Involvement of Src and Syk Tyrosine Kinases in HIV-1 T Lymphocytes

Involvement of Src and Syk Tyrosine Kinases in HIV-1 T Lymphocytes
The Journal of Immunology
Involvement of Src and Syk Tyrosine Kinases in HIV-1
Transfer from Dendritic Cells to CD4ⴙ T Lymphocytes1
Caroline Gilbert, Corinne Barat, Réjean Cantin, and Michel J. Tremblay2
Dendritic cells (DCs) are considered as key mediators of the early events in HIV-1 infection at mucosal sites. Although several
aspects of the complex interactions between DCs and HIV-1 have been elucidated, there are still basic questions that remain to
be answered about DCs/HIV-1 interplay. In this study, we examined the contribution of nonreceptor TKs in the known ability of
DCs to efficiently transfer HIV-1 to CD4ⴙ T cells in trans. Experiments performed with specific inhibitors of Src and Syk family
members indicate that these tyrosine kinases (TKs) are participating to HIV-1 transfer from immature monocyte-derived DCs
(IM-MDDCs) to autologous CD4ⴙ T cells. Experiments with IM-MDDCs transfected with small interfering RNAs targeting Lyn
and Syk confirmed the importance of these nonreceptor TKs in HIV-1 transmission. The Src- and Syk-mediated effect on virus
transfer was linked with infection of IM-MDDCs in cis-as monitored by quantifying integrated viral DNA and de novo virus
production. The process of HIV-1 transmission from IM-MDDCs to CD4ⴙ T cells was unaffected following treatment with protein
kinase C and protein kinase A inhibitors. These data suggest that Src and Syk TKs play a functional role in productive HIV-1
infection of IM-MDDCs. Additional work is needed to facilitate our comprehension of the various mechanisms underlying the
exact contribution of Src and Syk TKs to this phenomenon. The Journal of Immunology, 2007, 178: 2862–2871.
T
he involvement of dendritic cells (DCs)3 in the pathogenesis of HIV-1 infection was discovered very soon after the
identification of this retrovirus (1). These cells play a pivotal role not only in establishment and dissemination of HIV-1
infection but also in generating a virus-specific immune response
because they are recognized as the most potent APCs of the immune system. It is now well accepted that the initial attachment
step of HIV-1 to DCs is a complex process modulated by a large
variety of interactions between the virus and the target cell surface
(reviewed in Refs. 2 and 3). For example, the association between
the oligosaccharides found on the external envelope glycoprotein
gp120 and mannose C-type lectin receptors, such as the mannose
receptor (MR) (CD206), langerin (CD207), and DC-specific
ICAM3-grabbing nonintegrin (DC-SIGN), results in the capture
and transmission of HIV-1 to CD4⫹ T cells in an effective transinfectious mode (4 – 8). A viral entity that is bound to C-type lectin
receptors is rapidly taken up within endolysosomal vacuoles and
protected from degradation while remaining in an infectious state
Centre de Recherche en Infectiologie, Centre Hospitalier de l’Université Laval, and
Faculté de Médecine, Université Laval, Quebec, Canada
Received for publication December 28, 2005. Accepted for publication December
12, 2006.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was financially supported by operating grants to M.J.T. from the Canadian
Foundation for AIDS Research (Grant 015 026) and Canadian Institutes of Health Research under the HIV/AIDS Research Program (Grant MOP-79542). C.G. is the recipient
of a fellowship award from the Canadian Institutes of Health Research HIV/AIDS Research Program. J.M.T. holds the Tier 1 Canada Research Chair in Immuno-Retrovirology.
2
Address correspondence and reprint requests to Dr. Michel J. Tremblay, Laboratoire
d’Immuno-Rétrovirologie Humaine, Centre de Recherche en Infectiologie, RC709,
2705 Boulevard Laurier, Quebec G1V 4G2, Canada. E-mail address: michel.j.
tremblay@crchul.ulaval.ca
3
Abbreviations used in this paper: DC, dendritic cell; DC-SIGN, DC-specific ICAM3grabbing nonintegrin; IM-MDDC, immature monocyte-derived DC; M-MDDC, mature
monocyte-derived DC; MR, mannose receptor; PKA, protein kinase A; PKC, protein
kinase C; siRNA, small interfering RNA; PP2, pyrazolopyrimidine.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
www.jimmunol.org
for 1–3 days (9), which is approximately the time required for DCs
to migrate to lymph nodes (10). Following the contact between
DCs and CD4⫹ T cells, the internalized viruses are concentrated at
the virological synapse and are eventually transferred to the latter
cell type where a proficient virus production will ensue (11, 12).
Although some basic understanding of the role played by the
above-mentioned surface proteins in the interaction between
HIV-1 and DCs has been acquired, little is known as yet on the
contribution of intracellular signal transducers in virus capture and
transfer. The possible implication of nonreceptor tyrosine kinases
(nrTKs) in this process deserves some attention considering that
such molecules regulate a wide variety of basic cellular functions
such as proliferation (13, 14), migration (13), endocytosis (15),
antigenic presentation (14, 16), transport of Fc␥-chain to lysosomes (17), phagocytosis (18, 19), production of reactive oxygen
species (20), biosynthesis of lipid metabolites (21), intracellular
signaling (22, 23), and cellular communication (24). To date, the
most studied nrTKs are members of Src and Syk families. Src was
the first TK to be identified by Hunter in 1980 (25). This cellular
homolog of the Rous sarcoma virus-transforming protein (26) is
implicated in intra- and extracellular communication, cellular
growth, and embryonic development (24, 27). In addition to Src,
this family includes several other members such as Blk, Fgr, Fyn,
Lck, Lyn, and Yes, and their roles have been discussed previously
(reviewed in Ref. 23). It is now clear that aggregation or engagement of some specific receptors induces a rapid and transient activation of the Src family members, which then initiate several
downstream signaling pathways (15, 28 –31). Interestingly, similar
observations have been made for Syk family members (i.e., Syk
and Zap-70). Indeed, activation of Syk mediates several of the
responses mentioned above, and Syk also acts as a tumor suppressor in human breast carcinomas (13). Moreover, Syk is involved in
maturation of DCs and IL-12 production (32). Zap-70 is found
primarily in T lymphocytes, and there is still no report on its presence in myeloid cells, including DCs. In contrast, Syk is more
widely expressed because it has been found in hemopoietic cells
(33), epithelial cell lines (34), normal human breast tissue (34), and
endothelial cells (13).
