Download PDF
1 / 9 Pages
Original Article
Soluble isoforms of the DC-SIGN receptor can increase the
dengue virus infection in immature dendritic cells
Lailah Hor

acio Sales Pereira
a
,
b
, Amanda do Carmo Alves
a
,
Gabriela Francine Martins Lopes
b
,
*
, Brenda Fernandes da Silva
a
,
Mariana Sousa Vieira
c
,D

ebora de Oliveira Lopes
a
,
Jaqueline Maria Siqueira Ferreira
b
, Luciana Lara dos Santos
a
a
Universidade Federal de S
~
ao Jo
~
ao del-Rei (UFSJ), Laborat

orio de Biologia Molecular, Divin

opolis, MG, Brazil
b
Universidade Federal de S
~
ao Jo
~
ao del-Rei (UFSJ), Laborat

orio de Microbiologia M

edica, Divin

opolis, MG, Brazil
c
Universidade Federal de Minas Gerais (UFMG), Laborat

orio de Imunoparasitologia, Belo Horizonte, MG, Brazil
ARTICLE INFO
Article history:
Received 1 February 2024
Accepted 8 September 2024
Available online 25 September 2024
ABSTRACT
Dengue is a disease with a high-impact on public health worldwide. Many researches have
focused on the cell receptors involved in its pathogenesis. The role of soluble isoforms of
DC-SIGN (Dendritic Cell-Speci
fi
c ICAM-3 Grabbing Non-integrin) receptor in the process of
Dengue Virus (DENV) infection is not well understood. This work proposes to evaluate
changes in the infection process of Immature Dendritic Cells (iDCs) by DENV in the pres-
ence of DC-SIGN recombinant soluble isoforms 8, 10, and 12. The recombinant isoforms
were built by heterologous expression, the DENV-2 was multiplied in the
Aedes albopictus
C6/36 cells and quanti
fi
ed in BHK-21 cells, and the iDCs were produced from the THP-1
strain. Infection assays were performed in the presence of iDCs, DENV-2, and isoforms 8,
10, and 12 separately at 25, 50 and 100 ng/mL. The
fi
nal viral load was estimated by qPCR
and statistical analysis was performed by Kruskal-Wallis and ANOVA tests. The iDC pro
fi
le
was con
fi
rmed by increasing expression of CD11c, CD86, and CD209 surface markers and
maintaining CD14 expression. Infection assays demonstrated a 23-fold increase in DENV
viral load in the presence of isoforms 8 and 10 at 100 ng/mL compared to the viral control
(
p
<
0.05), while isoform 12 did not alter the viral load. It was possible to conclude that at
100 ng/mL isoforms (8 and 10) can interact with DENV, increasing viral infection, and
potentially acting as opsonins.
Ó
2024 Published by Elsevier España, S.L.U. on behalf of Sociedade Brasileira de Infectologia.
This is an open access article under the CC BY license
(
http://creativecommons.org/licenses/by/4.0/
)
Keywords:
sDC-SIGN
DC-SIGN
CD-209
Dengue
Viral pathogenesis
Introduction
Among the human viral diseases transmitted by arthropods
(arboviruses) dengue is considered the most important, being
the most prevalent and rapidly spreading according to the
* Corresponding author.
E-mail address:
gabrielafmlopes23@gmail.com
(G.F.M. Lopes).
https://doi.org/10.1016/j.bjid.2024.103873
1413-8670/
Ó
2024 Published by Elsevier España, S.L.U. on behalf of Sociedade Brasileira de Infectologia. This is an open access article
under the CC BY license (
http://creativecommons.org/licenses/by/4.0/
)
braz j infect dis.
2024;
28(6)
:103873
The Brazilian Journal of
INFECTIOUS DISEASES
www.elsevier.com/locate/bjid
World Health Organization.
1
The etiological agent of the dis-
ease, dengue virus (DENV), belongs to the Flaviviridae family,
and four serotypes, DENV-1, DENV-2, DENV-3, and DENV-4,
have been identi
fi
ed by the International Committee on Tax-
onomy of Viruses.
2
Immature Dendritic Cells (iDCs), macrophages, and circu-
lating iDCs are considered the primary targets for DENV infec-
tion and replication after a sting by the infected vector.
3-5
Interactions between DENV and Dendritic Cells (DCs) have
been shown to be crucial for the transport of viral particles to
secondary lymphoid organs and for the development of
acquired immunity. These interactions also appear to act as a
mechanism of deception to the immune system in which a
greater number of DCs are infected in the primary organs of
infection.
4-6
Glycoprotein E, the main
fl
avivirus surface protein, is
responsible for viral binding to the DC-SIGN, a type C lectin
receptor, on host iDCs and the endosome membrane through
well-de
fi
ned glycosylation sites.
3
,
7
,
8
DENV/DC-SIGN binding ini-
tiates viral particle adsorption (receptor-mediated endocyto-
sis). The glycoprotein E present in the DENV envelope interacts
with the Carbohydrate Recognition Domain (CRD) on the DC-
SIGN receptor, contributing to the binding and internalization
of the virus in host cells.
9
Some studies have proposed that the
sticky
function of DC-SIGN is an independent function of
infection, suggesting that even uninfected iDCs participate in
the infection process by presenting the pathogen adhered to
their cell surfaces to T-cells.
10
,
11
In contrast, it has been shown
by other studies that DC-SIGN adheres to and internalizes the
pathogen, whereas a receptor closely related to DC-SIGN, DC-
SIGNR or L-SIGN, acts only by adhering the pathogen to the sur-
face of DC without internalization capacity.
12
The CD209 gene, which codes for the DC-SIGN protein, has
six exons, and
fi
ve introns and goes through the alternative
splicing process, which generates varied membrane or solu-
ble isoforms.
12
,
13
Studies have shown that the same individ-
ual can express more than one isoform of transcripts and
synthesize their corresponding proteins.
14
However the exact
role of these soluble isoforms is not known. Protein isoforms
range from 168 (isoform 9) to 404 (isoforms 1 and 5) amino
acids.
12
,
15
The complete DC-SIGN protein consists of four
regions. CD209 exon I contain information for the cytoplasmic
region of the protein (N-terminal). The transcripts that hold
exon II contain information for the transmembrane domain,
encoding mDC-SIGN isoforms. If exon II is lost, soluble iso-
forms (sDC-SIGN) are produced. Exons III, IV, V, and VI encode
the extracellular portion of the molecule, which encompasses
two domains: the neck region and the CRD.
12
Exon III comprises seven and a half tandem repeats of
nucleotide sequences, which encode the neck region of the
protein.
16
,
17
The tetramerization generated by the neck region
also leads to the tetramerization of the CRD structure. The
neck region projects CRD beyond the cell surface and gives
DC-SIGN
fl
exibility comparable to that of immunoglobulins,
allowing it to bind to antigens on viral surfaces at different
distances.
18
The variation in expression levels and isoforms
generated between individuals may have important implica-
tions for dengue immunopathogenesis.
The function of soluble isoforms is not well known; some
studies have demonstrated the importance of these
recombinant DC-SIGN isoforms in blocking Human Immuno-
de
fi
ciency Virus (HIV),
