Interleukin 4 Is Inactivated Via Selective Disulfide-Bond Reduction by Extracellular Thioredoxin
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Interleukin 4 is inactivated via selective disulfide-bond reduction by extracellular thioredoxin Nicholas M. Plugisa,1, Nielson Wenga,b,c,1, Qinglan Zhaod, Brad A. Palanskia, Holden T. Maeckere, Aida Habteziond, and Chaitan Khoslaa,f,g,2 aDepartment of Chemistry, Stanford University, Stanford, CA 94305; bSchool of Medicine, Stanford University, Stanford, CA 94305; cMedical Scientist Training Program, Stanford University, Stanford, CA 94305; dDivision of Gastroenterology and Hepatology, Department of Medicine, School of Medicine, Stanford University, Stanford, CA 94305; eInstitute for Immunity, Transplantation and Infection, Stanford University, Stanford, CA 94305; fDepartment of Chemical Engineering, Stanford University, Stanford, CA 94305; and gStanford ChEM-H, Stanford University, Stanford, CA 94305 Edited by Peter Cresswell, Yale University School of Medicine, New Haven, CT, and approved July 27, 2018 (received for review March 28, 2018) Thioredoxin 1 (TRX), an essential intracellular redox regulator, is TG2 recognition in vivo (Fig. 1B) (13). These results motivated us also secreted by mammalian cells. Recently, we showed that TRX to harness the same tools to search for other physiological protein activates extracellular transglutaminase 2 via reduction of an substrates of extracellular TRX. During our investigation of TRX- allosteric disulfide bond. In an effort to identify other extracellular TG2 recognition, we noticed that in addition to TG2 activity, substrates of TRX, macrophages derived from THP-1 cells were macrophage morphology was also sensitive to TRX inactivation treated with NP161, a small-molecule inhibitor of secreted TRX. (13). Therefore, we sought to identify TRX substrates involved in NP161 enhanced cytokine outputs of alternatively activated macro- macrophage polarization. phages, suggesting that extracellular TRX regulated the activity of Macrophages play a critical role in the immune system based on interleukin 4 (IL-4) and/or interleukin 13 (IL-13). To test this their ability to engulf and destroy microorganisms while also hypothesis, the C35S mutant of human TRX was shown to form a serving as antigen-presenting cells that facilitate T-cell responses. mixed disulfide bond with recombinant IL-4 but not IL-13. Kinetic k K μ −1· −1 In response to environmental signals, macrophages acquire dis- analysis revealed a cat/ M value of 8.1 M min for TRX- tinct activated phenotypes and functions. Historically, two distinct mediated recognition of IL-4, which established this cytokine as polarization states of macrophages, “classically activated” (M1) the most selective partner of extracellular TRX to date. Mass spec- and “alternatively activated” (M2), have been recognized. More trometry 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 activ- recent work has refined this binary paradigm into a model of a ity. To demonstrate the physiological relevance of our biochemical phenotypic spectrum (16, 17). Classical activation can be achieved γ findings, recombinant TRX was shown to attenuate IL-4–dependent by exposure to interferon-gamma (IFN- ) and lipopolysaccharide proliferation of cultured TF-1 erythroleukemia cells and also to in- (LPS). In contrast, M2 macrophages result from exposure to ei- hibit the progression of chronic pancreatitis in an IL-4–driven mouse ther interleukin 4 (IL-4) or interleukin 13 (IL-13) (18). Our initial model of this disease. By establishing that IL-4 is posttranslationally screen revealed that M2 macrophages exposed to the TRX in- regulated by TRX-promoted reduction of a disulfide bond, our find- hibitor NP161 displayed increased secretion of cytokines, sug- ings highlight a novel regulatory mechanism of the type 2 immune gesting that IL-4 and/or IL-13 were the main targets of TRX. response that is specific to IL-4 over IL-13. Because IL-4 and IL-13 share structural homology, recep- tor subunits, and downstream effector functions (19, 20), any interleukin 4 | thioredoxin | disulfide bond | macrophages | M2 Significance ammalian thioredoxin 1 (TRX) is a ubiquitous protein Mcofactor that regulates redox homeostasis by promoting Macrophages are important regulators of the immune system. thiol–disulfide exchange reactions with oxidized cytosolic pro- They display remarkable phenotypic plasticity in response to teins (1). In the intracellular environment, oxidized TRX is environmental cues. Classical macrophage activation occurs in recycled via the activity of the NADPH-dependent enzyme thio- response to inflammatory signals, whereas alternative macro- redoxin reductase. Some mammalian cells are also known to se- phage activation results from exposure to IL-4 and/or IL-13. The crete TRX via a noncanonical export mechanism (1–4). While the mechanistic basis for differential regulation of macrophages by fate of oxidized TRX outside the cell is unclear, recent studies IL-4 and IL-13 remains poorly