Interleukin 4 is inactivated via selective disulfide-bondreduction by extracellular thioredoxinNicholas 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 ScientistTraining 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 ofChemical 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, isalso secreted by mammalian cells. Recently, we showed that TRXactivates extracellular transglutaminase 2 via reduction of anallosteric disulfide bond. In an effort to identify other extracellularsubstrates of TRX, macrophages derived from THP-1 cells weretreated with NP161, a small-molecule inhibitor of secreted TRX.NP161 enhanced cytokine outputs of alternatively activated macro-phages, suggesting that extracellular TRX regulated the activity ofinterleukin 4 (IL-4) and/or interleukin 13 (IL-13). To test thishypothesis, the C35S mutant of human TRX was shown to form amixed disulfide bond with recombinant IL-4 but not IL-13. Kineticanalysis revealed a kcat/KM value of 8.1 μM−1·min−1 for TRX-mediated recognition of IL-4, which established this cytokine asthe most selective partner of extracellular TRX to date. Mass spec-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-ity. To demonstrate the physiological relevance of our biochemicalfindings, recombinant TRX was shown to attenuate IL-4–dependentproliferation of cultured TF-1 erythroleukemia cells and also to in-hibit the progression of chronic pancreatitis in an IL-4–driven mousemodel of this disease. By establishing that IL-4 is posttranslationallyregulated by TRX-promoted reduction of a disulfide bond, our find-ings highlight a novel regulatory mechanism of the type 2 immuneresponse that is specific to IL-4 over IL-13.

interleukin 4 | thioredoxin | disulfide bond | macrophages | M2

Mammalian thioredoxin 1 (TRX) is a ubiquitous proteincofactor that regulates redox homeostasis by promotingthiol–disulfide exchange reactions with oxidized cytosolic pro-teins (1). In the intracellular environment, oxidized TRX isrecycled via the activity of the NADPH-dependent enzyme thio-redoxin reductase. Some mammalian cells are also known to se-crete TRX via a noncanonical export mechanism (1–4). While thefate of oxidized TRX outside the cell is unclear, recent studieshave led to the identification of a few extracellular substrates of itsreduced form. For example, TRX activates the TRPC ion channeland the HIV-1 envelope protein gp120 via reduction of allostericdisulfide bonds (5, 6). Elevated serum levels of TRX have beenreported in many pathological conditions associated with in-flammation including AIDS, rheumatoid arthritis, inflammatorybowel disease, and Sjögren’s syndrome (7–10).In previous studies, we demonstrated that TRX activates ex-

tracellular transglutaminase 2 (TG2) via reduction of an allostericdisulfide bond (11–13). In those experiments, we engineered andutilized two chemical biological tools. First, NP161 was identifiedas a potent and selective inhibitor of extracellular TRX in vitro(12) and in vivo (13). Because this small molecule deactivatesTRX via oxidation of its active-site cysteine residues, its effects arepresumably restricted to the extracellular environment, whereTRX reductase is not present (Fig. 1A). Second, we engineered anactive-site mutant of human TRX (C35S) that covalently traps itssubstrates (6, 14, 15), and used it to demonstrate selective TRX-

TG2 recognition in vivo (Fig. 1B) (13). These results motivated usto harness the same tools to search for other physiological proteinsubstrates of extracellular TRX. During our investigation of TRX-TG2 recognition, we noticed that in addition to TG2 activity,macrophage morphology was also sensitive to TRX inactivation(13). Therefore, we sought to identify TRX substrates involved inmacrophage polarization.Macrophages play a critical role in the immune system based on

