Download Pattern Generated by Oxidative Stress Th2

Reactive Carbonyls Are a Major
Th2-Inducing Damage-Associated Molecular
Pattern Generated by Oxidative Stress
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of June 17, 2017.
Amin E. Moghaddam, Kate H. Gartlan, Leopold Kong and
Quentin J. Sattentau
J Immunol published online 8 July 2011
http://www.jimmunol.org/content/early/2011/07/08/jimmun
ol.1003906
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Published July 8, 2011, doi:10.4049/jimmunol.1003906
The Journal of Immunology
Reactive Carbonyls Are a Major Th2-Inducing
Damage-Associated Molecular Pattern Generated by
Oxidative Stress
Amin E. Moghaddam, Kate H. Gartlan, Leopold Kong, and Quentin J. Sattentau
I
n addition to protecting the host from infection, the immune
system is involved in general homeostasis, exemplified by
roles played by the innate immune system during sterile tissue
stress and injury (1). It has therefore been proposed that the immune system can be alarmed by evolutionarily conserved endogenous structures termed damage-associated molecular patterns
(DAMPs), which either do not occur in, or are not released by,
healthy tissues (2). Since the coinage of this term subsequent to
that of pathogen-associated molecular patterns (3), several candidates have been proposed to act as DAMPs including those involved in the initiation of necrosis-induced inflammation (1).
Although the list is expanding, other classes of DAMPs specific to
homeostatic disturbances, such as commonly occurring oxidative
stress, await further elucidation.
Oxidation is both harnessed and tightly controlled in biological
systems, but when present in excess it can lead to oxidative stress,
a common source of tissue and cellular injury (4, 5). Carbohydrate
and lipid oxidation (glycoxidation and lipoxidation) generates a
variety of chemical species, of which aldehydes are a highly reSir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE,
United Kingdom
Received for publication November 29, 2010. Accepted for publication June 9, 2011.
This work was supported by the Leverhulme Trust and Dormeur Investment Service,
Ltd. Q.J.S. is a Jenner Vaccine Institute investigator and a James Martin senior fellow.
Address correspondence and reprint requests to Dr. Amin E. Moghaddam, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford
OX1 3RE, United Kingdom. E-mail address: [email protected]
Abbreviations used in this article: AGE, advanced glycation end product; BMDC,
bone marrow-derived dendritic cell; CML, N«-carboxymethyllysine; CpG ODN, CpG
oligodeoxynucleotide; DAMP, damage-associated molecular pattern; DC, dendritic
cell; FA, formaldehyde; FCA, Freund’s complete adjuvant; GA, glycolaldehyde; HEL,
hen egg lysozyme; HNE, 4-hydroxy-2-nonenal; MDA, malondialdehyde; RC, reactive
carbonyl; SPDC, splenic dendritic cell.
Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003906
active by-product (6). In addition to direct toxicity, aldehydic modification of biomolecules, particularly of proteins, gives rise to
a multitude of adducts that are increasingly implicated in the
pathology of a wide range of human diseases with prominent oxidative components (4–6). For many such conditions, including
atherosclerosis, diabetes, Alzheimer’s disease, and alcohol liver
disease, there exists a plethora of evidence in support of a critical
role for innate immune engagement by lipoxidation- and glycoxidation-derived products (5), in particular aldehyde-modified proteins and phospholipids (7, 8). Such extensive, and in many cases
proinflammatory, involvement of innate immunity is likely to provide fertile ground for the elicitation of adaptive immune responses. In support of this, T cell and B cell responses targeted to
both modified and unmodified endogenous Ags have been well
documented in oxidation-driven pathologies (9), exemplified by
autoantigen and antiadduct Abs observed in atherosclerosis and
alcohol liver disease (10, 11). However, far less is known about the
molecular and immunological pathways by which these adaptive
responses are initiated.
