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. 2018 Oct;80(4):e13032.
doi: 10.1111/aji.13032. Epub 2018 Aug 7.

Decidual stromal cell-derived PGE2 regulates macrophage responses to microbial threat

Lisa M Rogers  1 , Anjali P Anders  2 , Ryan S Doster  1 , Elizabeth A Gill  3 , Juan S Gnecco  4   5 , Jacob M Holley  1 , Tara M Randis  6   7 , Adam J Ratner  6   7 , Jennifer A Gaddy  1   8 , Kevin Osteen  4   5   8 , David M Aronoff  1   4   5
Affiliations

Decidual stromal cell-derived PGE2 regulates macrophage responses to microbial threat

Lisa M Rogers et al. Am J Reprod Immunol. 2018 Oct.

Abstract

Problem: Bacterial chorioamnionitis causes adverse pregnancy outcomes, yet host-microbial interactions are not well characterized within gestational membranes. The decidua, the outermost region of the membranes, is a potential point of entry for bacteria ascending from the vagina to cause chorioamnionitis. We sought to determine whether paracrine communication between decidual stromal cells and macrophages shaped immune responses to microbial sensing.

Method of study: Decidual cell-macrophage interactions were modeled in vitro utilizing decidualized, telomerase-immortalized human endometrial stromal cells (dTHESCs) and phorbol ester-differentiated THP-1 macrophage-like cells. The production of inflammatory mediators in response to LPS was monitored by ELISA for both cell types, while phagocytosis of bacterial pathogens (Escherichia coli and Group B Streptococcus (GBS)) was measured in THP-1 cells or primary human placental macrophages. Diclofenac, a non-selective cyclooxygenase inhibitor, and prostaglandin E2 (PGE2 ) were utilized to interrogate prostaglandins as decidual cell-derived paracrine immunomodulators. A mouse model of ascending chorioamnionitis caused by GBS was utilized to assess the colocalization of bacteria and macrophages in vivo and assess PGE2 production.

Results: In response to LPS, dTHESC and THP-1 coculture demonstrated enhancement of most inflammatory mediators, but a potent suppression of macrophage TNF-α generation was observed. This appeared to reflect a paracrine-mediated effect of decidual cell-derived PGE2 . In mice with GBS chorioamnionitis, macrophages accumulated at sites of bacterial invasion with increased PGE2 in amniotic fluid, suggesting such paracrine effects might hold relevance in vivo.

Conclusion: These data suggest key roles for decidual stromal cells in modulating tissue responses to microbial threat through release of PGE2 .

Keywords: chorioamnionitis; fetal membranes; infection; microfluidics; pregnancy; prostaglandins.

