Original research
published: 27 June 2018
doi: 10.3389/fimmu.2018.01494
Edited by:
Christel Vérollet,
UMR5089 Institut de
Pharmacologie et de Biologie
Structurale (IPBS), France
Reviewed by:
Arnaud Moris,
INSERM U1135 Centre
d’Immunologie et de Maladies
Infectieuses, France
Bin Su,
Capital Medical University,
China
*Correspondence:
Gabriela Turk
gturk@fmed.uba.ar
†
Present address:
María Julia Ruiz,
Centre de Recherche du Centre
Hospitalier de l’Université de
Montréal (CRCHUM), Montréal,
QC, Canada
Specialty section:
This article was submitted to
Microbial Immunology,
a section of the journal
Frontiers in Immunology
Received: 14 September 2017
Accepted: 15 June 2018
Published: 27 June 2018
Citation:
Trifone C, Salido J, Ruiz MJ, Leng L,
Quiroga MF, Salomón H, Bucala R,
Ghiglione Y and Turk G (2018)
Interaction Between Macrophage
Migration Inhibitory Factor and CD74
in Human Immunodeficiency Virus
Type I Infected Primary MonocyteDerived Macrophages Triggers the
Production of Proinflammatory
Mediators and Enhances Infection
of Unactivated CD4+ T Cells.
Front. Immunol. 9:1494.
doi: 10.3389/fimmu.2018.01494
interaction Between Macrophage
Migration inhibitory Factor and cD74
in human immunodeficiency Virus
Type i infected Primary MonocyteDerived Macrophages Triggers the
Production of Proinflammatory
Mediators and enhances infection
of Unactivated cD4+ T cells
César Trifone1, Jimena Salido1, María Julia Ruiz1†, Lin Leng 2, María Florencia Quiroga1,
Horacio Salomón1, Richard Bucala 2, Yanina Ghiglione1 and Gabriela Turk 1*
1
CONICET-Universidad de Buenos Aires, Instituto de Investigaciones Biomédicas en Retrovirus y Sida (INBIRS),
Buenos Aires, Argentina, 2 Department of Medicine, Yale University School of Medicine, New Haven, CT, United States
Understanding the mechanisms of human immunodeficiency virus type I (HIV-1)
pathogenesis would facilitate the identification of new therapeutic targets to control the
infection in face of current antiretroviral therapy limitations. CD74 membrane expression
is upregulated in HIV-1-infected cells and the magnitude of its modulation correlates
with immune hyperactivation in HIV-infected individuals. In addition, plasma level of
the CD74 activating ligand macrophage migration inhibitory factor (MIF) is increased in
infected subjects. However, the role played by MIF/CD74 interaction in HIV pathogenesis
remains unexplored. Here, we studied the effect of MIF/CD74 interaction on primary
HIV-infected monocyte-derived macrophages (MDMs) and its implications for HIV immunopathogenesis. Confocal immunofluorescence analysis of CD74 and CD44 (the MIF
signal transduction co-receptor) expression indicated that both molecules colocalized at
the plasma membrane specifically in wild-type HIV-infected MDMs. Treatment of infected
MDMs with MIF resulted in an MIF-dependent increase in TLR4 expression. Similarly,
there was a dose-dependent increase in the production of IL-6, IL-8, TNFα, IL-1β, and
sICAM compared to the no-MIF condition, specifically from infected MDMs. Importantly,
the effect observed on IL-6, IL-8, TNFα, and IL-1β was abrogated by impeding MIF
interaction with CD74. Moreover, the use of a neutralizing αMIF antibody or an MIF
antagonist reverted these effects, supporting the specificity of the results. Treatment
of unactivated CD4+ T-cells with MIF-treated HIV-infected MDM-derived culture supernatants led to enhanced permissiveness to HIV-1 infection. This effect was lost when
CD4+ T-cells were treated with supernatants derived from infected MDMs in which
CD74/MIF interaction had been blocked. Moreover, the enhanced permissiveness of
unactivated CD4+ T-cells was recapitulated by exogenous addition of IL-6, IL-8, IL-1β,
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MIF/CD74 Interaction in HIV Infection
and TNFα, or abrogated by neutralizing its biological activity using specific antibodies.
Results obtained with BAL and NL4-3 HIV laboratory strains were reproduced using
transmitted/founder primary isolates. This evidence indicated that MIF/CD74 interaction
resulted in a higher production of proinflammatory cytokines from HIV-infected MDMs.
This caused the generation of an inflammatory microenvironment which predisposed
unactivated CD4+ T-cells to HIV-1 infection, which might contribute to viral spreading
and reservoir seeding. Overall, these results support a novel role of the MIF/CD74 axis in
HIV pathogenesis that deserves further investigation.
Keywords: human immunodeficiency virus, cD74, macrophage migration inhibitory factor, primary monocytederived macrophages, cD4+ T-cells, immunopathogenesis
(8, 9) and Vpu (10) viral proteins. Moreover, accumulated data
suggest that Nef-mediated CD74 upregulation might play an
important role in HIV immunopathogenesis as: (i) this activity is
conserved among nef alleles from HIV-1 primary isolates, HIV-2,
and SIV (9, 11) as well as HIV-1 BF inter-subtype recombinant
forms (12), (ii) it has been documented in in vitro infected cell
lines [HeLa-CIITA, MelJuSo, and THP-1 (8, 13)] and also in primary CD4+ T-cells and monocyte-derived macrophages (MDMs)
(13, 14), and (iii) modulation levels differ among progressive
versus non-progressive infected individuals, both in adult (9) and
pediatric populations (13). Moreover, our group has demonstrated
that CD74 upregulation occurs on naturally infected MDMs
obtained directly from HIV+ subjects and that the magnitude of
this upregulation correlates with the level of immune activation
in those subjects, providing evidence for the contribution of the
HIV-mediated CD74 upregulation to immune damage during
the course of infection (15).
One of the alternative activities described for CD74 is its
ability to serve as the high-affinity binding component of the
heteromeric receptor for macrophage migration inhibitory factor
(MIF) (16–18). MIF is a proinflammatory cytokine that plays a
key role in anti-stress and anti-microbial responses. It is secreted
by different immune cells including T and B lymphocytes,
macrophages, monocytes, and dendritic cells among others (19).
MIF has been related to the pathogenesis of diverse inflammatory,
infectious, autoimmune, and metabolic diseases as well as different types of cancer (20–33). During HIV infection, increased
MIF plasma levels have been observed during the acute phase of
infection and remained elevated (34, 35). On the other hand, it
has been demonstrated that MIF was heavily produced by in vitro
infected peripheral blood mononuclear cells (PBMCs) and also
by uninfected gp120-stimulated PBMCs. Moreover, the addition of exogenous recombinant MIF to in vitro infected PBMCs
increased viral replication (34).
Despite the fact that MIF is a key component of the inflammatory immune response, that it is elevated in plasma from
HIV-infected subjects, and that the virus itself modulates the
surface expression of its receptor, no reports have explored
the role of the MIF/CD74 axis in HIV immunopathogenesis.
Thus, the aim of this work was to study the effect of MIF/CD74
interaction on the phenotype and the function of primary HIVinfected MDMs, and how this axis determines the environment
to modulate CD4+ T-cell permissiveness to infection.
inTrODUcTiOn
The pandemic of human immunodeficiency virus/acquired
immunodeficiency syndrome (HIV/AIDS) is still a major public
health concern worldwide. Combined antiretroviral therapy
(cART) can diminish the viral load (VL) to undetectable levels,
reducing not only morbidity and mortality but also transmission risks, with the subsequent impact on the dynamic of the
global epidemic (1). However, cART has several limitations like
the need of daily doses, the development of viral resistance, and
toxicity. More importantly, the rebound of VL levels in patients
who discontinue cART suggests the presence of long-lived viral
reservoirs that are resistant to cART, hampering the cure of the
infection. In addition, it is being increasingly clear that even
effectively treated HIV-infected individuals have a greater risk
of experiencing non-AIDS related morbidity and mortality
events than age-matched HIV-uninfected adults, indicating
that even effective cART cannot fully restore health. Most of
these complications are related to immune dysfunction and
inflammation and include gut-associated mucosal disruption,
lymphoid tissue damage, liver dysfunction, and monocyte/
macrophage activation which ultimately lead to the development of coagulopathies, atherosclerosis, vascular dysfunction,
and frailty, among other effects (2). Thus, understanding the
mechanisms underlying HIV persistence and irreversible
immune damage is extremely important to fight the infection
and its consequences.
CD4+ T-cells are the major targets of HIV infection followed
by macrophages. Productive viral replication is supported
mostly in activated CD4+ T-cells, which culminates in cell apoptosis. Conversely, macrophages are less permissive to HIV-1
infection albeit more resistant to virus-mediated cell killing,
thus viral replication proceeds for a longer time compared to
T cells (3, 4). Both cell types play an important role since the
onset of infection to the development of chronic inflammation
regardless of the different viral replication strategies maintained
in each cell type.
