Gasdermin D: Evidence of pyroptosis in spontaneous preterm labor with sterile intra-amniotic inflammation or intra-amniotic infection

Abstract

Problem: Pyroptosis, inflammatory programmed cell death, is initiated through the inflammasome and relies on the pore-forming actions of the effector molecule gasdermin D. Herein, we investigated whether gasdermin D is detectable in women with spontaneous preterm labor and sterile intra-amniotic inflammation or intra-amniotic infection.

Method of study: Amniotic fluid samples (n = 124) from women with spontaneous preterm labor were subdivided into the following groups: (a) those who delivered at term (n = 32); and those who delivered preterm (b) without intra-amniotic inflammation (n = 41), (c) with sterile intra-amniotic inflammation (n = 32), or (d) with intra-amniotic infection (n = 19), based on amniotic fluid IL-6 concentrations and the microbiological status of amniotic fluid (culture and PCR/ESI-MS). Gasdermin D concentrations were measured using an ELISA kit. Multiplex immunofluorescence staining was also performed to determine the expression of gasdermin D, caspase-1, and interleukin-1β in the chorioamniotic membranes. Flow cytometry was used to detect pyroptosis (active caspase-1) in decidual cells from women with preterm labor and birth.

Results: (a) Gasdermin D was detected in the amniotic fluid and chorioamniotic membranes from women who underwent spontaneous preterm labor/birth with either sterile intra-amniotic inflammation or intra-amniotic infection, but was rarely detected in those without intra-amniotic inflammation. (b) Amniotic fluid concentrations of gasdermin D were higher in women with intra-amniotic infection than in those with sterile intra-amniotic inflammation, and its expression in the chorioamniotic membranes was associated with caspase-1 and IL-1β (inflammasome mediators). (c) Decidual stromal cells and leukocytes isolated from women with preterm labor and birth are capable of undergoing pyroptosis given their expression of active caspase-1.

Conclusion: Pyroptosis can occur in the context of sterile intra-amniotic inflammation and intra-amniotic infection in patients with spontaneous preterm labor and birth.

Keywords: amniotic fluid; caspase-1; inflammasome; interleukin-1beta; parturition; pregnancy.

Figure 1.. Amniotic fluid gasdermin D concentrations in women with spontaneous preterm labor.

Extracellular gasdermin D (ng/mL) was measured in amniotic fluid of women with spontaneous preterm labor who delivered at term (n = 32) and those who delivered preterm without intra-amniotic inflammation (n = 41), with sterile intra-amniotic inflammation (n = 32), or with intra-amniotic infection (n = 19). Data are shown as box-plots, with boxes representing the interquartile range, and midlines representing the median. Whiskers extend to the most extreme data point which is no more than 1.5 times the interquartile range. Outliers are shown as individual dots.

Figure 2.. Gasdermin D expression in the chorioamniotic membranes of women who underwent spontaneous preterm labor without intra-amniotic inflammation.

Representative multiplex immunofluorescence images showing the brightfield view, cell segmentation map, nuclear staining (4′,6-diamidino-2-phenylindole, DAPI, blue), and protein expression of gasdermin D (green), caspase-1 (yellow), and IL-1β (red) in the chorioamniotic membranes from women who underwent preterm labor and delivered at term (upper rows, left merged image) or preterm (bottom rows, right merged image) without intra-amniotic inflammation. Merged images show the co-localization of gasdermin D, caspase-1, and IL-1β expression. Images taken at 200X magnification. Scale bars = 100 μm.

Figure 3.. Gasdermin D expression in the chorioamniotic membranes of women with spontaneous preterm labor and sterile intra-amniotic inflammation or intra-amniotic infection.

Representative multiplex immunofluorescence images showing the brightfield view, cell segmentation map, nuclear staining (4′,6-diamidino-2-phenylindole, DAPI, blue), and protein expression of gasdermin D (green), caspase-1 (yellow), and IL-1β (red) in the chorioamniotic membranes from women who underwent preterm labor and birth with sterile intra-amniotic inflammation (upper rows, left merged image) or intra-amniotic infection (bottom rows, right merged image). Merged images show the co-localization of gasdermin D, caspase-1, and IL-1β expression. Images taken at 200X magnification. Scale bars = 100 μm.

Figure 4.. Expression of active caspase-1 in decidual cells from women with preterm labor.

(A) Representative flow cytometry gating strategy showing the expression of active caspase-1 (FLICA) in non-leukocytes (CD45− cells) and leukocytes (CD45+ cells) isolated from the decidua of women with preterm labor. (B) Representative flow cytometry plots showing the expression of active caspase-1 (FLICA) in macrophages (CD45+CD14+ cells) and neutrophils (CD45+CD15+ cells) in the decidual tissues from women with preterm labor. Red quadrants indicate pyroptotic cells (active caspase-1+ and permeable cell membrane, DAPI+). N=5.