Nanoparticle-Exposure-Triggered Virus Reactivation Induces Lung Emphysema in Mice

Nanoparticles (NPs) released from engineered materials or combustion processes as well as persistent herpesvirus infection are omnipresent and are associated with chronic lung diseases. Previously, we showed that pulmonary exposure of a single dose of soot-like carbonaceous NPs (CNPs) or fiber-shaped double-walled carbon nanotubes (DWCNTs) induced an increase of lytic virus protein expression in mouse lungs latently infected with murine γ-herpesvirus 68 (MHV-68), with a similar pattern to acute infection suggesting virus reactivation. Here we investigate the effects of a more relevant repeated NP exposure on lung disease development as well as herpesvirus reactivation mechanistically and suggest an avenue for therapeutic prevention. In the MHV-68 mouse model, progressive lung inflammation and emphysema-like injury were detected 1 week after repetitive CNP and DWCNT exposure. NPs reactivated the latent herpesvirus mainly in CD11b+ macrophages in the lungs. In vitro, in persistently MHV-68 infected bone marrow-derived macrophages, ERK1/2, JNK, and p38 MAPK were rapidly activated after CNP and DWCNT exposure, followed by viral gene expression and increased viral titer but without generating a pro-inflammatory signature. Pharmacological inhibition of p38 activation abrogated CNP- but not DWCNT-triggered virus reactivation in vitro, and inhibitor pretreatment of latently infected mice attenuated CNP-exposure-induced pulmonary MHV-68 reactivation. Our findings suggest a crucial contribution of particle-exposure-triggered herpesvirus reactivation for nanomaterial exposure or air pollution related lung emphysema development, and pharmacological p38 inhibition might serve as a protective target to alleviate air pollution related chronic lung disease exacerbations. Because of the required precondition of latent infection described here, the use of single hit models might have severe limitations when assessing the respiratory toxicity of nanoparticle exposure.


Determination of cell viability by WST-1 assay
Cell viability was measured with WST-1 assay (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions.Briefly, Ana-1 or Ana-1/MHV-68 cells were seeded in a density of 10,000 cells / well in a 96-well plate and incubated in 37 °C incubator for 24 h.Cells were treated with indicated concentrations in 100 µl for proper time.For some experiments, cells were pretreated with inhibitors 30 min prior to treatments.After treatment, 10 µl WST-1 reagent was added to each well and mixed thoroughly, protected from light.Then, the plate was incubated for 1 h at 37 °C.The supernatant was transferred to a microcentrifuge tube and centrifuged at 14,000 rpm for 10 min to remove NP agglomerates.80 µl of supernatant was added to a new plate and the absorbance was measured using Microplate Reader (TECAN Group Ltd. Maennmedorf, Switzerland) at 450 nm.

LDH cytotoxicity assay
LDH assay (Roche Diagnostics, Mannheim, Germany) was used to assess the cytotoxicity of treatments by quantifying the release of lactate dehydrogenase (LDH) into medium due to cell membrane damage.Briefly, cells were seeded into a 24-well plate in a density of 200,000 cells / well and incubated in 37 °C incubator for 24 h.Cells were then exposed to CNP (50 µg/ml), DWCNT (50 µg/ml) or LPS (1 µg/ml) for 24 h.For some experiments, cells were pretreated with inhibitors 30 min prior to treatments.After 24 h, supernatants were collected and centrifuged at 14,000 rpm for 10 min to remove nanoparticles, then stored on ice for assay.
Cells lysed with 1% Triton X-100 in equal volume medium were used as a high control.30 µl of samples supernatant and 5 µl high control were taken and brought to 100 µl with serum-free medium.100 µl serum-free medium was used as background control.100 µl freshly prepared LDH working solution were added to each well.After incubation for 15 min at RT, 50 µl 1N HCl were added to stop the reaction.The absorbance was measured at a wavelength of 492 nm.

