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. 2022 Mar 25:13:807750.
doi: 10.3389/fimmu.2022.807750. eCollection 2022.

Placental Inflammasome mRNA Levels Differ by Mode of Delivery and Fetal Sex

Anya L Arthurs  1 , Melanie D Smith  1 , Mhyles D Hintural  1 , James Breen  2 , Dylan McCullough  1 , Francesca I Thornton  1 , Shalem Y Leemaqz  1 , Gustaaf A Dekker  3 , Tanja Jankovic-Karasoulos  1 , Claire T Roberts  1
Affiliations

Placental Inflammasome mRNA Levels Differ by Mode of Delivery and Fetal Sex

Anya L Arthurs et al. Front Immunol. .

Abstract

Parturition signals the end of immune tolerance in pregnancy. Term labour is usually a sterile inflammatory process triggered by damage associated molecular patterns (DAMPs) as a consequence of functional progesterone withdrawal. Activation of DAMPs recruits leukocytes and inflammatory cytokine responses in the myometrium, decidua, cervix and fetal membranes. Emerging evidence shows components of the inflammasome are detectable in both maternal decidua and placenta. However, the activation of the placental inflammasome with respect to mode of delivery has not been profiled. Placental chorionic villus samples from women delivering at term via unassisted vaginal (UV) birth, labouring lower segment caesarean section (LLSCS, emergency caesarean section) and prelabour lower segment caesarean section (PLSCS, elective caesarean section) underwent high throughput RNA sequencing (NextSeq Illumina) and bioinformatic analyses to identify differentially expressed inflammatory (DE) genes. DE genes (IL1RL1, STAT1, STAT2, IL2RB, IL17RE, IL18BP, TNFAIP2, TNFSF10 and TNFRSF8), as well as common inflammasome genes (IL1B, IL1R1, IL1R2, IL6, IL18, IL18R1, IL18R1, IL10, and IL33), were targets for further qPCR analyses and Western blotting to quantify protein expression. There was no specific sensor molecule-activated inflammasome which dominated expression when stratified by mode of delivery, implying that multiple inflammasomes may function synergistically during parturition. Whilst placentae from women who had UV births overall expressed pro-inflammatory mediators, placentae from LLSCS births demonstrated a much greater pro-inflammatory response, with additional interplay of pro- and anti-inflammatory mediators. As expected, inflammasome activation was very low in placentae from women who had PLSCS births. Sex-specific differences were also detected. Placentae from male-bearing pregnancies displayed higher inflammasome activation in LLSCS compared with PLSCS, and placentae from female-bearing pregnancies displayed higher inflammasome activation in LLSCS compared with UV. In conclusion, placental inflammasome activation differs with respect to mode of delivery and neonatal sex. Its assessment may identify babies who have been exposed to aberrant inflammation at birth that may compromise their development and long-term health and wellbeing.

