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Citation: Nesti´c, D.; Božinovi´c, K.;
Draškovi´c, I.; Kovaˇcevi´c, A.; van den
Bosch, J.; Kneževi´c, J.; Custers, J.;
Ambriovi´c-Ristov, A.; Majhen, D.
Human Adenovirus Type 26 Induced
IL-6 Gene Expression in an αvβ3
Integrin- and NF-κB-Dependent
Manner. Viruses 2022,14, 672.
https://doi.org/10.3390/
v14040672
Academic Editor: Akinori Takaoka
Received: 11 February 2022
Accepted: 22 March 2022
Published: 24 March 2022
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viruses
Article
Human Adenovirus Type 26 Induced IL-6 Gene Expression in
an αvβ3 Integrin- and NF-κB-Dependent Manner
Davor Nesti´c 1, Ksenija Božinovi´c 1, Isabela Draškovi´c 1, Alen Kovaˇcevi´c 1, Jolien van den Bosch 1,
Jelena Kneževi´c 2,3 , Jerome Custers 4, Andreja Ambriovi´c-Ristov 1and Dragomira Majhen 1, *
1Laboratory for Cell Biology and Signalling, Division of Molecular Biology, Ru ¯
der Boškovi´c Institute,
10000 Zagreb, Croatia; davor.nestic@irb.hr (D.N.); ksenija.bozinovic@irb.hr (K.B.); isabela.pehar@irb.hr (I.D.);
akovacevic90@gmail.com (A.K.); jolien.vdbosch@hotmail.com (J.v.d.B.);
andreja.ambriovic.ristov@irb.hr (A.A.-R.)
2Laboratory for Advanced Genomics, Division of Molecular Medicine, Ru ¯
der Boškovi´c Institute,
10000 Zagreb, Croatia; jelena.knezevic@irb.hr
3Faculty for Dental Medicine and Health, University of Osijek, 31000 Osijek, Croatia
4Janssen Vaccines and Preventions BV, 2333 CA Leiden, The Netherlands; jcuster1@its.jnj.com
*Correspondence: dmajhen@irb.hr
Abstract:
The low seroprevalent human adenovirus type 26 (HAdV26)-based vaccine vector was
the first adenovirus-based vector to receive marketing authorization from European Commission.
HAdV26-based vaccine vectors induce durable humoral and cellular immune responses and, as such,
represent a highly valuable tool for fighting infectious diseases. Despite well-described immuno-
genicity
in vivo
, the basic biology of HAdV26 still needs some refinement. The aim of this study
was to determine the pro-inflammatory cytokine profile of epithelial cells infected with HAdV26
and then investigate the underlying molecular mechanism. The expression of studied genes and
proteins was assessed by quantitative polymerase chain reaction, western blot, and enzyme-linked
immunosorbent assay. Confocal microscopy was used to visualize HAdV26 cell uptake. We found
that HAdV26 infection in human epithelial cells triggers the expression of pro-inflammatory cytokines
and chemokines, namely IL-6, IL-8, IL-1
β
, and TNF-
α
, with the most pronounced difference shown
for IL-6. We investigated the underlying molecular mechanism and observed that HAdV26-induced
IL-6 gene expression is
α
v
β
3 integrin dependent and NF-
κ
B mediated. Our findings provide new
data regarding pro-inflammatory cytokine and chemokine expression in HAdV26-infected epithelial
cells, as well as details concerning HAdV26-induced host signaling pathways. Information obtained
within this research increases our current knowledge of HAdV26 basic biology and, as such, can
contribute to further development of HAdV26-based vaccine vectors.
Keywords: human adenovirus type 26; vaccine vector; innate immune response; integrin αvβ3
1. Introduction
Vaccines, biological preparations that provoke specific immunity, are recognized as
one of the most cost-effective interventions for the prevention of infectious diseases. A
vaccine typically contains an agent that resembles a disease-causing microorganism, and is
often made from weakened or killed forms of the microbe, its toxins, or one of its surface
antigens. Recombinant adenovirus vectors are excellent shuttles for the delivery of antigens
since they mimic natural adenoviral infection and enable its intracellular expression. As
a consequence, a potent adjuvant effect can be exerted due to the adenovirus-induced
stimulation of various elements of innate and adaptive immunity, causing antigen-specific
immune response with protective antibody levels and adequate cellular responses [1].
Human adenoviruses (HAdVs) are non-enveloped double-stranded DNA viruses
spanning 67 human serotypes [
2
–
4
] classified in subgroups A–G. The molecular weight
of a functional HAdV particle is about 150 MDa and it has an icosahedral capsid of
Viruses 2022,14, 672. https://doi.org/10.3390/v14040672 https://www.mdpi.com/journal/viruses
Viruses 2022,14, 672 2 of 16
approximately 90 nm [
5
]. HAdV infection starts with binding to the primary receptor at the
cell surface followed by virus cell entry. HAdVs are highly immunogenic and, following
infection, can induce the production of numerous chemokines (macrophage inflammatory
protein 1, RANTES, IL-8, and monocyte chemo attractant protein 1) and cytokines (TNF-
α
,
IL-6, IL-1, IL-12, and type I interferons) that modulate the initiation of inflammation and
regulate the immune response. Immune responses to HAdV vectors can be induced by
the presence of the virus itself, via recognition through pathogen recognition receptors
or as a consequence of interactions with cell surface receptors. Signaling triggered by
interactions between HAdV fibers and coxsackie adenovirus receptor (CAR) activates the
downstream signaling of ERK 1/2, JNK, MAPK, and NF-
κ
B, leading to the upregulation of
IL-8, GRO-
α
, GRO-
γ
, or RANTES [
6
]. On the other hand, binding to CD46 and
α
v
β
3/5
integrins leads to Rac1, Pak1, and CtBP1 activation, which can interfere with the innate
immunity response [7].
