733: Mixed bacteria generate a differential intra-amniotic inflammatory response compared to infections with a single microbial species

inflammatory response compared to infections with a single microbial species Antonette Dulay, Catalin Buhimschi, Lydia Shook, Guomao Zhao, Mert Bahtiyar, Christian Pettker, Anna Sfakianaki, Heather Lipkind, Sonya Abdel-Razeq, Irina Buhimschi Yale University, Ob/Gyn & Reprod. Sci., New Haven, CT OBJECTIVE: Amniotic fluid (AF) TLR2 mediated innate immune signaling occurs following their engagement by Gram positive (GP), Mycoplasma and Ureaplasma spp. TLR4 recognizes Gram negative (GN) microbial ligands. Because cross-talk among TLR2 and TLR4 may generate a differential immune reaction, we explored in vivo and in vitro the intensity of an inflammatory response elicited by single vs mixed spp. STUDY DESIGN: We studied 133 preterm AF samples from amniocentesis in the following groups: single GN species (n 37, GA 26 3w), single GP (n 24, GA 26 3w), Mycoplasma or Ureaplasma (n 30, GA 26 4w), mixed TLR2 ligands (n 10, GA 26 2w), and mixed TLR2 and TLR4 ligands (n 32, GA 25 1w). IL6, TNF , IL8, and the anti-inflammatory IL10 were assessed by ELISA. Data was corrected for GA. A HEK293 Blue-4 cell line was used to address specific ligand effects with particular focus on TLR4 activation. HEK cells were incubated (n 5) with TLR4 (LPS), TLR2 (Lipoteichoic acid [LTA], Pam3Cys [PAM], Mycoplasma FSL-1) & mixed ligand groups (LPS PAM, LPS FSL-1, PAM LTA). RESULTS: In vivo, compared to all other possible combinations, single GN infections had the highest AF IL6, IL8, TNF and IL10 levels (P .001). There was no difference in the inflammatory response among various single TLR2 engaging spp. Conversely, mixed GPs augmented all cytokine responses at levels above single TLR2-engaging spp. (P .016). When GNs cohabitate in AF with GP microbes the cytokine response is above that elicited by single or mixed GPs but less than GNs alone (all P .05). In vitro, LPS induced the highest TLR4 activation (P .001), with the lowest response seen for FSL. LPS response was significantly dampened by PAM (12-fold decrease) and FSL-1 (10-fold decrease). LTA PAM gave a 2-fold increase in cell activation over either alone (all P .05). CONCLUSION: GN bacteria generate a robust AF inflammatory response, that could be either potentiated or dampened through their interaction with various GPs or Ureaplasma/Mycoplasma spp.