19
DENV
20
and Cytomegalovirus
(CMV)
21
infections. Soluble DC-SIGN isoforms are known not
to have the same functional activity in terms of binding to
ICAM-3 in CD4 T lymphocytes. A study with one sDC-SIGN
isoform showed that it was not secreted and was located in
the cytoplasm of producer cells with unknown function.
22
In order to better clarify the function of soluble isoforms in
the process of DENV infection in iDCs, this study deals with
three recombinant soluble DC-SIGN isoforms. The complete
recombinant soluble isoform (sDC-SIGN1B type I
isoform
10), an isoform without CRD alteration but with neck region
changes (sDC-SIGN1A type III
isoform 8), and an isoform
with changes in CRD, neck region, and other regions (sDC-
SIGN1B type III
isoform 12) were built. Their ability to bind
in mannose residues was veri
fi
ed previously.
23
The choice of
these three isoforms aimed to represent the variation of the
expression existing in the human organism and its possible
functions in the infectious process.
Material and methods
Protein expression, puri
fi
cation, purity, and function analysis
Recombinant sDC-SIGN isoforms 8, 10, and 12 were built by
heterologous expression. The nucleotide sequences were
obtained from GenBank, synthesized, and cloned into expres-
sion vectors with sequences encoding a histidine tail.
Escheri-
chia coli
BL21 Rosetta DE3 cells (Novagen) were used for
protein expression. The
E. coli
cells correctly transformed by
protein expression vector were grown in 2XYT, at OD
600
= 0.6.
The expression was induced with isopropyl-
b
-
D
-thiogalacto-
side for 4 h, when bacteria were collected and lysed by ultra-
sonic treatment. After, recombinant proteins expressed were
treated with urea 6 M to eliminate inclusion bodies, and then
denatured recombinant proteins were refolded in a refolding
buffer (pH = 7.4). Then, proteins were puri
fi
ed by af
fi
nity chro-
matography in a HiTrap column (GE Healthcare) and its func-
tion was con
fi
rmed by af
fi
nity chromatography in a
mannose-agarose column. Puri
fi
ed recombinant soluble pro-
teins were resolved by SDS-PAGE followed by western
blotting.
23
Cells, viruses, antibodies, and cytokines
Human peripheral blood acute monocytic leukemia cells THP-
1 (ATCC; number TIB-202) were cultured in RPMI 1640
medium (Gibco, Brazil) supplemented with 10 % FBS and 0.3 %
Penicillin-Streptomycin-Amphotericin (PSA) B solution
(Sigma-Aldrich, USA) maintained in a humidi
fi
ed atmosphere
oven with 5 % CO
2
at 37 °C. The continuous line C6/36 cells,
obtained from
Aedes albopictus
were grown in Leibovitz L-15
medium (Cultilab, Brazil) supplemented with 10 % FBS
(Sigma-Aldrich, USA) and PSA. The culture was incubated in a
Biochemical Oxygen Demand (BOD) oven at 28 °C until reach-
ing about 90 % con
fl
uency in the
fl
ask.
The C6/36 cells monolayer was infected with a Multiplicity
of Infection (MOI) of 0.01 and the culture was incubated in a
2
braz j infect dis.
2024;
28(6)
:103873
BOD oven at 28 °C for around four to seven days until the
appearance of a Cytopathic Effect (CPE).
The titer of DENV was determined in Baby Hamster Kidney
(BHK-21) cells (ATCC CCL-10) obtained from continuous line-
age from the Department of Microbiology of the Federal Uni-
versity of Minas Gerais. The virus titer was measured by
tissue culture infectious doses (TCID
50
/mL), calculated using
the Reed-Muench method.
24
The antibody was obtained from BD Biosciences, it used
anti-human CD14 (PE-Cy7), anti-CD86 (PERCP-Cy5.5), anti-
human CD209 (Paci
fi
c Blue), anti-CD11c (FITC), rh GM-CSF,
and rhIL-4 (BD
Biosciences, USA).
iDCs differentiation from THP-1 cells
For dendritic cell differentiation, 10
4
cells of THP-1 were
plated per well in a 96-well plate, with RPMI medium supple-
mented with 10 % BFS and cytokines for differentiation, GM-
CSF (50
h
g/mL; Immunotools), and IL4 (50 ng/mL; Immuno-
tools). The cells were incubated in a BOD with 5 % CO
2
,at37°
C for seven days. At each 72 h the differentiation, the
medium, and the cytokines were renewed.
Phenotyping of generated iDCs
For phenotypic determination of iDCs by
fl
ow cytometry, the
cells were stained with anti-CD11c, anti-CD86, anti-CD209,
and anti-CD14. The acquisition was performed in the
fl
ow
cytometer using a LSRFortessa with the software FACSDiva
(BD Biosciences), in the Interdisciplinary Laboratory of
Human Diseases Research, in the Department of Clinical and
Toxicological Analysis of the Faculty of Pharmacy of UFMG.
Data were analysed with FlowJo 10.0 software (Tree Stars
Inc.).
Infection assays
The iDCs obtained were used in the DENV-2 infection assays
in the presence of recombinant sDC-SIGN isoforms 8, 10, and
12 at concentration of 25, 50 and 100 ng/mL. The assays evalu-
ated the overall activity of the isoforms on the viral particles.
The cells were infected on the last day of differentiation.
Around 10
6
cells/well were infected with DENV-2 at 5
£
10
7
virus/mL and a MOI of 1. The infection assay was made using
the following steps: A) Plate 1 (Protein + iDCs): The iDCs were
incubated with 25, 50 or 100 ng/mL of each protein, sepa-
rately, for 30 min at 37 °C and 5 % CO
2
, in a total volume of 150
m
L of 5 % RPMI. B) Plate 2 (Protein + DENV): The recombinant
proteins (25, 50, or 100 ng/mL) were incubated with DENV-2
for 30 min in the same conditions as Plate 1. C) 150
m
L of Plate
2 was transferred to the cell plate (Plate 1) which was incu-
bated at 37 °C and 5 % CO
2
for two days. According to Alen et
al.,
25
the peak of infection occurs in 48 h. D) After 48 h of infec-
tion, the supernatant from each well was collected and the
extraction of viral RNA, Reverse Transcription (RT-PCR), qPCR
of cDNA, and ultrafreezer storage were made. Experiments
were performed in duplicate. All processes were repeated
three times in different months to evaluate the reproducibil-
ity of the results.
qPCR
The viral load was measured by absolute qPCR of DENV-2. The
DENV-2 RNA obtained from the cell culture supernatant was
extracted with a High Pure Viral Nucleic Acid Kit (Roche, Swit-
zerland) according to the manufacturer
s guidelines. The viral
RNA was quanti
fi
ed in an absorbance spectrophotometer and
submitted to the RT-PCR in a thermocycler (AB-Applied Bio-
system, Veriti thermal cycler, 2010). The cDNA production
was performed from approximately 1 ug of RNA, 10
£
Random
Primer, 10 mM dNTP
s, Depc water, 10
£
RT enzyme buffer
and the MultiScribe
Ò
Reverse Transcriptase enzyme with a
High Capacity cDNA Reverse Transcription Kit (Applied Bio-
systems, USA). The reaction conditions for cDNA synthesis
were: 01 cycle at 94°C for 1-minute; 30 cycles comprising three
steps of 94°C for 30 seconds, 57°C for 30 seconds, and 72°C for
30 seconds, and 01 cycle at 72°C for 10 minutes.
The cDNA was serially diluted (10