Determination of gene expression by qPCR
Whole cell RNA was isolated with NucleoSpin RNA Plus kit (MACHEREY-NAGEL, Duren, Germany) following the instructions of the manufacturer.Briefly, cells were cultured and treated in 24-well plates.After treatment, cell supernatant was collected and stored at -80 °C for further experiments.Then, 350 µl lysis buffer was added to lyse cells for 10 min at RT. Afterwards, cell lysate was transferred to genomic DNA removal column and centrifuged for 60 s at 11,000 x g.Then, RNA binding was adjusted followed by washing with wash buffer.
Finally, RNA was eluted with 30 µl RNase-free H 2 O for 60 s at 11,000 x g.This step was repeated one more time to get a final volume of 60 µl.RNA concentration was measured with Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA).Subsequently, RNA was reversetranscribed using superscript kit (Invitrogen, Waltham, MA, USA).Then, cDNA was used to analyze the target gene expression by real time quantitative PCR using SYBR Green PCR master mix (Thermo Fisher Scientific, Waltham, MA, USA).For viral gene expression determination, Open Reading Frame 50 (ORF50, expressed in the lytic phase of virus life cycle) and Open Reading Frame 73 (ORF73, continuously expressed in all phases of virus life cycle) were analyzed, and 60S ribosomal protein L8 (Rpl8) was used as a housekeeping gene.The primer pairs used in this study are shown in Table S3.The fold change in expression is shown

Flow cytometry
The expression of CD11b and CD11c (CD11b anti-human/mouse, PE, 130-113-235; CD11c anti-human/mouse, FITC, 130-110-837; Miltenyi Biotec.Bergisch Gladbach, Germany) on Ana-1 cells were detected by flow cytometry.Briefly, Ana-1 cells were collected after cultivation and centrifuged at 400 g for 5 min to get a pellet.Then the pellet was washed with FACS buffer and centrifuged at 300 g for 10 min at 4 °C.The pellet was resuspended in a density of 10*10 6 cells/ml in FACS buffer and transfer to FACS tubes.
Cells were incubated with CD11b-PE, CD11c-FITC antibody (1:50 dilution) on ice for 15 min, protect cells from light.Afterwards, cells were washed with 1 ml FACS buffer and centrifuged at 300 g for 5 min at 4 °C.The supernatant was discarded and cells were resuspended with 200 µl FACS buffer.Cells were ready for flow cytometry analysis.
Cells were acquired by FACScantoTM II flow cytometer and analyzed by FlowJo software (BD Biosciences, San Jose, CA, USA).Mean fluorescence Index (MFI) was used to show the intensity of CD11b or CD11c expression of the cells.

Western blot
Whole cell protein from NPs treated Ana-1/MHV-68 cells was extracted with RIPA buffer (contains 50 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.5% (w/v) deoxycholic acid, 0.1% (w/v) SDS, 0.5% (v/v) Nonidet P-40, pH 8.0) supplemented with protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA).Protein concentration was measured with BCA kit (Thermo Fisher Scientific, Waltham, MA, USA) following the instructions of the manufacturer.All protein samples were adjusted to equal concentrations.20 to 40 µg protein was denatured and loaded onto SDS-PAGE gel.Pre-stained precision protein ladder (Bio-Rad, Hercules, CA, USA) was used for evaluation of electrophoresis and protein size.
Electrophoresis was run at 100 V for 10 min and 120 V at least 60 min followed by blotting to PVDF membrane (Bio-Rad, Hercules, CA, USA) at 100 V for at least 60 min.

Transcriptomic study by microarray analysis
Total RNA was isolated followed by the same procedure described above.The Agilent 2100 Bioanalyzer was used to assess RNA quality and only high quality RNA (RIN > 7) was used for microarray analysis.Total RNA was amplified using the WT PLUS Reagent Kit (Thermo

Reactive oxygen species (ROS) detection
Intracellular ROS of Ana-1 cells were measured with DCFH-DA probe (Merck, Darmstadt, Germany).Briefly, Ana-1 cells were seeded in black 96-well plates and incubated for 24 h at 37 °C.Medium was removed and fresh medium containing 30 µM DCFH-DA was added in half volume and incubated for 30 min in the incubator.NACA (5 mM; Merck, Darmstadt, Germany) was added together with DCFH-DA.After 30 min, cells were treated with NPs (50 µg/ml) or H 2 O 2 (10 mM) in half volume containing 2 times concentration, equal volume medium was added as untreated group.Fluorescence with excitation / emission at 485 nm / 535 nm was measured after 10, 30 and 60 min.

Multiplex Cytokine/Chemokine Analysis
Cytokines and chemokines in bronchoalveolar lavage fluid were measured using the multiplex bead array system Bio-Plex Pro Mouse Chemokine Assay Panel 31-Plex (#12009159, Bio-Rad Laboratories GmbH), following the manufacturer's instructions.Data were acquired using the Luminex200 system with BioPlex Manager 6.1 software.Standard curves were fitted using the logistic-5PL regression type.

