Effects of P4 on the differentiation of APCs from BM.

<p>BM cell isolated from OVX mice were cultured in sex hormone-deficient medium with murine GM-CSF alone, or in the presence of varying concentrations of P4. Data in (A, B, C) are from cells collected at end of culture, and stained with Abs for CD11b and CD11c. Initial gating for monocytes as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153304#pone.0153304.g001" target="_blank">Fig 1A</a>. Representative dot plot data are shown in (A). Percentages of CD11b⁺CD11c⁺ APCs from cultures grown in presence of different concentrations of P4, treated with (dashed) or without (solid) LPS are shown in (B). Absolute numbers of CD11b⁺ CD11c⁺ APCs cultured with P4 are shown in (C). Data show Mean ± SD of 6 independent experiments. Statistical significance is indicated by * p<0.05; **p<0.01; *** p<0.001 compared with untreated control.</p

High doses of P4 reversed effects of E2 in presence or absence of LPS treatment.

<p>BM cells isolated from OVX mice were cultured with GM-CSF in the presence of E2 at a low (10^-11M) (A) or high (10^-9M) (B) concentration with varying doses of P4 (10^-9M to 10^-5M), with (dashed) or without (solid) LPS. At the end of culture, cells were harvested and stained with Abs to detect CD11b, CD11c, CD40 and MHCII. Data are percentages of CD11b⁺CD11c⁺ DCs (top panels); percentages of CD40⁺/CD11b⁺CD11c⁺DCs (middle panels); and percentages of MHCII⁺/CD11b⁺CD11c⁺ DCs (bottom panels). Data shown are Mean ± SD of 6 independent experiments. Statistical significance is indicated by *p<0.05; **p<0.01;*** p<0.001 compared with E2 only treated controls.</p

Effects of E2 and P4 on the uptake of FITC-Dextran by BMDC.

<p>BM cells isolated from OVX mice were cultured with GM-CSF in the presence of E2 (10^-11M and 10^-9M) or P4 (10^-8M and 10^-6M) or 10^-9M E2 + 10^-6M P4 and FITC-Dextran (200μg/ml) was added to the culture on day 5 for further 2 hrs at 37°C. Negative control was FITC-Dextran added to BMDCs and incubated at 4°C for 2 hrs. Cells were collected and stained with antibodies for CD11b and CD11c. Percentage of FITC-Dextran-positive CD11b⁺CD11c⁺ DCs were obtained by gating on CD11b⁺CD11c⁺ DCs. Representative flow cytometry data are shown in (A) and corresponding data graph are shown in (B). Data shown are Mean ± SD from 4 independent experiments. Statistical significance is indicated by *p<0.05; ** p<0.01; ***p<0.001 compared with untreated control or the comparison of combination treatment with single hormone treatment.</p

Effects of E2 on the differentiation of DC from BM.

<p>BM cell isolated from OVX mice were cultured in sex hormone-deficient medium with murine GM-CSF alone, or in the presence of varying concentrations of E2 (A, B, C, D). Initial gating for mononuclear cells is shown in (A). Data in (B, C, D) are from cells collected at end of culture, and stained with Abs for CD11b and CD11c. Representative dot plot data are shown in (B). Data for percentages of CD11b⁺CD11c⁺ DCs from cultures with different concentrations of E2 and treated with (dashed) or without (solid) LPS are shown in (C). Absolute numbers of CD11b⁺ CD11c⁺ DCs cultured with different concentrations of E2 are shown in (D). Data shown is Mean ± SD of 6 independent experiments. Statistical significance is indicated by * p<0.05; **p<0.01; *** p<0.001 compared with untreated control.</p

Effects of E2 and P4 on BM APC differentiation and maturation were mediated by their respective receptors.

<p>BM cells isolated from OVX mice (B, C, D) or ER KO mice (A) were cultured with GM-CSF in the presence of 10^-11M (A) or 10^-9M E2 (A, B) or (10^-6M) P4 (C, D). Varying concentrations of E2 inhibitor ICI 182 780 (B), P4 inhibitor RU-486 (C) or Glucocorticoid receptor AL082D06 (D) were used in E2- or P4-treated BM cultures, beginning at the first day of culture. At the end of culture, cells were collected and stained with antibodies against CD11b and CD11c (A, B) and plus MHC II (C, D). Percentage of CD11c+ cells are shown in (A, B) for E2 cultured cells and P4 cultured cells (C, D). Data show Mean ± SD of triplicate determination each time of 3 independent experiments. Statistical significance is indicated by *p<0.05,** p<0.01, ***p<0.001 compared with untreated control.</p

High dose of P4 inhibited the cytokine secretion from E2-treated BMDCs.

<p>BM cells isolated from OVX mice were cultured with GM-CSF in the presence of varying doses of E2 or P4 or combination of both. On day 6, 5ng/ml LPS was added to the culture for 24hrs. Culture treated with LPS alone (+LPS-E2) was negative control and untreated culture (-LPS -E2) was background. The protein transport inhibitor (BD Golgi Plug) was added for the last 6 hrs of culture. Cells were stained for intracellular IL-12, TNF- α and IL-6 with respective APC-conjugated antibodies. The top panel in A is gating scheme and isotype control. The rest of the panels in A are the percentage of IL-12-secreting CD11b⁺CD11c⁺ DCs by intracellular staining under experimental conditions. Percentage of CD11b+CD11c+ DCs positive for IL-12, TNF-α and IL-6 are showed in B, C and D, respectively. Data represent samples in triplicate from 2 independent determinations. *p<0.05; ** p<0.01; ***p<0.001 compared with non-hormone treated control or the comparison of combination treatment with single hormone treatment.</p

E2 and P4 induced different cytokine secretion profile from BMDCs.