Reactive carbonyl (RC) adduction to proteins is generally
considered a hallmark of direct or aldehyde-mediated oxidative
stress (12). RCs were first implicated as the active moieties in
driving adaptive immune responses against model Ags treated
with glycolaldehyde (GA), a product of glycoxidation and the
myeloperoxidase/H2O2/chloride pathway (13, 14), and NaIO4, a
common oxidizing agent (15). We subsequently extended this
observation by demonstrating a role for formaldehyde-generated
RCs in an exaggerated Th2-biased immune response implicated
in vaccine hypersensitivity (16). Similar to GA, 4-hydroxy-2nonenal (HNE) and malondialdehyde (MDA) are among the
most common bioactive aldehyde products of oxidative stress
(17), whose adducts have been implicated in various immune
pathologies (5, 18) and linked to induction of adaptive immunity
(19, 20). Because HNE and MDA attack on proteins can result
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Oxidative stress is widespread and entwined with pathological processes, yet its linkage to adaptive immunity remains elusive.
Reactive carbonyl (RC) adduction, a common feature of oxidative stress, has been shown to target proteins to the adaptive immune
system. Because aldehydes are important mediators of carbonylation, we explored the immunomodulatory properties of model Ags
modified by common bioactive aldehyde by-products of oxidative stress: 4-hydroxy-2-nonenal, malondialdehyde, and glycolaldehyde. Ag modification with all three aldehydes resulted in Ag-specific IgG1-dominated responses in adjuvant-free murine immunizations in an RC-dependent manner. The central role of RCs was confirmed, as their reduction into nonreactive groups abrogated
all adaptive responses, despite the presence of other well-known aldehyde-driven adducts such as N«-carboxymethyllysine and
glycolaldehyde–pyridine. Moreover, Ag-specific Ab responses robustly correlated with the extent of RC adduction, regardless of
the means of their generation. T cell responses mirrored the Th2-biased Ab isotypes by Ag-specific splenocyte production of IL-4,
IL-5, and IL-13, but not IFN-g. The RC-induced Th2 response was in sharp contrast to that induced by Th1/Th2 balanced or Th1biasing adjuvants and was maintained in a range of mouse strains. In vitro studies revealed that RC adduction enhanced Ag
presentation with Th2 polarization in the absence of conventional dendritic cell activation. Taken together, these data implicate
commonly occurring RC as an important oxidation-derived Th2 immunomodulatory damage-associated molecular pattern with
potentially important roles in health and disease. The Journal of Immunology, 2011, 187: 000–000.
2
in RC adduction (21, 22), we investigated the significance of
such structures and confirm here that RCs are indeed the central
immune-active moieties via which these aldehydes modulate Ag
immunogenicity. Furthermore, we show that RCs induce a profound Th2 biasing effect regardless of their mode of generation.
Taken together, these data underscore the importance of the RC
group as the main immune-active moiety of adducted structures
on oxidatively stressed Ags and highlight their role in the Th2
polarization of Ag-specific immune responses.
Materials and Methods
Mice
Female BALB/c, 129S6/SvEv, CBA, C57BL/6, and DO.11.10 3 SCID
mice were bred and maintained in accredited University of Oxford facilities and used for experimental procedures at ages 6–9 wk. All procedures
were authorized by institutional ethical review and conducted in accordance with the U.K. Home Office Animals (Scientific Procedures) Act
1986.
REACTIVE CARBONYLS INDUCE Th2 RESPONSES
OVA or hen egg lysozyme (HEL; 0.25–0.5 mg/ml) were incubated with 20
mM GA, MDA [released from bis salt according to the method of Kikugawa and Ido (23)], or HNE (Axxora, San Diego, CA) solutions in LPSfree PBS (Life Technologies, Carlsbad, CA) pH 7.4 for 3 h at 37˚C. For
dose-response experiment, OVA was modified with 2 or 20 mM GA or
formaldehyde (FA). Unbound aldehydes were removed by desalting via
Amicon Ultra or Microcon filters (Millipore). To reduce RCs, 20–50 mM
NaBH4 was used either at the time or after aldehyde treatment followed by
desalting as above. For oxidation, 0.25–0.5 mg/ml OVA was incubated
with 1 mM NaIO4 in sodium acetate buffer pH 4.5 at 4˚C in the dark for 90
min, followed by desalting and reduction as above. The protein concentration in samples was measured using a BCA Protein assay (Pierce,
Rockford, IL).
