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

Categories: Disease & Virus

March 18, 2025

hiPSC-SOs are susceptible to EV-A71 infection

We differentiated hiPSCs into skin organoids using an improved method according to the previously reported stepwise protocol¹⁴ (Supplementary Fig. 1A). Immunofluorescence staining analysis results validated the expression of the basal stem cell marker keratin 5 (KRT5), hair follicle stem cell markers keratin 15 (KRT15) and keratin 17 (KRT17), as well as the pan-neuronal marker tubulin β class III (TUJ1), in the hiPSC-SOs (Supplementary Fig. 1B). Electron microscopy showed the different hierarchical structures of hair follicles, secreted melanosomes, extracellular matrix, and desmosomes in the epidermis (Supplementary Fig. 1C). Next, to study the different cell types that are affected by EV-A71 infection, the hiPSC-SOs were subjected to EV-A71 infection and the proteomics analysis was performed on the infected organoids (Fig. 1A). With increasing EV-A71 infection time, the amount of viral replication in the hiPSC-SOs gradually increased, indicating that the hiPSC-SOs were susceptible to EV-A71 infection (Fig. 1B, C and Supplementary Fig. 2A). Whole-mount immunostaining of EV-A71-infected hiPSC-SOs revealed large amounts of virus protein appeared at different areas of hair follicles (KRT17, KRT82, and KRT71) and nerves (TUJ1 and S100 calcium binding protein B (S100B)) in organoids (Fig. 1D and Supplementary Fig. 2B), suggesting that the virus may infect different types of cells in hiPSC-SOs. Immunostaining analysis of organoid sections further validated that EV-A71 protein co-localized with the markers of hair follicle cells (KRT17, KRT82, and KRT71), as well as neurons (TUJ1) and S100B+ Schwann-like cells (Fig. 1E and Supplementary Figs. 2C, S3), indicating the virus may infect these types of cells.

Then, the quantitative proteomics analyses were performed on EV-A71-infected hiPSC-SOs over time and a total of 3128, 3198, 3124, and 3053 proteins were identified in hiPSC-SOs at 0 (control), 2, 4 and 8 days post-infection (dpi) (Supplementary Data 1). The proteome profiling showed that the protein expression pattern of the hiPSC-SOs without EV-A71 infection was quite different from that of the hiPSC-SOs infected with EV-A71 (Supplementary Fig. 4A, B). The pairwise comparisons were performed by a moderated t test to identify the differentially expressed proteins (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 (control group, n = 3). The DEPs between EV-A71-infected and control samples were determined based on a Benjamin–Hochberg (BH) adjusted p value < 0.01 and log2 (EV-A71 infected/Control) > 0.585 (upregulated) or < -0.585 (downregulated). Among these DEPs, we found 552, 335, and 371 upregulated proteins and 187, 244, and 364 downregulated proteins in the hiPSC-SOs infected with EV-A71 for 2, 4, and 8 days, respectively, compared to the hiPSC-SOs without EV-A71 infection (Fig. 2A, Supplementary Data 2). Biological process enrichment analysis of all the DEPs showed that the upregulated proteins were enriched in mRNA processing, translational initiation, viral entry into host cells, and DNA replication (Supplementary Fig. 4C), indicating that virus cycle-associated processes may be activated in EV-A71-infected hiPSC-SOs. The downregulated proteins were enriched in collagen fibril organization, basement membrane organization, keratinization, and fibroblast proliferation (Supplementary Fig. 4C), indicating that the abundance of skin cell- and extracellular matrix-related proteins was severely downregulated in EV-A71-infected hiPSC-SOs.

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

The above results suggest that hiPSC-SOs can be used as in vitro models of EV-A71 infection.