1
to 10

10
) to be used in
a subsequent qPCR to build a standard curve with primers as
previously described: Forward primer (F) 5
0
-TTA GAG GAG
ACC CCT CCC-3
0
and reverse primer (R) 5
0
-TCT CCT CTA ACC
TCT AGT CC

3
0
.
26
In the qPCR 2
m
L of diluted cDNA, 10 pmoL
of each primer, and 5
m
L of the HOT FIREPol
Ò
solution
EvaGreen
Ò
qPCR Supermix (Solis Biodyne, Estonia) were used.
The ampli
fi
cation conditions were: 01 cycle at 95°C for 12
minutes, 40 cycles comprising three steps of 95°C for 15 sec-
onds, 56°C for 20 seconds, and 72°C for 20 seconds. The analy-
ses of the Tm curves included in the qPCR were performed by
a denaturation step at 95°C for 15 s followed by 60°C for 1 m
and a ramp up to 94°C at a rate of 0.1°C/10 s with continuous
fl
uorescence measurement.
Statistical analysis
Data analysis was performed using the GraphPad Prism ver-
sion 7.04 statistical program. Non-parametric Kruskal-Wallis
tests were used for pre-testing and Dunn
s for post-testing.
Normally distributed samples were evaluated by ANOVA in a
pre-test and Tukey
s in a post-test. The signi
fi
cance interval
of
p
<
0.05 was considered for both tests.
Results
In the present study, a portion of THP-1 cells differentiated for
six days (144 h) with 50 ng/mL of IL-4 and 50 ng/mL of GM-CSF
showed morphological changes under inverted light micros-
copy when adhered. Another portion that remained in sus-
pension presented minor morphological changes. Cell
clusters possibly containing undifferentiated THP-1 cells and
non-adherent iDCs were observed. Differentiated iDCs, which
adhered and exhibited an elongated morphology with den-
drites, were also seen (
Fig. 1
).
The representative analysis of the phenotyping of iDC gen-
erated from the THP-1 cells and the percentages of each sur-
face marker in iDCs can be viewed in
Fig. 2
. Only singlet cells
were considered for analysis and the gates strategy is also
demonstrated. The gates were de
fi
ned from unmarked con-
trols and considered 10,000 events. Cells read too early or too
late were disregarded by the Time Gate to avoid reading
braz j infect dis.
2024;
28(6)
:103873
3
possible equipment bubbles or debris. iDCs cells (75.4 %)
showed up regulation of CD86, CD209, and CD11c markers
when compared to the THP-1 cells.
After obtaining DENV-2, producing the three isoforms by
heterologous expression, and generating the iDCs cells, the
DENV-2 iDC infection assays were performed in the presence
of soluble isoforms. The experiments were performed at the
concentrations of 25, 50, and 100 ng/mL of each isoform and a
MOI of 1, chosen according to the reference literature. It was
possible to quantify the replicates of infection and the
fi
nal
values obtained by qPCR reactions. These data are repre-
sented by the average of the results obtained in the three dif-
ferent infection assays. To cover the viral concentration
range found in the samples from the infection assays, the
Fig. 1
iDCs cells differentiated from THP-1 cells after 144 h. (A to D) Suspended cell clusters are observed with spicules (blue
arrows) and other adhering cells with elongated conformation (orange arrows), both typical of iDCs.
Fig. 2
Differentiation of THP-1 into iDC. Histograms and Dotplots obtained by
fl
ow cytometry. Blue histograms
unmarked
isotypes; Grey histograms
marked isotypes. The bars show the displacement of the marked population in relation to the
unmarked one. Before differentiation, among the labeled cells (iDCs) 58.3 % expressed the CD209 receptor, 50 % expressed the
CD86 receptor, and 40.1 % expressed the CD11c receptor. The joint expression of these three receptors demonstrated a pro
fi
le
closer to that of iDCs.
4
braz j infect dis.
2024;
28(6)
:103873
most diluted points of the standard curve were considered.
Higher mean viral concentration rates were observed just in
samples containing isoforms 8 and 10 at 100 ng/mL when
compared to the mean viral concentration rate of the viral
control. The values obtained can be observed in
Table 1
.
There was a statistically signi
fi
cant difference between
cellular and viral controls (
p
<
0.05) (
Table 1
). The cellular
control presented a high Ct (
Fig. 3
), but a different Tm dissoci-
ation curve from the virus-containing samples, and there was
no ampli
fi
cation in the negative control (
Fig. 4
).
The sample containing the isoform 12, with altered CRD, at
100 ng/mL presented a similar mean viral concentration to
that found in the viral control. The same happens in the con-
centration of 25 and 50 ng/mL.
Fig. 3
shows the ampli
fi
cation
graph of samples obtained from the infection assay at 100 ng/
mL.
Discussion
The differentiation results are similar to those described by
Guo et al.
27
with the phenotyping characteristics and mor-
phology equating to Berges et al.
28
As a monocytic lineage, THP-1 cells must have high CD14
expression; as observed in the literature, when differentiated
with IL-4, they continue to show high CD14 expression.
29
In
contrast, THP-1 cells have low CD86 and CD11 expression, as
expected in monocytic strains,
30
and iDCs show higher
expression of these markers.
31
In addition, THP-1 has a vari-
able (uptrend) expression of CD209, which should increase in
iDCs.
29
,
32
As predicted, not all iDC differentiated from the leukemia
cell model showed elongated phenotype although most cells
presented changes in round to slightly longer elongated
Table 1
Mean concentration and viral load obtained
from infection assays with 100 ng/mL of sDC-SIGN solu-
ble isoforms.
Isoform
(100 ng/mL)
Viral average
concentration
(ng/
m
L) by isoform
concentration
N

viral copies/
m
L
by isoform
concentration
8
8.3E-01
a
7.8E+21
a
10
5.4E-01
a
5.1E+21
a
12
6.0E-02
5.9E+20
Viral Control
3.0E-02
2.7E+20
Cellular Control
0.0E+00
0.0E+00
a
Statistical signi
fi
cance compared to viral control.
- There was a signi
fi
cant difference between all cellular and viral controls.
- There was a signi
fi
cant difference between all samples and the cell control.
- The means obtained by qPCR from replicates of infection assays are shown.
- The number of viral copies (copies/mL) found in the samples was calculated
using the formula: Copies/
m
L
=[g/
m
L
of RNA
£
6.022
1023]/[
transcript bp
£
660 g/
moL] * Average MM of 1 bp of DNA = 660 g/moL; 1 moL = 6.02
£
10
23
molecules.
Fig. 3
Viral ampli
fi
cation in samples treated with the three soluble isoforms at 100 ng/mL. Ampli
fi
cation plot of assays for iso-
forms 8, 10, and 12 at 100 ng/mL concentration. Grey: Standard curve dilutions from 10

