Dendritic cell activation and memory cell development are impaired among mice administered medroxyprogesterone acetate prior to mucosal herpes simplex virus type 1 infection

Abstract

Epidemiological studies indicate that the exogenous sex steroid medroxyprogesterone acetate (MPA) can impair cell-mediated immunity, but mechanisms responsible for this observation are not well defined. In this study, MPA administered to mice 1 wk prior to HSV type 1 (HSV-1) infection of their corneal mucosa impaired initial expansion of viral-specific effector and memory precursor T cells and reduced the number of viral-specific memory T cells found in latently infected mice. MPA treatment also dampened expression of the costimulatory molecules CD40, CD70, and CD80 by dendritic cells (DC) in lymph nodes draining acute infection, whereas coculture of such DC with T cells from uninfected mice dramatically impaired ex vivo T cell proliferation compared with the use of DC from mice that did not receive MPA prior to HSV-1 infection. In addition, T cell expansion was comparable to that seen in untreated controls if MPA-treated mice were administered recombinant soluble CD154 (CD40L) concomitant with their mucosal infection. In contrast, the immunomodulatory effects of MPA were infection site dependent, because MPA-treated mice exhibited normal expansion of virus-specific T cells when infection was systemic rather than mucosal. Taken together, our results reveal that the administration of MPA prior to viral infection of mucosal tissue impairs DC activation, virus-specific T cell expansion, and development of virus-specific immunological memory.

Figures

Fig. 1

Pretreatment reduces T cell expansion elicited by HSV-1 corneal infection. Ovx B6 mice were corneally infected with 105 PFU HSV-1 seven days after the insertion of 21-day sustained release pellets containing 50 mg MPA or matching placebo. Mice were euthanized at indicated time points, and various tissues excised to enumerate infiltrating virus-specific T cells by flow cytometry. (A) Absolute numbers of CD4+, CD8+, gB498-505-specific CD8+, and CD127+ (memory precursors) T cell subsets in TG 8 dpi. (B) Relative frequency of gB498-505-specific CD8+ and CD127+ T cells in TG 8 dpi. Data in (A) and (B) were pooled from 3 independent experiments (n = 18 per group). (C) Absolute numbers of gB498-505-specific CD8+ T cells in DLN at 5 and 8 dpi (n = 8–13 per group). (D) Absolute numbers of NK cells, NKT cells, and macrophages in TG 8 dpi (n = 10 per group). (E) Total number of live CD45+ cells (LIVE/DEAD−) in TG at 8, 15, and 35 dpi (n = 6–18 per group). (F) Absolute numbers of CD4+, CD8+, and gB498-505-specific CD8+ T cells in TG 15 and 35 dpi (pellets removed 14 dpi) (n = 6–18 per group). Data in (C–F) were pooled from 2 independent experiments for each panel. Comparisons were made using unpaired two-tailed Student t-test, except for comparisons of the number of gB498-505-specific CD8+ and CD4+ T cells per TG in (A), which were made using unpaired Mann-Whitney U test; (horizontal bars accordingly indicate mean and median values).

Fig. 2

Pretreatment impairs expansion of virus-specific CD8+ T cells induced by HSV-1 corneal infection in a dose-response manner. (A) Intact B6 mice were implanted with 21-day sustained release pellets containing 50 mg MPA, administered 1 or 4 mg DMPA, or were left untreated. 7 days later, all groups were corneally infected with 105 PFU HSV-1. Mice were euthanized 8 dpi, and TG excised to enumerate infiltrating HSV-1 specific T cells by flow cytometry. Absolute numbers of CD8+ T cells per TG are shown (n = 20–25 per group) (data shown pooled from 3 independent experiments). Of note, the numbers of CD4+ and gB498-505-specific CD8+ T cells were similarly reduced by pretreatment (data not shown). (B) Groups of mice administered MPA in exactly the same fashion as in (A) were sacrificed 7 days later to determine serum levels of MPA by radioimmunoassay (n = 5–8 per group) (** p < 0.01; *** p < 0.001 by one-way ANOVA and Tukey’s multiple comparison post test; horizontal bars indicate mean values).

