Defining a role for Interferon Epsilon in normal and complicated pregnancies

Abstract

Interferon epsilon (IFNe) is a recently described cytokine that is constitutively expressed in the female reproductive tract. However, the role of this hormonally regulated cytokine during human pregnancy is poorly understood. Moreover, whether IFNe participates in host immune response against bacteria-driven intra-amniotic infection or cervical human papillomavirus infection during pregnancy is unknown. Herein, using a unique set of human samples derived from multiple study cohorts, we aimed to uncover the role of IFNe in normal and complicated pregnancies. We showed that IFNe is expressed in the myometrium, cervix, and chorioamniotic membranes, and may therefore represent a constitutive element of host defense mechanisms in these tissues during pregnancy. The expression of IFNe in the myometrium and cervix appeared greater in late gestation than in mid-pregnancy, but did not seem to be impacted by labor. Notably, concentrations of IFNe in amniotic fluid, but not cervical fluid, were increased in a subset of women undergoing spontaneous preterm labor with intra-amniotic infection, indicating that IFNe could participate in anti-microbial responses in the amniotic cavity. However, stimulation with Ureaplasma parvum and/or lipopolysaccharide did not enhance IFNE expression by amnion epithelial or cervical cells in vitro, implicating alternative sources of this cytokine during intra-amniotic or cervical infection, respectively. Collectively, our results represent the first characterization of IFNe expression by human reproductive and gestational tissues during normal pregnancy and suggest a role for this cytokine in intra-amniotic infection leading to preterm birth.

Keywords: Amniotic fluid; Cervix; Chorioamniotic membranes; IFNE; Myometrium.

Figure 1

IFNe expression in the myometrium in mid-pregnancy and at term. (A) Experimental design for the determination of mRNA and protein expression of IFNe in the myometrium. (B) Hematoxylin & Eosin staining and immunohistochemistry staining for IFNe in myometrial tissues from a single case collected at 18 weeks of gestation. (C) IFNE expression in the myometrial tissues collected from women who delivered at term without (TNL, n = 13) or with (TIL, n = 11) labor. -ΔCt values and fold changes are shown. (D) Representative Hematoxylin & Eosin staining, immunohistochemistry staining for IFNe, and immunofluorescence staining for IFNe (orange) and nuclei (DAPI, blue) in myometrial tissues from women who delivered at term without or with labor (n = 5 per group). All images were taken at 200X magnification. Inset images show isotype control staining from the same case. Black arrowheads indicate areas of positive staining for IFNe. Scale bars represent 50 μm. GA, gestational age; TIL, term with labor; TNL, term without labor.

Figure 2

IFNe expression in the cervix in mid-pregnancy and late gestation. (A) Experimental design for the determination of IFNe protein expression in the cervix. (B) Hematoxylin & Eosin staining and immunohistochemistry staining for IFNe in cervical tissues from a single case collected at 18 weeks of gestation. Representative Hematoxylin & Eosin staining and immunohistochemistry staining for IFNe in myometrial tissues from women who delivered preterm or at term (C) without (n = 7) or (D) with (n = 5) labor. All images were taken at 200X magnification. Inset images show isotype control staining from the same case. Black arrowheads indicate cells with positive staining for IFNe. Scale bars represent 50 μm. GA, gestational age.

Figure 3

Intra-amniotic infection does not alter IFNe expression in cervical fluid or cervical cells. (A) Experimental design for the determination of IFNe concentrations in cervical fluid. (B) Percentage of cervical fluid samples with detectable IFNe from women who underwent spontaneous preterm birth without intra-amniotic inflammation (No IAI, n = 46), with sterile intra-amniotic inflammation (SIAI, n = 6), or with intra-amniotic infection (IAI, n = 10). (C) Concentrations of IFNe in cervical fluid samples collected from women who underwent spontaneous preterm birth with No IAI, SIAI, or IAI. (D) Concentrations of IFNe in cervical fluid samples grouped according to HPV negative (HPV-) or positive (HPV+) status. (E) Experimental design for the determination of IFNE expression by cervical cell lines. (F) IFNE expression (shown as –ΔC_T) in (left to right) ectocervical epithelial (n = 3 technical replicates), endocervical epithelial (n = 3 technical replicates), and cervical stromal cells (n = 2 cases with 3 technical replicates each) co-cultured with Ureaplasma parvum or control media. HPV, human papillomavirus.

Figure 4

Intra-amniotic infection increases IFNe concentrations in amniotic fluids from a subset of preterm birth cases. (A) Experimental design for the determination of IFNe concentrations in amniotic fluid. (B) Percentage of amniotic fluid samples with detectable IFNe from women who underwent spontaneous preterm birth without intra-amniotic inflammation (No IAI, n = 46), with sterile intra-amniotic inflammation (SIAI, n = 6), or with intra-amniotic infection (IAI, n = 10). (C) Concentrations of IFNe in amniotic fluid samples collected from women who underwent spontaneous preterm birth with No IAI, SIAI, or IAI. P-values were determined using the Kruskal-Wallis test followed by Dunn’s correction for multiple comparisons. (D) Concentrations of IFNe in amniotic fluid samples grouped according to HPV negative (HPV-) or positive (HPV+) status. (E) Experimental design for the co-culture of primary amnion epithelial cells with Ureaplasma parvum or lipopolysaccharide (LPS) to determine IFNE expression. (F) IFNE expression (shown as –ΔC_T) in primary amnion epithelial cells co-cultured with Ureaplasma parvum or control media. N = 3 cases with 3 technical replicates each. (G) IFNE expression (shown as –ΔC_T) in primary amnion epithelial cells co-cultured with LPS or PBS control. N = 3 cases with 3 technical replicates each. HPV, human papillomavirus.

Figure 5

IFNe expression in the chorioamniotic membranes is unaltered by intra-amniotic infection. (A) Experimental design showing the collection of the chorioamniotic membranes to determine IFNE expression. (B) IFNE expression in the chorioamniotic membranes collected from women who underwent spontaneous preterm labor without intra-amniotic inflammation (No IAI, n = 16), with sterile intra-amniotic inflammation (SIAI, n = 18), or with intra-amniotic infection (IAI, n = 17). -ΔC_T values and fold changes are shown. (C) Representative Hematoxylin & Eosin staining and immunohistochemistry staining for IFNe in chorioamniotic membranes from women who underwent spontaneous preterm labor with No IAI, SIAI, or IAI (n = 5 per group). All images were taken at 200X magnification. Inset images show isotype control staining from the same case. Black arrowheads indicate cells with positive staining for IFNe. Scale bars represent 50 μm.