Estradiol-regulated innate antiviral responses of human endometrial stromal fibroblasts

Abstract

Problem: The contribution of fibroblasts to innate immune protection of the human female reproductive tract (FRT) against viral pathogens is relatively unknown.

Method of study: Endometrial (EM), endocervical (Cx) and ectocervical (ECx) fibroblasts were isolated from hysterectomy patients and grown in vitro. Fibroblasts were treated with the viral mimic poly (I:C) in the presence or absence of the sex hormone estradiol (E₂ ), with gene expression measured by real-time RT-PCR and protein secretion by ELISA.

Results: Poly (I:C) induced the expression of the interferon-stimulated genes (ISG) MxA, OAS2 and APOBEC3G, and the cytokines MCP-1, IL-8, IL-6, CCL20, IFNβ and RANTES by fibroblasts from all three sites. ISG upregulation was dependent upon Type I IFN signaling. E₂ inhibited the poly (I:C)-induced upregulation of MxA and OAS2 in EM fibroblasts, but not Cx or ECx fibroblasts. E₂ upregulated SDF-1α by EM fibroblasts but had no effect on secretion of other cytokines either alone or in the presence of poly (I:C). Conditioned media (CM) from poly (I:C)-treated or E₂ -treated fibroblasts significantly reduced HIV infection of CD4+ T cells.

Conclusion: Stromal fibroblasts represent a level of innate immune protection against viral pathogens in the FRT beyond that seen with epithelial cells and immune cells. Our findings indicate that fibroblasts FRT are selectively responsive to E₂ , capable of initiating an antiviral response against viral pathogens and may play a role in preventing HIV infection of CD4+ T cells.

Figure 1:. FRT stromal fibroblast morphology and marker expression.

Matched endometrial, endocervical, and ectocervical stromal fibroblasts were isolated from tissue fragments and cultured in vitro prior to analysis. (A-C) Representative images of unstained stromal fibroblasts imaged under a light microscope. Flow cytometry analysis of intracellular vimentin (D-F), surface CD90 (G-I) and surface CD45 (J-L) expression in matched endometrium, endocervix, and ectocervix from a representative individual donor. (Red – Isotype control; blue – antibody). All panels are derived from the same patient and are representative of 8 individual donors.

Figure 2:. Constitutive pattern recognition receptors (PRRs) expression in FRT stromal fibroblasts.

mRNA from EM, Cx, and ECx stromal fibroblasts was analyzed for expression of PRRs by real-time RT-PCR (n=8). Bars and horizontal lines represent the mean relative mRNA expression +/− SEM respectively, normalized to the endogenous control β-Actin using matched EM, Cx and ECx from 8 individual donors. ND=Not detectable. * P<0.05; ** P<0.01 are significantly different when compared to the EM. Statistical analysis was performed using the non-parametric Friedmann with Dunn’s post-test correction for multiple comparison.

Figure 3:. Poly (I:C) dose-dependently induces IFNβ and antiviral gene expression in endometrial stromal fibroblasts.

mRNA from EM, Cx, and ECx stromal fibroblasts was analyzed for expression of IFNβ (A), MxA (B), OAS2 (C) and APOBEC3G (D) (n=3–4) after stimulation with 0.25, 2.5 and 25μg/ml of poly (I:C) for 24hr. Cellular mRNA was isolated and analyzed by real-time RT-PCR. Bars and horizontal lines represent the mean fold-change in mRNA expression +/− SEM respectively, from independent experiments with 3–4 individual donors with values normalized against the endogenous control β-Actin. Untreated (0) values are set to 1. * P<0.05; ** P<0.01 are significantly different compared to untreated samples. Statistical analysis was performed using the non-parametric Wilcoxon Signed Rank test.

Figure 4:. IFNβ induces antiviral gene expression.

mRNA from matched EM, Cx, and ECx stromal fibroblasts was analyzed for expression of IFNAR1 and IFNAR2 (A) by real-time RT-PCR (n=8). Bars and horizontal lines represent the mean relative mRNA expression +/− SEM respectively, normalized to the endogenous control β-Actin from 8 individual donors. Recombinant human IFNβ (rIFNβ) was used to stimulate cultured EM stromal fibroblasts for 24hr after which MxA, OAS2 and APOBEC3G expression was analyzed by real-time RT-PCR (B) (n=5). EM stromal fibroblasts were pretreated with an anti-IFNAR2 blocking antibody (αIFNAR2) (10μg/ml) or isotype control (ISO) for 1 hr followed by stimulation with poly (I:C) (25μg/ml) for 24 hours prior to mRNA expression analysis for MxA (C) and OAS2 (D) (n=5). Data shown in B-D is the mean fold-change in mRNA expression +/− SEM for independent experiments from 5 individual donors with values normalized against the endogenous control β-Actin. Bars and horizontal lines in all panels represent the mean +/− the SEM respectively. Statistical analysis was performed using the non-parametric Wilcoxon Signed Rank test.