Fig. 3

Pretreatment diminishes virus-specific CD8+ T cell effector function. Intact B6 mice given 4 mg DMPA 7 days prior to corneal infection with 105 PFU HSV-1 were euthanized 8 dpi. Excised TG were dispersed into single-cell suspensions and stimulated with HSV-1 infected targets to interrogate T cell effector function by flow cytometry. (A) Depiction of response profiles using Boolean analysis of 3 canonical CD8+ T cell effector functions (IFN-γ and TNF production and lytic activity as measured by CD107a surface expression) (n = 5 per group). (B) Pie charts compiled from bar graph data in (A) illustrate percentages of CD8+ cells that expressed 3, 2, 1 or 0 effector function markers (beginning at “12 o-clock” and moving clock-wise). Data shown in (A) and (B) are representative of 2 independent experiments (comparisons made using SPICE 5.2 [59]; horizontal bars and pie fractions indicate means). (C) Contour plots display expression of IFN-γ, TNF and CD107a by TG-resident CD8+ T cells stimulated ex vivo with infected targets. Data shown are representative results from each experimental group, and numbers indicate percentages within each quadrant.

Fig. 4

MPA does not affect HSV-1 replication. (A) The ability of MPA to alter in vitro HSV-1 replication was evaluated using multistep viral replication kinetics with Vero cell monolayers treated with MPA (10 μM) or vehicle (Ctrl) 24 h prior to infection (n = 9). Lower MPA concentrations were also tested, and none altered in vitro HSV-1 replication (data not shown). (B) Untreated or pretreated female B6 mice were infected with 105 PFU HSV-1 per eye, and eye swabs collected at indicated dpi to compare viral titers by standard plaque assay (n = 12 per group). In (A) and (B), viral titers are shown as mean ± SD, two-way ANOVA was used to compare groups. In other experiments, pretreated or untreated intact B6 mice or untreated B6 mice administered i.v. ACV 1 dpi were infected with 105 PFU HSV-1 per eye. Eye swabs were collected 2 dpi, and mice directly euthanized for corneal harvest (n = 18 per group). In (C), data points denote HSV-1 titers from corneal swabs of individual mice as determined by plaque assay (horizontal bars designate mean values). In (D), data bars denote mRNA expression in whole corneas of the immediate-early viral genes ICP0 and ICP4 and the leaky-late viral gene gH relative to expression of the housekeeping gene PCX as measured by quantitative real-time PCR (bars indicate mean ± SD). (In (C) and (D), *** p < 0.001 compared to all groups by one-way ANOVA and Tukey’s multiple comparison post test). In other experiments, pretreated and untreated B6 mice were infected, and sacrificed 8 dpi to excise TG. HSV-1 genome copy number per TG was determined by quantitative real-time PCR (n = 22 per group). Each data point represents the viral genome copy number from single TG. Comparison was performed using unpaired one-tailed Student t test, and horizontal bars indicate mean values. In all Fig. 4 experiments, mice were pretreated with 4 mg DMPA (all data shown are pooled results from 3 independent experiments).

Fig. 5

HSV burden in the acutely infected TG is increased only when TG-infiltrating CD8+ T cells are fully eliminated. Female B6 mice were corneally infected with 105 PFU HSV-1, and at 3 dpi were administered the indicated amounts of anti-CD8 mAb. Mice were euthanized 8 dpi, and TG excised to enumerate virus-specific T cells by flow cytometry and determine viral genome copy number per TG by quantitative real-time PCR (n = 5 per group). Each data point denotes the absolute number of CD8+ T cells or the HSV-1 genome copy number per TG (* p < 0.05; ** p < 0.01; *** p < 0.001 by one-way ANOVA and Tukey’s multiple comparison post test; horizontal bars indicate the mean values). Data shown are results from 1 of 2 independent experiments.

Fig. 6

Pretreatment reduces proliferation of virus-specific T cells. Pretreated (4 mg DMPA) or untreated intact female B6 mice were corneally infected with 105 PFU HSV-1. At 5 dpi, mice received i.v. BrdU, and 4 h later mice were euthanized. DLN were then processed into single-cell suspensions to determine BrdU incorporation by flow cytometry. (A) Figures show percentages of DLN CD4+, CD8+, and gB498-505-specific CD8+ T cells that incorporated BrdU (n = 12 per group). Data shown were pooled from 2 independent experiments, and comparisons were made using a one-tailed Mann-Whitney U test (except for the between-group comparisons of BrdU+CD4+ T cells, which were made using an unpaired one-tailed Student t test) (horizontal bars accordingly indicate mean and median values). (B) Contour plots illustrate BrdU incorporation by each examined T cell subset. Data shown are representative, and numbers indicate percentages of BrdU+ cells.