their ability to engulf and destroy microorganisms while alsoserving as antigen-presenting cells that facilitate T-cell responses.In response to environmental signals, macrophages acquire dis-tinct activated phenotypes and functions. Historically, two distinctpolarization states of macrophages, “classically activated” (M1)and “alternatively activated” (M2), have been recognized. Morerecent work has refined this binary paradigm into a model of aphenotypic spectrum (16, 17). Classical activation can be achievedby exposure to interferon-gamma (IFN-γ) and lipopolysaccharide(LPS). In contrast, M2 macrophages result from exposure to ei-ther interleukin 4 (IL-4) or interleukin 13 (IL-13) (18). Our initialscreen revealed that M2 macrophages exposed to the TRX in-hibitor NP161 displayed increased secretion of cytokines, sug-gesting that IL-4 and/or IL-13 were the main targets of TRX.Because IL-4 and IL-13 share structural homology, recep-

tor subunits, and downstream effector functions (19, 20), any

Significance

Macrophages are important regulators of the immune system.They display remarkable phenotypic plasticity in response toenvironmental cues. Classical macrophage activation occurs inresponse to inflammatory signals, whereas alternative macro-phage activation results from exposure to IL-4 and/or IL-13. Themechanistic basis for differential regulation of macrophages byIL-4 and IL-13 remains poorly understood. We show throughin vitro and in vivo experiments that thioredoxin 1, a redoxprotein cofactor, preferentially inactivates IL-4 over IL-13, byreduction of a specific disulfide bond. As extracellular levelsof thioredoxin are elevated in many pathological conditions,our results highlight a novel pharmacologically promisingimmunomodulatory mechanism.

Author contributions: N.M.P., N.W., Q.Z., B.A.P., H.T.M., A.H., and C.K. designed research;N.M.P., N.W., and Q.Z. performed research; B.A.P. contributed new reagents/analytictools; 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.,A.H., and C.K. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1N.M.P. and N.W. contributed equally to this work.2To whom correspondence should be addressed. Email: khosla@stanford.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1805288115/-/DCSupplemental.

Published online August 13, 2018.

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observed effect on M2 macrophages can be mediated througheither IL-4 and/or IL-13. While other studies have shown that areducing environment abrogates the downstream biological ef-fects of IL-4 (21–23), there is no evidence that this effect isunique to IL-4. In addition, no one has captured a direct in-teraction between any reducing factors and IL-4 or shown thatthis interaction is physiologically relevant. In this study, we haveidentified secreted IL-4 but not IL-13 as a preferred substrate ofextracellular TRX. In addition to characterizing the redoxmechanism of this regulatory process, we have also demon-strated its pathophysiological relevance in an animal model ofhuman disease.

ResultsExtracellular TRX Inhibition Enhances Cytokine Outputs of M2Macrophages Derived from THP-1 Cells. Because we noticed thatmacrophage morphology was sensitive to TRX inactivation, weperformed a cytokine screen to characterize the effect of TRX onmacrophages. We first evaluated an established cellular model in-volving the THP-1 human monocytic cell line (16). Specifically,THP-1 cells were differentiated into macrophages by exposure tophorbol 12-myristate 13-acetate (PMA); cells thus treated arecommonly referred to as unpolarized macrophages in the “M0state” (16). M0 macrophages can then be polarized into M1 mac-rophages with IFN-γ and LPS or into M2 macrophages with IL-4.To test the effect of endogenous extracellular TRX on mac-

rophage polarization, we added NP161, a small-molecule in-hibitor of extracellular TRX, to cultures of either M1 or M2macrophages. For this exploratory study, we looked for the mostprofound changes in the concentrations of secreted cytokines.Among the 62 cytokines screened, changes of at least fivefoldwere identified for 11 cytokines (Fig. 2). Notably, addition ofNP161 reduces secretion of these cytokines in M1 macrophages,whereas it increases cytokine levels in M2 macrophages.The above observations led us to suspect a role for extracel-

lular TRX in suppressing the M2 state of macrophages. BecauseIL-13 can elicit the same immune response as IL-4, our observedeffect could also be mediated through IL-13. Therefore, we hy-pothesized that extracellular TRX influenced this differential

polarization of macrophages by directly inactivating either IL-4or IL-13, two cytokines whose activities are known to require thepresence of disulfide bonds (24, 25). This hypothesis was directlytested through biochemical studies, as described below.