5
to 10

9
. Navy Blue: Sample treated
with isoform 8 at 100 ng/mL. Rose: Sample treated with isoform 10 at 100 ng/mL. Orange: Sample treated with isoform 12 at
100 ng/mL. Purple: infection controls from assays. Light blue: cellular controls of infection assays.
braz j infect dis.
2024;
28(6)
:103873
5
conformation, as described in the literature.
28
This fact can be
explained by the reasoning that 100% cell differentiation does
not occur and possibly, because there are non-adherent sus-
pended iDCs, this gives them a rounded shape when viewed
under inverted light microscopy.
THP-1 cells have been standardized as an easy, fast, and
reliable model for iDC differentiation, as corroborated by the
literature.
29
,
30
Chan et al.
33
differentiated THP-1 on iDC for
fi
ve
days with an initial density of 1
£
10
6
cells per well with 40 ng/
mL IL-4 and 40 ng/mL GM-CSF at 37 °C in a humidi
fi
ed environ-
ment with 5 % CO
2
. Cytokines were changed on the third day
and cells were labeled at the end of the
fi
fth day. At the end of
differentiation, THP-1 cells showing intracellular IL-10 expres-
sion, de novo expression of the CD86 costimulatory molecule,
and surface receptors indicative of iDCs CD11c, CD40, and
CD209 were observed. In addition, the cells did not show the
expression of the marker CD83, an indicator of iDC maturation.
Under these conditions, a THP-1 iDC differentiation rate of
75.4 % was obtained and the cells acquired functional proper-
ties of iDCs, such as increased receptor macromolecular endo-
cytosis and low T lymphocyte stimulatory capacity.
As already reported in the literature, IL-4 and GM-CSF are
suf
fi
cient to promote differentiation into functional iDC.
28
,
33
After differentiation, cells maintained the phenotypic proper-
ties of iDC for approximately six weeks by changing cytokine
differentiation factors every three days.
28
These data based
this work on the choice of differentiation and concentration
factors, and differentiation time and surface markers
observed at the end of differentiation.
Regarding mean concentration and viral load obtained
from infection assays with 100 ng/mL of sDC-SIGN soluble
isoforms, the supposed ampli
fi
cations that occurred in the
cell control samples did not correspond to the viral ampli
fi
ca-
tion, since these samples contained only cells and PBS. The
experiments were performed with the Eva green
fl
uorophore,
which interleaves on any double strand, not being speci
fi
c. In
this case, this incorporation clearly occurred in the primer
dimers used in the reaction that was not consumed, since the
virus was not present. This hypothesis can be con
fi
rmed by
the melting curve shown in
Fig. 3
, showing a different Tm for
these samples (light blue curves) and non-ampli
fi
cation of
the viral fragment.
Higher mean viral concentration rates were observed just
in samples containing isoforms 8 and 10 at 100 ng/mL when
compared to the mean viral concentration rate of the viral
control. Mean viral concentrations in these samples (
Table 1
)
were approximately 23 times higher than in the viral control.
There were noteworthy increases at 28 times higher for iso-
form 8 and 18 times higher for isoform 10, and the difference
in infection rate between isoforms was also signi
fi
cant
(
p
<
0.05).
One initial hypothesis to explain the increase in the viral
load when soluble isoform 8 and 10 are used is the possible
conjugation of recombinant soluble isoforms with membrane
isoforms, already existing in iDCs, that have not yet formed
stable (di-, tri-, or tetra-) multimers.
21
,
34
This conjugation
could contribute to increasing the infection, since the tetra-
meric structures of DC-SIGN, which increase the stability of
the DENV binding and other ligands, would be increased.
Given the results, we observed that the above theory may
have occurred: isoforms 8 and 10 associated with membrane
isoforms of differentiated cells forming multimers would
Fig. 4
Different Tm dissociation curve between cell control and other virus-containing samples. The melting temperature
found for the cell control samples (light blue) differs from the ampli
fi
cation temperature of the DENV-2 fragments, con
fi
rming
that the
fl
uorescence detected in the cell control was equivalent to primer dimers and not to ampli
fi
cation of viral fragments.
6
braz j infect dis.
2024;
28(6)
:103873
increase the avidity by circulating DENV with a consequent
increase in the rate of infection. This result was similar to
that found by Plazolles et al.,
21
who demonstrated increased
CMV infection in the presence of recombinant sDC-SIGN.
When they performed CMV infection testing on monocyte-
derived Dendritic Cells (moDC) with MOI = 1, for 24 to 48 h in
the presence of decreasing amounts of soluble isoforms 6
(sDC-SIGN1A type I) and 8 (sDC-SIGN1A type III) (400 to
12.5 ng/mL), they observed about double the infected DCs
than in infection control, with a concentration between
100 ng/mL and 50 ng/mL of protein. Concentrations greater
than 100 ng/mL and less than 50 ng/mL of the sDC-SIGN1A
type I recombinant isoform 6 showed no difference in infec-
tion rate.
Other studies have shown blockade of
S. aureus
and HIV
infection in the presence of soluble DC-SIGN isoforms. Kwon
et al.
19
and Navarro-Sanchez et al.
35
suggested DC-SIGN pro-
tein increases viral HIV and DENV infection only when
expressed in the cell membrane. In both studies, there was
blockade of infection by sDC-SIGN with MOI variable from 5
to 10; however, only CRD was produced and considered as sol-
uble DC-SIGN. Kwon et al. further demonstrated that mDC-
SIGN with the truncated cytoplasmic domain region is capa-
ble of capturing circulating viruses but is unable to internalize
them with low MOI.
19
This corroborates the highlighted
importance in our study that isoforms be fully studied, as
they exist
in vivo
, because all portions of the protein perform
functions that are still being discovered.
Another possible justi
fi
cation for the results found in the
present work is that at high concentration (100 ng/mL) there
is bioavailability of sDC-SIGN that complexes rapidly but
inef
fi
ciently to circulating viral particles acting as opsonins
