Download T Cells + Spread to CD4 Immature Dendritic Cells and Limits HIV

IFN-α Induces APOBEC3G, F, and A in
Immature Dendritic Cells and Limits HIV-1
Spread to CD4+ T Cells
This information is current as
of August 12, 2017.
Venkatramanan Mohanram, Annette E. Sköld, Susanna M.
Bächle, Sushil Kumar Pathak and Anna-Lena Spetz
J Immunol 2013; 190:3346-3353; Prepublished online 20
February 2013;
doi: 10.4049/jimmunol.1201184
http://www.jimmunol.org/content/190/7/3346
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References
The Journal of Immunology
IFN-a Induces APOBEC3G, F, and A in Immature Dendritic
Cells and Limits HIV-1 Spread to CD4+ T Cells
Venkatramanan Mohanram,1 Annette E. Sköld,1,2 Susanna M. Bächle,
Sushil Kumar Pathak,3 and Anna-Lena Spetz
I
mmature myeloid dendritic cells (DCs) are present in the
cervico-vaginal mucosa and have been proposed to be one of
the first target cells during HIV-1 transmission (1). They are
characterized by low expression of costimulatory molecules and
high capacity for Ag uptake. Upon encounter with Ags capable of
triggering myeloid DC maturation, DCs gain a migratory potential
and decrease uptake of additional Ags. Subsequently, the myeloid
DCs upregulate costimulatory signals required for effective T cell
priming (2, 3). Immature myeloid DCs are susceptible to HIV-1
infection, which can lead to productive infection of the cells or
hijacking by the virus (4). HIV-1 can use myeloid DCs as a Trojan
horse, which leads to effective dissemination of the virus from the
periphery to the local lymph nodes, enabling close contact with
T cells (5). Although myeloid DCs can become infected with HIV1, they do not allow replication of the virus to a high extent, which
may at least in part be explained by expression of restriction
factors that can limit viral replication.
Department of Medicine, Center for Infectious Medicine, Karolinska Institutet,
Karolinska University Hospital Huddinge, S-141 86, Stockholm, Sweden
1
V.M. and A.E.S. contributed equally to this work.
2
Current address: Department of Tumor Immunology, Nijmegen Centre for Molecular Life Sciences, Nijmegen, The Netherlands.
3
Current address: Department of Genetics, Microbiology, and Toxicology, Stockholm
University, Stockholm, Sweden.
Received for publication April 23, 2012. Accepted for publication January 17, 2013.
This work was supported by the European Commission (EC-FP7-242135 CHAARM
and EC-FP6-037611 EUROPRISE), the Hedlunds Foundation, SLL-ALF, and the
Swedish Foundation for Strategic Research. The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Address correspondence and reprint requests to Dr. Anna-Lena Spetz, Center for
Infectious Medicine, F59, Karolinska Institutet, Karolinska University Hospital Huddinge, S-141 86, Stockholm, Sweden. E-mail address: [email protected]
Abbreviations used in this article: A3G, APOBEC3G; APOBEC3, apolipoprotein B
mRNA–editing, enzyme-catalytic, polypeptide-like 3; DC, dendritic cell; env, envelope; MDDC, monocyte-derived DC; PEG, pegylated; Vif, Virion infectivity protein.
Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1201184
The apolipoprotein B mRNA–editing, enzyme-catalytic, polypeptide-like 3 family (APOBEC3) proteins comprise six members
of cytidine deaminases (6). The expression of APOBEC3G (A3G)
is elevated in monocyte-derived DCs (MDDCs) upon maturation,
leading to suppression of reverse transcription and introduction of
G-to-A hypermutations in the viral genome. Two other members
of the A3 family, A3F and A3A, were also shown to be expressed
in MDDCs and exert antiviral activity (6–8). Although humans have
evolved powerful retroviral restriction factors, HIV-1 has acquired
specific means to antagonize these antiviral defense mechanisms.
The HIV-1 Virion infectivity protein (Vif) promotes virion maturation and infectivity and was shown to counteract the activity of
A3G (6). In the absence of Vif, the antiviral effect of A3G is exerted
via its incorporation into newly formed virions in the virus-producing
cell. A successful incorporation leads to deamination of the subsequent viral DNA synthesized during the next round of the viral
life cycle (9, 10). However, deaminase-independent mechanisms
of viral inhibition were also described. In cases in which Vif is
present in sufficient amounts, A3G is targeted to an E3-ubiquiting
ligase complex, leading to its degradation in the virus-producing
cell. Hence, there will be a race between the target cell and the
incoming virus, in which the combat may depend on the expression levels of restriction enzymes already present upon encounter
with the virus. There are examples of reports suggesting that A3
proteins may indeed exert an inhibitory effect directly on incoming viruses when the amount of Vif is relatively low (11, 12).