CD74 (also known as invariant chain or Ii) is a non-polymorphic
type II integral membrane protein expressed by antigen-presenting
cells. It was first described to act as a major histocompatibility class
II-associated chaperone; but now, it is increasingly understood
as a versatile protein with multiple roles (5–7). In the context of
HIV-1 infection, surface CD74 expression is upregulated by Nef
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Monocyte-derived macrophages were infected with the
R5-tropic viruses (either with the BAL or the R5-tropic T/F
strain) using a ratio of 1 ng p24/106 cells. MDMs to be evaluated by immunofluorescence microscopy were infected with the
VSV-G pseudotyped X4-virus and CD4+ T-cells were infected
using the X4-tropic virus (either the NL4-3 or the dual-tropic
T/F strain) by spinoculation (1,200 g for 1.5 h at 37°C) using a
ratio of 150 ng p24/106 cells in both cases. After adsorption, the
inoculum was removed and cells were washed twice in RPMI
medium.
MaTerials anD MeThODs
Primary human MDM and cD4+ T-cell
Purification and culture
Buffy coats from healthy donors were used to obtain PBMCs
by Ficoll-Hypaque (GE Healthcare Life Sciences, USA) density
gradient centrifugation. Monocytes were then separated from
PBMCs by Percoll (GE Healthcare Life Sciences, USA) gradient
technique. Isolated monocytes (purity >80% measured by flow
cytometry) were further purified by adherence to plastic plates
in RPMI 1640 medium (HyClone, GE Healthcare Life Sciences,
USA). Non-adherent cells were removed after 2 h plating by means
of extensive washes. Adherent cells were allowed to differentiate
into MDMs in RPMI 1640 medium supplemented with 10% fetal
bovine serum (FBS, Gibco, Thermo Fischer Corporation, USA),
2 mM l-glutamine (Sigma-Aldrich, USA), 100 U/ml penicillin
(Sigma-Aldrich USA), 100 µg/ml streptomycin (Sigma-Aldrich,
USA), and 10 mM HEPES (Sigma-Aldrich) (from now on
complete RPMI medium) plus 20 ng/ml recombinant granulocyte monocyte-colony stimulating factor (GM-CSF, Miltenyi,
Germany) for 4 days. After differentiation, MDM purity was
analyzed by flow cytometry and only donors with >90% purity
were used in subsequent assays.
CD4+ T-cells were isolated from buffy coats by negative
selection using the RosetteSep kit (Stem cell, Canada). Purified
cells (>95% purity by flow cytometry) were cultured in complete
RPMI medium plus 25 ng/ml IL-2 (BioLegend, USA). Culture
plates were incubated at 37°C in a humidified atmosphere with
5% CO2.
human samples
Plasma from 13 HIV seronegative healthy donors (HIV−) and 13
individuals with recent HIV-1 infection (HIV+) were obtained.
Samples from HD were obtained from eligible voluntary blood
donors >18 years old who completed a survey on blood donation
which particularly excludes persons who had been exposed to
HIV; and were screened for serological markers before being
accepted as donors. HIV-infected subjects were enrolled as part
of an ongoing acute/early primary HIV infection cohort from
Argentina (40–45). This study was reviewed and approved by
two institutional review boards: Comité de Ética Humana,
Facultad de Medicina, Universidad de Buenos Aires and Comité
de Bioética, Fundación Huésped (Buenos Aires, Argentina). Both
HIV-infected participants and HD provided written informed
consents accepting to participate in this study in accordance with
the Declaration of Helsinki.
immunofluorescence Microscopy
Monocyte-derived macrophages obtained as mentioned above
were cultured over coverslips and infected either with the pseudotyped GFP-expressing Nef wild type (WT) virus or the pseudotyped GFP-expressing Nef-defective (ΔNef) virus. Uninfected
cells were used as controls. Three days post-infection, MDMs
were fixed and permeabilized with Cytofix-Cytoperm buffer
(BD Biosciences) following the manufacturer’s instructions and
blocked with Cytoperm wash buffer plus 2% FBS. MDMs were
subsequently stained overnight with an anti-CD44 antibody
(BioLegend). The following day, cells were washed three times
and stained with an Alexa546-conjugated anti-mouse antibody
(Jackson, MS, USA) during 1 h. Finally, cells were washed three
times, treated again with cytofix-cytoperm buffer, blocked,
and stained overnight with an APC-conjugated anti-CD74
antibody (BioLegend, USA). After three final washes, cells
were fixed and mounted with DAPI-Fluoromount-G (Thermo
Fisher Scientific) and analyzed in an Olympus FV-1000
(Olympus, Tokyo, Japan) microscope with a Plapon 60×/1.42
NA oil immersion objective and using FV10-ASW v.01.07.03.00
software. Cross-sectional quantitation of mean fluorescence
intensity (mFI) was performed using Image J software. Single
stained controls were performed in order to exclude channel
spillover (cells stained only with APC-conjugated anti-CD74
antibody or the anti-CD44 antibody followed by Alexa546conjugated anti-mouse antibody staining). Also, individual
isotype controls were performed in order to exclude unspecific
antibody binding and cross-reactivity with the secondary
antibody.
Virus Production and infections
GFP-expressing X4-tropic HIV-1 virus stock was produced
by transfecting 293 T cells using the X-tremeGENE 9 DNA
transfection reagent (Roche, Switzerland) with the pBR43IeGnef+plasmid (kindly provided by Dr. Michael Schindler). This
plasmid encodes the full-length HIV genome plus the reporter
protein GFP (pBR-NL4-3 nef-IRES-eGFP, NefWT virus).
Similarly, a Nef-defective virus (ΔNef) was produced using
the pBR43IeG-nefSTOP plasmid. When stated, a pseudotyped
X4-tropic virus, generated by adding a plasmid encoding the
vesicular stomatitis virus (VSV) protein G to the transfection
solution, was used. Also, an R5-tropic HIV-1 viral stock was
produced by infecting primary MDMs from healthy donors
with the HIV-1 BAL strain. Finally, an R5- and a dual (R5X4)tropic transmitted/founder (T/F) infectious molecular clones
(IMCs) were selected from the full panel of T/F IMCs available at the NIH AIDS Reagent program [Division of AIDS,
NIAID, NIH: Cat #11746 and 11744, respectively, from Dr.
John Kappes (36–39)]. Both T/F viral stocks were produced
by transfecting 293 T cells using the X-tremeGENE 9 DNA
transfection reagent. Culture supernatants were harvested 48 h
post-transfection (for the NL4-3 and T/F viruses) or 14 days
post-infection (for the BAL R5-tropic stock). In all cases, culture supernatants were clarified by centrifugation at 600 g for
15 min at 4°C, fractioned and stored at −80°C until use. Viral
titer was estimated by p24 antigen quantitation by ELISA (Sino
Biological Inc., China).
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First, single cells were gated in a forward scatter (FSC)-height
(FSC-H) versus an FSC-area (FSC-A) plot. Then, gating was
performed on living MDMs in an FSC versus a side scatter
(SSC) plot. Infected cells were identified in an SSC versus FL-1
(FITC) plot as p24 positive events (Figure S1 in Supplementary
Material). Bystander cells were identified as the p24-negative
population on the same plot. TLR4 median fluorescence intensity
(MFI) was determined for uninfected, infected, and bystander
cells. Modulation of TLR4 expression was calculated as the ratio
between MFI corresponding to infected or bystander versus
uninfected cells.
evaluation of cD74 Modulation
After infection with the WT or ΔNef GFP-expressing viruses,
MDMs were detached with trypsin (Gibco) and stained with
an anti-CD74-PE (Santa Cruz Biotechnology, USA). Cells were
washed and analyzed in a BD FACSCanto flow cytometer (BD
Biosciences) using the FACSDiva v8.0.1 software (BD Biosciences)
or FlowJO v10 (Data Analysis Software, LLC). HIV-mediated
CD74 upregulation was calculated as the ratio between the FL2
median fluorescence intensity (MFI) of infected (GFP+) versus
uninfected (GFP−) cells.
recombinant cytokines and antibodies
cytokine Quantitation
Recombinant human MIF (rhMIF) was prepared as described
elsewhere (46) (endotoxin content < 0.1 EU/ml). MIF antagonist
MIF098 [3-(3-hydroxybenzyl)-5-methylbenzooxazol-2-one] was
dissolved in DMSO at a concentration of 149 µM (47). The
neutralizing anti-MIF monoclonal antibody (clone NIHlllD.9)
was obtained from ascites after purification using protein A/G
spin column and resuspended at 5.15 mg/ml (48, 49). A CD74
blocking antibody (BD Pharmingen, clone LN2), the recombinant human cytokines IL-6, IL-8, IL-1β (BioLegend), and TNFα
(MiltenyiBiotec), and the cytokine neutralizing antibodies
anti-IL-8 (R&D Systems), anti-IL-6, anti-IL-1β, and anti-TNFα
(BioLegend) were obtained.