The most common and best-described HAdV so far is human adenovirus type 5
(HAdV5), which is very efficient in terms of transduction efficacy; however, it is dis-
advantaged by high pre-existing immunity that may limit the efficacy of HAdV-based
vectors [
8
]. Thus, development of new strategies to evade undesired anti-vector–host
immune responses is needed, such as the use of vectors based on low seroprevalent human
adenoviruses type 26 (HAdV26) or 35 (HAdV35). HAdV26 has emerged as a promising
platform for vaccine vector development. Vaccine vectors based on HAdV26 are listed as
the intervention in more than 30 clinical trials, among which several are phase III. Very
recently, an important milestone in HAdV26-based vaccine vector research was reached
when the European Medicines Agency approved two vector vaccines based on HAdV26,
namely Ad26.ZEBOV against Ebola and Ad26.COV2.S against COVID-19 [
9
]. HAdV26-
based vaccine vectors have an acceptable safety profile in humans and are able to induce
neutralizing [
10
] and binding antibodies [
11
], CD4+ and CD8+ T cell responses [
12
], and
a Th1-biased immune response in animals and humans [
13
]. Vaccination with HAdV26
induced also substantially high levels of antiviral (IFN-
γ
, IP-10) and pro-inflammatory
(IL-1RA, IL-6) cytokines on day 1 following immunization [14].
While HAdV26 immunogenicity
in vivo
is rather well described and understood, the
basic biology of this virus is much less clear. To date, no reports are available describing
the host signaling activity induced by HAdV26 infection of epithelial cells. Likewise, there
are no studies characterizing the host response to HAdV26 in the context of receptor usage.
However, there is evidence that specific immunomodulatory factors can be released after
epithelial infection with adenoviruses and that these factors can be involved in virus-
induced inflammation [
15
]. Furthermore, in the context of vaccination, antigens expressed
by epithelial cells that receive AdV vector vaccine can be indirectly presented by antigen
presenting cells to CD4+ T cells or via cross-presentation to CD8+ T cells [
1
], indicating a
role for epithelial cells in vaccine efficacy. Therefore, in the current study, we determined the
pro-inflammatory cytokine profile of epithelial cells infected with HAdV26 and investigated
the underlying molecular mechanism. We found that HAdV26 infection of human epithelial
cells triggers the expression of pro-inflammatory cytokines, namely IL-6, IL-8, IL-1
β
, and
TNF-
α
. In order to investigate the possible mechanism underlying HAdV26 induced
inflammation, we further explored expression of IL-6 and the corresponding signaling
mechanism in relation to HAdV26 infection. By using
β
3 integrin-specific siRNA we
observed that the HAdV26-induced IL-6 gene expression is
α
v
β
3 integrin dependent.
We have also shown that IL-6 gene expression triggered by HAdV26 infection is NF-
κ
B
mediated. Our findings provide new data regarding pro-inflammatory cytokine expression
in HAdV26-infected epithelial cells, as well as details concerning the HAdV26-induced host
signaling pathways. Information obtained within this research allows better understanding
of HAdV26 basic biology and, as such, can contribute to further development of HAdV26-
based vaccine vectors.
Viruses 2022,14, 672 3 of 16
2. Materials and Methods
Cells, viruses, antibodies and chemicals. HEK293 (human embryonic kidney: ATCC
CRL-1573), A549 (human lung carcinoma: ATCC CCL-185), and SK-OV-3 (human ovar-
ian carcinoma: ATCC HTB-77) cells were obtained from ATCC Cell Biology Collection
and were cultured according to manufacturer’s instructions. Replication-incompetent
recombinant adenoviral vectors based on adenovirus type 5, 26, and 35 were previously
constructed [
16
,
17
]. Viruses were propagated on HEK293 cells and purified by a CsCl
gradient. Enhanced green fluorescent protein (eGFP) gene, encoded by adenoviral vector
genome and driven by the CMV promoter, served as a reporter gene. Fluorescent labelling
of adenoviruses with Alexa Fluor 488 was previously described [
18
]. Antibodies used for
western blot analyses were: p-p38 (Santa Cruz Biotechnology, sc-166182, Dallas, TX, USA);
p38 (Santa Cruz Biotechnology, sc-535), pERK (Santa Cruz Biotechnology, sc-7383), ERK
(Santa Cruz Biotechnology, sc-94), pJNK (Santa Cruz Biotechnology, sc-6254), JNK (Santa
Cruz Biotechnology, sc-7345), I
κ
B-
α
(Santa Cruz Biotechnology, sc-1643). For inhibiting
the nuclear factor-kappa B (NF-kB)-mediated signaling pathway cells were pretreated (1 h,
37
◦
C) with Bay 11-7082 (Sigma-Aldrich, Burlington, MA, USA) and infected with HAdV26
(10,000 vp/cell) in presence of Bay 11-7082. Final Bay 11-70082 concentrations used were
10 µM for SK-OV-3 and 20 µM for A549.
