Leishmania induces survival, proliferation and elevated cellular dNTP levels in human monocytes promoting acceleration of HIV co-infection.
ABSTRACT Leishmaniasis is a parasitic disease that is widely prevalent in many tropical and sub-tropical regions of the world. Infection with Leishmania has been recognized to induce a striking acceleration of Human Immunodeficiency Virus Type 1 (HIV-1) infection in coinfected individuals through as yet incompletely understood mechanisms. Cells of the monocyte/macrophage lineage are the predominant cell types coinfected by both pathogens. Monocytes and macrophages contain extremely low levels of deoxynucleoside triphosphates (dNTPs) due to their lack of cell cycling and S phase, where dNTP biosynthesis is specifically activated. Lentiviruses, such as HIV-1, are unique among retroviruses in their ability to replicate in these non-dividing cells due, at least in part, to their highly efficient reverse transcriptase (RT). Nonetheless, viral replication progresses more efficiently in the setting of higher intracellular dNTP concentrations related to enhanced enzyme kinetics of the viral RT. In the present study, in vitro infection of CD14+ peripheral blood-derived human monocytes with Leishmania major was found to induce differentiation, marked elevation of cellular p53R2 ribonucleotide reductase subunit and R2 subunit expression. The R2 subunit is restricted to the S phase of the cell cycle. Our dNTP assay demonstrated significant elevation of intracellular monocyte-derived macrophages (MDMs) dNTP concentrations in Leishmania-infected cell populations as compared to control cells. Infection of Leishmania-maturated MDMs with a pseudotyped GFP expressing HIV-1 resulted in increased numbers of GFP+ cells in the Leishmania-maturated MDMs as compared to control cells. Interestingly, a sub-population of Leishmania-maturated MDMs was found to have re-entered the cell cycle, as demonstrated by BrdU labeling. In conclusion, Leishmania infection of primary human monocytes promotes the induction of an S phase environment and elevated dNTP levels with notable elevation of HIV-1 expression in the setting of coinfection.
- SourceAvailable from: PubMed Central[show abstract] [hide abstract]
ABSTRACT: Among parasitic diseases, morbidity and mortality caused by leishmaniasis are surpassed only by malaria and lymphatic filariasis. However, estimation of the leishmaniasis disease burden is challenging, due to clinical and epidemiological diversity, marked geographic clustering, and lack of reliable data on incidence, duration, and impact of the various disease syndromes. Non-health effects such as impoverishment, disfigurement, and stigma add to the burden, and introduce further complexities. Leishmaniasis occurs globally, but has disproportionate impact in the Horn of Africa, South Asia and Brazil (for visceral leishmaniasis), and Latin America, Central Asia, and southwestern Asia (for cutaneous leishmaniasis). Disease characteristics and challenges for control are reviewed for each of these foci. We recommend review of reliable secondary data sources and collection of baseline active survey data to improve current disease burden estimates, plus the improvement or establishment of effective surveillance systems to monitor the impact of control efforts.PLoS Neglected Tropical Diseases 02/2008; 2(10):e313. · 4.57 Impact Factor
Article: Leishmaniasis and poverty.[show abstract] [hide abstract]
ABSTRACT: Leishmaniasis, a neglected tropical disease, has strong but complex links with poverty. The burden of leishmaniasis falls disproportionately on the poorest segments of the global population. Within endemic areas, increased infection risk is mediated through poor housing conditions and environmental sanitation, lack of personal protective measures and economically driven migration and employment that bring nonimmune hosts into contact with infected sand flies. Poverty is associated with poor nutrition and other infectious diseases, which increase the risk that a person (once infected) will progress to the clinically manifested disease. Lack of healthcare access causes delays in appropriate diagnosis and treatment and accentuates leishmaniasis morbidity and mortality, particularly in women. Leishmaniasis diagnosis and treatment are expensive and families must sell assets and take loans to pay for care, leading to further impoverishment and reinforcement of the vicious cycle of disease and poverty. Public investment in treatment and control would decrease the leishmaniasis disease burden and help to alleviate poverty.Trends in Parasitology 01/2007; 22(12):552-7. · 5.51 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: A thorough understanding of the transmission mechanism of any infectious agent is crucial to implementing an effective intervention strategy. Here, our current understanding of the mechanisms that Leishmania parasites use to ensure their transmission from sand fly vectors by bite is reviewed. The most important mechanism is the creation of a "blocked fly" resulting from the secretion of promastigote secretory gel (PSG) by the parasites in the anterior midgut. This forces the sand fly to regurgitate PSG before it can bloodfeed, thereby depositing both PSG and infective metacyclic promastigotes in the skin of a mammalian host. Other possible factors in transmission are considered: damage to the stomodeal valve; occurrence of parasites in the salivary glands; and excretion of parasites from the anus of infected sand flies. Differences in the transmission mechanisms employed by parasites in the three subgenera, Leishmania, Viannia and Sauroleishmania are also addressed.International Journal for Parasitology 09/2007; 37(10):1097-106. · 3.64 Impact Factor
Leishmania Induces Survival, Proliferation and Elevated
Cellular dNTP Levels in Human Monocytes Promoting
Acceleration of HIV Co-Infection
David J. Mock1.*, Joseph A. Hollenbaugh2., Waaqo Daddacha2, Michael G. Overstreet3,
Chris A. Lazarski4, Deborah J. Fowell3, Baek Kim2*
1Department of Biomolecular Genetics, University of Rochester Medical Center, Rochester, New York, United States of America, 2Department of Microbiology and
Immunology, University of Rochester Medical Center, Rochester, New York, United States of America, 3Center of Vaccine Biology and Immunology, University of
Rochester Medical Center, Rochester, New York, United States of America, 4GenVec, Inc., Gaithersburg, Maryland, United States of America
Leishmaniasis is a parasitic disease that is widely prevalent in many tropical and sub-tropical regions of the world. Infection
with Leishmania has been recognized to induce a striking acceleration of Human Immunodeficiency Virus Type 1 (HIV-1)
infection in coinfected individuals through as yet incompletely understood mechanisms. Cells of the monocyte/
macrophage lineage are the predominant cell types coinfected by both pathogens. Monocytes and macrophages contain
extremely low levels of deoxynucleoside triphosphates (dNTPs) due to their lack of cell cycling and S phase, where dNTP
biosynthesis is specifically activated. Lentiviruses, such as HIV-1, are unique among retroviruses in their ability to replicate in
these non-dividing cells due, at least in part, to their highly efficient reverse transcriptase (RT). Nonetheless, viral replication
progresses more efficiently in the setting of higher intracellular dNTP concentrations related to enhanced enzyme kinetics of
the viral RT. In the present study, in vitro infection of CD14+ peripheral blood-derived human monocytes with Leishmania
major was found to induce differentiation, marked elevation of cellular p53R2 ribonucleotide reductase subunit and R2
subunit expression. The R2 subunit is restricted to the S phase of the cell cycle. Our dNTP assay demonstrated significant
elevation of intracellular monocyte-derived macrophages (MDMs) dNTP concentrations in Leishmania-infected cell
populations as compared to control cells. Infection of Leishmania-maturated MDMs with a pseudotyped GFP expressing
HIV-1 resulted in increased numbers of GFP+ cells in the Leishmania-maturated MDMs as compared to control cells.
