key: cord-0332498-t6rexhmh
authors: Brügger, Melanie; Démoulins, Thomas; Barut, G. Tuba; Zumkehr, Beatrice; Oliveira Esteves, Blandina I.; Mehinagic, Kemal; Haas, Quentin; Schögler, Aline; Rameix-Welti, Marie-Anne; Eléouët, Jean-François; Moehrlen, Ueli; Marti, Thomas M.; Schmid, Ralph A.; Summerfield, Artur; Posthaus, Horst; Ruggli, Nicolas; Hall, Sean R. R.; Alves, Marco P.
title: Pulmonary mesenchymal stem cells are engaged in distinct steps of host response to respiratory syncytial virus infection
date: 2021-07-04
journal: bioRxiv
DOI: 10.1101/2021.05.12.443770
sha: 97efc9d17db73ecd3cd5fcc0f35a0b35cf55591e
doc_id: 332498
cord_uid: t6rexhmh

Lung-resident (LR) mesenchymal stem and stromal cells (MSCs) are key elements of the alveolar niche and fundamental regulators of homeostasis and regeneration. We interrogated their function during virus-induced lung injury using the highly prevalent respiratory syncytial virus (RSV) which causes severe outcomes in infants. We applied complementary approaches with primary pediatric LR-MSCs and a state-of-the-art model of human RSV infection in lamb. Remarkably, RSV-infection of pediatric LR-MSCs led to a robust activation, characterized by a strong antiviral and pro-inflammatory phenotype combined with mediators related to T cell function. In line with this, following in vivo infection, RSV invades and activates LR-MSCs, resulting in the expansion of the pulmonary MSC pool. Moreover, the global transcriptional response of LR-MSCs appears to follow RSV disease, switching from an early antiviral signature to repair mechanisms including differentiation, tissue remodeling, and angiogenesis. These findings demonstrate the involvement of LR-MSCs during virus-mediated acute lung injury and may have therapeutic implications. AUTHOR SUMMARY This work identifies a novel function of lung-resident MSCs during virus-induced acute lung injury. These findings contribute to the understanding of host response and lung repair mechanisms during a highly prevalent clinical situation and may have therapeutic implications.

To determine if LR-MSCs can be a target for RSV in vivo, we designed a FCM assay allowing the 200 identification of the pulmonary epithelial (CD31 -CD45 -panCTK + ) and mesenchymal (CD31 -CD45 -201 panCTK -CD29 + CD44 + ) compartments, as well as detection of RSV infection in vivo (Fig. 4A) . In 202 13-80% of the infected animals, both RSV-positive epithelial cells and LR-MSCs were detected 203 in the lung cell suspensions 3, 6 and 14 days p.i. (Fig. 4B, S6) . When RSV-positive LR-MSCs 204 were plotted as a function of RSV-positive epithelial cells, a significant positive association was 205 found, suggesting that the spread of RSV infection to LR-MSCs is linked to the extent of 206 replication in the pulmonary epithelium (Fig 4C) . Next, MSCs derived from lung tissue and BALs, 207

