Nanoparticle exposure reactivates latent herpesvirus and restores a signature of acute infection
Sattler et al. Particle and Fibre Toxicology
Nanoparticle exposure reactivates latent herpesvirus and restores a signature of acute infection
Christine Sattler 1 4
Franco Moritz 3
Shanze Chen 1 4
Beatrix Steer 0 2 7 8
David Kutschke 1 4
Martin Irmler 6
Johannes Beckers 5 6 9
Oliver Eickelberg 1 4
Philippe Schmitt-Kopplin 3
Heiko Adler 0 2 7 8
Tobias Stoeger 1 4
0 Comprehensive Pneumology Center, Research Unit Lung Repair and Regeneration, Helmholtz Zentrum München - German Research Center for Environmental Health (GmbH) , Marchioninistrasse 25, D-81377 Munich , Germany
1 Helmholtz Zentrum München - German Research Center for Environmental Health (GmbH), Comprehensive Pneumology Center, Institute of Lung Biology and Disease, Ingolstädter Landstr. 1, D-85764 Neuherberg , Germany
2 Comprehensive Pneumology Center, Research Unit Lung Repair and Regeneration, Helmholtz Zentrum München - German Research Center for Environmental Health (GmbH) , Marchioninistrasse 25, D-81377 Munich , Germany
3 Helmholtz Zentrum München - German Research Center for Environmental Health (GmbH), Research Unit BioGeoChemistry, Ingolstädter Landstr. 1, D-85764 Neuherberg , Germany
4 Helmholtz Zentrum München - German Research Center for Environmental Health (GmbH), Comprehensive Pneumology Center, Institute of Lung Biology and Disease, Ingolstädter Landstr. 1, D-85764 Neuherberg , Germany
5 German Center for Diabetes Research (DZD), Ingolstädter Landstr. 1, D-85764 Neuherberg , Germany
6 Helmholtz Zentrum München - German Research Center for Environmental Health (GmbH), Institute of Experimental Genetics, Ingolstädter Landstr. 1, D-85764 Neuherberg , Germany
7 Comprehensive Pneumology Center, Member of the German Center of Lung Research (DZL) , D-81377 Munich , Germany
8 University Hospital Grosshadern, Ludwig-Maximilians-University , D-81377 Munich , Germany
9 Technische Universität München, Chair of Experimental Genetics , D-85354 Freising , Germany
Background: Inhalation of environmental (nano) particles (NP) as well as persistent herpesvirus-infection are potentially associated with chronic lung disease and as both are omnipresent in human society a coincidence of these two factors is highly likely. We hypothesized that NP-exposure of persistently herpesvirus-infected cells as a second hit might disrupt immune control of viral latency, provoke reactivation of latent virus and eventually lead to an inflammatory response and tissue damage. Results: To test this hypothesis, we applied different NP to cells or mice latently infected with murine gammaherpesvirus 68 (MHV-68) which provides a small animal model for the study of gammaherpesvirus-pathogenesis in vitro and in vivo. In vitro, NP-exposure induced expression of the typically lytic viral gene ORF50 and production of lytic virus. In vivo, lytic viral proteins in the lung increased after intratracheal instillation with NP and elevated expression of the viral gene ORF50 could be detected in cells from bronchoalveolar lavage. Gene expression and metabolome analysis of whole lung tissue revealed patterns with striking similarities to acute infection. Likewise, NP-exposure of human cells latently infected with Epstein-Barr-Virus also induced virus production. Conclusions: Our results indicate that NP-exposure of persistently herpesvirus-infected cells - murine or human - restores molecular signatures found in acute virus infection, boosts production of lytic viral proteins, and induces an inflammatory response in the lung - a combination which might finally result in tissue damage and pathological alterations.
Carbonaceous nanoparticles (CNP); Double-walled carbon nanotubes (DWCNT); Intratracheal instillation; Persistent virus infection; Virus reactivation; Phospholipids
The rapid expansion of nanotechnology is expected to
bring considerable benefit to mankind, but at the same
time, newly developed materials might pose new and
unknown risks to exposed people. Inhalation of high levels
of spherical carbonaceous nanoparticles (CNP) –
surrogates for combustion derived nanoparticles – has
been shown to induce an inflammatory phenotype in the
lungs of healthy mice  as well as acute extra-pulmonary
cardiovascular distress . Comparing a panel of different
CNPs revealed particle surface related oxidative stress to
be the common driver of the acute response to particles
of low solubility and low toxicity [3, 4]. At moderate
doses, CNP-triggered acute inflammation has been shown
to resolve within several days after exposure in
noncompromised mice . Yet, repeated inflammatory events or
exposure of individuals with higher susceptibility to
NPassociated adverse effects, such as asthmatics , might
provoke severe damage to the lung tissue. Recent research
indicates that at equal surface dose, fiber shaped carbon
nanotubes (CNT) – which due to their rapidly increasing
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
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mass production gain increasingly more environmental
importance – also generate an acute inflammatory
response via oxidative stress pathways, but in contrast to
spherical particles, the CNT-induced pulmonary
inflammation persists over weeks . Inhaled nanoparticles
generally deposit efficiently and persistent in the respiratory
tract, and may due to their pro-inflammatory potency
represent one environmental factor contributing to the
development of lung diseases including asthma, chronic
obstructive lung disease (COPD) and potentially even
interstitial pulmonary fibrosis or cancer . An additional
environmental factor driving the susceptibility for chronic
lung disease could be virus infection. A number of studies
imply that especially herpesviruses might contribute to
the development of lung diseases such as idiopathic
pulmonary fibrosis (IPF). In lungs of patients affected from
IPF, DNA of one, two or even more herpesviruses has
been detected by PCR, suggesting an association between
chronic virus infection and IPF [9, 10]. In particular,
proteins and DNA from Human Cytomegalovirus (HCMV)
and Epstein-Barr-Virus (EBV) have frequently been
detected. Due to the species-specificity of HCMV and EBV,
pathogenic studies of the human infections are restricted.
Thus, animal models are needed and the murine
gammaherpesvirus 68 (MHV-68) provides such an animal model
[11, 12]. MHV-68 has been shown to act as a cofactor in
bleomycin-induced fibrosis [13, 14] and to exacerbate
fluorescein isothiocyanate-induced pulmonary fibrosis [14, 15].
Furthermore, infection of Th2-biased mice with MHV-68
induces the development of progressive lung fibrosis with
pathological features also seen in IPF . Upregulation of
profibrotic or proinflammatory factors in infected cells and
repeated virus reactivation followed by lytic replication
events are supposed to be important factors in the
development or exacerbation of IPF in these models [17, 18].
Control of viral latency and prevention of productive virus
replication depends on a highly complex balance between
immune surveillance and regulation of viral and cellular
gene expression [12, 19, 20]. Both the immune response
and the metabolism are important players in surveillance of
viral latency and regulation of immune responses within
this context. Viruses have been shown to alter metabolic
pathways of their host cells in a highly specific manner to
generate optimal conditions not only for virus replication
and production of new virus particles but also for
maintenance of viral latency [21, 22]. Simultaneously, immune
responses and metabolism are increasingly considered to be
closely linked, e.g. by sharing pathways and being
crossregulated [23, 24], suggesting a well-established balance
between metabolic alterations caused by the virus and
modifications due to counteractions by the host. We
hypothesize that inhalation of NP as a second hit disrupts
this balance and interferes with the ability to control viral
infection, which might finally result in a non-resolving,
chronic inflammation and even fibrosis. At present, no
information about the health relevance of a suchlike liaison
of NP and persistent virus infection is available.
In this study, we show that the presence of NP induces
the production of lytic virus from persistently infected
murine cells in vitro. In vivo, exposure to NP by
intratracheal instillation leads to an increase in the
expression of lytic viral proteins in lungs of mice persistently
infected with MHV-68 and creates transcriptome and
metabolome signatures in the lung with considerable
parallels to the ones observed during the acute phase of
virus infection. NP exposure of human cells that are
latently infected with EBV also induces reactivation of latent
virus, indicating that the NP-effect is not limited to the
murine system. Taken together, our results suggest that
the combination of persistent herpesvirus infection and
NP exposure disrupts the immune control of viral latency
by altering cellular metabolism and gene expression.