Interestingly, a sub-population of Leishmania-maturated MDMs was found to have re-entered the cell cycle, as
demonstrated by BrdU labeling. In conclusion, Leishmania infection of primary human monocytes promotes the induction
of an S phase environment and elevated dNTP levels with notable elevation of HIV-1 expression in the setting of coinfection.
Citation: Mock DJ, Hollenbaugh JA, Daddacha W, Overstreet MG, Lazarski CA, et al. (2012) Leishmania Induces Survival, Proliferation and Elevated Cellular dNTP
Levels in Human Monocytes Promoting Acceleration of HIV Co-Infection. PLoS Pathog 8(4): e1002635. doi:10.1371/journal.ppat.1002635
Editor: Michael Emerman, Fred Hutchinson Cancer Research Center, United States of America
Received November 14, 2011; Accepted February 24, 2012; Published April 5, 2012
Copyright: ? 2012 Mock et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by the following NIH grants. Deb J. Fowell was supported by NIAID R01 AI072690. Michael G. Overstreet was supported by
F32 AI089079-01A1. Waaqo Daddacha was supported by F31 GM095190. Joseph A. Hollenbaugh was supported by T32 DA07232. Baek Kim was supported by
A1077401. The authors have no competing interests. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com (DJM); firstname.lastname@example.org (BK)
. These authors contributed equally to this work.
Leishmaniasis has recently been recognized to be both one of
the world’s most neglected and most important parasitic diseases,
threatening an estimated 350 million people worldwide [1,2].
Surveys have estimated that approximately 12 million people are
currently infected with 2 million new cases reported yearly,
primarily afflicting the world’s poorest populations in some 88
countries . Leishmaniasis is transmitted to humans by the bite
of the female Phlebotomine sandfly upon taking a blood meal .
Infection results in three basic clinical presentations. Cutaneous
and mucocutaneous leishmaniasis are disfiguring and even
mutilating diseases, while visceral leishmaniasis (VL) is character-
ized by fever, massive hepatosplenomegaly, pancytopenia, and a
wasting syndrome called Kala-azar, which is nearly uniformly fatal
without treatment [5,6].
Early after the emergence of the global Human Immunodefi-
ciency Virus Type 1 (HIV-1) epidemic, clinicians recognized that
reciprocal activation of each pathogen by the other frequently
occurred. It was noted, on the one hand, that infection with HIV-1
modifies the natural history of leishmaniasis, leading to 100–2,230
times increase in the risk of developing VL and reducing the
likelihood of a therapeutic response [7–11]. At the same time, VL
was shown to induce activation of latent HIV-1, increase viral
load, and cause a striking acceleration in the progression of
asymptomatic HIV-1 infection to AIDS that corresponded to a
reduction of life expectancy in patients [12–15]. Similarly, it was
recognized that monocytes and macrophages are the primary cell
types coinfected with both HIV-1 and Leishmania. Initial studies
demonstrated that Leishmania coinfection reactivated HIV-1
replication in latently infected monocytoid cell lines .
Subsequent studies in primary MDMs coinfected with L. infantum
PLoS Pathogens | www.plospathogens.org1 April 2012 | Volume 8 | Issue 4 | e1002635
and HIV-1 also found enhanced HIV-1 replication associated with
increased secretion of the pro-inflammatory cytokines TNF-a, IL-
1a, and IL-6. In these experiments, HIV-1 replication, as
measured by p24 ELISA, was reduced in the presence of either
chemical inhibitors or blocking antibodies to these three cytokines
Human monocytes circulate in the blood and reside in bone
marrow and spleen and are generally believed not to proliferate in
the steady state [18,19]. However there is an emerging awareness
that human monocytes possess far greater heterogeneity than
originally perceived, and subpopulations of monocytes have
recently been described that can re-enter the cell cycle in response
to both Macrophage- and Granulocyte Macrophage-Colony
Stimulating Factors (M-CSF and GM-CSF, respectively) [20–
22]. Proliferation of these presumably immature peripheral blood
monocyte subpopulations has been demonstrated by multiple
techniques including uptake of 5-bromo-29-deoxyuridine (BrdU)
and CFSE labeling, leading to this population being termed
‘‘proliferative monocytes’’ [23,24].
Such cellular proliferative capacity has important implications
because cellular dNTP levels correlate directly with the replicative
capacity of mammalian cells . Consistent with this observa-
tion, a variety of studies, including those from our laboratory, have
reported that dNTP levels are consistently higher in dividing
versus non-dividing cells [25–31]. Among the retroviruses, HIV-1
possesses the unique ability to infect both dividing (activated CD4+
T cells) and non-dividing cells (macrophages). This ability is due,
at least in part, to the evolutionary adaptation of its reverse
transcriptase (RT) to function under conditions of extremely
limited dNTP availability . However, as noted for the
replicative capacity of mammalian cells, HIV-1 replication
efficiency is also directly correlated with cellular dNTP concen-
trations and proceeds with far greater efficiency in both tumor cells
and PHA-stimulated CD4+ T cells, in which the average dNTP
levels are 150–225 times higher than that of non-dividing MDMs
[32,33]. Several recent studies have shown that HIV-2 Vpx
protein promotes the degradation of the SAMHD1, a host anti-
viral restriction factor [34–37]. Recently, SAMHD1 was shown to
function as a dNTP hydrolase [38,39], limiting the cellular dNTP
pool and restricting HIV-1 replication in cells of myeloid lineage
. Moreover, our recent paper shows a direct connection
between SAMHD1 degradation, an increase in dNTP levels and
enhanced transduction of HIV-1 in myeloid cells .