TRX Preferentially Reduces IL-4 over IL-13. The cytokines IL-4 andIL-13 are homologous helical proteins that signal through ashared receptor, IL-4Rα (Fig. 3). Both cytokines harbor threedisulfide bonds. To directly test the hypothesis that TRX regu-lates IL-4 and/or IL-13 function by reduction of an allostericdisulfide bond, we first needed to produce sufficient quantities ofboth recombinant proteins. Genes encoding the mature humanIL-4 and IL-13 were expressed in Escherichia coli, and the pro-teins were isolated as inclusion bodies. As detailed in Materialsand Methods, each cytokine was refolded, purified, and demon-strated to have comparable biological activity to authentic stan-dards in a TF-1 cell-proliferation assay. The ED50 values of IL-4and IL-13 in this proliferation assay were 1.7 and 0.5 μg/mL,respectively (SI Appendix, Fig. S1).To test whether recombinant human TRX was able to rec-

ognize and react with recombinant human IL-4 or IL-13, we tookadvantage of the active-site C35S mutant (Fig. 1B) that has beenpreviously used to trap mixed disulfide adducts between TRXand its substrates (6, 15). This mutant protein (13 kDa) waspurified 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-tant TRX was incubated with IL-4; under identical conditions,

Fig. 1. Molecular tools to investigate the biology of extracellular TRX. (A)NP161 inactivates TRX by oxidizing its C32XXC35 active site via disulfide-bondformation. Whereas oxidized TRX in the cytosol is rapidly regenerated bythioredoxin reductase in an NADPH-dependent manner, extracellular TRXhas no known mechanism of regeneration; therefore, this mechanism ofinactivation is selective for extracellular TRX (12, 13). (B) The C35S mutant ofhuman TRX enables covalent trapping of its extracellular substrates. A mixeddisulfide intermediate is formed between C32 and one of the two Cys resi-dues comprising a disulfide bond in a target protein substrate. Whereas thecorresponding 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 whileinhibiting secretion in M1 cells. THP-1 cells were differentiated into M0macrophages with PMA for 48 h, followed by polarization into M1 macro-phages with IFN-γ (20 ng/mL) and LPS (1 ng/mL) or into M2 macrophageswith IL-4 (20 ng/mL) for 36 h. Then, M1 or M2 cells were exposed to vehicleor NP161 (33 μM). Fold changes in secreted cytokine concentrations (pg/mL)are plotted in binary-logarithmic scale in a heatmap. Cytokines that arechanged by fivefold or more by NP161 are shown for either M1 or M2macrophages. For a full list of cytokine measurements, see SI Appendix,Table S1. Cytokine and chemokine levels were determined by a multiplexedLuminex assay. Data are the means from three biological replicates.

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only a small amount of the putative adduct was observed be-tween the C35S TRX mutant and IL-13 (Fig. 4A).To quantify the specificity of TRX for IL-4 versus IL-13, we

adopted an established kinetic assay for TRX activity, using insulinas a reference substrate (11). Steady-state kinetic analysis revealedthat TRX had significantly higher specificity toward IL-4 than IL-13 (Fig. 4B and Table 1). Kinetic parameters for insulin recogni-tion by TRX were in agreement with previously reported data (11,26). Notably, to our knowledge, IL-4 appears to be the mostpreferred extracellular substrate of TRX identified to date.To verify the preference of TRX for IL-4 over IL-13, the

TRX-promoted rate of IL-4 deactivation was compared in thepresence or absence of an initially equal concentration of IL-13.To simulate physiological conditions, substantially lower cyto-kine concentrations were employed in these assays than thoseused for the estimation of kinetic parameters. As predicted,addition of IL-13 had negligible effect on the rate of oxidativedeactivation of IL-4 (Fig. 4C). By way of confirmation, the extentof TRX reduction of the two cytokines was directly quantified at

5 min (Fig. 4D), and was found to be in line with the specificity ofTRX for IL-4 over IL-13, as measured above.