Keywords: inflammasome; inflammation; labour; parturition; placenta; pregnancy.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Analysis of differential gene expression determined by RNA sequencing between labouring lower segment Caesarean section (LLSCS) (n = 7) and prelabour lower segment Caesarean section (PLSCS) (n = 3) in male-bearing pregnancies only. Volcano plot shows the level of change (log transformed normalised counts); differentially expressed genes (false discovery rate (FDR) < 0.05 & log fold change (logFC) > |1|) indicated in red and green. Horizontal dotted line corresponds to FDR of 0.05 (-log10 scaled). Vertical dotted lines correspond to logFC > |1|.
Figure 2
Figure 2
The mRNA expression of (A) Interleukin 1β (IL1B), (B) Interleukin 1 Type 1 Receptor (IL1R1), (C) Interleukin 1 Type 2 Receptor (IL1R2), (D) Interleukin 18 (IL18), (E) Interleukin 18 Binding Protein (IL18BP) and (F) Interleukin 18 Type 1 Receptor (IL18R1) in placentae from female- and male-bearing pregnancies classed by delivery mode (as determined by qPCR). Data are presented as a 10-90 percentile interleaved box-and-whisker plot. The same letter above bars indicates that groups are not different from each other. A different letter above bars indicates that groups are different (all p < 0.05). Dark grey colour denotes placenta samples from female fetal sex; light grey colour denotes placenta samples from male fetal sex. (Number of samples: unassisted vaginal (UV) 20/fetal sex; labouring lower segment Caesarean section (LLSCS) = 10/fetal sex; prelabour lower segment Caesarean section (PLSCS) = 5/fetal sex).
Figure 3
Figure 3
(A) Interleukin 6 (IL6), (B) Interleukin 2 Receptor β (IL2RB), (C) Interleukin 17 Receptor E (IL17RE), (D) Tumour Necrosis Factor Alpha Induced Protein 2 (TNFAIP2), (E) Tumour Necrosis Factor Super Family Member 10 (TNFSF10) and (F) Tumour Necrosis Factor Receptor Super Family Member 8 (TNFRSF8) mRNA expression in placentae from female- and male-bearing pregnancies classed by delivery mode (qPCR). Data are presented as a 10-90 percentile interleaved box-and-whisker plot. The same letter above bars indicates that groups are not different from each other. A different letter above bars indicates that groups are different (all p < 0.05). Dark grey colour denotes placenta samples from female fetal sex; light grey colour denotes placenta samples from male fetal sex. (Number of samples: unassisted vaginal (UV) 20/fetal sex; labouring lower segment Caesarean section (LLSCS) = 10/fetal sex; prelabour lower segment Caesarean section (PLSCS) = 5/fetal sex).
Figure 4
Figure 4
(A) Signal transducer and activator of transcription 1 (STAT1) and (B) 2 (STAT2) mRNA expression (qPCR) in placentae from female- and male-bearing pregnancies classed by delivery mode. Data are presented as a 10-90 percentile interleaved box-and-whisker plot. The same letter above bars indicates that groups are not different from each other. A different letter above bars indicates that groups are different (all p < 0.05). Dark grey colour denotes placenta samples from female fetal sex; light grey colour denotes placenta samples from male fetal sex. (Number of samples: unassisted vaginal (UV) 20/fetal sex; labouring lower segment Caesarean section (LLSCS) = 10/fetal sex; prelabour lower segment Caesarean section (PLSCS) = 5/fetal sex).
Figure 5
Figure 5
The mRNA expression (as determined by qPCR) of Interleukin 10 (IL10) in placentae from female- and male-bearing pregnancies classed by delivery mode. Data are presented as a 10-90 percentile interleaved box-and-whisker plot. The same letter above bars indicates that groups are not different from each other. A different letter above bars indicates that groups are different (all p < 0.05). Dark grey colour denotes placenta samples from female fetal sex; light grey colour denotes placenta samples from male fetal sex. (Number of samples: unassisted vaginal (UV) 20/fetal sex; labouring lower segment Caesarean section (LLSCS) = 10/fetal sex; prelabour lower segment Caesarean section (PLSCS) = 5/fetal sex).
Figure 6
Figure 6
(A) Interleukin 33 (IL33) and (B) Interleukin 1 Receptor Like 1 (IL1RL1) mRNA expression in placentae from female- and male-bearing pregnancies classed by delivery mode, and by indication of fetal distress (FD) [(C) IL33 in FD, (D) IL1RL1 in FD)] during labour (qPCR). Data are presented as a 10-90 percentile interleaved box-and-whisker plot. The same letter above bars indicates that groups are not different from each other. A different letter above bars indicates that groups are different (all p < 0.05). Dark grey colour denotes placenta samples from female fetal sex; light grey colour denotes placenta samples from male fetal sex. (Number of samples: unassisted vaginal (UV) 20/fetal sex; labouring lower segment Caesarean section (LLSCS) = 10/fetal sex; prelabour lower segment Caesarean section (PLSCS) = 5/fetal sex. Samples with FD = 9, samples with no FD = 51).
Figure 7
Figure 7
The mRNA expression of Interleukin 1β (IL-1β) (A, C) and Interleukin 18 (IL-18) (B, D) protein in placentae from female- and male-bearing pregnancies classed by delivery mode. For (A, B), Data are presented as a 10-90 percentile interleaved box-and-whisker plot. The same letter above bars indicates that groups are not different from each other. A different letter above bars indicates that groups are different (all p < 0.05). Dark grey colour denotes placenta samples from female fetal sex; light grey colour denotes placenta samples from male fetal sex. (C, D) show representative images of staining with an IL-1β antibody (molecular weight ~35 kDa) and an IL-18 antibody (molecular weight ~20 kDa), respectively. Below this is a representative image of stain-free Total Protein staining as a control. (Number of samples: unassisted vaginal (UV) = 6/fetal sex; labouring lower segment Caesarean section (LLSCS) = 4/fetal sex; prelabour lower segment Caesarean section (PLSCS) = 2/fetal sex).
Figure 8
Figure 8
Diagrammatic representation of the interactions of inflammasome and inflammatory molecules explored in this study.
See this image and copyright information in PMC

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