Fig. 3

Chemokine ligand expression in vaginal tissue. WT female C57BL/6 mice were immunized with a single nasal (n.i.) or intraperitoneal (i.p.) dose comprising 10⁵ PFU of live attenuated HSV-2 TK⁻. Vaginal tissue samples were harvested at 1 week after immunization. a RT-qPCR analysis of CCL3, CCL4, and CCL5 expression in vaginal tissues. Chemokine mRNA expression levels were normalized to that of β-actin. Values are means ± 1 SD (n = 3 or 4 mice per group). *P < 0.05 versus nonimmunized mice (nasal PBS) group. Data are representative of two independent experiments. b CCL5 expression in frozen sections of vaginal tissue was evaluated by using immunofluorescence assays. Tissue sections were prepared and stained with anti-CCL5 (red), anti-CD45 Ab (green), and DAPI (4′, 6-diamidino-2-phenylindole; blue). Arrows indicate co-localization of CCL5 and CD45. Data are representative of two independent experiments (n = 3 mice per group). The merged-color (yellow) cells in two random fields of the stained image were counted; the total numbers of CD45⁺ CCL5⁺ cells are presented in a table. c CCL5 expression in vaginal tissue after intraperitoneal (i.p.) and nasal immunization (n.i.) of mice with HSV-2 TK⁻. At 7 days after the immunization, CD45⁻ and CD45⁺ cells from vaginal tissues were enriched by magnetic activated cell sorting. The levels of CCL5-specific mRNA expression were assessed by RT-qPCR analysis and normalized to that of β-actin. Values are given as means ± 1 SD (n = 3 or 4 mice per group). *P < 0.05 versus nonimmunized (nasal PBS) mice. Data are representative of two independent experiments. d, e Col-GFP transgenic mice were immunized with a single nasal dose comprising 10⁵ PFU of live HSV-2 TK⁻ (or PBS, nonimmune controls). Vaginal tissue samples were harvested at 1 week after immunization. d CCL5 expression in frozen sections of vaginal tissue from each mice group was detected by using immunofluorescence assays. Tissue sections were prepared and stained with anti-CCL5 (red) and DAPI (4′, 6-diamidino-2-phenylindole; blue). GFP⁺ (green) cells indicate type I collagen-producing cells. Arrows indicate cells where co-localization of collagen-GFP with CCL5 occurs. Data are representative of two independent experiments (n = 2 or 3 mice per group). The merged-color (yellow) proportion of randomly selected field of stained image was quantified by using ImageJ software (National Institutes of Health, Bethesda, MD). Values are given as means ± 1 SD (n = 3 fields). *P < 0.05 versus nonimmunized (nasal PBS) mice. e RT-qPCR analysis of CCL5 expression in CD45⁻ GFP⁺ cells from vaginal tissues of col-GFP transgenic mice after nasal immunization. Live CD45⁻GFP⁺ cells were sorted by flow cytometry. The CCL5 mRNA expression level was normalized to that of β-actin. Values are means ± 1 SD (n = 3 or 4 mice per group). *P < 0.05 versus nonimmunized col-GFP transgenic mice. Samples were pooled from three independent experiments