The Journal of Immunology
Activation of Src and Syk TKs in nondividing myeloid cells
such as neutrophils, monocytes, and DCs occurs also by aggregation of cell surface receptors such as MR, Fc␥, complement, dectin, integrin, and several other receptors lacking intrinsic protein
TK activity. In these cells, processes such as endocytosis, phagocytosis, antigenic presentation, and maturation are all regulated by
activation of both TK families (16, 18, 32, 35–39). It has been
shown that Lyn and Syk sequentially bind to Fc␥RII upon phagocytosis-mediated signaling events (35, 40), as they do with the
BCR, the high-affinity Fc␥R, and the TCR (14, 31, 41, 42). Fusion
with lysosomes and targeting of Fc␥-chain to lysosomal degradation have also been attributed to activation of Src and Syk TKs (17,
36). Moreover, these TKs play an important role in lymphocyte
activation in response to ligation of various receptors such as
CD16 (43), TCR (41), Fc␥RII (42), and CD40 (44). Signal transduction events mediated through FcRs, BCRs, or TCRs involve
recruitment of Syk to their ITAMs, and this process is also required for certain subsequent cellular responses such as the targeting of ␥-chains to lysosomes (17, 45).
It has been reported that the uptake of HIV-1 by DCs and its
eventual transfer to more susceptible target cells require some defined receptors, the best known of which are MR and DC-SIGN
(46). Interestingly, it is known that MR activates Src TKs (37). In
addition, tyrosine residues located in the internalization dileucine
motif of the DC-SIGN cytoplasmic tail have been described as
playing a major role in internalization and transmission of HIV-1
(47). More recently, Lyn and Syk were both found to be associated
with DC-SIGN in DCs (48). Moreover, different receptors at the
surface of DCs can mediate signal transduction through TKs (i.e.,
CD4, FcRs, MR, and integrin). This previous published information led us to scrutinize the possible involvement of TKs in the
intricate interactions between HIV-1 and DCs through the use of
some pharmacological inhibitors and small interfering RNA
(siRNA). We demonstrate here that treatment of immature monocytederived DCs (IM-MDDCs) with the Src family inhibitor pyrazolopyrimidine (PP2) and the Syk inhibitor piceatannol or transfection with
siRNAs targeting Lyn and Syk leads to a greater HIV-1 production in
a coculture system consisting of IM-MDDCs and autologous CD4⫹ T
lymphocytes. These findings are not due to an effect on CD4⫹ T cells
because HIV-1 replication in this cell type is diminished upon pretreatment with PP2 or piceatannol. Additional studies indicate that the
TK inhibitors affect de novo HIV-1 production in IM-MDDCs. These
data suggest that Src and Syk TKs play a dominant role in the multifaceted interplay between DCs and HIV-1.
Materials and Methods
Reagents
3⬘-Azido-3⬘-deoxythymidine, PHA-L, LPS, LPS-free DMSO, and transstilbene were purchased from Sigma-Aldrich. Piceatannol, PP2, PP3, H89,
and Ro-318220 were obtained from Calbiochem. IL-2 and Efavirenz were
obtained through the AIDS Repository Reagent Program. IL-4 and IFN-␥
were purchased from R&D Systems, whereas GM-CSF was a gift from
Cangene (Winnipeg, Canada). The culture medium consisted of RPMI 1640
medium supplemented with 10% FBS, penicillin G (100 U/ml), streptomycin
(100 U/ml), and glutamine (2 mM), which were all purchased from Wisent.
Antibodies
The monoclonal anti-ICAM-1 Ab RR1/1.1.1 was supplied by R. Rothlein
(Boehringer Ingelheim; Ridgefield, CT). The anti-CD3 (OKT3, specific for the
␨-chain), anti-CD11a (TS1/22.1), anti-CD18 (TS1/18.1), and anti-HLA-DR
(L243) hybridomas were obtained from the American Type Culture Collection. The anti-CD86 (BU-63) was supplied by D. L. Hardie (University of
Birmingham, Birmingham, United Kingdom). The anti-DC-SIGN Ab DC28,
which recognizes the DC-SIGN repeat region and cross-reacts with DCSIGNR (49), was obtained from the AIDS Repository Reagent Program.
The anti-CD19 (LT19) and anti-CD14 (MEM-18) were obtained from
EXBIO Praha, whereas the anti-CD83 (HP15E) was purchased from Re-
2863
search Diagnostics. PE-conjugated goat anti-mouse IgG was purchased
from Jackson ImmunoResearch Laboratories.
Cells
The human DCs were generated from monocytes (i.e., CD14⫹ cells).
Briefly, peripheral blood was obtained from normal healthy donors, and
PBMCs were prepared by centrifugation on a Ficoll-Hypaque density gradient as we described previously (50, 51). Next, CD14⫹ cells were isolated
by using a monocyte-positive selection kit according to the manufacturer’s
instructions (MACS CD14 microbeads from StemCell Technologies).
CD14⫹ cells were cultured in 6-well plates at a density of 106 cells/ml. To
generate IM-MDDCs, purified monocytes were cultured in complete culture medium that was supplemented every other day with GM-CSF (1000
U/ml) and IL-4 (200 U/ml) for 7 days. The maturation of IM-MDDCs was
induced on the fifth day by culturing them for 48 h with the above-described cytokines supplemented with IFN-␥ (1000 U/ml) and LPS (100
ng/ml). The final phenotype of IM-MDDCs and mature monocyte-derived
DCs (M-MDDCs) was monitored by flow cytometry (data not shown).
