Lactobacillus rhamnosus GR-1 stimulates colony-stimulating factor 3 (granulocyte) (CSF3) output in placental trophoblast cells in a fetal sex-dependent manner

Abstract

Bacterial vaginosis is associated with a 1.4-fold increased risk of preterm birth. We have shown previously that Lactobacillus rhamnosus GR-1 supernatant up-regulates interleukin 10 and down-regulates tumor necrosis factor-alpha output in lipopolysaccharide (LPS)-treated human primary placenta cultures in a fetal sex-dependent manner. We hypothesize that lactobacilli also exert their anti-inflammatory effect by up-regulation of colony-stimulating factor 3 (granulocyte) (CSF3), which is secreted from both immune and placental trophoblast cells, and that this activity is dependent on the sex of the fetus. Placental trophoblast cells were isolated from term elective cesarean section placentae using a Percoll gradient and separated from CD45(+) cells using magnetic purification. Cells were treated with LPS in the presence or absence of pretreatments with L. rhamnosus GR-1 supernatant or chemical inhibitors of the intracellular signaling pathways. Phosphorylations of mitogen-activated protein kinase 14 (MAPK14, previously known as p38) and signal transducer and activator of transcription (STAT) 3 were measured by Western blot analysis, and levels of CSF3 were determined by ELISA. CSF3 output was increased only in the placental trophoblast cells of female fetuses treated with LPS, GR-1 supernatant, and a combination of both treatments. The GR-1 supernatant up-regulated the phosphorylation of STAT3 and MAPK14. CSF3 output was inhibited by both Janus kinases (JAK) and MAPK14 inhibitors. None of the treatments was able to increase CSF3 output in either the pure trophoblast or the CD45(+) cell preparations alone. These results suggest an underlying mechanism for the sex difference in incidence of preterm birth and provide potential evidence for a therapeutic benefit of lactobacilli in reducing the risk of preterm labor.

Figures

FIG. 1.

Effect of fetal sex and L. rhamnosus GR-1 supernatant on concentration of anti-inflammatory CSF3 in LPS and L. rhamnosus GR-1-stimulated placental trophoblast cells. Histogram shows CSF3 concentration (fold-increase relative to control) for different treatment groups in media from placental trophoblast cell cultures. Results are presented as the mean ± SEM and are relative to control (n = 5 males and 6 females). Concentration of CSF3 in the media of the control cultures was 231 pg/ml. Asterisks indicate statistical difference (***P < 0.001). Different letters indicate statistical significance (P < 0.001) within gender groups (male: a; female: a′, b′, and c′) as determined by two-way ANOVA.

FIG. 2.

The relationship between CSF3 and the MAPK and JAK/STAT pathways in human placental trophoblast cells of the female fetuses. A) Representative Western blots of phospho-STAT3-Tyr705 (86 kDa, n = 4), total STAT3 (86 kDa, n = 4), phospho-MAPK14 (43 kDa, n = 5), total MAPK14 (43 kDa, n = 5), and ACTB (43 kDa) expression under control, LPS, L. rhamnosus GR-1 supernatant, and LPS plus L. rhamnosus GR-1 supernatant as well as addition of a CSF3 neutralizing antibody to these four basic treatments. Western blots were normalized to ACTB. Histograms show CSF3 (B; n = 5) and TNF (C; n = 4) concentration (fold-increase relative to control) for different treatment groups in media from placental trophoblast cell cultures. Results are presented as the mean ± SEM and are relative to control. Different letters indicate statistical significance (P < 0.001) as determined by one-way ANOVA.

FIG. 3.

Effect of fetal sex on CSF3R expression in placental trophoblast cells. A) Relative optical density (ROD) of CSF3R expression under control conditions in placentae of male or female fetuses normalized to ACTB (P > 0.05, n = 8 males and 5 females). B) Representative Western blot of CSF3R (92 kDa) and ACTB (43 kDa) expression under LPS, L. rhamnosus GR-1 supernatant, as well as LPS plus L. rhamnosus GR-1 supernatant treatments of placental trophoblast cells from pregnancies carrying a male or a female fetus. C and D) ROD of CSF3R Western blot normalized to ACTB for pregnancies carrying a male or female fetus (C) and total samples (D; n = 13). Results are presented as the mean ± SEM and are relative to control. Different letters indicates statistical significance within gender groups (C; male: a; female: a′ and b′; P = 0.007 by two-way ANOVA) and across treatments (D; a and b; P = 0.001 by one-way ANOVA).

FIG. 4.

CSF3 concentration in mixed, pure trophoblast, and CD45+ culture sets. CSF3 concentrations (fold-increase relative to control) for control and for LPS-, GR-1-, and GR-1/LPS-treated cells from (A) both placentae of male or female fetuses (n = 7), (B) placentae of male fetuses (n = 3), and (C) placentae of female fetuses (n = 4) are shown. Results are presented as the mean ± SEM and are relative to control. Asterisks indicate statistical difference (*P = 0.015, **P = 0.004, ***P < 0.001). Different letters indicate statistical significance (mixed cells: a, b, and c; pure trophoblasts: a′; CD45+ cells: a″) as determined by three-way ANOVA as follows: A) relative to control, P < 0.001 for LPS and P = 0.002 for GR-1 alone as well as combined with LPS; B) P > 0.05; and C) P = 0.048.