NP exposure in vitro boosts lytic virus replication in
pseudo-latently infected cells
MHV-68 such as all herpesviruses is characterized by its
complex life cycle consisting of an acute lytic infection
and a lifelong maintained latent infection. In this study,
we wanted to analyze the influence of NP on both
phases of the viral life cycle. To investigate lytic virus
growth after primary infection in the presence or
absence of NP, we performed multistep growth curves in
murine alveolar epithelial cells (LA-4 cell line) and
alveolar macrophages (MH-S cell line). Alveolar
macrophages act as the first defense against inhaled NP 
whereas alveolar epithelial cells create the large
respiratory surface area of the lung and are known to support
acute lytic virus replication after intranasal infection
. As stimulating carbonaceous NP, we used Printex
90 (CNP) which represents a widely used and well
characterized type of carbon black with well described effects
on inflammatory and oxidative stress related processes
[26, 27]. We used doses of 50 μg/ml as aqueous
dispersions with an averaged agglomerate diameter (Z-Ave) in
medium of 337 ± 5 nm (see also Additional file 1: Fig.
S1), which did not reduce cell viability to less than 80%
on average neither in MH-S nor in LA-4 cells, as tested
by WST-assay (Additional file 1: Fig. S2a and b). Both
cell lines supported lytic virus growth and virus titers
increased by 3 (MH-S cells) to 5 logs (LA-4 cells) over a
period of 96 h (Fig. 1a and b). However, no changes in
virus titers in the presence of CNP could be observed.
Therefore, we suppose that CNP do not affect lytic virus
growth in de novo infected cells. In the next step, we
established a cell culture assay to detect low-level lytic
virus replication in pseudo-latently infected cells by a
slight modification of a previously published method
Fig. 1 Exposure to NP does not alter lytic virus replication after primary infection in vitro but boosts lytic replication in persistently infected cells: The
alveolar macrophage cell line MH-S a or the alveolar epithelial cell line LA-4 b were infected for 2 h with MHV-68 wildtype virus at an MOI of 1 and then
treated with 50 μg/ml CNP. Cells and cell culture supernatants were harvested at different time points post infection, and titers were determined by plaque
assay on BHK-21 cells. Data shown are the means ± SD from two independent experiments. c and d: MH-S cells were infected with MHV-68 overnight,
treated with 50 μg/ml CNP c or DWCNT d and incubated for another two hours. Free lytic virus was inactivated by incubation with citrate buffer (pH =
3.0). After plating of serial cell dilutions on indicator cells, the amount of cytopathic effect (CPE) was determined. Relative values, normalized to the genomic
load in the infected cells, were calculated. The value for untreated cells was set as “1”. Symbols represent values from individual experiments and the bars
represent the mean. Asterisks indicate a statistically significant difference to the untreated control (*: P < 0.05)
 to evaluate if CNP can boost low-level lytic virus
replication, an event that can be observed in latently
infected individuals at regular intervals . As
macrophages have been shown to harbor latent MHV-68 ,
we again used MH-S cells as target cells in these
experiments. MH-S cells were first infected with MHV-68 at a
low multiplicity of infection (MOI = 0.01) and then
treated with NP. Remaining extracellular lytic virus was
inactivated and serial dilutions of cells were plated on
indicator cells to study the development of a cytopathic
effect (CPE). Control assays performed with cells that
were mechanically disrupted by two freeze-thaw cycles
before plating yielded an insignificant amount of CPE,
indicating that the lytic virus detected in this assay was
actively produced by living MH-S cells and not due to
remaining input virus. Exposure of infected cells to CNP
increased the amount of lytic virus in three out of four
experiments (Fig. 1c). To test if this observation was
specific for the type of carbonaceous NP, we also
analyzed the effect of fiber-shaped double-walled carbon
nanotubes (DWCNT). Addition of DWCNT dispersions
(Z-Average of 26 ± 1 nm; see also Additional file 1: Fig.
S1) to the cells, at a dose of 50 μg/ml, induced
significantly higher amounts of lytic virus when compared to
untreated controls (Fig. 1d). The used concentration of
DWCNT showed some impact on cell survival and
reduced cell viability by roughly 30% (Additional file 1:
Fig. S2b). Our results demonstrate that exposure to NP
boosts the release of lytic virus from pseudo-latently
infected cells irrespective of the type of NP.
NP exposure of persistently infected cells reactivates
Since the described system used with MH-S cells is not
a true “latency-model”, we performed additional
experiments with two latently infected cell lines to investigate
if NP-exposure can actually reactivate latent virus. First,
the B cell line S11 was used, which has been isolated
from the spleen of a mouse with MHV-68-associated
lymphoproliferative disease . Second, we established
a persistently infected macrophage cell line by infecting
the bone marrow-derived cell line ANA-1 with a
recombinant virus carrying a hygromycin-resistance as
described in materials and methods (= ANA-1/MHV-68).
The establishment of a latently infected MH-S cell line
by this method was technically not feasible, probably
due to the fact that MH-S cells support lytic virus
replication to a too high extent (see also results in Fig. 1a).
We incubated both cell lines with three different doses
of NP (5 μg/ml, 20 μg/ml and 50 μg/ml) to determine
the optimal dose. Lytic virus was determined in the
supernatant by plaque assay after 72 h (Additional file 1:
Fig. S3a and b). Treatment with
12-O-tetradecanoylphorbol-13-acetate (TPA) was used as a positive control
for the induction of virus reactivation in S11 B cells ,
and treatment with lipopolysaccharide (LPS) in ANA-1/
MHV-68 macrophages . Exposure to 50 μg/ml NP
resulted in an increase in virus titers in the supernatants
of both cell lines, indicating virus reactivation. At this
dose, both NP showed a slight effect on cell survival and
reduced viability by 11% (CNP) or 29% (DWCNT) in
uninfected ANA-1 cells (Additional file 1: Fig. S2c). A
dose of 50 μg/ml NP was used in the following
experiments. S11 and ANA-1/MHV-68 cells were exposed to
NP for 72 h and virus titers in the supernatant were
determined (Fig. 2a and b). An increase of virus titers
could be observed after exposure to both CNP and
DWCNT, but particularly in ANA-1/MHV-68 cells
DWCNT had a bigger impact than CNP. As DWCNT
have a two times higher surface area compared to CNP,
we also tested a surface-adapted amount of CNP in
further experiments (100 μg/ml), but the effect of
DWCNT was still higher than the one induced by CNP
(Additional file 1: Fig. S4). We further analyzed the
expression of the viral genes ORF50 and ORF73 72 h after
Fig. 2 Exposure to NP reactivates latent virus in persistently infected cells in vitro: The S11 B-cell line and the persistently with MHV-68 infected
macrophage cell line ANA-1/MHV-68 were incubated with 50 μg/ml NP and lytic virus was determined in the supernatant by plaque assay after
72 h (panels a and b). Expression of the viral genes ORF50 (specific for the lytic phase) and ORF73 (expressed during lytic and latent phase) –
shown as the ratio ORF50/ORF73 - (panels c and d) and of genes that have been shown to be associated with reactivation of latent virus (panels
e and f) was analyzed by RT-PCR 72 h after NP exposure in S11 and in ANA-1/MHV-68 cells. The values in untreated cells were set as “1” and the
values for cells after NP treatment were calculated relative to the control. Treatment with TPA or LPS, respectively, was used as a positive control.
Data shown are the means + SD from three (S11 cells; panels a, c, e) or four independent experiments (ANA-1/MHV-68 cells; panels b, d, f).