In addition, as the HIV-1 replication in MDDCs is relatively low,
this could theoretically give more time for the restriction enzymes
to act. However, it is presently not fully explored how different
members of the APOBEC family of proteins are regulated in
MDDCs to achieve protection against HIV-1 infection.
APOBEC family proteins can be induced by type I IFNs, such
as IFN-a (6). There are several members of the IFN-a protein
family, but they bind a common cellular receptor to exert antiviral activities (13). Different type I IFNs stimulate different anti-
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Cytokines and IFNs, such as TNF-a and IFN-a, upregulate costimulatory molecules in monocyte-derived dendritic cells (MDDCs),
enabling effective Ag presentation to T cells. This activation of MDDCs is often accompanied by upregulation of apolipoprotein B
mRNA–editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) (A3) family proteins that are able to restrict HIV-1 replication in
MDDCs by inducing hypermutations in the viral genome. In this study, we show that TNF-a upregulates costimulatory molecules
and are able to restrict HIV-1BaL replication in MDDCs without significant induction of A3G, A3A, or A3F. Conversely, low
quantities of IFN-a failed to upregulate costimulatory molecules, did not induce IL-12p40 or migration, but significantly induced
A3G, A3A, and A3F mRNA expression and restricted viral replication in MDDCs. We also showed that transmission of HIV-1 from
MDDCs to autologous T cells was significantly reduced in the presence of IFN-a. Sequence analyses detected the induction of high
frequency of G-to-A hypermutations in the env genes from HIV-1BaL–infected MDDCs treated with low quantities of IFN-a2b. These
findings show that low quantities of IFN-a can induce functional A3 family proteins and restrict HIV-1 replication in MDDCs while
keeping an immature nonmigratory phenotype, supporting further investigations of modalities that enhance retroviral restriction
factors. In addition, the findings highlight the role of IFN-a as a double-edged sword in HIV-1 infection, and we show that IFN-a can
be powerful in reducing HIV-1 infection both in MDDCs and T cells. The Journal of Immunology, 2013, 190: 3346–3353.
The Journal of Immunology
Materials and Methods
Human subjects and blood collection
Buffy coats from healthy human blood donors were obtained from the blood
bank at Karolinska University Hospital Huddinge. Ethical approval was
obtained from the medical ethics committee in Stockholm.
MDDC cultures
CD14+ monocytes were enriched from buffy coats by negative selection
using RosetteSep Human Monocyte Enrichment (1 ml/10 ml blood; Stem
Cell Technologies, Vancouver, BC, Canada). Monocytes were separated
using Lymphoprep density gradient (Nycomed, Oslo, Norway) and were
cultured for 6 d in MDDC medium containing RPMI 1640 (Life Technologies, Paisley, U.K.), supplemented with 1% HEPES [N-(2-hydroxyethyl)piperazine-N9-2-ethanesulfonic acid] (Life Technologies), 1 mM
sodium pyruvate (Life Technologies), 2 mM L-glutamine (Life Technologies), 1% streptomycin (Life Technologies), 1% penicillin (Life Technologies), and 10% endotoxin-free FBS (Life Technologies) with the
addition of recombinant human cytokines IL-4 (6.5 ng/ml; R&D Systems,
Minneapolis, MN) and GM-CSF (250 ng/ml; PeproTech; London, U.K.) to
obtain immature DCs (23). Immature MDDCs were left untreated (medium) or treated either with LPS (100 ng/ml; Sigma-Aldrich, St. Louis,
MO) or with TNF-a at single-dose increasing concentration (0.3, 3, 30 ng/ml;
R&D Systems) or with different subtypes of IFN-a (pegylated [PEG] IFNa2b, IFN-a2a, IFN-aA/D) at single-dose increasing concentration (102, 103,
104 U/ml; IntronA; Schering-Plough, R&D Systems). MDDCs were analyzed by flow cytometer for CD80 and CD86 expression 2 d after IFN-a or
TNF-a treatment.
Phenotypic characterization of MDDCs
MDDCs were stained with the following anti-human mAbs: CD1a (clone
NA1/34; DAKO, Glostrup, Denmark), CD14 (clone TÜK4; DAKO), CD3
(clone SK7; BD Biosciences), CD80 (clone L307.4; BD Biosciences), and
CD86 (clone 2331/FUN-1; BD Biosciences). Cell surface expression was
measured by a FACSCalibur flow cytometer (BD Biosciences). MDDCs
were gated on CD1a+, CD32, and CD142 cells, and 5 3 104 cells/sample
were acquired.