The levels of the following cytokines were evaluated in MDM
supernatants using commercially available ELISA sets: IL-8,
IL-6, IL-1β, TNFα, IL-10 (ELISA MAX Deluxe kits, BioLegend)
and sICAM (DouSet ELISA, R&D Systems). MIF plasma levels
were evaluated using an in-house ELISA constructed with an
anti-human MIF antibody pair and an MIF standard obtained
from BioLegend.
Permissiveness induction in Unactivated
cD4+ T-cells
Unactivated CD4+ T-cells were incubated with supernatants
(1/2,000 dilution) from MIF-treated or untreated MDMs, for
72 h at 37°C. Before incubation, supernatants were clarified for
15 min at 600 g and UV-inactivated for 30 min (253.7 nm, 15 cm
away from the light source). After that, cells were washed and
infected with an X4-tropic HIV. Supernatants were collected
daily for p24 antigen quantitation till day 7 post-infection.
Phytohemagglutinin- (PHA, 2.5 ng/ml, Sigma-Aldrich, USA)
and RPMI-treated CD4+ T-cells were used as positive and negative controls, respectively.
Alternatively, unactivated CD4+ T-cells were stimulated with
recombinant IL-1β, IL-6, IL-8, and TNF-α, either alone or in
combination, for 72 h prior to infection.
MDM stimuli
Monocyte-derived macrophages were infected with the R5-tropic
HIV and the infection was left to progress. At day 11, infection percentage was evaluated by p24 intracellular staining
as described in the following paragraph (Figure S1 in Supplementary Material). After that, MDMs were washed twice with
PBS 1× (Sigma) and rhMIF was added to a final concentration
of 1, 10, or 25 ng/ml. Cells were incubated at 37°C for 8 h until
the supernatant was collected. When denoted, pretreatment
with the αCD74 blocking antibody (or the appropriate isotype
control) was performed at 5 ng/ml for 30 min. In some experiments (TLR4 expression), Fc receptors were blocked for 10 min
before the addition of the αCD74 blocking antibody (or its
isotype-matched control) with an Fc blocking reagent from BD
Biosciences.
cD4+ T-cell Phenotype, Viability,
and infection Percentage
The expression of CD38, CD69, HLA-DR, CD25, PD-1, and
CD28 surface molecules were analyzed by flow cytometry after
CD4+ T-cell stimulation with MDMs-derived supernatants for
72 h. Percentages of cells expressing the markers mentioned
above as well as their MFI were recorded. Initial gating was
performed on lymphocytes followed by gating on CD4+ events.
Isotype-matched non-specific antibodies were used in each
sample to set the corresponding negative populations accurately.
In addition, CD4+ T-cells were harvested from day 1 to 7
post-infection and both cell viability and infection percentages
were evaluated by flow cytometry. First, single cells were gated
in an FSC-H versus an FSH-A plot. Then, living lymphocytes
were gated an FSC versus an SSC plot (%viability). Subsequently,
infected cells were identified in an FSC-H versus GFP plot (Figure
S1 in Supplementary Material). Data acquisition was performed
in a BD FACSCanto flow cytometer using the BD FACSDiva
software and analyzed subsequently with FlowJO v10 software
(Data Analysis Software, LLC).
evaluation of Tlr4 expression
After MIF stimulation, infected and uninfected MDMs were
harvested and stained with a PE-conjugated anti-TLR4 antibody
(BioLegend) for 30 min at 4°C. Following incubation, cells were
washed, fixed, and permeabilized using the Cytofix/Cytoperm
kit (BD Biosciences) following the instructions provided by
the manufacturer. Then, intracellular p24 antigen was stained
using an anti-p24-FITC antibody (KC57-FITC, Coulter-clone,
Beckman Coulter, USA) for 30 min at 4°C. Cells were then
washed, fixed, and acquired in a BD FACSCanto flow cytometer.
Data acquisition was performed using the BD FACSDiva software
and analyzed subsequently with FlowJO v10 software (Data
Analysis Software, LLC). An isotype-matched FITC-conjugated
non-specific antibody was used to set the p24-negative population accurately.
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MIF/CD74 Interaction in HIV Infection
panel). In the cultures infected with the WT virus, CD74 MFI
was significantly higher in GFP-expressing cells (i.e., infected)
when compared to GFP-negative cells (i.e., uninfected). On the
contrary, no differences were observed in cultures infected with
the Nef-defective virus when comparing infected versus uninfected cells. Figure 1B compiles the upregulation magnitude
from three different donors (relative to the Nef-defective virus).
On the other hand, it has been reported that the plasma levels
of the CD74 ligand MIF were elevated during HIV infection
(34, 35). To confirm this, MIF concentration was evaluated in
plasma from recently HIV-infected subjects enrolled as part of
the Grupo Argentino de Seroconversión study group. In line with
the previous reports, our results indicated that the MIF plasma
level during the acute HIV infection (<6-month post-infection)
was 30-fold higher when compared to uninfected individuals
(Figure 1C).
Data analysis
Experiments were performed at least three independent times
and analyzed using parametric tests, unless otherwise stated (see
exact number of independent experiments in each figure legend).
Data normality was assessed using the Shapiro–Wilk test. All tests
were considered significant when p < 0.05 (GraphPad Prism 7
Software).
resUlTs
cD74 is Upregulated in In Vitro hiVinfected MDMs and This effect is
accompanied by higher MiF Plasma
levels in hiV-infected subjects,
compared to hiV-negative Donors
Nef-mediated CD74 upregulation is a well-described phenomenon. More specifically, this was shown to occur in in vitro
HIV-infected primary MDMs (13) and in an ex vivo model of
MDMs obtained from HIV-infected subjects (15). Figure 1A
depicts surface CD74 expression in one representative MDM
donor. CD74 expression was monitored in uninfected cells
(UN, left panel) as well as in cells infected with a Nef-defective
virus (ΔNef, middle panel) or a Nef-expressing virus (WT, right
Plasma Membrane expression of cD74
and cD44, the signaling component of the
MiF receptor complex, are increased in
WT hiV-infected MDMs
We hypothesized that the increased CD74 expression found
in HIV-1 infected MDMs may translate into enhanced MIF
FigUre 1 | CD74 upregulation in human immunodeficiency virus (HIV)-infected monocyte-derived macrophages (MDMs) and macrophage migration inhibitory
factor (MIF) plasma levels in HIV+ subjects. (a) Flow cytometry analysis of CD74 surface expression in primary uninfected MDMs (left panel); infected MDMs with a
Nef-defective virus expressing the reporter molecule GFP (ΔNef HIV-1, middle panel); and infected with a wild type (WT) HIV-1 also expressing the reporter molecule
GFP (WT HIV, right panel). The plots show CD74 versus GFP expression (HIV-1 infection) on MDMs [gated previously in a forward scatter (FSC) versus side scatter
plot]. In each dot plot, two different populations were gated: the HIV-1 negative population (GFP negative) and the HIV-1 positive population (GFP positive). One
representative healthy donor, out of three donors, is shown. (B) Quantitation of Nef-mediated upregulation of CD74, calculated as the ratio between FL-2 MFI
obtained for cells infected with the WT virus and the FL-2 MFI obtained for cells infected with the ΔNef virus. Each black dot represents one out of three
independent experiments (donors). Horizontal red bars stand for the mean value. (c) MIF concentration in plasma obtained from HIV-negative (HIV−, N = 13) and
HIV-positive (HIV+, N = 13) donors. Each plasma was evaluated in duplicate. Dots represent the average of duplicates for each donor. Data were normally
distributed and analyzed by two-tailed unpaired Student’s t-test. Horizontal lines within boxes represent the median and whiskers extend from min to max.
****p < 0.0001.
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receptor availability and signal transduction. Thus, the cellular
localization of CD74 and the CD44 signaling co-receptor was
evaluated in infected primary MDMs by confocal immunofluorescence microscopy. In consonance with previous reports on
HeLa-CIITA cells (8, 50), CD74 staining was observed mainly in
intracellularly both in uninfected cells and in cells infected with
the Nef-defective virus (Figure 2A). More specifically, CD74
staining was mostly located in membranous compartments
within the cytoplasm. This could be visualized in the images
but it also could be inferred from cross-sectional quantitation
of mean fluorescence intensity (mFI) by image processing
(Figure 2B) where an uneven mFI profile characterized by different cytoplasmic peaks was obtained. Conversely, an intense
CD74 signal comprising the plasma membrane was observed in
MDMs infected with the WT virus (Figures 2A,B, lower panels).
This is consistent with the ability of Nef to reduce the rate of
CD74 internalization (12, 51). Surprisingly, CD44 distribution
mirrored that of CD74 in all conditions. Particularly, both CD44
and CD74 were strongly co-expressed at the plasma membrane
of WT HIV-infected cells.
