Myometrial immune cells contribute to term parturition, preterm labour and post-partum involution in mice

Abstract

This study aimed to determine the mechanism of uterine activation during labour, both term (TL) and preterm (PTL). We hypothesized that the peripheral leucocytes are recruited to uterine tissues by locally produced cytokines where they contribute to the initiation of parturition. Mouse uteri were collected (i) during gestation, TL and post-partum (PP), (ii) during PTL initiated by intrauterine infusion of LPS (125 μg) or (iii) injection of the progesterone receptor antagonist RU486 and analysed for multiple cytokine expression levels by real-time polymerase chain reaction (RT-PCR) and 23-plex Cytokine assay or enzymatically dispersed for assessment of immune cell populations. Markers of myeloid cell differentiation (Gr1, Neu7/4 and F4/80) were evaluated by FACS to define tissue macrophages (Macs), monocytes (M) and neutrophils (N) and by immunohistochemistry to detect tissue Macs and N. Our results indicate that: (1) Macs were elevated in mouse myometrium before TL (P < 0.05) followed by an increase in M and N; these changes were accompanied by an increase in multiple pro-inflammatory cytokines/chemokines genes. The expression of corresponding proteins increased PP. (2) TL and RU486-PTL models showed similar gene/protein expression profiles, (3) LPS-PTL was characterized by strong pro-inflammatory response and massive influx of N in myometrial tissues showing a pattern different from TL and RU486-PTL, (4) The PP period appears similar in all three models, with elevated myometrial cytokine levels and high infiltration of immune cells. We concluded that leucocytes infiltrate myometrium around the time of parturition implicating their potential role in labour activation (both term and preterm) and major role in PP uterine involution.

Fig. 1

Changes in cytokine mRNA levels in the mouse myometrium during normal gestation, term labour and post-partum (TL group), LPS-induced PTL and post-partum (LPS PTL group) and RU486-induced PTL and post-partum (RU PTL group). (A) Pro-inflammatory (Il1b, Il6, Il12b, TNF) and anti-inflammatory (Il10) cytokines (B) Chemokines Ccl2 (Mcp1), Ccl3 (Mip1a), Ccl4 (Mip1b), Csf2 (Gmscf), Cxcl1 (KC or Groa) and Cxcl2 (Mip2a) gene expression were detected by real-time PCR (RT-PCR). Shown are non-labouring samples (GD15, RU Vehicle or LPS Sham, white bars), term and preterm labouring samples (grey bars) or myometrium samples collected 2–6 hrs post-partum (black bars). Results were expressed as an average ± SEM (n = 4–5). One-way anova was utilized followed by Newman-Keuls post-test (for Il10, Ccl2) and Kruskal-Wallis non-parametric test followed by Dunns post-test (for all other cytokines). Significant difference with GD15/RU486 Vehicle/Sham LPS is indicated by *(P < 0.05), **(P < 0.01) and ***(P < 0.001).

Fig. 2

Changes in cytokine protein levels in the mouse myometrium during normal gestation, term labour and post-partum (TL group), LPS-induced PTL and post-partum (LPS PTL group) and RU486-induced PTL and post-partum (RU PTL group). (A) Pro-inflammatory (Il1b, Il6, Il12(p40), TNF-α) and anti-inflammatory (Il10) cytokines (B) Chemokines Ccl2 (Mcp1), Ccl3 (Mip1a), Ccl4 (Mip1b) and Cxcl1 (KC or Groa) protein expression were detected by multiplex magnetic bead assay. Shown are non-labouring samples (GD15, RU Vehicle or LPS Sham, white bars, n = 8 for all groups), term (n = 10) and preterm (n = 8) labouring samples (grey bars) or myometrium samples collected 2–6 hrs post-partum (n = 8, black bars). One-way anova was utilized followed by Newman-Keuls post-test for all cytokines in TL group except for Il6. Results were expressed as mean ± SEM. Kruskal-Wallis non-parametric test was utilized for cytokine analysis in LPS PTL and RU PTL groups followed by Dunns post-test. Results were expressed as an average ± SEM. Significant difference with GD15/RU486 Vehicle/Sham LPS is indicated by *(P < 0.05), **(P < 0.01) and ***(P < 0.001).

Fig. 3

Genes up-regulated during term labour (TL) are induced by RU486 treatment (RU PTL) and intrauterine infusion of LPS (LPS PTL). Connexin 43 (Gja1), oxytocin receptor (Oxtr) and prostaglandin synthase 2 (Ptgs2, formerly Cox2) expression were assessed by RT-PCR in mouse myometrium. Shown are non-labouring samples (GD15, RU Vehicle or LPS Sham, white bars), term and preterm labouring samples (grey bars) or myometrium samples collected 2–6 hrs post-partum (black bars). One-way anova was utilized followed by Newman-Keuls post-test. Significant difference with GD15/RU486 Vehicle/Sham LPS is indicated by *(P < 0.05), **(P < 0.01) and ***(P < 0.001). Data represent mean ± SEM of four myometrium samples.

Fig. 4

Recruitment of myeloid cells in the mouse myometrium. Myometrial suspensions were stained with anti-CD45, F4/80, Gr1 and Neu7/4. Leucocytes were gated on CD45 and further analysed on the Neu7/4 versus Gr1 dot plot. Macrophages were defined as F4/80⁺⁺ and Neu7/4⁻. Monocytes were identified as Neu 7/4⁺⁺ and Gr1⁻ to Gr1^+/−. Neutrophils were defined as Neu7/4⁺, Gr1⁺⁺ [18]. Myometrium was analysed during normal gestation, term labour and post-partum (TL), LPS-induced PTL (LPS PTL) and RU486-induced PTL (RU PTL). Shown are non-labouring samples (GD15, LPS Sham surgery or RU Vehicle, white bars, n = 6–8), GD18 (term not in labour, striped bars, n = 6), term labouring (n = 7) and preterm labouring samples (grey bars, n = 4) or post-partum samples (n = 6 black bars). Data represent mean ± SEM. Significant difference from GD15/RU486 Vehicle/Sham LPS is indicated by asterisk (*, P < 0.05).

Fig. 5

Neutrophil and macrophage infiltration into the mouse myometrium during TL and PTL. Neutrophils were identified using anti-Neu7/4 antibody and macrophages were identified using anti-F4/80 antibody. NewCast software was used to quantify neutrophil and macrophage numbers relative to total cell numbers in the myometrium during normal gestation, term labour and post-partum (TL), LPS-induced PTL and post-partum (LPS PTL) and RU486-induced PTL and post-partum (RU PTL). Shown are non-labouring samples (GD15, LPS Sham or RU Vehicle, white bars), GD18 (term not in labour, striped bars), term and preterm labouring samples (grey bars) or post-partum samples (black bars). Results were expressed as mean ± SEM (n = 4–12). Neutrophil infiltration data from the LPS PTL mouse model was transformed using the natural logarithm (ln) function to obtain a normal distribution. One-way anova was utilized followed by Newman-Keuls post-test. Significant difference with GD15/RU486 Vehicle/Sham LPS is indicated by *(P < 0.05).