Microbiota-independent antiviral protection conferred by aminoglycoside antibiotics

Smita Gopinath; Myoungjoo V. Kim; Tasfia Rakib; Patrick W. Wong; Michael van Zandt; Natasha A. Barry; Tsuneyasu Kaisho; Andrew L. Goodman; Akiko Iwasaki

EtOH/Water Neomycin Table S2 . Depo-treated Tlr7 -/-mice were treated intravaginally with neomcyin (1mg/day) for 6 days and then infected with HSV-2 and disease scores monitored (a) and vaginal viral titer measured (b). Error bars represent SEM and statistical signi cance was calculated using 2-way ANOVA (a,b). Speci c p values are reported in Table S2 . Type I IFN receptor knockout mice (Ifnar1 -/-) and accompanying WT controls were treated subcutaneously with Depo-Provera and treated intravaginally with neomycin (1mg) or PBS daily for 6 days and vaginal gene expression measured via qPCR (a). In an independent experiment neomycin-treated Ifnar1 -/mice and PBS-treated controls were also infected with HSV-2, disease score monitored daily (b) and vaginal viral titers measured (c). Error bars represent SEM and signi cance was calculated using either unpaired t-tests (a) or 2-way ANOVA (b,c) correcting for multiple comparisons. Exact p values for all comparisons are reported in Table S2 . Mice treated subcutaneously with Depo-Provera were inoculated intravaginally with PBS or neomycin (1mg) for one week. Vaginal tissue from neomycin-treated and PBS control mice was xed, embedded and stained with hematoxylin and eosin. Images are magni ed 200X (a). Splenocytes were treated with 2mg/ml kasugamycin for 6 hours and dead cells were quanti ed using xable live/dead stain as a frequency of total leukocytes (c) or total cells (d). In an independent experiment kasugamycin-treated splenocytes were depleted of DCs and ISG expression quanti ed (e). Error bars represent SEM and exact p values are reported in Table S2 . Splenocytes were treated with2mg/ml kasugamycin for 12 hours, washed thoroughly, stained with cell trace violet and incubated with DCs isolated from WT or Tlr3 -/splenocytes. After 6 hours of incubation, DCs (cell trace violet dim and negative cells) were sorted, RNA extracted and ISG expression quanti ed. Error bars represent SEM and speci c p values are detailed in Table S2 .