Dual inhibition of intercellular adhesion molecule-1 and nucleolin reduces RSV infection efficiency in human respiratory organoids

Figures

• 

  Figure 1. RSV-A efficiently infects human iPSC-derived respiratory organoids (A) The expression levels of FOXJ1, KLF5, MUC20, and TP63 in human iPSC-derived respiratory organoids and undifferentiated iPSCs were measured by RT-qPCR (iPSCs = 1.0). Data are shown as mean ± SD (n = 3, technical replicates); Two-tailed Student’s t test (∗∗p < 0.01, compared with iPSCs). (B) The expression levels of ICAM-1, EGFR, NCL, and IGF1R in respiratory organoids and human adult lung were measured by RT-qPCR (human adult lung = 1.0). Data are shown as mean ± SD (n = 3, technical replicates); Two-tailed Student’s t test (∗p < 0.05, ∗∗p < 0.01, compared with human adult lung). (C) TPM values of ICAM-1, EGFR, NCL, and IGF1R in respiratory organoids. (D–F) Human iPSC-derived respiratory organoids were infected with RSV-A at 0.1 TCID50/cell (8 × 104 TCID50/well) and cultured for 96 h. (D) The cell culture supernatants of RSV-A-infected respiratory organoids were collected and TCID50 assay was performed. Data are shown as mean ± SD (n = 3, three infection batches). (E) RSV nucleoprotein (N) gene expression was analyzed by RT-qPCR. Data are shown as mean ± SD (n = 3, three infection batches). (F)…

• 

  Figure 2. ICAM-1- and EGFR-KO iPSCs were established using CRISPR-Cas9 system (A) ICAM-1-knockout (KO) and EGFR-KO iPSCs were generated using the CRISPR-Cas9 system. Bar plots represent the indel distribution for ICAM-1 and EGFR. (B) Phase images of wild-type (WT), ICAM-1-KO, and EGFR-KO iPSCs. Scale bars, 100 μm. (C) Immunofluorescence analysis of OCT3/4 (red) expression in WT, ICAM-1-KO, and EGFR-KO iPSCs. Nuclei were counterstained with DAPI (blue). Scale bars, 100 μm. (D) The expression levels of ICAM-1 and EGFR in WT, ICAM-1-KO, and EGFR-KO iPSC-derived respiratory organoids were measured by RT-qPCR. Data are shown as mean ± SD (n = 3, technical replicates); two-tailed Student’s t test (∗∗p < 0.01). (E) The expression levels of ICAM-1, EGFR, and β-actin in WT, ICAM-1-KO, and EGFR-KO iPSC-derived respiratory organoids were measured by the capillary-based immunoassay.

• 

  Figure 3. RSV-A infection experiment using ICAM-1 and EGFR-KO respiratory organoids (A) WT, ICAM-1-KO, and EGFR-KO iPSC-derived respiratory organoids were infected with RSV-A at 0.1 TCID50/cell (8 × 104 TCID50/well) and cultured for 96 h. (B) At 96 hpi, the expression levels of RSV N in WT, ICAM-1-KO, and EGFR-KO respiratory organoids were measured by RT-qPCR. Data are shown as mean ± SD (n = 3, three infection batches); Two-tailed Student’s t test (∗∗p < 0.01). (C and D) The expression levels of MX1 and ISG15 in RSV-A-infected and uninfected WT, ICAM-1-KO (C), and EGFR-KO (D) respiratory organoids were measured by RT-qPCR. Data are shown as mean ± SD (n = 3, three infection batches); one-way analysis of variance (ANOVA) with Tukey’s post hoc test (∗∗p < 0.01). (E and F) The expression levels of IL-6 and IL-8 in RSV-A-infected and uninfected WT, ICAM-1-KO (E), and EGFR-KO (F) respiratory organoids were measured by RT-qPCR. Data are shown as mean ± SD (n = 3, three infection batches); one-way ANOVA with Tukey’s post hoc test (∗p < 0.05, ∗∗p < 0.01).

• 

  Figure 4. RSV-A infection experiment using NCL and IGF1R neutralizing antibodies (A) WT iPSC-derived respiratory organoids were treated with anti-NCL antibodies (10 μg/mL) or anti-IGF1R antibodies (10 μg/mL), or control antibodies (10 μg/mL). After 1 h of incubation, respiratory organoids were infected with RSV-A at 0.1 TCID50/cell (8 × 104 TCID50/well) and cultured for 96 h. (B) At 96 hpi, the expression levels of RSV N in anti-NCL, anti-IGF1R, and control antibodies-treated respiratory organoids were measured by RT-qPCR. Data are shown as mean ± SD (n = 3, three infection batches); Two-tailed Student’s t test (∗∗p < 0.01). (C and D) The expression levels of MX1 and ISG15 in RSV-A-infected and uninfected respiratory organoids treated with control, anti-NCL (C), and anti-IGF1R (D) antibodies or isotype control were measured by RT-qPCR. Data are shown as mean ± SD (n = 3, three infection batches); one-way ANOVA with Tukey’s post hoc test (∗∗p < 0.01). (E, F) The expression levels of IL-6 and IL-8 in RSV-A-infected and uninfected respiratory organoids treated with control, anti-NCL (E), and anti-IGF1R (F) antibodies were measured by RT-qPCR. Data are shown as mean ± SD (n = 3, three…

• 

  Figure 5. ICAM-1 and NCL cooperatively reduce RSV-A infection efficiency in human iPSC-derived respiratory organoids (A) ICAM-1-KO iPSC-derived respiratory organoids were treated with anti-NCL antibodies (10 μg/mL) or an isotype control (10 μg/mL). After 1 h of incubation, respiratory organoids were infected with RSV-A at 0.1 TCID50/cell (8 × 104 TCID50/well) and cultured for 96 h. (B) At 96 hpi, the expression levels of RSV N were measured by RT-qPCR in WT, ICAM-1-KO, and anti-NCL antibody-treated ICAM-1-KO respiratory organoids. Data are shown as mean ± SD (n = 3, three infection batches); one-way ANOVA with Tukey’s post hoc test (∗∗p < 0.01). (C) At 96 hpi, the cell culture supernatants of RSV-A-infected WT, ICAM-1-KO, and anti-NCL antibody-treated ICAM-1-KO respiratory organoids were collected and TCID50 assay was performed. Data are shown as mean ± SD (n = 3, three infection batches); one-way ANOVA with Tukey’s post hoc test (∗p < 0.05, ∗∗p < 0.01). (D) Immunofluorescence staining of RSV F protein (red) in infected WT, ICAM-1-KO, and anti-NCL–treated ICAM-1-KO respiratory organoids. Cell nuclei were counterstained with DAPI (blue). Scale bars, 50 μm.