IM-MDDCs express HLA-DR, CD86, DC-SIGN, and low levels of CD14,
whereas M-MDDCs express CD83 and high amounts of ICAM-1, HLADR, and CD86 but lower amounts of DC-SIGN and CD14 than IMMDDCs. Expression of CD3 and CD19 was measured to assess contamination with T and B cells, respectively. Autologous CD4⫹ T cells were
isolated using a negative selection kit according to the manufacturer’s instructions (StemCell Technologies). These cells were activated with
PHA-L (1 ␮g/ml) and maintained in complete culture medium supplemented with IL-2 (30 U/ml) at a density of 2 ⫻ 106 cells/ml. Experiments
were performed with cell preparations that were devoid of contamination
(i.e., DCs: purity, 95%; CD4⫹ T cells: purity, 98%).
Production of virus stocks
Virions were produced by transient transfection in human embryonic kidney 293T cells as described previously (52). Plasmids used include pJRCSF (R5-tropic), pNL4-3balenv (R5-tropic), and pNL4-3 (X4-tropic).
Progeny viruses were also produced upon acute infection of PBMCs for 7
days with various laboratory and clinical HIV-1 isolates (i.e., JR-CSF,
NL4-3balenv, 93TH054/R5-tropic, 91US056/R5-tropic, 92TH026/R5tropic, 93US151/R5-tropic, and 92HT599/X4-tropic). The pNL4-3balenv
vector (provided by R. Pomerantz, Thomas Jefferson University, Philadelphia, PA) was generated by replacing the env gene of the T-tropic HIV-1
strain, NL4-3, with that of the macrophage-tropic HIV-1 Bal strain, thus
resulting in an infectious molecular clone with macrophage-tropic properties (53). The other molecular constructs and HIV-1 strains were obtained
through the AIDS Repository Reagent Program. The virus-containing supernatants were filtered through a 0.22-␮m cellulose acetate syringe filter
and normalized for virion content using an in-house-sensitive double-Ab
sandwich ELISA specific for the viral p24gag protein (54).
Transfer studies
DCs (105 cells in 100 ␮l) were either left untreated or treated with the
tested pharmacological inhibitors before being pulsed with virus preparations (2 or 10 ng of p24gag) for 60 min at 37°C. Next, the virus-cell mixture
was washed three times with PBS to remove untrapped virions. DCs were
cocultured with autologous activated CD4⫹ T lymphocytes (ratio 1:3) in
complete RPMI 1640 medium supplemented with IL-2 (30 U/ml) in 96well plates in a final volume of 200 ␮l. Every 2 days, half of the medium
was removed and kept frozen at ⫺20°C, and fresh medium was added to
the culture. Virus production was estimated by measuring p24gag levels in
culture supernatants.
Gene silencing of Lyn or Syk with siRNAs
siRNAs targeting Lyn or Syk as well as control siRNAs (containing scrambled sequences) were obtained from Dharmacon Research and dissolved in
an appropriate buffer. IM-MDDCs were washed with OptiMEM (Invitrogen Life Technologies) without serum and antibiotics. The tested siRNAs
were transfected at a final concentration of 200 pMol/well using Oligofectamine according to the manufacturer’s instructions (Invitrogen Life
Technologies). Control cells were treated either with Oligofectamine alone
or Oligofectamine plus scrambled sequences. Forty hours following transfection, a coculture made of IM-MDDCs and autologous CD4⫹ T cells was
initiated as described above. Silencing efficiency of Lyn or Syk was monitored by western blot analysis as described previously (55).
Infection of CD4⫹ T cells
Purified CD4⫹ T cells (3 ⫻ 105 cells) were either left untreated or treated
with the tested pharmacological inhibitors before infection with HIV-1
2864
ROLE OF Src AND Syk IN HIV-1 TRANSFER
(15 ng p24gag) (JR-CSF, NL4-3balenv, or NL4-3). Cells were exposed to
HIV-1 particles for 2 h at 37°C and then washed three times with PBS. The
cells were maintained in complete RPMI containing IL-2 (30 U/ml) for 3
days and virus production was measured by assessing p24gag levels in the
culture supernatants.
Real-time PCR test
The amount of integrated viral DNA was measured with a real-time PCR
approach as described by Suzuki and coworkers (56). Briefly, the DNA in
IM-MDDCs was extracted at day 12 postinfection with the Qiagen DNeasy
tissue kit according to the manufacturer’s instructions. Briefly, a first round
of PCR amplification was conducted using 100 ng of DNA and TaqDNA
polymerase (Promega). An Alu-specific sense primer (5⬘-TCC CAG CTA
CTC GGG AGG CTG AGG-3⬘) was used in combination with an antisense
HIV-1 specific primer (M661) (5⬘-CCT GCG TCG AGA GAT CTC CTC
TG-3⬘, 673– 695). Cycling conditions included an initial denaturation step
(94°C for 3 min), followed by 22 denaturation cycles (94°C/30 s), annealing (66°C/30 s), and extension (70°C/10 min) followed by a final extension
(72°C/10 min). The first PCR products were diluted 5-fold and subjected to
a real-time PCR assay targeting the HIV-1 R/U5 region. The specific sense
primer (M667) (5⬘-GGC TAA CTA GGG AAC CCA CTG C-3⬘, 496 –517)
coupled to an antisense primer (AA55) (5⬘-CTG CTA GAG ATT TTC
CAC ACT GAC-3⬘, 612– 635) were used with the fluorogenic probe
TaqMan 5⬘-(FAM) TAG TGT GTG CCC GTC TGT TGT GTG AC (BHQ1)-3⬘ (Biosearch Technologies). Finally, PCR was performed using the
Rotor-Gene 3000 four-channel multiplexing system (Corbett Research).
Cycling conditions included 40 denaturation cycles (95°C/20 s) and extension (60°C/1 min). NL4-3 DNA was used for the standard curve (i.e.,
from 469 to 30,000 copies). HIV-1 standards contained 1 ng of DNA from
uninfected cells as a carrier.