HIV-1 growth and preparation
The HIV-1 isolate HIV-1BaL (which uses CCR5 receptors) was obtained
from the National Institutes of Health AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases (National Institutes of Health, Bethesda, MD). HIV-1
isolates were grown in PBMC cultures stimulated with PHA (2.5 mg/ml;
Sigma-Aldrich) and IL-2 (75 IU/ml; Chiron, Emeryville, CA). Primary
virus was isolated from HIV-infected patient blood by coculture with PHAactivated donor PBMCs. To concentrate the virus and to minimize the
presence of bystander activation factors in the supernatant that could induce
MDDC maturation, virus stocks were ultracentrifuged (138,000 3 g for 30
min at 4˚C) (Beckman L-80 Ultra-centrifuge, rotor 70.1; Beckman Coulter,
Fullerton, CA), and the virus pellets were resuspended in RPMI 1640 with
10% FBS to obtain a virus concentrate. The titers of virus stocks were 1.7 3
106/ml 50% tissue culture infectious dose for HIV-1BaL.
HIV-1 infection of MDDCs
For infection of MDDCs, 3000–6000 50% tissue culture infectious dose/
million cells of HIV-1BaL were used (19). The percentage of infected
MDDCs was determined by intracellular p24 staining after 7 d of infection,
as previously described (23, 24). Cells were first stained for cell surface
markers (CD1a and CD86) and then fixed in 2% formaldehyde (SigmaAldrich). Cells were washed in saponin solution (PBS with 2% FBS, 2%
HEPES, and 0.1% saponin; Sigma-Aldrich), to allow permeabilization of
the cell surface membrane, and then incubated for 1–2 h at 4˚C with a p24
mAb (clone KC57; Coulter, Hialeah, FL) or the corresponding isotype
control Ab. p24 expression was assessed by a FACSCalibur flow cytometer
(BD Biosciences), and data were analyzed using FlowJo software (Tree
Star). Gates were set on CD1a+ cells, and 5 3 104 cells were acquired per
sample. Supernatants from MDDC cultures were harvested 6, 8, and 10 d
after HIV-1 infection, and RT activity was quantified using the Lenti-RTactivity kit (Cavidi Tech, Uppsala, Sweden).
HIV-1 MDDC–T cell transfer assay
Monocytes and CD4+ T cells were prepared from the same donors using
RosetteSep enrichment kits (Stem Cell Technologies). The T cells were
frozen at 280˚C in 10% DMSO, 20% FCS, and 70% complete RPMI 1640
medium. On day 6, MDDCs were infected with HIV-1BaL and treated with
PEG IFN-a2b at single-dose increasing concentration (102, 103, 104 U/ml),
whereas the T cells were thawed and cultured in complete RPMI 1640
supplemented with IL-2 (75 IU/ml; Chiron) for 2 d. Two days postinfection, the virus was thoroughly washed away from the MDDC cultures and
the autologous IL-2–treated T cells were added to the MDDCs in a 2:1
ratio. Cells were cultured for 4 d in the presence or absence of PEG IFNa2b at single-dose increasing concentration (102, 103, 104 U/ml). The cells
were then stained with CD1a, CD3, CD86, and p24 mAbs, and 1 3 105
cells/sample were acquired in a FACSCalibur.
Cytokine detection
Supernatants from HIV-1BaL cultures were treated with 0.5% Triton X-100
to inactivate free virus. Secretion of human IL-12p40 was thereafter
measured with an ELISA kit (Mabtech AB, Stockholm, Sweden).
Real-time PCR analysis of A3A, A3F, and A3G
RNA was extracted using the RNeasy Mini Kit (Qiagen), either from freshly
isolated MDDCs or from MDDCs treated with LPS or with TNF-a or with
different subtypes of IFN-a, as described above. RNA was reverse transcribed
using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems).
Amplification of A3A, A3F, A3G, and 18S RNA was performed using the
7500 Real-Time PCR System (Applied Biosystems) and FAM dye-labeled
TaqMan MGB probes and primers (Applied Biosystems) (25, 26). We did
a homology BLAST for the primer probe, which is highly specific to the
above-mentioned A3 molecules. Cycle threshold values for A3 were normalized to the value for 18S in control experiments. Data are presented as
fold changes in mRNA copy number in the MDDCs treated with cytokines
as compared with mRNA in MDDCs cultured in medium only.
