Cytokine production by CD4⁺ T cells in the vagina post intravaginal HSV-2 challenge.

<p>Cytokine production by CD4⁺ T cells in the vagina post intravaginal HSV-2 challenge.</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

E2 conditions vaginal DCs to induce T_h17 responses through an IL-1-dependent pathway.

<p>(A) Tissue cells from the vagina, lung and intestine were pulsed with OVA peptide and co-cultured with CD4⁺ T cells (TC+CD4). IL-6, IL-23 and TGF-β were measured in co-culture supernatants by ELISA. (B) Intracellular staining of vagina co-cultures on day 2 of co-culture to examine IL-1β and IL-6 production by vaginal DCs (CD11c⁺ cells) and macrophages (CD11c⁻ CD11b⁺ F4/80⁺ Gr-1⁻). (C) IL-17 levels compared among vaginal TC co-cultures from IL-6 KO, IL-1β KO and WT control mice. (D) IL-17 levels compared in co-cultures conducted with TC or CD11c⁺ DCs purified from the vagina of IL-6 KO mice and WT controls. 40 ng/ml of rIL-6 was added to co-cultures as indicated on X-axis. (E) IL-17 levels were compared in co-cultures conducted with TC or CD11c⁺ DCs purified from the vagina of IL-1β KO mice and WT controls. 100 ng/ml rIL-1β was added to co-cultures as indicated on X-axis. (F) Vaginal cells were cultured overnight without any stimulation, and intracellular staining was conducted to identify IL-1β production in CD11c⁺ DCs from OVX mice implanted with E2, P4 or placebo (mock) pellets. G) Matched experimental data showing the proportion of IL-1β⁺ DCs. Significance was calculated by a ratio paired t test and * p = 0.0284. (H) CD11c⁺ DCs compared among E2-, P4- or placebo (mock)-treated mice from three independent experiments. Data for all cytokine measurements is represented as mean±SD of three separate co-culture wells. Data is a representative of at least two separate experiments with similar results, and significance was calculated by two-way ANOVA. (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001).</p

E2 conditioned T_h17 responses are not altered by the addition of CpG.

<p>(A) OVX mice were implanted with E2 or placebo (mock) pellets, and 14 days later, vaginal cells were isolated and incubated overnight unstimulated (UNS), or stimulated with the TLR ligand CpG. ICS was conducted to examine the expression of IL-1β in CD11c+ vaginal DCs. (B) Vaginal cells from E2- or placebo (mock)-treated mice, and CD11c+ DCs flow sorted from E2-treated mice were pulsed with OVA peptide in the presence or absence of CpG for 12h, and co-cultured with OT-II Tg CD4+ T cells for 3.5 days. IL-17 levels in supernatants were measured by ELISA. Significance was calculated by comparing mean±SD of three separate co-culture wells per condition, by two-way ANOVA (* p<0.05, **** p<0.0001), and data is representative of two similar experiments.</p

E2 pre-treatment enhances protection against genital HSV-2 challenge in intranasally immunized mice.

<p>WT (C57Bl/6) OVX mice treated with E2 or placebo pellets, were intranasally immunized 1 week and 3 weeks later with 1x10³ TK⁻ HSV-2, or 5μg HSV-2 gD + 30μg CpG, or 1x10⁴ pfu HI HSV-2 333 + 30μg CpG (n = 5–10 mice/hormone group for each vaccine formulation). Five weeks following the second immunization, all groups of mice were intravaginally challenged with 5x10³ pfu/mouse WT HSV-2 333. (A) Survival curves showing the percentage of mice that survived WT HSV-2 challenge in all vaccine formulations. Significance in difference in survival was calculated using the log-rank (Mantel-Cox) test (* p<0.05, ** p<0.01). (B) Pathology scores in these mice were graded on a 1–5 scale as described in the Materials and Methods section, and plotted. Data points superimposed on X-axis indicate mice without genital pathology, and the % indicates maximum number of mice that showed pathology. (C) Vaginal washes were collected for 5 days post-challenge, and HSV-2 viral shedding (bar indicates mean pfu/mL of shed virus) was calculated by conducting viral titrations with a vero-cell based assay. Dashed line indicates the lower detection limit of this assay, and data points on this line indicate undetectable viral shedding. The % indicates maximum number of mice that shed virus between days 1 to 5 post-challenge. Each symbol in B and C represents a single animal and data has been pooled from two separate experiments with similar results.</p