Mass spectrometry and carbonyl assays
Protein samples for mass spectrometry were extensively buffer exchanged
into dH2O and then acidified with formic acid before being purified with
a C4 reverse-phase Ziptip (Millipore, Bedford, MA). The eluted samples
were vacuum-dried and then reconstituted in acetonitrile before injection
into an LTQ Orbitrap mass spectrometer (Thermo Scientific, Hertfordshire,
U.K.). All data were analyzed with XCalibur Software (Thermo Scientific).
Protein modification
RC measurement
DNPH-based colorimetric and ELISA assays were carried out as published
previously (16). Briefly, for ELISA, protein samples were reacted with 10
mM DNPH solution in 2 M HCl for 45 min followed by coating on ELISA
plates (Greiner bio-one, Frikenhausen, Germany) overnight in pH 8.5
NaHCO3 buffer and subsequent detection of DNPH-tagged RCs using
FIGURE 1. Immunogenicity of RC-adducted Ags. A, Primary amines on proteins (P) are attacked by aldehydes and via various intermediates (pathway
1) result in RC, commonly detected using DNPH (pathway 2). RC can be reduced to nonreactive hydroxy-alkyl adducts (pathway 3) or could yield other
end products (pathway 4). B, Protein RC adduction by HNE, MDA, and GA and their reduced adducts. C, HEL was aldehyde-treated under nonreducing
(HNE, MDA, GA) or reducing (-R) conditions, and RC content (carbonyl) was measured using DNPH ELISA (top graph). Ten micrograms of preparations
in PBS were used to immunize BALB/c mice (n = 5), and HEL-specific IgG isotypes were monitored at week 8 by ELISA (middle and bottom graphs). D,
Mice (as in C) were immunized with 10 mg OVA, unmodified or RC-adducted through treatment with 2 or 20 mM GA or FA or by NaOI4 oxidation, and
corresponding reduced forms. RC content was measured using a quantitative colorimetric method, and Ab responses were monitored as in C. All groups
were then pooled and used for regression analysis. Carbonyl data are mean + SD, and immunization plots are box and whiskers with minimum and
maximum. Data are representative of at least two independent experiments. *p # 0.05, **p # 0.01, ***p # 0.001.
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Chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless
otherwise stated. Protein Ags were either cleared of endotoxin using DetoxiGel endotoxin removing gel (Pierce, Rockford, IL) or purchased as an
endotoxin-free product (EndoGrade OVA; Hyglos). The endotoxin levels
were externally tested (Lonza, Verviers, Belgium) and reported as ranging
from very low (#0.2 EU/ml) to undetectable. For aldehyde modification,
The Journal of Immunology
a biotinylated anti-DNPH Ab (Molecular Probes, Eugene, OR) (16).
Colorimetric detection of tagged DNPH was used for quantitative measurements as described previously (16) using molar absorption coefficient
of DNPH (22,000 M21cm21) in reacted samples.
Immunization
Six- to nine-week-old mice were immunized s.c. in the flank using 10–50 mg
of unmodified or modified proteins in 100 ml LPS-free PBS (Life Technologies) or with unmodified protein adjuvanted 1:1 in 50 ml Imject Alum
(Pierce) containing 2 mg aluminum hydroxide, 50 ml Freund’s complete
adjuvant (FCA; Sigma-Aldrich, St. Louis, MO) 1:1, or 20 mg ODN1826
CpG oligonucleotide (InvivoGen, San Diego, CA) where stated. In some
experiments, mice were further boosted with 2–50 mg of either unmodified
or modified adjuvant-free protein preparations between week 6 and 12
postpriming as indicated. Mice were bled prior to and after immunizations
and sacrificed for final blood samples and spleen harvests.
Ab ELISA and titer calculation
Statistical analysis
Statistical analyses were performed using Prism software version 5.0
(GraphPad Software). In vivo data were handled using non-parametric tests
(Mann–Whitney U test; Kruskal–Wallis test and Dunn post hoc test for
multiple comparisons; Spearman test for correlation). All other data were
tested using Student t test or one-way ANOVA and appropriate post hoc
tests (Dunnett test for multiple comparisons with control). Means and
standard deviations are presented for normally distributed data sets,
whereas for in vivo data, no assumption of normality was made and data
are depicted by median and range.