Figure 5:. TLR3 blockade inhibits APOBEC3G expression.

EM stromal fibroblasts were pretreated with an anti-TLR3 blocking antibody (αTLR3) (20μg/ml) or isotype control (ISO) for 1 hr followed by stimulation with poly (I:C) (25μg/ml) for 24hr prior to mRNA expression analysis for MxA (A) and OAS2 (B) and APOBEC3G (C) (n=3). Bars and horizontal lines represent the mean fold-change in mRNA expression +/− SEM respectively, for independent experiments from 3 individual donors with values normalized against the endogenous control β-Actin. Statistical analysis was performed using the non-parametric Wilcoxon Signed Rank test.

Figure 6:. E₂ inhibits poly (I:C)-induced MxA and OAS2 mRNA expression in endometrial stromal fibroblasts.

Effect of poly (I:C) (25μg/ml) and E₂ (5×10⁻⁸M) on MxA and OAS2 mRNA expression in matched EM, Cx and ECx stromal fibroblasts. Cells were pretreated for 48hr with control media or media containing E₂, washed out and retreated with E₂ or control media for a subsequent 24hr in the presence or absence of poly (I:C) at 25μg/ml. After 24hr the total cellular mRNA was recovered and analyzed for changes in gene expression (n=4). Bars and horizontal lines represent the mean fold change in gene expression +/− SEM respectively, for independent experiments with 4 individual donors using matched EM, Cx and ECx, with β-Actin as the endogenous control. Untreated control is set to 1. * P<0.05, ** P<0.01. Statistical analysis was performed using the non-parametric Wilcoxon Signed Rank test.

Figure 7:. Poly (I:C) upregulates cytokine secretion by FRT stromal fibroblasts.

Stromal fibroblasts from matched EM, Cx and ECx were pretreated for 48hr with E₂ (5×10⁻⁸M) and subsequently retreated with poly (I:C), E₂, or a combination of both for a further 24hr. Conditioned media was recovered and analyzed for SDF-1α (A), MCP-1 (B), CCL20 (C), IL-6 (D), IL-8 (E), RANTES (F) and IFNβ (G) by ELISA (n=4). Bars and horizontal lines represent the mean cytokine concentration +/− SEM respectively, for independent experiments using matched EM, Cx and ECx from 4 different donors.

Figure 8:. E₂ stimulates SDF-1α secretion by endometrial fibroblasts.

EM stromal fibroblasts were pretreated for 48hr with E₂ (5×10⁻⁸M) and subsequently retreated with E₂ for a further 24hr. Conditioned media was recovered and analyzed for SDF-1α by ELISA (A) (n=4). Cultured EM stromal fibroblasts were pretreated for 1 hr using the ERα antagonist Raloxifene (Rx) (5×10⁻⁶M), followed by addition of E₂ (5×10⁻⁸M) for a further 48hr during which Rx was maintained in the culture media. Conditioned media was recovered and SDF-1α concentration determined by ELISA (B). Data shown in (A) is the mean +/− SEM of individual experiments from 4 individual donors while (B) shows the mean +/− SEM of a single donor that is representative of independent experiments from 3 individual donors. ** P<0.01, ***P<0.001. Bars and horizontal lines in both panels represent the mean +/− the SEM respectively.

Figure 9:. Endometrial stromal fibroblast conditioned media inhibits HIV infection.

Conditioned media (CM) was recovered from EM stromal fibroblasts, diluted 1:1 in X-Vivo 15 media and incubated with R5-tropic HIV-1 BaL (A) or X4-tropic HIV-1 IIIB (D) (MOI=0.1) for 1hr prior to addition to CD4+ T cells for 1hr, followed by extensive washout, and incubation with fresh media. Infection was determined by assessing levels of secreted p24 by ELISA after 7 days. * P<0.05, ** P<0.01 compared to CM from untreated cells. Bars and horizontal lines represent the percent mean HIV infection +/− SEM respectively, of independent experiments from 6 different individual donors. Statistical analysis was performed using the non-parametric Wilcoxon Signed Rank test.