TRX Reduces the C46–C99 Disulfide Bond in IL-4. IL-4 contains threedisulfide bonds. The high specificity of TRX for IL-4 and theobservation of a single adduct between C35S TRX and IL-4suggested that a unique disulfide of IL-4 was targeted by TRX.Mass spectrometric analysis was therefore used to identify thisrecognition site (SI Appendix, Scheme S1). As summarized inTable 2, the disulfide bond between C46 and C99 of IL-4 wasexclusively reduced by TRX. Given that this disulfide bond isessential for cytokine function (24), our findings suggest thatTRX recognition of IL-4 has the potential to be a biologicallyrelevant regulatory mechanism.

TRX Selectively Inactivates the Cytokine Activity of IL-4. A cellularmodel was used to assess the biological relevance of the observedprotein–protein recognition between IL-4 and TRX. Proliferationof the TF-1 erythroleukemia cell line was evaluated in the pres-ence of TRX. Growth of this cell line requires IL-4, IL-13, or GM-CSF in the culture medium (27). The IC50 of TRX was 50 nM incultures containing IL-4 whereas, under equivalent conditions, theIC50 of TRX was 2.2 μM in cultures containing IL-13. TRX hadno effect on TF-1 cell growth in the presence of GM-CSF (Fig. 5).

TRX Mitigates IL-4–Driven Pathological Conditions in Chronic Pancreatitis.In light of the above findings in vitro, we sought to assess the path-ophysiological relevance of TRX-mediated IL-4 inactivation in vivo.To do so, we took advantage of a recent study highlighting the role ofIL-4 signaling in a mouse model of chronic pancreatitis (28).Chronic pancreatitis is characterized by progressive irrevers-

ible damage to the pancreas (29). Some of the key histologicalfeatures include inflammation, fibrosis, and acinar cell death(30), which in part are promoted by M2 macrophages. Becausepharmacological inhibition of the IL-4 receptor decreases thesepathological phenotypes and halts chronic pancreatitis progres-sion (28), we used these pathological features as evidence for IL-4 inactivation in vivo. To that end, we first induced chronicpancreatitis in C57BL/6J mice by repetitive injection withcerulein over 4 wk (six injections per d, 3 d/wk). At the beginningof week 3, we initiated dosing of recombinant TRX to diseased

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-helixbundles 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 TRXwas incubated with recombinant IL-4 or IL-13, andthe resulting protein mixtures were analyzed vianonreducing SDS/PAGE. (B) Steady-state kineticanalysis of TRX-mediated reduction of insulin: areference substrate of TRX (triangle), IL-4 (circle),and IL-13 (square). In each case, the data points werefitted to the Michaelis–Menten equation. Dataare mean ± SEM of two replicates from threeindependent experiments. (C ) TRX-mediated re-duction 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-4was measured, and the data were fitted to a first-order rate law. The negative control (gray) con-tained no TRX. Data are mean ± SEM; n = 3 pergroup. (D) Direct measurements of IL-4 and IL-13concentrations of samples withdrawn at 5 min intothe experiment corresponding to C. In C and D, theconcentrations of IL-4 and IL-13 were measured byELISA. The antibodies against human IL-4 and IL-13used for these measurements were specific for oxi-dized, active IL-4 and IL-13, respectively, and displayno cross-reactivity (SI Appendix, Fig. S2). Data aremean ± SEM; n = 3 per group.

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mice. Compared with the control group, TRX-treated mice showed amarked reduction in the amount of active, oxidized IL-4 in pancreatictissue (Fig. 6B). Moreover, TRX treatment limited pancreatic fibrosis,as shown by increased pancreas weight, histology, and decreasedfibrosis-associated gene expression (Fig. 6 A and C–H), providingfurther support for the ability of TRX to inactivate IL-4 in vivo.