Statistical analysis
Statistical analyses were conducted according to the methods outlined in
Zar (57). Means were compared using either the Student’s t test or a single
factor ANOVA followed by Dunnett’s multiple comparison when more
than two means were considered. When a same test had to be performed
five times or more for a same experiment, a sequential Bonferroni correction was applied to minimize the probability of type I errors (58). Values
of p ⬍ 0.05 were deemed statistically significant. For all figures, an asterisk
(ⴱ) denotes a p value of ⬍0.05, whereas two asterisks (ⴱⴱ) denotes a p
value of ⬍0.01. Calculations were made with the GraphPad Prism
software.
Results
Src and Syk TKs modulate HIV-1 transfer by IM-MDDCs
Our initial series of investigations were focused on Src family
kinases because they are cytoplasmic protein TKs known to be
essential for many cell functions. To test the relative contribution
of Src TKs in the process of HIV-1 transmission from DCs to
CD4⫹ T cells, IM-MDDCs as well as M-MDDCs were first treated
with the selective Src family inhibitor PP2 for 10 min before pulsing such cells with a R5-tropic HIV-1 strain (i.e., JR-CSF). The
cell-virus mixture was next cocultured with autologous CD4⫹ T
lymphocytes that were used as receptor cells. Transmission of
HIV-1 was found to be slightly more rapid and efficient when
using M-MDDCs as compared with IM-MDDCs (Fig. 1), which is
in agreement with the data found in the literature (50, 59). Interestingly, the process of trans-infection was increased upon treatment of IM-MDDCs with PP2 (Fig. 1A). The Src inhibitor had no
such comparable effect on HIV-1 transfer by MMDDCs (Fig. 1B). It should be noted that similar observations
were made in transfer experiments when using IM-MDDCs pulsed
with various R5-tropic clinical HIV-1 isolates amplified in primary
human cells (i.e., 93TH054, 91US056, 92TH026, and 93US151)
(data not shown). Virus transfer was not affected when X4-tropic
laboratory and clinical HIV-1 variants (NL4-3 and 92HT599, respectively) were used to pulse IM-MDDCs similarly treated with
PP2 (data not shown). Therefore, subsequent experiments were
performed exclusively with IM-MDDCs in combination with R5
viruses. The use of IM-MDDCs is also prompted by the concept
that immature DCs will be most likely the first cell type to come
FIGURE 1. Involvement of Src kinases in HIV-1 transmission from IMMDDCs to CD4⫹ T cells. IM-MDDCs (A) and M-MDDCs (B) (1 ⫻ 105
cells) were either left untreated or preincubated with PP2 (10 ␮M) for 10
min before pulsing with JR-CSF (2 ng of p24gag) for 60 min at 37°C. After
three washes with PBS, IM-MDDCs and M-MDDCs were cocultured with
autologous CD4⫹ T cells at a DC/T cell ratio of 1:3. Cell-free supernatants
were collected at the indicated times and assayed for p24gag. Data shown
correspond to the means ⫾ SD of triplicate samples and are representative
of three independent experiments. C, IM-MDDCs (1 ⫻ 105 cells) were
either left untreated or preincubated with PP2 or its inactive analog PP3 (10
␮M) for 10 min before pulsing with JR-CSF (10 ng of p24gag) for 60 min
at 37°C. After three washes with PBS, IM-MDDCs were cocultured with
autologous CD4⫹ T cells at a DC/T cell ratio of 1:3. Cell-free supernatants
were collected at the indicated times and assayed for p24gag. Data shown
correspond to the means ⫾ SD of triplicate samples and are representative
of three independent experiments. Virus production detected at day 2 following initiation of the coculture is presented in the insert. For A and B,
means for individual time points were compared using the Student’s t test,
whereas for C, means for individual time points were compared using
single factor ANOVAs followed by Dunnett’s multiple comparisons. For
all three panels, sequential Bonferroni corrections were applied.
The Journal of Immunology
FIGURE 2. Transfer of HIV-1 by IM-MDDCs is also affected by Syk
TKs. IM-MDDCs (1 ⫻ 105 cells) were either left untreated or preincubated
with piceatannol or its inactive analog trans-stilbene (10 ␮M) for 10 min
before pulsing with JR-CSF (10 ng of p24gag) for 60 min at 37°C. After
three washes with PBS, IM-MDDCs were cocultured with autologous
CD4⫹ T cells at a DC/T cell ratio of 1:3. Cell-free supernatants were
collected at the indicated times and assayed for p24gag. Data shown correspond to the means ⫾ SD of triplicate samples and are representative of
three independent experiments. Virus production detected at day 2 following initiation of the coculture is presented in the insert. Means for individual time points were compared using single factor ANOVAs followed by Dunnett’s multiple comparisons and a sequential Bonferroni
correction.
in contact with the virus because they reside in mucosal tissues.
The next virus transfer experiments were also performed in the
presence of IL-2 to assure a constant virus production at earlier
time points following initiation of coculture and also to eliminate
the possibility that several rounds of virus replication in autologous CD4⫹ T lymphocytes could mask an initial difference in
transfer by IM-MDDCs. The specificity of the observed effects
was tested by performing transfer studies with PP3, which is an
inactive analog of PP2. As illustrated in Fig. 1C, transmission of
HIV-1 from IM-MDDCs to autologous CD4⫹ T cells was still
augmented upon treatment of DCs with PP2 but was not affected
by a similar treatment with the inactive analog PP3, thereby confirming the validity of our results. The PP2-induced modulatory
effect was rapid because an increase was already detected as early
as 2 days following coculture of virus-pulsed IM-MDDCs with
autologous CD4⫹ T cells (Fig. 1C, small inset).
Subsequently, we studied the possible implication of Syk because this TK acts more downstream of Src family members in
several endocytosis/phagocytosis-signaling pathways and DCSIGN-mediated signal transduction events (16, 18, 32, 35, 36, 40,
48). To this end, IM-MDDCs were first treated with the Syk inhibitor 3,4,3⬘,5⬘-tetrahydroxy-trans-stilbene (piceatannol) before
virus exposure, followed by a coculture with autologous CD4⫹ T
lymphocytes. Pretreatment of IM-MDDCs with such a Syk-selective TK inhibitor resulted in a significant increase in HIV-1
transfer (Fig. 2, small inset). The specificity of the piceatannoldependent effect was confirmed by the inability of the inactive
analog trans-stilbene to induce a similar enhancement of virus
transmission.