Time-dependent molecular features of EV-A71-infected hiPSC-SOs

To investigate the dynamic changes in EV-A71-infected hiPSC-SOs over time, we examined the proteomics profiling at 2, 4 and 8 dpi (Supplementary Fig. 4A, B). The specifically highly and lowly expressed proteins at different infection timepoint were defined as those with BH adjusted one-way ANOVA 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 in all other samples. The results showed specific high and low expression patterns of protein abundances at the assessed time points (Supplementary Fig. 4D, Supplementary Data 3). Four high expression patterns of protein abundances were found at 0, 2, 4, and 8 days after EV-A71 infection. The functional annotation of these proteins revealed the proteomic signatures at each timepoint. At 2 dpi (early stages of infection), the proteins with high abundance were mainly functionally enriched in virus processes, such as response to virus and viral genome replication, as well as DNA replication, such as prereplicative complex assembly involved in nuclear cell cycle DNA replication and DNA replication initiation, indicating that in the early stages of hiPSC-SO EV-A71 infection, the main biological processes of host cells appear to help virus replication (Fig. 2B). For example, the DEAD box protein DDX21, an RNA helicase, promotes ribosomal RNA processing and transcription from polymerase II 1 and 2 and is highly expressed at 2 dpi (Fig. 2C). Glycolytic processes (ALDOA, ALDOC, ENO2, GPI, and HK2) and glucose import across the plasma membrane (SLC2A1 and SLC2A3) seem to occur at 4 dpi (Fig. 2B, C), thereby suggesting enormous cellular energy expenditure at this point during the infection. Finally, the proteins with high abundance were enriched in immune responses, such as cell chemotaxis (CCL20, CXCL12, DEFB103A, and TPBG), responses to cytokines (ACP5, PTGES, and TIMP1), and responses to hypoxia (ATP1B1, ENG, and PLOD2), at 8 dpi, thereby indicating that immune and hypoxic responses occur in the later stages of EV-A71 infection (Fig. 2B, C).

Similarly, we obtained the specific low expression patterns of protein abundances via expression profile clustering (Supplementary Fig. 4D). Hair follicle morphogenesis (KRT25, KRT27, KRT34, and KRT71) and hair differentiation (KRT33A, KRT36, and KRT40)-associated proteins seemed to be downregulated at 4 dpi (Fig. 2B, C), indicating that hair follicle may be an important structure and its function would be affected after EV-A71 infection. Immunostaining analysis further validated that EV-A71 can directly infect hair follicle cells at the different timepoint assessed (Supplementary Fig. 4E). We further observed that proteins involved in epidermal development, such as basal stem cell markers (KRT5 and KRT15) and epithelium markers (IVL and KRT12) were significantly dysregulated at 8 dpi in EV-A71-infected hiPSC-SOs (Fig. 2B, C). In addition, the main components of the basement membrane (COL7A1, COL17A1, LAMA1, LAMB1, LAMC1, NID1, and NID2), the basal stem cell markers, the specialized anchoring complexes of skin, hemidesmosomes (ITGB4 and ITGA6) and adhesion receptors (ITGA3) were also downregulated significantly at 8 dpi in EV-A71-infected hiPSC-SOs (Fig. 2B, C). The ECM organization-associated proteins such as collagen fibril-related proteins (COL1A1, COL1A2, COL2A1, COL3A1, COL5A1, COL11A1, COL14A1, and LUM) and elastic fiber assembly-related proteins (LOX and EMILIN1) were also downregulated in EV-A71-infected hiPSC-SOs at 8 dpi (Fig. 2B, C).

In conclusion, the above data showed the processes of dynamic interactions of EV-A71 with hiPSC-SOs and indicated that the viral infection can lead to the disorder of specific physiological structures and functions of the skin.

EV-A71 infection depletes reticular fibroblasts

Next, we performed scRNA-seq of EV-A71-infected hiPSC-SOs to investigate the cell tropism of EV-A71 and its impact on the different cell types within hiPSC-SOs (Fig. 3A, Supplementary Data 4). scRNA-seq data of the hiPSC-SOs revealed the presence of fibroblast (COL5A1 and COL6A3), nerve cell (HES1 and PTN), keratinocyte (KRT5 and KRT14), melanocyte (PMEL and TYRP1), merkel cell (KRT8 and TTR), chondrocyte (CHI3L2 and SOX5), and cycling progenitor cell (TOP2A and CDK1) (Fig. 3B, Supplementary Data 4). The key factors involved in EV-A71 entry into host cells, including ANXA2²¹, FN1²², NCL²³, PHB2²⁴, PPIA²⁵, SCARB2²⁶, and VIM²⁷, were found to be expressed in main types of cells identified in scRNA-seq data (Fig. 3C). Correspondingly, the numbers of fibroblasts, nerve cells, and epidermal cells showed greatest changes before and after EV-A71 infection (Fig. 3D).