Results
RC adduction to proteins by common aldehyde by-products of
oxidative stress enhances Ag immunogenicity
Adduction of RCs to Ags (Fig. 1A, reaction 1) by GA, FA, and
NaIO4 treatment has previously been shown to result in enhanced
Ag immunogenicity (15, 16). HNE and MDA are among the most
studied aldehyde by-products of oxidative stress (5) and have been
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Sera from immunized mice were serially diluted, starting at 1/20, on ELISA
plates (Greiner bio-one) coated with 5–10 mg/ml unmodified OVA or HEL,
and Ag-specific isotype reactivity was detected using appropriate antimouse isotype HRP conjugates (Total IgG, Serotec, Oxford, U.K.; IgG1
and IgG2a, BD Biosciences, Oxford, U.K.; IgG2c, GeneTex). Plates were
developed using a TMB (3,39,5,59-tetramethylbenzidine) substrate (Pierce)
and absorbance (A450) determined using a SpectraMax M5 microplate
reader (Molecular Devices, Berkshire, U.K.). End-point titers were extrapolated, using cutoff points determined based on preimmune sera reactivity means at 1/20 6 3 SD, from sigmoidal dose-response curves (4PL
or 5PL) upon log transformation of data in Prism software version 5.0
(GraphPad Software) and expressed as reciprocal titers. For dose-response
units, OD values at a serum dilution of 1/100 were normalized to the
background response elicited by carbonyl-free unmodified OVA samples.
3
Splenocyte cytokine secretion
Splenocytes were harvested from immunized mice at indicated time points,
cultured at the density of 2 3 106/ml in 24-well plates (Corning) in the
absence or presence of 50 mg/ml unmodified Ag. Supernatants were collected at days 1–5 and frozen at 280˚C. Cytokines were quantified using
Bio-Plex mouse cytokine kits (BioRad, Herts, U.K.) acquired and quantified on a Luminex 100 reader (Luminex, Austin, TX) according to the
manufacturer’s instructions. Wells without Ag had cytokine levels below
the detection limit of the assay ranging from 3 to 5 pg/ml.
Dendritic cell pulsing and T cell proliferation
Dendritic cells (DCs) were purified from BALB/c spleen by enzymatic
digestion and density gradient centrifugation as published previously (24),
followed by CD11c MACS positive selection (Miltenyi Biotec, Surry,
U.K.) achieving purities $95% CD11c+MHC II+. Purified DCs were then
pulsed with native or modified OVA at 100 mg/ml for 3 h at 37˚C for Ag
presentation assays or 12 h at 37˚C for cytokine production and cell surface analysis by FACS. CD4+ T cells were isolated from DO11.10 3 SCID
mice by MACS negative selection (Miltenyi Biotec) and labeled with 2.5
mM CFSE (Invitrogen, Paisley, U.K.). T cells (5 3 105) were then coincubated with 1 3 105 pulsed DCs (3 h) for a further 60–72 h. Supernatants
were collected for cytokine analysis, and CD3+ T cell proliferation was
assessed by flow cytometric CFSE dilution analysis using FACSCalibur
(BD Biosciences) and FlowJo software (Tree Star, Ashland, OR). Immature DCs (bone marrow-derived dendritic cells; BMDCs) were derived by
culturing murine bone marrow in the presence of 20 ng/ml rGM-CSF
(R&D, Abingdon, U.K.) for 6 d (adapted from Ref. 25) to $85%
CD11c+CD11b+ purity before being pulsed and characterized as described
for splenic dendritic cells (SPDCs). Cytokine analysis was performed using Bio-Plex as specified earlier, and for FACS analysis directly fluoresceinated Abs (BD Biosciences; Serotec, Oxford, U.K.) were employed
using CellQuest software (BD Biosciences).