In order to quantitate CD44 and CD74 expression at different
subcellular localization across all conditions, MFI cross-sectional
quantifications corresponding to the regions encompassing only
the plasma membrane (Figure 2C) or the cytoplasm (Figure 2D),
both for CD74 and CD44, were performed in all infection conditions. Results again indicated that there is a substantial overlap
between both molecules in all conditions but that the staining
pattern was significantly different in WT-infected cells compared
both to uninfected and ΔNef-infected cells, being the expression
of both molecules concentrated at the plasma membrane. These
FigUre 2 | CD74 and CD44 expression in uninfected and infected monocyte-derived macrophages (MDMs). (a) Confocal immunofluorescence microscopy of
primary uninfected MDMs (UN, upper panels); primary ΔNef human immunodeficiency virus (HIV)-infected MDMs (ΔNef, middle panels); and primary wild type (WT)
HIV-infected MDMs (WT, lower panels). From left to right: bright field, GFP (HIV-1 infection), CD74 staining and CD44 staining are shown, in one representative cell
for each condition. (B) Plots show cross-sectional mean fluorescence intensity (mFI) for CD74 (left axis, cyan line) and CD44 (right axis, red line) corresponding to
the depicted cells (in the lower panel, cross-sectional mFI was evaluated in the cell pointed with an arrow). The black lines indicate the area comprising the cell
according to the DIC. (c,D) Cross-sectional mFI quantification for CD44 (upper panel) and CD74 (lower panel) intensity at plasma membrane (c) and cytoplasm
(D). Quantifications were performed in 15 individual cells for each condition. Bars represent mean ± SD. Data were analyzed by one-way ANOVA followed by
Dunnett’s post-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
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data support the notion that the expression of the components
of the main MIF receptor might be enhanced in Nef-expressing
HIV-infected cells which in turn would be translated into higher
responsiveness to MIF by HIV-infected cells.
reduction found in TLR4 expression when cells were treated
with the anti-CD74 blocking antibody or the isotype-matched
control represented a non-specific response to Fc engagement.
Collectively, these results indicate that exogenous MIF had an
effect on TLR4 up-modulation, which was only evident in IN
cells (not UN or By cells) but this effect could not be blocked by
interfering MIF binding to CD74.
MiF Modulates Tlr4 expression in hiVinfected MDMs in a cD74-independent
Fashion
interaction Between cD74 and MiF
Triggers the Production of
Proinflammatory Mediators specifically
From hiV-infected MDMs
We next investigated the well-documented action of MIF to
upregulate TLR4 expression in MDMs. To elucidate if this
activity was affected by HIV infection, and if it was dependent of CD74 engagement, MDMs were infected with the
R5-tropic HIV strain. Uninfected cells were used as controls.
At day 11, cells were treated with 1, 10, or 25 ng/ml of MIF.
These concentrations were chosen as reported previously to
represent those observed in plasma from healthy volunteers,
pathophysiological fluids, or plasma from HIV+ individuals,
respectively (34, 52). In this model, cell viability at day 11 was
83 ± 3.7% in uninfected wells and 66.8 ± 2.5% in infected wells
post-treatment. Percentage of infection was 48.48 ± 16.8%, and
p24 production in culture supernatant was 20.53 ± 12.7 ng/ml.
These parameters did not differed significantly across the different MIF concentrations evaluated here (not shown). To study
the expression of TLR4 in uninfected (UN), bystander (By),
and productively infected (IN) cells, the gating strategy shown
in Figure S1 in Supplementary Material and Figure 3A was
used. Representative results from one donor can be observed in
Figure 3B. There, it can be observed that TLR4 expression was
not modified by MIF treatment either in uninfected of bystander
cells. However, TLR4 expression increased with increasing MIF
concentration in productively infected cells. When results from
four independent donors were expressed relative to their corresponding UN condition and then combined (Figure 3C) it
could be observed that TLR4 expression peaked at 25 ng/ml MIF
specifically in HIV-infected cells, almost doubling in magnitude
when compared to the “no-MIF” condition. This can also be
observed in the overlaid histograms shown in Figure 3D, where
the MFI for the “IN plus 25 ng/ml MIF” condition is the highest.
In order to elucidate whether an interaction between CD74 and
MIF was responsible for a higher TLR4 expression, cells were
pre-incubated with a neutralizing anti-CD74 antibody (or an
isotype-matched control antibody), prior to MIF treatment at
peak effect concentration (25 ng/ml MIF). TLR4 expression was
significantly reduced both in the CD74-blocked condition but
also in the control condition (Figure 3C, gray box). As MDMs
may constitutively release MIF in low levels (53), this result may
reflect the saturating action of autocrine/paracrine stimulation
by endogenously released MIF or, alternatively, a non-specific
action of Fc receptor engagement. To test the latter hypothesis,
Fc receptors were blocked in this model (Figure 3E). When
the FcR block was applied prior to the treatment with the antiCD74 blocking antibody or the isotype-matched control and
the cells were treated with 25 ng/ml MIF, the TLR4 expression
reverted to the levels detected in cells only treated with MIF.
Moreover, no differences were observed between the CD74
blocked and isotype control conditions. This indicates that the
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Next, we aimed at investigating the requirement of CD74/MIF
interaction in the production of proinflammatory cytokines
from HIV-infected versus uninfected MDMs following treatment with MIF. MDMs were infected with the R5-tropic HIV
strain and uninfected cells were used as controls. At day 11 (peak
infection), cells were treated with different MIF concentrations.
Cell viability, infection percentages, and culture supernatant
p24 production were as described in the previous section.
Figures 4A,B show the raw data from one representative donor
and the compiled data from six donors, respectively. Except
for IL-10, most supernatants obtained from infected MDMs
showed higher production of cytokines when treated with MIF,
compared to the uninfected MIF-treated counterpart. While
no-MIF effect was observed in uninfected cultures, an MIFdependent production of IL-8, IL-6, IL-1β, TNF-α, and sICAM
was detected in infected cells. Compiled results (N = 6) indicated
that the greatest effect of MIF on IL-8 production occurred at
10 ng/ml (twofold increase). Similar observations were found for
IL-6 (a peak fold increase of 2 and 2.5 at 10 and 25 ng/ml MIF,
respectively), IL-1β (a peak fold increase of 8 at 25 ng/ml MIF),
TNF-α (a peak fold increase of 26 at 1 ng/ml MIF), and sICAM
(a peak fold increase of 2 at 1 ng/ml MIF). By contrast, no-MIF
effect was observed in IL-10 production. These results suggest
that MIF drive the production of proinflammatory mediators
and that this effect is specific for HIV-infected cells. In order to
elucidate whether an interaction between CD74 and MIF was
responsible for the higher production of these cytokines, cells
were pre-incubated with an anti-CD74 blocking antibody prior
to MIF treatment at the peak effect concentrations (e.g., 25 ng/ml
MIF for IL-8, IL-6, and IL-1β and 1 ng/ml MIF for TNF-α
and sICAM; Figures 4A,B, gray boxes). In all cases, except for
sICAM, CD74 blockade resulted in diminished levels of cytokine
production similar to those observed in the no-MIF condition.
Conversely, this effect was not observed when cells were preincubated with the corresponding isotype control antibody.
Thus, CD74/MIF interaction was a necessary condition for the
higher production of the studied cytokines in infected cells. Of
note, this result was not recapitulated for sICAM, suggesting an
alternative mechanism for this mediator.
Altogether, these results reveal that MIF favors the production of proinflammatory mediators specifically from HIVinfected cells and demonstrate that the interaction with CD74
is needed to achieve this effect. Moreover, they suggest a joint
contribution of MDM infection, HIV-mediated upregulation of
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FigUre 3 | TLR4 expression after macrophage migration inhibitory factor (MIF) stimulation in primary human immunodeficiency virus (HIV)-infected monocytederived macrophages (MDMs). (a) TLR4 expression in uninfected (UN, upper panel), bystander (By, lower panel), and productively infected cells identified on the
bases of intracellular p24 staining (In, lower panel). Living MDMs were gated previously on a forward scatter versus side scatter dot plot. An isotype-matched
FITC-conjugated antibody was used to accurately set the p24-negative population. (B) TLR4 MFI in uninfected MDMs (Un), in productively infected MDMs
(p24 positive population within the well inoculated with the virus, In) and in the bystander uninfected MDMs (p24-negative population within the well inoculated
with the virus, By) after MIF treatment. These data represent the results obtained from one representative donor. (c) Ratio between the TLR4 MFI of the infected
(or bystander population) and the TLR4 MFI of the uninfected cells after treatment with MIF, with or without CD74 blockade with an anti-CD74 antibody. Fold up
from four independent donors, evaluated in duplicate are shown collectively. Data represent the mean ± SD. (D) Flow cytometry histogram overlay for TLR4
expression on Un, By, and In MDMs all treated with 25 ng/ml MIF (e) Ratio between the TLR4 MFI of the infected (or by-stander population) and the TLR4 MFI
of the uninfected cells using the different CD74-blocking conditions represented in the x-axis. Data were analyzed by two-way ANOVA followed by Tukey’s
post-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
surface CD74, and MIF stimulation to promote the production
of a proinflammatory environment.