<p>BM cells isolated from OVX mice were cultured with GM-CSF in the presence of varying doses of E2 or P4. On day 6, 5ng/ml LPS was added to the culture for further 24h culture and supernatants were collected and cytokine concentrations were determined (IL-12, IL-10, IL-8, IL-10, TNF-α, IFN-γ and IL-1β) by multi-analyte cytokine and chemokine assays. TGF-β in the supernatants was determined by ELISA. The values (pg/ml, mean ±SD) were normalized to the percentage of CD11b⁺ CD11c⁺ DCs in each treatment. Data represent triplicate samples from 2 independent experiments. Statistical significance is indicated by *p<0.05; ** p<0.01; ***p<0.001 compared with untreated control.</p

Effects of E2 and P4 on BM APC maturation.

<p>BM cell isolated from OVX mice were cultured in sex hormone-deficient medium with murine GM-CSF alone, or in the presence of varying concentrations of E2 (A, B) or P4 (C, D). At end of culture, cells were collected and stained with Abs for CD11b, CD11c, MHC II and CD40 antibodies. Cell culture were treated with (dashed) or without (solid) LPS for last 24 hrs. Y axis represents the percentage of CD40+ (A, C) or MHCII+ (B, D) CD11b⁺CD11c⁺ DCs. Data shown are Mean± SD of 6 independent experiments. Statistical significance is indicated by * p<0.05; **p<0.01 compared with untreated control.</p

IL-17 KO mice are more susceptible to intravaginal HSV-2 challenge.

<p>OVX IL-17 KO (n = 5 mice) and WT mice (n = 4 mice) were intravaginally exposed to a sub-lethal dose of HSV-2 333 (10² pfu/mouse), and 8 weeks later, intravaginally challenged with a lethal dose of HSV-2 333 (5x10³ pfu/mouse). (A) Survival curves for IL-17 KO and WT mice showing percentage of mice that survived challenge. Significance in difference in survival was calculated using the log-rank (Mantel-Cox) test (* p<0.05). (B) Genital pathology graded on a 1–5 scale for both groups of mice for 12 days post-challenge. Data points superimposed on X-axis indicate mice without genital pathology, and the % indicates maximum number of mice that showed pathology. (C) HSV-2 viral shedding (pfu/mL) in vaginal washes collected for 6 days post-challenge, was measured by conducting viral titers using vero cells. The dashed line indicates the lower detection limit of this assay. The % indicates maximum number of mice that shed virus between days 1 to 6 post challenge. Each symbol represents a single animal, and data points on the lower limit indicate mice that do not show detectable viral shedding in vaginal washes. The survival curves, pathology and viral titers are from a single representative of three separate experiments with similar results. (D) Vaginal tissues from stage-matched WT and IL-17 KO mice were isolated, pulsed with OVA peptide and co-cultured for 3.5 days with OT-II Tg CD4⁺ T cells. IFN-γ levels produced by vaginal tissue cells alone (TC) and co-cultures (TC+CD4) were measured by ELISA. Data is represented as mean±SD of three co-culture wells, and is a representative of three separate experiments with similar trends. Significance was calculated by two-way ANOVA (**** p<0.0001).</p

E2 pre-treatment enhances the recruitment of CD103⁺ CD44⁺ CD4⁺ T cells in the vagina, and is related to increased proportions of T_h1 and T_h17 cells, post-challenge.

<p>WT OVX mice implanted with E2 or placebo pellets (mock) (n = 5–10 mice/group in all three time points: D1, D3 and D5 p.c.), were immunized intranasally with 1x10³ pfu/mouse TK⁻ HSV-2, and five weeks later, challenged intravaginally with 5x10³ pfu WT HSV-2 333. Vaginal tissues isolated at D1, D3 and D5 post challenge (p.c.), from each group were pooled, processed and stained with a panel of antibodies against CD3, CD4, CD8, CD44, CD103, IL-17 and IFN-γ according to protocols detailed in the Materials and Methods section, and examined by flow cytometry. (A) CD8^- CD4⁺ T cells were gated among total CD3⁺ T cells in the vagina. (B) The proportion of mucosal memory CD103⁺ CD44⁺ T cells from tissues isolated on days 1, 3 and 5 p.c. were compared between E2- and placebo (mock)-treated mice. (C) For intracellular staining of IL-17 and IFN-γ, vaginal cells pooled from n = 5 mice per group, at days 1, 3 and 5 p.c, were incubated in the presence of golgi inhibitors alone to examine the <i>in vivo</i> response to HSV-2 challenge, or stimulated <i>in vitro</i> with cell stimulation cocktail (CSC) containing golgi inhibitors and PMA + ionomycin, for 18h. Intracellular staining for IL-17 and IFN-γ was used to examine the differentiation of CD4⁺ T cells into T_h17 and T_h1 cells, respectively. A representative of this data from day 3 p.c. is shown. Data is representative of two independent experiments.</p