Our next series of investigations were performed with increasing doses of the specific Src and Syk inhibitors and their appropriate inactive analogs using a highly infectious R5-tropic virus
isolates (i.e., NL4-3balenv). Data shown in Fig. 3 indicate that
there is a dose-dependent PP2- and piceatannol-mediated increase
in virus transfer with a peak reached in both instances at a 10 ␮M
2865
FIGURE 3. Dose-dependent effect of the tested Src- and Syk-specific
inhibitors. IM-MDDCs (1 ⫻ 105 cells) were either left untreated or preincubated with PP2 (A) or piceatannol (B) at the indicated concentrations
for 10 min before pulsing with NL4-3balenv (10 ng of p24gag) for 60 min
at 37°C. After three washes with PBS, IM-MDDCs were cocultured with
autologous CD4⫹ T cells at a DC/T cell ratio of 1:3. Cell-free supernatants
were collected at day 2 following initiation of the coculture and assayed for
p24gag. Data shown correspond to the means ⫾ SD of triplicate samples
and are representative of three independent experiments. Means were compared using single factor ANOVAs followed by Dunnett’s multiple
comparisons.
concentration. The inactive analogs (i.e., PP3 and trans-stilbene)
were still unable to affect HIV-1 transmission from IM-MDDCs to
autologous CD4⫹ T cells (data not shown). It should be noted that
all concentrations tested were found to be noncytotoxic using a
colorimetric assay (data not shown). Importantly, HIV-1 transmission was again increased upon treatment with both PP2 and piceatannol when using this time a R5-tropic clinical isolate of HIV-1
(i.e., 91US056) (Fig. 4), which provides physiological significance
FIGURE 4. Transfer of a clinical virus isolate is also affected by Srcand Syk-specific inhibitors. IM-MDDCs (1 ⫻ 105 cells) were either left
untreated or preincubated with the listed inhibitors or their inactive analogs
for 10 min before pulsing with clinical isolates 91US056 (10 ng of p24gag)
for 60 min at 37°C. After three washes with PBS, IM-MDDCs were cocultured with autologous CD4⫹ T cells at a DC/T cell ratio of 1:3. Cell-free
supernatants were collected at day 8 following initiation of the coculture
and assayed for p24gag. Data shown correspond to the means ⫾
SD of triplicate samples and are representative of three independent experiments. Means were compared using single factor ANOVAs followed
by Dunnett’s multiple comparisons.
2866
ROLE OF Src AND Syk IN HIV-1 TRANSFER
FIGURE 5. Virus transfer is increased upon siRNA-mediated gene silencing of Lyn or Syk. IM-MDDCs (1 ⫻ 105 cells) were first transfected with
control siRNAs or siRNAs targeting Lyn or Syk. Next, cells were pulsed with NL4-3Balenv (10 ng of p24gag) for 60 min at 37°C. After three washes with
PBS, IM-MDDCs were cocultured with autologous CD4⫹ T cells at a DC/T cell ratio of 1:3. Cell-free supernatants were collected at day 2 (A) and day
3 (B) before monitoring p24gag. The immunoblot of cellular lysates revealed with anti-Lyn and anti-Syk Abs is presented in the inset of A. Data shown
correspond to the means ⫾ SD of triplicate samples and are representative of four independent experiments. Means were compared using single factor
ANOVAs followed by Dunnett’s multiple comparisons.
to these observations.
Next, we have used the siRNA technology to confirm the implication of Src and Syk TKs in the observed phenomenon and also
to circumvent nonspecific effect(s) mediated by chemical inhibitors. More specifically, siRNAs targeting Lyn and Syk were introduced within IM-MDDCs before exposure to HIV-1 and initiation
of the coculture. As depicted in Fig. 5, a more important HIV-1
transfer from IM-MDDCs to autologous CD4⫹ T cells was seen
when protein expression of Lyn or Syk was diminished through the
use of siRNAs. A reduced expression of Lyn and Syk was confirmed by western blot analysis (Fig. 5, small inset) and intracellular flow cytometry (data not shown).
HIV-1 infection in cis of IM-MDDCs is increased upon
inhibition of Src and Syk TKs
To shed light on the putative mechanism(s) by which Src and Syk
kinases can regulate the ability of IM-MDDCs to transfer HIV-1
particles to CD4⫹ T cells in trans, we measured the effect of Srcand Syk-selective inhibitors on the initial interactions between
DCs and HIV-1. Flow cytometry analyses demonstrated that cell
surface expression of DC-SIGN and MR was not modulated upon
treatment with PP2 or piceatannol (data not shown). Moreover,
results from virus internalization studies revealed that similar
amounts of viruses were found inside IM-MDDCs that were either
left untreated or pretreated with PP2 or piceatannol. An average of
4% of the IM-MDDCs were found to be positive for intracellular
p24gag after 60 min of pulsing under our experimental conditions
(data not shown).
Previous reports have shown that HIV-1 is transferred from DCs
to CD4⫹ T cells via a process involving two distinct phases (9, 60).
An initial transfer phase occurs whereby the virus located within
endosomal compartments in IM-MDDCs is transported to the
DCs-T cell synapse (i.e., early transfer). This event is followed by
a second kinetic phase that is dependent on productive infection of
IM-MDDCs and eventual transfer of progeny virus to CD4⫹ T
cells (i.e., late transfer). To define whether the Src- and Syk-mediated action on virus transfer is affecting the early and/or late
transfer phase, IM-MDDCs were either left untreated or treated
with the antiretroviral drug Efavirenz before initiation of the coculture. This treatment blocks productive virus infection of IM-
MDDCs and allows only the early transfer phase to occur. A significant diminution in HIV-1 is detected when the late transfer
phase is abolished (Fig. 6A). More importantly, under such experimental conditions, no increase in virus transfer was observed upon
treatment with PP2 or piceatannol, thus suggesting that the early
phase transfer is not affected upon treatment with the tested TK
inhibitors. Similar observations were made when the antiviral
agent 3⬘-azido-3⬘-deoxythymidine was used under comparable experimental conditions (data not shown). To confirm that the Src
and Syk TKs are affecting primarily the late phase transfer, IMMDDCs were either left untreated or treated with the studied
chemical drugs before acute infection with a highly infectious R5tropic viral strain (i.e., NL4-3balenv). Virus production was evaluated at day 12 postinfection, a time lapse sufficient enough to
measure the release of viruses from HIV-1-infected IM-MDDCs.
