Fig 2: LPS titration experiments showing dose-dependent changes in mRNA expression levels of pro-inflammatory markers in BV2 cells. Murine BV2 cells were serum starved for 24 h prior to treatment with 0.1 µg/mL, 1.0 µg/mL and 10 µg/mL of LPS for 24 h, shown with no LPS controls. RNA was extracted by TRISURE reagent and reverse transcribed into cDNA. The mRNA levels of (A) IL-1ß, (B) IL-6, (C) IL-10, (D) Itgam, (E) AIF1, (F) Adgre1, (G) CD68 and (H) NOS2 were determined by real-time qPCR, using S18 as an internal housekeeping gene. Levels were quantified using the Delta-CT method. Data represent means of 4–6 samples for each group. Results are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control as determined by one-way ANOVA followed by Dunnett’s post hoc test. (Gene: protein acronyms): IL-1ß: Interleukin 1ß, IL-6: Interleukin 6, IL-10: Interleukin 10, Itgam: CD11b, AIF1: Iba1, Adgre1: F4/80, CD68: Cluster of differentiation 68 (CD68), NOS2: inducible nitric oxide synthase (iNOS), S18: ribosomal protein S18.

Fig 3: Effects of treatment with PACAP or VIP on LPS-induced microglia activation markers in BV2 cells. Real-time qPCR and Western blot analyses depicting AIF1/Iba1 and NOS2/iNOS mRNA and protein expression levels in cells exposed to LPS (1 µg/mL) and after co-treatment with either PACAP or VIP at 24 h. mRNA levels of AIF1 (C) and NOS2 (E) and related proteins (Iba1 and iNOS (A,B,D)) are shown. qPCR data represents means of n = 5–6 biological replicates for each group. Results are expressed as mean ± SEM. ** p < 0.01 or *** p < 0.001 vs. control; # p < 0.05, ## p < 0.01 or ### p < 0.001 vs. LPS only, as determined by one-way ANOVA followed by the Tukey post hoc test. Western blot data represent the mean of four independent experiments, each run in duplicate. AIF1: Ionized calcium binding adaptor molecule 1 (Iba1), GAPDH: Glyceraldehyde3-phosphate deyhydrogenase, S18: ribosomal protein subunit.

Fig 4: iPSC-EV regulated macrophage polarisation and cytokine secretion. A. Representative western blot image showing the expression of a M2 marker (CD206) and M1 markers (iNOS, and TNF-a) in M0, M0EV+, M1, and M1EV+. B.-D. Quantification of protein abundance from western blot results (n ?= ?3) E-G. ELISA analysis of cytokines secreted by macrophages under different conditions (n ?= ?3) *Significant difference to control: **p ?< ?0.01. #Significant difference between groups: #p ?< ?0.05, ##p ?< ?0.01).Abbreviations: PSC-EV: induced pluripotent stem cell-derived extracellular vesicles; IFN-?: Interferon gamma; iNOS: inducible nitric oxide synthase; TNF-a: tumour necrosis factor alpha

Fig 5: Gene expression of a panel of pro- and anti-inflammatory genes in BV2 microglial cells after 1000 µM AZE treatment for 6 and 24 h. Relative fold-changes were calculated using the ?Ct method after normalization to the S18 housekeeping gene. Box-and-whisker plots depict the differential expression of pro-inflammatory (A–F) IL-1ß, IL-6, NOS2, Itgam, CD68 and MHC-2a, (G) extracellular matrix (ECM) degradation (MMP-9) and (H–I) anti-inflammatory IL-10 and Arg1 transcripts. Results are presented as mean fold changes with respect to no treatment (Ctrl) ± SEM. Data represents n = 6 samples per group. * p < 0.05, ** p < 0.01 or **** p < 0.0001; as determined by ANOVA followed by Sidak’s post hoc test. Ns = not significant.