Asterisks indicate a statistically significant difference to the untreated control (*: P < 0.05; **: P < 0.01)
NP exposure by RT-PCR (Fig. 2c and d and Additional
file 1: Fig. S2c and d). Expression of ORF73 served for
normalization since it is expressed throughout all phases
of the viral life cycle . ORF50, also referred to as the
replication and transcription activator Rta, is exclusively
expressed during lytic virus replication . Thus, an
increase in the relative ratio of ORF50/ORF73 indicates
that lytic virus production is induced. Consistent with
the finding of higher virus titers in the supernatants of
cells after NP-exposure, the relative ratio of ORF50/
ORF73 was increased in NP-exposed cells, indicating
that ORF50 expression and thereby lytic virus
replication is induced. A dose-dependency could be
observed to a certain extent at least in ANA-1/MHV-68
cells treated with DWCNT and S11 cells treated with
CNP (Additional file 1: Fig. S3c and d). A number of
cellular genes have been described to be potentially
involved in virus reactivation and therefore we
analyzed if some of these genes are regulated by
exposure of latently infected cells to NP. Five genes were
chosen for further investigation by RT-PCR 72 h after
exposure of S11 or ANA-1/MHV-68 cells to NP.
Ribonucleoside-diphosphate reductase large subunit
(Rrm1) is described as being upregulated in the
context of reactivation of the human gammaherpesviruses
EBV and Kaposi’s sarcoma-associated herpesvirus
(KSHV)  while Fructosamine-3-kinase (Fn3k),
Tousled-like Kinase 1 (Tlk1), TGFbeta-activated
kinase 1/MAP3K7-binding protein 1 (Tab1), and Sirtuin
1 (Sirt1) have been found to be downregulated [35,
36]. Consistent with reactivation of latent virus,
Rrm1, Fn3k and Sirt1 were up- or downregulated as
described for EBV and KSHV, albeit to a variable
degree depending on the cell line and the type of NP
(Fig. 2e and f ). Tab1 and Tlk1 proved to be
nonregulated in both cell lines.
In summary, our in vitro results show that exposure of
persistently infected cell lines to carbonaceous NP induces
virus reactivation and alters the expression of cellular
genes associated with virus reactivation independently of
the NP’s type or aspect ratio, whereas acute virus growth
in cells after de novo infection is not affected.
Treatment of latently infected mice with NP induces the
expression of lytic virus proteins in the lung
To test our in vitro findings also in vivo, we infected
mice i.n. with MHV-68 (experimental setup see Fig. 3).
At day 28 post infection – at the time when latent
infection is known to be established in B cells, pulmonary
epithelial cells and macrophages and lytic virus is no
longer detectable [29, 37] – the mice were instilled
intratracheally either with CNP or DWCNT or left untreated.
Tissue samples of the lung were harvested for analysis
24 h later. A dose of 50 μg per mouse was chosen, since
such a dimension of pulmonary carbon deposition could
be achieved at maximal occupational settings (5 day and
8 h per day exposure at a carbon black occupational
exposure limit of 3.5 mg/m3 , with a clearance of less
than 3% per day for inhaled NP ). Lung sections
were stained with a polyclonal serum against lytic
proteins of MHV-68 and expression of lytic virus proteins
could be detected in latently infected mice treated with
NP (Fig. 4c and d) but not in mice with latent virus only
(Fig. 4b). Similar to the acute infection situation, the
presence of lytic virus seemed to be locally restricted,
albeit it did not reach the dimensions observed in acute
virus infection (Fig. 4a).
We further analyzed if this induction of lytic viral
proteins leads to an increase in infectious virus or virus
genomes. However, no lytic virus could be detected in
lysates of whole lung tissue by plaque assay or by
TCID50 assay, and the viral genomic load was found to
be similar in all groups (Fig. 5a). We furthermore
determined the expression of the viral genes ORF50 and
ORF73 by RT-PCR in cells from bronchoalveolar lavage
(BAL) and whole lung tissue as already described for the
in vitro experiments. In whole lung tissue, the ORF50/
ORF73 ratio increased slightly in mice after treatment
with NP, indicating that ORF50 expression and thus lytic
virus production might be induced (Fig. 5b), but it did
not reach the values seen in acutely infected mice (Virus
6d: 3.2fold increase vs. Virus 29d). In BAL cells, the
ORF50/ORF73 ratio showed a significant increase after
exposure to CNP and a visible but not statistically
significant increase after exposure to DWCNT, indicating
that virus reactivation occurs in BAL cells (Fig. 5c). BAL
cell analysis revealed the well-known acute inflammatory
response to high surface area carbon nanoparticles and
accordingly, the BAL cell composition changed most
significantly by an increase in neutrophilic granulocytes
from virtually zero for day 29 virus infected mice to
25.6 ± 10.7% for additionally CNP and 9.0 ± 1.5% for
additionally DWCNT treated mice (Fig. 5d). In contrast,
the lymphocyte percentage raised from 7.3 ± 2.3 to 8.9 ±
2.3% and 14.0 ± 2.2%, respectively. Taken together, our
results suggest that NP exposure of latently infected
mice apparently induced an abortive virus reactivation
which led to an increased expression of lytic viral
proteins but not to the generation of new infectious virus.
Short-time treatment of latently infected mice with NP
creates a transcriptome signature with parallels to acute
Next, we investigated if there is a match in the lung
transcriptome between acutely infected mice and latently
infected mice treated with NP. For that reason, total
RNA from lung samples was isolated, processed and
analyzed with the Illumina-MouseRef-8v2.0 Expression
Fig. 3 Schematic overview of the experimental setup in vivo: C57BL/6 mice were infected intranasally with 5 × 104 PFU of MHV-68 (①). At day 28
post infection, when virus latency is established, the mice were instilled with 50 μg in 50 μl volume of spherical carbon nanoparticles (CNP) or
double-walled fiber shaped carbon nanoparticles (DWCNT) after non-surgical intubation (②). Tissue samples of the lung were harvested 24 h after
instillation for analysis of the metabolome, transcriptome, histology and viral load (③)
BeadChip as described in Materials and Methods. We
scanned the data for genes that were both regulated in
the acute infection situation and in latent infection plus
NP compared to untreated mice and mice with latent
virus alone. In the group “latent infection plus CNP”, we
found an overlap between this group and the group of
acutely infected mice of 208 significantly altered genes
(116 upregulated and 92 downregulated) from which 34
genes were at least 1.5-fold changed compared to the
control groups (30 upregulated, 4 downregulated; Fig. 5e).
Some of the induced genes (such as Saa3, Timp1, Cxcl1,
Slc26a4, Lcn2, Ch25h and Cd14) have recently been
described to be also induced by instillation of a single high
dose of 162 μg CNP in the lungs of mice . 18 genes
Fig. 4 Immunohistochemical staining shows increased expression of lytic viral proteins in lungs persistently infected with MHV-68 and subsequently
exposed to NP: Lung sections were stained with a rabbit-polyclonal serum directed against lytic proteins of MHV-68. Arrows indicate positive staining
in the positive control a and in latently infected mice treated subsequently with NP c and d. No positive staining was detected in mice with latent
virus only b. Scale bar: 200 μm. Representative stainings from 3 mice per group are shown
Fig. 5 (See legend on next page.)
(See figure on previous page.)
Fig. 5 Treatment of latently infected mice with NP restores features resembling the ones seen in acute infection: The viral genomic load in whole lung
tissue was measured by qPCR a. The expression of the viral genes ORF50 (specific for lytic replication) and ORF73 (expressed throughout all phases of the
viral life cycle) was analyzed in whole lung tissue b and in BAL cells c by RT-PCR. The mean ratio between ORF50 and ORF73 expression detected in mice
with virus for 29d only was set as “1” and all other values were expressed as relative values. Differential cell counts were made to examine the
composition of BAL cells d. The transcriptome of whole lung tissue was analyzed with the Illumina-MouseRef-8v2.0 Expression BeadChip. Genes that were
both regulated in the acute infection situation and in latent infection plus NP treatment compared to untreated control mice or to mice with latent virus
or NP only were searched for in the transcriptome data. Only genes that were at least 1.5-fold up- or downregulated compared to all control groups
were considered as differentially regulated. An overlap of 15 upregulated and 3 downregulated genes was found in the group subsequently treated with
CNP e, and 33 upregulated plus 5 downregulated genes in the group treated with DWCNT f. Regulated pathways that were identified by IPA are shown
in g for CNP and in h for DWCNT as a second hit in latently infected mice. All data were obtained from a single experiment with 6 mice per group for
BAL cells and 3 mice per group for whole lung tissue, except for the control group in panels e and f, where the expression values of 2 mice are shown.