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Figures

Figure 1:
Figure 1:. Co-culture alters the inflammatory response of dTHESCs and THP-1 cells to lipopolysaccharide (LPS) driven by dTHESC conditioned media (CM) paracrine signaling.
Macrophages, stromal cells, direct co-cultures, or dTHESC conditioned media cultures were stimulated with 1 μg/mL LPS for 24 h and supernatants were collected for ELISA analysis. A. Heat map of inflammatory mediator analysis results from 6-well plates containing 1 × 105 THP-1 cells, 1 × 106 dTHESCs, or co-culture combined in 1 mL media. B. TNF-α production (pg/mL; n=13) from 6-well plates containing 1 × 105 THP-1 cells, 1 × 106 dTHESCs, or co-culture combined in 1 mL media. C. TNF-α production (pg/mL; n=6) from 96-well plates containing 1 × 105 THP-1 cells using 200 μL dTHESC CM. STATISTICS: (B-C) 1-way RM ANOVA with Tukey’s post-test; mean ± SEM; *p<0.05, **p<0.01.
Figure 2:
Figure 2:. TNF-α profile of THP-1 cells, decidualized THESCs, and co-culture cells cultured in single-chamber chip devices.
2 × 106/mL THP-1 cells, 5 × 106/mL dTHESCs, or co-culture cells were loaded into chip devices and rested for 1 h before being stimulated with 1 μg/mL LPS for 24 h. A. Photographic image and schematic of the device. B. TNF-α analysis by ELISA (pg/mL; n=5). C. Representative fluorescent images of cells within the chip devices (10×; blue represents NucBlue Live (Hoechst 33342), green represents CellTracker Green, and red represents anti-Vimentin). STATISTICS: (B) 1-way RM ANOVA with Tukey’s post-test; mean ± SEM; ***p<0.001.
Figure 3:
Figure 3:. The heat-tolerant immunomodulatory lipid PGE2 plays a role in the LPS-induced suppression of TNF-α seen in coculture cells in a paracrine manner.
dTHESC conditioned media (CM) was heat-inactivated at 85°C for 10 m before being placed onto 1 × 105 THP-1 cells in a 96-well plate for 1 h (200 μL). Cells were then stimulated with 1 μg/mL LPS for 24 h before supernatants were harvested for TNF-α ELISA analysis. A. Heat-inactivated dTHESC CM suppresses LPS-induced TNF-α (pg/mL; n=3). B. Validation that PGE2 remained in the dTHESC CM after heat-inactivation (pg/mL; n=3). C. 1 × 105 THP-1 cells in a 96-well plate were pretreated for 15 m with 1 μM exogenous PGE2 before being stimulated with 1 μg/mL LPS for 24 h. PGE2 appears to be a potent suppressor of LPS-induced TNF-α (pg/mL; n=4). D. 1 × 105 THP-1 cells in a 96-well plate were pretreated for 15 m with 1 μM exogenous PGE2 before being stimulated with live GBS at a multiple of infection (MOI) of 10:1 for 24 h. PGE2 appears to be a potent suppressor of GBS-induced TNF-α (pg/mL; n=7). E. Treatment of 1 × 106 dTHESCs in a 6-well plate with the cyclooxygenase inhibitor diclofenac significantly prevented PGE2 synthesis (pg/mL; n=3), and F. CM from diclofenac-treated dTHESC cells lost the capacity to limit macrophage TNF-α following 24 h LPS stimulation (pg/mL; n=3) (1 × 105 THP-1 cells in a 96-well plate with 200 μL). STATISTICS: (A, C, D, E, F) 1-way ANOVA followed by Tukey’s post-test or (B) paired student t-test; mean ± SEM for all graphs; *p<0.05, **p<0.01, and ***p<0.001.
Figure 4:
Figure 4:. Prostaglandins from conditioned dTHESC medium and exogenous PGE2 impair macrophage phagocytosis of Streptococcus agalactiae (GBS) and E. coli.
2 × 105 THP-1 cells in a 384-well plate were treated for 30 m with dTHESC-generated CM (3 μg/mL diclofenac or 0.1% solvent control) before being stimulated with A. heat-killed FITC-GBS at MOI 150:1 (n=3), or B. FITC-E.coli bioparticles at MOI 50:1 (n=3) for 3 h. C. 2 × 105 THP-1 cells or D. placental macrophages were treated with 1 μM exogenous PGE2 for 15 m before being stimulated with heat-killed FITC-GBS at MOI 150:1 for 3 h. E. 2 × 105 THP-1 cells or F. placental macrophages were treated with 1 μM exogenous PGE2 for 15 m before being stimulated with FITC-E.coli bioparticles at MOI 50:1 for 3 h. G. 2 × 105 placental macrophages were treated for 30 m with dTHESC-generated CM (3 μg/mL diclofenac or 0.1% solvent control) before being stimulated with FITC-E.coli bioparticles at MOI 50:1 (n=3) for 3 h. Phagocytosis was quantified by fluorescence. Data are presented as Phagocytic Activity (% of untreated control). STATISTICS: (A, B, G) 1-way ANOVA followed by Tukey’s post-test and (C-F) paired student t-test; mean ± SEM for all graphs; *p<0.05, **p<0.01, and ***p<0.001.
Figure 5:
Figure 5:. Ex vivo model of chorioamnionitis results in GBS membrane invasion, PGE2 secretion and macrophage recruitment.
Using a 12-mm punch biopsy culture system of human gestational membranes, conditioned media (CM) supernatant was generated for use on THP-1 cells. A. 24 h GBS-infected gestational membrane biopsies secrete PGE2 ex vivo in response to 1 × 106 CFU GBS (GB37) or B. GB1084 (fold change compared to control; n=7–8). C. 24 h uninfected CM obtained from the gestational membrane punch biopsies significantly reduced TNF-α production by 1 × 105 THP-1 cells in a 96-well plate after 1 h pretreatment and 24 h stimulation with 1 μg/mL LPS (pg/mL; n=4). D. Human gestational membranes were organized into transwell contructs and infected with GBS (1 × 106 CFU GB37) on the choriodecidual surface for 48 h before tissue processing and immunohistochemistry analysis. Tissues were stained with anti-CD68 for macrophages (top panels) or anti-GBS (bottom panels). Tissue analysis shows invasion of human membrane tissues in vitro by GBS and the presence of macrophages within the choriodecidua (CD) at the site of infection. AE = amniotic epithelium. STATISTICS: (A, B) paired student t-test; mean ± SEM; *p<0.05 and **p<0.01; (C) 1-way ANOVA with Tukey’s post-test; mean ± SEM; **p<0.01.
Figure 6:
Figure 6:. Mouse model of ascending chorioamnionitis results in GBS gestational tissue invasion, PGE2 production and macrophage recruitment.
Pregnant mice at day E13.5 were intravaginally inoculated with 1 × 103 CFU GBS (GB37) and sacrificed on E15.5 (48 h after infection). Placental tissues were dissected and evaluated by immunohistochemistry for GBS invasion and the presence of F4/80+ macrophages. Tissue analysis demonstrates that after 48 h of ascending infection, GBS invades into (A) gestational membrane (B) and placental tissues (bottom panels). F4/80+ macrophages traffic to the site of tissue infections (top panels of A and B). C. Amniotic fluid was isolated from control- and GBS-infected mice and assayed for PGE2 following 48 h GBS (GB37) vaginal infection (pg/mL; n=3). Bacterial infection results in a statistically significant increase in amniotic fluid PGE2. D. Pregnant mice at day E13.5 were intravaginally inoculated with 1 × 107 CFU GBS (GB1084) and sacrificed on E17.5 (96 h after infection). Placental tissues were dissected and evaluated by immunohistochemistry for GBS invasion and the presence of F4/80+ macrophages. Immunohistochemistry from D. sham and E. GBS-infected mouse gestational tissues demonstrate that GBS invades placental tissues and F4/80+ macrophages influx into infected tissues. STATISTICS: (C) paired student t-test; mean ± SEM; **p<0.01.
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