As the largest population in target cells of EV-A71, we found that the number of fibroblasts decreased after virus infection in hiPSC-SOs (Fig. 3D and Supplementary Fig. 5A). Next, we found that fibroblasts consisted of different cell types, such as proliferative fibroblast (FGF7 and NRN1), reticular fibroblast (MFAP5 and COL11A1), MKI67 PC (CENPW and F2R), and chondrocyte (COL2A1 and ACAN) (Supplementary Fig. 5B–D, Supplementary Data 4). Among them, the number of reticular fibroblasts, which are a type of fibroblast that synthesizes collagen alpha-1 (III) to produce and maintain the thin fibrous networks that are the foundation of most lymphoid organs, markedly decreased after EV-A71 infection (Supplementary Fig. 5E, F), indicating that dermal tissue lost the ability to provide structural support after EV-A71 infection and that the balance of the immune response was disrupted. The biological process enrichment analysis was performed on the upregulated differentially expressed genes (DEGs) in reticular fibroblasts and proliferative fibroblasts between the EV-A71-infected hiPSC-SOs (8 dpi) and control hiPSC-SOs (0 dpi). The results showed that both reticular fibroblasts and proliferative fibroblasts exhibited a significant enrichment in host-virus interaction, DNA repair, apoptosis, and aging related proteins (Fig. 3E). Reticular fibroblasts were enriched in more processes related to host of viral transcription and response to virus, indicating that reticular fibroblasts are the main target of EV-A71 virus infection (Fig. 3E). In addition, immune response pathways, such as TNF-α and IL-1 mediated signaling pathways, were enriched in EV-A71-infected reticular fibroblasts (Fig. 3E), leading to the upregulation of IL-6 and CXCL8 (Fig. 3F). Activation of the TNF pathway (TNFR1, CREB3, and C/EBPβ) leads to upregulation of matrix metalloproteinases (MMPs), which can degrade several collagens^28,29. Results showed that MMP1, MMP3, and MMP13 were upregulated in the EV-A71-infected reticular fibroblasts (Fig. 3F), which may be the key pathway for the massive reduction of extracellular matrix components such as collagen in previous proteomic results (Figs. 2C, 3F). Loss of dermal collagen accompanied by the production of pro-inflammatory factors will contribute to skin senescence³⁰. These results suggested that EV-A71 infection depletes fibroblast subsets and spreads senescence and thus may trigger skin aging.

EV-A71 infection promotes NNMT + SFRP1+ progenitor cells’ proliferation through the Hippo-YAP/TAZ signaling pathway

To further investigate the impact of EV-A71 on epidermal cells, we analyzed the scRNA-seq data of epidermal cell subtypes including basal stem cell, mature epidermal cell (mature EpC), and NNMT + SFRP1+ progenitor cell (NNMT + SFRP1 + PC) (Fig. 4A–D, Supplementary Data 4). Results showed that genes associated with host-virus interaction, mRNA processing, positive regulation of transcription, viral translational termination-reinitiation, aging, and the innate immune response were upregulated in these three epidermal cell types (Fig. 4E), indicating disruption of epidermal cell homeostasis by viral infection. In addition, genes associated with skin development, skin barrier establishment, collagen fibril organization, elastic fiber assembly, and tissue regeneration were downregulated in epidermal cells (Fig. 4E), which is consistent with our proteome results, indicating that EV-A71 infection of hiPSC-SOs affects the growth and barrier function of epidermal cells, as well as their matrix environment in the dermis. Interestingly, we found the number of NNMT + SFRP1+PCs nearly doubled after virus infection (Fig. 4D). Immunofluorescence staining confirmed the surge of NNMT + SFRP1+PCs in a time-dependent manner after viral infection (Fig. 4F). Several upregulated genes associated with viral processes, such as antiviral defense, viral genome replication, defense response to virus were enriched in the NNMT + SFRP1+PCs of the EV-A71-infected hiPSC-SOs, compared to the control group (Fig. 4E). KEGG pathway analysis showed that the upregulated genes enriched in the NNMT + SFRP1+PCs were associated with EGF/EGFR signaling (MAP3K2, MAP2K2, etc.), TGF-beta signaling (MMP1, TNC, etc.), integrin-mediated cell adhesion (ITGB1, ITGAV, etc.), tight junction (ACTR2, ACTR3, etc.), as well as Hippo signaling (FZD8, TAZ, etc.) pathways (Fig. 4G). Activation of the Integrin and Hippo signaling pathways in NNMT + SFRP1+PCs promotes YAP/TAZ expression, which may be an important pathway leading to increased NNMT + SFRP1 + PC numbers after EV-A71 infection (Fig. 4H). The PC marker NNMT plays a key role in the development and progression of several cancers³¹. Therefore, in addition to the aberrant proliferation of PCs, activation of YAP/TAZ could also induce cancer cell phenotypes of PCs³². The above results suggested that EV-A71 infection may promote NNMT + SFRP1+PCs’ proliferation through the Hippo-YAP/TAZ signaling pathway.

