Human Vascularized Macrophage-Islet Organoids to Model Immune-Mediated Pancreatic β cell Pyroptosis upon Viral Infection

Abstract

There is a paucity of human models to study immune-mediated host damage. Here, we utilized the GeoMx spatial multi-omics platform to analyze immune cell changes in COVID-19 pancreatic autopsy samples, revealing an accumulation of proinflammatory macrophages. Single cell RNA-seq analysis of human islets exposed to SARS-CoV-2 or Coxsackievirus B4 (CVB4) viruses identified activation of proinflammatory macrophages and β cell pyroptosis. To distinguish viral versus proinflammatory macrophage-mediated β cell pyroptosis, we developed human pluripotent stem cell (hPSC)-derived vascularized macrophage-islet (VMI) organoids. VMI organoids exhibited enhanced marker expression and function in both β cells and endothelial cells compared to separately cultured cells. Notably, proinflammatory macrophages within VMI organoids induced β cell pyroptosis. Mechanistic investigations highlighted TNFSF12-TNFRSF12A involvement in proinflammatory macrophage-mediated β cell pyroptosis. This study established hPSC-derived VMI organoids as a valuable tool for studying immune cell-mediated host damage and uncovered mechanism of β cell damage during viral exposure.

Figure 1.. Macrophage accumulation in islets of COVID-19 pancreatic autopsy samples.

(A) Representative images illustrating morphology marker and selection of ROIs using GeoMx platform. 004: islet area; 005: ductal area; 006: exocrine area. Scale bar= 1 mm or 50 μm. (B) Representative images illustrating the insulin (INS) staining in COVID-19 (N=7) and control (N=8) pancreatic autopsy samples. Dotted lines encircle the islet regions. Scale bar=75 μm. (C) Quantification of areas of islets and percentages of INS⁺ β cells per islet in COVID-19 (N=7) and control (N=8) pancreatic autopsy samples. (D) 3D PCA plot of data from human islet areas of COVID-19 (N=7) and control (N=8) pancreatic autopsy samples. (E) Volcano plot of transcriptome sequencing data highlighting the pathways enriched in human islet areas of COVID-19 (N=7) versus control (N=8) pancreatic autopsy samples. (F) Heatmap of the CIBERSORT analysis of immune cells (LM22) using the GeoMx whole transcriptome sequencing data of human islet areas of COVID-19 (N=7) and control (N=8) pancreatic autopsy samples. (G) Normalized counts (Log2) of marker proteins associated with macrophages from human islet areas of COVID-19 (N=7) and control (N=8) pancreatic autopsy samples. Each dot represents one count in each ROI. (H) Box plot of normalized counts of macrophage associated targets in human islet areas of COVID-19 (N=7) and control (N=8) pancreatic autopsy samples. Each dot represents one count in each ROI. (I and J) Immunohistochemistry staining (I) and quantification (J) of CD80 in COVID-19 (N=3) and control (N=3) pancreatic autopsy samples. Dotted lines encircle the regions of the islets. Scale bar=20 μm. P values were calculated by unpaired two-tailed Student’s t test. n.s., no significance; *P < 0.05, **P < 0.01, ***P < 0.001. See also Figure S1.

Figure 2.. Single cell RNA-seq analysis of human islets upon SARS-CoV-2 or CVB4 exposure.