In panels a, b, c and d, the means + SD are shown
including the aforementioned showed upregulation in
mice with CNP only in our experiments as well, even after
exposure to the comparatively moderate dose of 50 μg
(Additional file 1: Table S1). As differential expression of
these genes is also detected during acute virus infection,
they apparently are representatives of general pathways of
inflammation and immune response that are both induced
during acute infection and other inflammatory events. To
test if the differentially expressed genes indeed
represented specific cellular pathways, data were analyzed by
the use of Ingenuity Pathway Analysis (IPA, https://
www.ingenuity.com). As expected, functional pathways
found to be differentially regulated in latently infected
mice treated with CNP as well as in acutely infected mice
involved immune functions such as “activation of
leukocytes” or “invasion of cells”, but also pathways associated
with cell proliferation (Fig. 5g). In the group “latent
infection plus DWCNT”, the overlap with the group of acutely
infected mice consisted of 369 significantly altered genes
(156 upregulated, 213 downregulated) and expression of
49 of these genes was at least 1.5-fold changed compared
to the control groups (43 upregulated; 6 downregulated;
Fig. 5f ). Similarly to the results observed in mice treated
with CNP, 19 of these 49 genes were also regulated by
DWCNT alone (Additional file 1: Table S1). Data analysis
using IPA showed an increase in inflammatory pathways
and a reduction in “import of D-glucose” (Fig. 5h).
Additionally, we analyzed the transcriptome data by an
unbiased approach for differential regulation of genes
after combination of latent virus and particle exposure
compared to all control groups, and heatmaps showing
these genes were generated (Additional file 1: Fig. S5). In
the group treated with virus and CNP, 257 differentially
regulated genes were detected (110 upregulated, 147
downregulated) and 38 of these genes showed at least
1.5-fold changed expression values compared to all
control groups (35 upregulated, 3 downregulated; Additional
file 1: Fig. S5a). Data analysis using IPA revealed
differential regulation of pathways associated with migration,
activation and proliferation of cells (Additional file 1:
Fig. S5c). In the group treated with virus and DWCNT,
881 differentially regulated genes were found (288
upregulated, 593 downregulated), and 197 of these genes were at
least 1.5-fold changed compared to controls (92
upregulated, 105 downregulated; Additional file 1: Fig. S5b). Data
analysis with IPA depicted regulation of pathways involved
in proliferation and organization of cells, but also
tumorassociated pathways (Additional file 1: Fig. S5d). In
contrast, the IPA pathways assigned to only CNP or DWCNT
(24 h) treated mice depicted mainly the inflammatory
pathways: inflammatory response, cell movement,
leukocyte migration and recruitment of leukocytes (data
not shown). The results obtained with the Illumina
expression BeadChip were validated by quantitative
realtime PCR for 6 selected genes (Additional file 1: Fig. S6).
Our transcriptome data indicates that exposure of
latently infected lungs to NP creates a unique expression
profile which cannot be observed in untreated controls
or in latently infected mice without a second hit and
only to some extent in mice that were exposed to NP
only. The observed transcriptome signature shows
similarities to the one seen in acute virus infection and is
characterized by the stimulation of inflammatory
processes and the induction of an immune response.
Short-time treatment of latently infected mice creates a
metabolite pattern in lung tissue with high similarity to
the metabolite composition in acute virus infection
In order to examine chemical similarities between the
experimental groups, comparisons were performed on
subsets of the mass spectrometric data using principal
component analysis (PCA; see also Additional file 2).
Loadings into the first two PCs highlighted a strong
impact of glycerophospholipids into the acute viral
infection metabotype. The first PC loadings of the
secondhit-scenario “Virus 28d + CNP 24 h” (covering 20% of
covariance) correlated with the acute virus infection
metabotype (Fig. 6a; p = 2.37 × 10−4). A more detailed
view of the compound class pattern shown in Fig. 6a as
defined by the corresponding PC-loadings is shown in
Additional file 1: Fig. S7. Querying the HMDB 3.5
database  for all 2697 annotated molecular formulas
Fig. 6 Exposure of latently infected mice to NP produces a metabolite composition in the lung resembling the one seen in acute virus infection:
PCA was used to compare chemical similarities in the examined groups, and significant observations on PC-scores are shown in panel a. Mass
difference networks were created to visualize the chemical similarity of the combined virus plus CNP treatment and the control groups. Figure 6
b gives an overview of the compound classes shown in panels c-e. The mass difference networks of the two control groups “virus alone” and
“CNP alone” appeared exactly the same but proved to be entirely contraindicative once performed separately with compounds that were
upregulated in one group being downregulated in the other group c. Green color stands for unchanged compounds. Blue color represents
metabolites that are upregulated in “virus 29d” and downregulated in “CNP 24 h”. Red color depicts metabolites that are upregulated in “CNP
24 h” and downregulated in “virus 29d”. The pattern observed in “virus 28d + CNP 24 h” d showed upregulation of specifically one compound
group. This profoundly resembles the pattern seen in acute virus infection e. In panels d and e, blue color stands for downregulated metabolites
while red color depicts upregulated ones. The most pronounced changes in the metabolic profile were seen in the group of phospholipids. All
data are obtained from 3 mice per group
resulted in 642 hits (covering 23.8% of all annotated
molecular formulas) and revealed that the acute virus
infection phenotype is majorly characterized by the
overrepresentation of glycerophospholipids and organic
phosphoric acids as well as an underrepresentation of
monosaccharides, carboxylic acids, peptides, steroids,
amino acids and prenol lipids.
In order to illustrate the involvement of their
corresponding compound classes with the found multivariate
associations, a mass difference network (MDiN) was
created (Fig. 6c-e) in which nodes are the metabolite
candidates and edges are formula differences (potential
biochemical reactions). Fig. 6b visualizes the compound
classes shown in the metabolite networks. As implied by
the similarity of their loadings with the acute viral
infection metabotype, the combined treatment with latent
virus plus CNP showed high similarity to the pattern
seen in acute virus infection, characterized by an
upregulation of phospholipids, but not to the control groups
(Fig. 6d and e and Additional file 1: Fig. S7).
More detailed information on the involved building
blocks could be mined using mass difference enrichment
analysis (MDEA; as described by Moritz et al. ). In
MDEA, MDiN-edges are interpreted as biochemical
reactions or building blocks, whose association to nodes of
interest can be investigated by the discrete Fisher’s exact
test. MDEA results implied the induction of various fatty
acid pathways as the major building blocks of acute viral
infection and markers were C18-C22 saturated and
polyunsaturated fatty acids (Z-Scores and p-values ranged
from 5.87 to 9.94 and 0 to 8.68 × 10−8, respectively).
Confirming the data shown in the MDiN, one major
upregulated compound class was found to be
glycerophospholipids (see also Additional file 1: Fig. S7).
Furthermore, arachidonic acid and eicosatrienoic acid
molecular formula were determined significantly frequent,
indicating a non-random usage of these substances and
similar tendencies could also be found for other
unsaturated fatty acids such as linoleic acid. On the other hand,
down-regulated, or extensively consumed, metabolites
were found to be composed of typical metabolites of
glycolysis pathways. DWCNT exposure of latently infected
mice caused a similar response to a certain extent, which
was also accompanied by upregulation of phospholipids
(Additional file 1: Fig. S8), but the response was not
significant. This might be due to the strong hydrophobic
nature of DWCNT which might adsorb compound groups
such as phospholipids and therefore detract them from
analysis. Taken together, the profound match of the
metabolite pattern observed between acutely infected mice
and latently infected mice treated with CNP suggests that
– similar to our aforementioned observations –
shorttime treatment of latently infected mice with NP induces
a boost of lytic virus replication and restores features of
an acute virus infection in these mice.