Cumulative pathology scores for HSV-2 pre-exposed WT and IL-17 KO mice challenged with WT HSV-2.

<p>Cumulative pathology scores for HSV-2 pre-exposed WT and IL-17 KO mice challenged with WT HSV-2.</p

E2 can influence the differentiation of CD4⁺ T cells in vaginal APC-T cell co-cultures.

<p>(A) OVX WT mice were implanted with E2 or placebo (mock) pellets, and two weeks later, vaginal tissue cells (TC) were isolated and pulsed with 5x10⁵ pfu/ml UV-inactivated HSV-2 for 16h. These TC were co-cultured for 3.5 days with HSV-2 specific CD4⁺ T cells (TC + HSV-2 CD4) isolated by MACS from the draining lymph nodes and vaginal tracts of HSV-2 immunized and challenged mice. For control co-cultures, naïve CD4⁺ T cells (TC + control CD4) were isolated from the spleen of uninfected OVX mice. IL-17 levels in co-culture supernatants were measured by ELISA. (B) Vaginal tissue cells (TC) (5x10⁵ cells/ml) from OVX mice (n = 6 mice/group) implanted with E2, P4, or placebo pellets (mock) were pulsed with OVA peptide and co-cultured with OT-II Tg CD4⁺ T cells (TC+CD4) (5x10⁵ cells/ml) for 3.5 days. Proliferation of CD4⁺ T cells was compared among co-cultures conducted with TC from E2-, P4- or placebo (mock)-treated mice. (C) IL-17 and IFN-γ levels in co-culture supernatants were measured by ELISA. Data is mean±SD of three individual co-culture wells from one of three separate experiments with similar trends, and significance was calculated by two-way ANOVA (* p<0.05, **** p<0.0001). (D) Intracellular staining of vaginal co-cultures to identify the cellular source of IL-17. On day 2 of co-culture, 2 ul/mL of CSC was added, and 18h later, co-cultures were stained with antibodies against CD3, CD4, IL-17 and IFN-γ, and analyzed on a flow cytometer. (E) Vaginal tissues from ovary-intact mice were pooled depending on the stage of their reproductive cycle (n = 6 mice/stage) (E2-dominant: Estrus, and P4-dominant: Diestrus, and OVX controls), pulsed with OVA-peptide, and co-cultured with OT-II Tg CD4⁺ T cells for 3.5 days. IL-17 levels in co-culture supernatants were measured by ELISA. Data is mean+SD of three individual co-culture wells, representative from one of three separate experiments with similar results, and significance was calculated by two-way ANOVA (**** p<0.0001). (F) IL-17 levels were compared among WT and ERKO vaginal and spleen tissue co-cultures. Data is representative of two separate experiments with similar results, and significance was calculated by two-way ANOVA (*** p = 0.0005).</p

IRF4 expression is not critical for E2-mediated priming of vaginal T_h17 responses.

<p><b>(</b>A) Vaginal cells from WT OVX mice implanted with E2, P4 or placebo (mock) pellets for 14 days were cultured in media overnight without any stimulation (12h), and stained for antibodies against IRF4 and DCs (CD11c, CD11b). ICS was conducted according to protocols in Materials and Methods to identify IRF4 expression in total vaginal cells and vaginal CD11c⁺ DCs. (B) Spleens from IRF4 KO mice and their WT littermates were isolated, and cultured overnight without stimulation. IRF4 expression in CD4⁺ T cells was compared between IRF4 KO mice and WT littermates by ICS. (C) Vaginal cells from reproductive cycle stage matched WT and IRF4 KO mice were pulsed with OVA peptide, and co-cultured with OT-II Tg CD4+ T cells for 3.5 days. IL-17 levels in co-cultures were measured by ELISA, and expressed as mean±SD of three replicate wells from one of two different experiments. Analysis was conducted by two-way ANOVA.</p

Vaginal CD11c⁺ DCs are the primary inducers of T_h17 responses, and are more potent inducers than other mucosal DCs.

