Figures and data
Rubella virus infects primary human microglia in cultured brain slices.
A. Schematic for brain slice infection. Mid-gestation (GW18-23) human brain slices were infected with RV for 72 hours. B,C. Immunostaining for RV capsid and Iba1 on cultured cortical slices at 72 hpi, at 10x with scale bar 100 μm (B) and at 40x objective with scale bar 50 μm (C). D. Quantification of RV capsid-positive cells co-labeled with microglial marker Iba1: 764/819 (93.3%) of RV+ cells were microglia based on Iba1 staining across four biological replicates. Error bars denote standard deviation. E. Diagram of viral genome of GFP-expressing RV (RV-GFP). Cortical brain slices were infected with RV-GFP for 72 hours. F. Examples of GFP fluorescence and Iba1 immunostaining at 72 hpi of cultured cortical slices with GFP-RV, at 62x with scale bar 20 μm. GFP expression of modified rubella virus is localized to Iba1-positive microglia cells (arrows).
Rubella virus infection of microglia is dependent on the presence of other cells.
A. Schematic of rubella infection. Primary prenatal brain tissue was dissociated and different cell types were purified using MACS. Microglia cells were cultured alone or in combination with neurons, glial cells, or all cell types. 2D cultures were infected with RV for 72 hours and processed for immunostaining. B-E. Representative images of microglia cultured with different cell types. Cell cultures were stained for microglia marker Iba1 (red), RV capsid (green) and DAPI (grey; on the overlay Merge channel). B. Purified microglia only. C. Microglia and neurons (purified with PSA-NCAM magnetic beads) co-cultured at 1:5 ratio. D. Microglia and glial cell types (flow through fraction after PSA-NCAM magnetic beads) cultured together at 1:5 ratio. E. Microglia cultured with non-microglial cells (flow-through after CD11b magnetic beads; mixed cell populations) at 1:5 ratio. F. Quantification of RV capsid immunopositivity among microglia (Iba1+) for conditions in B-E. FT: flow through after microglia MACS purification. Error bars denote SEM. Each data point represents a field of view from the same experimental batch. G. Quantification of microglia (Iba1+) among RV capsid-positive cells.
Direct cell-cell contact is not required for microglia infection by rubella virus.
A. Schematic for experimental set up. Primary human brain tissue was dissociated, and microglia were cultured with or without microglia-depleted flow through portion. Cells were co-cultured in direct contact or in solution-permeable chambered transwells (TW). B. Representative images of microglia-enriched cultures (top row), microglia cultured with other cell types in the same well (middle row), and microglia cultured in the bottom compartment with other cell types cultured in a permeable transwell chamber (bottom row) infected with RV for 72 hours. C. Quantification of RV capsid immunopositivity among microglia (Iba1+). Three fields of view across the same experiment were quantified for each condition. Error bars represent SEM. p-value between microglia and co-culture condition is 0.0479. p-value between microglia and trans well condition is 0.0159. D. Quantification of microglia (Iba1+) among RV capsid-positive cells.
Microglia in neuroimmune organoids are infected with rubella virus.
A. Primary human microglia were transplanted into brain organoids and resulting neuroimmune organoids were exposed to RV. After 72 hours or 2 weeks organoids were processed for immunofluorescence validation (this figure) or scRNAseq analysis (Figure 5). B. Representative immunofluorescence images of brain organoids without microglia subjected to RV exposure for 72 hrs. Radial glial cells are labeled with Sox2 (cyan), microglia are labeled with Iba1 (red) and RV is labeled with anti-RV capsid antibody (green). Scale bar is 50 μm. C. Representative immunofluorescence images of brain organoids with microglia at 72 hrs (top panel) or 2 weeks (bottom panel) after RV exposure. Radial glial cells are labeled with Sox2 (cyan), microglia are labeled with Iba1 (red) and RV is labeled with anti-RV capsid antibody (green). Dashed boxes represent zoomed-in examples of microglia cells. Scale bar is 50 μm.
Rubella virus exposure of brain organoids leads to interferon response.
A. Single cell RNA sequencing analysis identified 13 clusters, including neurons and glial cells (Div.: dividing cells, RG: radial glia, Astros: astrocytes, IPC: intermediate progenitor cells). B. Dot plot depicting cluster marker genes for each cluster. C. UMAP plots of organoids colored by the presence or absence of microglia. D. UMAP plots of organoids colored by the presence or absence of RV treatment. E. Feature plot for expression levels of IFITM3. F. IFITM3 expression in all cells across different conditions. G. Representative images of IFITM3 immunofluorescence in brain organoids with microglia with wild type RV (bottom panel) or heat-inactivated control (top panel) 72 hours post-infection. IFITM3 is labeled in magenta, microglia are labeled with Iba1 (green), cell nuclei are labeled with DAPI (grey). H. Quantification of fluorescence intensity of IFITM3 normalized to DAPI intensity per organoid. Columns represent mean of four organoids. Dots represent averages across several section for each individual organoid. Error bars represent SEM. Unpaired parametric student t-test was used to compare the two groups in H-I. p-value=0.04 I. Quantification of fluorescence intensity of DAPI staining per organoid. p-value=0.22.
NOVA1 expression is reduced in response to rubella virus exposure.
A. Differentially expressed genes in different cell types in response to RV treatment without (top panel) and with microglia (bottom panel). IPC – intermediate progenitor cells. In the presence of microglia, fewer differentially expressed genes in response to RV treatment were identified across all major cell types. In organoids with microglia, NOVA1 trended towards a decrease in IPC and neurons (labeled in blue in the panel). Kolmogorov-Smirnov test was used on DEGs with p-value<0.05. *** <0.001, NS – not significant, * <0.05. B. Violin plot for NOVA1 that is differentially expressed in response to RV and presence of microglia. IPC – intermediate progenitor cells, RG – radial glia, Div. – dividing cells, EN – excitatory neurons. C. Representative images of RV-exposed organoids with microglia at 2 weeks post-exposure, stained with DAPI for cell nuclei (blue), NOVA1 (magenta), NeuN for neurons (green) and EOMES for intermediate progenitor cells (cyan). D. Cell number quantification for NeuN+ neurons that were also positive for NOVA1 in control (heat-inactivated RV) or RV condition. Unpaired parametric student t-test was used to compare the two groups in D-G. p-value=0.088. E. Cell number quantification for EOMES+ intermediate progenitors that were also positive for NOVA1 in control (heat-inactivated RV) or RV condition. p-value=0.0042. F. Cell number quantification for NeuN+ neurons per organoid area displayed in 1,000cells x mm2 or organoid surface area in control (heat-inactivated RV) or RV condition. p-value=0.0004. G. Cell number quantification for EOMES+ intermediate progenitor cells per organoid area displayed in 1,000cells x mm2 or organoid surface area in control (heat-inactivated RV) or RV condition. p-value=0.86.