Gel electrophoresis
One to five micrograms of native or modified HEL or OVA was prepared and
loaded onto Novex minigels (Invitrogen) and run under reducing conditions
according to the manufacturer’s protocol. Gels were subsequently stained
using a silver-staining kit (Amersham International, Buckinghamshire,
U.K.) or SimplyBlue SafeStain (Invitrogen) following the manufacturer’s
protocols.
FIGURE 2. The role of RC versus other oxidation-derived structures
in enhanced Ag immunogenicity. A, GA-treated HEL (HEL/GA, top) was
further reduced (HEL/GA-R, bottom), and the major adduct peaks were
determined using Orbitrap mass spectrometry. B, Samples from A (GA and
GA-R) along with unmodified HEL (Unmod) were run on SDS-PAGE and
visualized using Coomassie blue-based staining and tested for their RC
content (carbonyl) before they were used to immunize BALB/c mice (n = 4
and 5). Anti-HEL IgG1 responses were determined by ELISA at week 12.
C, NaIO4-oxidized OVA (Ox) was subsequently reduced (Ox-R), and
samples were characterized and tested as in B using silver staining. Carbonyl data are mean + SD, and immunization plots are box and whiskers
with minimum and maximum. *p # 0.05, **p # 0.01.
4
REACTIVE CARBONYLS INDUCE Th2 RESPONSES
FIGURE 3. Ab isotype bias by RC-adducted Ags in comparison with other well-known immunomodulators. A, BALB/c mice (n = 5) were immunized
s.c. with 10 mg HEL, RC-adducted by GA treatment, or formulated in aluminum hydroxide (Alum) or administered in FCA. At week 10, all groups
received a boost of 2 mg unmodified HEL, and Ab titers were determined a week later. B, 129S6/SvEv mice (n = 5) were immunized with 10 mg HEL,
unmodified, treated with GA, or admixed with CpG, and boosted 6 wk later with 10 mg unmodified HEL. Ab responses determined as before. C, Mice (n =
5 and 6) were immunized with 50mg GA-treated HEL once (CBA) or twice 3 wk apart (C57BL/6), and Ab responses were determined as before. Plots are
box and whiskers with minimum and maximum. Data are representative of at least two independent experiments. *p # 0.05.
FIGURE 4. In vivo Th polarization profiles
of RC-adducted Ag. A, BALB/c (n = 5) were
immunized with 10mg HEL, unmodified
(Unmod), GA-treated (GA), or GA-treated
and reduced (GA-R), and boosted once with
2 mg HEL 6 wk later. At week 12, splenocyte
cultures were pulsed ex vivo with HEL, and
day 1 and 5 supernatants were assayed for
cytokines using the Luminex multiplex system. B, IL-4 and IFN-g secretion from the
HEL/FCA immunized mice, run in the same
experiment as A, is compared with other
groups. C, Splenocyte cytokine profile of
HNE and MDA modified HEL, or HEL in
alum, run as in A. D, Splenocyte cytokine
profile of 129S6/SvEv or C57BL/6 mice (n =
4 and 5), immunized and boosted with 10 or
50 mg GA-treated HEL, respectively. Box
and whiskers are minimum and maximum.
Data are representative of at least two independent experiments. *p # 0.05, **p #
0.01.
appeared to be related to the HEL RC content (Fig. 1C). To investigate this further, we adducted RCs to a second model Ag, the
glycoprotein OVA, by aldehyde treatment (2 or 20 mM FA or GA)
or by NaIO4 oxidation of the glycan (16, 27, 29). After adjuvantfree immunization of BALB/c mice, OVA-specific total IgG
responses, pooled irrespective of the method of modification,
correlated robustly with the moles of adducted RCs on OVA (r =
0.71, p = 0.036; Fig. 1D). Given that these methods can also
generate distinct arrays of other products, such a correlation
confirms the data for HEL (Fig. 1C) and highlights the importance
of RC in the observed Ag immunogenicity.