Bronchoalveolar lavage (BAL) preparation and cell differentiation
After 24 h of CNP instillation, mice were anesthetized with midazolam/medetomidine/fentanyl (MMF).Immediately after sacrification, BAL was performed by cannulating the trachea and infusing the lungs six times with 1.0 ml PBS without calcium and magnesium, as described previously. 4The first two times lavage were collected separately and centrifuged at 425 x g for 20 min at 4 °C to separate fluids for cytokine detection.The pellet was also collected with the rest lavage to finally get all cells.The pellets were suspended in RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, PAN Biotech, Aidenbach, Germany), total living cell number was counted with trypan blue dye and 30,000 cells were used for each cytospin.Cytospin slides were stained with May-Grünwald-Giemsa solution followed by macrophages, neutrophils and lymphocytes number counting.

Immunofluorescence (IF) staining and ZEISS scanning
Anti MHV-68 immunofluorescence double staining together with macrophage markers CD11b and CD11c were performed and used further for ZEISS scanning.
Lung tissue slices were deparaffinized and rehydrated by dipping slices into Xylene, 100% Ethanol, 90% Ethanol, 80% Ethanol, 70% Ethanol in order and rinsed with distilled H 2 O. Heatinduced epitope retrieval (HIER) was done by incubation of slices in Citrate pH = 6.0 buffer.
Later, slices were incubated in blocking solution (5% goat serum + 0.03% Triton X-100 in PBS) at RT for 1 h, followed by incubation with a primary antibody (polyclonal rabbit serum directed against lytic proteins of MHV-68, described before with 4% PFA for 15 min at RT and washed two times with PBS, followed by incubation with primary antibodies and secondary antibodies as usual.

TUNEL assay and quantification
DNA fragmentation and cell death of lung tissue was assessed using the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay according to manufacturer's instruction (ab66110, Abcam, Cambridge, Massachusetts) on paraffinembedded sections.dUTP-labeled DNA was visualized directly using Olympus BX51 fluorescence microscopy.Nuclei were counterstained with DAPI.TUNEL staining was quantified by taking 6 random fields of view per mouse lung, using an Olympus BX51 fluorescence microscope.TUNEL positive cells were counted and normalized to the DAPI events for each field.3 mice per group were analyzed were the mean of the 6 fields per lung indicated at the individual dots in the graph.

Quantification of interstitial inflammation score and MCL
Airspace enlargement (MCL) and inflammation score was assessed by hematoxylin and eosin (H&E) staining.Design-based stereology was used to analyze sections using an Olympus BX51 light microscope equipped with a computer-assisted stereological toolbox (CAST) software Visopharm Integrator System (VIS) v6.0.0.1765 (newCAST, Visiopharm) on H&E stained lung slides for assessing the airspace enlargement and the tissue inflammation volume.Air space enlargement was assessed by quantifying mean chord length (MCL) on 30 field of view per lung, a line grid was superimposed on lung section images taken with the ×20 objective.
Intercepts of lines with alveolar septa and points hitting airspace were counted to calculate MCL applying the formula MCL = ∑P air × L(p)/∑I septa × 0.5.P air are the points of the grid hitting air spaces, L(p) is the line length per point, and I septa is the sum of intercepts of alveolar septa with grid lines.To quantify interstitial inflammation in the lung, a line grid was superimposed on lung section images taken with the ×20 objective and were analyzed on lung sections across at least 30 random fields per lung.Intercepts of lines crossing with airways and vessels were counted to calculate the interstitial inflammation (µm 3 /µm 2 ) = ∑P inflamed area × L(p)/∑I intercept(A+V) .
P inflamed area are the points of the grid hitting inflammatory tissue area, L(p) is the line length per point, and I intercept(A+V) is the sum of intercepts of airways and vessels to normalize the inflammatory area.