rather than infection blockers. Thus, soluble isoforms at
high concentrations could lead to a high number of immobi-
lized viral particles to capture and internalize by iDCs, favor-
ingfavouring infection. As already described by Mikloska et
al.,
36
this hypothesis presents th
e need for another receptor
that favors iDC opsonization, such as CD11b has been shown
to facilitate HIV opsonization by iDC mDC-SIGN-depen-
dence. It is also possible that these aggregates (virus + sDC-
SIGN) may associate with mDC-SIGN and somehow facilitate
viral penetration into cells. These data indicate that the
virus
opsonized
by sDC-SIGN is more effectively captured
by iDCs than free viruses. sDC-SIGN molecules capable of
interacting with infectious agents at high serum concentra-
tions could potentiate the severity of diseases such as Den-
gue, since infection itself can alter the expression pattern of
isoforms.
21
,
36
DC-SIGN molecules in tetramers are known to bind to
better af
fi
nity N-glycan residues, with the neck region of the
protein essential in this oligomerization.
3
,
14
Although iso-
form 8 has unchanged CRD, its neck region is altered and it
is well demonstrated that it is through this region that inter-
action with other soluble and membrane isoforms occurs.
14
Even so, in our results a signi
fi
cant increase in infection was
also found in the trials with the presence of this isoform
(
Table 1
). One possible explanation for this is that remaining
amino acid residues in the neck region of isoform 8 (three
and a half tandem repeats) are suf
fi
cient to promote interac-
tion with other neck regions of membrane isoforms also
forming the multimers responsible for the increase of avid-
ity for DENV-2 and, consequently, increasing the infection
rate. Moreover, because it is a smaller molecule than the
complete isoform, isoform 8 linked to DENV could be more
easily internalized.
The second hypothesis was that recombinant soluble iso-
forms capable of binding to DENV could interact with each
other to form stable multimers that could neutralize circulat-
ing viral particles and, consequently, the binding, internaliza-
tion, and infection of iDCs. This hypothesis was discarded in
experiments with isoforms 8 and 10 at 100 ng/mL.
Besides the possibility of observing this process
in vitro
assays, these events could also occur with circulating sDC-
SIGN, naturally or via therapeutic administration, neutraliz-
ing the infection, and therefore, it is important to observe the
necessary concentrations of circulating isoforms to provide
blockade. Schmid and Harris
37
proposed that the skin is an
important site for therapeutic actions or even for intradermal
vaccination, since DCs and macrophages are the primary tar-
get of DENV infection with additional monocyte recruitment
for further differentiation into iDCs susceptible to infection
and subsequent antigen presentation.
DENV causes a diverse spectrum of disease ranging from
asymptomatic infection and mild febrile illness to more seri-
ous complications, including hemorrhage and shock. The
associations between host genetics, DENV infection and clini-
cal outcome are complex and may involve more than one fac-
tor such as age, ethnicity, primary or secondary infection,
patient
s metabolic conditions and even genetic factors that
lead to the expression of proteins involved in the process. of
infection.
38
,
39
Studies demonstrate the relationship between the severity
of the disease and the different polymorphism pro
fi
les of
genes that express proteins associated with DENV infection,
such as DC-SIGN.
40
,
41
This demonstrates the importance of
the protein structure of receptors for viral infection. There-
fore, therapeutic strategies targeting protein structures
involved in the DENV infection process, associated with a
higher degree of infection, as observed in the present study
for DC-SIGN isoforms 8 and 10, are promising.
Infection experiments in the presence of soluble isoform
12 presented different results from other isoforms. This iso-
form does not appear to interact with circulating viral par-
ticles at any concentration, neither increasing nor decreasing
the infection rate compared to the infection control. The
results obtained were statistically similar to those found in
viral control. This result is in agreement with those found in
the mannose column binding experiments which demon-
strated the inability of this soluble isoform to bind to these
residues.
23
Thus, isoform 12 would be unable to bind to DENV
glycoprotein E, apparently not interfering in any way with the
infection process.
Unlike isoforms 8 and 10, isoform 12 has an altered CRD
region, which is essential for the binding and internalization
of DENV in the cell.
9
Therefore, it can be inferred that the
absence of this region prevents the interaction of the virus
with isoform 12, maintaining infection levels similar to those
of the viral control.
Finally, an important aspect to be analysed in new stud-
ies is the relationship between viral infection and variation
braz j infect dis.
2024;
28(6)
:103873
7
at the expression level of soluble isoforms, since we
observed that infection rates were increased at the tested
concentration, which may suggest a mechanism to
aid
viral
particles.
Conclusion
DC-SIGN soluble isoforms with intact CRD (8 and 10) studied
in this work maintain the ability to bind to DENV mannose
residues and potentiate infection rates in 100 ng/mL iDCs.
DC-SIGN soluble isoform 12 with altered CRD lost its ability
to interact with DENV mannose residues and did not generate
signi
fi
cant changes in mean viral load at the concentration
tested.
The amino acid residues that constitute the neck region
seem to allow the polymerization of the isoforms, even in
smaller repetitions than in the canonical isoform. Isoform 8,
which has an altered neck region but intact CRD, was also
able to increase the rate of infection. In addition, the smaller
size of this isoform seems to favor the internalization of the
sDC-SIGN-DENV complex.
Funding
This study was funded by the Coordena
̧
c
~
ao de Aperfei
̧
coa-
mento de Pessoal de Nível Superior
Brazil (CAPES)
Funding
Code 001, Conselho Nacional de Desenvolvimento Cientí
fi
co e
Tecnol