RCs but not other oxidation-related adducts account for
increased protein immunogenicity
Apart from RC-containing derivatives, aldehyde attack on proteins
can give rise to a multitude of other adducts (6), some of which,
such as N«-carboxymethyllysine (CML) (Fig. 1A, reaction 4) (13),
have been proposed to possess immune-activating properties (30,
31). Selective elimination of RCs from aldehyde–protein adducts
through borohydride reduction [Fig. 1A (reaction 3), 1B (“Reduced Adducts”)] serve as a reliable control in the investigation of
immune-activating roles played by RC moieties (15, 16). These
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implicated in immune modulation (19, 20), but the chemical nature of the immune-modulating activity remains inconclusive.
Because RCs are commonly adducted by HNE and MDA (Fig.
1B) (21, 22), we investigated their effects on model Ag immunogenicity. Treatment of HEL with HNE and MDA resulted in
significantly increased RC content that was comparable with that
adducted by GA (Fig. 1C), as determined by DNPH-based ELISA
(Fig. 1A, reaction 2) (26). Subcutaneous administration to BALB/c
mice of RC-adducted Ag in the absence of adjuvant resulted in
enhanced immunogenicity of HNE, MDA, and GA-modified HEL,
as determined by native Ag-specific IgG responses, comprising
high IgG1 titers but with undetectable or low titers of IgG2a
across groups (Fig. 1C).
RCs can be eliminated via reduction into less reactive alcohol
groups by borohydrides such as NaBH4 (Fig. 1A, reaction 3) (27,
28). Using this method, we eliminated the RCs from HNE- and
MDA-treated HEL (Fig. 1B, 1C), and, similar to GA, this reduced
all HEL immunogenicity to the levels elicited with unmodified
HEL (Fig. 1C). These results suggested that RC may be the central
immune-activating moiety, irrespective of the diversity of HNE,
MDA, or GA adducts present (13, 21, 22). This was consistent
with the observation that the median size of Ab responses
The Journal of Immunology
levels of cross-linked protein species but, similar to unmodified
HEL, it only induced low Ab titers (Fig. 2B). Furthermore, RC
adduction of OVA by NaIO4 oxidation rendered the protein immunogenic despite minimal cross-linking visualized by sensitive
silver staining (Fig 2C), and a subsequent reducing step significantly reduced the Ab responses to low titers (Fig. 2C). These
results highlight the central role of RC moieties in eliciting observed adaptive humoral responses, independent of the adduct
types or of cross-linking.
RCs drive robust Th2-biased responses independently of mouse
genetic background
The Ab responses we observed were all of dominant IgG1 isotypes
(Figs. 1, 2), indicative of Th2-biased immunity (34). To explore
this further, we amplified the humoral responses with a more
extensive prime/boost immunization of BALB/c mice and used
GA treatment of Ag as a particularly efficient means of RC adduction (Fig. 1B, 1C). As before, RC-adducted HEL consistently
elicited high titers of IgG1 but only modest IgG2a titers, similar to
Ag formulated in an aluminum-based adjuvant (alum) (Fig. 3A).
By contrast, HEL administered in FCA induced comparable levels
of IgG1 to RC-adducted Ag but significantly higher IgG2a titers
(Fig. 3A). Certain strains of inbred mice have been attributed with
inherent adaptive immune biases, with BALB/c generally regarded
as Th2-biased (35). To address this, we compared RC-adducted
HEL to HEL adjuvanted in CpG oligodeoxynucleotide (CpG
FIGURE 5. In vitro effects of Ag RC adduction on DC maturation and Ag presentation to T cells. A, DO11.10 T cells were incubated with BALB/c
SPDCs pulsed with OVA, unmodified, GA-treated (GA), or treated and reduced (GA-R), and proliferation was measured by CFSE assay. The representative
histogram (left panel) depicts a typical response from four independent experiments (pooled data, right panel). B, DO11.10 T cell proliferation was
performed as in A along with OVA/CpG mix. Supernatants were collected from T cell/SPDC cultures and cytokines measured using the Luminex multiplex
system. C and D, Surface marker expression (C) and cytokine secretion (D) was determined in SPDCs pulsed as in A along with OVA mixed with LPS or
CpG using flow cytometry and the Luminex multiplex system. Data presented in B–D are means + SD of three independent experiments. *p # 0.05, **p #
0.01, ***p # 0.001.