understood. We show through have led to the identification of a few extracellular substrates of its in vitro and in vivo experiments that thioredoxin 1, a redox reduced form. For example, TRX activates the TRPC ion channel protein cofactor, preferentially inactivates IL-4 over IL-13, by and the HIV-1 envelope protein gp120 via reduction of allosteric reduction of a specific disulfide bond. As extracellular levels disulfide bonds (5, 6). Elevated serum levels of TRX have been of thioredoxin are elevated in many pathological conditions, reported in many pathological conditions associated with in- our results highlight a novel pharmacologically promising flammation including AIDS, rheumatoid arthritis, inflammatory immunomodulatory mechanism. bowel disease, and Sjögren’s syndrome (7–10). Author contributions: N.M.P., N.W., Q.Z., B.A.P., H.T.M., A.H., and C.K. designed research; In previous studies, we demonstrated that TRX activates ex- N.M.P., N.W., and Q.Z. performed research; B.A.P. contributed new reagents/analytic tracellular transglutaminase 2 (TG2) via reduction of an allosteric tools; N.M.P., N.W., Q.Z., B.A.P., H.T.M., A.H., and C.K. analyzed data; and N.M.P., N.W., disulfide bond (11–13). In those experiments, we engineered and A.H., and C.K. wrote the paper. utilized two chemical biological tools. First, NP161 was identified The authors declare no conflict of interest. as a potent and selective inhibitor of extracellular TRX in vitro This article is a PNAS Direct Submission. (12) and in vivo (13). Because this small molecule deactivates Published under the PNAS license. TRX via oxidation of its active-site cysteine residues, its effects are 1N.M.P. and N.W. contributed equally to this work. presumably restricted to the extracellular environment, where 2To whom correspondence should be addressed. Email: [email protected]. A TRX reductase is not present (Fig. 1 ). Second, we engineered an This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. active-site mutant of human TRX (C35S) that covalently traps its 1073/pnas.1805288115/-/DCSupplemental. BIOCHEMISTRY substrates (6, 14, 15), and used it to demonstrate selective TRX- Published online August 13, 2018. www.pnas.org/cgi/doi/10.1073/pnas.1805288115 PNAS | August 28, 2018 | vol. 115 | no. 35 | 8781–8786 Downloaded by guest on September 29, 2021 polarization of macrophages by directly inactivating either IL-4 or IL-13, two cytokines whose activities are known to require the presence of disulfide bonds (24, 25). This hypothesis was directly tested through biochemical studies, as described below. TRX Preferentially Reduces IL-4 over IL-13. The cytokines IL-4 and IL-13 are homologous helical proteins that signal through a shared receptor, IL-4Rα (Fig. 3). Both cytokines harbor three disulfide bonds. To directly test the hypothesis that TRX regu- lates IL-4 and/or IL-13 function by reduction of an allosteric disulfide bond, we first needed to produce sufficient quantities of both recombinant proteins. Genes encoding the mature human IL-4 and IL-13 were expressed in Escherichia coli, and the pro- teins were isolated as inclusion bodies. As detailed in Materials and Methods, each cytokine was refolded, purified, and demon- strated to have comparable biological activity to authentic stan- Fig. 1. Molecular tools to investigate the biology of extracellular TRX. (A) dards in a TF-1 cell-proliferation assay. The ED50 values of IL-4 NP161 inactivates TRX by oxidizing its C32XXC35 active site via disulfide-bond formation. Whereas oxidized TRX in the cytosol is rapidly regenerated by and IL-13 in this proliferation assay were 1.7 and 0.5 μg/mL, thioredoxin reductase in an NADPH-dependent manner, extracellular TRX respectively (SI Appendix, Fig. S1). has no known mechanism of regeneration; therefore, this mechanism of To test whether recombinant human TRX was able to rec- inactivation is selective for extracellular TRX (12, 13). (B) The C35S mutant of ognize and react with recombinant human IL-4 or IL-13, we took human TRX enables covalent trapping of its extracellular substrates. A mixed advantage of the active-site C35S mutant (Fig. 1B) that has been disulfide intermediate is formed between C32 and one of the two Cys resi- dues comprising a disulfide bond in a target protein substrate. Whereas the previously used to trap mixed disulfide adducts between TRX corresponding mixed disulfide bond with wild-type TRX is highly transient, and its substrates (6, 15). This mutant protein (13 kDa) was the complex involving the C35S mutant is more stable (13). purified and incubated with IL-4 (17 kDa) or IL-13 (15 kDa), and the protein mixtures were analyzed via nonreducing SDS/ PAGE. A prominent ∼30-kDa adduct was observed when mu- observed effect on M2 macrophages can be mediated through tant TRX was incubated with IL-4; under identical conditions, either IL-4 and/or IL-13. While other studies have shown that a reducing environment abrogates the downstream biological ef- fects of IL-4 (21–23), there is no evidence that this effect is unique to IL-4.