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

Collectively, these results suggested that EV-A71 mediates abnormal epidermal phenotypes by simultaneously disrupting the normal ecological balance of epidermal cells and inducing overproliferation of PCs, which may be associated with tumorigenesis.

Drug blocks EV-A71 replication

Next, in order to deeply investigate the reasons for the increased proliferation of NNMT + SFRP1+PCs after EV-A71 infection and screen candidate antiviral targets, we performed the Wilcoxon rank-sum test to identify DEGs in NNMT + SFRP1+PCs from the scRNA-seq data between the EV-A71-infected hiPSC-SOs and control samples. The genes with Bonferroni-adjusted p value < 0.05 and log2 (EV-A71 infected/Control) > 0.25 or < -0.25 are considered as upregulated or downregulated DEGs. There were 744 upregulated and 472 downregulated DEGs in the hiPSC-SOs infected with EV-A71 compared to those without EV-A71 infection (Supplementary Fig. 6A). Within the upregulated DEGs in NNMT + SFRP1+PCs, 12 genes were overlapped with the proteins that were simultaneously upregulated in EV-A71-infected hiPSC-SOs at 2, 4, and 8 dpi (Supplementary Fig. 6B and Fig. 5A). Among them, HMGB1 (high mobility group box 1), a protein associated with autophagy³³ and viral infection^34,35, aroused our interest. Furthermore, we detected the gene expression level of HMGB1 based on scRNA-seq data and found that it mainly expressed in fibroblasts, nerve cells, epidermal cells, and hair follicle cells, which was consistent with the expression levels of some of the EV-A71 receptor (Supplementary Fig. 6C and Fig. 3C). In addition, HMGB1 showed increased expression levels in all cell types after EV-A71 infection; then the knockdown experiments were performed on HMGB1. The results showed knockdown of HMGB1 can prevent EV-A71 replication in cell lines (Fig. 5B and Supplementary Fig. 6D).

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

Moreover, we found that the expression level of HMGB1 increased in a time-dependent manner with the increase of virus infection time (Supplementary Fig. 7A), consistent with previous proteomic and scRNA-seq results. To identify drug candidates capable of inhibiting EV-A71 replication, cell lines and hiPSC-SOs were treated with an FDA-approved HMGB1 inhibitor (NSC167409, 0.3-10 μM). The cells were treated with NSC167409 both immediately (Fig. 5C, D) and several hours (Supplementary Fig. 7B) after being inoculated with EV-A71; the results showed that the inhibition rate of EV-A71 levels increased both in rhabdomyosarcoma (RD) cells and Vero cells with a dose-dependent manner of drug treatment (Fig. 5C, D and Supplementary Fig. 7B). The inhibitory effect of NSC167409 on EV-A71 was also observed in hiPSC-SOs; and the drug showed a more significant inhibitory effect when treated at 24 hours post-infection (hpi) than at 72 hpi (Supplementary Fig. 7C). The immunofluorescence results further validated the inhibitory effect of NSC167409 on EV-A71 replication (Fig. 5E). To evaluate the drug activities in vivo, we performed the prophylactic treatment on humanized mice carrying hiPSC-SOs with NSC167409 (2 mg/kg) before intraxenograft inoculation with the EV-A71 virus (Fig. 5F). EV-A71 staining was significantly decreased in the epidermal cells of hiPSC-SO xenografts from humanized mice treated with NSC167409 and EV-A71, suggesting that the drug successfully prevented the virus from multiplying in vivo (Fig. 5G). The above results indicated that NSC167409 can effectively inhibit virus replication both in vitro and in vivo.