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reducing agents, when applied at the time of aldehyde reaction
with protein (frequently with the ε-amine of lysine), drive the
reactions toward alcohol groups (Fig. 1A, reaction 3) (27), which
prevents the development of other end products (Fig. 1A, reaction 4). However, reduction post-aldehyde treatment should only
selectively reduce the aldehydic RC groups, leaving other derived
stable products, such as CML, intact. To test this, we first treated
HEL with GA and then reduced the resulted adducts using NaBH4
and analyzed the samples by mass spectrometry. We identified
three major GA–lysine derived adducts: aldoamine, CML, and
GA–pyridine (Fig. 2A, “HEL/GA”) (13, 27, 32). Upon reduction,
the RC-containing aldoamine peak was replaced by the hydroxyethyl alcohol form, whereas the CML and GA–pyridine peaks
remained intact (Fig. 2A, “HEL/GA-R”). Adjuvant-free immunization of BALB/c mice with these samples resulted in HELspecific IgG1 dominant responses only in the sample containing
RC (aldoamine) and not hydroxyethyl, carboxymethyl, and GA–
pyridine lysine derivatives (Fig. 2A, 2B). These results confirm
that RCs are the major immune-modulating adducts of aldehyde
treatment.
RCs can mediate intermolecular cross-linking, potentially giving
rise to multimers that may contribute to enhanced immunogenicity
(33). However, it was shown previously that the monomeric RCbearing fraction of Ag remained as immunogenic as the nonfractionated material containing multimers (15). In this study, HEL
reduced subsequent to GA treatment still contained comparable
5
6
to RC-adducted Ag, we stimulated immature BMDCs with unmodified or GA-treated RC-adducted HEL and OVA. As with the
SPDCs, we found no significant upregulation of CD40, OX40L,
CD80, or CD86 or cytokine release by RC-adducted compared with
unmodified proteins (Fig. 6A, 6B, data not shown). These data
collectively suggest a lack of requirement for conventional activation of APCs for their role in RC-driven Th2 immune enhancement.
Discussion
Oxidative stress is thought to be a major contributor to a diverse
range of human pathologies, yet despite its interactions with innate
immunity, the links to adaptive immune responses have remained
ill-defined. Aldehyde-modified proteins, commonly occurring under oxidative stress, have long been reported to be targeted to a
range of immune-related cells (37–39) and to exhibit immunemodulating activities (40, 41). Allison and Fearon (15) reported
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ODN) in a more balanced strain of mice, 129S6/SvEv (36). As
before, RC adduction induced a robust IgG1-dominated response
in contrast with the Th1-biasing CpG ODN, which exhibited the
expected IgG2a-biased response (Fig. 3B). Finally, the RC-driven
Th2 isotype profile was further reproduced in mouse strains
generally considered to elicit an intrinsically Th1-biased response,
CBA and C57BL/6 (36) (Fig. 3C).
Parallel studies of T cell responses confirmed the RC-driven Th2
bias. Splenocytes from BALB/c mice immunized with GA-treated
HEL, but not the unmodified or GA-treated and reduced samples,
responded to ex vivo restimulation by secreting significant amounts
of IL-2, IL-3, IL-4, IL-5, IL-6, and IL-13, but only trace levels of
TNF-a and no detectable IFN-g (Fig. 4A). This was in contrast to
the FCA-adjuvanted HEL, which showed dominant IFN-g with
minimal IL-4 secretion in the same mouse strain (Fig. 4B). Further, splenocytes from BALB/c mice immunized with MDA or
HNE-treated HEL showed the same Th2 bias in their cytokine
secretion, similar to that of alum-adjuvanted HEL immunized
mice (Fig. 4C). Immunization of the more balanced or Th-1 biased
mouse strains 129S6/SvEv and C57BL/6, respectively, with HEL–
GA similarly elicited IL-5 and IL-13 dominant responses with
minimal TNF-a and IFN-g (Fig. 4D). These data confirm the
profound Th2 biasing property of RC adduction on Ags. To explore this effect at the cellular level, we next investigated RC–Ag–
induced immune modulation in an in vitro Ag presentation model.