were isolated from each animal at the different time points p.i. and expanded in culture. When 208 testing the presence of RSV within the lung-derived MSC cultures, viral RNA was detectable in 209 most cultures from the animals at 3 and 6 days p.i. and in none of the cultures isolated from 210 animals at 14 and 42 days p.i. In line with the higher viral loads in the BAL cellular fraction 211 compared to the lung tissue, we detected high levels of RSV RNA in all BAL-derived MSCs from 212 animals at 3 and 6 days p.i. and from 3 out of 7 animals isolated at 14 days p.i. RSV RNA levels 213 were undetectable in all cultures isolated from animals at 42 days p.i. (Fig. 4D) . Interestingly, we 214 noticed giant multinucleated cells in cultures derived from infected animals which were never seen 215 for mock animal-derived cultures (Fig. 4E) . Given that RSV spread in foci and infectious virus 216 was rarely detected in the supernatants of infected human and ovine LR-MSCs cultures, we 217 hypothesized that these phenotypically distinct cells were RSV-infected MSCs. This was 218 confirmed by confocal microscopy visualization (Fig. 4F) and by the infection of cultures from 219 human and ovine LR-MSCs with RSV-mCherry (Fig. S7A, B) . Together, these results indicate 220 that the lung MSC compartment is a target for RSV infection in vivo during the early phase of 221 respiratory disease. 222 convalescence phases of RSV disease. As a mild to asymptomatic RSV disease control, adult 227 animals were infected for a period of 14 days (Fig. 5A) . Contrary to RSV-infected neonates, 228 infection of adults did not lead to any notable clinical manifestation nor macroscopic lesions, while 229 rare foci of interstitial thickening with leukocyte infiltrates and mild alveolar type 2 hyperplasia 230 were visible histologically (Fig. 5B) . A colony-forming unit-fibroblast (CFU-F) assay was applied 231 to lung-derived cells from infected and mock-infected animals. This assay is commonly used to 232 assess the proliferative activity of MSCs and their ability to form discrete fibroblast-like colonies 233 [37, 38] . The analysis revealed a significant effect of RSV infection on the proliferative properties 234 of LR-MSCs by increasing their activity already 3 days p.i. compared to mock control (Fig. 5C) . 235

Quantitative analysis confirmed significantly increased CFU-F counts during the acute phase of 236 infection 3 and 6 days p.i. Interestingly, there was still a nonsignificant tendency of more CFU-F 237 counts during later phases of RSV disease 14 and 42 days p.i. compared to age-matched non-238 infected animals (Fig. 5D) . CFU-F counts were around 20 times lower for control adults (average 239 CFU-F count of 0.6) in comparison to control neonates (average CFU-F count of 14.2), indicating 240 an age-dependent effect on the steady-state proliferative properties of LR-MSCs. However, RSV 241 infection led to a significant increase of these colonies 14 days p.i. with an average count of 6.3 242 (average CFU-F count of 29.9 for infected neonates 14 days p.i.) (Fig. 5E, F) LR-MSCs isolated from infected animals long after viral clearance revealed processes such as 292 myogenesis, epithelial-mesenchymal transition (EMT), angiogenesis, p53 pathway, and TGF-β 293 signaling, suggesting that LR-MSCs are involved in tissue repair and regeneration following virus-294 induced injury (Fig. 6E) . To sum up, these results show that LR-MSCs, are activated during virus-295 induced lung injury by an increase in their proliferative activity and by mounting dynamic 296 transcriptional profiles leading to an early antiviral and inflammatory response followed by 297 mechanisms associated with tissue remodeling, repair and regeneration (Fig. 6F) The ovine lung is a classical model of the human respiratory tract due to similarities in size, 384 structure, development and immune system [71] . However, some limitations should be 385 mentioned. First, we observed a high animal-to-animal variability in most endpoints measured, 386 probably due to the outbred nature of the animals. While this might look like a limitation, we believe 387 it reflects best the situation in humans compared to inbred models. Secondly, we used the isolated 388

This might explain why we didn't detect RSV-positive cells for some animals for whom productive 392 infection was demonstrated by the assessment of the viral loads. 393

In summary, our data demonstrate the involvement of pulmonary MSCs during respiratory virus 394 infection. While being a target for RSV, these cells can respond to infection by switching to an AgPath-ID™ One-Step RT-PCR Reagents (ThermoFisher), according to the manufacturer's 500

