A skin organoid-based infection platform identifies an inhibitor specific for HFMD

Abstract

The EV-A71 poses a serious threat to the health and lives of children. The EV-A71 can be transmitted by direct and indirect skin contact. Therefore, there is an urgent need to create novel skin models using human-derived cells to study the biology and pathogenesis of the virus and facilitate drug screening. Here, we use human induced pluripotent stem cells-derived skin organoids (hiPSC-SOs) as a model for EV-A71 infection and find that multiple cell types within the skin organoids, including epidermal cells, hair follicle cells, fibroblasts, and nerve cells, express EV-A71 receptors and are susceptible to EV-A71 infection. We elucidate the specific response of different cell types to EV-A71 and reveal that EV-A71 infection can degrade extracellular collagen and affect fibroblasts. We find that EV-A71 can mediate epidermal cell damage through autophagy and Integrin/Hippo-YAP/TAZ signaling pathways, thereby promoting hyperproliferation of progenitor cells. Based on this finding, we identify an autophagy-associated protein as a drug target of EV-A71 and discover an EV-A71 replication inhibitor. Altogether, these data suggest that hiPSC-SOs can be used as an infectious disease model to study skin infectious diseases, providing a valuable resource for drug screening to identify candidate virus therapeutics.

Fig. 1. hiPSC-SOs are susceptible to EV-A71 infection.

A Workflow of EV-A71 virus-infected human skin organoids (hiPSC-SOs) and proteomics analysis. B Immunofluorescence of EV-A71 virus protein in the infected hiPSC-SOs (scale bar: 50 μm). C Evaluation of the EV-A71 viral infection efficiency at 2, 4, and 8 days post-infection (dpi). The quantification analysis is performed from three images for each group. The data are shown as the mean ± SD (n = 3 per group). Significant differences are determined by unpaired two-tailed t test. D Whole-mount staining of EV-A71 virus protein KRT17, and TUJ1 in one side and the other side of infected hiPSC-SOs at 8 dpi (scale bar: 200 μm). E Immunofluorescence of EV-A71 virus protein with different cellular markers, KRT71, KRT82, TUJ1, and S100, in infected hiPSC-SOs at 8 dpi (scale bar: 20 μm). The white triangles indicate where the viruses co-localize with corresponding cellular markers. The immunofluorescence experiments are repeated three times. hiPSC-SOs: human induced pluripotent stem cell-derived skin organoids. Source data are provided as a Source Data file.

Fig. 2. Quantitative expression profiles and biological characteristics of proteins identified in EV-A71-infected hiPSC-SOs.

A Volcano plots of –log10 p value vs. log2 protein abundance comparisons between the EV-A71-infected hiPSC-SOs (2, 4, and 8 dpi) and control hiPSC-SOs (0 dpi) (n = 3 for each group). The pairwise comparisons are performed by the two-sided moderated t test to identify the DEPs between the hiPSC-SOs with EV-A71 infection at 2 (n = 3), 4 (n = 3), and 8 (n = 3) dpi and the hiPSC-SOs without EV-A71 infection (0 dpi, n = 3). The DEPs between EV-A71-infected and control samples are determined based on a BH adjusted p value < 0.01 and log2 (EV-A71 infected/Control) > 0.585 (upregulated) or < -0.585 (downregulated), and labeled in red (upregulated) or blue (downregulated), respectively. B Biological process enrichment analysis of proteins that are specifically highly and lowly expressed in EV-A71-infected hiPSC-SOs at 2 (n = 3), 4 (n = 3), and 8 (n = 3) dpi compared to the controls (0 dpi, n = 3). The specific highly and lowly expressed proteins are determined with a BH -adjusted p value < 0.01 and log2FC > 0.585 or < -0.585, respectively. The fold change (FC) represents the ratio of the normalized intensity of the protein identified in the hiPSC-SOs at the indicated infection time to those at all other samples. The enrichment analysis is performed using the one-sided hypergeometric test and the p values are provided in source data. The enriched terms of high and low expressed proteins are indicated in red and blue, respectively. The size of the dot indicates the number of proteins belonging to each term. The color scale indicates the enrichment p value. C Expression heatmap of the proteins that are involved in specific biological functions of EV-A71-infected hiPSC-SOs at 0, 2, 4, and 8 dpi. The red and blue boxes indicate proteins with increased and decreased abundance levels, respectively. The clusters of proteins associated with similar biological processes are grouped and labeled. BH: Benjamin–Hochberg; DEP: differentially expressed protein; dpi: days post-infection; hiPSC-SOs: human induced pluripotent stem cell-derived skin organoids. Source data are provided as a Source Data file.

Fig. 3. EV-A71 infection depletes reticular fibroblasts.