In the present study, we found that in vitro infection of freshly
isolated, undifferentiated CD14+ primary human monocytes with
Leishmania consistently led to maturation into macrophages and to
higher cell numbers over time as compared to uninfected control
cells. In addition to the inhibition of apoptosis previously reported
in Leishmania-infected MDMs, we also report the unexpected
finding that a sub-population of CD14+ human MDMs proliferate
in response to Leishmania, as measured by BrdU incorporation at
days 12–14 after infection. As the efficiency of HIV-1 RT DNA
synthesis and subsequent viral replication are directly dependent
on cellular dNTP concentration, we subsequently employed a
highly sensitive single nucleotide incorporation assay that was
recently developed in our laboratory to measure cellular dNTP
concentration [32,42,43]. We found a marked increase in the
content of dNTPs in Leishmania-maturated MDMs as compared to
uninfected control cells. Consistent with this observation, elevated
levels of ribonucleotide reductase (RNR), the rate-limiting enzyme
for dNTP synthesis, was also found in Leishmania-maturated
MDMs as compared to control cells. Finally, we found
significantly enhanced expression and transcription of a GFP-
expressing pseudotyped HIV-1 (HIV-1 D3 GFP) in Leishmania-
maturated MDMs as compared to control cultures as assayed by
FACS analysis of HIV-1 D3 GFP expressing cells and qPCR for 2
LTR-circle copy number.
As noted above, previous studies have suggested a role for
Leishmania infection of monocytes causing the induction of pro-
inflammatory cytokines as a stimulus to HIV-1 replication in
coinfected cells. Our data support a novel model whereby
Leishmania infection stimulates monocytes’ differentiation and cell
division. Consistent with the increased proliferation capacity,
Leishmania infection increases cellular dNTP concentrations that
facilitate enhanced HIV-1 coinfection.
Kinetic analysis of cell survival
The effect of Leishmania infection on cell survival of primary
human monocytes was examined over a time course of 28 days
from eleven individual donors. Preliminary experiments were
performed to examine potential effects of both heat-inactivated
Leishmania (also applied to monocytes at an MOI=7) and day 7
conditioned medium from Leishmania-infected monocytes re-
applied to freshly isolated monocytes. These experiments demon-
strated no significant effects on monocyte cell survival, maturation,
or proliferation (data not shown). In parallel experiments,
Leishmania labeled with the vital dye PKH showed that at an
MOI=7 virtually all monocytes within the culture became
infected (Figure S1). This MOI is well within the range of those
previously published [16,17].
Purified human monocytes were cultured at 16106cells/well in
6 well dishes, and three wells from each of three culture conditions
were combined and counted: 1) RPMI media with 10% FBS
(‘‘control cells’’), 2) RPMI media with 10% FBS plus 5 ng/ml
human recombinant GM-CSF (‘‘GM-CSF’’), or 3) RPMI media
with 10% FBS with Leishmania major (MOI=7) at the time of
plating (‘‘Leishmania’’). The GM-CSF-treated monocytes differen-
tiate into MDMs and were used as a positive control for all the
studies. Medium was changed at day 7 and then weekly, replating
any non-adherent cells into their respective wells. As illustrated in
Figure 1A, a marked decline in the cell numbers was seen at day 3
after initial plating in all three conditions, though more notably in
the control monocytes as compared to either GM-CSF-treated or
Leishmaniasis is a parasitic disease that infects several
human host immune cells, including neutrophils, mono-
cytes, and macrophages. Moreover, while HIV-1 infects
monocytes and macrophages, only the infected macro-
phages productively release viral progenies. Importantly,
patients coinfected with both pathogens progress more
rapidly to AIDS. In this study, we examine how Leishmania
major changes the cellular environment of monocytes in
vitro. We found that Leishmania-infected monocytes
actively mature into macrophages in the absence of GM-
CSF, and that these cells up-regulate the expression of
ribonucleotide reductase, an enzyme that catalyzes the
formation of deoxynucleoside triphosphates (dNTPs). We
confirmed the elevation of dNTP concentrations using a
very sensitive dNTP assay for monocytes and monocyte-
maturated macrophages. Collectively, these data support a
model in which infection of monocytes with Leishmania
elevates the intracellular dNTP pools, which is one of the
natural anti-viral blocks to HIV-1 infection in monocytes
and macrophages in patients.
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Leishmania-infected monocytes. Cell numbers fell from 36106at
day 0 in all three conditions and were consistently lower in control
cells as compared to either GM-CSF-treated (positive control) or
Leishmania-infected cells at all times tested from day 3 to day 28.
Control monocyte numbers declined until day 7, when their
numbers stabilized through day 28. Cell numbers for GM-CSF-
treated and Leishmania-infected groups remained significantly
higher than control monocytes at all time points from day 3 to
day 28 (Friedman test; p,0.05).
Next, we examined the different cell populations using light
microscopy. The control cells largely retained a small, mostly
rounded morphology (Figure 1B) at day 14 as compared to either
GM-CSF-treated (Figure 1C; positive control) or Leishmania-
maturated MDMs (Figure 1D). For both treatments, the
monocytes were larger, more adherent and spread out with some
processes, which is characteristic of mature macrophages.
Using FACS analysis, the GM-CSF-treated and Leishmania-
maturated MDMs were larger (as assayed by forward scatter) with
greater cellular complexity (assayed by side scatter) as compared to
control monocytes (Figure S2). These findings were further
confirmed and quantitated by FACS analysis of cell surface
CD14 expression from six independent donors. This demonstrated
a decreased cell surface expression of CD14 (CD14low) in both day
14 GM-CSF-treated and Leishmania-infected MDMs as compared
to control monocytes (CD14high), again consistent with monocytes
to macrophages maturation in the GM-CSF and Leishmania-
infected cultures (Figure S2). FACS analysis for both Annexin V
and propidium iodide also showed pronounced reduction in cell
death for the Leishmania-infected monocytes compared to unin-
fected controls (Figure S3) Collectively, these data suggest that
Leishmania infection of monocytes leads to less cell death and
increased cellular maturation towards a macrophage phenotype
compared to control monocytes.
The effect of Leishmania infection on human monocyte
While performing the kinetic studies of Leishmania-infected
monocytes, we observed clusters of small cells lying on top of
Figure 1. Determining cell viability. A) 16106monocytes/well were plated and then left untreated (control), GM-CSF-treated or Leishmania-
infected at an MOI=7. At various days afterwards, three wells/condition were collected, pooled and the cell numbers determined. Nine independent
donors were examined. B–D) Images were captured using bright field microscope. Control cells had very few adherent cells as compared to GM-CSF-
treated and Leishmania-infected cell cultures.
Co-Infection Mechanism of HIV and Leishmania
PLoS Pathogens | www.plospathogens.org3 April 2012 | Volume 8 | Issue 4 | e1002635
larger, more differentiated appearing macrophages in both the
Leishmania-infected and GM-CSF-treated (positive control) cultures
but not for the control cell culture. Although, as noted above,
human monocytes are generally believed not to proliferate once
released from the bone marrow [18,19], it has been more recently
recognized that these cells possess far greater heterogeneity than
originally believed and subpopulations of monocytes have been
recently described that can re-enter the cell cycle in response to M-
CSF and GM-CSF [20–22]. Proliferation of these monocyte
subpopulations has been demonstrated by multiple techniques
including uptake of BrdU and CFSE labeling [23,24]. Thus, we
next asked whether their presence might also be induced in the
setting of Leishmania infection. To address this, we did a time-
course analysis at days 3, 7, 10, and 14, examining BrdU uptake at
48 hours after treatment for the Leishmania-infected groups .