Latently infected B cells and macrophages are
differentially affected by TiO2 NP and diesel exhaust
To investigate if other types of commonly investigated
low-toxicity low-solubility particles show a similar
effect as described for CNP and DWCNT, we exposed
latently infected cell lines to aqueous dispersions of
commercial titanium dioxide NP (TiO2 NP, P25) or to
diesel exhaust particles (DEP, SRM 2975) (Z-Average
and PdI see Additional file 1: Fig. S1). The cells were
treated with TiO2 NP or DEP for 72 h and virus titers
in the supernatant and the ratio of ORF50/ORF73
expression were determined. Differences between the cell
lines concerning the impact of the different types of
NP were detected. Significant effects on virus
reactivation were, however, only observed for DEP and the
expression of the lytic switch protein ORF50 in S11 B
lymphocytes (Additional file 1: Fig. S9a and c). Under
these conditions, TiO2 NPs induced a similar pattern
but did not reach statistical significance (p > 0.05). For
ANA-1 macrophages, neither NP caused significant
virus reactivation, i.e. increased the production of lytic
virus and the expression of the viral gene ORF50
(Additional file 1: Fig. S9b and d). These comparisons
suggest that – depending on the cell type – different
nanomaterials might have different potencies to affect
the maintenance and control of viral latency.
Carbon NP reactivate EBV from latently infected human
We next analyzed whether reactivation of latent virus by
exposure to NP also occurs in human cells latently
infected with the human gammaherpesvirus EBV. As it
is a well-known phenomenon in EBV-biology that
different EBV-infected cell lines can vary highly in their
response to stimuli [43, 44], we tested five different
lymphoblastoid cell lines (LCL) that were either infected
with recombinant EBV or EBV wildtype. The cells were
incubated with CNP, DWCNT or TPA (used as a
positive control) for 72 h, and viral genomes in the
supernatant were quantified by qPCR. In addition, expression
of the viral genes BZLF1 (= Zta), which reactivates the
EBV lytic cycle  and EBNA1 (episome maintenance
protein), which is particularly important in viral latency
but is expressed throughout all phases of the viral life
cycle , was determined by RT-PCR. Relative
expression of BZLF1 as a marker for the induction of lytic
virus production increased both after exposure to CNP
and to DWCNT (Fig. 7b, e and h). Although the strength
of the effect varied from cell line to cell line, the BZLF1/
EBNA1 ratio was always increased after NP treatment.
Consistent with the BZLF1/EBNA1 ratio, increased
amounts of viral genomes in the supernatant of cells
treated with CNP were detected by qPCR, confirming that
new virus particles are produced (Fig. 7a, d and g).
Surprisingly, no increase in viral genomes could be observed
after treatment with DWCNT. This seems to contradict
the results found for the BZLF1/EBNA1 ratio, but we
assume that it might be a false-negative result as the
strongly hydrophobic DWCNT might capture the DNA
during the isolation procedure and thereby exclude it from
the downstream analysis. This is in line with already
published observations demonstrating the ability of DNA to
bind to carbon nanotubes through pi-stacking and the
strong hypochromic interactions of DNA with carbon
nanotubes in aqueous media [47, 48]. We tried additional
DNA isolation methods but so far, we were not able to
overcome this problem. We furthermore tested the
expression of the five previously described cellular genes
with potential roles in virus reactivation (Rrm1, Fn3k,
Sirt1, Tlk1 and Tab1). All genes showed the expected
down- or upregulation (Fig. 7c, f and i), but to a varying
extent and with some variation between the experiments.
Therefore, statistically significant differences could only be
found for some genes and conditions. Nevertheless, our
results clearly indicate that the effect of carbon NP is not
limited to murine cells or tissues but that exposure to NP
also reactivates EBV from latently infected human cells.
Since both inhalation of environmental NP and
persistent herpesvirus-infection have been implicated to
contribute to the development of chronic lung disease, we
hypothesized that the combination of both might lead to
a different outcome than each factor alone. Given that
virtually every human being is persistently infected with
Fig. 7 (See legend on next page.)
herpesviruses, NP exposure of an already infected
individual can be easily envisaged as a practically relevant
scenario. At present, no information about the health
relevance of a suchlike liaison of NP and persistent virus
infection is available.
There are several publications providing evidence that
the presence of carbon-based NP has an influence on
virus infection in mice or in cells. For example, it has
been demonstrated that exposure of cells to
singlewalled carbon nanotubes increases the susceptibility of
(See figure on previous page.)
Fig. 7 Exposure of persistently infected human cells to carbon NP induces reactivation of EBV: two lymphoblastoid cell lines (LCL) infected with
recombinant EBV (panels a–c and d–f) and three LCL infected with EBV WT (panels g–i) were incubated with 50 μg/ml CNP or DWCNT for 72 h.
Virus genomes in the supernatant were determined by qPCR for the viral gene EBNA1 after 72 h (panels a, d and g; individual values and means).
Expression of the viral genes BZLF1 (specific for the lytic phase) and EBNA1 (expressed in all phases of EBV life cycle) – shown as the ratio BZLF1/
EBNA1 (panels b, e and h; individual values and means) – and expression of genes that have been shown to be associated with reactivation of
latent virus (panels c, f and i; means + SD) was analyzed by RT-PCR 72 h after NP exposure. The values in untreated cells were set as “1” and the
values for cells after NP treatment were calculated relative to the control. The symbols shown in panels a–c depict two, and in panels d-e three
independent experiments. In panels g-h, the symbols reflect three different cell lines tested in a single experiment. Asterisks indicate a statistically
significant difference to the untreated control (*: P < 0.05)
these cells to infection with influenza H1N1 .
Additionally, it has been shown that preexposure of mice to
carbon black prior to infection with respiratory syncytial
virus (RSV) induces an inflammatory milieu that
promotes disease exacerbation . Furthermore, treatment
of RSV-infected mice with ultrafine carbon black
enhances the expression of various chemokines that are
associated with virus infection, and leads to an enhanced
RSV-induced airway hyperresponsiveness to
methacholine . In this paper, we show that NP exposure of
persistently herpesvirus-infected cells in vitro reactivates
latent virus. Interestingly, we observed differences
depending on the cell type (B cells, macrophages) and on
the type of NPs (CNP, DWCNT, TiO2, or DEP). For
example, the effect of DWCNT was more pronounced
than the one by CNP in most of our experiments. This
does not seem to be a consequence of the higher surface
area but might be due to the fact that DWCNT, apart
from inducing oxidative stress and activating acute
inflammatory responses, affect a number of additional
cytotoxicity pathways . The triggers of herpesvirus
reactivation and the underlying molecular mechanisms
are – taken as a whole – only incompletely understood
[53, 54], and along the same line, we so far can neither
depict by which mechanisms/pathways NP might induce
reactivation of latent virus nor which are the target cells
for this interaction in vivo. In our in vivo experiments
we could show that NP exposure of persistently infected
mice leads to the expression of lytic viral proteins and
restores a signature observed during acute virus
infection. Although we observed the induction of lytic viral
proteins after NP treatment of latently infected mice, no
increase in infectious virus or virus genomes could be
detected. This might indicate that only small amounts of
new infectious virus were produced which were below
the detection limits of the assays used. Another
possibility is that NP treatment induces an abortive reactivation
which leads to re-expression of lytic virus proteins –
serving as potential targets for the immune system – but
does not lead to the completion of the replication cycle
and the production of new infectious virus. Such a
repetitive appearance of viral proteins induced by
NPexposure might nevertheless provoke the infiltration of
immune cells and finally cause a chronic aberrant
inflammatory response even in the absence of
completely assembled infectious virus. For example, it has
been shown that the CD8+ T cell response against
MHV-68 antigens can mediate inflammation and altered
cellular recruitment to the lung, finally resulting in
immunopathology and fibrosis . The increased
appearance of glycerophospholipids which was found by
metabolome analysis in the lungs of acutely infected
mice as well as in the lungs of latently infected mice
treated with NP is another indicator for the presence of
lytic virus replication – irrespective of successful
completion of the replication cycle or not. Noteworthy, the
phospholipid pattern was observed in association with
the detection of lytic MHV-68 proteins only, but not
with the acute inflammatory response caused by NP
exposure alone. As shown by Sutter et al., who investigated
the role of phospholipids in infection with Herpes
simplex virus, virus infection triggers the production of
phospholipids to maintain cellular membrane integrity
and to deliver membrane components for envelopment
of virus capsids and formation of transport vacuoles
during virus production . Other substances detected by
metabolome analysis such as arachidonic acid,
eicosatrienoic acid, and linoleic acid imply upregulation of mediator
molecules of immunomodulatory and of oxidative stress
related pathways after exposure of latently infected mice to
CNP. Particularly arachidonic and eicosatrienoic acid have
been described to have an impact on primary infection
with herpesviruses as well as on virus reactivation .