ogico (CNPq) and Funda
̧
c
~
ao de Amparo

a Pesquisa do
estado de Minas Gerais (FAPEMIG).
Con
fl
icts of interest
The authors declare no con
fl
icts of interest.
CRediT authorship contribution statement
Lailah Hor

acio Sales Pereira:
Conceptualization, Investiga-
tion, Methodology, Software, Validation, Visualization, Writ-
ing
original draft, Writing
review & editing.
Amanda do
Carmo Alves:
Investigation, Methodology, Writing
review &
editing.
Gabriela Francine Martins Lopes:
Methodology, Vali-
dation, Writing
review & editing.
Brenda Fernandes da
Silva:
Methodology, Validation, Writing
review & editing.
Mariana Sousa Vieira:
Methodology, Validation, Writing
review & editing.
D

ebora de Oliveira Lopes:
Funding acquisi-
tion, Supervision.
Jaqueline Maria Siqueira Ferreira:
Concep-
tualization, Formal analysis, Funding acquisition,
Investigation, Methodology, Project administration, Resour-
ces, Supervision, Validation, Writing
original draft, Writing
review & editing.
Luciana Lara dos Santos:
Conceptualiza-
tion, Data curation, Formal analysis, Funding acquisition,
Investigation, Methodology, Project administration, Resour-
ces, Software, Supervision, Writing
review & editing.
Acknowledgments
Professors Dr. Danielle da Gl

