recombinant human TRX was incubated with recombinant IL-4 or IL-13, and the resulting protein mixtures were analyzed via nonreducing SDS/PAGE. (B) Steady-state kinetic analysis of TRX-mediated reduction of insulin: a reference substrate of TRX (triangle), IL-4 (circle), and IL-13 (square). In each case, the data points were fitted to the Michaelis–Menten equation. Data are mean ± SEM of two replicates from three independent experiments. (C) TRX-mediated reduction of IL-4 in the absence (black) or presence (red) of an initially equimolar concentration of IL-13. At each time point, the concentration of active IL-4 was measured, and the data were fitted to a first-order rate law. The negative control (gray) contained no TRX. Data are mean ± SEM; n = 3 per group. (D) Direct measurements of IL-4 and IL-13 concentrations of samples withdrawn at 5 min into the experiment corresponding to C. In C and D, the concentrations of IL-4 and IL-13 were measured by ELISA. The antibodies against human IL-4 and IL-13 used for these measurements were specific for oxidized, active IL-4 and IL-13, respectively, and display no cross-reactivity (SI Appendix, Fig. S2). Data are mean ± SEM; n = 3 per group.

Fig. 5.

TRX specifically abrogates the cytokine activity of IL-4. TF-1 cells were stimulated with 8 ng/mL IL-4 (red squares), IL-13 (blue circles), or GM-CSF (green triangles), and varying amounts of TRX were added. Viable cells were counted after 48 h by flow cytometry (forward scatter/side scatter). Data are mean ± SEM from two biological replicates with three technical replicates.

Fig. 6.

TRX inactivates IL-4 and ameliorates established chronic pancreatitis (CP). Thioredoxin (i.p., 250 mg/kg, two times per d, 3 d/wk) was administered to mice 3 wk after starting CP induction and mice were killed after 4 wk of cerulein injections. (A) Relative pancreas weights from CP- and TRX-treated mice are shown. Means ± SEM; n = 10 per group. (B) ELISA analysis of relative pancreatic tissue at the IL-4 level in control (Con), CP-, and TRX-treated mice are shown. For serum IL-4 levels, see SI Appendix, Fig. S3A. Mouse IL-4 ELISA used for this study can only detect active IL-4 (SI Appendix, Fig. S4). Means ± SEM. (C) Representative pancreatic histological slides by H&E and Trichrome staining. (Scale bar, 100 μm.) More sections included in this study are shown in SI Appendix, Fig. S3B. (D) Quantitative analysis of fibrosis using the images from Trichrome staining. Means ± SEM. (E–H) RT-PCR analysis of αSMA, Col1α1, Fn1, and TGF-β expression in the pancreas of the indicated mice. Means ± SEM; n = 5 per group. ns, not significant.