Vaginal progesterone, but not 17α-hydroxyprogesterone caproate, has antiinflammatory effects at the murine maternal-fetal interface

Abstract

Objective: Progestogen (vaginal progesterone or 17-alpha-hydroxyprogesterone caproate [17OHP-C]) administration to patients at risk for preterm delivery is widely used for the prevention of preterm birth (PTB). The mechanisms by which these agents prevent PTB are poorly understood. Progestogens have immunomodulatory functions; therefore, we investigated the local effects of vaginal progesterone and 17OHP-C on adaptive and innate immune cells implicated in the process of parturition.

Study design: Pregnant C57BL/6 mice received vaginal progesterone (1 mg per 200 μL, n = 10) or Replens (control, 200 μL, n = 10) from 13 to 17 days postcoitum (dpc) or were subcutaneously injected with 17OHP-C (2 mg per 100 μL, n = 10) or castor oil (control, 100 μL, n = 10) on 13, 15, and 17 dpc. Decidual and myometrial leukocytes were isolated prior to term delivery (18.5 dpc) for immunophenotyping by flow cytometry. Cervical tissue samples were collected to determine matrix metalloproteinase (MMP)-9 activity by in situ zymography and visualization of collagen content by Masson's trichrome staining. Plasma concentrations of progesterone, estradiol, and cytokines (interferon [IFN]γ, interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, keratinocyte-activated chemokine/growth-related oncogene, and tumor necrosis factor-α) were quantified by enzyme-linked immunosorbent assays. Pregnant mice pretreated with vaginal progesterone or Replens were injected with 10 μg of an endotoxin on 16.5 dpc (n = 10 each) and monitored via infrared camera until delivery to determine the effect of vaginal progesterone on the rate of PTB.

Results: The following results were found: (1) vaginal progesterone, but not 17OHP-C, increased the proportion of decidual CD4+ regulatory T cells; (2) vaginal progesterone, but not 17OHP-C, decreased the proportion of decidual CD8+CD25+Foxp3+ T cells and macrophages; (3) vaginal progesterone did not result in M1→M2 macrophage polarization but reduced the proportion of myometrial IFNγ+ neutrophils and cervical active MMP-9-positive neutrophils and monocytes; (4) 17OHP-C did not reduce the proportion of myometrial IFNγ+ neutrophils; however, it increased the abundance of cervical active MMP-9-positive neutrophils and monocytes; (5) vaginal progesterone immune effects were associated with reduced systemic concentrations of IL-1β but not with alterations in progesterone or estradiol concentrations; and (6) vaginal progesterone pretreatment protected against endotoxin-induced PTB (effect size 50%, P = 0.011).

Conclusion: Vaginal progesterone, but not 17OHP-C, has local antiinflammatory effects at the maternal-fetal interface and the cervix and protects against endotoxin-induced PTB.

Keywords: decidua; endotoxin; interleukin-1β; macrophages; matrix metalloproteinase-9; myometrium; neutrophils; preterm birth; preterm labor; regulatory T cells.

FIGURE 1. Animal model and identification of decidual CD4+ Tregs and CD8+CD25+Foxp3+ cells

A, Vaginal progesterone administration scheme. B, Gating strategy used to identify CD4+ Tregs (CD4+CD25+Foxp3+ cells) and CD8+CD25+Foxp3+ cells in decidual tissues. CD3+ T cells were gated within the total lymphocyte gate (FSC vs SSC). The green histogram represents the autofluorescence control. CD4+ Tregs and CD8+CD25+Foxp3+ cells were gated within the CD4+ and CD8+ gates, respectively. C, Proportions of decidual CD4+ Tregs in mice treated with vaginal progesterone or Replens (control; Lil’ Drug Store Products, Inc). D, Proportions of decidual CD8+CD25+Foxp3+ cells in mice treated with vaginal progesterone or Replens (control). E, Proportions of decidual CD4+ Tregs in mice injected with 17OHP-C or castor oil (control). F, Proportions of decidual CD8+CD25+Foxp3+ cells in mice injected with 17OHP-C or castor oil (control) (n = 10 each). Data are represented as mean ± SEM. FSC,; 17OHP-C, 17-alpha-hydroxyprogesterone caproate; SSC, saline sodium citrate; Treg, regulatory T cell.

FIGURE 2. Proportions of myometrial CD4+ Tregs and CD8+CD25+Foxp3+ cells

Proportions of myometrial CD4+ Tregs (CD4+CD25+Foxp3+ cells) and CD8+CD25+Foxp3+ cells in mice treated with vaginal progesterone or Replens (control; Lil’ Drug Store Products, Inc) (n = 10 each). Data are represented as mean ± SEM. Treg, regulatory T cell.