As expected, we detected a few cells that were uniformly BrdU+
(green) and nuclei counterstained with DAPI (blue) (Figure 2A).
We detected a progressive increase in the numbers of BrdU+ cells
over time, with maximal numbers of BrdU+ cells observed at day
14 of cell culture. Lastly, we co-labeled primary human monocytes
with PKH-labeled L. major (orange) and then pulsed with BrdU
(Figure 2A, bottom right panel Day 21). BrdU+ nuclei were seen
in Leishmania-infected cells suggesting that infection may promote
re-entry into the cell cycle for a sub-population of cells. This may
be of importance to the dissemination of Leishmania within a host
because macrophages are generally considered terminally differ-
entiated, non-dividing cells .
We subsequently performed quantitative FACS analyses to
compare the percentages of BrdU+ cells. As shown in a
representative FACS plot, Figure 2B, a relatively large sub-
population of BrdU+ cells was seen in both Leishmania-infected
(13.4%) and GM-CSF-treated cells (14.8%) but not in control cells
(,1.0%). Figure 2C summarizes results for 48 hour BrdU
incorporation for seven independent donors between days 12–
14. Leishmania-maturated MDMs demonstrated highly statistically
significant (p,0.01) elevations of the percentage of BrdU+ cells as
compared to control cells while GM-CSF-maturated MDMs were
significantly (p,0.05) higher. We also CSFE-labeled fresh
monocytes and found at least one cell division in a small
subpopulation of cells for the GM-CSF-treated and Leishmania-
infected groups (data not shown). Collectively our results are
consistent with previous studies of a proliferative monocyte sub-
population that can be stimulated to enter cell division by the
related monokine M-CSF [21,24]. However, of greater relevance
is the demonstration that L. major infection of monocytes can
induce an S phase environment as assayed here by BrdU
incorporation. Whether this promotes cell division in vivo, allowing
for greater dissemination of Leishmania, remains unclear.
The impact of Leishmania infection on human monocyte
intracellular dNTP concentration
We employed the highly sensitive HIV-1 RT based assay for
measuring cellular dNTP content [32,42–45]. As depicted in
Figure 3A, HIV-1 RT is bound to a template/primer complex.
HIV-1 RT can extend the primer by one nucleotide, depending
on the template nucleotide (N) present at the 59 end of the
template. This assay allows for the determination of differences
between cellular extracts for a specific cellular dNTP. Using this
assay, we compared the cellular content of dGTP (purine) and
dTTP (pyrimidine) for the different treatment groups. Figure 3B
shows a representative result for primer extension of dGTP (left
panel) and dTTP (right panel). Summary results for nine
individual donors are presented in graph form in Figure 3C and
are summarized below.
In Figure 3B, left side panel, dGTP levels were assayed at days 7
and 13, while the right side panel shows dTTP analysis for the
same days. In lanes 1 for both dGTP and dTTP analysis, no
dNTPs were added to the reaction, leading to no extension
product of the labeled primer (open arrow). In lanes 2, exogenous
dNTPs were added as a positive control to show extension of all
primers in the reactions (closed arrow). In lanes 3–8, days 7 and 13
cellular extracts were analyzed. Content of dGTP were notably
higher in GM-CSF- and Leishmania-maturated MDMs as com-
pared to untreated control cells at day 7 (lanes 4 and 5) and day 13
(lanes 7 and 8) after treatment. In comparison, dTTP concentra-
tions at day 7 were slightly higher for the GM-CSF-maturated
MDMs (lanes 4, positive control) as compared to the control and
Leishmania-maturated MDMs. At day 13, we detected much higher
Figure 2. BrdU analysis. A) Monocytes were infected with Leishmania
at day 0. BrdU reagent was added 48 hours before fixing, processing
and capturing the images. B) BrdU incorporation as monitored by FACS
analysis. At day 13 of maturation, the cell populations from different
groups were harvested and BrdU incorporation was determined. One of
six independent donors is displayed for each group. C) The six
independent donors were graphed and displayed as mean and SEM for
BrdU+ cells. The GM-CSF-treated and Leishmania-infected MDMs have
significantly higher BrdU incorporation as compared to control
monocytes (*=p,0.05; **=p,0.01).
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Co-Infection Mechanism of HIV and Leishmania
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dTTP concentrations in the GM-CSF and Leishmania-maturated
MDMs at day 13 (lanes 7 and 8) as compared to the control group
(lanes 6). These data demonstrate that Leishmania infection can lead
to notable increases in cellular dNTP concentrations and this
conclusion is fully validated by quantification of the assay results in
nine individual donors (Figure 3C). Results for dGTP (Figure 3C,
upper panels) demonstrated statistically significant increases for
GM-CSF-matured and Leishmania-infected MDMs as compared to
control monocytes at day 7; by day 13 dGTP increases were now
highly significantly elevated in Leishmania and still significantly
elevated in the GM-CSF-maturated MDM groups as compared to
controls. The results for dTTP at day 7 (Figure 3C, lower left
panel) trended higher in Leishmania-maturated MDMs as com-
pared to monocyte controls but only reached significance in GM-
CSF-maturated MDMs. However at day 13, (Figure 3C, lower
right panel) Leishmania-maturated MDMs were significantly
increased in dTTP concentrations as compared to monocyte
controls. These data demonstrate that Leishmania infection of
monocytes induces elevation of both purine and pyrimidine
concentrations in the host cell. The finding of elevated purine
levels is particularly intriguing in light of the fact that Leishmania
species are entirely dependent on host cell synthesis for their
supply of purine nucleotides .
Exposure of primary human monocytes to Leishmania
induces elevated levels of RNR subunits
Mammalian RNR is a dimeric enzyme essential for catalyzing the
direct reduction of relatively large intracellular pools of ribonucle-
otides into the corresponding deoxyribonucleotides for DNA
synthesis. The catalytic enzyme is a heterodimer, containing two
subunits of R1 and either two subunits of R2 or p53R2. Expression
of the R2 subunit is strictly limited to the S phase of the cell cycle
. As shown in Figure 4A, western blot analyses were done for R2
and p53R2 on cell extracts using freshly isolated monocytes, day 13
GM-CSF or Leishmania-maturated MDMs. As shown in Figure 4B,
we quantitated the western blots for four independent donors and
found that R2 was significantly (p,0.05) increased in the Leishmania-
maturated MDMs over control monocytes. For the p53R2, we
found a significant increase in the GM-CSF-treated cells but the
increase failed to reach significance for the Leishmania-infected cells
when compared to monocytes, which were set to 1. Moreover, the
R2 and p53R2 antibodies were specific for human ribonucleotide
reductase and did not cross-react with L. major (data not shown).