DiscussionThe immune response to danger signals involves a complex or-chestration of cells and secreted molecules, and is characterizedby an initial response that is amplified by the activation and re-cruitment of effector cells followed by resolution of the response.In broad terms, two types of immune responses (TH1 and TH2)have been extensively described (31, 32). Macrophages are anessential component of both of these responses, becoming acti-vated in response to tissue microenvironmental signals contrib-uted by microbial components, the innate and adaptive immunesystems, and damaged cells and tissues (18). Classically activated(M1) macrophages promote TH1-type inflammatory responsesalong with strong microbicidal and tumoricidal activity, whereasalternatively activated (M2) macrophages are associated withTH2-type antiinflammatory responses, wound healing, and res-olution of inflammation. While substantial progress has beenmade in defining the molecular networks underlying macrophagepolarization, there is considerable interest in the identification ofmolecules that regulate macrophage polarization.Two cytokines, IL-4 and IL-13, are the primary regulators of the

type 2 immune response (33). Through exogenous administration,overexpression, and knockout studies, these two closely relatedextracellular proteins have been shown to have overlapping (34–39) but nonredundant (33) roles in immunity. While IL-4 signalingis initiated through both type I and II receptors, IL-13 only signalsthrough binding of type II receptors (20). The two cytokines ap-pear to be differentially regulated, but the underlying mechanismsof differential regulation of immune responses by IL-4 and IL-13remain poorly understood. The only known way that the two cy-tokines can exhibit unique effects is through exclusive receptorsubunit binding and segregation of expression among differentcellular and tissue sources (33). Here we have identified anddissected a redox mechanism that differentially regulates the de-activation of IL-4 versus IL-13. Specifically, extracellular TRXselectively recognizes the C46–C99 disulfide of IL-4, leading to

inactivation of its cytokine activity. This posttranslational regula-tory mechanism was shown to attenuate macrophage polarizationtoward an alternatively activated state.The observed specificity of TRX for IL-4 over IL-13 is un-

precedented. In retrospect, the existence of such an endogenousregulatory mechanism should not be surprising, given that experi-ments involving mice deficient in cytokines, cytokine-producingcells, or receptor subunits have repeatedly shown that IL-4 and IL-13 play distinct roles in allergic immunity in vivo (33). Our work hasprovided a molecular mechanism by which immune and non-immune cells can regulate local cytokine concentration and effect.Finally, in addition to opening a new window to an immune

regulatory mechanism in mammals, our findings may also havepromise for immunotherapy, given the role of IL-4 in a variety ofdisease states. A number of preclinical and clinical studies havedemonstrated that exogenously administered TRX is generally well-tolerated in mammals (40). By inactivating IL-4 with TRX, we wereable to ameliorate pancreatic fibrosis in a mouse model of chronicpancreatitis. More generally, inhibition of IL-4 may also provide aclinical benefit for diseases such as atopic dermatitis, allergic rhi-nitis, asthma, chronic obstructive pulmonary disease, inflammatorybowel disease, autoimmune disease, and fibrotic disease (19). Thus,administration of TRX could represent a potential alternativeapproach to monoclonal antibody-based IL-4 inhibition.

Materials and MethodsChemicals and Other Reagents. Unless otherwise specified, reagents werefrom Sigma-Aldrich. DTT was from Invitrogen, SDS/polyacrylamide gradientgels (4 to 20%) were from Bio-Rad, Ni-NTA resin was from Qiagen, the HiTrapQ anion-exchange column was from GE Healthcare, and 7-kDa molecularmass cutoff spin columns were from Pierce. Cell-culture medium, FBS, anti-biotics, and sterile PBS were from Invitrogen. Glutamine was from Lonza.

Macrophage Polarization. The human THP-1 monocytic cell line was main-tained in RPMI 1640 culture medium containing 10% heat-inactivated FBSand penicillin/streptomycin. Cells were seeded at 106 cells per mL and dif-ferentiated into M0 macrophages by incubation for 48 h in the presence of100 ng/mL phorbol 12-myristate 13-acetate (P8139; Sigma). These M0 cellswere then maintained in the same state for an additional 72 h in RPMImedium. THP-1–derived M0 macrophages were polarized into M1 macro-phages by incubation for 36 h with 20 ng/mL IFN-γ (285-IF; R&D Systems) and1 ng/mL LPS (tlrl-pb5lps; InvivoGen). Alternatively, macrophages were polarizedinto the M2 state by incubation for 36 h with 20 ng/mL interleukin 4 (I4269;Sigma) or 20 ng/mL interleukin 13 (50-813-223; Fisher). When needed, 33 μMNP161 (a small-molecule inhibitor of TRX) was added.