To provide further insight into the role of MIF in these observations, infected and uninfected MDMs from three independent
donors were treated with 25 ng/ml of MIF plus different concentrations of an anti-MIF neutralizing monoclonal antibody (range
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3.125–100 ng/ml, Figure 5A) or the MIF antagonist MIF098
(range 5–100 nM, Figure 5B). Neither the anti-MIF neutralizing
monoclonal antibody nor the MIF antagonist affected cell viability in the concentration range tested. After incubation, production of IL-8, IL-6, IL-1β, and IL-10 was monitored. As expected,
a significant reduction in IL-8, IL-6, and IL-1β production was
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FigUre 4 | Continued
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FigUre 4 | Expression of cytokines after macrophage migration inhibitory factor (MIF) stimulation in primary human immunodeficiency virus (HIV)-infected and
uninfected monocyte-derived macrophages (MDMs). (a) Expression of IL-8, IL-6, IL-1β, TNF-α, sICAM, and IL-10 in supernatants from HIV-infected (In) and
uninfected (Un) MDMs obtained from one representative healthy donor. (B) Data combined from six independent experiments (donors), each evaluated in triplicate.
Here, data are shown as the ratio between cytokine concentrations found under the infection condition versus the uninfected counterpart. Cells were stimulated
with MIF as follows: 0, 1, 10, or 25 ng/ml. Data shown in the gray boxes depict CD74 blocking (10 ng/ml of αCD74 or the corresponding isotype control) followed
by MIF stimulation (1 or 25 ng/ml as denoted). Data represent the mean ± SD. Data were analyzed by one-way ANOVA followed by Tukey’s post-test. *p < 0.05,
**p < 0.01, ***p < 0.001.
with the corresponding MIF concentration. Overall, results
indicate that, at 7 days post-infection, viral production from
initially unactivated CD4+ T-cells is significantly higher upon
exposure to supernatants derived from infected MDMs treated
with 1 ng/ml MIF, compared to exposure to supernatants derived
from uninfected MDMs. Similarly, unactivated CD4+ T-cells
sensitized with supernatants from infected MDMs treated with
25 ng/ml MIF showed a significant, albeit transient, increase in
viral production at day 4 post-infection, compared to the uninfected condition, which was later downmodulated.
Despite sustained viral production is observed from CD4+
T-cells treated with supernatants derived from the 25 ng/ml
MIF-treated infected MDMs (see Figure 6B, dark red line), the
peak effect observed at day 4 in Figure 6D is lost at later time
points. This might be indicating that the supernatant from
infected MDMs treated with 25 ng/ml might be enhancing cell
permissiveness and/or favoring an earlier viral production from
these unactivated cells.
In order to rule out the possibility of inefficient viral inactivation by UV of MDM-derived supernatants, CD4+ T-cells were
incubated with the UV-inactivated supernatants and left to
proceed as described previously but without infecting them. At
days 4 and 7, p24 antigen was quantified, and no viral production was detected under either condition. This indicates that no
viral carry-on from infected MDMs occurred. We also explored
whether CD4+ T-cell viability and infection percentages were
affected by the addition of supernatants derived from MDMs
treated under the different MIF conditions. No differences in cell
viability (Figure 6E) was observed along time for CD4+ T-cell
treated with supernatants obtained from uninfected MDMs
treated with the different MIF concentrations (black, dark gray,
and light gray lines). As expected, CD4+ T-cell treated with
supernatants obtained from infected MDMs showed a reduction
in cell viability, compared to the uninfected condition, no differences were observed across the different MIF concentrations
(pink, red, and dark red lines). This indicates that cell viability
most likely does not account for the differences observed in viral
production. On the other hand, the infection percentage was
higher after PHA treatment (Figure 6F). All other conditions,
including the RPMI control, showed infection percentages <2%
and no significant differences across treatments were observed.
This might be indicating that the treatments with the MDMderived supernatant might enhance viral production from
those few infected cells rather than promoting infection. Then,
the effect of blocking CD74/MIF interaction in MDMs on the
observed results was studied. For this, unactivated CD4+ T-cells
were incubated with supernatants derived from MDMs in which
the interaction between CD74 and MIF had been blocked with
observed upon MIF inhibition when using either the neutralizing
antibody or the small molecule MIF antagonist. Serial dilution
of both of these CD74/MIF interaction inhibitors reconstituted
cytokine expression. Indeed, significant dose-dependent effects
were observed, further supporting a role for MIF in promoting
the production of proinflammatory mediators specifically from
HIV-infected cells. No significant MIF or anti-MIF effects were
observed on uninfected cells. Similarly, no changes were observed
in IL-10 production.
cD74/MiF-Dependent Production of
Proinflammatory Factors From hiVinfected MDMs enhances Viral Production
From Unactivated cD4+ T-cells
Then, we reasoned that conditioned media (supernatants) from
MIF-treated HIV-infected MDMs could have an enhancing
effect on the permissiveness of unactivated CD4+ T-cells to HIV
infection, which are mostly naturally resistant to HIV. To test this
hypothesis, supernatants obtained from infected and uninfected
MDMs, treated or not with MIF, were UV inactivated and used
to stimulate purified unactivated CD4+ T-cells. RPMI- or PHAtreated CD4+ T-cells were used as negative and positive controls,
respectively. At 72 h post-treatment, CD4+ T-cells were infected
with an X4-tropic GFP-expressing viral strain, and infection
was monitored during 7 days by flow cytometry to evaluate % of
infected cells (GFP+, see Figure S1 in Supplementary Material)
and by ELISA (p24 antigen) to evaluate viral production.
Figures 6A,B depict the viral production kinetics observed in
CD4+ T-cells treated with supernatants derived from uninfected
and HIV-infected MDMs, respectively, treated with 0, 1, and
25 ng/ml MIF (the 10 ng/ml condition was not evaluated).
Viral production was very low when CD4+ T-cells were preincubated with supernatants derived from uninfected MDMs
and it was independent of MDM MIF treatment (Figure 6A).
A similar kinetic was observed in CD4+ T-cells pre-incubated
with supernatants from infected MDMs treated with 0 MIF
(Figure 6B, pink line). Conversely, viral production from CD4+
T-cells pre-incubated with supernatants from infected MDMs
treated with 1 ng/ml MIF (Figure 6B, red line) and 25 ng/ml MIF
(Figure 6B, dark red line) tended to increase over time reaching
maximal viral production at day 7 for the 1 ng/ml MIF condition
and at day 4 for the 25 ng/ml MIF condition. This can be better
observed in Figures 6C,D. Here, viral production from CD4+
T-cells pre-incubated with supernatants from infected MDMs
treated with 1 ng/ml (Figure 6C) and 25 ng/ml (Figure 6D) is
shown relative to the viral production from CD4+ T-cells preincubated with supernatants from uninfected MDMs treated
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FigUre 5 | Effect of macrophage migration inhibitory factor (MIF) neutralization in the expression of cytokines from primary human immunodeficiency virus-infected
and uninfected monocyte-derived macrophages (MDMs). Serial dilutions of a neutralizing αMIF antibody (clone NIHlllD.9) (a), and the MIF antagonist MIF098
(B) were used to inhibit MIF activity in infected (In, red lines) and uninfected (Un, gray lines) MDMs at a constant concentration of this cytokine (25 ng/ml). Data
represent three independent experiments (donors), each evaluated in duplicate. Data represent the mean ± SD. Data were analyzed by two-way ANOVA followed
by Tukey’s post-test (intragroup analysis, In group only; ****p < 0.0001) or by Sidak’s post-test (intergroup, In versus Un; #1p < 0.05, #2p < 0.01, #3p < 0.001,
#4
p < 0.0001). Asterisks corresponding to the intragroup analysis are shown above the horizontal bars, and those from the intergroup analysis are shown above
points corresponding to each antagonist or antibody dilution.