HIV-1 env sequencing and analysis
Total cell DNA was isolated from cells treated with PEG IFN-a2b, 72 h
postinfection with a QIAamp DNA mini kit (Qiagen). HIV-1 envelop (env)
V1–V5 gene region was amplified by performing high-fidelity nested PCR
using an EasyA kit (Stratagene). The primers used were as follows: round
1 (product size 1415 bp), forward 59-TTGCAATAGAAAAATTCTCCTC-39,
and reverse 59-CCTGGTGGGTGCTACTCCTA-39); round 2 (product size
1098 pb), forward 59-CCATGTGTAAAGTTAACCCC-39, and reverse, 59ATGAGGGACAATTGAGAAGTGTCTAG-39; PCR condition as described
in (27). Amplicons were purified by using a high pure PCR product purification kit (Roche), and cloned into the TA cloning vector provided in a
TOPO TA cloning kit (Invitrogen) in accordance with the manufacturer’s
instructions. Plasmids were isolated using Genejet plasmid mini prep
kit (Fermentas), and the plasmids were sent to Eurofins MWG (Operon,
Germany) for sequencing. Primers M13 forward (59-GTAAAACGACGGCCAG-39) and M13 reverse (59-CAGGAAACAGCTATGAC-39) were
used for sequencing. G-to-A mutations were analyzed by using the program
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viral, antiproliferative, and clinical responses (14). The IFN receptor is coupled to a tyrosine kinase, which phosphorylates STAT
proteins (15). IFN-a and LPS were shown to induce MDDC maturation and upregulate A3G in MDDCs (16–18). We previously
showed that TNF-a plays a significant role in HIV-1BaL replication restriction in MDDCs that were cocultured with activated
apoptotic T cells (19). The coculture with activated apoptotic T cells
also led to induction of MDDC maturation as measured by upregulation of costimulatory molecule CD80 and CD86 expression
(19, 20). Induction of MDDC maturation was previously shown
to be accompanied by induction of A3G and A3F expression in
MDDCs (7), and maturation suppresses HIV-1 infection through
multifaceted mechanisms that involve decreased viral fusion (4),
a block of reverse transcription (21), and a postintegration restriction that have been proposed to exist at the transcriptional level
(22). In this study, we set out to investigate whether inducers of
MDDC maturation such as TNF-a and IFN-a result in similar induction of expression of A3G, A3F, and A3A with subsequent inhibition of HIV-1 replication. We also measured whether this treatment
is accompanied by increased MDDC maturation in terms of expression of costimulatory molecules, secretion of IL-12p40, and migratory
capacity, as these are central functional properties of MDDCs.
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APOBEC3G, F, AND A INDUCTION IN IMMATURE DENDRITIC CELLS
Hypermut 2.0 that is freely available at http://www.hiv.lanl.gov/content/
sequence/HYPERMUT/hypermut.html (28).
Migration assay
Transwell chambers (8 mm; BD Biosciences) were inserted into wells of
24-well plates containing 750 ml complete RPMI 1640 supplemented with
CCL21 (250 ng/ml; R&D Systems) and MDDCs were added in the upper
well. The cell concentration in the lower chamber was assessed after 4 h by
trypan blue exclusion of nonviable cells in an Auto T4 Cellometer
(Nexcelom Bioscience, Lawrence, MA).
Results
Induction of CD80 and CD86 expression in MDDCs after
treatment with IFN-a or TNF-a
MDDC maturation can be triggered by proinflammatory cytokines
such as TNF-a and IFN-a (29). To investigate whether exogenously
added TNF-a and IFN-a can trigger MDDC maturation in the
present culture system, immature MDDCs were treated with TNF-a
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FIGURE 1. CD80 and CD86 expression as well as
IL-12p40 secretion in human MDDCs. Human in vitro
differentiated monocytes cultured for 6 d in the presence of IL-4 and GM-CSF were used as the source of
human immature MDDCs. Immature MDDCs were
exposed to HIV-1BaL, treated with cytokines for 48 h,
and then analyzed for expression of CD80 and CD86
molecules by flow cytometry. Gates were set on live
large CD1a+CD32 cells. LPS, which is a potent DC
activator, was used as a positive control, and MDDCs
cultured in medium only were used as a negative
control. Immature MDDCs were cultured in medium
alone or in the presence of LPS or with TNF-a at
single-dose increasing concentration (0.3, 3, 30 ng/ml)
or with different subtypes of IFN-a (PEG IFN-a2b,
IFN-a2a, IFN-aA/D) at single-dose increasing concentration (102, 103, 104 U/ml) in at least three independent experiments. (A) The average percentages of
CD80+ MDDCs 6 SD for 11 donors. (B) The average
percentages of CD80+ MDDCs 6 SD for 7 donors. (C)
The average percentages of CD86+ MDDCs 6 SD for
11 donors. (D) The average percentages of CD86+
MDDCs 6 SD for 7 donors. (E) MDDCs were treated
with increasing concentrations of TNF-a (0.3, 3, 30 ng/
ml), PEG IFN-a2b (102, 103, 104 U/ml), or with LPS as
a positive control, in the presence of HIV-1BaL. The
secretion of IL-12p40 was measured by ELISA in
supernatants taken at 24 h of culture. The data are
presented as pg/ml 6 SD for 6 donors. Significant
differences compared with medium control were assessed
by the paired one-way ANOVA with Bonferroni´s
multiple comparison test, and significance is indicated by **p , 0.01 and ***p , 0.001.
