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Co-culture led to additive LPS-stimulated responses for IL-10, IL-1, MIC-1, TGF- 1 and CCL5, which were not statistically different than the mathematical sum from the individual cell types exposed to LPS

Co-culture led to additive LPS-stimulated responses for IL-10, IL-1, MIC-1, TGF- 1 and CCL5, which were not statistically different than the mathematical sum from the individual cell types exposed to LPS. PGE2 production. Results: In response to LPS, dTHESC and THP-1 co-culture demonstrated enhancement of most inflammatory mediators, but a potent suppression of macrophage KT185 TNF- 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 (GBS) strain NCTC 1084 (1169-NT1; serotype V, provided by V. Nizet), the strain UTI89 / UPEC (provided by M. Mulvey) or the GBS strain 37 (GB37; serotype V, sequence type ST-1, provided by S. Manning) were used as indicated. Mouse chorioamnionitis model Animal experiments were performed in accordance with the Animal Welfare Act, U.S. federal law, and NIH guidelines. All experiments were carried out under a protocol approved by either Vanderbilt University Institutional Animal Care and Use Committee (IACUC; M/14/034), a body that has been accredited by the Association of Assessment and Accreditation of Laboratory Animal Care (AAALAC), or Columbia University IACUC (AC-AAAE3951, AC-AAAD1332). GBS infection of pregnant mice and subsequent analyses were performed as previously published 34 with minor modifications 35. Mouse tissue immunohistochemistry Pregnant animals were sacrificed on day E17.5 (96 h infection) and the entire fetal-placental unit (most proximal to cervix, right side) was removed, separated and fixed in 4% paraformaldehyde and paraffin-embedded. NYU Experimental Pathology Immunohistochemistry Core Laboratory performed macrophage staining and imaging. A subset of pregnant animals was sacrificed on day E15.5 (48 h infection) immunohistochemistry staining was KT185 performed by the Vanderbilt Translational Pathology Shared Resource. Human gestational membrane culture and placental macrophage isolation Human gestational membranes were excised from placental tissues from women who delivered healthy, full term infants by cesarean section without labor. De-identified tissue Rabbit Polyclonal to CGREF1 samples were provided by the Cooperative Human Tissue Network, which is funded by the National Cancer Institute. All tissues were collected in accordance with Vanderbilt University Institutional Review Board (approval #131607) and Declaration of Helsinki. Gestational membranes were processed into 12-mm punch biopsies as published 36 or mounted on transwell devices as we have previously described 37, 38. Placental macrophages were isolated and cultured according to our previously published protocol 39. Culture and decidualization of telomerase-immortalized human endometrial stromal cells Telomerase-immortalized human endometrial stromal cells, THESC (American Type Culture Collection (ATCC) CRL-4003; Manassas, VA) can be decidualized into cells mimicking primary decidual stromal cells 40. Decidualized THESC cells (dTHESCs) were generated as originally published 40 using 0.5 mM 8-Bromo-cAMP (Tocris Bioscience; Minneapolis, MN), 1 M medroxyprogesterone acetate (MPA; Sigma), and 10 nM estradiol (E2; 17-estradiol-acetate; Sigma). The stromal cells were assessed for decidualization by measurement of human insulin-like growth factor binding protein-1 (IGFBP-1) and prolactin by ELISA (Alpha Diagnostic International; San Antonio, TX). THP-1 macrophage culture and differentiation The human monocytic leukemia-derived cell line, THP-1 cells (ATCC #TIB202), was maintained in RPMI 1640 (Invitrogen), 10% charcoal-stripped FBS, and 1% antibiotic/antimycotic solution (referred to as RPMI+/+) in T-75 flasks until use in experiments. Phorbol myristate acetate (PMA, 5 ng/mL; Sigma) was used to differentiate the nonadherent monocytes into macrophage-like cells on the day prior to co-culture with dTHESCs (day 7). THP-1 and dTHESC co-culture experiments Inflammatory responses of individual cell cultures were performed in parallel with the co-culture of dTHESCs and PMA-differentiated THP-1 KT185 cells. Using a ratio of 1 1 THP-1 cell to 10 dTHESCs, THP-1 cells (1 105/well) or dTHESCs (1 106/well) were plated alone and in co-culture (1 106 dTHESCs + 1 105 THP-1 cells) in DMEM/F12+/+ media. Briefly, after THESC decidualization on day 8, PMA-differentiated KT185 THP-1 cells were added to the dTHESC wells for co-culture (or cells were plated individually as described above). Each of the cell groups (THP-1 alone,.