4 is inactivated via selective -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 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, derived from THP-1 cells were 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 outputs of alternatively activated macro- . phages, suggesting that extracellular TRX regulated the activity of Macrophages play a critical role in the immune system based on (IL-4) and/or (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 -gamma (IFN- ) and 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 , 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 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 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 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. 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 is rapidly regenerated by and IL-13 in this proliferation assay were 1.7 and 0.5 μg/mL, 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. In addition, no one has captured a direct in- teraction between any reducing factors and IL-4 or shown that this interaction is physiologically relevant. In this study, we have identified secreted IL-4 but not IL-13 as a preferred substrate of extracellular TRX. In addition to characterizing the redox mechanism of this regulatory process, we have also demon- strated its pathophysiological relevance in an animal model of human disease. Results Extracellular TRX Inhibition Enhances Cytokine Outputs of M2 Macrophages Derived from THP-1 Cells. Because we noticed that macrophage morphology was sensitive to TRX inactivation, we performed a cytokine screen to characterize the effect of TRX on macrophages. 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 to phorbol 12-myristate 13-acetate (PMA); cells thus treated are commonly referred to as unpolarized macrophages in the “M0 state” (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 M2 macrophages. For this exploratory study, we looked for the most profound changes in the concentrations of secreted cytokines. Fig. 2. TRX inhibitor NP161 stimulates cytokine secretion in M2 cells while Among the 62 cytokines screened, changes of at least fivefold inhibiting secretion in M1 cells. THP-1 cells were differentiated into M0 were identified for 11 cytokines (Fig. 2). Notably, addition of macrophages with PMA for 48 h, followed by polarization into M1 macro- NP161 reduces secretion of these cytokines in M1 macrophages, phages with IFN-γ (20 ng/mL) and LPS (1 ng/mL) or into M2 macrophages whereas it increases cytokine levels in M2 macrophages. with IL-4 (20 ng/mL) for 36 h. Then, M1 or M2 cells were exposed to vehicle μ The above observations led us to suspect a role for extracel- or NP161 (33 M). Fold changes in secreted cytokine concentrations (pg/mL) are plotted in binary-logarithmic scale in a heatmap. Cytokines that are lular TRX in suppressing the M2 state of macrophages. Because changed by fivefold or more by NP161 are shown for either M1 or M2 IL-13 can elicit the same immune response as IL-4, our observed macrophages. For a full list of cytokine measurements, see SI Appendix, effect could also be mediated through IL-13. Therefore, we hy- Table S1. Cytokine and levels were determined by a multiplexed pothesized that extracellular TRX influenced this differential Luminex assay. Data are the means from three biological replicates.

8782 | www.pnas.org/cgi/doi/10.1073/pnas.1805288115 Plugis et al. Downloaded by guest on September 29, 2021 5 min (Fig. 4D), and was found to be in line with the specificity of TRX for IL-4 over IL-13, as measured above.

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

TRX Selectively Inactivates the Cytokine Activity of IL-4. A cellular model was used to assess the biological relevance of the observed protein–protein recognition between IL-4 and TRX. Proliferation Fig. 3. Structure of IL-4 and IL-13. Interleukin 4 (A) [ (PDB) of the TF-1 erythroleukemia cell line was evaluated in the pres- ID code 1HIK] and interleukin 13 (B) (PDB ID code 3LB6) are four-helix bundles that each possess three disulfide bonds (shown in yellow). 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 in cultures containing IL-4 whereas, under equivalent conditions, the only a small amount of the putative adduct was observed be- IC50 of TRX was 2.2 μM in cultures containing IL-13. TRX had tween the C35S TRX mutant and IL-13 (Fig. 4A). no effect on TF-1 in the presence of GM-CSF (Fig. 5). To quantify the specificity of TRX for IL-4 versus IL-13, we adopted an established kinetic assay for TRX activity, using insulin TRX Mitigates IL-4–Driven Pathological Conditions in Chronic Pancreatitis. as a reference substrate (11). Steady-state kinetic analysis revealed In light of the above findings in vitro, we sought to assess the path- that TRX had significantly higher specificity toward IL-4 than IL- ophysiological relevance of TRX-mediated IL-4 inactivation in vivo. 13 (Fig. 4B and Table 1). Kinetic parameters for insulin recogni- To do so, we took advantage of a recent study highlighting the role of tion by TRX were in agreement with previously reported data (11, IL-4 signaling in a mouse model of chronic pancreatitis (28). 26). Notably, to our knowledge, IL-4 appears to be the most Chronic pancreatitis is characterized by progressive irrevers- preferred extracellular substrate of TRX identified to date. ible damage to the pancreas (29). Some of the key histological To verify the preference of TRX for IL-4 over IL-13, the features include , , and acinar cell death TRX-promoted rate of IL-4 deactivation was compared in the (30), which in part are promoted by M2 macrophages. Because presence or absence of an initially equal concentration of IL-13. pharmacological inhibition of the IL-4 receptor decreases these To simulate physiological conditions, substantially lower cyto- pathological phenotypes and halts chronic pancreatitis progres- kine concentrations were employed in these assays than those sion (28), we used these pathological features as evidence for IL- used for the estimation of kinetic parameters. As predicted, 4 inactivation in vivo. To that end, we first induced chronic addition of IL-13 had negligible effect on the rate of oxidative pancreatitis in C57BL/6J mice by repetitive injection with deactivation of IL-4 (Fig. 4C). By way of confirmation, the extent cerulein over 4 wk (six injections per d, 3 d/wk). At the beginning of TRX reduction of the two cytokines was directly quantified at of week 3, we initiated dosing of recombinant TRX to diseased

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 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-4 was 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 = 3per group. (D) Direct measurements of IL-4 and IL-13 concentrations of samples withdrawn at 5 min into the experiment corresponding to C.InC 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 oxi- dized, active IL-4 and IL-13, respectively, and display

no cross-reactivity (SI Appendix, Fig. S2). Data are BIOCHEMISTRY mean ± SEM; n = 3 per group.

