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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: [email protected].
This article contains supporting information online at
www.pnas.org/lookup/suppl/doi:10.1073/pnas.1805288115/-/DCSupplemental.
Published online August 13, 2018.
www.pnas.org/cgi/doi/10.1073/pnas.1805288115 PNAS | August 28,
2018 | vol. 115 | no. 35 | 8781–8786
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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
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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.).
1. Holmgren A, Lu J (2010) Thioredoxin and thioredoxin
reductase: Current researchwith special reference to human disease.
Biochem Biophys Res Commun 396:120–124.
2. Nakamura H (2004) Thioredoxin as a key molecule in redox
signaling. Antioxid RedoxSignal 6:15–17.
3. Rubartelli A, Bajetto A, Allavena G, Wollman E, Sitia R
(1992) Secretion of thioredoxinby normal and neoplastic cells
through a leaderless secretory pathway. J Biol
Chem267:24161–24164.
4. Lillig CH, Holmgren A (2007) Thioredoxin and related
molecules—From biology tohealth and disease. Antioxid Redox Signal
9:25–47.
5. Xu S-Z, et al. (2008) TRPC channel activation by
extracellular thioredoxin. Nature 451:69–72.
6. Azimi I, Matthias LJ, Center RJ, Wong JWH, Hogg PJ (2010)
Disulfide bond that constrainsthe HIV-1 gp120 V3 domain is cleaved
by thioredoxin. J Biol Chem 285:40072–40080.
7. Nakamura H, et al. (2001) Chronic elevation of plasma
thioredoxin: Inhibition ofchemotaxis and curtailment of life
expectancy in AIDS. Proc Natl Acad Sci USA 98:2688–2693.
8. Maurice MM, et al. (1999) Expression of the
thioredoxin-thioredoxin reductase systemin the inflamed joints of
patients with rheumatoid arthritis. Arthritis Rheum
42:2430–2439.
9. Tamaki H, et al. (2006) Human thioredoxin-1 ameliorates
experimental murine colitis inassociation with suppressed
macrophage inhibitory factor production.
Gastroenterology131:1110–1121.
10. Kurimoto C, et al. (2007) Thioredoxin may exert a protective
effect against tissuedamage caused by oxidative stress in salivary
glands of patients with Sjögren’s syn-drome. J Rheumatol
34:2035–2043.
11. Jin X, et al. (2011) Activation of extracellular
transglutaminase 2 by thioredoxin. J BiolChem 286:37866–37873.
12. DiRaimondo TR, Plugis NM, Jin X, Khosla C (2013) Selective
inhibition of extracellularthioredoxin by asymmetric disulfides. J
Med Chem 56:1301–1310.
13. Plugis NM, Palanski BA, Weng CH, Albertelli M, Khosla C
(2017) Thioredoxin-1 se-lectively activates transglutaminase 2 in
the extracellular matrix of the small intestine:Implications for
celiac disease. J Biol Chem 292:2000–2008.
14. Schwertassek U, et al. (2007) Selective redox regulation of
cytokine receptor signalingby extracellular thioredoxin-1. EMBO J
26:3086–3097.
15. Wu C, et al. (2014) Identification of novel nuclear targets
of human thioredoxin 1.Mol Cell Proteomics 13:3507–3518.
16. Chanput W, Mes JJ, Wichers HJ (2014) THP-1 cell line: An in
vitro cell model for im-mune modulation approach. Int
Immunopharmacol 23:37–45.
17. Spiller KL, et al. (2016) Differential gene expression in
human, murine, and cell line-derived macrophages upon polarization.
Exp Cell Res 347:1–13.
18. Mosser DM, Edwards JP (2008) Exploring the full spectrum of
macrophage activation.Nat Rev Immunol 8:958–969.
19. May RD, Fung M (2015) Strategies targeting the IL-4/IL-13
axes in disease. Cytokine 75:89–116.
20. LaPorte SL, et al. (2008) Molecular and structural basis of
cytokine receptor pleiotropyin the interleukin-4/13 system. Cell
132:259–272.
21. Peterson JD, Herzenberg LA, Vasquez K, Waltenbaugh C (1998)
Glutathione levels inantigen-presenting cells modulate Th1 versus
Th2 response patterns. Proc Natl AcadSci USA 95:3071–3076.
22. Jeannin P, et al. (1995) Thiols decrease human interleukin
(IL) 4 production and IL-4-induced immunoglobulin synthesis. J Exp
Med 182:1785–1792.