oria de Souza, Dr. Erna Geessien
Kroon (Universidade Federal de Minas Gerais, Brazil), and Dr.
Luiz Felipe Leomil Coelho (Universidade Federal de Alfenas,
Brazil) for providing cell lines, Professors Dr. Andrea Tavares
and Dr. Carlos Eduardo Calzavarra for providing some of the
antibodies (Instituto Rene Rachou, Fiocruz, Brazil).
references
1.
WHO (World Health Organization). Dengue and Severe
Dengue. Geneva: World Health Organization; 2023.
2.
ICTV (International Committee on Taxonomy of Viruses).
Virus Taxonomy Pro
fi
le: Flaviviridae. International
Committee on Taxonomy of Viruses; 2020.
3.
Lozach PY, Burleigh L, Staropoli I, et al. Dendritic cell-speci
fi
c
intercellular adhesion molecule 3-grabbing non-integrin (DC-
SIGN)-mediated enhancement of dengue virus infection is
independent of DC-SIGN internalization signals. J Biol Chem.
2005;280:23698
708
.
4.
Schmid MA, Diamond MS, Harris E. Dendritic cells in dengue
virus infection: targets of virus replication and mediators of
immunity. Front Immunol. 2014;5:1
10.
5.
Wu SJ, Grouard-Vogel G, Sun W, et al. Human skin Langerhans
cells are targets of dengue virus infection. Nat Med.
2000;6:816
20.
6.
Marovich M, Grouard-Vogel G, Louder M, et al. Human
dendritic cells as targets of dengue virus infection. J Investig
Dermatol Symp Proc. 2001;6:219
24.
7.
Lindenbach BD, Thiel HJ, Rice CM. Flavivirus: the virus and
their replication. In: Knipe DM, Howley PM, eds. Fields
Virology, Philadelphia: Lippincott Williams & Wilkins;
2007:1101
52
.
8.
Pokidysheva E, Zhang Y, Battisti AJ, et al. Cryo-EM
reconstruction of dengue virus in complex with the
carbohydrate recognition domain of DC-SIGN. Cell.
2006;124:485
93.
9.
Tassaneetrithep B, Burgess TH, Granelli-Piperno A, et al. DC-
SIGN (CD209) mediates dengue virus infection of human
dendritic cells. J Exp Med. 2003;197:823
9.
10.
Steinman RM. DC-SIGN: a guide to some mysteries of
dendritic cells. Cell. 2000;100:491
4.
11.
P
ohlmann S, Baribaud F, Doms RW. DC-SIGN and DC-SIGNR:
helping hands for HIV. Trends Immunol. 2001;22:643
6.
12.
Mummidi S, Catano G, Lam LA, et al. Extensive repertoire of
membrane-bound and soluble dendritic cell-speci
fi
c ICAM-3-
grabbing nonintegrin 1 (DC-SIGN1) and DC-SIGN2 isoforms:
inter-individual variation in expression of DC-sign transcripts.
J Biol Chem. 2001;276:33196
212
.
13.
Thierry-Mieg D, Thierry-Mieg J. AceView: a comprehensive
cDNA-supported gene and transcripts annotation. Genome
Biol. 2006;7:1
14.
14.
Feinberg H, Mitchell DA, Drickamer K, Weis WI. Structural
basis for selective recognition of oligosaccharides by DC-SIGN
and DC-SIGNR. Science. 2001;294:2163
6.
15.
Berman HM, Westbrook J, Feng Z, et al. The protein data bank.
Nucleic Acids Res. 2000;28:235
42.
16.
Mason CP, Tarr AW. Human lectins and their roles in viral
infections. Molecules. 2015;20:2229
71.
17.
Bernhard OK, Lai J, Wilkinson J, Sheil MM, Cunningham AL.
Proteomic analysis of DC-SIGN on dendritic cells detects
tetramers required for ligand binding but no association with
CD4. J Biol Chem. 2004;279:51828
35.
8
braz j infect dis.
2024;
28(6)
:103873
18.
Feinberg H, Guo Y, Mitchell DA, Drickamer K, Weis WI.
Extended neck regions stabilize tetramers of the receptors DC-
SIGN and DC-SIGNR. J Biol Chem. 2005;280:1327
35.
19.
Kwon DS, Gregorio G, Bitton N, Hendrickson WA, Littman DR.
DC-SIGN-mediated internalization of HIV is required for
trans-enhancement of T cell infection. Immunity.
2002;16:135
44.
20.
Navarro-Sanchez E, Altmeyer R, Amara A, et al. Dendritic-cell-
speci
fi
c ICAM3-grabbing non-integrin is essential for the
productive infection of human dendritic cells by mosquito-
cell-derived dengue viruses. EMBO Rep. 2003;4:723
8
.
21.
Plazolles N, Humbert J-M, Vachot L, Verrier B, Hocke C, Halary
F. Pivotal advance: the promotion of soluble DC-SIGN release
by in
fl
ammatory signals and its enhancement of
cytomegalovirus-mediated cis-infection of myeloid dendritic
cells. J Leukoc Biol. 2011;89:329
42.
22.
Martinez O, Brackenridge S, El-Idrissi MEA, Prabhakar BS. DC-
SIGN, but not sDC-SIGN, can modulate IL-2 production from
PMA- and anti-CD3-stimulated primary human CD4 T cells.
Int Immunol. 2005;17:769
78.
23.
Pereira LHS, de Souza TPP, Camargos VN, et al. Assays with
recombinant soluble isoforms of DC-SIGN, a dengue virus
ligand, show variation in their ability to bind to mannose
residues. Arch Virol. 2019;164:2793
7.
24.
Reed LJ, Muench H. A simple method of estimating
fi
fty
percent endpoints. Am J Epidemiol. 1938;27:493
7.
25.
Alen MMF, de Burghgraeve T, Kaptein SJF, Balzarini J, Neyts J,
Schols D. Broad antiviral activity of carbohydrate-binding
agents against the four serotypes of dengue virus in
monocyte-derived dendritic cells. PLoS One. 2011;6:e21658.
26.
Chutinimitkul S, Payungporn S, Theamboonlers A,
Poovorawan Y. Dengue typing assay based on real-time PCR
using SYBR Green I. J Virol Methods. 2005;129:8
15.
27.
Guo Q, Zhang L, Li F, Jiang G. The plasticity and potential of
leukemia cell lines to differentiate into dendritic cells. Oncol
Lett. 2012;4:595
600.
28.
Berges C, Naujokat C, Tinapp S, et al. A cell line model for the
differentiation of human dendritic cells. Biochem Biophys Res
Commun. 2005;333:896
907.
29.
Puig-Kr
oger A, Serrano-G