Plugis et al. PNAS | August 28, 2018 | vol. 115 | no. 35 | 8783 Downloaded by guest on September 29, 2021 inactivation of its cytokine activity. This posttranslational regula- Table 1. Kinetic parameters for TRX-promoted reduction of tory mechanism was shown to attenuate macrophage polarization insulin, IL-4, and IL-13 toward an alternatively activated state. −1 −1 −1 Substrate kcat/KM, μM ·min kcat,min KM, μM The observed specificity of TRX for IL-4 over IL-13 is un- precedented. In retrospect, the existence of such an endogenous Insulin 2.3 130 56 regulatory mechanism should not be surprising, given that experi- Interleukin 4 8.1 370 46 ments involving mice deficient in cytokines, cytokine-producing Interleukin 13 1.6 110 65 cells, or receptor subunits have repeatedly shown that IL-4 and IL- 13 play distinct roles in allergic immunity in vivo (33). Our work has provided a molecular mechanism by which immune and non- mice. Compared with the control group, TRX-treated mice showed a immune cells can regulate local cytokine concentration and effect. marked reduction in the amount of active, oxidized IL-4 in pancreatic Finally, in addition to opening a new window to an immune tissue (Fig. 6B). Moreover, TRX treatment limited pancreatic fibrosis, regulatory mechanism in , our findings may also have as shown by increased pancreas weight, histology, and decreased promise for , given the role of IL-4 in a variety of fibrosis-associated expression (Fig. 6 A and C–H), providing disease states. A number of preclinical and clinical studies have further support for the ability of TRX to inactivate IL-4 in vivo. demonstrated that exogenously administered TRX is generally well- tolerated in mammals (40). By inactivating IL-4 with TRX, we were Discussion able to ameliorate pancreatic fibrosis in a mouse model of chronic The immune response to danger signals involves a complex or- pancreatitis. More generally, inhibition of IL-4 may also provide a chestration of cells and secreted molecules, and is characterized clinical benefit for diseases such as , allergic rhi- by an initial response that is amplified by the activation and re- nitis, , chronic obstructive pulmonary disease, inflammatory cruitment of effector cells followed by resolution of the response. bowel disease, autoimmune disease, and fibrotic disease (19). Thus, In broad terms, two types of immune responses (TH1 and TH2) administration of TRX could represent a potential alternative have been extensively described (31, 32). Macrophages are an approach to monoclonal antibody-based IL-4 inhibition. essential component of both of these responses, becoming acti- Materials and Methods vated in response to tissue microenvironmental signals contrib- uted by microbial components, the innate and adaptive immune Chemicals and Other Reagents. Unless otherwise specified, reagents were from Sigma-Aldrich. DTT was from Invitrogen, SDS/polyacrylamide gradient systems, and damaged cells and tissues (18). Classically activated gels (4 to 20%) were from Bio-Rad, Ni-NTA resin was from Qiagen, the HiTrap (M1) macrophages promote TH1-type inflammatory responses Q anion-exchange column was from GE Healthcare, and 7-kDa molecular along with strong microbicidal and tumoricidal activity, whereas mass cutoff spin columns were from Pierce. Cell-culture medium, FBS, anti- alternatively activated (M2) macrophages are associated with biotics, and sterile PBS were from Invitrogen. Glutamine was from Lonza. TH2-type antiinflammatory responses, , and res- olution of inflammation. While substantial progress has been Macrophage Polarization. The human THP-1 monocytic cell line was main- made in defining the molecular networks underlying macrophage tained in RPMI 1640 culture medium containing 10% heat-inactivated FBS 6 polarization, there is considerable interest in the identification of and penicillin/streptomycin. Cells were seeded at 10 cells per mL and dif- molecules that regulate macrophage polarization. ferentiated into M0 macrophages by incubation for 48 h in the presence of 100 ng/mL phorbol 12-myristate 13-acetate (P8139; Sigma). These M0 cells Two cytokines, IL-4 and IL-13, are the primary regulators of the were then maintained in the same state for an additional 72 h in RPMI type 2 immune response (33). Through exogenous administration, medium. THP-1–derived M0 macrophages were polarized into M1 macro- overexpression, and knockout studies, these two closely related phages by incubation for 36 h with 20 ng/mL IFN-γ (285-IF; R&D Systems) and extracellular proteins have been shown to have overlapping (34– 1 ng/mL LPS (tlrl-pb5lps; InvivoGen). Alternatively, macrophages were polarized 39) but nonredundant (33) roles in immunity. While IL-4 signaling into the M2 state by incubation for 36 h with 20 ng/mL interleukin 4 (I4269; is initiated through both type I and II receptors, IL-13 only signals Sigma) or 20 ng/mL interleukin 13 (50-813-223; Fisher). When needed, 33 μM through binding of type II receptors (20). The two cytokines ap- NP161 (a small-molecule inhibitor of TRX) was added. pear to be differentially regulated, but the underlying mechanisms of differential regulation of immune responses by IL-4 and IL-13 remain poorly understood. The only known way that the two cy- tokines can exhibit unique effects is through exclusive receptor subunit binding and segregation of expression among different cellular and tissue sources (33). Here we have identified and dissected a redox mechanism that differentially regulates the de- activation of IL-4 versus IL-13. Specifically, extracellular TRX selectively recognizes the C46–C99 disulfide of IL-4, leading to

Table 2. Mass spectrometric analysis of the mixed disulfide adduct formed between IL-4 and C35S TRX IAA, relative IAM, relative Residue intensity intensity

Cys3 0 100 Cys24 0 100 Cys46 12.2 87.8 Cys65 0 100 Cys99 11.2 88.8 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 Iodoacetic acid (IAA) was used to label free Cys residues when the two (green triangles), and varying amounts of TRX were added. Viable cells were proteins were coincubated. Iodoacetamide (IAM) was used to label all other counted after 48 h by flow cytometry (forward scatter/side scatter). Data are Cys residues following complete reduction of the protein mixture. mean ± SEM from two biological replicates with three technical replicates.

8784 | www.pnas.org/cgi/doi/10.1073/pnas.1805288115 Plugis et al. Downloaded by guest on September 29, 2021 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 Ap- pendix,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,andTGF-β expression in the pancreas of the indicated mice. Means ± SEM; n = 5 per group. ns, not significant.