Collectively, these data show that 1) R2 subunit expression, which is
S phase linked, is significantly increased upon Leishmania infection,
and 2) that infection indirectly leads to an increase in the p53R2
subunit, which is involved in increasing cellular dNTP concentra-
tions in non-dividing cells.
Analysis of Ribonucleotide reductase R2 and p53R2
Next, quantitative reverse transcriptase quantitative PCR (qRT-
PCR) using Taqman analysis was performed in three individual
donors to examine whether the observed increase in RNR R2
subunit and P53R2 protein expression showed transcriptional
regulation (Figure 4C). Consistent with the significantly increased
protein expression of the RNR R2 subunit seen by western blot,
significantly increased transcription was seen in Leishmania-infected
monocytes as compared to GM-CSF-treated MDM and control
monocytes. It is also possible that these results may be due, at least
in part, to an increase in RNR R2 transcript stability. In contrast,
increased expression of p53R2 protein likely occurs due to post-
transcriptional regulation as no significant elevation of transcrip-
tion was seen in either the GM-CSF-treated or Leishmania-infected
MDMs as compared to control monocytes.
Results of HIV-1 D3 GFP transduction for the different
As noted above, cellular dNTP levels serve as a biomarker for
the replicative capacity of mammalian cells, a finding corroborated
by the presence of consistently higher dNTP levels in dividing cells
as compared to non-dividing cells [25–30]. HIV-1 replication
efficiency is also directly correlated with the cellular dNTP
concentration, and we and others have reported that it proceeds
with far greater efficiency in tumor cells or PHA-stimulated CD4+
T cells in which the average dNTP level is 150–225 times higher
than in non-dividing monocytes/macrophages [32,33]. Given our
findings that Leishmania infection induces both significant elevation
of dNTP levels and replication capacity in MDMs, we examined
whether transduction of Leishmania-maturated MDMs with a VSV-
g pseudotyped HIV-1 vector, designated HIV-1 D3 GFP, resulted
in accelerated HIV-1 expression, as determined by GFP
expression. Six days after isolation, control cells, GM-CSF
maturated MDMs, and PKH-labeled (red) Leishmania-maturated
MDMs were transduced in 6-well dishes with equal amounts of
HIV-1 D3 GFP vector. We examined cells by bright field and
fluorescence microscopy 24 hours later (Figure 5A). HIV-1 D3
GFP expression (‘‘GFP’’ [green-top 3 panels]) was markedly
enhanced, relative to control cells, in both the GM-CSF- and
Leishmania-maturated MDMs (upper middle and right-sided
panels, respectively), consistent with both strikingly increased
intensity and numbers of HIV-1 D3 GFP transduced cells in these
two conditions relative to control cells (Figure 5A; top left panel).
Only a rare control cell appeared to express HIV-1 D3 GFP. GM-
CSF- and Leishmania-maturated MDMs had many more cells
expressing GFP as compared to control cells. Leishmania maturated
MDMs labeled with PKH showed comparable numbers of GFP+
cells/field as compared to GM-CSF-maturated MDMs (Figure 5A;
middle and top right panels). We next quantified the three
different groups by FACS analysis (Figure 5B and Table 1). For
these studies, the Leishmania were not labeled with PKH dye. As
shown in Table 1, cells from four independent donors were
examined at 24 and 48 h after the addition of the HIV-1 D3 GFP
vector. The percent of GFP+ cells for Leishmania-maturated MDMs
were consistently higher as compared to the control cell group
with a somewhat weaker trend to higher percentage of GFP+ cells
Figure 3. HIV-1 RT based dNTP assay. A) Diagram shows how a single nucleotide extension assay is done. The reaction contains the template, 59
32P-end-labeled primer, HIV-1 RT and cellular dNTP extract. After the reactions are completed, they are resolved on a polyacrylamide gel to determine
product formation. The concentrations of dGTP and dTTP in the cellular extract will determine the amount of primer extension. B) For the different
groups, primer extension products are shown for day 7 and day 13 cellular extracts. Control monocytes, GM-CSF-treated and Leishmania-infected cells
formation is indicated with a filled arrow.Thenegative controlscontain no dNTPsandareshown in lane 1.Positive controls(lanes2) contained50 mM of
exogenous dNTP mix. Lanes 3–8 are cellular extracts from the different treatment groups. C) Graphs plotting the percent extension of dGTP and dTTP.
From the primer extension assays, data was plotted for days 7 and 13 for dGTP (n=9; top graphs) and for dTTP (n=6; bottom graphs). Significantly
different groups are displayed as *=p,0.05, **=p,0.01 and ***=p,0.001 for the different groups as compared to control monocytes.
Co-Infection Mechanism of HIV and Leishmania
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also found in the GM-CSF-maturated MDMs as compared to
Real Time PCR analysis for 2LTR circles
Next, we examined 2LTR circles, an indicator for the
completion of DNA synthesis by HIV-1 reverse transcriptase
but a failure of the DNA to integrate into the host genome. As
shown in Figure 6, the 2LTR circles copy number ratio was
significantly higher (*, p,0.05) in the Leishmania maturated
MDMs group as compared to control cell group (set to 1.0). The
2LTR circle number ratio for GM-CSF-maturated MDMs group
is higher than the controls cells, but did not achieve statistical
significance. Collectively, these data indicate that Leishmania
infection promotes a pro-HIV-1 environment within the cell,
leading to higher dNTP concentrations that allow for more
efficient viral infection.
In mutually endemic areas of the world, Leishmania species and
HIV-1 primarily co-infect mononuclear phagocytes of infected
mammalian hosts. It is widely believed that Leishmania infection
found concurrently with HIV-1 induces a state of chronic immune
activation leading to subsequent increased HIV-1 viral load and
accelerated progression to AIDS . Although the mechanisms
underlying this phenomenon are incompletely understood, in vitro
studies to date have implicated a variety of Leishmania-induced pro-
inflammatory cytokines including TNF-a, IL-1, and IL-6, in
stimulating HIV-1 replication in both monocytoid cell lines and
macrophages [17,49–52]. For example, the induction of TNF-a is
known to activate HIV-1 replication through mechanisms
involving transcriptional activation of nuclear factors binding to
NF-kB sequences in the HIV-1 LTR , while IL-6 and IL-1
Figure 4. Western blot analysis of ribonucleotide reductase. A) Cellular lysates from different treatment groups were analyzed for
ribonucleotide reductase R2 and p53R2 subunits. Afterwards, blots were stripped and re-probed for actin. Freshly isolated monocytes (Mo), day 13
maturated GM-CSF (GM-CSF) and Leishmania (Leish) MDMs are shown. B) Quantitiation of western blots was done. Freshly isolated monocytes were
set to 1 and increases in R2 and p53R2 expression levels for GM-CSF- and Leishmania-maturated MDMs groups are shown. Mean and SEM are
displayed for four independent donors. C) qRT-PCR analysis was done on total cellular RNA extracts. mRNA fold changes for the different treatment
groups (n=3) are graphed as mean and SEM. Significantly different groups (p,0.05) as compared to monocyte control group are indicated with an
Co-Infection Mechanism of HIV and Leishmania
PLoS Pathogens | www.plospathogens.org7 April 2012 | Volume 8 | Issue 4 | e1002635
appear to promote HIV-1 replication through less well-defined
mechanisms [51,52]. In this study, a novel mechanism is described
in which Leishmania infection of HIV-1 infected CD14+ primary
human monocytes promotes accelerated HIV-1 expression by
induction of MDMs RNR with subsequent elevation of intracel-
lular dNTP concentrations.
This same mechanism could explain numerous previous in vitro
and in vivo observations of accelerated HIV-1 replication in AIDS
clinical trials for patients treated with GM-CSF . Soon after
the recognition that HIV-1 was the etiologic agent of AIDS, it was
recognized that physiological stimuli, including GM-CSF, could
exert an inductive effect on HIV-1 replication in infected
monocytoid cells, though the potential mechanisms for this
induction have remained unknown . Most subsequent studies
have largely confirmed this original observation [55–61], although
some others have demonstrated opposite results with the
suppression of HIV-1 replication [62,63]. In vivo, however, the
results of four clinical trials using GM-CSF therapy in HIV-1
infected patients not treated with anti-retroviral drugs all
demonstrated increased plasma levels of HIV-1 RNA and p24
antigen as compared to control patients [64–67]. Most recently,
the results of the previous negative in vitro studies, in which
treatment with GM-CSF may have lowered HIV-1 replication,
Figure 5. HIV-1 D3 vector analysis. A) Control, GM-CSF-treated and Leishmania-infected cells were transduced with HIV-1 D3 GFP vector. Twenty-
four hours after transduction, the cells were examined for GFP expression (top row). Very few monocytes were GFP+, whereas more cells were GFP+
for the GM-CSF-treated and Leishmania-infected groups. For this experiment, the L. major were labeled with PKH dye and showed that the monocytes
were infected. Bright field images were captured for the different groups. B) FACS analysis was done of three different cell populations. Data is
representative of four different donors done at 24 h. Complete data sets for 24 h and 48 h are shown in Table 1.
Co-Infection Mechanism of HIV and Leishmania
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may be reconciled: the majority of results showed that up-
regulation of viral replication was generally enhanced in GM-
CSF-maturated MDMs when grown at low densities, whereas
more crowded cultures of MDMs and excessive acidification of the
medium led to suppressed viral replication .
Although GM-CSF treatment promotes maturation of mono-
cytes into macrophages, which are terminally differentiated, non-
dividing cells, there is an emerging awareness that, although
human monocytes do not proliferate in the steady state, a
proliferative monocyte sub-population exists that can re-enter the
cell cyclein response to
have been found to be able to produce a variety of colony
stimulating factors, most notably GM-CSF [70–72]. Here we
confirm that monocyte sub-populations treated with GM-CSF are
able to re-enter the cell cycle and show, for the first time, that
Leishmania infection promotes an S-phase environment in normally
quiescent monocyte sub-populations. Statistically significant ele-
vated percentages of BrdU+ cells were found in Leishmania-infected
MDMs compared to uninfected controls (Figure 2C). Further,
both monocyte maturation and proliferation occurred through a
mechanism independent of GM-CSF as treatment with a high
concentration of neutralizing antibody, fully sufficient to block the
effects of 5 ng/ml added GM-CSF had no effect on the Leishmania-
infected cells (Figure S4). These findings are in accord with newly
described rodent data demonstrating local in situ proliferation of
tissue macrophages in response to infection with a rodent filarial
nematode  and a previous study demonstrating in situ
proliferation of macrophages in the lungs of hookworm-infected
The promotion of monocyte proliferation by both GM-CSF
treatment and Leishmania infection has profound implications for
monocyte cell biology. We now report the quite novel finding that
monocyte proliferation, induced by the presence of GM-CSF and,
more potently by infection with L. major, also promotes
significantly higher dNTP levels at days 7 and 13 in culture as
compared to freshly isolated peripheral blood monocytes. Elevated
synthesis of the purine, dGTP in particular, was highly statistically
significant in day 13 Leishmania-infected MDMs compared to levels
in control monocytes (Figure 3C). In addition, induction of cellular
RNR, the enzyme catalyzing the direct reduction of ribonucleo-
tides to their corresponding dNTPs was found to be significantly
elevated in the Leishmania-maturated MDMs. Specifically, an
approximately 40-fold increase in RNR protein levels was
observed in immunoblots of day 13 Leishmania-maturated MDMs
versus freshly isolated monocytes using an antibody directed
against the R2 subunit of human RNR (Figure 4B). That this
induction of RNR R2 is regulated at the transcriptional level is
supported by the similarly statistically significant elevation of RNR
R2 RNA assayed by qRT-PCR (Figure 4C). These findings are
particularly intriguing in that the expression of the R2 subunit is
known to be strictly and specifically restricted to the S phase of the
cell cycle , consistent with the observed induction of cell cycle
re-entry in both Leishmania- and GM-CSF-maturated MDMs.
The present demonstration that Leishmania infection of human
monocytes induces elevated dNTP concentrations also has far-
reaching implications for Leishmania pathogenesis. Unlike their
mammalian hosts, Leishmania lack the metabolic machinery needed
for purine nucleotide synthesis. They must therefore rely on the
host cell production of purines and have evolved an obligatory
purine salvage pathway for this purpose . The dimeric enzyme
ribonucleotide reductase is the major source of dNTPs in
mammalian and other cells, forming them from the far more
Table 1. Percent HIV-1 D3 GFP Transduction.