instructions. The data were analyzed using the SDS software (Applied Biosystems). Relative copy numbers were interpolated from a standard curve generated with the serial dilution of a 504 plasmid containing the cDNA of the RSV L gene or the housekeeping 18S rRNA. The sequence 505 of all the primers and probes is summarized in Table S3 . Table S4 . Confocal microscopy analysis was performed with a Nikon Eclipse Ti 544 microscope (Nikon). All images were acquired using a 63X oil-immersion or a 40X objective. The 545 images were analyzed with IMARIS 7.7 software (Bitplane) with threshold subtraction and gamma 546 correction. To monitor the spread of RSV-mCherry infection in WD-AEC in comparison to MSC 547 cultures, a Nikon BioStation CT was used (Nikon). The cultures were followed over a period of 2 548 days with a 10X objective and images were acquired in an automated sequence every 4 to 8 549 hours following infection. Reporter expression of RSV-mCherry-infected pediatric LR-MSCs, 550 ovine LR-MSCs, and RSV-GFP-infected ovine PCLS was assessed using an Evos FL Auto 2 cell 551 imaging system (ThermoFisher) or a Leica TCS-SL, respectively. The micrographs from the trans-552 differentiation assays were captured using an inverted microscope ECLIPSE TS100 with a Nikon 553 DS-Fi3 camera using a DS-L4 application v.1.5.03 (all form Nikon). Lung specimens were fixed in 4% buffered formalin for 48 h, embedded in paraffin and routinely 587 processed for histology. Histological sections of 3 µm were stained with hematoxylin and eosin 588 coloration and observed by light microscopy. For immunohistochemistry, deparaffinization of the 589 sections was done with xylol for 5 minutes followed by rehydration in descending concentrations 590 of ethanol (100, 95, 80, and 75%). H2O2 (3.25% in methanol, 10 min at room temperature) 591 inhibited endogenous peroxidase activity. Then, the slides were incubated in boiling citrate buffer 592 (pH 6.0) for 10 min for antigen retrieval. 1% BSA (10 min) was used for blocking of nonspecific 593 antibody binding, followed by an overnight incubation at 4°C with the primary antibody targeting 594 RSV (ThermoFisher). For secondary antibody incubation and signal detection LSAB and AEC 595 Kits (DakoCytomation) were used following the manufacturers protocol. Counterstaining was 596 done with Ehrlich hematoxylin and cover slips were mounted using Aquatex (Merck) [77] . 597 598

One specimen per lung region (cranial, middle, and caudal) were pooled and dissociation was 600 done using a collagenase I and II and a DNase I enzyme mix (all from BioConcept) and the 601 gentleMACS Octo Dissociator (Miltenyi Biotec). Following this mechanical and enzymatical 602 dissociation, the samples were applied to a sieve, to remove any remaining particulate matter. 603

The cell suspensions were passed through cell strainers (100 and 70 µm pore-size, Falcon) and 604 centrifuged at 350g for 10 min at 4°C to obtain single-cell suspensions. For the isolation of cells 605 from BALs, the lungs were isolated with the trachea, which was clamped before cutting, to prevent 606 blood from entering the lungs. Then, a PBS-containing antibiotic solution with 100 units/ml of 607 penicillin and 100 µg/ml streptomycin (both Sigma), and 2.5 µg/ml Amphotericin B was poured 608 into the lungs through a sterile funnel (200-500 ml). The cell suspensions were then passed 609 through cell strainers (100 and 70 µm pore-size, Falcon) and centrifuged at 350g for 10 min at 610

4°C to obtain single cell suspensions. If needed, red blood cells were lysed by resuspending the 611 pellet with 2 ml of H2O and washed immediately in cold PBS before centrifugation at 350g for 10 at 37°C with 5% CO2 until reaching ~80% of confluence. Regular media changes were performed 619 twice a week. 620 621 Colony-forming unit-fibroblast assay 622

Lung single-cell suspensions were seeded at a density of 3.5x10 4 cells per cm 2 in a six-well plate 623 as described previously [3] . Cells were fixed after 7-14 days with ice-cold methanol for 20 min 624 and then washed with PBS. The cells were stained with Giemsa stain for 6 min, rinsed with H2O, 625 and air-dried. Images were captured using an ImmunoSpot analyzer (CTL). Two experienced 626 investigators performed the CFU-F counts independently. 627