Luminex Assay. This multiplexed assay for secreted proteins was performed at IL-4 and IL-13 Competition Assay. Before use, recombinant human TRX was the Human Immune Monitoring Center at Stanford University. Human 62-plex freshly reduced with a 10-fold molar excess of DTT on ice. The excess DTT was was from Affymetrix, and used according to the manufacturer’srecommen- removed by passing the solution through a 7-kDa molecular mass cutoff spin −1 −1 dations with modifications, as detailed elsewhere (41). Briefly, antibody-linked column. TRX concentration was determined by A280 (e = 7,570 M ·cm ), and beads were added to a 96-well plate and washed in a BioTek ELx405 washer. the protein was freshly used within 2 h. Human recombinant IL-4 (200-04) Culture supernatants were added to these wells and incubated at room and recombinant IL-13 (200-13) were from PeproTech. Kinetic analysis of temperature for 1 h followed by overnight incubation at 4 °C with shaking TRX-mediated reduction of IL-4 was performed via a coupled assay con- (500 to 600 rpm on an orbital shaker). Plates were washed in a BioTek ELx405 taining 1 μM TrxR, 5 nM TRX, 60 μM NADPH, 50 nM IL-4, and 50 nM IL-13 in washer, and the biotinylated detection antibody was added for 75 min at PBS buffer (pH 7.4). The rate of oxidative inactivation of IL-4 was compared room temperature with shaking. Plates were washed again as above, and with that in the presence of equimolar IL-13 as a competitive substrate. At streptavidin-phycoerythrin was added. After incubation for 30 min at room specific time points, aliquots were withdrawn and diluted 5,000-fold into temperature, plates were washed once more and reading buffer was added to cold PBS (4 °C). Cytokine concentrations were determined via ELISAs. the wells. Plates were read on a Luminex 200 instrument with a lower bound of 50 beads per sample per cytokine. Each sample was measured in duplicate. Mass Spectrometric Determination of the Target Disulfide Bond in IL-4. A60-μL Control beads (Radix BioSolutions) were added to all wells. solution containing IL-4 (34 μg, 20 μM) and C35S TRX (26 μg, 20 μM) was incubated for 30 min at room temperature in 20 mM Tris·HCl, 1 mM EDTA Cross-Linking of C35S TRX and IL-4 or IL-13. IL-4 (10 μM) or IL-13 (10 μM) was (pH 7.6). The solution was then diluted with 7.5 μL of 8 M urea in 100 mM μ incubated for 30 min at room temperature with C35S TRX (10 M) in 20 mM NH4HCO3 and incubated with iodoacetic acid (320 μg, 25 mM) for 1 h at Tris·HCl, 1 mM EDTA (pH 7.6). Samples were diluted with 2× Laemmli sample room temperature in the dark. The reaction was buffer-exchanged three buffer (Bio-Rad) and applied to a nonreducing 4 to 20% SDS/polyacrylamide times on a Zeba desalting column (Thermo Scientific) into 20 mM Tris, 1 mM

gel (Bio-Rad). EDTA, 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) Thioredoxin Activity Assay. Before use, recombinant human TRX was freshly was added to the samples and allowed to incubate at room temperature in reduced with a 10-fold molar excess of DTT on ice. The excess DTT was re- the dark for 30 min. Reconstituted trypsin solution (20 mg/mL in resus- moved by passing the solution through a 7-kDa molecular mass cutoff spin pension buffer; Promega) was added to a final concentration of 6 μg/mL. −1 −1 column. TRX concentration was determined by A280 (e = 7,570 M ·cm ), and The samples were digested for 4 h in a 37 °C water bath, after which the the protein was freshly used within 2 h. Steady-state kinetic analysis of TRX- digestion was quenched by adding formic acid to a final concentration of mediated reduction of insulin and IL-4 was performed via a coupled assay 7.5% (vol/vol). Peptides were desalted using C18 StageTips (42), lyophilized containing 6 μM TrxR, 10 nM TRX, and 0.3 mM NADPH in a buffer containing overnight, resuspended in 5% formic acid in water, and analyzed by mass 50 mM Tris·HCl and 2 mM EDTA (pH 7.5). The reaction rate was calculated spectrometry on an Orbitrap Elite Mass Spectrometer (Thermo Scientific) in from the slope of the absorbance curve at 340 nm, using the extinction data-dependent acquisition mode, where the top 10 peaks per acquisition cycle − − coefficient of NADPH (6,220 M 1·cm 1). Michaelis–Menten parameters were were selected for collision-induced fragmentation. Peptides were identified by BIOCHEMISTRY determined by fitting the kinetic data using GraphPad Prism 6. searching the spectra against the human proteome using the SEQUEST