Table 2. Mass spectrometric analysis of the mixed disulfideadduct formed between IL-4 and C35S TRX

ResidueIAA, relativeintensity

IAM, relativeintensity

Cys3 0 100Cys24 0 100Cys46 12.2 87.8Cys65 0 100Cys99 11.2 88.8

Iodoacetic acid (IAA) was used to label free Cys residues when the twoproteins were coincubated. Iodoacetamide (IAM) was used to label all otherCys residues following complete reduction of the protein mixture.

Fig. 5. TRX specifically abrogates the cytokine activity of IL-4. TF-1 cells werestimulated 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 werecounted after 48 h by flow cytometry (forward scatter/side scatter). Data aremean ± SEM from two biological replicates with three technical replicates.

Table 1. Kinetic parameters for TRX-promoted reduction ofinsulin, IL-4, and IL-13

Substrate kcat/KM, μM−1·min−1 kcat, min−1 KM, μM

Insulin 2.3 130 56Interleukin 4 8.1 370 46Interleukin 13 1.6 110 65

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Luminex Assay. This multiplexed assay for secreted proteins was performed atthe Human Immune Monitoring Center at Stanford University. Human 62-plexwas from Affymetrix, and used according to the manufacturer’s recommen-dations with modifications, as detailed elsewhere (41). Briefly, antibody-linkedbeads were added to a 96-well plate and washed in a BioTek ELx405 washer.Culture supernatants were added to these wells and incubated at roomtemperature for 1 h followed by overnight incubation at 4 °C with shaking(500 to 600 rpm on an orbital shaker). Plates were washed in a BioTek ELx405washer, and the biotinylated detection antibody was added for 75 min atroom temperature with shaking. Plates were washed again as above, andstreptavidin-phycoerythrin was added. After incubation for 30 min at roomtemperature, plates were washed once more and reading buffer was added tothe wells. Plates were read on a Luminex 200 instrument with a lower boundof 50 beads per sample per cytokine. Each sample was measured in duplicate.Control beads (Radix BioSolutions) were added to all wells.

Cross-Linking of C35S TRX and IL-4 or IL-13. IL-4 (10 μM) or IL-13 (10 μM) wasincubated for 30 min at room temperature with C35S TRX (10 μM) in 20 mMTris·HCl, 1 mM EDTA (pH 7.6). Samples were diluted with 2× Laemmli samplebuffer (Bio-Rad) and applied to a nonreducing 4 to 20% SDS/polyacrylamidegel (Bio-Rad).

Thioredoxin Activity Assay. Before use, recombinant human TRX was freshlyreduced with a 10-fold molar excess of DTT on ice. The excess DTT was re-moved by passing the solution through a 7-kDa molecular mass cutoff spincolumn. TRX concentration was determined byA280 (e = 7,570 M

−1·cm−1), andthe protein was freshly used within 2 h. Steady-state kinetic analysis of TRX-mediated reduction of insulin and IL-4 was performed via a coupled assaycontaining 6 μM TrxR, 10 nM TRX, and 0.3 mM NADPH in a buffer containing50 mM Tris·HCl and 2 mM EDTA (pH 7.5). The reaction rate was calculatedfrom the slope of the absorbance curve at 340 nm, using the extinctioncoefficient of NADPH (6,220 M−1·cm−1). Michaelis–Menten parameters weredetermined by fitting the kinetic data using GraphPad Prism 6.