(A) UMAP of immune cell populations in human islets exposed to mock, SARS-CoV-2 (MOI=1) or CVB4 (2X10⁶ PFU/ml) . (B) UMAP and violin plots of immune cell markers. (C) Dot plot analysis of proinflammatory macrophage-associated genes in macrophages of human islets exposed to mock or SARS-CoV-2 (MOI=1). (D and E) Confocal images (D) and quantification (E) of the relative expression of CD80 in CD68⁺ cells in human islets exposed to mock or SARS-CoV-2 (MOI=0.5). The white arrows highlight the CD68⁺CD80⁺ cells. Scale bar= 50 μm. (F) Pathway enrichment analysis of cell death pathways in β cell cluster of human islets exposed to mock or SARS-CoV-2 (MOI=1). (G) Dot plot analysis of pyroptosis associated genes in β cell cluster of human islets exposed to mock or SARS-CoV-2 (MOI=1). (H and I) Confocal images (H) and quantification (I) of the relative expression of CAPS1 in human islets exposed to mock or SARS-CoV-2 (MOI=0.5). The yellow arrows highlight the expression of CASP1 in SARS-N⁺INS⁺ cells while the white arrows highlight the expression of CASP1 in SARS-N⁻INS⁺ cells. Scale bar= 50 μm. (J) Dot plot analysis of proinflammatory macrophage-associated genes in macrophage cluster of human islets exposed to mock or CVB4 (2×10⁶ PFU/ml). (K and L) Confocal images (K) and quantification (L) of the relative expression of CD80 in CD68⁺ cells in human islets exposed mock or CVB4 (2×10⁶ PFU/ml). The white arrows highlight the CD68⁺CD80⁺ cells. Scale bar= 50 μm. (M) Dot plot analysis of pyroptosis pathway associated genes in β cell cluster of human islets exposed to mock or CVB4 (2×10⁶ PFU/ml). (N and O) Fluorescent images (N) and quantification (O) of the relative expression of CAPS1 in human islets exposed to mock or CVB4 (2×10⁶ PFU/ml). The yellow arrows highlight the expression of CASP1 in SARS-N⁺INS⁺ cells while the white arrows highlight the expression of CASP1 in SARS-N⁻INS⁺ cells. Scale bar= 50 μm. N=3 independent biological replicates. Data was presented as mean ± STDEV. P values were calculated by unpaired two-tailed Student’s t test. *P < 0.05, **P < 0.01. See also Figure S2 and S3.

Figure 3.. Construction of hPSC-derived VMI organoids.

(A) Schematic representation of VMI organoids construction. (B) Phase contract images of VMI organoids at day 14 after reaggregation and human islets. Scale bar= 200 μm. (C) Composite Z-stack confocal images of live VMI organoids at day 14 after reaggregation. β cells: INS-GFP; macrophages: RFP; and endothelial cells: Far Red. Scale bar= 200 μm. (D) Composite Z-stack confocal images of VMI organoids at day 14 after reaggregation stained with antibodies against INS, CD68 and PECAM1 (CD31). Scale bar= 100 μm. (E) Composite Z-stack confocal images of VMI organoids at day 14 after reaggregation stained with antibodies against INS, CD68, GCG, SST and PECAM1 (CD31). Scale bar= 100 μm. (F) Transmission electron microscope (TEM) images of human islets, VMI organoids at day 14 after reaggregation, and endothelial cells without reaggregation. Arrows indicate fenestrae. Scale bar= 500 nm. (G) Dynamic glucose stimulated insulin secretion of VMI organoids at day 14 after reaggregation and hPSC-derived endocrine cells. LG (low glucose): 2 mM D-glucose; HG (high glucose): 20 mM D-glucose; KCl: 30 mM KCl. Quantification was performed using the areas under curve of KCl stimulation from 86 min to 90 min. (H) Composite Z-stack confocal images of VMI organoids at day 7 after reaggregation upon CVB4 infection (2×10⁶PFU/ml). β cells: INS-GFP, macrophages: RFP. Scale bar= 50 μm. Arrows highlight RFP⁺ macrophages that have phagocytosed damaged INS-GFP⁺ β cells. N=3 independent biological replicates. Data was presented as mean ± STDEV. P values were calculated by unpaired two-tailed Student’s t test. **P < 0.01. See also Figure S4.

Figure 4.. Single cell multi-omics analysis of VMI organoids.

(A) Integrative UMAP of scRNA-seq and snATAC-seq analysis of VMI organoids at day 7 after reaggregation and separately cultured cells. (B) Dot plot displaying cell markers of each cluster using scRNA-seq dataset. (C) Individual UMAP of scRNA-seq and snATAC-seq analysis of VMI organoids at day 7 after reaggregation and separately cultured cells. (D) Pie chart showed the relative percentages of each cell types in VMI organoids at day 7 after reaggregation. (E) Volcano plot of DE genes in β cell cluster of VMI organoids at day 7 after reaggregation versus separately cultured cells. (F) Dot plot analysis of β cell associated genes in β cell cluster of VMI organoids at day 7 after reaggregation and separately cultured cells. (G) Chromatin accessibility signals of SLC2A1, INS, PDX1 in β cell cluster of VMI organoids at day 7 after reaggregation and separately cultured cells. The normalized signal shows the averaged frequency of sequenced DNA fragments within a genomic region. The fragment shows the frequency of sequenced fragments within a genomic region for individual cells. (H) Dot plot analysis of endothelial cell associated genes in endothelial cell cluster of VMI organoids at day 7 after reaggregation and separately cultured cells. See also Figure S4.

Figure 5.. Construction and multi-omics analysis of VMI organoids containing unstimulated and proinflammatory macrophages.