For mRNA sequencing, total RNA was extracted from ovine LR-MSCs using TRIzol reagent 630 (ThermoFisher) in combination with the Nucleospin RNA Kit (Machery-Nagel) as previously 631 described [78] . In short, cells were lysed with 1 ml of TRIzol reagent and kept at -70°C until further 632 processing. After thawing, 0.2 ml chloroform was added to the TRIzol lysate and the samples 633 were mixed vigorously and incubated for 2-3 min at room temperature. The extractions were then 634 centrifuged at 12'000g for 15 min at 4°C. The aqueous phase was collected and mixed with 500 635 µl 75% ethanol and the RNA precipitated for 10 min at room temperature. The RNA precipitate 636 was further purified with the Nucleospin RNA kit according to the manufacturer's instructions. The 637 quantity and quality of the extracted RNA was assessed using a ThermoFisher Scientific 

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PubMed PMID: 26771021; PubMed Central 1037 PMCID: PMCPMC4707969. 1038 1039 1040 on pediatric LR-MSCs. (B) Representative phase-contrast (PC) micrograph showing morphology 1044 in culture and demonstrates plastic adherence

A) 1046 differentiation, respectively. Magnification 40X (PC, O) and 200X (C, A). (C) mRNA expression 1047 levels of RSV receptors relative to 10 6 18S. A Mann-Whitney U test was applied to compare the 1048 two cell types (AECs versus LR-MSCs). Boxplots indicate the median value (centerline) and 1049 interquartile ranges

Each symbol represents an individual donor (n=5). *p<0.05, **p<0.01. (D) Intracellular viral loads 1051 in AECs and LR-MSCs over time following infection with RSV-A2 at a MOI of 1 PFU/cell 1052 expressed as RSV copies per 10 12 18S copies

LR-MSCs were 1054 infected with 0.1 PFU/cell with RSV-A2 (n=3) or a clinical isolate RSV-ON1-H1 (n=4-6)

MSCs infected with 0.1-0.5 PFU/cell with RSV-mCherry and followed over time. The micrographs 1057 were taken at 16, 32, and 48 h p.i

confocal microscopy evaluation of LR-MSCs noninfected (mock) or infected with RSV-A2 at 1

PFU/cell 24 to 72 hours p.i. RSV, green; DAPI

Scale bar

Supernatants of infected LR-MSCs or apical washes of infected WD-AEC cultures were analyzed 1061 by a PFU assay. Cells were infected with RSV-ON1-H1 at a MOI of 0.1 PFU/cell

U test was applied to compare the two cell types (WD-AECs, n=3 versus LR-MSCs, n=5-6)

Extracellular RSV RNA load over time in supernatants of infected LR-poly(I:C) 10 µg/ml, RSV-A2, or RSV-ON1-H1 for 24 h at 1 PFU/cell. Boxplots indicate the median 1073 value (centerline) and interquartile ranges (box edges), with whiskers extending to the lowest and 1074 the highest values

The data were compared with the Kruskal-Wallis test followed by

) and IFN-l1/3 (D) protein levels in 1077 supernatants of LR-MSCs treated with mock control, poly(I:C) 10 µg/ml, RSV-A2, or RSV-ON1-1078 H1 for 24 h and 72 h at 1 PFU/cell. Boxplots indicate median value (centerline) and interquartile 1079 ranges (box edges), with whiskers extending to the lowest and the highest values. Each symbol 1080 represents an individual donor (mock, n=6; poly(I:C), n=5

The detection limits of the assays are indicated with the 1083 dotted line at 7.7 pg/mL and 79.8 pg/mL for IFN-b and IFN-l1/3, respectively. (E, F) Multiplex 1084 assay of supernatants of LR-MSCs (E) or basolateral medium of WD-AECs (F), 24 and 72h after 1085 treatment with mock control, 10 µg/ml poly(I:C)