or PEG IFN-a2b at single-dose increasing concentrations (0.3, 3,
30 ng/ml and 102, 103, 104 U/ml, respectively). We used a PEG IFN,
as the addition of PEG may delay clearance of the protein and
is used in the clinic for treatment of hepatitis C in combination
with ribavarin (30). The MDDCs were collected and stained for
CD80 and CD86 for flow cytometry analysis 48 h post–TNF-a or
PEG IFN-a2b treatment. LPS served as a positive control. We
detected a significant increase in the expression of CD80 and CD86
in a dose-dependent manner in the cells that were treated with
TNF-a (Fig. 1A, 1C). Similar data were obtained for PEG IFN-a2b
displaying significant induction of CD80 and CD86 after stimulation with the highest dose of PEG IFN-a2b, but not when using
the lower concentrations (102 or 103 U/ml) (Fig. 1A, 1C). Additional phenotypic analyses showed induction of CD83 and HLADR using the highest concentrations of IFN-a or TNF-a. The 7aminoactinomycin D staining did not reveal any toxic effects on
the cells even at higher concentration of the cytokines (data not
shown). To investigate whether the pegylation may have affected
The Journal of Immunology
the IFN-a2b from inducing the expression of costimulatory molecules, we further treated MDDCs with non-PEG IFN-a (IFN-a2a,
IFN-aA/D) at single-dose increasing concentrations (102, 103, 104
U/ml). We confirmed that different IFN-a subtypes (with or without
PEG), at high doses, can induce expression of costimulatory molecules (Fig. 1B, 1D).
Mature MDDCs produce cytokines to influence the subsequent
adaptive immune response. One cytokine important for Th1 induction is IL-12, and we therefore investigated whether IL-12p40
release into the supernatant correlated with the expression of
maturation markers following HIV-1BaL infection and single-dose
increasing concentrations of TNF-a or IFN-a (0.3, 3, 30 ng/ml and
102, 103, 104 U/ml, respectively). The release of IL-12p40 correlated with TNF-a treatment, but was not induced by PEG IFNa2b (Fig. 1E). Significant high quantity of IL-12p40 was only
induced after stimulation with the positive control LPS.
Restriction of HIV-1BaL replication in MDDCs upon treatment
either with TNF-a or IFN-a
(Fig. 2A). Thus, our data suggest that both TNF-a and IFN-a can
significantly reduce viral replication in MDDCs.
Reduced HIV-1 transfer from MDDCs to autologous T cells in
the presence of PEG IFN-a2b, but not TNF-a
We next investigated the viral transfer from HIV-1–infected
MDDCs to cocultured autologous IL-2–activated T cells. MDDCs
were infected with HIV-1BaL and treated with increasing concentrations of PEG IFN-a2b (102, 103, 104 U/ml) or the highest
concentration of TNF-a for 2 d. Thereafter, the cultures were extensively washed, to prevent further infection from unbound virus.
IL-2–activated autologous T cells were added to the MDDCs in a
ratio of 2:1 and either left untreated (Fig. 3A) or continuously
treated with PEG IFN-a2b (Fig. 3B) for 4 d. The p24 expression
was then investigated in both T cells and MDDCs. MDDCs pretreated with the highest dose of PEG IFN-a2b (104 U/ml) displayed significantly reduced viral transfer to T cells even without
addition of new PEG IFN-a2b (Fig. 3A). An even more impressive
reduction of viral transfer was measured if the PEG IFN-a2b (102,
103, 104 U/ml) treatment was continued also with the addition of
T cells. A significant reduction of the viral transfer was measured
for both the intermediate and the higher concentration of PEG IFNa2b (103 and 104 U/ml) (Fig. 3B). Similar results were obtained for
the MDDC infection in the coculture (data not shown). However,
pretreating the MDDCs with the TNF-a concentration that protected the MDDCs from HIV-1BaL infection (30 ng/ml) (Fig. 2A)
did not significantly alter the viral transfer to autologous T cells as
compared with nontreated control (Fig. 3). Hence, TNF-a protects
MDDCs from high HIV-1BaL infection by the induction of maturation, but this is not protecting interacting T cells from the infection. However, PEG IFN-a2b protects MDDCs from high HIV1BaL infection without inducing a mature phenotype, and also
protects interacting T cells, even in the absence of exogenously
added PEG IFN-a2b. Therefore, we continued to compare the induction of host-restriction factors in MDDCs treated with either
PEG IFN-a2b or TNF-a at single-dose increasing concentrations
(102, 103, 104 U/ml and 0.3, 3, 30 ng/ml, respectively).