Results depicted in Fig. 6B indicate that de novo virus production
in IM-MDDCs is increased by a pretreatment with PP2 or piceatannol, therefore corroborating the idea that Src and Syk TKs influence the late transfer phase (i.e., productive infection of IMMDDCs). As expected, HIV-1 infection in cis of IM-MDDCs was
unaffected upon a pretreatment with the inactive analogs (data not
shown). Moreover, cellular DNA was extracted from such virusinfected IM-MDDCs at day 12 postinfection. Thereafter, the
amount of integrated viral DNA was quantified using a real-time
PCR test that provides a measure of viral integration into the host
cell chromatin. This technical strategy uses primer sets designed to
encompass both cellular (i.e., Alu) and HIV-1-specific DNA sequences. The number of integrated viral DNA copies was higher in
IM-MDDCs that were pretreated with PP2 or piceatannol as compared with untreated cells (Fig. 6C). The amounts of integrated
HIV-1 DNA copies were not affected by the inactive analogs (data
not shown).
Effect of other kinases on HIV-1 transfer by IM-MDDCs
We next tested whether HIV-1 transmission by IM-MDDCs could
be affected by other signal transducers such as the serine/threonine
kinases protein kinase C (PKC) and protein kinase A (PKA) because they are known to play essential roles in the fine-tuning of
signaling cascades of several cellular process, including Ag uptake
The Journal of Immunology
FIGURE 6. Src and Syk family members are regulating HIV-1 infection
in cis of IM-MDDCs. A, IM-MDDCs (1 ⫻ 105 cells) were pretreated with
DMSO (control) or Efavrirenz (50 nM) for 10 min. Next, cells were either
left untreated or preincubated with PP2 or piceatannol (10 ␮M) for 10 min
before pulsing with NL4-3balenv (10 ng of p24gag) for 60 min at 37°C.
After three washes with PBS, IM-MDDCs were cocultured with autologous CD4⫹ T cells at a DC/T cell ratio of 1:3. Cell-free supernatants were
collected at day 2 following initiation of the coculture and assayed for
p24gag. B and C, IM-MDDCs (1 ⫻ 106 cells) were either left untreated or
preincubated with PP2 or piceatannol (10 ␮M) for 10 min before pulsing
with NL4-3balenv (100 ng of p24gag) for 60 min at 37°C. After three
washes with PBS, IM-MDDCs were cultured for 12 days in complete
RPMI 1640 medium supplemented with GM-CSF and IL-4 every 3 days.
B, Cell-free supernatants were collected at day 12 following initial virus
infection and assayed for p24gag. C, Total cellular DNA was extracted at
day 12 following HIV-1 infection and subjected to a first PCR with Alu and
M661 primers to amplify integrated proviral DNA. Next, the first PCR
products were subjected to real-time PCR using AA55 and M667 primers
to further amplify the integrated HIV-1 LTR. The number of viral DNA
copies was determined by using a standard curve prepared with the NL4-3
vector. Data shown correspond to the means ⫾ SD of triplicate samples
and are representative of three independent experiments. Means were compared using single factor ANOVAs followed by Dunnett’s multiple
comparisons.
and formation of the immunological synapse (61, 62). Pharmacological inhibition of PKC and PKA was achieved through the use
of the selective inhibitors Ro-318220 and H89, respectively. In
contrast to what is seen with Src and Syk inhibitors, the process of
HIV-1 transfer from IM-MDDCs to autologous CD4⫹ T cells was
unaltered by both Ro-318220 and H89 (Fig. 7A). Virus infection of
IM-MDDCS, which corresponds to de novo virus production, was
also unaffected upon pretreatment with Ro-318220 and H89 (Fig.
7B). These findings were corroborated when estimating integrated
2867
FIGURE 7. Virus transfer and infection of IM-MDDCs in cis are not
affected by PKC and PKA inhibitors. IM-MDDCs (1 ⫻ 105 cells) were
either left untreated or preincubated with Ro-318220 (1 ␮M), H89 (30
␮M), or piceatannol (10 ␮M) for 10 min before pulsing with NL4-3balenv
(10 ng of p24gag) for 60 min at 37°C. A, After three washes with PBS,
IM-MDDCs were cocultured with autologous CD4⫹ T cells at a DC/T cell
ratio of 1:3. Cell-free supernatants were collected at day 2 following initiation of the coculture and assayed for p24gag. B, After three washes with
PBS, IM-MDDCs were cultured for 12 days in complete culture RPMI
1640 medium supplemented with GM-CSF and IL-4 every 3 days. Next,
cell-free supernatants were collected and assayed for p24gag. C, After three
washes with PBS, IM-MDDCs were cultured for 12 days in complete culture RPMI 1640 medium supplemented with GM-CSF and IL-4 every 3
days. Next, total cellular DNA was extracted and subjected to the real-time
PCR test. Data shown correspond to the means ⫾ SD of triplicate samples
and are representative of three independent experiments. Means were compared using single factor ANOVAs followed by Dunnett’s multiple
comparisons.
viral DNA copies in either untreated, Ro-318220-treated, or H89treated IM-MDDCs (Fig. 7C). In this set of experiments, piceatannol was used as a positive control. Altogether, these results indicate that PKC and PKA do not play a functional role in HIV-1
transfer from IM-MDDC to CD4⫹ T cells.