REACTIVE CARBONYLS INDUCE Th2 RESPONSES
RC adducts enhance Ag presentation and T cell proliferation
with a Th2 bias in the absence of conventional costimulation
Aldehyde-treated proteins have been reported to be targeted for
increased uptake and processing (37, 38), resulting in enhanced
T cell proliferation (39), in an RC-dependent manner (15).
However, neither the effects of RC-adducted Ag on DC activation
and maturation nor their subsequent polarization of T cells have
been studied. To investigate this, freshly isolated murine SPDCs
were pulsed with unmodified, RC-adducted OVA via GA treatment, or reduced GA-treated OVA then tested for their ability to
induce proliferation of DO11.10 (OVA-TCR transgenic) CD4+
T cells. Significantly enhanced OVA-specific T cell division, as
demonstrated by flow cytometric analysis of CFSE-labeled cells,
was observed for RC-containing samples above that of unmodified
or GA-treated and reduced preparations (Fig. 5A), coordinate with
levels of IL-2 secretion (Fig. 5B). Significant IL-4 and a trend for
increased IL-13 secretion were induced by RC-adducted OVA,
whereas IFN-g was dominantly induced with OVA formulated in
CpG ODN (Fig. 5B). These opposing cytokine profiles corroborate
our earlier immunization results (Figs. 3, 4) lending further support to the Th2-polarizing nature of RC adduction.
To investigate whether RC adducts mediate DC activation,
SPDCs were exposed to modified or unmodified OVA preparations or OVA formulated with LPS or CpG ODN and analyzed by
flow cytometry for surface markers and multiplex assay for cytokine release. Whereas OVA administered with LPS or CpG ODN
resulted in significant upregulation of CD40, CD80, and CD86, no
detectable increases were seen for the RC-adducted sample, which
displayed a phenotype similar to that of unmodified and GA-treated
and reduced OVA (Fig. 5C). Similarly, OVA/LPS or CpG ODN
elicited high levels of IL-1b, IL-6, IL-12 p70, TNF-a, and KC,
whereas RC adduction was associated with weak DC cytokine
responses, indistinguishable from unmodified and GA-treated and
reduced OVA (Fig. 5D).
SPDCs may be partially activated as a result of ex vivo manipulation, probably explaining the high level of MHC class II expression across all groups (Fig. 5C, data not shown). Because it is
plausible that partial activation may mask subtle SPDC responses
FIGURE 6. Impact of RC adduction on BMDC activation. A, BMDCs
(BALB/c) were pulsed with unmodified or RC-adducted HEL (GA-treated)
or with LPS. Surface marker expression was determined using flow
cytometry. Histograms are representative of three independent experiments. B, Cytokine secretion in culture supernatants from A using the
Luminex multiplex system. Data represent means + SD from three independent experiments. *p # 0.05, **p # 0.01, ***p # 0.001.
The Journal of Immunology
Our perception of oxidative damage and disease has substantially shifted toward a more immune-oriented perspective.
Oxidative stress is a collective term implicating various reactions
with distinct chemical modifications, which would inevitably interact, one way or another, with homeostatic maintenance. Although there is some evidence in the literature for other oxidationderived immunomodulators (49), we provide robust evidence in
this study that RCs are a major ubiquitous Th2-biasing oxidationassociated DAMP. From an evolutionary point of view, a selective
advantage for RCs in bridging innate to adaptive immunity is not
yet clear. An alternative nonselective explanation could be that
a heightened state of oxidation, for example due to postindustrial
changes in our diet and environment, can overwhelm the clearance
and repair systems of innate immunity leading to unwanted enhanced Ag presentation and adaptive responses. Although there is
some evidence to suggest that aldehyde/RC-driven Th2 responses
can play both pathogenic (16) and protective roles (50, 51)
depending on the context, a clearer perspective will await more
detailed studies of relevant disease models.
Acknowledgments
We thank Ben Thomas for help with mass spectrometry.
Disclosures
The authors have no financial conflicts of interest.
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26.
REACTIVE CARBONYLS INDUCE Th2 RESPONSES