PEG IFN-a2b, but not TNF-a, upregulates mRNA expression
of A3G, A3F, and A3A
IFN-a is known to upregulate A3 molecules that possess cytidine
deaminase activity in various cell types (32, 33). A3 molecules are
FIGURE 2. Frequency of HIV-infected MDDCs. Immature MDDCs were exposed to HIV-1BaL, in the presence of LPS or with TNF-a at single-dose
increasing concentration (0.3, 3, 30 ng/ml) or with different subtypes of IFN-a (PEG IFN-a2b, IFN-a2a, IFN-aA/D) at single-dose increasing concentration (102, 103, 104 U/ml). The percentage of infected MDDCs was assessed by flow cytometry analysis of intracellular p24 staining after 6–7 d in at least
three independent experiments. (A) The average percentage of p24+ MDDCs 6 SD after 6–7 d after treatment with cytokines for 10 donors. (B) The
average percentage of p24+ MDDCs 6 SD after 6–7 d after treatment with non-PEG IFN-a for 6 donors. Significant differences compared with HIV-1BaL–
infected control were assessed by the paired one-way ANOVA with Bonferroni´s multiple comparison test, and significance is indicated by ***p , 0.001.
(C) MDDCs were infected with HIV-1BaL. After 2 d, the cells were thoroughly washed and further cultured for 8 d. Supernatants from 4 donors were taken
at 6, 8, and 10 d postinfection, and the reverse-transcriptase activity was measured with a Lenti RT activity kit.
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We previously showed that TNF-a plays a significant role in restriction of HIV-1BaL replication in MDDCs that are cocultured
with activated apoptotic T cells (19). In addition, a-IFNs are known
to play a major role in antiviral activity (13, 14, 31). To further
investigate the role for TNF-a and IFN-a in inhibition of viral
replication, HIV-1–infected MDDCs were treated either with TNF-a
at single-dose increasing concentration (0.3, 3, 30 ng/ml) or with
three different subtypes of IFN-a (PEG IFN-a2b, IFN-a2a, IFNaA/D) (102, 103, 104 U/ml), respectively. The cells were collected
and quantified for expression of the HIV-1 structural protein p24 by
flow cytometry 6 d after TNF-a and IFN-a treatments (Fig. 2A,
2B). The release of viral particles into the supernatant of infected
cells did not increase after this time point (Fig. 2C). We have
previously shown that the intracellular detection of p24 requires de
novo synthesis of viral proteins and fails to detect capture of HIV-1
virions (23). There was large variability between donors with
regard to HIV-1 infection efficiency, ranging from 8.9 to 59.4%
after 6 d, as previously reported (19). However, there was a significant decrease in percentage of p24+ in cells treated with IFN-a
in a dose-dependent manner (Fig. 2A, 2B). All three concentrations
of different subtypes of IFN-a could effectively reduce p24 percentage. TNF-a, in contrast, could reduce the frequency of p24positive MDDCs only at the highest concentration (i.e., 30 ng/ml)
3349
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APOBEC3G, F, AND A INDUCTION IN IMMATURE DENDRITIC CELLS
potent in inducing mutations of a broad spectrum of retroviruses,
including HIV-1 (34–37). A3A was recently reported to be expressed in MDDCs and inhibited early phases of HIV-1 infection
(8). In this study, we investigated whether A3G, A3F, and A3A
were upregulated in MDDCs exposed to HIV-1 and treated with
TNF-a and PEG IFN-a2b at single-dose increasing concentration
(0.3, 3, 30 ng/ml and 102, 103, 104 U/ml), respectively. RT-PCR
was performed 24 h post–TNF-a and IFN-a treatment. There was
a significant upregulation of A3G, A3F, and A3A, when the cells
were treated with PEG IFN-a2b, even at the lowest concentration. However, no significant upregulation of these molecules was
detected even at the higher concentration of TNF-a (Fig. 4). Hence,
we confirm a previous study showing expression of A3A in
MDDCs and further show that a low concentration (102 U/ml) of
FIGURE 4. A3G, A3F, and A3A mRNA expression after treatment with
TNF-a and PEG IFN-a2b. Real-time PCR was used to determine fold
changes in mRNA for A3G (A), A3F (B), and A3A (C) in MDDCs exposed
to HIV-1BaL and treated with LPS, TNF-a at single-dose increasing concentration (0.3, 3, 30 ng/ml), or with PEG IFN-a2b at single-dose increasing concentration (102, 103, 104 U/ml). mRNA levels were compared
among MDDCs cultured in medium for 24 h. Data represent the mean 6 SD
from four different donors. Significant differences compared with medium
control were assessed by the nonparametric Mann–Whitney U test and are
indicated as *p , 0.05.