<p>Vaginal cells from WT mice (n = 13 mice) were pooled and sorted by FACS, and total vaginal cells, as well as sorted populations, were pulsed with OVA peptide and co-cultured with 5x10⁵ cells/ml OT-II Tg CD4⁺ T cells at the indicated ratios. (A) CD4⁺ T cell proliferation in total vaginal tissue cell co-cultures, CD11c⁺ DC co-cultures and macrophage co-cultures. (B) IL-17 levels in co-culture supernatants were measured by ELISA and represented as mean±SD of three separate wells per co-culture condition. Statistical analysis was done by one-way ANOVA, to calculate significant differences in IL-17 levels between total vaginal co-cultures and the indicated cell-specific co-cultures at each given ratio of APCs:T cells. Data is representative of two separate experiments with similar results. (C) CD11c DTR mice (n = 5 mice/group) were treated with 400ng DT (200ng IVAG + 200ng IP) or PBS, and 18h later, vaginal tissues and spleen from each group were pooled, and 5x10⁵ tissue cells/ml were pulsed with OVA peptide, and co-cultured with OT-II Tg CD4⁺ T cells in a 1:1 ratio for 3.5 days. IL-17 and IL-22 levels in vaginal co-cultures, and IL-17 levels in spleen tissue co-cultures, were compared between DT-treated and PBS-control groups. Data is represented as mean±SD of three separate culture wells from one of two separate experiments with similar trends, and significance was calculated by two-way ANOVA (** p<0.01, *** p<0.001). (D) The vagina, lung and small intestine from WT mice were isolated, pooled (n = 7 mice) and processed into a cell suspension. Total tissue cells (5x10⁵ cells/ml) were cultured alone (TC) or co-cultured with 5x10⁵ OT-II Tg CD4⁺ T cells for 3.5 days, and IL-17 and IL-22 levels in culture supernatants were measured by ELISA. Data is represented as mean±SD of three separate culture wells from one of three separate experiments with similar trends, and significance was calculated by one-way ANOVA (*** p<0.001). (E) CD11c⁺ DCs and CD11c⁻ cells were sorted by FACS from the lungs and vagina of WT mice, and total tissue cells (5x10⁵ cells/ml), or purified cells (2.5x10⁵ cells/ml), were OVA peptide pulsed and co-cultured with OT-II Tg CD4⁺ T cells (5x10⁵ cells/ml). For heterologous mixed co-cultures, CD11c⁺ cells (2.5x10⁵ cells/ml) from the vagina or lung were mixed with CD11c⁻ cells (2.5x10⁵ cells/ml) from the other tissue, pulsed with OVA peptide, and co-cultured with CD4⁺ T cells (5x10⁵ cells/ml). IL-17 levels in supernatants were measured by ELISA. Significance was calculated by comparing mean±SD of three separate co-culture wells per condition, by one-way ANOVA (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001). Data is representative from two separate experiments with similar trends. (F) CD11c+ DCs (2.5x10⁵ cells/ml) sorted from the vaginal tissues of OVX mice implanted with E2 or placebo (mock) pellets (n = 20 mice/group), were pulsed with OVA peptide, and co-cultured with CD4+ T cells (5x10⁵ cells/ml) for 3.5 days. IL-17 levels in co-culture supernatants were measured by ELISA. Significance was calculated by comparing mean±SD of 6 replicates per condition, by a two-tailed unpaired T test with Welch’s correlation (* p = 0.026).</p