In this paper, we show that exposure of latently infected
cells or tissues to NP leads to reactivation of latent virus
accompanied by an increase in viral proteins and
metabolome- and transcriptome-signatures that can also be
found in acute virus infection. Concerning the health
relevance for humans it should be considered that
application of a single dose of NP, as in our experiments,
does only partially reflect the occupational settings
where the applied amount might be gradually
accumulated over one working week. Nevertheless, repetitive
appearance of even a small amount of viral proteins,
induced by exposure to NP, might be sufficient to trigger
a chronic aberrant immune response and consequently
lead to tissue damage. The question whether the
combined exposure to NP and virus de facto causes disease
aggravation needs to be further investigated and will be
a focus of subsequent studies.
Four different types of NP were used in this study (see
also Table 1): carbonaceous spherical NP (CNP;
Printex90, Degussa, Frankfurt, Germany), double-walled
carbon nanotubes (DWCNT; Nanocyl, Auvelais, Belgium),
TiO2 NP (P25, Degussa, Frankfurt, Germany), and diesel
exhaust particles (DEP; SRM 2975, National Institute of
Standards and Technology, Gaithersburg, MD, USA).
CNP, TiO2 and DEP were suspended in pyrogen-free
water using a previously described method . For in
vitro experiments, DWCNT were suspended in medium,
incubated in an ultrasonic bath and subsequently
sonicated on ice for 1 min prior to use, using a Bioruptor
(Diagenode, Liege, Belgium). For in vivo experiments,
DWCNT were dispersed in pyrogen-free, distilled water
supplemented with 1% Pluronic F-127 (Sigma-Aldrich,
Germany) – an FDA approved surfactant to facilitate
dispersion quality - as described earlier . The average size
(Z-Ave) and size distribution (represented by the
polydispersity index = PdI) of nanoparticles dispersed in medium
and in water was determined by photon correlation
spectroscopy using a Dynamic Laser Scatter (DLS) Zetasizer
Nano ZS. (Malvern Instruments Ltd., Malvern, UK) as
described in the literature . The dispersion quality is
shown in Additional file 1: Fig. S1. The absence of
endotoxin from the particle preparations was approved by
LIMULUS assay (<0.05 EU/μg CNP or DWCNT), and
even more relevant by in vitro studies using an
LPSsensitive alveolar macrophage cell line which showed no
pro-inflammatory activation (e.g. TNFα gene and protein
expression) for concentrations up to 150 ug/ml CNP or
BHK-21 cells (ATCC: CCL-10) were grown in
GlasgowMEM (PAN Biotech, Aidenbach, Germany)
supplemented with 5% fetal calf serum (FCS; PAN Biotech,
Aidenbach, Germany), 5% tryptose phosphate broth,
2 mM L-glutamine, 100 U/ml Penicillin and 100 μg/ml
Table 1 Nanoparticles used in this study
Streptomycin. NIH 3T3 cells (ATCC: CRL-1658) were
grown in DMEM High Glucose (Gibco, Darmstadt,
Germany) supplemented with 5% FCS, 2 mM
L-Glutamine, 100 U/ml Penicillin and 100 μg/ml Streptomycin.
The persistently with MHV-68 infected B cell line S11
, the two macrophage cell lines ANA-1 [60, 61] and
MH-S (ATCC: CRL-2019), and human LCL cell lines
(kindly provided by Bettina Kempkes and Josef Mautner,
Helmholtz Zentrum Muenchen, Munich, Germany)
were cultivated in RPMI (Gibco, Darmstadt, Germany)
supplemented with 15% fetal calf serum (FCS; PAN
Biotech, Aidenbach, Germany), 2 mM L-glutamine, 1%
non-essential amino acids (Gibco, Darmstadt, Germany),
100 U/ml Penicillin and 100 μg/ml Streptomycin. For
S11 and MH-S cells, 50 μM 2-Mercaptoethanol
(Bioconcept, Allschwil, Switzerland) was added to the medium.
Determination of cell viability by WST assay
Cell viability after exposure to NP was measured by
WST assay, which determines the activity of
mitochondrial succinate dehydrogenase in cells, according to the
manufacturer’s instructions (Roche Diagnostics,
Mannheim, Germany). Briefly, cells were plated and incubated
with the indicated concentrations of NP for 72 h. WST
reagent was added and incubated with the cells for 2 h
at 37 °C. The plates were centrifuged to remove the bulk
of NP agglomerates and supernatants were transferred
to a new plate prior to analysis. Enzymatic conversion of
WST reagent was determined using an ELISA-reader at
430 nm with 630 nm as reference.
Analysis of lytic virus growth in vitro after NP treatment
To test lytic growth of MHV-68 in vitro after NP
treatment, MH-S cells or LA-4 cells were infected with a
MOI of 1 for 2 h. After removing the inoculum (=0 h),
cells were washed two times with PBS and then treated
for 2 h with 50 μg/ml NP (NP preparation see section
“Nanoparticles”). After the incubation period, the
inoculum was removed, the cells were washed two times with
PBS and then incubated with fresh medium at 37 °C and
5% CO2 until the supernatants or the supernatants
together with the cells were harvested at different time
points after infection. Virus titers were determined by
plaque assay on BHK-21 cells.
Overview of features of the nanoparticles used in this study (all data according to manufacturer’s information; Size a: average primary particle size)
In vitro assay for measurement of low level virus
replication in infected macrophages
To measure low-level virus replication in infected
macrophages, a modification of a previously described
method for a limiting dilution in vitro reactivation assay
was used . Briefly, MH-S cells (alveolar macrophage
cell line) were plated in a 6-well plate and infected with
MHV-68 o.N. at an MOI of 0.01. The inoculum was
removed and each well was washed two times in PBS. The
cells were then either left untreated or incubated with
50 μg/ml CNP or DWCNT (NP preparation see section
“Nanoparticles”). After 2 h, the medium containing the
NP was removed and each well was washed two times
with PBS. The cells were incubated for 1 min with an
acidic citrate buffer (pH = 3.0) to remove remaining lytic
virus from the inoculum and washed three times with
medium. Serial threefold dilutions of infected MH-S
cells were plated on monolayers of 7 × 103 low-passage
NIH 3T3 cells per well in 96-well tissue culture plates.
Twenty-four wells were plated per dilution (starting with
1 × 103 MH-S cells). NIH 3T3 cells were screened
microscopically for a viral cytopathic effect for up to 2 weeks.
To differentiate between freshly produced virus and
residual lytic virus from the inoculum, serial threefold
dilutions of MH-S cells were plated before or after mechanical
disruption of viable cells (by two freeze-thaw cycles).
Frequencies of cells supporting lytic virus replication were
calculated on the basis of the Poisson distribution by
determining the cell number at which 63.2% of the wells
scored positive for CPE. To compensate for variations in
the infection efficiency, the viral genomic load was
determined as described below and taken account of when
calculating the frequency of cells producing lytic virus.