Light-sheet fluorescence (LSFM) microscopy
Mouse lungs were perfused and fixed with 4% PFA at 4°C overnight and then washed with PBS for two times.Lung lobes were blocked and permeabilized in PBSGT (0.2% gelatin, 0.5% TritonX-100 in PBS, 0.01% thimerosal) for 3 days with rotation at RT.Then, lobes were incubated with primary antibodies in PBSGT containing 0.1% saponin (10 µg/mL) for 7 days with rotation at RT. Lobes were later rinsed in washing buffer (PBS + 0.5% Triton X-100) 6 times during 1 day at RT with rotation.Then, lobes were incubated with secondary antibodies in PBSGT containing 0.1% saponin (10 µg/mL) at RT for 3 days.Secondary antibody solution was passed through 0.22 µm filter to avoid secondary antibody precipitates in solution.Lobes were rinsed again in washing buffer 6 times during 1 day at RT with rotation.Then, lobes were incubated in 50% Tetrahydrofuran (THF) in H 2 O overnight subsequently with 50%, 80% and 100% THF for 1 h.Next, lobes were incubated in 100% THF overnight and 100% THF 1 h.
Then, lobes were incubated in Dichloromethane (DCM) for 20 to 40 min followed by incubation in Dibenzyl Ether (DBE) solution for at least 4 to 6 h until imaging.Images were taken with UltraMicroscope II (Miltenyi Biotec, Bergisch Gladbach, Germany).

as 2 -
ΔΔCt  , where ΔCt = Ct target gene -Ct housekeeping gene , and ΔΔCt reflects ΔCt of each treatment normalized to control.Thus, the mean value for control is 1, and the value for treatment is shown as the fold change compared to control.Results of three or four independent experiments are shown.

Fisher
Scientific Inc., Waltham, MA, USA).Amplified cDNA was hybridized on Mouse Clariom S arrays (Thermo Fisher Scientific, Waltham, MA, USA).Staining and scanning (GeneChip Scanner 3000 7G) was done according to manufacturer's instructions.The Transcriptome Analysis Console (TAC; version 4.0.1.36;Thermo Fisher Scientific, Waltham, MA, USA) was used for quality control and to obtain annotated normalized SST-RMA genelevel data.Statistical analyses were performed by utilizing the statistical programming environment R (v4.0.4).Genewise testing for differential expression was done employing the limma t-test and regulated gene sets were defined by raw p-value < 0.05.To reduce background, gene sets were filtered using DABG p-value < 0.05 in more than half of the samples in at least one group per comparison.Heatmaps were done in R with pheatmap package.Pathway analysis were generated through the use of Gene Set Enrichment Analysis (GSEA) software (v4.1.0,Broad Institute, Cambridge, MA, USA) and clusterProfiler package in R (version 4.0.4),comparing the Gene Ontology Biological Process (GOBP) database. 2-3Dotplot, barplot and treeplot were used to visualize enriched terms according to the use of ggplot2 package.Array data has been submitted to the GEO database at NCBI (GSE223818).

Figure S6 .
Figure S6.CNP exposure caused airspace inflammation and is not attenuated by p38

In vitro lytic virus protein detection by immunofluorescence staining
Agilent Technologies, Santa Clara, CA, USA) on microscope slides.Slides were dried at RT and stored at 4 °C.Images were taken with Leica Laser Scanning Confocal fluorescence microscope (SP5-II, Leica, Wetzlar, Germany) virus protein expression in mouse lungs was detected by immunohistochemistry (IHC) staining.Briefly, mouse lung tissue was fixed in 4% PFA at 4 °C and washed with PBS two times.Then, lung tissue was embedded in paraffin and cut to 3 μm slices.Deparaffinization and rehydration were done by dipping slices into Xylene, 100% Ethanol, 90% Ethanol, 80% Ethanol, 70% Ethanol in order and rinsed with distilled H 2 O. Slices were then incubated in 1.8% H 2 O 2 -Methanol solution for 20 min to block peroxidase and rinsed with distilled H 2 O, followed by heat-induced epitope retrieval (HIER) with Citrate pH = 6.0 buffer.Later, slices were incubated with 1 x blocking buffer (Rodent Block M; Biocare Medical/Zytomed Systems, Berlin, followed by 2 times Xylene incubation.Finally, slices were mounted with Entellan solution and dried under the chemical hood for at least 1 h before imaging.Design-based stereology was used to analyse IHC slides using a light microscope (Olympus BX51) equipped with a computer-assisted stereological toolbox (newCAST, Visiopharm) running Visiopharm SP5-II, Leica, Wetzlar, Germany) for high resolution images or Zeiss Axio Scan.Z1 (Carl Zeiss, Jena, Germany) for slices scanning.Later analysis was performed with ZEN Blue 2.3 software.For BAL cells cytospin staining, cytospin slides were firstly fixed After washing, slices were mounted with Dako fluorescence mounting medium (Agilent Technologies, Santa Clara, CA, USA).Images were taken either by LEICA confocal fluorescence microscope (