IL-4 and IL-13 Competition Assay. Before use, recombinant human TRX wasfreshly reduced with a 10-fold molar excess of DTT on ice. The excess DTT wasremoved by passing the solution through a 7-kDa molecular mass cutoff spincolumn. TRX concentration was determined byA280 (e = 7,570M

−1·cm−1), andthe protein was freshly used within 2 h. Human recombinant IL-4 (200-04)and recombinant IL-13 (200-13) were from PeproTech. Kinetic analysis ofTRX-mediated reduction of IL-4 was performed via a coupled assay con-taining 1 μM TrxR, 5 nM TRX, 60 μM NADPH, 50 nM IL-4, and 50 nM IL-13 inPBS buffer (pH 7.4). The rate of oxidative inactivation of IL-4 was comparedwith that in the presence of equimolar IL-13 as a competitive substrate. Atspecific time points, aliquots were withdrawn and diluted 5,000-fold intocold PBS (4 °C). Cytokine concentrations were determined via ELISAs.

Mass Spectrometric Determination of the Target Disulfide Bond in IL-4. A 60-μLsolution containing IL-4 (34 μg, 20 μM) and C35S TRX (26 μg, 20 μM) wasincubated for 30 min at room temperature in 20 mM Tris·HCl, 1 mM EDTA(pH 7.6). The solution was then diluted with 7.5 μL of 8 M urea in 100 mMNH4HCO3 and incubated with iodoacetic acid (320 μg, 25 mM) for 1 h atroom temperature in the dark. The reaction was buffer-exchanged threetimes on a Zeba desalting column (Thermo Scientific) into 20 mM Tris, 1 mMEDTA, 1 M urea, 12.5 mM NH4HCO3 and then incubated with DTT (93 μg,10 mM) for 30 min at room temperature. Iodoacetamide (320 μg, 25 mM)was added to the samples and allowed to incubate at room temperature inthe dark for 30 min. Reconstituted trypsin solution (20 mg/mL in resus-pension buffer; Promega) was added to a final concentration of 6 μg/mL.The samples were digested for 4 h in a 37 °C water bath, after which thedigestion was quenched by adding formic acid to a final concentration of7.5% (vol/vol). Peptides were desalted using C18 StageTips (42), lyophilizedovernight, resuspended in 5% formic acid in water, and analyzed by massspectrometry on an Orbitrap Elite Mass Spectrometer (Thermo Scientific) indata-dependent acquisition mode, where the top 10 peaks per acquisition cyclewere selected for collision-induced fragmentation. Peptides were identified bysearching the spectra against the human proteome using the SEQUEST

Fig. 6. TRX inactivates IL-4 and ameliorates establishedchronic pancreatitis (CP). Thioredoxin (i.p., 250 mg/kg,two times per d, 3 d/wk) was administered to mice3 wk after starting CP induction and mice were killedafter 4 wk of cerulein injections. (A) Relative pancreasweights from CP- and TRX-treated mice are shown.Means ± SEM; n = 10 per group. (B) ELISA analysis ofrelative pancreatic tissue at the IL-4 level in control(Con), CP-, and TRX-treated mice are shown. For serumIL-4 levels, see SI Appendix, Fig. S3A. Mouse IL-4 ELISAused for this study can only detect active IL-4 (SI Ap-pendix, Fig. S4). Means ± SEM. (C) Representativepancreatic histological slides by H&E and Trichromestaining. (Scale bar, 100 μm.) More sections includedin this study are shown in SI Appendix, Fig. S3B.(D) Quantitative analysis of fibrosis using the imagesfrom Trichrome staining. Means ± SEM. (E–H) RT-PCRanalysis of αSMA, Col1α1, Fn1, and TGF-β expression inthe pancreas of the indicated mice. Means ± SEM; n = 5per group. ns, not significant.

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algorithm (43), with a false discovery rate cutoff of 1% at the peptide level. Therelative amounts of peptides containing Cys3, Cys24, Cys46, Cys65, and Cys99were determined by integration of their precursor (MS1) peak intensities.