A UMAP plots of scRNA-seq data generated from EV-A71-infected hiPSC-SOs at 0 and 8 dpi. A total of 33,971 cells are represented. The major cell groups are manually annotated and labeled with different colors. FB: fibroblast; NC: nerve cell; EpC: epidermal cell; Mel: melanocyte; Mer: merkel cell; Cho: chondrocyte; PC: progenitor cell. B Expression and percentage distribution of the key gene markers of different cell clusters. The statistical significance of cell type marker genes is determined using the two-sided Wilcoxon rank-sum test with Bonferroni-adjusted p value < 0.05. C Gene expression levels of key receptors (ANXA2, FN1, NCL, PHB2, PPIA, SCARB2, and VIM) involved in EV-A71 entry in scRNA-seq data of FB (n = 22,649), NC (n = 3,712), EpC (n = 4,779), Mel (n = 1,077), Mer (n = 639), Cho (n = 336), and PC (n = 599). Boxes represent the median values and the first and third quartiles. The upper and lower whiskers represent ranges that extending up to 1.5 the interquartile range. The outliers with the values below the first quartile or above the third quartile are represented with stars. D The proportion of each cell type in normal control and EV-A71-infected hiPSC-SOs. E Biological process enrichment analysis of upregulated genes between EV-A71-infected and normal hiPSC-SOs in proliferative fibroblast and reticular fibroblast. The DEGs between EV-A71-infected and control hiPSC-SOs are determined based on a BH-adjusted p value of <0.05 and log2 (EV-A71 infected/Control) > 0.25 (upregulated). The enrichment analysis is performed using the one-sided hypergeometric test and the p values are provided in source data. F Mechanism diagram of EV-A71 infecting reticular fibroblasts. When reticular fibroblasts are infected by viruses, TNF-mediated signaling pathway (TNFR1, CREB3, and C/EBPβ) is activated, producing pro-inflammatory factors (IL6 and CXCL8) and MMPs (MMP1, MMP3, and MMP13) that metabolize extracellular collagen. Ultimately, skin inflammation and collagen loss combine to cause the onset of skin aging. BH, Benjamin–Hochberg; DEG: differentially expressed gene; dpi: days post-infection; hiPSC-SOs: human induced pluripotent stem cell-derived skin organoids. Source data are provided as a Source Data file.

Fig. 4. EV-A71 infection promotes NNMT + SFRP1 + PCs’ proliferation through the Hippo-YAP/TAZ signaling pathway.

A UMAP plots showing four subpopulations of epidermal cells in EV-A71-infected hiPSC-SOs at 0 and 8 dpi. B Dot plot showing the expression of representative marker genes for NNMT + SFRP1 + PC, basal stem cell, and mature EpC. The statistical significance of cell type marker genes is determined using the two-sided Wilcoxon rank-sum test with Bonferroni-adjusted p value of <0.05. C Expression distribution of representative marker genes for NNMT + SFRP1 + PC (NNMT and SFRP1), basal stem cell (TP63 and COL17A1), and mature EpC (KRT1 and IVL). D Proportions of different types of epidermal cell in EV-A71-infected and normal hiPSC-SOs. E Biological process enrichment analysis of the DEGs in NNMT + SFRP1 + PC, basal stem cell, and mature EpC between EV-A71-infected and normal hiPSC-SOs. The enriched terms of upregulated and downregulated DEGs are indicated in red and blue, respectively. The size of the dot indicates the number of genes belonging to each term. The color scale indicates the enrichment p value. F Immunofluorescence of the EV-A71 virus protein and NNMT + SFRP1 + PC markers (NNMT and SFRP1) in the EV-A71-infected hiPSC-SOs at 0, 4, and 8 dpi (scale bar: 100 μm). The bar charts quantify the expression levels of NNMT + SFRP1+PCs’ markers NNMT and SFRP1 in the EV-A71-infected hiPSC-SOs with infection time. The data are shown as the mean ± SD (n = 3 per group). G KEGG pathway analysis of the upregulated genes in the NNMT + SFRP1 + PC of EV-A71-infected hiPSC-SOs at 8 dpi compared with normal controls. The enrichment analysis is performed using the one-sided hypergeometric test and the p values are provided in source data. H Mechanism diagram of EV-A71 infection-induced proliferation of NNMT + SFRP1 + PC. When NNMT + SFRP1+PCs are infected by viruses, intergrin-mediated signaling pathway (ITGAV and ITGB1) and following Hippo signaling pathway (YAP, TAZ, TEAD1, and BIRC2) are activated to promote proliferation and invasion of NNMT + SFRP1+PCs. The immunofluorescence experiments are repeated three times. dpi: days post-infection; EpC: epidermal cell; hiPSC-SOs: human induced pluripotent stem cell-derived skin organoids; PC: progenitor cell. Source data are provided as a Source Data file.