(A and B) Composite Z-stack confocal images (A) and quantification (B) of INS intensity in INS⁺ cells of VMI organoids at day 7 after reaggregation containing unstimulated or proinflammatory macrophages stained with the antibodies against INS, CD68 and PECAM1 (CD31). Scale bar= 50 μm. (C) Measurements of cytokine secretions in the supernatant of VMI organoids at day 5 after reaggregation containing unstimulated or proinflammatory macrophages. (D) Integrative UMAP of VMI organoids at day 7 after reaggregation containing unstimulated or proinflammatory macrophages. (E) Percentage of cells in β cell cluster in VMI organoids at day 7 after reaggregation containing unstimulated or proinflammatory macrophages. (F) Volcano plot of DE genes in β cell cluster of VMI organoids at day 7 after reaggregation containing proinflammatory versus unstimulated macrophages. (G) Dot plot analysis of β cell identity associated genes in β cell cluster of VMI organoids at day 7 after reaggregation containing unstimulated or proinflammatory macrophages. (H) Dot plot analysis of pyroptosis pathway associated genes in β cell cluster of VMI organoids at day 7 after reaggregation containing unstimulated or proinflammatory macrophages. (I) Chromatin accessibility signals of CASP1, CASP9, IL1B and NLRP3 in β cell cluster of VMI organoids at day 7 after reaggregation containing unstimulated or proinflammatory macrophages. The normalized signal shows the averaged frequency of sequenced DNA fragments within a genomic region. The fragment shows the frequency of sequenced fragments within a genomic region for individual cells. (J and K) Immunostaining (J) and quantification (K) of CASP1 staining in INS+ cells of VMI organoids at day 7 after reaggregation containing unstimulated or proinflammatory macrophages. β cells: INS-GFP, macrophages: RFP, endothelial cells: Far Red. CASP1: grey. Scale bar= 25 μm. N=3 independent biological replicates. Data was presented as mean ± STDEV. P values were calculated by unpaired two-tailed Student’s t test. *P < 0.01, **P < 0.05, ***P < 0.001. See also Figure S5.

Figure 6.. TNFSF12-TNFRSF12A as a candidate pathway that contributes to proinflammatory macrophage-mediated β cell pyroptosis.

(A) Dot plot showed the differential signaling from macrophages to β cells in VMI organoids containing unstimulated or proinflammatory macrophages at day 7 after reaggregation. (B) Dot plot showed the differential signaling from macrophages to β cells in human islets exposed to mock or CVB4 virus (2×10⁶ PFU/ml). (C) Dot plot of the expression level of TNFSF12 in macrophage of human islets exposed to mock or SARS-CoV-2 virus (MOI=1). (D and E) Confocal images (D) and quantification (E) of CASP1 expression in INS⁺ cells in control or 10 ng/ml TNFSF12 treated human islets. Scale bar= 25 μm. (F and G) Confocal images (F) and quantification (G) of CASP1 expression in INS⁺ cells in control or 10 ng/ml TNFSF12 treated VMI organoids at day 7 after reaggregation. Scale bar= 50 μm. (H and I) Confocal images (H) and quantification (I) of CASP1 expression in INS⁺ cells of SARS-CoV-2 (MOI=0.5) or CVB4 (2×10⁶ PFU/ml) exposed human islets treated with control, 10 μg/ml TNFSF12 blocking antibody, 5 μg/ml IL-1β blocking antibody or 10 μg/ml TNFSF12 + 5 μg/ml IL-1β blocking antibodies. Scale bar= 25 μm. (J and K) Confocal images (J) and quantification (K) of CASP1 expression in INS⁺ cells of VMI organoids containing proinflammatory macrophages at day 7 after reaggregation and treated with control, 10 μg/ml TNFSF12 blocking antibody, 5 μg/ml IL-1β blocking antibody or 10 μg/ml TNFSF12 + 5 μg/ml IL-1β blocking antibodies. Scale bar= 50 μm. (L and M) Confocal images (L) and quantification (M) of the CASP1 expression in INS⁺ cells of pancreas autopsy samples from control (N=3) and COVID-19 (N=4) subjects. The insert shows a high magnification of cells. Scale bar= 50 μm. N=3 independent biological replicates. Data was presented as mean ± STDEV. P values were calculated by unpaired two-tailed Student’s t test or one-way ANOVA with a common control. n.s., no significance; *P < 0.05, **P < 0.01, ***P < 0.001. See also Figure S6.