23. Curbo S, et al. (2009) Regulation of interleukin-4 signaling
by extracellular reductionof intramolecular disulfides. Biochem
Biophys Res Commun 390:1272–1277.
24. Kruse N, Lehrnbecher T, Sebald W (1991) Site-directed
mutagenesis reveals the im-portance of disulfide bridges and
aromatic residues for structure and proliferativeactivity of human
interleukin-4. FEBS Lett 286:58–60.
25. Eisenmesser EZ, et al. (2000) Expression, purification,
refolding, and characterizationof recombinant human interleukin-13:
Utilization of intracellular processing. ProteinExpr Purif
20:186–195.
26. Holmgren A (1979) Thioredoxin catalyzes the reduction of
insulin disulfides by di-thiothreitol and dihydrolipoamide. J Biol
Chem 254:9627–9632.
27. Lefort S, Vita N, Reeb R, Caput D, Ferrara P (1995) IL-13
and IL-4 share signal trans-duction elements as well as receptor
components in TF-1 cells. FEBS Lett 366:122–126.
28. Xue J, et al. (2015) Alternatively activated macrophages
promote pancreatic fibrosisin chronic pancreatitis. Nat Commun
6:7158.
29. Klöppel G, Maillet B (1995) Development of chronic
pancreatitis from acute pancre-atitis: A pathogenetic concept.
Zentralbl Chir 120:274–277.
30. Klöppel G (2007) Chronic pancreatitis, pseudotumors and
other tumor-like lesions.Mod Pathol 20(Suppl 1):S113–S131.
31. Mosmann TR, Coffman RL (1989) TH1 and TH2 cells: Different
patterns of lymphokinesecretion lead to different functional
properties. Annu Rev Immunol 7:145–173.
32. Wynn TA (2015) Type 2 cytokines: Mechanisms and therapeutic
strategies. Nat RevImmunol 15:271–282.
33. Liang HE, et al. (2011) Divergent expression patterns of
IL-4 and IL-13 define uniquefunctions in allergic immunity. Nat
Immunol 13:58–66.
34. Urban JF, Jr, et al. (1998) IL-13, IL-4Ralpha, and Stat6 are
required for the expulsion of thegastrointestinal nematode parasite
Nippostrongylus brasiliensis. Immunity 8:255–264.
35. Finkelman FD, et al. (2004) Interleukin-4- and
interleukin-13-mediated host pro-tection against intestinal
nematode parasites. Immunol Rev 201:139–155.
36. Fallon PG, et al. (2002) IL-4 induces characteristic Th2
responses even in the combinedabsence of IL-5, IL-9, and IL-13.
Immunity 17:7–17.
37. McKenzie GJ, Fallon PG, Emson CL, Grencis RK, McKenzie AN
(1999) Simultaneousdisruption of interleukin (IL)-4 and IL-13
defines individual roles in T helper cell type 2-mediated
responses. J Exp Med 189:1565–1572.
38. Rankin JA, et al. (1996) Phenotypic and physiologic
characterization of transgenicmice expressing interleukin 4 in the
lung: Lymphocytic and eosinophilic inflammationwithout airway
hyperreactivity. Proc Natl Acad Sci USA 93:7821–7825.
39. Fallon PG, Emson CL, Smith P, McKenzie ANJ (2001) IL-13
overexpression predisposesto anaphylaxis following antigen
sensitization. J Immunol 166:2712–2716.
40. Yodoi J, Matsuo Y, Tian H, Masutani H, Inamoto T (2017)
Anti-inflammatory thio-redoxin family proteins for medicare,
healthcare and aging care. Nutrients 9:1081.
41. Whiting CC, et al. (2015) Large-scale and comprehensive
immune profiling andfunctional analysis of normal human aging. PLoS
One 10:e0133627.
42. Rappsilber J, Mann M, Ishihama Y (2007) Protocol for
micro-purification, enrichment,pre-fractionation and storage of
peptides for proteomics using StageTips. Nat Protoc2:1896–1906.
43. Eng JK, McCormack AL, Yates JR (1994) An approach to
correlate tandem massspectral data of peptides with amino acid
sequences in a protein database. J Am SocMass Spectrom
5:976–989.
44. Lerch MM, Gorelick FS (2013) Models of acute and chronic
pancreatitis. Gastroenterology144:1180–1193.
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