% GFP+ + cells (24 h)
DonorControl GM-CSF Leishmania
1 0.027.21 7.81
3 0.100.59 1.79
% GFP+ + cells (48 h)
1 0.02 0.251.31
2 0.073.08 6.26
42.14 7.69 6.27
Figure 6. 2LTR circle copy number ratio. The different treatment
groups were treated with the D3 GFP vector. At 48 h after transduction,
total cellular DNA was collected and analyzed by real-time PCR. The
control group for each donor was set to 1 and then compared to GM-
CSF- and Leishmania-maturated MDMs groups. Mean and SEM are
plotted. The significantly different group (p,0.05), as compared to
control, is indicated with an asterisk. Seven independent donors were
Co-Infection Mechanism of HIV and Leishmania
PLoS Pathogens | www.plospathogens.org9 April 2012 | Volume 8 | Issue 4 | e1002635
abundant pool of rNTPs by the removal of the 29 OH on the
ribose sugar moiety . Our finding that Leishmania infection of
(Figure 4B) is fully consistent with the elevated dNTP concentra-
tions noted above and represents an elegant evolutionary
adaptation by which Leishmania can salvage necessary host purines
(and pyrimidines). A more recent consequence of Leishmania-
mediated induction of host RNR and elevated dNTP concentra-
tions has been to provide a highly permissive environment for
HIV-1 replication in the setting of co-infection. These findings are
especially significant in light of data that HIV-1 proviral DNA
synthesis in non-dividing cells is slower than in dividing cells ,
and can be accelerated by experimentally elevating the intracel-
lular dNTP concentration . They may also be of particular
relevance in the setting of infection with Leishmania, in which rapid
proliferative expansion of local splenic and bone marrow
monocyte/macrophage progenitor populations has been described
. In this setting, elevated dNTP concentration would also be
expected with accompanying enhancement of HIV-1 replication
in such dividing cells.
HIV D3 GFP transduction, a model for HIV-1 infection, is also
markedly enhanced in these matured cells (Figures 5A, 5B, and
Table 1). Both the fluorescent microscopic and flow cytometry
results demonstrated substantially increased numbers of HIV-1 D3
GFP+ transduced cells in the setting of Leishmania infection. These
findings were further confirmed by a statistically significant
elevation of the 2LTR circle copy number ratio in Leishmania
infected MDM compared to control monocytes by qPCR
Our results for MDMs maturated by GM-CSF treatment or
infection with Leishmania conform well to the majority of studies
showing enhanced HIV-1 replication, most likely due to
monocytes maturating into macrophages. This is a critical finding
in that we have recently reported that HIV replication efficiencies
in a wide variety of relevant cell types, including monocytes and
macrophages, is directly related to the relative intra-cellular dNTP
concentrations [31,32]. Thus, the finding of elevated dNTP levels
in both GM-CSF- and Leishmania-maturated human MDMs, as
compared to both freshly isolated monocytes and untreated
control cells, offers a novel mechanism to explain both the present
results as well as prior in vitro and in vivo studies that demonstrate
accelerated HIV-1 replication in both GM-CSF-treated and
Leishmania co-infected patients [55,67,77,78]. These results are
consistent with the 200–1500 times decrease in replication
competence of wild-type HIV-1 in monocytes as compared to
the corresponding differentiated MDMs .
The present study represents the first demonstration that
Leishmania promotes both maturation and proliferation phenotypes
in primary human monocytes. During this process we detected
elevated intracellular dNTP pools in Leishmania-infected cells,
which allows more efficient replication of intracellular co-infected
HIV-1. This observation of enhanced pathogen expression in co-
infected target cells may be a more generalized phenomenon. For
example, the course of HIV-1 related immunodeficiency is also
known to be accelerated by active infection with Mycobacterium
tuberculosis (MTB) , and in vitro studies have demonstrated that
MTB-infection of MDMs subsequently infected with HIV-1
produce increased levels of virus as compared to MDMs
uninfected with MTB . In matched CD4+ T cell cohorts,
both HIV-1 viral load and heterogeneity are increased by MTB
infection. In addition, infection of monocytes/macrophages with
two other clinically relevant Mycobacterium was found to enhance
HIV-1 replication both in vitro and in situ [81–83]. Conversely,
patients co-infected with HIV-1 and MTB have altered granulo-
mas within the lung . Also higher bacterial burden was
detected for HIV-1 and MTB co-infection of MDMs in vitro .
Our data suggests that we are just beginning to understand the
synergy between virus and parasite co-infections of human cells.
Materials and Methods
These experiments used primary human primary monocytes
obtained from human buffy coats (New York Blood Services, Long
Island, NY). These are pre-existing materials that are publicly
available, and there is no subject-identifying information associ-
ated with the cells. As such, the use of these samples does not
represent human subjects research because: 1) materials were not
collected specifically for this study, and 2) we are not able to
identify the subjects.
Primary human monocytes were isolated from the peripheral
blood buffy coats by positive selection using MACS CD14+ beads
as previously described . Three culture condition were used: 1)
RPMI 1640 containing 10% FCS and Penicillin/Streptomycin
antibiotics without further supplements indicating ‘‘control’’
monocytes, 2) RPMI containing 10% FCS, Pen/Strep antibiotics
and 5 ng/ml human recombinant GM-CSF (R&D Systems)
indicating ‘‘GM-CSF-treated’’ monocytes, or 3) RPMI 1640
containing 10% FCS, Penicillin/Streptomycin antibiotics and
Leishmania major (MOI=7) indicating ‘‘Leishmania-infected’’ mono-
cytes. Leishmania major promastigotes (strain WHOM/IR/–/173)
were grown to stationary phase culture and infectious metacyclic
promastigotes were isolated by negative selection using peanut
agglutinin . L. major were labeled with 2 mM PKH26
fluorescent cell dye (Sigma) as per manufacturer’s protocol.
HIV-1 D3 GFP vector generation: HIV-1 D3 GFP vector encodes
the HIV-1 NL4-3 genome with the eGFP gene in place of the
HIV-1 nef gene and has a deleted envelope . To generate
virus, 293T cells in T225 flasks were transfected with 60 mg pD3-
HIV and 10 mg pVSV-g plasmids using 140 ml polyethyenimine
(1 mg/ml) in 37 ml DMEM media/flask. At day 1 of HIV-1
production, media was discarded and replaced with fresh DMEM
media. At day 2, media was harvested and replaced with fresh
DMEM media. The media was centrifuged at 2500 RPM for
7 minutes to remove cellular debris, and then stored at 4uC in T75
flask. Day 3 media was harvested and processed as described for
day 2. HIV-1 D3 GFP was concentrated using ultracentrifugation
(22K RPM for 2 h in a SW28 rotor). Viral pellets were DNase I
digested for 1 h at 37uC. Afterwards, debris was removed by
centrifugation (14K for 5 minutes). Sample aliquots were frozen at
280uC until used. Different groups were transduced with HIV-1
D3 GFP and then the samples were analyzed using Accuri C6 flow
cytometer monitoring GFP expression at 24 h or 48 h after
transduction. Data files were analyzed using FlowJo software
Primer extension assay
Nucleotide incorporation assay employs a 19-mer DNA
template (39-CAGGGAGAAGCCCGCGGTN-59). The N indi-
cates the change in template for detecting a specific dNTP within
the cellular extract. The template is annealed to a 59 end32P-
labeled 18-mer DNA primer (59-GTCCCTGTTCGGGCGCCA-
39). HIV-1 RT is used for this reaction . 16106cells for
control monocytes, GM-CSF-treated MDMs, and Leishmania-
infected MDMs were collected and lysed with 60% cold methanol.