Confocal microscopy. Intracellular localization of HAdV26 in SK-OV-3 and A549 cells
was assessed by confocal microscopy. Cells were seeded in 24-well tissue culture plates
on coverslips (2
×
10
4
cells/well), and 48 h later incubated with Alexa Fluor 488 labelled
HAdV26 (10,000 vp/cell) for 1 h at 37
◦
C. Non-internalized viruses were removed by
washing the cells twice with PBS, and cells were fixed with 2% paraformaldehyde in PBS
for 12 min at room temperature. Next, cells were permeabilized (0.1% Triton X-100 in PBS
for 2 min at room temperature) and incubated with Alexa Fluor 555 Phalloidin (Thermo
Fischer Scientific, Waltham, MA, USA) for 15 min at room temperature. Coverslips were
mounted in Fluoromount G (Southern Biotech, Birmingham, AL, USA) containing DAPI for
nuclei staining. Leica TCS SP8 X inverted confocal microscope (Leica Microsystems, Wetzlar,
Germany) with 63x/1.40 oil-immersion objective was used for imaging. The images were
analyzed using LAS X (Leica Microsystems, Wetzlar, Germany) software and they show
maximum projections of confocal stacks. The qualification of HAdV internalization in cells
is presented as the number of virus particles per cell with mean and standard deviations.
The N number represents the number of cells analyzed in each sample.
Reverse transcription quantitative polymerase chain reaction (RT-qPCR). Gene expression of
pro-inflammatory cytokines was assessed by quantitative polymerase chain reaction (qPCR)
after reverse transcription (RT) using isolated total RNA. Cells were seeded in 6-well tissue
culture plates (3
×
10
5
cells/well), and 24 h later infected with HAdV
(10,000 vp/cell)
.
Cells were collected and total RNA was isolated using High Pure RNA Isolation kit (Roche,
Mannheim, Germany) 1 h, 3 h, or 6 h post infection (p.i.). RNA was reverse-transcribed
into first strand complementary DNA (cDNA) by a High-Capacity cDNA Reverse Tran-
scription kit (Applied Biosystems, Bedford, MA, USA). cDNA was analyzed by qPCR
on a StepOnePlus
TM
Real-Time PCR System (Thermo Fischer Scientific, Waltham, MA,
USA) using Sybr
®
Green (Applied Biosystems, Bedford, MA, USA). To determe the Ct
values, StepOne Software (v 2.3) was used (Applied Biosystems, Bedford, MA, USA). qPCR
conditions were: initial denaturation for 10 min at 95
◦
C, followed by 40 cycles of 15 s
at 95
◦
C and 1 min at the annealing temperature of 60
◦
C. Housekeeping gene used for
assessing
∆∆
Ct was GAPDH. Fold changes were calculated using the standard 2
−∆∆Ct
method [19]. PCR primers for analyzed genes are listed in Table 1.
Viruses 2022,14, 672 4 of 16
Table 1. Primer sequences for qPCR analysis of IL-6, IL-8, IL-1β, and TNF-αgene expression.
Gene ID Primer Sequences
IL-6 F: 50CAATGAGGAGACTTGCCTGG30
R: 50GCACAGCTCTGGCTTGTTCC30
IL-8 F: 50GTTTTTGAAGAGGGCTGAGAATTC30
R: 50ATGAAGTGTTGAAGTAGATTTGCTTG30
IL-1βF: 50TGGCAATGAGGATGACTTGTTC30
R: 50CTGTAGTGGTGGTCGGAGATT30
TNF-αF: 50AACCTCCTCTCTGCCATCAA30
R: 50GGAAGACCCCTCCCAGATAG30
Enzyme-linked immunosorbent assay (ELISA). Amounts of secreted cytokines were mea-
sured by ELISA. Cells were seeded in 6-well tissue culture plates (3
×
10
5
cells/well), and,
24 h later, infected with HAdV. Supernatants used for ELISA were collected at 6 h or 24 h
p.i. Human IL-6 Uncoated Elisa and Human IL-8 Uncoated Elisa (all Invitrogen, Waltham,
MA, USA) were used according to the manufacturer’s instructions.
Western blot. Activation of MAP kinases or NF-
κ
B was assessed by western blot. Cells
seeded in 6-well tissue culture plates (3
×
10
5
cells/well) were infected with HAdV and
at 1 h or 6 h p.i. lysed with Laemmli buffer (heated to 95
◦
C), scraped off the plate, soni-
cated, and boiled (95
◦
C) for 3 min. Proteins were separated using 10% polyacrylamide
gel by SDS-PAGE and transferred to nitrocellulose membrane (Amersham Protran, GE
Healthcare, Chicago, IL, USA). Membranes were blocked with 5% non-fat dry milk in
Tris-buffered saline containing 1% Tween-20 and probed with specific primary antibod-
ies. Following incubation, blots were visualized with appropriate anti-species adapted
horseradish peroxidase-conjugated IgG antibody. Detection was performed with Pierce
™
ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, USA), while
signals were detected using ChemiDoc
™
Imaging System (Bio-Rad, Hercules, CA, USA).
Densitometry was performed with ImageJ software (J52.A). Proteins were normalized to
the corresponding total protein level or total protein level stained with amido black. The
results were presented as relative expression or activation of proteins in HAdV infected
cells compared to non-infected cells (n.i.).
siRNA experiments. To downregulate
β
3 integrin we used two specific siRNA: Silencer
Select Predesigned siRNAs
β
3 integrin siRNA ID s 7581 (hereafter referred to as si(
β
3)_1)
from Thermo Fisher Scientific and Mission esiRNA human itgb3: EHU051661 from Merck
(hereafter referred to as si(
β
3)_2). Scrambled siRNA #1 (si(-)), catalog No. 4390844, was from
Thermo Fisher Scientific. Cells were transfected at a confluency of 70% using Lipofectamine
RNAiMAX reagent (Invitrogen, USA) according to the manufacturer’s protocol and used
for experiments at 48 h after transfection. Gene expression results assessed by qPCR
were presented as relative expression of investigated genes after HAdV infection in cells
transfected with si(β3) compared to cells transfected with si(-).
Flow cytometry. After
β
3 integrin downregulation, flow cytometry was used to analyze
the expression of
α
v
β
3 integrin. Cells were detached by trypsin and washed twice with
PBS; then, 3
×
10
5
cells/sample were incubated on ice for 1 h with anti-
α
v
β
3 integrin
antibody (MAB1976, LM609, Merck Milipore, Darmstadt, Germany) or isotype control
(IgG, M5284, Sigma-Aldrich, Burlington, MA, USA). Binding of the unlabeled primary
antibody was revealed by incubation with FITC-conjugated anti-mouse IG (554001, BD
Biosciences, Franklin Lakes, NJ, USA) as a secondary antibody on ice for 1 h. Analysis was
performed on FACSCalibur (BD Biosciences, Franklin Lakes, NJ, USA), and cell acquisition
was measured using BD CellQuest software package (BD Biosciences, Franklin Lakes, NJ,
USA). Data were analyzed using FCS Express 3 (De Novo Software, Pasadena, CA, USA).
Viruses 2022,14, 672 5 of 16
Statistical analysis. All experiments were performed at least three times in duplicate or
triplicate, except flow cytometry experiments, which were performed twice. All analyses
and graphs were created in GraphPad Prism (GraphPad Software Inc., San Diego, CA,
USA). Data were analyzed by unpaired Student’s t-test, and expressed as mean
±
standard
deviation (SD). * p< 0.05; ** p< 0.01; *** p< 0.001; **** p< 0.0001; ns = non-significant
(p> 0.05).
3. Results
3.1. HAdV26 Infection Triggers Expression of Pro-Inflammatory Cytokines in Human Epithelial
Cells In Vitro
Innate cytokine profiles induced
in vivo
by HAdV26 have been described in detail. It
has been demonstrated that in rhesus monkeys, HAdV35, HAdV26, and HAdV48, which
are vectors which do not use CAR as their primary receptor, trigger innate cytokine profiles
characterized by higher levels of antiviral and pro-inflammatory cytokines than those
triggered by HAdV5 vectors [
14
]. Recently, we reported that HAdV26 uses
α
v
β
3 integrin
as a receptor for infecting epithelial cells [
18
]. The gene expression of pro-inflammatory
cytokines induced by HAdV26 infection in epithelial cells has not been previously assessed;
therefore, we determined the gene expression of the most common adenovirus induced
pro-inflammatory cytokines [
20
] in A549 and SK-OV-3 cells infected with HAdV26, on the
basis that A549 and SK-OV-3 cells differ in expression of
α
v
β
3 integrin and consequently in
transduction efficiency. Namely, A549 cells have very low amounts of
α
v
β
3 integrin while
SK-OV-3 express
α
v
β
3 integrin, allowing better transduction efficiency with HAdV26 [
18
].
Of note, both of these cell lines exhibit epithelial characteristics. A549 is an adenocarcinomic
human alveolar basal epithelial cell line and SK-OV-3 is a human ovarian cancer cell line
with epithelial-like morphology.
In this study, we measured host gene expression in the early phase of HAdV26
infection, when the resulting signaling will be due only to HAdV26 detection by cellular
receptors. In order to determine the cellular uptake of HAdV26 in A549 and SK-OV-3
cells, we fluorescently labelled HAdV26 and observed its localization in studied cells at 1 h
p.i. (Figure 1). The average amount of HAdV26 particles per cell was 1.7-fold higher in
SK-OV-3 than in A549 cells, indicating that presence of
α
v
β
3 integrin increases HAdV26
cell uptake.
Viruses 2022,14, 672 6 of 16
Figure 1.
HAdV26 uptake in SK-OV-3 and A549 cells. Cells were incubated with Alexa Fluor 488-
labelled HAdV26 (10,000 vp/cell) for 1 h at 37
◦
C. Non-internalized viruses were rinsed away, and
the cells were fixed with 2% paraformaldehyde (PFA). Green, Alexa Fluor 488-labeled virus; blue,
nuclei stained with DAPI; red, actin cytoskeleton stained with phalloidin. The images are maximum
projections of confocal stacks. Representative confocal images are shown in the left panel. Scale
bars = 25 µm
. Quantification of virus internalization efficiency, expressed as virus number per cell, is
shown in the right panel. The horizontal bars represent means, and the error bars indicate standard
deviations; N represents the numbers of cells analyzed, n.i. denotes non-infected, *** p< 0.001.
We further examined expression of pro-inflammatory cytokines on gene expression
level after infection with HAdV26. As benchmark viruses, we used HAdV5 which is so
far the best studied adenovirus, and low-seroprevalent HAdV35, used as vaccine vector
candidate like HAdV26. As shown in Figure 2, the infection of A549 and SK-OV-3 with
HAdV26 induced changes in the expression of the IL-6, IL-8, IL-1
β
, and TNF-
α
genes. For
all studied genes, the most pronounced change was observed at 1 h p.i. The expression
of the IL-8, IL-1
β
, and TNF-
α
genes in HAdV26
−
infected cells is comparable to that in
HAdV5-infected cells. Infection with HAdV35 caused increased expression of IL-6, IL-1
β
,
TNF-
α
, and especially IL-8 in both A549 and SK-OV-3 cells. The most noticeable change in
gene expression after infection with HAdV26 was seen for IL-6. Infection with HAdV26
caused a 5.7-fold increase in IL-6 gene expression in A549 cells and a 16.7-fold increase in
SK-OV-3 cells at 1 h after infection in comparison to non-infected cells. In addition, the
expression of IL-6 after HAdV26 infection in A549 and SK-OV-3 cell lines was higher than
that induced by HAdV5 infection. These data indicate that HAdV26 infection of epithelial
cells triggers the gene expression of pro-inflammatory cytokines, especially IL-6. Thus, in
order to investigate the possible mechanism underlying HAdV26-induced inflammation
in the current study, we chose IL-6 as a representative inflammatory effector and further
explored its expression in relation to HAdV26 infection.
Viruses 2022,14, 672 7 of 16
Figure 2.
Relative gene expression of pro-inflammatory cytokines in human epithelial cells upon
infection with HAdV5, HAdV26, or HAdV35. A549 and SK-OV-3 cells were transduced with
10,000 vp/cell of virus for 1, 3, and 6 h. Relative gene expression was determined by RT-qPCR.
Data are shown as mRNA levels relative to the non-infected (n.i.) cells, plus standard deviations.
Representative data from three independent experiments yielding similar results are shown (n = 3).
*p< 0.05; ** p< 0.01; *** p< 0.001; **** p< 0.0001.
Viruses 2022,14, 672 8 of 16
3.2. HAdV26 Triggered IL-6 Gene Expression Is αvβ3 Integrin Dependent
Next, we wanted to examine the differences in the HAdV26-induced gene expression
of pro-inflammatory genes between SK-OV-3 and A549 cells in the context of
α
v
β
3 integrin.
As can be seen in Figure 3, HAdV26 infection in SK-OV-3 cells resulted in increased
expression of IL-6 and moderately increased expression of TNF-
α
compared to A549 cells.
Due to the higher amount of
α
v
β
3 integrin, HAdV26 enters (Figure 1) and infects [
18
] SK-
OV-3 cells more efficiently than A549, which led us to hypothesis that triggering expression
of IL-6 and TNF-
α
might be correlated to the amount of
α
v
β
3 integrin, i.e., the receptor of
HAdV26, known for mediating immune responses [21,22].
Figure 3.
Gene expression of pro-inflammatory cytokines in A549 and SK-OV-3 cells upon infection
with 10,000 vp/cell of HAdV26 for 1 h. Data correspond to those shown in Figure 2. * p< 0.05;
** p< 0.01.
To further investigate this assumption, we downregulated
α
v
β
3 integrin in SK-OV-
3 cells using specific siRNA for the
β
3 integrin subunit, and subsequently determined
the gene expression of IL-6, IL-8 and TNF-
α
following HAdV26 infection. We used two
β
3 integrin-specific siRNA, si(
β
3)_1 and si(
β
3)_2. Both these integrin-specific siRNAs
decreased the expression of
α
v
β
3 integrin on the surface of SK-OV-3 cells (Figure 4A,B).
Downregulating
α
v
β
3 integrin using si(
β
3)_1 decreased the HAdV26-induced expression
of the IL-6 gene, but had no influence on the expression of IL-8 or TNF-
α
(Figure 4C,E,G).
Downregulating
α
v
β
3 integrin using si(
β
3)_2 decreased the HAdV26-induced expression
of all three cytokines, namely IL-6, IL-8, and TNF-
α
(Figure 4D,F,H). Compared to si(
β
3)_1,
si(
β
3)_2 is composed of a heterogeneous pool of siRNA that all target the same mRNA
sequence; thus, the effect is more pronounced both on the level of
α
v
β
3 integrin expression
and the expression of the studied cytokines. Data obtained here indicate that the difference
in the HAdV26-induced IL-6 profile occurs as the result of enhanced virus uptake in
SK-OV-3 cells due to the higher amount of αvβ3 integrin on the cell surface.
Viruses 2022,14, 672 9 of 16
Figure 4.
Relative gene expression of IL-6, IL-8, and TNF-
α
upon infection with HAdV5, HAdV26,
or HAdV35 in SK-OV-3 cells with decreased expression of
α
v
β
3 integrin. (
A
,
B
) Expression of
α
v
β
3 integrin in SK-OV-3 cells after downregulating expression of
β
3 integrin using si(
β
3)_1 and
si(
β
3)_2, respectively. Thin-line histograms represent isotype controls (IgG) and filled histograms the
expression of
α
v
β
3 integrin. Representative data from two independent experiments yielding similar
results are shown (n = 2). (
C
,
E
,
G
) Relative gene expression of IL-6, IL-8, and TNF-
α
upon infection
with HAdV5, HAdV26, or HAdV35 in SK-OV-3 cells transfected with si(
β
3)_1.
(D,F,H) Relative
gene
expression of IL-6, IL-8, and TNF-
α
upon infection with HAdV5, HadV26, or HAdV35 in SK-OV-3
cells transfected with si(
β
3)_2. Cells transfected with si(
β
3)_1 or si(
β
3)_2 were transduced with
10,000 vp/cell of virus for 1 h. Relative gene expression was determined by RT-qPCR. Data are
shown as mRNA levels relative to the cells transfected with scrambled siRNA (si(-)), plus standard
deviations. Representative data from three independent experiments yielding similar results are
shown (n = 3). * p< 0.05; ** p< 0.01; *** p< 0.001; **** p< 0.0001; ns = non-significant (p> 0.05).
Viruses 2022,14, 672 10 of 16
3.3. HAdV26-Induced IL-6 Gene Expression Is NF-κB Mediated
The early host response to adenovirus vectors includes activation of MAP kinases
and NF-
κ
B signaling pathways [
23
]. To examine which signaling pathway is affected after
HAdV26 infection, we determined the activation status of the following representative
MAP kinases: the extracellular signal-regulated kinases (ERK), the p38 kinases (p38), and
the c-Jun NH2-terminal kinases (JNK). In addition, the activation of NF-
κ
B, known to be
involved in regulation of cytokine expression, was measured by determining the amount
of I
κ
B
α
, a key factor in NF-
κ
B activation, where signal-induced degradation of I
κ
B
α
is the
effect of NF-
κ
B activation. HAdV-induced host signaling was assessed 1 h and 6 h p.i. The
obtained data are shown in Figure 5and Supplementary Figure S1. We observed a decrease
in the activation of ERK in A549 cells at 1 h p.i. with HAdV26. A decrease in ERK activation
in SK-OV-3 cells was observed at a later time point, namely 6 h p.i. Infection with HAdV26
had no influence on p38 and JNK activation in A549 cells, but increased p38 activation at 1 h
p.i. and decreased JNK activation 6 h p.i. in SK-OV-3 cells. Infection with HAdV26 caused
a significant decrease in the amount of I
κ
B
α
. reflecting the activation of NF-
κ
B signaling.
The decrease in I
κ
B
α
amount in A549 cells was present only at 1 h p.i., but persisted until
6 h p.i. in SK-OV-3 cells. HAdV5 and HAdV35 have a similar pattern in activation status of
MAP kinases compared to HAdV26; HAdV35 ERK activation was decreased within 1 h
p.i., while for HAdV5 and HAdV26 it decreased only after 6 h p.i. I
κ
B
α
expression was
less reduced after HAdV5 infection, indicating decreased NF-
κ
B activation compared to
infection with HAdV26 and HAdV35.
Figure 5.
Western blot and densitometric analysis of MAP kinases and NF-
κ
B activation status
in HAdV5-, HAdV26-, or HAdV35-infected A549 and SK-OV-3 cells. Cells were transduced with
10,000 vp/cell of virus for 1 h or 6 h. Relative protein amount was determined by western blot.
Densitometric analysis is shown between the corresponding protein lines. Proteins were normalized
to the corresponding total protein level or total protein level stained with amido black. The results
were presented as relative expression or activation of proteins in HAdV-infected cells compared to
non-infected cells (n.i.). Amido black staining was also used as control for gel loading. Representative
data from three independent experiments yielding similar results are shown (n = 3).
It is known from the literature that NF-
κ
B mediates the induction of pro-inflammatory
cytokines such as TNF-
α
and IL-6 [
24
]. In order to examine if NF-
κ
B signaling is involved
in the HAdV26-induced expression of pro-inflammatory cytokines IL-6, IL-8, and TNF-
α
,
Viruses 2022,14, 672 11 of 16
we infected A549 and SK-OV-3 cells with HAdV26 in the presence of an NF-
κ
B inhibitor,
Bay 11-7082 [
25
]. As shown in Figure 6, inhibiting the NF-
κ
B signaling pathway decreased
the HAdV26-induced expression of the IL-6 gene by more than 50% in A549 cells and by
more than 30% in SK-OV-3 cells. The expression of the IL-8 gene was increased by 50%
in both cell lines, while the expression of the TNF-
α
gene was not affected by the NF-
κ
B
inhibitor. Our data suggest that the HAdV26 infection in epithelial cells could increase IL-6
expression through the NF-kB signaling pathway.
Figure 6.
Relative gene expression of IL-6, IL-8, and TNF-
α
upon infection with HAdV26 in A549
and SK-OV-3 cells treated with NF-
κ
B inhibitor, Bay 11-7082 (10
µ
M for SK-OV-3, 20
µ
M for A549).
Cells were transduced with 10,000 vp/cell of virus for 1 h. Relative gene expression was determined
by qPCR. Data are shown as mRNA levels relative to the cells transfected with scrambled siRNA,
plus standard deviations. Representative data from three independent experiments yielding similar
results are shown (n = 3). * p< 0.05; *** p< 0.001; ns = non-significant (p> 0.05).
3.4. HAdV26-Induced Production of IL-6 Is αvβ3 Integrin Dependent
Since we observed increased expression of several pro-inflamatory cytokine genes
after infecting epithelial cells with HAdV26, we then wanted to examine if this increased
gene expression resulted in increased secretion of the corresponding protein. The amount
of secreted IL-6 and IL-8 protein was measured at 6 h and 24 h p.i. in A549 and SK-OV-3
cells. As can be seen in Figure 7, a modest increase in IL-6 protein was observed at 6 h
p.i. in SK-OV-3 cells and at 24 h p.i. in A549 cells. A meagre increase in IL-8 protein was
observed only in SK-OV-3 cells at 6 h p.i. Of note, infecting A549 and SK-OV-3 cells with
HAdV35 resulted in significantly increased amounts of IL-6 and IL-8 at both time points
p.i. compared to non-infected cells (data not shown). Even though the increase in the
IL-6 protein in HAdV26-infected SK-OV-3 cells at 6 h p.i. is moderate, it can be correlated
with a higher amount of
α
v
β
3 integrin in these cells, since we did not observe the same in
A549 cells.
Viruses 2022,14, 672 12 of 16
Figure 7.
Amount of IL-6 and IL-8 secreted from A549 and SK-OV-3 cells upon infection with
10,000 vp/cell of HAdV26 for 6 h and 24 h. Amount of secreted cytokines was determined by ELISA
assay. Data are shown as total amount of secreted protein, plus standard deviations. Representative
data from three independent experiments yielding similar results are shown (n = 3). * p< 0.05.
In order to confirm the role of
α
v
β
3 integrin in the production of IL-6 and IL-8 after
infection with HAdV26 we measured the amounts of those cytokines at 6 h and 24 h
p.i. in SK-OV-3 cells transfected with
β
3 integrin-specific siRNA, si(
β
3)_2. The amount
of secreted IL-6 in HAdV26-infected SK-OV-3 cells with decreased expression of
α
v
β
3
integrin was significantly lower than that in the control cells. At 6 h p.i., the amount of
IL-6 secreted 6 was 3.1 times lower in the cells with decreased expression of
α
v
β
3 integrin
compared to cells transfected with si(-), and 1.4 times lower at 24 h p.i. The amount of IL-8
secreted in HAdV26-infected SK-OV-3 cells with decreased expression of
α
v
β
3 integrin
was comparable to the amount secreted from the control cells (Figure 8).
Figure 8.
Amount of IL-6 and IL-8 secreted from SK-OV-3 cells with decreased expression of
α
v
β
3
integrin upon infection with 10,000 vp/cell of HAdV26 for 6 h and 24 h. Amount of secreted cytokines
was determined by ELISA assay. Data are shown as fold change of total amount of secreted protein
in the non-infected (n.i.) cells, plus standard deviations. Representative data from three independent
experiments yielding similar results are shown (n = 2).
4. Discussion
Cell types that can receive AdV vector following intramuscular vaccination include
antigen presenting cells, dendritic cells, muscle cells, fibroblasts, and epithelial cells [
26
].
Viruses 2022,14, 672 13 of 16
The antigen encoded by the AdV-based vaccine expressed by infected muscle or epithelial
cell can be presented by antigen presenting cell to CD4+ T cells or via cross-presentation to
CD8+ T cells [
1
], thus influencing vaccine efficacy. Host immune system can detect aden-
ovirus capsid, DNA, or infection itself during almost all steps of the adenovirus infection
pathway, from binding and endocytosis to intracellular trafficking. The initial phase of
infection, receptor binding and cell uptake, can trigger the expression of pro-inflammatory
cytokines that can subsequently influence all cells in the surroundings. Epithelial cells
possess pattern recognition receptors that enable them to sense and respond to incoming
pathogens, thus triggering pro-inflammatory signals soon after virus interactions. It has
been reported that the co-culture of mouse lung epithelial cells and macrophages synergis-
tically augmented its inflammatory responses to AdV infection compared to the epithelial
cells or macrophages alone, indicating that the interactions between these two cell types are
critical for the AdV-induced innate immune response [
27
]. Anti-vector adaptive immune
response, i.e., neutralizing antibodies against vector itself, may limit the efficacy of a viral
vector; hence, the potential role of epithelial cells in initiating the innate immune response
to adenoviral vectors deserves careful investigation [28].
In this study, we investigated expression of pro-inflammatory cytokine genes in
epithelial cells infected with HAdV26 and found that HAdV26 infection of human epithelial
cells triggers the expression of pro-inflammatory cytokines, namely IL-6, IL-8, IL-1
β
,
and TNF-
α
. By further exploring the mechanism responsible for HAdV26-induced IL-
6 expression, we observed that it was
α
v
β
3 integrin dependent and NF-
κ
B mediated.
To the best of our knowledge, this is the only study describing early host signaling in
HAdV26-infected cells.
Here, we determined the expression of pro-inflammatory genes in A549 and SK-OV-3
cells infected by HAdV26. The infection of A549 and SK-OV-3 with HAdV26 induced
changes in the expression of the IL-6, IL-8, IL-1
β
, and TNF-
α
genes; namely the expression
of all four genes was increased in HAdV26-infected A549 cells, while HAdV26 infection
resulted in increased expression of IL-6 and TNF-
α
in SK-OV-3 cells. The most pronounced
increase after infection with HAdV26 was observed for IL-6, whose expression was in-
creased 6 times in A549 cells and more than 15 times in SK-OV-3 cells. Our results are in line
with those from the literature, where the increased expression of IL-6 was reported in BEAS-
2B cells, a human bronchial epithelial cell line, after infection with HAdV3 and HAdV7 [
29
]
,
and in primary human bronchial epithelial cells infected with HAdV-B14p1 [
30
]. With
regard to the expression of IL-8, increased expression of this cytokine was reported in A549
cells infected with HAdV7 [
31
,
32
] and human corneal fibroblasts [
33
] and keratinocytes [
34
]
infected with HAdV19. It is necessary to emphasize that the internalization of HAdV26
is significantly decreased compared to that of HAdV5 and HAdV35 in both A549 and
SK-OV-3 cells [
18
], but that it results in greater expression of pro-inflammatory genes than
HAdV5 and comparable expression to HAdV35.
Previously, we demonstrated that due to higher expression of
α
v
β
3 integrin, HAdV26
infects SK-OV-3 cells more efficiently than A549 cells [
18
]. Here, we presented that HAdV26
cell uptake is also increased in cells with higher expression of
α
v
β
3 integrin; thus, we
hypothesized that there might also be differences in the HAdV26-induced innate immune
response between these two cell lines. We compared the expression of IL-6, IL-8, IL-1
β
,
and TNF-
α
between HAdV26-infected A549 and SK-OV-3 cells and observed the biggest
difference in the expression of IL-6, for which we saw a three-fold increase in expression
in SK-OV-3 cells compared to A549 cells. In order to investigate role of this receptor
in the HAdV26-induced expression of IL-6 and TNF-
α
, we downregulated the amount
of
α
v
β
3 integrin by using specific siRNA in SK-OV-3 cells prior to HAdV26 infection.
Downregulating
α
v
β
3 integrin decreased the HAdV26-induced expression of IL-6, IL-8,
and TNF-
α
genes, but had only a significant influence only on the production of IL-6.
Namely, the decreased expression of
α
v
β
3 integrin caused a decrease in the secretion of
IL-6 after infection with HAdV26. The role of integrins in mediating the HAdV-induced
production of pro-inflammatory cytokines and chemokines was studied in macrophages,
Viruses 2022,14, 672 14 of 16
where it was shown that in response to HAdV5, macrophage-derived IL-1a triggered
the IL-1RI-dependent production of a defined set of pro-inflammatory cytokines and
chemokines. The IL-1a-mediated response required the selective interaction of virus RGD
motifs with macrophage
β
3 integrins [
35
]. Comparable results were obtained
in vivo
by
using a model of integrin
β
3
−
/
−
mice in which it was shown that cytokine induction by
oncolytic adenovirus was
β
3 integrin dependent. Namely, adenovirus-induced cytokines
(IL-1
β
, IL-6, and TNF-
α
) were significantly higher in the circulation of WT compared with
β
3
−
/
−
animals [
36
]. The latter data regarding IL-6 are in good agreement with our results
indicating that HAdV-induced IL-6 expression and production are β3 integrin dependent
both in epithelial cells
in vitro
and in animal models. Besides HAdV,
α
v
β
3-integrin is
involved in recognizing another DNA virus, herpes simplex virus-1, i.e.,
α
v
β
3-integrin
has been reported as a major sensor and activator of innate immunity to herpes simplex
virus-1 [21].
Adenovirus infection can activate host cell signaling, which most commonly involves
the MAP kinase pathway. Here, we explored if HAdV26 infection triggers early events in
the signal transduction pathways that induce the synthesis of cytokines and thus contribute
to the inflammatory response. We therefore determined activation status of ERK, p38, and
JNK in epithelial cells following infection with HAdV26. We saw that HAdV26 infection
decreased ERK phosphorylation but had no or minor influence on the activation of p38
or JNK. However, we did observe increased activation of NF-
κ
B, which was also required
for increased expression of the IL-6 gene. The role of MAP kinases in pro-inflammatory
cytokine induction due to the adenovirus infection was reported for different HAdV
serotypes. It has been shown that the Ras/Raf/MEK/ERK pathway is necessary for the
induction of IL-8 by HAdV7 [
32
], while this same virus triggers the expression of IL-6 in
human airway epithelial cells via the p38/NF-
κ
B signaling pathway [
37
]
,
which was in
line with our results. NF-
κ
B activation was equally important for the expression of the
chemokine CXCL10 induced by HAdV5 infection in kidney epithelium-derived cells [
38
].
Interestingly, NF-
κ
B was also found to mediate the leaky expression of adenovirus genes
from the HAdV5 vector genome and the inhibition of NF-
κ
B leads to the suppression of
HAdV5 gene expression and hepatotoxicity following transduction with HAdV5-based
vectors [
39
]. In this study, NF-
κ
B inhibition decreased HAdV26-induced IL-6 expression,
which could potentially correlate to decreased toxicity. Correlation between NF-
κ
B and
IL-6 was also reported in the case of Kaposi’s sarcoma herpesvirus infection. MicroRNA
miR-K12-1, encoded by this virus, activates transcription factor STAT3 indirectly through
inducing NF-
κ
B activation and NF-
κ
B-dependent expression of IL-6 by repressing the
expression of the NF-
κ
B inhibitor I
κ
B
α
[
40
]. Similarly, activation of the transcription factor
NF-
κ
B, involving the small GTPase Rac1, was required for IL-6 production and subsequent
STAT3 activation in human papillomavirus infection [
41
], indicating that link between
NF-κB and IL-6 might be a common feature of DNA viruses.
5. Conclusions
Even though vaccine vectors can benefit from the activation of host innate immune re-
sponse, undesired inflammation can limit their efficacy. Thus, it is important to understand
the host response to HAdV vectors in order to further optimize and customize vaccine
vector design aimed at specific diseases. Our findings presented in this study may provide a
mechanistic link between HAdV26–
α
v
β
3 integrin interactions and the activation of the NF-
κ
B signaling pathway that triggers IL-6 expression, i.e., innate immune and inflammatory
responses, and represents an expansion of the current knowledge of HAdV26 biology.
Supplementary Materials:
The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/v14040672/s1, Figure S1: Quantification of the protein bands
obtained by densitometric analysis from at least three independent Western blot experiments. Data
are presented as means
±
SD compared to non-infected cells. * p< 0.05; ** p< 0.01; *** p< 0.001;
**** p< 0.0001; ns = non-significant (p> 0.05).
Viruses 2022,14, 672 15 of 16
Author Contributions:
Conceptualization, D.N. and D.M.; methodology, D.N., K.B., I.D., A.K.,
J.v.d.B., J.K., J.C., A.A.-R.; writing—original draft preparation, D.M.; writing—review and editing,
D.N., K.B., I.D., J.K., J.C., A.A.-R.; supervision, D.M.; project administration, D.M.; funding acquisition,
D.M. All authors have read and agreed to the published version of the manuscript.
Funding:
This research was funded by the Croatian Science Foundation, grant number IP-2019-04-6048.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article.
Acknowledgments: The authors would like to thank Marina Šutalo for her technical assistance.
Conflicts of Interest: The authors declare no conflict of interest.
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