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an anti-CD74 neutralizing antibody (Figure 6G). This assay was
performed using supernatants from MDMs treated with the
25 ng/ml condition and viral production was evaluated at day
4 to reproduce the peak result observed in Figure 6D. Notably,
unactivated CD4+ T-cells treated with supernatants derived from
“CD74-blocked” MDMs were not able to recapitulate the increase
FigUre 6 | Continued
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FigUre 6 | Induction of permissiveness to human immunodeficiency virus type I (HIV-1) infection in primary CD4+ T-cells after stimulation with macrophage
migration inhibitory factor (MIF)-treated monocyte-derived macrophages (MDMs)-derived supernatants. (a,B) Seven-day kinetics of HIV p24 antigen production
from primary unactivated CD4+ T-cell incubated with supernatants from uninfected (a) and infected (B) MDMs treated with 0, 1, or 25 ng/ml MIF. (c,D) Ratio of
p24 production from unactivated CD4+ T-cell incubated with supernatants from uninfected MDMs and infected MDMs treated with 1 ng/ml MIF (c) or 25 ng/ml MIF
(D) over the no-MIF condition. (e) Percentage of living CD4+ T-cells stimulated with supernatants derived from uninfected (black, dark gray, and light gray lines)
and infected (pink, red, and dark red lines) MDMs. (F) Percentage of infected (GFP+) CD4+ T-cells after stimulation with MDM-derived supernatants obtained
from MIF-treated uninfected MDMs (black, dark gray, and light gray lines), infected (pink, red, and dark red lines) MDMs, RPMI (negative control, black line with
diamonds), or PHA (positive control, black line with triangles). (g) Ratio of p24 production from unactivated CD4+ T-cell incubated with supernatants from uninfected
MDMs and infected MDMs treated with 25 ng/ml MIF, with or without CD74 blockade with an anti-CD74 antibody. (h) Expression of surface markers on CD4+
T-cells subjected to 72 h stimulation with supernatants derived from infected and uninfected MDMs and exposed or not to MIF treatment (0 and 25 ng/ml MIF).
Data represent the mean ± SD from six independent donors evaluated in duplicate. In (c,D), data were analyzed by two-way ANOVA followed by Sidak’s post-test.
In (g), data were analyzed by two-way ANOVA followed by Tukey’s post-test. *p < 0.05, ****p < 0.0001.
in viral production observed in the “non-blocked” condition. In
summary, these results suggested that soluble factors released
after MIF treatment in infected MDMs enhanced permissiveness
of unactivated CD4+ T cell. Moreover, the production of these
factors appears to be dependent on CD74/MIF interaction as the
effect was abrogated by immunoneutralization of CD74.
Finally, we investigated if preincubation with the supernatants derived from MDMs induced CD4+ T-cell activation
differentially as it is known that the level of cell activation
correlates with HIV-1 infection efficiency. To assess this, the
phenotype of CD4+ T-cells was studied after stimulation with
MDMs-derived supernatants. The expression of CD38, CD69,
HLA-DR, CD25, PD-1, and CD28 markers was evaluated by
flow cytometry after 72 h. Supernatants from infected or uninfected MDMs treated (25 ng/ml) or not with MIF were used. No
differences were detected in the percentage of cells expressing
the different membrane markers (Figure 6H) or their MFI
(not shown) among treatments. In sum, the improved viral production observed in CD4+ T-cells after treatment with supernatants derived from 25 ng/ml MIF-treated infected MDMs
could not to be explained by differential cell viability, infection
percentage, or cell activation (measured by surface markers).
In summary, we identified that the production of TNFα,
IL-6, IL-8, and IL-1β increased significantly after CD74/MIF
interaction in infected MDMs. Moreover, conditioned media
from MIF-treated infected MDMs significantly enhanced viral
production from unactivated CD4+ T-cells. Thus, the next
step was to study a possible link between cytokines secreted
by infected MDMs in an MIF-dependent manner and viral
production from unactivated CD4+ T-cells. To do this analysis,
recombinant IL-1β, IL-6, IL-8, and TNFα were used to stimulate
primary unactivated CD4+ T-cells in the absence of any other
stimuli at concentrations that resemble those found in MDM
supernatants stimulated with 25 ng/ml MIF (peak effect). RPMI
alone and PHA-supplemented RPMI were used as negative and
positive controls, respectively. After 72 h, cells were infected, and
viral p24 antigen was quantified. CD4+ T-cells treated with single
cytokines or dual combinations did not alter viral production,
regardless of the cytokines involved (data not shown). Only
when treating CD4+ T-cells with three or four cytokines simultaneously viral production increased significantly compared to the
RPMI control (Figure 7A). No differences in cell viability and
infection percentages were observed across treatments (except
for PHA) (Figures 7B,C). Finally, MDMs-derived supernatants
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were incubated with anti-IL-8, -IL-6, -IL-1β, and -TNFα neutralizing antibodies and used as CD4+ T-cells activation stimuli.
In line with our hypothesis, a significant reduction in viral
production was observed under this condition, compared to the
non-neutralized and isotype control supplemented supernatants
(Figure 7D).
Overall, IL-1β, IL-6, IL-8, and TNFα were identified as factors
secreted from MIF-treated HIV-infected MDMs that, in combination, exerted a transient enhancing effect on viral production
from unactivated CD4+ T-cells.
In Vitro infections With Transmitted/
Founder (T/F) hiV strains reproduced
Both the effect of MiF on the Production
of Proinflammatory Mediators From hiVinfected MDMs and also the enhanced
Viral Production From Unactivated cD4+
T-cells stimulated With conditioned
Media Derived From MiF-Treated
hiV-infected MDMs
To examine whether the findings reported here could be
extended to other HIV-1 strains, we generated viral stocks
from selected transmitted/founder (T/F) IMCs. These clones
were derived from full-length transmitted HIV-1 genomes and
represent viruses actually responsible for productive clinical
infection. Thus, these are instrumental tools for studying different aspects of HIV pathogenesis (36–39).
First, MDMs were infected with the R5 T/F virus. At day
11, both infected and uninfected cells were treated with 0, 1, or
25 ng/ml MIF and the production of cytokines was evaluated in
cell supernatants. As observed for the HIV BAL strain, an MIFdependent effect was observed for IL-1β, IL-6, and IL-8, which
was significantly marked in infected cells while the production
of IL-10 was unaltered across conditions (Figure 8A). In particular, peak IL-6 and IL-8 effects were observed at 25 ng/ml
MIF while for IL-1β, the effect was already evident at 1 ng/ml
MIF. Contrary to our initial observations using the BAL strain,
the production of TNF-α and sICAM was not affected by MIF
(not shown).
Then, the effect of MDM supernatants on viral production
from unactivated CD4+ T-cells was again tested but using the dualtropic T/F virus to infect the CD4+ T-cells. Thus, supernatants
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from MIF-treated BAL-infected and uninfected MDMs were
used to stimulate unactivated CD4+ T-cells during 72 h. Viral
production was evaluated at 4 and 7 days post-infection. Results
FigUre 7 | Identification of cytokines as responsible for enhancing human
immunodeficiency virus type I (HIV-1) infection in unactivated CD4+ T-cells.
(a) Unactivated CD4+ T-cells were stimulated with different combinations of
cytokines for 72 h. Then, cells were infected and p24 antigen production was
evaluated at days 4 and 7 post-infection. Each condition was compared with
the corresponding RPMI condition (negative control). As a positive control,
PHA stimulation was used. Percentage of living CD4+ T-cells (B) and
percentage of infected (GFP+) CD4+ T-cells (c) after stimulation with the
denoted treatments are shown. Data represent mean ± SD from four
independent donors evaluated in duplicate. Concentrations of cytokines used
in these experiments corresponded to the average concentrations found in
monocyte-derived macrophage (MDM) supernatants stimulated with 25 ng/
ml macrophage migration inhibitory factor (MIF) (peak effect) as follows:
250 pg/ml IL-6, 9,000 pg/ml IL-8, 1,400 pg/ml TNF-α, and 20 pg/ml IL-1β.
(D) Neutralization of IL-8, IL-6, IL-1 β, and TNFα biological activity with
monoclonal neutralizing antibodies. Primary CD4+ T-cells were incubated with
supernatants derived from the 25 ng/ml MIF-treated HIV-infected MDM
neutralized previously with 18 µg/ml anti-IL-8, 20 ng/ml anti-IL-6, 2 µg/ml
anti-IL-1β, and 2 µg/ml anti-TNFα antibodies. Non-neutralized and isotype
control antibody conditions were tested for comparison. Also, RPMI and PHA
controls were included. Viral production was evaluated at day 4 postinfection. Data were analyzed by one-way ANOVA followed by Dunnett’s
post-test (all conditions versus the corresponding RMPI control) in (a) and
Tukey’s post-test in (D). *p < 0.05.
indicated that production of a dual-tropic T/F virus from unactivated CD4+ T-cells sensitized with supernatants derived from
25 ng/ml MIF-treated infected MDMs was significantly higher
compared to the uninfected counterpart (Figure 8B, right panel).
Contrary, no effect was observed when using supernatants derived
from 1 ng/ml MIF-treated MDMs (Figure 8B, left panel). These
results partially recapitulated those obtained when infecting
unactivated CD4+ T-cells with the X4-tropic NL4-3 laboratory
strain: an enhancing effect on viral production was observed
when sensitizing cells with MIF-treated infected MDM-derived
supernatants although the kinetics seems to be different for the
T/F strain.