FIGURE 3. Immunophenotyping of innate immune cells in decidual tissues

A, Gating strategy used to identify NK cells (CD45+CD49b+ cells), DCs (CD45+CD11c+ cells), neutrophils (CD45+Ly6G+ cells), and macrophages (CD45+F4/80 cells) in decidual tissues. B, Proportions of decidual macrophages in mice treated with vaginal progesterone or Replens (control; Lil’ Drug Store Products, Inc). C, Proportions of decidual macrophages in mice injected with 17OHP-C or castor oil (control) (n = 10 each). Data are represented as mean ± SEM. DC, dendritic cell; FSC,; NK, natural killer; 17OHP-C, 17-alpha-hydroxyprogesterone caproate; SSC, saline sodium citrate.

FIGURE 4. M1- and M2-like macrophages in decidual tissues

A, Gating strategy used to identify M1-like (CD11b+ Ly6G-F4/80+ IFNγ -positive or iNOS-positive cells) and M2-like (CD11b Ly6G-F4/80+ IL4-positive or Arg1-positive cells) macrophages. The green histogram represents the autofluorescence control. B and C, Proportions of M1-like (CD11b+Ly6G-F4/80+ IFNγ-positive or iNOS-positive cells) macrophages in decidual tissues from mice treated with vaginal progesterone or Replens (control; Lil’ Drug Store Products, Inc). D and E, Proportions of M2-like (CD11b+Ly6G-F4/80+ IL4-positive or Arg1-positive cells) macrophages in decidual tissues from mice treated with vaginal progesterone or Replens (control) (n = 10 each). Data are represented as mean ±SEM. IFN, interferon; IL, interleukin; iNOS, inducible nitric oxide synthase.

FIGURE 5. Macrophages and neutrophils in myometrium

A, Proportions of myometrial macrophages in mice treated with vaginal progesterone or Replens (control; Lil’ Drug Store Products, Inc). B, Proportions of myometrial neutrophils in mice treated with vaginal progesterone or Replens (control). C, Proportions of myometrial IFNγ -positive neutrophils (CD11b+Ly6G+F4/80− cells) in mice treated with vaginal progesterone or Replens (control) (n = 10 each). Data are represented as mean ±SEM. IFN, interferon.

FIGURE 6. MMP-9 activity and collagen content in cervical tissues

A, MMP-9 activity (green staining) in mice treated with vaginal progesterone or Replens (control; Lil’ Drug Store Products, Inc). B, MMP-9 activity (green staining) in mice injected with 17OHP-C or castor oil (control). Nuclei were stained with DAPI. White arrows represent active MMP-9-positive cells. Scale bars in ×10 and ×40: 200 μm and 50 μm, respectively. C, Masson’s trichrome staining of the cervical tissues from mice treated with vaginal progesterone or Replens (control). Scale bars in ×10 and ×40: 200 μm and 50 μm, respectively. D, Masson’s trichrome staining of the cervical tissues from mice injected with 17OHP-C or castor oil (control). Collagen fibers are stained in blue. Scale bars in ×10 and ×40: 200 μm and 50 μm, respectively. E, Semiquantification of active MMP-9-positive cells in the cervices from mice treated with vaginal progesterone or Replens (control). F, Semiquantification of active MMP-9-positive cells in cervices from mice injected with 17OHP-C or castor oil (control) (n = 5 each). Data are represented as mean ±SEM. G, Magnified image of active MMP-9-positive neutrophils and monocytes in cervical tissues from control mice. Scale bars: 20 μm. DAPI, 4′,6-diamidino-2-phenylindole; MMP, matrix metalloproteinase; 17OHP-C, 17-alpha-hydroxyprogesterone caproate.

FIGURE 7. Plasma concentrations of progesterone and estradiol

A, Progesterone and estradiol concentrations in mice treated with vaginal progesterone or Replens (control; Lil’ Drug Store Products, Inc). B, Progesterone and estradiol concentrations in mice injected with 17OHP-C or castor oil (control). Plasma samples were collected at 18.5 dpc (n = 10 each). Data are represented as mean ± SEM. dpc, days postcoitum; 17OHP-C, 17-alpha-hydroxyprogesterone caproate.

FIGURE 8. Plasma concentration of IL-1β

A, IL-1β concentrations in mice treated with vaginal progesterone or Replens (control; Lil’ Drug Store Products, Inc). B, IL-1β concentrations in mice injected with 17OHP-C or castor oil (control). Plasma samples were collected at 18.5 dpc (n = 10 each). Data are represented as mean ± SEM. dpc, days postcoitum; IL, interleukin; 17OHP-C, 17-alpha-hydroxyprogesterone caproate.