Palmitate and group B Streptococcus synergistically and differentially induce IL-1β from human gestational membranes

Abstract

Introduction: Rupture of the gestational membranes often precedes major pregnancy complications, including preterm labor and preterm birth. One major cause of inflammation in the gestational membranes, chorioamnionitis (CAM) is often a result of bacterial infection. The commensal bacterium Streptococcus agalactiae, or Group B Streptococcus (GBS) is a leading infectious cause of CAM. Obesity is on the rise worldwide and roughly 1 in 4 pregnancy complications is related to obesity, and individuals with obesity are also more likely to be colonized by GBS. The gestational membranes are comprised of several distinct cell layers which are, from outermost to innermost: maternally-derived decidual stromal cells (DSCs), fetal cytotrophoblasts (CTBs), fetal mesenchymal cells, and fetal amnion epithelial cells (AECs). In addition, the gestational membranes have several immune cell populations; macrophages are the most common phagocyte. Here we characterize the effects of palmitate, the most common long-chain saturated fatty acid, on the inflammatory response of each layer of the gestational membranes when infected with GBS, using human cell lines and primary human tissue.

Results: Palmitate itself slightly but significantly augments GBS proliferation. Palmitate and GBS co-stimulation synergized to induce many inflammatory proteins and cytokines, particularly IL-1β and matrix metalloproteinase 9 from DSCs, CTBs, and macrophages, but not from AECs. Many of these findings are recapitulated when treating cells with palmitate and a TLR2 or TLR4 agonist, suggesting broad applicability of palmitate-pathogen synergy. Co-culture of macrophages with DSCs or CTBs, upon co-stimulation with GBS and palmitate, resulted in increased inflammatory responses, contrary to previous work in the absence of palmitate. In whole gestational membrane biopsies, the amnion layer appeared to dampen immune responses from the DSC and CTB layers (the choriodecidua) to GBS and palmitate co-stimulation. Addition of the monounsaturated fatty acid oleate, the most abundant monounsaturated fatty acid in circulation, dampened the proinflammatory effect of palmitate.

Discussion: These studies reveal a complex interplay between the immunological response of the distinct layers of the gestational membrane to GBS infection and that such responses can be altered by exposure to long-chain saturated fatty acids. These data provide insight into how metabolic syndromes such as obesity might contribute to an increased risk for GBS disease during pregnancy.

Keywords: gestational membranes; group B Streptococcus; inflammation; obesity; palmitate; pregnancy; preterm birth; preterm prelabor rupture of membranes.

Figure 1

Long chain saturated fatty acid palmitate induces Group B Streptococcus growth, synergizes with GBS infection to induce IL-1β and MMP9 from human decidual stromal cells. (A) OD₆₀₀ reading of GB112 grown for 24 hours in THB broth with and without added palmitate. IL-1β secretion measured by ELISA from the uterine stromal cell line THESC decidualized over 7 days with estrogen, progesterone, and cAMP (referred to as DSCs) (B), the CTB cell line HTR-8 (C), and the macrophage cell line THP.1 (D) infected with GBS for 24 hours at MOIs of 1, 10, and 100. (E) IL-1β and (F) MMP9 secretion measured by ELISA from uninfected or GBS-infected DSCs with or without added palmitate for 24 hours. For all experiments in Figure 1 , N = 3 or more separate experiments with averaged independent replicates. *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s T-test (A–D), or 2-way ANOVA (E, F). ns, not significant; L.O.D., limit of detection.

Figure 2

LCFA palmitate induces MMP9 secretion and synergizes with GBS to induce IL-1β secretion from human cytotrophoblasts. (A) IL-1β and (B) MMP9 secretion measured by ELISA from cytotrophoblast cell line HTR8 uninfected or GBS-infected with or without added palmitate for 24 hours. N = 3 or more separate experiments with averaged independent replicates. **p < 0.01, ***p < 0.001, ****p < 0.0001 by 2-way ANOVA. ns, not significant.

Figure 3

LCFA palmitate synergizes with GBS to induce IL-1β secretion from macrophages but not MMP9. (A) IL-1β and (B) MMP9 secretion measured by ELISA from PMA-stimulated human monocyte cell line THP-1 uninfected or GBS-infected with or without added palmitate for 24 hours. N = 5 or more separate experiments with averaged independent replicates. *p < 0.05 by 2-way ANOVA. ns, not significant.

Figure 4

Palmitate and GBS synergize to induce IL-1β secretion from DSC-Mφ and CTB-Mφ co-cultures, while MMP9 secretion is more complicated. (A–C) IL-1β and (D–F) MMP secreted protein by ELISA in co-cultures of cell line and primary structural and immune cells. (A, D) ELISA data of co-cultures of DSCs with THP-1 Mφ over 24hrs of palmitate treatment and GBS infection. (B, E) Co-cultures of HTR8 CTBs and THP-1 Mφs over 24hrs of palmitate treatment and GBS infection. (C, F) primary placental CTBs co-cultured with primary placental Mφ over 24hrs of palmitate treatment and GBS infection. N = minimum of 4 separate experiments with averaged independent replicates. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by 2-way ANOVA. ns, not significant.

Figure 5

Whole and separated human extraplacental membrane punches have differential IL-1β and MMP9 responses to palmitate and GBS. (A–C) IL-1β and (D–F) MMP9 secretion from whole and separated human extraplacental membrane punches after 24 hours of culture with GBS and palmitate. (A, D) Whole membrane punches show increase in IL-1β and MMP9 during GBS infection that is not tied to palmitate treatment. (B) GBS and palmitate synergize in the choriodecidua to induce IL-1β, while there is no induction of MMP9 secretion (E) from any treatment. (C, F) Amnion punches do not induce IL-1β or MMP9 significantly under any treatments. N = minimum of 3 separate experiments with averaged independent replicates. *p < 0.05, **p < 0.01, ***p < 0.001 by 2-way ANOVA.

Figure 6

Primary human amnion epithelial cells (AECs) do not induce IL-1β or MMP9 under any treatments. (A) IL-1β and (B) MMP9 secretion measured by ELISA from uninfected or GBS-infected AECs with or without added palmitate for 24 hours. For all experiments, N = 4 or more separate experiments with averaged independent replicates. ns, not significant 2-way ANOVA. L.O.D., limit of detection.

Figure 7

Oleate ameliorates the synergistic induction of IL-1β from palmitate and GBS dual stimulation in DSC, CTB, and Mj cell lines. IL-1β secretion measured by ELISA from (A) DSC, (B) CTB, and (C) THP-1 Mφ when infected with GBS and treated with palmitate and/or oleate. N = minimum of 4 separate experiments with averaged independent replicates. *p < 0.05, ***p < 0.001, ****p < 0.0001 by 2-way ANOVA. ns, not significant.