Overall, MIF effect on the production of proinflammatory
mediators from HIV-infected MDMs and also the enhancing
effect of the conditioned media (derived from MIF-treated HIVinfected MDMs) on viral production from unactivated CD4+
T-cells could be reproduced when using T/F viral strains. This
provides further support to the notions presented in this work
pointing toward a relevant role of the MIF/CD74 axis in HIV
pathogenesis.
DiscUssiOn
It has become increasingly clear that signaling events downstream of MIF/CD74 interaction are key components in the
regulation of immune responses that are involved in the pathogenesis of different inflammatory and immune-mediated diseases. However, whether this axis participates in HIV-mediated
immune dysfunction has not been elucidated yet. Several
lines of evidence suggest that this might be the case, based on
the fact that CD74 expression is modulated in HIV-infected
cells and that MIF plasma levels are elevated throughout the
course of infection in HIV-infected subjects. Results depicted
in this study provide support to this hypothesis by showing
FigUre 7 | Continued
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FigUre 8 | Infection of monocyte-derived macrophages (MDMs) and unactivated CD4+ T-cells with T/F viruses reproduce the results obtained with the R5-tropic
(BAL) and the X4-tropic (NL4-3) laboratory strains. (a) Expression of IL-1β, IL-8, IL-6, and IL-10 in supernatants from uninfected (Un) and R5-tropic T/F-infected (In)
MDMs. Data represent mean ± SD from three independent donors. Data were analyzed by two-way ANOVA followed by Tukey’s post-test. *p < 0.05, **p < 0.01,
****p < 0.0001. (B) Human immunodeficiency virus (HIV) p24 antigen production from primary unactivated CD4+ T-cells incubated with supernatants from uninfected
and infected MDMs treated with 1 ng/ml (left panel) or 25 ng/ml (right panel) macrophage migration inhibitory factor (MIF). Treated CD4+ T-cells were infected with a
dual-tropic T/F virus and viral production was evaluated at 4 and 7 days post-infection. Data represent mean ± SD from five independent donors. Data were
analyzed by two-way ANOVA followed by Sidak’s post-test. *p < 0.05.
that production of soluble inflammatory factors by primary
HIV-infected MDMs was increased in an MIF dose-dependent
manner and that CD74/MIF interaction was necessary for
this effect. Moreover, the conditioned environment generated
by MIF/CD74 interaction in infected MDMs promotes CD4+
T-cell permissiveness to infection.
In an initial report, CD74 was described as the central component of the MIF cell surface receptor (54). However, whereas the
CD74 intracellular domain was shown to undergo intracellular
phosphorylation upon engagement of the CD74 ectodomain by
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MIF, its short non-canonical structure suggested the involvement
of a recruited co-receptor. A subsequent study demonstrated
that CD44 was a necessary component for MIF signaling (16).
CD74 surface expression has been shown to be upregulated in
HIV-infected cells, and we show herein that this was also accompanied by surface CD44 upregulation which translated into an
overlapping cell surface expression pattern observed specifically
in WT HIV-infected cells. This allowed us to hypothesize that
this phenomenon may translate into higher MIF receptor availability and enhanced receptor activation by MIF in these cells.
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It is worth highlighting that only few reports describe the effect
of HIV infection in CD44 expression in myeloid cells (55–57).
Other molecules proposed to act as MIF coreceptors together
with CD74, including CXCR2, CXCR4, and CXCR7 (17, 18, 58),
were not analyzed here.
This evidence led us to study the MIF/CD74 interaction in
HIV infection. First, we decided to study MIF-mediated modulation of TLR4 in infected cells. This was based on the observation that detectable plasma LPS levels are common in HIV
infection (2, 59), thus modulation of one of the components of
LPS receptor complex, TLR4, might contribute to disease progression. Also, endogenous MIF has been shown to modulate
TLR4 expression in murine macrophages (60, 61). Here, the
effect of exogenously added MIF was studied to unravel how its
interaction with CD74 might have an impact on TLR4 modulation. In our system, a modest effect was observed particularly
in infected cells at 25 ng/ml MIF with no evidence of CD74
participation. A more recent report indicated that exogenously
added MIF could modulate TLR4 in murine fibroblasts but
only at 375 ng/ml MIF (15-fold higher concentration than
in our system) (62). Regardless MIF stimuli, it is also worth
pointing that TLR4 expression was lower in bystander cells,
compared to the uninfected condition. We speculate that this
might be the consequence of factors produced by productively
infected cells that affect, directly or indirectly, the phenotype
and/or function of the neighboring non-productively infected
cells (63).
The capability of HIV-infected MDMs to secrete different
proinflammatory cytokines in response to MIF treatment was
examined later. The fact that MIF is able to stimulate the secretion
of proinflammatory cytokines in different settings is a phenomenon well-documented (19, 64–66). Moreover, many of these
events have been reported to occur after CD74 engagement and
by activating multiple intracellular signaling pathways (7, 29,
67–69). We add new evidence on the role of MIF in the clinically
important HIV infection scenario. Quantitation of IL-1β, IL-6,
IL-8, TNFα, and sICAM in MDMs supernatants demonstrated
that MIF stimulation led to an augmented production of the
proinflammatory cytokines studied. The first three cases showed
a dose dependence with the MIF stimuli with maximum expression when using the highest MIF concentration tested. On the
other hand, TNFα and sICAM production peaked at the lowest
MIF concentration tested. Even more, enhanced production of
IL-1β, IL-6, and IL-8 was also observed in MIF-treated MDMs
infected with a T/F virus, indicating that this effect could be
reproduced with clinically relevant viral strains. These results
led to a direct link between the secretion of proinflammatory
cytokines and MDM exposure to MIF. Even more relevant, the
effect was maximum in infected cells compared to uninfected
cells, pointing to a differential effect on HIV-infected cells.
According to our hypothesis, this outcome could be explained
by the higher availability of membrane CD74 molecules in
HIV-infected cells that translate into greater MIF binding and
signal transduction. To confirm that the MIF/CD74 axis was
required for these effects, the interaction was blocked with a
αCD74 immunoglobulin. The production of most mediators (all
but sICAM) was inhibited by this treatment. In sum, our results
Frontiers in Immunology | www.frontiersin.org
provide support to the hypothesis that links the MIF/CD74
interaction and the differential production of proinflammatory
molecules such as IL-1β, IL-6, IL-8, and TNFα expression from
HIV-infected cells. Of note, sICAM was proposed to promote
interactions between B and T cells that ultimately render resting T-cells permissive to HIV infection (70). Thus, the finding
regarding MIF-mediated induction of sICAM production was of
special interest. Results indicated that sICAM response peaked
at 1 ng/ml MIF and was then downmodulated at higher exogenous MIF concentrations. Again, this result might be reflecting
the saturating action of autocrine/paracrine stimulation by
endogenously produced MIF or the involvement of alternative
mechanisms yet to be elucidated.
During the last years, the concept of macrophage polarization
has gained special focus, thus distinguishing different MDM
subsets with different functionalities (71). Here, unpolarized
(i.e., differentiated from blood monocytes only in the presence
of GM-CSF) MDMs were used throughout the study. This was
based on bibliography indicating that M1 and M2 polarized
MDMs are less efficient to support productive HIV infection
compared to unpolarized cells due to different blocks imposed at
different levels of the replicative cycle (72–74). On the other hand,
it has been reported that MDM infection with HIV results in
polarization toward an M1-like phenotype. Moreover, infection
sensitized macrophage responses to TLR ligands (75). Despite
TLR ligands were not assayed here, a parallelism between these
and our findings can be proposed since, according to our results,
HIV infection renders MDMs more reactive to a proinflammatory stimuli such as MIF.
The hallmarks of HIV infection include the gradual decline
in the number of CD4+ T-lymphocytes and the chronic
and persistent inflammation and immune activation. HIVmediated immunopathogenesis is a complex process involving a dynamic interplay between viral and host molecules.
Activation of T cells is driven directly by HIV replication but
also by indirect mechanisms such as the breakdown in the gut
mucosa and dysfunction of immunoregulatory factors, among
others (2). Concomitantly, immune activation plays a key role
in the systemic spread of the infection. HIV efficiently infects
activated CD4+ T-cells leading to a productive infection state.
However, it has been recently documented that the infection
of unactivated CD4+ T-cells also occurs, resulting mostly in
a latent infection (76). We therefore raised the question of
whether MIF-treated MDM-derived supernatants could promote the infection of unactivated primary CD4+ T-cells in the
absence of other stimuli. Results indicated that viral production
was significantly enhanced by conditioned media obtained
from MIF-treated HIV-infected MDMs (compared to MIFtreated uninfected MDMs). The effect was highest as early as
4 days post-infection when using the 25 ng/ml MIF-stimulated
MDMs while it occurred at day 7 post-infection for the 1 ng/ml
MIF condition. This pattern in viral production mirrors the
MIF-dependent cytokine production from infected MDMs:
a peak production of IL-1β, IL-6, and IL-8 was observed with
25 ng/ml MIF and a peak in TNFα with 1 ng/ml. Particularly,
TNFα showed the highest modulation magnitude when
infected MDMs were compared with those uninfected but at
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Trifone et al.
