Fig 1.
The HLOs used in this study replicated the 3-dimensional architecture and cell composition of human fetal lungs such as ciliated cell, mesenchymal cells, goblet and club cells. The HLO shown in the left panel was 50 days old. Scale bar left panel 300μm, scale bars in the right three panels are 25μm.
Fig 2.
Dose-dependent RSV infection of HLOs.
Increasing volumes of rrRSV suspension (107 pfu/ml) or sterile medium were used to infect HLOs using a microinjector system. (A) Red fluorescence was expressed 5 days after inoculation by the replication of rrRSV in a dose-dependent manner. (B) After inoculation of control sterile medium or increased amounts of rrRSV suspension (107 pfu/ml) into the of HLOs lumen, little infection was noted at 24 h, whereas increasing infectivity was noted at 48 and 72 hours post-infection. (C) ddPCR detection of viral RNA in culture medium. (D) copy number of RSV nucleocapsid (N) genes in culture medium, confirming a dose-dependent infection.
Fig 3.
RSV effect on key protein expression.
(A) RSV infection increased expression of the Club cells’ marker CC10, as well as the transient receptor potential vanilloid 1 (TRPV1) calcium channel in mesenchymal cells. In contrast, the expression of ciliagenesis marker FoxJ1 was inhibited. While the expression of β2ARs was reduced, its phosphorylated fraction increased after RSV infection. (B) RSV infection did not affect the total expression of the epithelial cells’ marker E-cadherin, smooth muscle cells’ marker actin, mesenchymal cells’ marker vimentin, and basal cells’ marker p63. Graphs below are densitometry of images of a minimum 5 organoids for each control or RSV for each stain.
Fig 4.
RSV-induced Remodeling of cytoskeletal proteins.
(A) At 5 days after RSV infection, immunohistochemical staining for F-actin shows extensive remodeling and rearrangement of the HLO’s cytoskeletal architecture. This pattern is consistent with the role played by actin and profilin in virus-mediated cell fusion and viral maturation. HLOs at 50 and 100 days in culture, infected with 8X103 pfu rrRSV for 72 hours, and immunohistochemical staining for (B) Tubulin and RSV (C) vimentin and RSV and (D) smooth muscle actin and RSV shows viral infection is greater in 50 days HLOs. Scale bars 25μm.
Fig 5.
Proteomic analysis of RSV-infected organoids.
Heatmaps of dysregulated proteins exhibiting ≥2-fold change in RSV-infected vs. non-infected control organoids, using discrete color to represent proteins abundance after log10-transform. (A) Proteins are uniquely identified in either control or RSV groups. Blue label means no signals were being captured for the corresponding proteins. (B) Proteins were identified in both control and RSV groups but with at least two-fold changes between groups.
Fig 6.
Gene ontology annotation of differentially expressed proteins, and PANTHER pathway analysis.
(A) The 55 proteins exhibiting significant changes were searched against GO categories from the UniProt database, including biological process, molecular function, and cellular component. Proteins were counted in each ontology they belonged to, and percentages indicate the proportion of the 55 proteins belonging to each class. Bar charts indicate the number of proteins differentially expressed after RSV infection that belong to a specific PANTHER (B) Protein Class ontology or (C) Pathway. Three proteins (cofilin-1, profilin-1, and tubulin β-chain) were associated with the “Cytoskeletal Regulation by Rho GTPase” pathway. Five other pathways matched two proteins, and 19 pathways matched a single protein.
Table 1.
Primary antibodies used for immunofluorescence experiments.