Fig 2: ORF8 interacts with hIL-17RA to activate inflammatory responses. (A) Hypothesis model of ORF8-mediated IL-17RA signaling. (B) Purified ORF8-Flag interacts with 293T-expressing hIL-17RA. At 24 h posttransfection with vector or hIL-17RA expression vector, 293T cells were subsequently incubated for 30 min with Flag-tagged ORF8 or untreated, followed by flow cytometry with anti-Flag antibody. (C) THP-1 cells express high levels of surface hIL-17RA. THP-1 cells were stained with hIL-17RA antibody and followed by flow cytometry. (D) Purified ORF8-Flag protein interacts with endogenous IL-17RA in THP-1 cells. THP-1 cells were incubated with purified ORF8-Flag proteins for 2 h, and cell lysates were immunoprecipitated with anti-Flag, followed by immunoblotting with indicated antibodies. (E) ORF8 induces the phosphorylation of p65 and IκΒα. THP-1 cells were treated with or without ORF8 (50 ng/mL) for the indicated times, followed by immunoblots with the indicated antibodies. (F) ORF8 treatment increases inflammatory cytokine gene expression. THP-1 cells were treated with ORF7b or ORF8 at the indicated concentrations for 16 h, followed by RT-qPCR. (G) ORF8 neutralizing antibody blocks ORF8 function. THP-1 cells were treated with purified ORF8 (20 ng/mL) and its antibody at the indicated concentrations for 16 h, followed by RT-qPCR. (H) IL-17RA neutralizing antibody blocks ORF8 function. THP-1 cells were treated with control IgG or IL-17RA neutralizing antibody at the indicated concentrations for 2 h and then stimulated with ORF8 for 16 h, followed by RT-qPCR. P values were calculated by two-way ANOVA with Tukey’s test in panel F or Bonferroni’s test in panels G and H for multiple comparisons. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.0001.

Fig 3: ORF8 natural variants for hIL-17R binding and inflammatory signaling activity. (A) S24, V62, and L84 residues in SARS-CoV-2 ORF8 structure. (B) Binding activity of ORF8 natural variants to THP-1 cells. THP-1 cells were incubated with mock treatment or ORF8 WT or variants for 2 h, followed by flow cytometry with anti-Flag antibody. (C, D) Binding activity of ORF8 natural variants to hIL-17RA (C) or hIL-17RC (D). At 24 h posttransfection with hIL-17RA (C) or hIL-17RC (D) expression plasmids, 293T cells were subsequently incubated for 30 min with mock treatment or purified ORF8 WT or variants, followed by flow cytometry with anti-Flag antibody. (E) Signaling activity of ORF8 variants to induce inflammatory cytokine gene expressions. THP-1 cells were mock treated or treated with ORF8 WT or variants for 16 h, followed by RT-qPCR to measure the mRNA levels of indicated genes, with horizontal lines indicating median values of the mRNA levels of indicated genes in mock-treated THP1 cells observed. (F) L84S variant shows the loss of IL-17RA/RC heterodimerization. At 24 h posttransfection with HA-hIL-17RA and Myc-hIL-17RC, NIH 3T3 cells were treated with the ORF8 WT or L84S variant for 30 min, followed by PLA. (G) Predicted structural representation of the Y42, Y106, and L84 residues at the binding interface between ORF8 and IL-17RA. P values were calculated by one-way ANOVA with Dunnett’s posttest in panels E and F. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.0001.

Fig 4: IL-17A or Poly(I:C) treatment of the placenta cell lines. Cell morphologies were investigated by IL-17A or Poly(I:C) treatment for 24 h (A). Cell viabilities were measured in a dose-dependent manner for 24 h and in a time-dependent manner by MTT assay (B). Activation of IL-17RA, IL-17RC, IL-6, IFNγ, and TNFα mRNA expression in placental cell lines (C). IL-17RA, PPARγ protein expression in cells treated with 100 ng/mL IL-17A or 100 µg/mL Poly(I:C) (D). GAPDH was used for loading control. Calculated by t-test (B,C) or one-way ANOVA with Tukey’s test.

Fig 5: Human NPCs express IL17RA and respond to exogenous IL-17a by modulating the activity of ERK1/2 and mTORC1 signaling pathways. (A) Relative transcript levels of IL17RA in iPSC-derived NPCs of control (n = 4) and ASD (n = 7) subjects. (B) Relative protein levels of IL17RA in iPSC-derived NPCs of control (n = 5) and ASD (n = 7) subjects. β-actin was used as a loading control and representative immunoblot images are shown. Human NPCs endogenously express IL17RA. (C) Relative protein levels of IL17RA in iPSC-derived NPCs of control (n = 2–3) and ASD (n = 5) subjects after treatment with either vehicle (–) or rhIL-17a (10 and 50 ng/ml) for 48 h. The data are expressed as fold change in IL17RA expression relative to vehicle-treated samples. β-actin was used as a loading control and representative immunoblot images are shown. No significant differences in the protein levels of IL17RA were observed between rhIL-17a-treated and untreated human NPCs. (D) Time course analysis of pERK1/2 expression in iPSC-derived NPCs of control (n = 4) and ASD (n = 6) subjects after treatment with either vehicle (–) or rhIL-17a (50 ng/ml). Total-ERK was used as a loading control and the data are expressed as fold change in normalized pERK1/2 expression relative to vehicle-treated samples. Representative immunoblot images are shown. (E) Time course analysis of pRPS6 expression in iPSC-derived NPCs of control (n = 3) and ASD (n = 4) subjects after treatment with either vehicle (–) or rhIL-17a (50 ng/ml). β-actin was used as a loading control and the data are expressed as fold change in normalized pRPS6 expression relative to vehicle-treated. Representative immunoblot images are shown. Exogenous IL-17a led to a significant increase in pERK1/2 levels and significant decrease in pRPS6 levels in human NPCs. *p ≤ 0.05, **p ≤ 0.01, and ****p ≤ 0.0001. (F) Time course analysis of pNF-kB65 expression in iPSC-derived astrocytes (used as positive control of pNF-kB expression) and in iPSC-derived NPCs of control (n = 2) and ASD (n = 1) subjects after treatment with either vehicle (–) or rhIL-17a (50 ng/ml). Total-NF-kB and β-actin were used as a loading controls and representative immunoblot images are shown. While iPSC-derived astrocytes express pNF-kB65, no detectable expression of this protein was observed in human NPCs.