Cellular debris was cleared by 14K centrifugation. Supernatant
Co-Infection Mechanism of HIV and Leishmania
PLoS Pathogens | www.plospathogens.org10April 2012 | Volume 8 | Issue 4 | e1002635
was dried. Pellet was resuspended in 20 ml reaction buffer (50 mM
Tris-HCl, pH 8 and 10 mM MgCl2). Two microliters were used
in the primer extension assay.
Forty-eight hours before harvesting, cells were pulsed with
300 mM BrdU. For microscope analysis, media was removed and
the 6-well plate was washed once with PBS. Cells were fixed for
using 4% paraformaldehyde for 20 minutes and then washed with
PBS. Two milliliters of Target Retrieval Solution (Dako) was
added and plates were heated in a rice cooker for 15 minutes at
95uC. Afterwards the plates were removed and allowed to cool.
Cells were stained with rat anti-BrdU-FITC antibody (AbD
Serotec) for 20 minutes at 4uC. Images were captured using a
Zeiss microscope. For FACS analysis, on the day of harvest, the
free cells were collected while the adherent cells were Trypsin
treated for 30 minutes before scraping the 6-well plate. Both free
and adherent cell populations were pooled, centrifuged at
1200 RPM for 5 minutes. Supernatant was removed and the cells
were fixed using 4% paraformaldehyde for 20 minutes. After
fixing, the cells were washed once with PBS. The cells were stored
at 4uC until processing for BrdU staining. For BrdU staining, cells
were transferred to a 6-well plate containing 2 ml of Target
Retrieval Solution and heated in a rice cooker for 15 minutes at
95uC. Afterwards the plates were removed and allowed to cool.
Cells were transferred to tubes and cells washed once with PBS.
Next the cells were stained with rat anti-BrdU-FITC antibody for
20 minutes at 4uC. The sample data were collected using an
Accuri C6 flow cytometer.
Western blot analysis
Samples were processed in RIPA buffer containing 1 mM DTT,
10 mM PMSF, 10 ml/ml phosphatase inhibitor (Sigma) and 10 ml/ml
pulses, to ensure complete lysis. Cellular debris was removed by 15K
RPM centrifugation for 10 minutes. Supernatants were stored at
280uC before use. Cell lysates (25 mg) were resolved on an 8% SDS-
PAGE gel. Proteins were transferred to a nitrocellulose membrane.
The membrane was blocked with 2% non-fat milk in TBST for 1 h,
followed by the addition of primary goat anti-R2 antibody (Santa
Cruz Biotechnology) and incubation overnight at 4uC. The next day,
the membrane was washed (3X, 20 minutes with TBST) followed by
membrane was washed 36 with TBST and developed using
SuperSignal West Femto Kit (Thermo Scientific). The immunoblot
a BioRad ChemiDoc Imager.
Analysis of Ribonucleotide reductase R2 and p53R2
mRNAs by quantitative reverse transcriptase PCR
46106cells were lysed and RNA prepared using the RNeasy
Mini Protocol as per the manufacturers’ instructions (Qiagen,
Valencia, CA). Pre-mixed Taqman primer/probe sets for RNR R2
and p53R2 were obtained from Life technologies (Cat numbers
Hs01072069_gi and Hs00968432_m1, respectively). Template
RNA was diluted to 80 ng/ml and 4 ml from each sample, mixed
with Express One-Step SuperScript qRT-PCR reagents, was ran in
triplicate using an Applied Biosystems 7300 Real Time thermo-
cycler. Data were normalized to GAPDH mRNA.
2LTR circle analysis
Genomic extracts were prepared using QuickGene-810 Nucleic
Acid Isolation System (FujiFilm Global). The DNA was assayed for
2LTR circles by real time PCR using the following primers: 59-LTR
region — 59-GTGCCCGTCTGTTGTGTGACT-39 and 39LTR
region — 59-CTTGTCTTCTTTGGGAGTGAATTAGC-39, and
the probe 59-6-carboxylfluorsecein-TCCACACTGACTAAAAGG-
All samples were normalized to total DNA. The control samples for
each donor were set to 1.0 and 2LTR circle copy number ratio was
Graphing and statistical analysis
Prism software was used for plotting the data. All the data sets
were compared for significant difference using ANOVA analysis
cytes. Three independent donors were plated in 6-well plates at 1
million monocytes alone or with 7 million PKH-labeled
Leishmania. At days 2 and 4 after plating, PKH+ cells were
monitored using FACS analysis. Data for the different donors are
plotted as mean and SEM.
Monitoring Leishmania infection of mono-
tions. Three independent donors were examined by FACS analysis
at day 7 of maturation. FSC and SSC for the different populations,
wereexamined forfreshlyisolated,floating (F) andadherent(A) cells.
As clearly shown, freshly isolated monocytes have a smaller FCS and
SSC as compared to the other groups; day 7 control monocytes,
GM-CSF-treated and Leishmania infected cells. Next, we examined
CD14 expression levels by FACS analysis having a high and low
gating for mean fluorescent intensity (MFI). For the floating cells,
CD14 MFI was highest for the control monocytes (Mo(F)), and was
reduced for the remaining cell subsets. Finally, the percentage of
CD14 low and CD14 high cells were plotted. Floating cells were
CD14 high, whereas adherent cells were CD14 low.
Phenotypic analysis of different cell popula-
cytes, GM-CSF maturated and Leishmania maturated
MDMs. Annexin V and propidiumiodide (PI) staining of total cells
(both adherent and non-adherent) were monitored in all experi-
ments by FACS analysis. A) The untreated control monocytes had
33.2% apoptotic cell death as measured by annexin V and 21%
necrotic cell death as assayed by propidium iodide. B) The GM-
CSF-maturated MDMs, positive control, had 1.65% apoptotic cell
death and roughly 2% necrotic death, and C) the Leishmania-
maturated MDMs had roughly 2.1% apoptotic cell death and 4%
necrotic cell death. Our findings suggest that infection of monocytes
with Leishmania promoted cell survival comparable to the positive
control monocytes treated with GM-CSF.
Determining apoptosis for control mono-
Leishmania-infected maturation of monocytes. GM-
CSF-treated and Leishmania-infected monocyte cultures were
treated with isotype control or anti-GM-CSF antibodies (10 mg/
ml). Images were captured at day 5 of culture.
Anti-GM-CSF treatment does not block
Conceived and designed the experiments: DJM JAH DJF BK. Performed
the experiments: DJM JAH WD. Analyzed the data: DJM JAH WD.
Contributed reagents/materials/analysis tools: CAL MGO. Wrote the
paper: DJM JAH DJF BK.
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