Hyaluronan in cervical epithelia protects against infection-mediated preterm birth

Abstract

Increased synthesis of cervical hyaluronan (HA) from early to late pregnancy has long been proposed to play an essential role in disorganization of the collagen-rich extracellular matrix to allow for maximal compliance and dilation of the cervix during the birth process. Here, we show that HA is not essential for increased cervical distensibility during late pregnancy. Rather, cervicovaginal HA plays an unanticipated important role in epithelial barrier protection of the lower reproductive tract. Specifically, HA depletion in the cervix and vagina resulted in inappropriate differentiation of epithelial cells, increased epithelial and mucosal permeability, and strikingly increased preterm birth rates in a mouse model of ascending vaginal infection. Collectively, these findings revealed that although HA is not obligatory for cervical compliance, it is crucial for maintaining an epithelial and mucosal barrier to limit pathogen infiltration of the lower reproductive tract during pregnancy and thereby is protective against infection-mediated preterm birth.

Figure 7. HA depletion compromises epithelial and mucosal barrier functions.

(A) siRNA-mediated Has3 silencing increased permeability of confluent ECT-1 cells. However, treatment with exogenous HA did not restore siRNA-treated cells to control levels. FACE analysis confirmed 60%–70% HA knockdown in Has3 siRNA–treated groups. (B) Fluorescent PSPEGs (red) penetrated deeper into the mucus layer in Has1/2/3 KO mice than in WT controls. TFF1 staining (green) marks mucus and mucosal epithelia. Nuclei were stained with DAPI (blue). n = 4–5 per group. *P < 0.05, 1-way ANOVA with pairwise multiple comparisons by Tukey test. Scale bars: 50 μm.

Figure 6. HA depletion compromises epithelial barrier function and increases susceptibility to infection-mediated PTB.

Gene expression of classical (Ptgs2, Il1a, and Tnfa) and alternative (Ym1 and Arg) immune response after 24-hour live vaginal E. coli exposure (10⁵ CFU) in WT and Has1/2/3 KO (KO) mice. n = 4 per group. *P < 0.05 vs. uninfected, 1-way ANOVA with pairwise multiple comparisons by Tukey test.

Figure 5. HA depletion diminishes occludin expression in CD44⁺ epithelia.

(A) CD44 (red) and occludin (Occ; green) revealed decreased occludin expression in the CD44⁺ epithelial cells of Has1/2/3 KO versus WT mice at term (d18). (B) The luminal ectocervical epithelia and contiguous vaginal epithelia displayed reduced volume of mucus-laden vacuoles and increased occludin expression in d18 Has1/2/3 KO versus WT mice. Nuclei were stained with DAPI (blue). 4 cervices per genotype were evaluated for each stain. Scale bars: 20 μm.

Figure 4. Aberrant organization of epithelia in HA-depleted cervices does not result from increased proliferation or apoptosis.

Proliferation of cervical basal epithelial cells, as assessed by Ki67 staining (green), indicated similar proliferation rates between WT and Has1/2/3 KO mice. Apoptosis was assessed by TUNEL (green) counterstained with nuclear marker (red). Arrows denote basal epithelial cells lining the basement membrane. 3 cervices per genotype were evaluated for each stain. Scale bars: 20 μm.

Figure 3. HA depletion results in aberrant organization and differentiation of cervical epithelia.

Ectocervical sections from term (late d18) WT and Has1/2/3 KO mice were evaluated. Alcian blue stained mucus vacuoles (blue) and nonmucosal cells (pink); arrows denote basal epithelial cells lining the basement membrane. Masson trichrome stained keratin and muscle fibers (red), cytoplasm (pale red), collagen (blue), and nuclei (dark brown/black). E-cadherin (green) was present on the cell surface of epithelial cells. TFF1 (green) immunofluorescence was localized to the mucus present in vacuoles of luminal epithelia and secreted mucus. In E-cadherin and TFF1 images, nuclei were stained with DAPI (blue). 4 cervices per genotype were evaluated for each stain. Scale bars: 20 μm.

Figure 2. HA is not obligatory for increased tissue compliance during cervical ripening and dilation.

(A) Biomechanical assessment of cervical stiffness indicated similar declines in WT and Has1/2/3 KO cervices at term (d18) compared with the nonpregnant (NP) WT cervix. n = 5–8 per group. (B) Collagen fibers (blue) exhibited similar organization between genotypes, although they appeared more condensed in Has1/2/3 KO than in WT mice. 4 cervices per genotype were evaluated for Masson trichrome stain. (C) TEM of collagen fibrils within the cervical stroma, which were similar in diameter between WT and Has1/2/3 KO animals. At least 4 different areas in each of the 3 term cervices per genotype were evaluated for the micrographs. *P < 0.05, 1-way ANOVA with pairwise multiple comparisons by Tukey test. Scale bars: 20 μm (B); 200 nm (C).

Figure 1. The majority of cervical HA is synthesized by Has2, with ≥93% depletion in Has2 KO and Has1/2/3 KO mice.

(A) HA and CS/DS GAGs (sGAG) were quantified by FACE in d18 cervices from WT, Has2 KO, Has1/3 KO, and Has1/2/3 KO mice. n = 3 per genotype. (B) Decreased Has2 expression in Has2 KO and Has1/2/3 KO cervix. Has1 and Has3, while expressed at low levels in the cervix, did not increase to compensate for Has2 loss. n = 4 per genotype. (C and D) Visualization of HA (green) and cell nuclei (DAPI; blue) in d18 cervices. While HA was completely depleted from Has1/2/3 KO epithelia and mucus (C), low levels of HA were detectable in the stroma of Has2 KO (not shown) and Has1/2/3 KO mice (D), likely derived from progesterone receptor–negative cells. 4 cervices per genotype were evaluated for immunofluorescence. S, stroma; ME, mucosal epithelia (defined as terminally differentiated epithelia with visible mucus-laden vacuoles); NME, nonmucosal epithelia (defined as epithelia with no visible mucus-laden vacuoles); M, mucus; L, cervicovaginal lumen. *P < 0.05, 1-way ANOVA with pairwise multiple comparisons by Tukey test. Scale bars: 20 μm.