Plugis et al. PNAS | August 28, 2018 | vol. 115 | no. 35 | 8785 Downloaded by guest on September 29, 2021 algorithm (43), with a false discovery rate cutoff of 1% at the peptide level. The Quantitative RT-PCR. Total RNA was isolated from pancreatic tissue using TRIzol relative amounts of peptides containing Cys3, Cys24, Cys46, Cys65, and Cys99 reagent (Invitrogen) according to the manufacturer’s instructions. In brief, were determined by integration of their precursor (MS1) peak intensities. cDNA was generated using the GoScript Reverse-Transcription System (Promega). Quantitative PCR was performed with an ABI 7900 Sequence Thioredoxin-Mediated Inhibition Assay. TF-1 cells (American Type Culture Detection System (Applied Biosystems) using designed specific TaqMan α ′ Collection) were cultured in RPMI medium 1640 (Gibco) supplemented with probes and primers as follows: SMA (forward, 5 -CTCCCTGGAGAA- GAGCTACG-3′;reverse,5′-TGACTCCATCCCAATGAAAG-3′;probe,5′-AAAC- 5% (vol/vol) FBS, 10 mM Hepes, 1 mM sodium pyruvate, and penicillin/ GAACGCTTCCGCTGCC-3′); collagen 1A1 (forward, 5′-AGAAGGCCAGTCTGGAGAAA-3′; streptomycin. TF-1 cells were washed with PBS and seeded at 3 × 105 cells per reverse, 5′-GAGCCCTTGAGACCTCTGAC-3′;probe,5′-TGCCCTGGGTCCTCCTGGTC-3′); mL in 96-well plates (0.2 mL per well) in medium supplemented with 8 ng/mL fibronectin (forward, 5′-TGGTGGCCACTAAATACGAA-3′;reverse,5′-GGAGGGC- recombinant IL-4, IL-13, or GM-CSF. Cells were incubated with TRX concen- TAACATTCTCCAG-3′;probe,5′-CAAGCAGACCAGCCCAGGGA-3′); TGF-β (forward, μ trations ranging from 0 nM to 10 M at 37 °C and 5% CO2 for 48 h. Cells 5′-CCCTATATTTGGAGCCTGGA-3′; reverse, 5′-CTTGCGACCCACGTAGTAGA-3′; were counted on a BD Accuri C6 Cytometer (BD Biosciences). Data were probe, 5′-CCGCAGGCTTTGGAGCCACT-3′); and GAPDH (forward, 5′- collected in triplicate and fit to a log(inhibitor) vs. response (three- TGTGTCCGTCGTGGATCTGA-3′; reverse, 5′-CCTGCTTCACCACCTTCTTGA-3′; parameter) equation using GraphPad Prism 6. probe, 5′-CCGCCTGGAGAAACCTGCCAAGTATG-3′). Samples were normalized to GAPDH and displayed as fold induction over untreated controls, unless Pancreatitis Model and Treatment. Chronic pancreatitis was induced by re- otherwise stated. petitive cerulein injections (44). In brief, mice were given six hourly i.p. injections − of 50 μg·kg 1 body weight of cerulein (Sigma-Aldrich) 3 d/wk for a total of 4 wk. Statistical Analysis. Unpaired Student’s t test was used to determine statis- Mice were then killed 3 d after the last cerulein injection, and pancreatic tissues tical significance between two groups. Values are expressed as mean ± SEM were analyzed. For the TRX therapy, all mice were given cerulein injections 3 d/wk (Prism 7; GraphPad Software). for 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 times ACKNOWLEDGMENTS. The authors thank E. Arner (Karolinska Institutet) for per d, 3 d/wk for 1 wk) until being killed at the fourth week. The Stanford generously providing the pSUABC plasmid for expression of sel genes (selA, selB, selC) and Yi Wei for technical assistance. The authors acknowledge Institutional Animal Care and Use Committee (IACUC) approved all animal technical support from the Stanford Human Immune Monitoring Center studies, and animals were housed in an Association for Assessment and with the Luminex assay. This research was supported by NIH Grants R01 Accreditation of Laboratory Animal Care (AAALAC)-accredited facility. DK063158 (to C.K.) and R01 DK105263 (to A.H.).

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