Nanoparticle exposure reactivates latent herpesvirus and restores a signature of acute infection
- Christine Sattler1,
- Franco Moritz2,
- Shanze Chen1,
- Beatrix Steer3, 4, 5,
- David Kutschke1,
- Martin Irmler6,
- Johannes Beckers6, 7, 8,
- Oliver Eickelberg1,
- Philippe Schmitt-Kopplin2,
- Heiko Adler†3, 4, 5Email author and
- Tobias Stoeger†1Email authorView ORCID ID profile
© The Author(s). 2017
Received: 24 June 2016
Accepted: 15 December 2016
Published: 10 January 2017
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.
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.
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.
KeywordsCarbonaceous 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 NP-associated 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 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
NP exposure of persistently infected cells reactivates latent virus
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
Short-time treatment of latently infected mice with NP creates a transcriptome signature with parallels to acute virus infection
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 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 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 tumor-associated 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 real-time 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 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 poly-unsaturated 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 – short-time 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 particles (DEP)
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 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 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 single-walled carbon nanotubes increases the susceptibility of 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 NP-exposure 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.
Nanoparticles used in this study
Size a [nm]
Evonik Degussa GmbH
10 × 1000
Evonik Degussa GmbH
Diesel exhaust particles
BHK-21 cells (ATCC: CCL-10) were grown in Glasgow-MEM (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 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.
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 SphI-SacI 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 time PCR
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).
Primer sets used in this study
Forward primer (5’–3’)
Reverse primer (5’–3’)
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 and 67/2015).
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 ice-cold 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 in-house 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 normalization.
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 184.108.40.206. 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 using Gephi-0.8.2.
Immunohistochemical staining for lytic virus proteins
Expression of MHV-68 lytic proteins in the lung of NP-treated 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-rodent-AP-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 MouseRef-8_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 “transcriptome analysis”.
Diesel exhaust particles
Double-walled carbon nanotubes
Idiopathic pulmonary fibrosis
Kaposi’s sarcoma-associated herpesvirus
Lymphoblastoid cell line
Mass difference enrichment analysis
Mass difference network
Murine gammaherpesvirus 68
Principal component analysis
Reverse transcription polymerase chain reaction
Tissue culture infective dose (amount of a pathogenic agent that will produce pathological change in 50% of cell cultures inoculated)
We are grateful to the members of our animal facilities for expert technical assistance. We also thank the members of our institutes for helpful discussions.
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.
Availability of data and materials
Microarray data has been submitted to the GEO database at NCBI (GSE79501).
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.
The authors declare no competing financial interest.
Consent for publication
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).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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