Effect of Src and Syk inhibitors on HIV-1 infection
of CD4⫹ T cells
Experiments were subsequently performed to estimate whether
part of the observed enhancement of HIV-1 transfer might be due
to a transport of the tested drugs from DCs, which would next act
at the level of autologous CD4⫹ T lymphocytes. This scenario was
addressed by pretreating CD4⫹ T cells with the Src- and Sykselective inhibitors before infection with HIV-1. In sharp contrast to what is seen when measuring infection in trans from
2868
FIGURE 8. HIV-1 production in CD4⫹ T lymphocytes is reduced upon
treatment with PKC and PKA inhibitors. Purified CD4⫹ T cells (3 ⫻ 105
cells) were either left untreated or preincubated with PP2 or piceatannol
(10 ␮M) for 10 min before pulsing with JR-CSF (A) or NL4-3balenv (B)
(10 ng of p24gag) for 60 min at 37°C. After three washes with PBS, CD4⫹
T cells were cultured in complete culture RPMI 1640 medium supplemented with IL-2. Cell-free supernatants were collected at day 6 following
virus infection and assayed for p24gag. Data shown correspond to the
means ⫾ SD of triplicate samples and are representative of three independent experiments. Means were compared using single factor ANOVAs
followed by Dunnett’s multiple comparisons.
IM-MDDCs to autologous CD4⫹ T lymphocytes, data shown in
Fig. 8 demonstrate that replication of R5-tropic JR-CSF (Fig. 8A)
and NL4-3balenv (Fig. 8B) was significantly reduced upon pretreatment of CD4⫹ T lymphocytes with PP2 and piceatannol, respectively. These findings were confirmed when using X4-tropic
isolates of HIV-1 produced in PBMCs (i.e., NL4-3 and 92HT599)
(data not shown). A colorimetric assay revealed that the studied
compounds were not cytotoxic at the concentration tested (data not
shown).
Discussion
It has been well established that nrTKs are involved in regulating
many key cellular responses and particularly the uptake and/or
endocytosis of some specific ligands. Considering that the capture of HIV-1 by DCs and its eventual transmission to more
susceptible target cells such as CD4⫹ T cells involve endocytosis, we have investigated the participation of nrTKs in virus
transfer. Here, we present evidence that Src and Syk TKs can limit
HIV-1 transmission from IM-MDDCs to autologous CD4⫹ T
cells. Our results confirmed the recently described two-phase virus
transfer model proposed by Turville et al. (9) and indicated that Src
and Syk TKs can diminish HIV-1 transfer by decreasing acute
infection of IM-MDDCs. The Src- and Syk-related modulation of
IM-MDDCs cis-infection was observed only with HIV-1 R5-tropic
variants of HIV-1 and was not seen with X4 strains. This is in
accordance with the fact that IM-MDDCs do not support efficient
in vitro cis-replication of X4-tropic viral isolates (63). Moreover,
we have not been able to detect a similar process in M-MDDCs
because productive HIV-1 infection occurs almost exclusively in
IM-MDDCs rather than in M-MDDCs (59, 64). In contrast to the
data obtained when using inhibitors specific for Src or Syk, no
decrease in neither infection of IM-MDDCs in cis nor HIV-1 trans-
ROLE OF Src AND Syk IN HIV-1 TRANSFER
fer could be detected following a pretreatment with selective PKC
and PKA inhibitors.
Quantitative measurements of internalized virions in IMMDDCs indicated that HIV-1 entry is not affected upon a treatment with the studied Src- and Syk-specific inhibitors. For example, an average of 4% of the IM-MDDCs that were either left
untreated or treated with the studied chemical compounds were
found to be positive for p24gag following virus exposure. Such a
low percentage of cells capturing HIV-1 particles compared with
previously published studies can be explained by differences in
experimental methodologies. For example, a virus input corresponding to 2–3 ␮g of p24gag per 106 DCs was used in the work
described by Turville et al. (9) compared with a virus input of 100
ng of p24 per 106 DCs in the present study. This also help to
explain why we have performed quantitative analyses of proviral
DNA in IM-MDDCs at 12 days postinfection compared with 3
days postinfection in the study by Turville et al. (9). Additionally,
we have used a real-time PCR test that permits to estimate the
amount of integrated proviral DNA copies while Turville et al. (9)
have performed proviral DNA quantification using a real-time
quantitative PCR test that does not allow to discriminate between
unintegrated and integrated proviral DNA copies.
Three distinct and not mutually exclusive hypotheses can be
proposed to explain the augmentation of productive HIV-1 infection in IM-MDDCs and enhancement of virus transfer that are seen
following treatment with Src- and Syk-specific chemical compounds (Fig. 9). First, Src and Syk TKs could alter the virus entry
route by favoring endocytosis of the incoming virions. This is
based on the demonstrated capacity of Src and Syk TKs to promote
internalization of ligands and possibly viruses by a direct action on
phagocytosis/endocytosis pathways (16, 18, 32, 35, 36, 40). It is
now recognized that the HIV-1 entry mode into myeloid cells can
result either in cytosolic delivery that results in productive infection (65– 67), preservation in intracellular vesicles in an infectious
state for a subsequent transmission through the virological synapse
(7, 12, 68, 69), or degradation by lysosomal enzymes in the endosomal apparatus (9). In view of this, we propose that virions can
remain for a longer time period on the surface of IM-MDDCs
without being internalized when Src and Syk TKs are inhibited.
This process will favor interactions between gp120 and a complex
made of CD4 and an appropriate coreceptor (e.g., CCR5) and the
release of the viral material in the cytosol as a consequence of a
pH-independent fusion of viral and cellular membranes. Interestingly, it is known that this mechanism of HIV-1 entry into target
cells results in productive infection (65– 67). This postulate is supported by the ability of CCR5 antagonists to inhibit HIV-1 infection in cis of IM-MDDCs (data not shown). Second, it can be
proposed that Src and Syk TKs promote the degradation of virions
in the endosomal apparatus. This scenario is validated by the previous observation that members of Src and Syk families target
several cell surface receptors for degradation inside the lysosome
(17). Interestingly, it is now clear that a significant lysosomal degradation occurs following uptake of HIV-1 by IM-MDDCs (9).
