Imiquimod

fcMSCs progenitor cells were plated at a density of 3 x 10⁵ cells/well in 0.1% gelatin coated 6 well-plates. Cells were pre-treated for 1 h with 5 μg/ml of poly I:C (Sigma-Aldrich) or 5 μg/ml of imiquimod (Enzo Life Sciences), then cells were infected with 0.1, 1, or 10 MOI of ZIKV. ZIKV replication was assessed using qRT-PCR for ZIKV NS1 mRNA 48 h post infection. For TLR dose-dependent activation, fcMSCs were pretreated with 0.1, 1, or 10 μg/ml of either poly I:C or imiquimod, cells were then infected with 0.1 MOI of ZIKV for 6 h. Cells were harvested 72 h post-infection, fixed, permeabilized, and intracellular staining of ZIKV was quantified by flow cytometry as described above. Following virus exposure period in both experiments, media was aspirated, cells were washed with 1X PBS, and fresh media added.

For stereotaxic surgery, 3–5-months-old GR^DATiCre, GR^LysMCre, GR^CX3CR1CreER2 mutant male mice and GR^loxp/loxp control littermates were anesthetized by an i.p. injection of 100 mg kg⁻¹ ketamine and 10 mg kg⁻¹ xylazine. The scalp was shaved and a small hole was made at the surface for injection into the left substantia nigra using the following stereotaxic coordinates in mouse Paxinos atlas (David Kopf Instruments, Tujunga, CA, USA) from Bregma −2.8 mm anteroposterior, +1.3 mm mediolateral, and –4 mm dorsoventral. In all, 0.5, 1, and 1.5 μg of TLR4 agonist LPS (LPS Escherichia coli, serotype 055:B5; Sigma, St. Quentin Fallavier, France), 1 μg of TLR2 agonist (Pam3Cys-Ser- (Lys) 4 Trihydrochloride; Abcam, ab142085), 1 μg of TLR7 agonist, Imiquimod (Enzo Life Sciences, ALX-420-039-M100), 0.96 μg (1 μM) of TLR9 agonist ODN 1826 (TLRgradeTM; 5′-TCCATGACpGTTCCTGACpGTT-3′) (Enzo Life Sciences, ALX-746-002-T100) or a control ODN sequence (5′-TCCATGAGCTTCCTGAGCTT-3′) dissolved in PBS were injected using 10 μl Hamilton syringe into the substantia nigra over a 5-min period. For Adeno-associated vector (AAV) serotype 6 PGK-A53T-α-synuclein injections, the stock of 1.97 × 10¹³ gp (genomic particles/ml) was diluted to 2.5 × 10⁹ gp μl⁻¹ in saline. 2 μl of AAV-PGK-A53T α-synuclein was injected and mice sacrificed after 8 weeks.