Fig. 5. NSC167409 blocks EV-A71 replication both in vivo and in vitro.

A Overlap of the upregulated DEGs in NNMT + SFRP1+PCs with the upregulated DEPs in the EV-A71-infected hiPSC-SOs at 2, 4, and 8 dpi compared to 0 dpi. The red boxes present the log2 average expression of genes in NNMT + SFRP1+PCs between the EV-A71-infected hiPSC-SOs and controls. B Western blot analysis of HMGB1, the EV-A71 virus protein (EV-A71-VP1), and tubulin in the siHMGB1-1, siHMGB1-2, siControl, and normal control (NC) samples of Vero cells infected with EV-A71 (1 MOI) for 48 h. The quantitative data are shown as the mean ± SD (n = 3 per group). Significant differences are determined by unpaired two-tailed t test and the p values are provided in source data. C Western blot analysis of the EV-A71 virus protein in EV-A71-infected (1 MOI) Vero cells that treated with NSC167409. The samples are treated with NSC167409 immediately after being infected with EV-A71 and collected at 30 hpi for analysis. D Antiviral activity and cytotoxicity analysis of NSC167409 on RD and Vero cells. The samples are treated with NSC167409 immediately after being infected with EV-A71 (100 TCID₅₀/ml) and collected at 72 hpi for analysis. The antiviral activity and cytotoxicity of NSC167409 are measured using CellTiter-Glo cell viability assay kit. The data are shown as the mean ± SD (n = 3 per group). E Immunofluorescence of EV-A71 virus protein and KRT5 in the EV-A71-infected hiPSC-SOs at 0 and 8 dpi with or without NSC167409-treatment (scale bar: 500 μm). F Workflow of in vivo transplantation of hPSC-SOs, drug administration, and EV-A71 infection on mice. The prophylactic treatment is performed on mice with NSC167409 (2 mg/kg) by subcutaneous injection four hours before EV-A71 infection. G Immunofluorescence of the EV-A71 virus protein and KRT5 in the EV-A71-infected hiPSC-SO xenografts at 0 and 8 dpi with or without NSC167409-treatment (scale bar: 100 μm). The immunofluorescence experiments are repeated three times. DEG: differentially expressed gene; DEP: differentially expressed protein; dpi: days post-infection; hiPSC-SOs: human induced pluripotent stem cell-derived skin organoids; hpi: hours post-infection. Source data are provided as a Source Data file.

Fig. 6. EV-A71 mediates epidermal cell dysfunction via autophagy pathways in hiPSC-SOs.

A Violin plots illustrating the expression of HMGB1 in selected subclusters of epidermal cell and fibroblast of the EV-A71-infected hiPSC-SOs at 8 dpi. B Pathway enrichment analysis of DEGs in NNMT + SFRP1 + PC, basal stem cell, and mature EpC in the EV-A71-infected hiPSC-SOs between 8 dpi and 0 dpi. The upregulated and downregulated DEGs are indicated in red and blue, respectively. The size of the dot indicates the percentage of cells with gene expression in a cell cluster, while the color indicates the average expression level of the gene. The DEGs of the EV-A71-infected hiPSC-SOs between 8 dpi and 0 dpi are determined based on the two-sided Wilcoxon rank-sum test with Bonferroni-adjusted p value of <0.05 and log2 (EV-A71 infected/Control) > 0.25 (upregulated) or < −0.25 (downregulated). C Immunofluorescence of the EV-A71 virus protein, NNMT, TAZ, YAP, TEAD1, LC3B, and HMGB1 in the EV-A71-infected hiPSC-SOs at 0, 4 and 8 dpi with or without NSC167409-treatment (scale bar: 10 μm). The experiments are repeated three times. D Mechanistic diagram of the role of HMGB1 in EV-A71 infection of NNMT + SFRP1+PCs. HMGB1 positively correlates with viral replication. HMGB1 promotes autophagy (LC3B) and the production of pro-inflammatory factors (IL6, IL8, and MMP1), and enhances YAP/TAZ signaling to promote the NNMT + SFRP1+PCs’ proliferation. When an inhibitor (NSC167409) is used to inhibit the expression of HMGB1, not only is viral replication and its concomitant inflammation suppressed, but autophagy and YAP/TAZ signaling are also suppressed. DEG: differentially expressed gene; dpi: days post-infection; hiPSC-SOs: human induced pluripotent stem cell-derived skin organoids; EpC: epidermal cell; FB: fibroblast; PC: progenitor cell. Source data are provided as a Source Data file.