MIF/CD74 Interaction in HIV Infection
the lowest MIF concentration tested. In line with the dependence on CD74/MIF interaction for MDM cytokine production, blockade of MIF/CD74 engagement in infected MDMs
abrogated the effect observed in CD4+ T cells. In order to
better support the impact of cytokines produced from infected
MDMs downstream of the MIF/CD74 interaction on the permissiveness of unactivated CD4+ T-cells, recombinant cytokines
were used as direct stimuli. When a combined treatment with
IL-1β, IL-6, IL-8, and/or TNFα was attempted, viral production
increased. In the same line, neutralizing the biological activity
of these cytokines in MDM-derived supernatants resulted in
diminished CD4+ T-cell permissiveness, resembling the same
scenario obtained in the negative control. Finally, supernatants
from MIF-treated HIV-infected MDMs could enhance viral
production from unactivated CD4+ T-cells infected with a T/F
virus suggesting that the proposed mechanism extends not
only to laboratory strains but also to primary viral isolates.
The fact that cytokines enhance viral replication but, more
importantly, promote the infection of resting CD4+ T-cells is
not new (77). CCL19, CCL21, IL-7, and IL-15 are known to
promote latent infection in resting CD4+ T-cells (78–80). Also,
IL-6 and TNFα has been shown to facilitate infection of resting
CD4+ T-cells and to induce productive infection (77, 81). In a
particularly relevant report, soluble factors (sCD23 and sICAM)
released by infected MDMs promoted the efficient infection
of resting lymphocytes although the presence of B cells was a
requisite for this effect (70). Nevertheless, it resulted interesting
that the effect on resting CD4+ T-cell permissiveness mediated
by sCD23 and sICAM, occurred without promoting cell activation and proliferation, which is in line with the observations
described in this work. In a recent report, Morris et al. (81)
described that IL-6 produced from endothelial cells increased
productive HIV infection in resting CD4+ T-cells. Even more,
this effect was not accompanied by an increase in the expression of T cell activation markers, mirroring our own results.
Although the mechanism underlying this phenomenon was not
studied by the authors, it could be associated with the capacity of
IL-6 to favor CD4+ T-cell cycling and survival (82). On the other
hand, IL-8 and TNFα have been reported to directly enhance
the rate of productive infection in activated T cells (82, 83).
In particular, binding of TNFα to its receptor triggers several
signaling cascades, including NF-κB, MAPK, ERK, and JNK
pathways, which directly enhances transcription from the LTR
promoter both on models of productive HIV infection and also
in latently infected cells [reviewed in Ref. (82)].
Thus, in our model we suggest that IL-6, IL-8, TNFα, and IL-1β
might be acting synergistically at different levels (i.e., modifying
the cellular environment and/or by enhancing transcriptional
and/or post-transcriptional mechanisms) to promote, at least
transiently, the productive infection of unactivated CD4+ T cells.
This could explain that no effect was observed when cells were
treated with a single or a dual combination of the cytokines
studied. It will be a matter of subsequent studies to investigate if
this also results in a higher rate of latent infection in these cells.
Finally, the role of IL-1β is less clear since there is no definite
report suggesting a direct mechanism of IL-1β-mediated modulation of HIV replication in T-cells.
Frontiers in Immunology | www.frontiersin.org
Taken together, we postulate that modulation of CD74 by
HIV infection in MDMs leads to the enhanced susceptibility of
these cells to MIF stimulation, which may have an impact on the
spread of HIV infection and the enhancement of viral-mediated
pathogenesis. The current results indicate that the expression
of proinflammatory cytokines was significantly higher in MIFstimulated infected MDMs compared to MIF-stimulated uninfected MDMs. In addition, this proinflammatory microenvironment, conditioned positively primary unactivated CD4+ T cells
to HIV-1 infection. The methodological strengths of this work
include the exclusive use of primary cells, emphasizing that treatment effects can be observed despite interdonor variability, the use
of both laboratory and T/F viral strains, and the employment of
physiological MIF concentrations as stimuli. On the other hand,
interdonor variability and the use of a limited number of donors
might have masked differences across conditions, representing
an important limitation of the study. Also, macrophages exhibit
significant heterogeneity in vivo, as already discussed and this fact
should not be overlooked. Thus, results might not be extended to
polarized MDMs, tissue macrophages, or other HIV susceptible
myeloid cells such as dendritic cells. For instance, it would be
very interesting to evaluate CD74/MIF axis in microglial cells. If a
similar hypothesis was confirmed in this model, this mechanism
could be associated with the development of HIV-related neurological complications.
Overall, this work provides further insights in the role of macrophages in HIV infection, not only as a cell type which supports
viral replication itself but also as a source of soluble factors that
facilitate viral dissemination. Evidence gathered here suggests
that CD74/MIF interaction could be implicated in modulating
viral reservoir seeding, persistent viremia and inflammation—all
key aspects of HIV immunophatogenesis. Data presented here
support further studies to fully understand how this mechanism
operates in HIV infection and to explore the possibility to target
CD74/MIF axis as a therapy aimed at reducing inflammation and
reservoir size during HIV infection.
eThics sTaTeMenT
Samples from HIV-infected subjects used in this study were enrolled
as part of an ongoing acute/early primary HIV infection cohort from
Argentina (Grupo Argentino de Seroconversión study group). This
study was reviewed and approved by two institutional review boards
(IRB): Comité de Ética Humana, Facultad de Medicina, Universidad
de Buenos Aires and Comité de Bioética, Fundación Huésped
(Buenos Aires, Argentina). Both HIV-infected participants and HD
provided written informed consents accepting to participate in this
study in accordance with the Declaration of Helsinki.
aUThOr cOnTriBUTiOns
YG and GT conceived the study and designed the experiments; CT, JS, MR, and YG performed experiments; LL and
RB contributed with reagents; CT, RB, MQ, HS, YG, and GT
analyzed and interpreted the data; CT and GT wrote the manuscript. All authors read and approved the final version of this
manuscript.
17
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MIF/CD74 Interaction in HIV Infection
GlaxoSmithKline (PICT2012, Grant #0475 and PICTO-GSK2013,
Grant #0006), from Fundación Alberto J. Roemmers (2013/2015)
and from the Universidad de Buenos Aires (UBACyT 2013–2016,
Grant #20020120200263BA) to GT; by a grant from ANPCyT to
MQ (PICT2012, Grant #0549), and by the US NIH NIAID (RB
and LL). The funders had no role in study design, data collection
and interpretation, or the decision to submit the manuscript for
publication.
acKnOWleDgMenTs
We would like to acknowledge Dr. Philippe Benaroch (Institut
Curie, INSERM U932, Paris, France) and Dr. Michael Schindler
(University Hospital Tübingen, Tübingen, and German Research
Center for Environmental Health, Neuherberg, Germany) for
providing reagents to perform the study and for intellectual input.
We also thank María Noé García and Daniel Grasso [Instituto
de Bioquímica y Medicina Molecular (IBIMOL), Universidad
de Buenos Aires/CONICET, Argentina] for technical assistance
with fluorescence microscopy image analysis; Matías Ostrowski
(INBIRS) and María Victoria Delpino (Instituto de Inmunología,
Genética y Metabolismo (INIGEM), Universidad de Buenos
Aires/CONICET, Argentina) for helpful suggestions during the
course of the study; and Grupo Argentino de Seroconversión study
group for providing plasma samples. Finally, we thank Mr. Sergio
Mazzini for language assistance during manuscript preparation.
sUPPleMenTarY MaTerial
The Supplementary Material for this article can be found online at
https://www.frontiersin.org/articles/10.3389/fimmu.2018.01494/
full#supplementary-material.
FigUre s1 | Gating strategy used for flow cytometry analysis of monocytederived macrophages (MDMs) (a) and CD4+ T-cells (B). First, doublets were
excluded in a forward scatter (FSC)-height (FSC-H) versus an FSH-area (FSH-A)
plot. Then, living cells were gated an FSC-A versus a side scatter (SSC) plot.
Subsequently, infected cells were identified in an FSC-H versus FITC plot
(MDMs) or versus GFP plot (CD4+ T-cells). Data acquisition was performed in a
BD FACSCanto flow cytometer using the BD FACSDiva software and analyzed
subsequently with FlowJO v10 software (Data Analysis Software, LLC).
FUnDing
This work was supported by grants from the Agencia Nacional
de Promoción Científica y Tecnológica (ANPCyT) and
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Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Copyright © 2018 Trifone, Salido, Ruiz, Leng, Quiroga, Salomón, Bucala, Ghiglione
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