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

To further investigate the specific role of HMGB1 in EV-A71 infection, we analyzed HMGB1 expression levels in different cellular subpopulations before and after EV-A71 infection. We found that HMGB1 was highly expressed in the NNMT + SFRP1+PCs, mature EpCs, basal stem cells, proliferative fibroblasts, and reticular fibroblasts in scRNA-seq data of EV-A71-infected hiPSC-SOs compared to the control group (Fig. 6A), indicating that HMGB1 may be related to EV-A71 infection in these cells. Among them, we found that HMGB1 expression level was highest in NNMT + SFRP1+PCs and proliferative fibroblasts (Fig. 6A). Since the number of NNMT + SFRP1+PCs increased specifically after viral infection (Figs. 4D, 6A), suppression of HMGB1 with NSC reduced the number of NNMT + SFRP1+PCs, indicating that the specific high expression level of HMGB1 was closely related to the number of NNMT + SFRP1+PCs (Supplementary Fig. 7D).

By gene enrichment analysis, we found that several signaling pathways were activated, such as HIF-1 (EGLN3, EIF4BP1, ALDOC, ALDOA, EGLN1, CUL2, SLC2A1, EGFR, VEGFA, and ELOB) and autophagy (HMGB1, HIF1A, TANK, VMP1, RB1CC1, DDIT4, and NRBF2) in epidermal cells in EV-A71-infected hiPSC-SOs (Fig. 6B), indicating that EV-A71 replication and EV-A71-host interactions may be achieved through HIF-1 and autophagy signaling pathways. Immunostaining analysis further validated that over time, EV-A71 infection can upregulate the expression of microtubule-associated protein 1A/1B light chain 3B (LC3B), which is associated with the formation of autophagosomes, accompanied by an increase of the viral load in mature EpCs, basal stem cells and NNMT + SFRP1+PCs of the EV-A71-infected iPSC-SOs, as well as the humanized mice carrying hiPSC-SOs with EV-A71 infection (Fig. 6C and Supplementary Fig. 7A, E, F, 8A). Moreover, the expression of the autophagy inhibitor BCL2 was downregulated, further validating the omics analysis results. Suppression of HMGB1 with NSC reduced the expression of LC3B in vitro and vivo (Fig. 6C and Supplementary Fig. 8B), indicating that EV-A71 may replicate through the autophagy pathway.

In previous study, we found EV-A71 infection promotes NNMT + SFRP1+PCs’ proliferation through the Hippo-YAP/TAZ signaling pathway. Results also showed that YAP, TAZ, and TEAD1 of Hippo-YAP/TAZ signals were highly expressed in a time-dependent, accompanied by an increase of the viral load in EV-A71-infected organoids (Fig. 6C). Further, suppression of HMGB1 with NSC can reduced the expression of them, accompanied by decreased expression of LC3B and HMGB1 and the number of NMT + SFRP1+PCs (Fig. 6C). Following inhibition of YAP/TAZ signaling by 5 μΜ Verteporfin, we found that the expression of NNMT was significantly decreased in infected organoids (Supplementary Fig. 9). These results indicated that the increase in the number of NNMT + SFRP1+PCs may be related to autophagy-YAP signaling (Fig. 6D), which is also the signaling pathways in cancer progression^36,37.

Gene enrichment analysis also showed that immune response-related pathways, such as the IL-6 signaling pathway (TIMP1, PRDM1, NR2F6, IL6, and IL6ST), IL-2 signaling pathway (MAP2K2, SHC1, RPS6KB2, PTPN11, CBL, HRAS, FYN, SOS1, and JAK1), IL-17 signaling pathway (CXCL8, MMP1, MMP3, MMP13, S100A9, S100A8, S100A7, S100A7A, JUND, MAPK6, and HSP90B1) and TGFβ signaling pathway (MAP2K3, ROCK1, ITGA2, KLF6, ZFYVE16, COPS5, SNW1, SKIL, PAK2, ITGB1, TNC, DAB2, and ZEB1), were activated in EV-A71-infected hiPSC-SOs (Fig. 6B), indicating that EV-A71 infection may induce inflammation in epidermal cells. Immunofluorescen indicated that HMGB1 inhibitor also attenuated inflammatory response caused by viral infection due to the downregulated expression levels of IL6, IL8, MMP1, and MMP3 in the EV-A71-infected xenografts with NSC treatment in mice, compared to those without NSC treatment (Supplementary Fig. 10). These results suggested that by suppressing the HMGB1, epidermal cell dysfunction and inflammatory responses caused by viral infections were effectively alleviated. Our data illustrated an important mechanism for NSC as a potential drug for inhibiting epidermal PC abnormal proliferation by anti EV-A71 replication.