Generation of a persistently infected macrophage cell line
To generate a persistently infected macrophage cell line,
we constructed a recombinant MHV-68 containing a
hygromycin-resistance cassette by a two-step
mutagenesis procedure using the BAC-technology [62, 63]. To
this end, a 2.4 kb expression cassette containing the
coding sequence of hygromycin phosphotransferase driven
by the SV40 early-enhancer promoter, was excised from
vector pRTS-1  (kindly provided by Bettina
Kempkes, Helmholtz Zentrum Muenchen, Munich,
Germany) and cloned blunt end into the PmlI site
(nucleotide position 46.347 of the MHV-68 genome) of the
plasmid pST76K-SR already containing a 4.0 kb
SphISacI fragment of MHV-68 (nucleotide positions 44.301
to 48.346). As a result, the hygromycin
phosphotransferase expression cassette is flanked on both sides by
homologous sequences as needed for homologous
recombination during the two-step mutagenesis
procedure. BHK-21 cells were transfected with 1.5 μg of BAC
MHV-68-Hygro DNA using X-treme GENE HP DNA
Transfection Reagent (Roche, Mannheim, Germany) to
reconstitute recombinant MHV-68-Hygro. A virus stock
was generated and the virus titer was determined by
plaque assay on BHK-21 cells. To establish a permanently
infected cell line, the bone marrow derived macrophage
cell line ANA-1 was infected with MHV-68-Hygro at an
MOI of 1. Hygromycin B (Sigma-Aldrich, Seelze,
Germany) at a final concentration of 500 μg/ml was added
24 h after infection and persistently infected cells were
expanded under permanent selection. As the BAC-sequence
in the virus genome contains the GFP-gene, the cells
could be monitored under the fluorescence microscope
and more than 90% of the cells proved to be GFP positive.
Treatment of persistently infected cell lines with NP
To analyze the effect of NP exposure on persistently
infected murine cells, the B cell line S11 and the
macrophage cell line ANA-1/MHV-68 were used. To
investigate the effect of NP on persistently infected
human cells, human lymphoblastoid cell lines (LCL) were
used. NP were suspended as described above and added
to the cells at a concentration of 5 μg/ml or 50 μg/ml.
After 72 h, supernatants were harvested for analysis of
virus titer by Plaque assay (murine cells) or qPCR
(human cells), and cells were harvested for RNA isolation
and subsequent RT-PCR.
Measurement of viral genomic load by quantitative real
DNA was isolated from lung tissue samples that were
homogenized by using the FASTPREP®-24 instrument (MP
Biomedicals, Heidelberg, Germany), from cell culture
supernatants, or from infected cell lines with the QIAmp
DNA Mini Kit (Qiagen, Hilden, Germany). The viral
genomic load in infected murine cells or in murine lung tissue
was determined by quantitative real-time PCR using the
ABI 7300 Real Time PCR System (Applied Biosystems,
Foster City, CA) as described previously . The amount
of viral genomes in cell culture supernatants from LCL
was analyzed by real time quantitative PCR for the viral
gene EBNA1 using the Taqman SYBR green PCR master
mix (Applied Biosystems, Foster City, CA).
mRNA was isolated from cells or tissues using the
RNeasy MiniKit (Qiagen, Hilden, Germany) including
DNase digestion of remaining genomic DNA. RNA was
reverse-transcribed using the High Capacity cDNA
Reverse Transcription Kit (Applied Biosystems, Foster City,
CA). The cDNA was analyzed for the expression of
selected genes by real time quantitative PCR using the
Taqman SYBR green PCR master mix (Applied
Biosystems, Foster City, CA). The used primer sets are
depicted in Table 2. The fold change in expression of
Table 2 Primer sets used in this study
each target mRNA relative to beta-Actin (Actb, murine
cells) or HPRT (human cells) was calculated based on
the threshold cycle (Ct) as 2-ΔCt, where ΔCt = Ctspecific
gene – Ctbeta-Actin. The mean value for untreated controls
was set as 1, and all values were calculated relative to
In vivo experiments
C57BL/6 mice were purchased from Charles River
Laboratories (Sulzfeld, Germany) and housed in
individually ventilated cages (IVC) during the MHV-68 infection
period. Mice were anesthetized with ketamine and
xylazine and infected i.n. with 5 × 104 PFU MHV-68. After
28 days, mice were either left untreated or instilled with
50 μg of spherical (CNP) or double-walled fibre-shaped
carbon nanoparticles (DWCNT) per mouse as described
earlier [5, 58] (NP preparation see section
“Nanoparticles”). Lung tissue was harvested after 24 h for
transcriptome and metabolome analysis as well as for
determination of viral genomic load and for histology.
Bronchoalveolar lavage (BAL) cells for RNA isolation
and analysis of viral gene expression were collected by
cannulating the trachea and rinsing the lung six times
with PBS as described earlier . All animal
experiments were in compliance with protocols approved by
the local Animal Care and Use Committee (District
Government of Upper Bavaria; permit numbers 124/08
For the metabolomics analysis, lung tissue samples (three
mice per group) were processed by using the
FASTPREP®24 instrument (MP Biomedicals, Heidelberg, Germany).
50 mg of lung tissue were homogenized in 1 ml of
icecold methanol (LC/MS grade, Sigma-Aldrich, Steinheim,
Germany). The homogenates were centrifuged at
maximum speed in an Eppendorf centrifuge to remove debris.
The supernatants were stored at −80 °C and diluted 1:50
in methanol prior to metabolome analysis. Samples were
injected in a randomized order at a flowrate of 120μLh−1
using a Gilson autosampler system (Gilson, Inc.,
Meddleton, WI, USA). Electrospray ionization (ESI) was
performed using an APOLLO II ion source (Bruker Daltonics
GmbH, Bremen, Germany) in negative ionization mode
with capillary voltage and spray shield voltage being set to
−3000 V and 500 V, respectively. Drying gas flow rate and
temperature were set to 4Lmin−1 and 200 °C. The
nebulizer pressure was set to 1.1 bar. Ultra-high resolution
and accuracy mass spectrometra were recorded using a
Bruker (Bruker Daltonics GmbH, Bremen, Germany)
solariX Fourier Transform Ion Cyclotron Resonance Mass
Spectrometer (FT-ICR-MS) equipped with a 12 T
superconducting magnet. External calibration of the mass
spectrometer is performed daily using a 1 mgL−1 arginine/
methanol solution. Linear calibration on at least 4 arginine
clusters is accepted once the standard deviation of m/z
error was minor 100 ppb. Mass spectra were recorded
over a mass range of 129 m/z to 1000 m/z. The time
domain was set to 4 M words (MW) and the resolution at
m/z = 400 was R = 430,000.
Raw mass spectra were pre-processed using Data
Analysis Version 4.1 (Bruker Daltonics GmbH, Bremen,
Germany). All mass spectra were uploaded and peak
picking was performed using a signal to noise cutoff of
S/N = 4. Linear calibration was performed using an
inhouse generated list of 390 metabolite m/z values that
are frequently occurring across species and bio-fluids.
The central error-m/z distribution was visualized within
the Data Analysis calibration functionality and centered
on zero ppm given a linear calibration function. The
overall error standard deviation was found to be <
100 ppb. All calibrated mass spectra were exported as
ASCI file and aligned with the in-house written software
‘Matrix Generator’ given an error window of 1 ppm.
M/z signals which were not once detected within a full
triplicate were omitted. M/z peaks were subjected to
combinatorial formula assignment at ±0.5 and given
elemental counts of C1-100O0-70N0-20S0-3P0–3 using an
in-house written software. Masses were filtered for the
Senior rules and for diverse approximations of elemental
relationships. Formulae were then run through a strict
isotopic pattern matching algorithm assuming infinite
resolution and given a noise level that was set to be the
minimum of all maximal m/z intensities across all
samples. Validated formulae were used as starting points for
mass difference network-based formula assignment to
low abundance peaks (> > 95% of a dataset) . The
initial dataset of >200.000 m/z values was reduced to
6890 m/z values by removal of non-triplicate features
and reduced to 2697 sum formula annotations.