The concentration ranges of the different cytokines are indicated (lower detection limit-1087 highest concentration measured). An asterisk is present when the concentration of the sample 1088 was higher than the upper range of the assay. Each column represents a different donor (mock, 1089 n=6; poly(I:C), n=5

RSV-mediated 1091 activation of LR-MSCs, characterized by an antiviral and pro-inflammatory phenotype combined 1092 with cytokines promoting T helper cell (Th) polarization. and RSV-infected lambs at 3, 6, 14, and 42 days p.i. Scale bar, 200 µm. (C) Histological lung 1100 sections from animals 6 days p.i. stained for RSV (red) and counterstained with haematoxylin 1101 (blue). From left to right panels, scale bars 50 µm, 100 µm and 20 µm, respectively. (D) Viral load 1102 in lungs and the cellular fraction of the BAL of

Comparison of viral loads between BAL and lung tissues was done with a one-way ANOVA and 1104 the Tukey post-hoc test. Each symbol represents an individual animal

F) Frequency of cleaved caspase 3 (CASP3)-positive cells in the lung (E) and BAL 1106 (F) of infected animals 3 to 42 days p.i. Boxplots indicate the median value (centerline) and 1107 interquartile ranges

Multiple comparison 1109 was done with a one-way ANOVA and the Tukey post-hoc test. *p<0.05. (G) Total BAL cell counts 1110 in mock and RSV-infected animals 3, 6, and 14 days p.i. pooled. Boxplots indicate the median 1111 value (centerline) and interquartile ranges (box edges), with whiskers extending to the lowest and 1112 the highest values

CD31 -CD45 -panCTK + ) (panCTK, 1117 pan-cytokeratin) and LR-MSCs (CD31 -CD45 -panCTK -CD29 + CD44 + ). (G1, gate 1) (B) Percentage 1118 of animals with RSV-positive epithelial and LR-MSCs 3 to 42 days p.i. The fractions indicate the 1119 number of infected animals where infection was detected by FCM compared to the total number Representative confocal microscopy evaluation of LR-MSCs derived from BALs expanded in 1128 culture from noninfected (mock) or infected

Scale bars, 15 µm (left and middle panels) and 10 µm for the 3D capture 1130

RSV infection leads to the expansion of the pulmonary MSC niche in vivo 1133 (A) Newborn or adult (average age of 29 months) animals were trans-tracheal

Newborns were euthanized 3, 6, 14, 1135 and 42 days p.i. and adults 6 and 14 days p.i. and lung tissue was harvested for CFU-F assay

Lung tissue of adults was harvested for histopathological evaluation (B) Representative H&E 1137 stained histopathological sections of the lung tissue from noninfected (mock) and RSV-infected 1138 adults at 6 and 14 days p.i. scale bar, 200 µm. (C, E) Representative images of the CFU-F assay 1139 for mock control and RSV-infected neonates (C) and adults (E) 3 to 42 days p.i. and 6 and 14 1140 days p.i., respectively. Each image represents an individual animal

CFU-F assay for each neonate (D) and adult (F) animals, given as CFU-F count relative to 1142

33X10 5 nucleated cells over time. Boxplots indicate the median value (centerline) and 1143 interquartile ranges

Each symbol represents an individual animal (neonates, per timepoint: mock

8; adults: mock, n=14; RSV, n=3 per timepoint). A one-way ANOVA and the Holm-Sydak post-1146 hoc test was applied to compare differences between groups

Normalized enrichment score of significant (false discovery rate, FDR<0.05) hallmark gene 1155 sets for the comparisons of RSV infection and mock control 6, 14, and 42 days p.i. 1156 (downregulated, blue and upregulated, red) (F) Cartoon summarizing the different functions of

LR-MSCs during distinct steps of RSV disease

Poly (I:C)

These authors contributed equally to this work # Acknowledgment Mock RSV