PEG IFN-a2b is sufficient to induce up to a 100-fold (range fold
increase 9.5–285) increase of A3A as well as significant induction
of A3G and A3F.
PEG IFN-a2b induces G-to-A hypermutation in MDDCs
infected with HIV-1BaL
To investigate whether there were signs of A3 family protein activity, we screened for G-to-A hypermutation in the HIV-1 env
genes that were amplified from the DNA obtained from the infected MDDCs treated with PEG IFN-a2b (10 2 U/ml). The env
region was chosen for analyses because several studies have confirmed that A3 exhibits a pronounced 59 to 39 editing gradient,
resulting in a particular susceptibility to A3 cytidine deaminase
activity (38, 39). Sixty different clones were sequenced, and the
total numbers of bp investigated were 51,000. We observed a high
frequency of G-to-A hypermutations in the DNA that were obtained from the cells treated with the relatively low concentration
Downloaded from http://www.jimmunol.org/ by guest on August 12, 2017
FIGURE 3. Viral transfer from HIV-1–exposed MDDCs to autologous
IL-2–activated T cells in the presence of TNF-a or PEG IFN-a2b. MDDCs
were treated with PEG IFN-a2b at single-dose increasing concentration
(102, 103, 104 U/ml) or with TNF-a at 30 ng/ml for 2 d. The cells were then
extensively washed and thereafter cocultured with autologous T cells that
had been treated with IL-2 (75 IU/ml) for 2 d. The PEG IFN-a2b was either
washed away (A) or replenished in the same concentration as during the
HIV-1 infection (B). After additional 4 d, the cultures were assessed for p24
expression and phenotypical markers in at least three independent experiments. The average percentage of CD3+ p24+ T cells 6 SD for 10 donors is
displayed. Significant differences compared with HIV-1BaL–infected control
were assessed by the paired one-way ANOVA with Bonferroni´s multiple
comparison test, and significance is indicated by *p , 0.05 and **p , 0.01.
The Journal of Immunology
3351
Table I. G-to-A mutation frequencies in HIV-1 env sequences obtained from HIVBaL-infected MDDCs treated with PEG IFN-a2b
Item Analyzed
MDDC PEG IFN-a2b–Treated
102 U/ml (env V1, V2, V4, V5)
Total no. of clonesa
Total no. of bp
Total no. of clones Fisher exact
p value ,0.05
Total no. of G-to-A mutations
No. of G-to-A mutations (GG context)
No. of G-to-A mutations (GA context)
Average no. of G-to-A mutations/100 bp
MDDC PEG IFN-a2b–Treated
102 U/ml (env V3 Region)
60
33,240
9
60
17,760
5
1,615
496
831
4.85
860
245
360
4.84
MDDC PEG IFN-a2b–Treated
102 U/ml (Total env)
60
51,000
14
2,475
741
1,191
4.85
a
Representative clones from three donors were sequenced.
cells were used as a background control, and LPS-treated cells
were used as a positive control for migration. For some donors,
CCL21 was instead added in the top chamber as a control, but the
number of migrating cells did not differ from untreated cells (data
not shown). In accordance with the relative lack of costimulatory
molecules, MDDCs treated with PEG IFN-a2b did not migrate
toward the CCL21 gradient (Fig. 5). The LPS-treated cells, however, migrated efficiently after pretreatment, but not when stimulated just prior to the migration assay.
MDDC migration is not induced by PEG IFN-a2b treatment
In this study, we measured the induction of A3G, A3F, and A3A
antiviral restriction factors and activation/maturation of MDDCs
after HIV-1 infection and treatment with TNF-a or IFN-a. Our
study shows significant inhibition of HIV-1BaL replication and induction of A3G, A3F, and A3A in MDDCs after incubation with
relatively low concentration of PEG IFN-a2b (102 U/ml). The
lower PEG IFN-a2b concentrations of 102 U/ml and 103 U/ml did
not induce MDDC maturation, as we did not detect significant
induction of the costimulatory molecules CD80 and CD86, or secretion of IL-12p40. The presence of 103 U/ml PEG IFN-a2b was,
however, sufficient to protect autologous IL-2–activated T cells
from high HIV-1 infection in a MDDC–T cell transmission setting.
None of the investigated PEG IFN-a2b concentrations induced a
migratory phenotype in MDDCs. However, higher concentrations
of PEG IFN-a2b induced expression of costimulatory molecules
and a dose-dependent induction of APOBEC3 family proteins.
Notably, the relatively low concentration of 102 U/ml was sufficient
to induce significant G-to-A mutations (4.85 per 100 bp) in env
sequences obtained from the MDDCs.