Third, based on previous studies by Sedlik et al. (32) and
Wilflingseder et al. (70), it can also be postulated that Src and Syk
TKs might be involved in maturation of DCs. Indeed, treatment
with Src- and Syk-specific inhibitors could block the maturation
process of IM-MDDCs, thus resulting in a more efficient infection
in cis of DCs based on the idea that M-MDDCs do not support
active HIV-1 replication as it is the case for IM-MDDCs (59, 64).
However, this postulate is unlikely because uninfected IMMDDCs that were treated with the tested TK inhibitors were not
frozen in an immature state (as monitored by flow cytometry
The Journal of Immunology
2869
FIGURE 9. Proposed hypothetical model.
Three distinct mechanisms can be proposed for
the Src- and Syk-mediated effect on susceptibility of IM-MDDCs to HIV-1 infection in cis.
In the first scenario, treatment of IMMDDCs with Src- and Syk-specific inhibitors
will modulate the virus entry mode by reducing internalization via endocytosis and favoring fusion of viral and cellular membranes.
This event will promote the cytosolic delivery
of viral material, which is known to result in
productive infection. The second possibility
deals with a reduced degradation of viral constituents within lysosomes and accumulation
of viral material in the cytosol in the presence
of Src- and Syk-specific inhibitors. The third
and last postulate suggests that IM-MDDCs
treated with Src- and Syk-specific inhibitors
are frozen in an immature state.
through the use of anti-CD83, anti-CD86, anti-DC-SIGN, and antiHLA-DR Abs) (data not shown).
In contrast to the situation prevailing in IM-MDDCs, Src- and
Syk-specific inhibitors resulted in a significant diminution of virus
production in CD4⫹ T cells. Until now, there was a paucity of data
concerning the susceptibility of primary human CD4⫹ T lymphocytes to HIV-1 infection in the presence of TK inhibitors. The
significant inhibition of virus production in this cell type might be
linked to the previously described properties of Src family members. For example, PP2 could block p56lck activity, a process of
prime importance in CD4-mediated signaling (71). It should be
noted that HIV-1 infection is affected when the natural association
between CD4 and p56lck is prevented (72). Additionally, PP2
might affect the interaction between Hck and Nef (73), a virusencoded regulatory protein that has a positive effect on viral infection and replication (74). Last, it is also possible that inhibition
of Fyn activity might contribute to the observed effect due to its
role in the M phase of the cell cycle (75). Regarding the implication of Syk/Zap-70 in the marked inhibition of virus production in
CD4⫹ T cells, it can be proposed that such TKs might favor the
recruitment of the CD4/p56lck complex near the TCR as shown
previously (76). Piceatannol might also modify this process and
thereby affect virus-mediated signaling events, which in turn might
influence HIV-1 replication. Alternatively, although piceatannol
does not affect HIV-1 entry inside CD4⫹ T cells (77), it can modulate virus gene expression by its ability to modulate NF-␬B induction by various inflammatory agents through inhibition of both
I␬B␣ kinase and p65 phosphorylation (78).
Different technical approaches can be undertaken to study the
involvement of signal transducers in a definite process. Small-molecular inhibitors have emerged as indispensable tools for studying
signal transduction. We have therefore deliberately selected an experimental strategy that consists of treating cells with specific
pharmacological inhibitors to study the implication of nrTKs in the
complex interplay occurring between DCs and HIV-1. Experiments performed with increasing doses of the tested inhibitors and
appropriate inactive analogues permit to validate findings made
with chemical tools. We are aware that one cannot completely rule
out that pharmacological compounds can alter other known or un-
known targets at the cellular level. Therefore, experiments were
also conducted with siRNAs targeting Lyn and Syk to exclude
indirect effects exerted by the studied chemical compounds. This
experimental strategy corroborated the implication of Src, and
more precisely of Lyn, and Syk in the control of HIV-1 replication
in IM-MDDCs.
We report that Src and Syk family members influence HIV-1
infection of IM-MDDCs (infection in cis) and virus transfer to
autologous CD4⫹ T cells (infection in trans). A better understanding of the precise details on how this is achieved is essential because it might bear consequence for the loading of virus Ags and
the mounting of a specific immune response. Indeed, DCs are considered as professional Ag-capturing and -presenting cells able to
trigger strong and effective immune responses both in vitro and in
vivo (79, 80). There is accumulating evidence that viral pathogens
such as HIV-1 can exploit DCs to subvert the immune response
and establish a persistent infection in the host by mechanisms that
are still unresolved. For example, the uptake of HIV-1 induces,
without replication, a specific cytotoxic T lymphocyte activity (81,
82). On the other hand, viral Ags located onto infected DCs and
cell-free virions captured and processed by DCs can activate virusspecific CD4⫹ and CD8⫹ T cell activities (81, 83, 84).
In summary, this work provides new insights into the complex
interconnections between HIV-1 and DCs. Altogether, our results
reveal that members of Src and Syk families limit HIV-1 replication in IM-MDDCs. This is in sharp contrast to what is seen when
infection is allowed to proceed in primary human CD4⫹ T cells
pretreated with specific Src and Syk inhibitors. Thus, the intricate
relationships that are established between HIV-1 and host cells
will vary depending of the cell type. Further studies are needed to
elucidate how Src and Syk family members can modulate the susceptibility of IM-MDDCs to HIV-1 infection in cis.
Acknowledgments
We express our gratitude to Lahlou Hadji, Chantal Burelout, and Philippe
Desaulnier for critical and constructive comments for this study. We thank
Sylvie Méthot and Lahlou Hadji for their excellent technical assistance in
writing this manuscript and also Marc Bergeron for his expertise in statistics. We are grateful to Michael Imbeault and Mélanie Tardif for their
2870
assistance with the real-time PCR assay. We appreciate the excellent technical contributions of Maurice Dufour, Odette Simard, and Caroline Côté.
Disclosures
The authors have no financial conflict of interest.
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