Fig. 7

Pretreatment impairs DC activation and T cell priming. (A) Pretreated (4 mg DMPA) and untreated female B6 mice were corneally infected with 105 PFU HSV-1. Mice were euthanized 2 dpi. DLN were excised and rendered into single-cell suspensions, to determine expression levels for MHC and several costimulatory molecules in DC and B cells by flow cytometry. CD70, CD80, CD86, TNFSF4 (OX40L), MHC-I, MHC-II, and CD40 iMFI are shown as a relative measure of the amount of each protein expressed by the indicated DC subsets (n = 10 per group, data pooled from 2 independent experiments) (comparisons made with unpaired one-tailed Student t-tests; bars indicate mean ± one SD). Bottom right of panel shows representative contour plots for CD40 expression by various DC subsets (numbers indicate percentages of CD40+ cells). (B) Representative contour plots illustrate gating strategies that were used to define 4 main DLN DC subsets. (C) 2.5 × 104 CD11c+ cells that were isolated from the DLN of pretreated or untreated B6 female mice 2 dpi were co-cultured 72 h with 5 × 104 CD8+ splenic cells from pretreated or untreated naive gBT-I.1 mice labeled with a cell-tracing reagent, and the proliferation of CD8α+CD90.1+Vα2+ cells was determined by flow cytometry. Histograms show percentages of proliferating cells for the indicated culture conditions (data shown are representative results from 1 of 3 independent experiments).

Fig. 8

Reduced CD40 expression by pretreated mice inhibits T cell expansion. Untreated B6 mice, untreated B6 mice administered α-CD154 mAb, and pretreated (4 mg DMPA) B6 mice were corneally infected with 105 PFU HSV-1 (another group of pretreated mice were also administered rsCD154 concomitant with infection as indicated). Mice were euthanized 8 dpi, and TG excised to interrogate T cell infiltrates by flow cytometry. (A) Absolute numbers of total CD45+, CD8+, and CD4+ T cells are shown (n = 13–15 per group). (B) Panel depicts percentages of CD8+ T cells that expressed GzmB (n = 10 per group). (C) Relative expression of CD69 by CD4+ and CD8+ T cells (n = 10 per group) (data shown are pooled from 2 independent experiments). (D–G) To confirm results above using α-CD154 mAb, we corneally infected untreated BALB/cJ controls, pretreated BALB/cJ mice, and CD154−/− BALB/cJ mice with 105 PFU HSV-1, and harvested TG 10 dpi (n = 10 per group). (D) Absolute numbers of TG-resident CD45+ cells and CD8+ and CD4+ T cells. (E) Percentage of CD8+ T cells that express GzmB in TG. (F) Relative expression of CD69 by CD8+ and CD4+ T cells in TG 10 dpi (data are pooled from 2 independent experiments) (* p < 0.05; ** p < 0.01; *** p < 0.001 by one-way ANOVA and Tukey’s multiple comparison post test; horizontal bars indicate means). (G) Contour plots show CD69 and GzmB expression by CD8+ T cells infiltrating TG during acute infection of untreated BALB/cJ, pretreated BALB/cJ, and CD154−/− BALB/cJ mice (representative data; numbers indicate percentages in each quadrant).

Fig. 9

MPA-mediated reduction of virus-specific T cell expansion is not observed with systemic HSV-1 infection. Untreated or pretreated (4 mg DMPA) intact female B6 mice were i.p. or i.v. infected with 106 PFU HSV-1. At 6 dpi, mice were administered 1 mg BrdU (i.v.). 4 h later mice were euthanized, spleens processed into single-cell suspensions, and (A) percentages of BrdU+ cells and (B) numbers of gB498-505-specific CD8+ T cells were determined by flow cytometric analysis. Comparisons were made using unpaired one-tailed Student t-tests; horizontal bars indicate means (n = 5 per group). Data shown are representative results from 1 of 2 independent experiments.