Thioredoxin-Mediated Inhibition Assay. TF-1 cells (American Type CultureCollection) were cultured in RPMI medium 1640 (Gibco) supplemented with5% (vol/vol) FBS, 10 mM Hepes, 1 mM sodium pyruvate, and penicillin/streptomycin. TF-1 cells were washed with PBS and seeded at 3 × 105 cells permL in 96-well plates (0.2 mL per well) in medium supplemented with 8 ng/mLrecombinant IL-4, IL-13, or GM-CSF. Cells were incubated with TRX concen-trations ranging from 0 nM to 10 μM at 37 °C and 5% CO2 for 48 h. Cellswere counted on a BD Accuri C6 Cytometer (BD Biosciences). Data werecollected in triplicate and fit to a log(inhibitor) vs. response (three-parameter) equation using GraphPad Prism 6.

Pancreatitis Model and Treatment. Chronic pancreatitis was induced by re-petitive cerulein injections (44). In brief, mice were given six hourly i.p. injectionsof 50 μg·kg−1 body weight of cerulein (Sigma-Aldrich) 3 d/wk for a total of 4 wk.Mice were then killed 3 d after the last cerulein injection, and pancreatic tissueswere analyzed. For the TRX therapy, all mice were given cerulein injections 3 d/wkfor a total of 4 wk as above, and 3 wk following start of the cerulein injections,mice were either given vehicle control (PBS) or TRX (i.p., 250 mg/kg, two timesper d, 3 d/wk for 1 wk) until being killed at the fourth week. The StanfordInstitutional Animal Care and Use Committee (IACUC) approved all animalstudies, and animals were housed in an Association for Assessment andAccreditation of Laboratory Animal Care (AAALAC)-accredited facility.

Quantitative RT-PCR. Total RNAwas isolated frompancreatic tissue using TRIzolreagent (Invitrogen) according to the manufacturer’s instructions. In brief,cDNA was generated using the GoScript Reverse-Transcription System(Promega). Quantitative PCR was performed with an ABI 7900 SequenceDetection System (Applied Biosystems) using designed specific TaqManprobes and primers as follows: αSMA (forward, 5′-CTCCCTGGAGAA-GAGCTACG-3′; reverse, 5′-TGACTCCATCCCAATGAAAG-3′; probe, 5′-AAAC-GAACGCTTCCGCTGCC-3′); collagen 1A1 (forward, 5′-AGAAGGCCAGTCTGGAGAAA-3′;reverse, 5′-GAGCCCTTGAGACCTCTGAC-3′; probe, 5′-TGCCCTGGGTCCTCCTGGTC-3′);fibronectin (forward, 5′-TGGTGGCCACTAAATACGAA-3′; reverse, 5′-GGAGGGC-TAACATTCTCCAG-3′; probe, 5′-CAAGCAGACCAGCCCAGGGA-3′); TGF-β (forward,5′-CCCTATATTTGGAGCCTGGA-3′; reverse, 5′-CTTGCGACCCACGTAGTAGA-3′;probe, 5′-CCGCAGGCTTTGGAGCCACT-3′); and GAPDH (forward, 5′-TGTGTCCGTCGTGGATCTGA-3′; reverse, 5′-CCTGCTTCACCACCTTCTTGA-3′;probe, 5′-CCGCCTGGAGAAACCTGCCAAGTATG-3′). Samples were normalizedto GAPDH and displayed as fold induction over untreated controls, unlessotherwise stated.

Statistical Analysis. Unpaired Student’s t test was used to determine statis-tical significance between two groups. Values are expressed as mean ± SEM(Prism 7; GraphPad Software).

ACKNOWLEDGMENTS. The authors thank E. Arner (Karolinska Institutet) forgenerously providing the pSUABC plasmid for expression of sel genes (selA,selB, selC) and Yi Wei for technical assistance. The authors acknowledgetechnical support from the Stanford Human Immune Monitoring Centerwith the Luminex assay. This research was supported by NIH Grants R01DK063158 (to C.K.) and R01 DK105263 (to A.H.).

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