Data normalization was performed as follows: The
inter quartile ranges (IQRs) of all non-zero entries per
sample were calculated. The sample-wise Euclideans of
all m/z feature intensities that were elements of their
corresponding IQR were calculated and used for
Data was separated into two data sets which were
composed of the second hit experiments (Virus 28d +
CNP 24 h or Virus 28d + DWCNT 24 h), their
corresponding time matched controls and a mouse-group
with acute virus infection (Virus 6d).
Both datasets were scaled by Z-transformation and
subjected to principal component analysis (PCA) using
the Perseus software, version 126.96.36.199. The scores of the
first three components of each dataset were tested for
significant differences between combined exposure,
single exposures and untreated controls using Student’s T
test in Microsoft Excel 2016. Mass difference networks
were reconstructed and mass difference enrichment
analyses were performed using Matlab R2011 (as described
in detail by Moritz et al.). The theoretical mass
difference network was reconstructed using 490 reaction
equivalent mass differences (REMDs), part of which
were derived from the KEGG metabolic maps and part
of which were a manually curated extension and
correction of data published previously by Breitling et al. .
Given theoretical molecular masses derived from
molecular formula assignment, REMDs, which here are
interpreted as building blocks, were used as edges to
connect all annotations (nodes). REMDs were then
tested for significant associations to metabolic features
(molecular formulas) of interest using Fisher’s exact test,
which assumes a hypergeometric distribution. This
technique is used to include features that could not be
matched to e.g. the human metabolome database
(HMDB ). Box-plots were generated using ggplot2 in
RStudio Version 0.99.489. Networks were visualized
Immunohistochemical staining for lytic virus proteins
Expression of MHV-68 lytic proteins in the lung of
NPtreated or control animals was examined by standard
immunohistochemical methods. Since all lungs were
divided for different assays, collapsed lungs had to be used
for histology. Lung tissue was embedded in paraffin and
cut into 4 μm sections. Slides were incubated with 3%
hydrogen peroxide to bleach tissues and produce a
better contrast for the alkaline phosphatase staining
procedure. Following epitope retrieval by heating the
sections in citrate buffer (pH = 6.0), the sections were
incubated with blocking buffer (Rodent Block M; Biocare
Medical/Zytomed Systems, Berlin, Germany) and labeled
with a polyclonal rabbit serum directed against lytic
proteins of MHV-68 (described previously by Steer et al.
; 1:500 dilution). After washing,
Rabbit-on-rodentAP-polymer was added (Biocare Medical/Zytomed
Systems, Berlin, Germany), and finally, the phosphatase
reaction was visualized using the Vulcan Fast Red
Chromogen Kit (fuchsin-red reaction product; Biocare
Medical/Zytomed Systems, Berlin, Germany). All
sections were counterstained with hematoxylin.
Total RNA from lung tissue was isolated employing the
RNeasy MiniKit (Qiagen, Hilden, Germany) including
DNase digestion of remaining genomic DNA. The
Agilent 2100 Bioanalyzer was used to assess RNA quality
and only high quality RNA was used for microarray
analysis. 300 ng of high quality total RNA were amplified
using the Illumina TotalPrep RNA Amplification kit
(Ambion, Life Technologies, Carlsbad, CA, USA).
Amplified cRNA was hybridized to Mouse Ref-8 v2.0
Expression BeadChips (Illumina, San Diego, CA, USA).
Staining and scanning were done according to the Illumina
expression protocol. Data was processed using the
GenomeStudioV2010.1 software (gene expression module
version 1.6.0) in combination with the
MouseRef8_V2_0_R3_11278551_A.bgx annotation file. The
background subtraction option was used and an offset to
remove remaining negative expression values was
introduced. Data normalization (quantile) was performed by
utilizing the statistical programming environment R
implemented in CARMAweb [69, 70]. Genewise testing for
differential expression was done employing the limma t-test
and Benjamini-Hochberg multiple testing correction (FDR
< 10%). Heatmaps showing genes that were at least 1.5fold
regulated in mice treated with latent virus and NP
compared to untreated control mice were generated with
CARMAweb. Pathway enrichment analyses were done with
the Ingenuity pathway analysis software (IPA®, Qiagen,
Redwood City, CA, USA,
https://www.qiagen.com/ingenuity). For genes that were detected by more than one probe,
only one representative value is shown. Array data has been
submitted to the GEO database at NCBI (GSE79501).
Datasets were analyzed by Student’s t-test using the
GraphPad Prism software, vs5 (GraphPad Software, Inc.,
San Diego, CA, USA). Results with a p-value < 0.05 were
considered significant. Statistical analysis of
transcriptome data was performed as described in the section
Additional file 1: Figure S1. Average Size and size distribution of the
used NP. Figure S2. Measurement of cell viability. Figure S3. Exposure
to NP reactivates lytic virus in persistently infected cells in vitro in a dose
dependent manner. Figure S4. Exposure to NP reactivates lytic virus in
persistently infected cells independently of the particle aspect ratio.
Figure S5. Short-time exposure of latently infected mice to NP differentially
regulates gene expression in whole lung tissue cells independently of the
particle aspect ratio. Figure S6. Confirmation of gene expression data by
real-time quantitative PCR for selected genes. Figure S7. Exposure of
latently infected mice to CNP leads to an increase in glycerophospholipids.
Figure S8. Exposure of latently infected mice to DWCNT leads to an
increase in glycerophospholipids. Figure S9. Exposure of persistently
infected cells to TiO2 NP or DEP has differential effects on virus reactivation
in vitro. Table S1. Gene expression values of selected genes (PDF 1767 kb)
Additional file 2: Supplementary Material (XLSX 7772 kb)
BAL: Bronchoalveolar lavage; CNP: Carbonaceous nanoparticles;
CPE: Cytopathic effect; d: Day; DEP: Diesel exhaust particles; DWCNT:
Double-walled carbon nanotubes; EBV: Epstein-Barr-virus; h: Hour;
HCMV: Human cytomegalovirus; IPF: Idiopathic pulmonary fibrosis;
KSHV: Kaposi’s sarcoma-associated herpesvirus; LCL: Lymphoblastoid cell
line; LPS: Lipopolysaccharide; MDEA: Mass difference enrichment analysis;
MDiN: Mass difference network; MHV-68: Murine gammaherpesvirus 68;
NP: Nanoparticles; PCA: Principal component analysis; RT-PCR: Reverse
transcription polymerase chain reaction; TCID50: Tissue culture infective
dose (amount of a pathogenic agent that will produce pathological
change in 50% of cell cultures inoculated); TiO2: Titanium dioxide; TPA:
This work was supported by intramural funding for Environmental Health
projects of Helmholtz Zentrum München – German Research Center for
Environmental Health to T.S., H.A. and P.S.-K., and by grants from the
Helmholtz Portfolio Theme ‘Metabolic Dysfunction and Common Disease’
and the Helmholtz Alliance ‘Imaging and Curing Environmental Metabolic
Diseases, ICEMED’ to J.B.
C.S. designed and performed the experiments, analyzed the data, wrote and
edited the paper. F.M. designed and performed the metabolome analysis,
analyzed the data, wrote and edited the paper. S.C., B.S. and D.S. performed
the experiments. M.I. and J.B. performed microarray analysis and analyzed
the data. O.E. participated in coordination of the study and acquiring
funding. P.S.-K. participated in acquiring funding, supervised metabolome
analysis and gave conceptual advice. H.A. and T.S. participated in acquiring
funding, supervised the project, designed the experiments and analyzed the
data, wrote and edited the paper.
Ethics approval and consent to participate
All animal experiments were in compliance with protocols approved by the
local Animal Care and Use Committee (District Government of Upper
Bavaria; permit numbers 124/08 and 67/2015).
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