Fig 1: 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. ß-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. ß-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). ß-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 ß-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.
Fig 2: Exogenous IL-17a does not affect the proliferation and migration of human NPCs. (A) Line graph showing cell proliferation curves of iPSC-derived NPCs of control (n = 5) and ASD (n = 6) subjects cultured in the presence of either vehicle or rhIL-17a (10 and 50 ng/ml) for 24 and 48 h. (B) Line graph showing cell proliferation curves of iPSC-derived NPCs of control (n = 5) and ASD (n = 6) subjects cultured in the presence of either vehicle, or rhIL-17a (10 and 50 ng/ml), or rapamycin (5 nM, used as control), or rhIL-17a (50 ng/ml) plus rapamycin (5 nM) for 72 and 144 h. While rapamycin-treated human NPCs showed significantly decreased proliferation, **p = 0.01, no significant differences were observed in the proliferation rates between rhIL-17a-treated and untreated human NPCs. (C) Line graph of mean% relative wound density over time in iPSC-derived NPCs of control (n = 5) and ASD (n = 3) subjects cultured in the presence of either vehicle or rhIL-17a (10 and 50 ng/ml) for 24 h. No significant differences were observed in the migration rates between rhIL-17a-treated and untreated human NPCs.
Fig 3: Exosomes do not affect PMA-induced NETs and IL-17 release by neutrophils. (A) Representative images of NET formation by neutrophils PMA stimulation, in the presence or absence of different concentrations of exosomes. Cells were stained for nuclei (DAPI, blue) and H3 Citrullinate R2/8/17 (green). Scale bar, 25 µm. (B) Quantification of NET release by neutrophils pooled from three independent experiments performed using neutrophils from three unique donors. At least 100 cells were quantified from each experiment. The neutrophils used in these experiments were from different donors than those in Figures 1, 2 and 4. Data shown as mean ± SEM (n = 3). ***P < 0.0001, two-way ANOVA. (C) mRNA expression levels of il17a relative to hprt1 in neutrophils upon PMA stimulation, in the presence or absence of different concentrations of exosomes (n = 5). Five independent experiments were performed using neutrophils from five unique donors. The neutrophils used in these experiments were from different donors than those in Figures 1, 2 and 4. *P = 0.03, unpaired two-tailed Student's t test. (D) Neutrophils were stimulated with PMA in the presence or absence of different concentrations of exosomes, and culture supernatants were assayed for IL-17 by ELISA (n = 3). Three independent experiments were performed using neutrophils from three unique donors. **P = 0.01, unpaired two-tailed Student's t test. (Color version of figure is available online.)
Fig 4: Exosomes suppress C5b-9–induced NETs and IL-17 release by neutrophils. (A) Representative images of NET formation by neutrophils upon assembly of C5b-9, in the presence or absence of different concentrations of exosomes. Cells were stained for nuclei (DAPI, blue) and H3 Citrullinate R2/8/17 (green). Scale bar, 25 µm. (B) Quantification of NET release by neutrophils pooled from three independent experiments performed using neutrophils from four unique donors. At least 100 cells were quantified from each experiment. The neutrophils used in these experiments were from the same donors as those in Figures 1 and 4. Data shown as mean ± SEM (n = 4). *P = 0.03, **P = 0.006, 0.004, 0.005 (from left to right), two-way ANOVA. (C) mRNA expression levels of il17a relative to hprt1 in neutrophils upon assembly of C5b-9, in the presence or absence of different concentrations of exosomes (n = 3). Three independent experiments were performed using neutrophils from three unique donors. The neutrophils used in these experiments were from the same donors as those in Figures 1 and 4. *P = 0.02, one-way ANOVA. *P = 0.02, **P = 0.007, unpaired two-tailed Student's t test. (D) C5b-9 were assembled on neutrophils in the presence or absence of different concentrations of exosomes, and culture supernatants were assayed for IL-17 by ELISA (n = 3). Three independent experiments were performed using neutrophils from three unique donors. The neutrophils used in these experiments were from the same donors as those in Figures 1 and 4. *P = 0.02, one-way ANOVA. *P = 0.03, unpaired two-tailed Student's t test. (Color version of figure is available online.)
Fig 5: Complement C5b-9 complex induces NET formation and IL-17 release by neutrophils. (A) Flow cytometric analysis of C5b-9 assembly on neutrophils. (B) Representative images of NET formation by neutrophils upon assembly of complements C5b-9. Cells were stained for nuclei (DAPI, blue) and H3 Citrullinate R2/8/17 (green). Scale bar, 25 µm. (C) Quantification of NET release by neutrophils pooled from three independent experiments performed using neutrophils from four unique donors. At least 100 cells were quantified from each experiment. Data shown as mean ± SEM (n = 4). **P = 0.006, two-way ANOVA. (D) mRNA expression levels of il17a relative to hprt1 in neutrophils upon assembly of C5b-9 (n = 3). Three independent experiments were performed using neutrophils from three unique donors. **P = 0.002, unpaired two-tailed Student's t test. (E) C5b-9 were assembled on neutrophils, and culture supernatants were assayed for IL-17 by ELISA (n = 3). Three independent experiments were performed using neutrophils from three unique donors. *P = 0.02, unpaired two-tailed Student's t test. (F) mRNA expression levels of s100a8 and s100a9 relative to hprt1 in neutrophils upon assembly of C5b-9 (n = 4). Four independent experiments were performed using neutrophils from four unique donors. (Color version of figure is available online.)
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