Cell-free HIV-1 virions can be relatively poor inducers of type I
IFN production compared with HIV-1–infected cells, as suggested
in a recent study showing that HIV-1–infected cells induced 10–
100 times more IFN-a (mean 600 U/ml) than cell-free HIV-1
virions in vitro (40). It remains to be elucidated whether a local
IFN-a release at the site of transmission, without concomitant
proinflammatory responses, can induce sufficient restriction factors
to protect from infection. In a recent study, significant induction of
A3G and A3F as well as reduction (20.921 [60.858] log10 copies/
ml) of viral load was demonstrated in HIV-1/hepatitis C–coinfected
patients undergoing treatment with PEG IFN-a and ribavarin (30).
The plasma concentrations measured in patients after s.c. injection
of PEG IFN-a are in the same order (mean week 1, 1710 pg/ml or
week 4, 2380 pg/ml) (41, 42) as the lower doses used in the current
study (102 U/ml equals 1430 pg/ml PEG IFN-a2b). However, it
remains to be elucidated whether PEG IFN-a can effectively
penetrate into tissue compartments to modulate HIV-1 restriction
factors in DCs and macrophage reservoirs. Although IFN-a has
antiviral activity and can induce factors that limit viral replication
(such as APOBEC molecules, TRIM-5 a, tetherin, and SAMHD1)
Because PEG IFN-a2b did not induce strong maturation of
MDDCs, but upregulated sufficient amounts of A3 molecules to
reduce HIV-1 infection, we investigated whether a migratory
phenotype was induced by the cytokine. MDDCs were treated with
single-dose increasing concentrations (102, 103, 104 U/ml) of PEG
IFN-a2b, either overnight or directly before addition to the upper
well in a Transwell setting (Fig. 5). To stimulate migration of
activated cells, the medium below the Transwell contained the
chemokine CCL21, and the MDDCs were allowed to migrate for
4 h before the number of migrated cells was counted. Untreated
FIGURE 5. Migration of MDDCs toward CCL21. MDDCs were treated
with PEG IFN-a2b at single-dose increasing concentration (102, 103, 104
U/ml) either overnight (o.n.) or directly before addition to Transwells.
LPS-treated cells were used as positive control and medium treated as a
negative control. The lower wells contained CCL21 as a migratory gradient. The number of migrated cells in the lower chamber was counted 4 h
after addition of cells. Data represent the mean 6 SD from eight different
donors analyzed in at least three independent experiments. Significant
differences compared with medium control were assessed by the nonparametric Wilcoxon matched-pairs signed rank test and are indicated as
**p , 0.01.
Discussion
Downloaded from http://www.jimmunol.org/ by guest on August 12, 2017
of PEG IFN-a2b (Table I). There was a similar distribution within
the V3 env region as compared with other variable parts of env, in
total, 14 of the 60 clones harbored significant mutation rates (Fisher
exact p value ,0.05). The total numbers of G-to-A mutations were
2,475, and of these 741 were in GG context, whereas 1,191 were in
GA context. The average number of G-to-A mutations per 100 bp
was 4.85. Our data suggest that even a relatively low concentration
of PEG IFN-a2b, which is not able to induce full MDDC maturation, is yet able to inhibit viral replication by upregulating A3
molecules, which in turn induce G-to-A hypermutation in the viral
genome, causing the viruses to become replication incompetent.
3352
APOBEC3G, F, AND A INDUCTION IN IMMATURE DENDRITIC CELLS
that patients with low viral set point had significantly higher levels
of A3G mRNA both pre- and postinfection compared with patients
that have high viral set point (56). Furthermore, HIV-1–exposed
seronegative individuals were reported to have higher A3G expression levels than healthy controls (58), and PBMCs of HIV-1–
exposed seronegative individuals have higher A3G expression and
decreased susceptibility to infection of HIV-1 in vitro (59). Hence,
it remains to be further explored which levels of APOBEC family
proteins are required for effective block of HIV-1 acquisition
in vivo and means to induce their expression without providing fuel
for HIV-1 infection. It can be noted that highly exposed seronegative women were reported to have upregulated IFN-a expression
in the cervix as quantified by immunohistochemistry (60). In addition, immunomodulatory or immunization strategies inducing
A3G expression were reported to –correlate with reduced viral load
or protection against infection in macaques (26, 61, 62). In this
study, we showed that relatively modest quantity of IFN-a can
significantly induce expression of A3G, A3F, and, in particular,
A3A, without inducing signals that induce MDDC maturation and
migration in vitro. We also showed that transmission of HIV-1 from
MDDCs to autologous T cells is significantly reduced in the presence of IFN-a. This study supports future development of interventions that can enhance APOBEC expression and suggests that
this can be achieved in myeloid DCs without inducing maturation
and migration.
Disclosures
The authors have no financial conflicts of interest.
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