omez D, Caparr

os E, et al. Regulated
expression of the pathogen receptor dendritic cell-speci
fi
c
intercellular adhesion molecule 3 (ICAM-3)-grabbing
nonintegrin in THP-1 human leukemic cells, monocytes, and
macrophages. J Biol Chem. 2004;279:25680
8
.
30.
Chen R-F, Wang L, Cheng J-T, Yang KD. Induction of IFN
a
or IL-
12 depends on differentiation of THP-1 cells in dengue
infections without and with antibody enhancement. BMC
Infect Dis. 2012;12:340.
31.
Wieder E. Dendritic cells : a basic review. Int Soc Cell Ther.
2003: 1
6.
32.
Pont

en F, Jirstr
om K, Uhlen M. The human protein atlas
a
tool for pathology. J Pathol. 2008;216:387
93.
33.
Chan WK, Cheung CC, Law HK, Lau YL, Chan GC. Ganoderma
lucidum polysaccharides can induce human monocytic
leukemia cells into dendritic cells with immuno-stimulatory
function. J Hematol Oncol. 2008;1:9.
34.
Mitchell DA, Fadden AJ. Drickamer K. A novel mechanism of
carbohydrate recognition by the C-type lectins DC-SIGN and
DC-SIGNR. Subunit organization and binding to multivalent
ligands. J Biol Chem. 2001;276:28939
45.
35.
Navarro-Sanchez E, Altmeyer R, Amara A, et al. Dendritic-
cell-speci
fi
c ICAM3-grabbing non-integrin is essential for
the productive infection
of human dendritic cells by
mosquito-cell-derived dengue viruses. EMBO Rep.
2003;4:723
8
.
36.
Mikloska Z, Bosnjak L, Cunningham AL. Immature monocyte-
derived dendritic cells are productively infected with herpes
simplex virus type 1. J Virol. 2001;75:5958
64.
37.
Schmid MA, Harris E. Monocyte recruitment to the dermis and
differentiation to dendritic cells increases the targets for
dengue virus replication. PLoS Pathog. 2014;10:e1004541.
38.
Guzman MG, Harris E. Dengue. Lancet. 2015;385:453
65.
39.
Bhatt P, Sabeena SP, Varma M, Arunkumar G. Current
understanding of the pathogenesis of dengue virus infection.
Curr Microbiol. 2021;78:17
32.
40.
Noecker CA, Amaya-Larios IY, Galeana-Hern

andez M, Ramos-
Casta
~
neda J, Martínez-Veja RA. Contrasting associations of
polymorphisms in Fc
g
RIIa and DC-SIGN with the clinical
presentation of dengue infection in a Mexican population.
Acta Trop. 2014;138:15
22.
41.
Xavier-Carvalho C, Gibson G, Brasil P, et al. Single nucleotide
polymorphisms in candidate genes and dengue severity in
children: a case
control, functional and meta-analysis study.
Infect Genet Evol. 2013;20:197
205
.
braz j infect dis.
2024;
28(6)
:103873
9

Other users also viewed these articles

CARACTERÍSTICAS CLÍNICAS E SOCIODEMOGRÁFICAS EM POPULAÇÃO COM SUSPEITA DE ARBOVIROSE EM GOIÂNIA: ESTUDO CASO-CONTROLE Raquel da Silva Carvalho; Jéssica Barletto de Sousa Barros; Fernanda de Oliveira Feitosa de Castro; Arthur Antonucci Vieira Morais; Raisa Melo Lima; Antonio Márcio Teodoro Cordeiro Silva; Irmtraut Araci Hoffmann Pfrimer;
Braz J Infect Dis. 2024;28 Supl 1:
PERFIL EPIDEMIOLÓGICO DA DENGUE NO CENTRO-OESTE: DA ENDEMIA À EPIDEMIA Manuela Zaidan Rodrigues; Larissa Bevilaqua Sampaio Contreiras; Leandra Lucas Nogueira; Katharina Rezende Esterl; Maria Eduarda Barbosa de Sousa; Júlia Anastácio Furtado; Lucas Fruet Sperandio; Pedro Paulo Cruz de Oliveira Silva; Melissa Gomes Carvalho; Letícia Olivier Sudbrack;
Braz J Infect Dis. 2024;28 Supl 1:
ANÁLISE DOS CASOS DE DENGUE NO PRIMEIRO TRIMESTRE DE 2023 E 2024 NO ESTADO DE GOIÁS Janaina Fontes Ribeiro; Vitor Hugo Pereira Jardim; Jade Oliveira Vieira; Luiz Gustavo Vieira Gonçalves; Anna Luiza Silva Carvalho; Divina D'arc Cândida de Araújo Bezerra; Laíza Barbosa Guimarães; Mariana Rodrigues Sandes da Silva; Maysa Aparecida de Oliveira; Edna Joana Cláudio Manrique;
Braz J Infect Dis. 2024;28 Supl 1: