Project II

Thrombin effects on decidual TLR expression

The decidua acts in an immunologically paradoxical fashion (i.e., accepting the semi-allogenic placenta, while maintaining host defenses against an array of microbial pathogens). Placental abruption/decidual hemorrhage is a major cause of perinatal morbidity and mortality and preterm delivery (PTD) and is associated with both aseptic inflammatory responses as well as increased rates of frank chorioamnionitis (CAM). Abruption results in intense decidual thrombin generation and, in severe cases, hypofibrinogenemia and disseminated intra-vascular coagulation. Recurrent abruption is also associated with a 7-fold higher rate of preterm premature rupture of the membranes (PPROM). Our previous studies demonstrated that thrombin, acts via protease activated receptors (PARs) to increase decidual matrix metalloproteinase (MMP) and interleukin-8 (IL-8) expression, factors strongly linked to PPROM. We now reveal that 1) decidualized stromal cells, i.e., decidual cells (DCs) and human endometrial endothelial cells (HEECs) express specific Toll-like Receptors (TLRs) and their intermediate signaling proteins; 2) thrombin alters DC expression of TLR2, TLR3 and TLR4 whereas in HEECs, thrombin alters the expression of several TLR pathway intermediates without affecting TLR gene expression. TLRs recognize and respond to conserved pathogen-associated molecular patterns (PAMPs) expressed by microbes and stress proteins released by dying cells. Activation of TLRs is necessary to promote innate immunity. The underlying hypothesis of this study is that: 

  • Excess thrombin alters TLR expression and signaling in decidual and endothelial cells.
  • Alteration of TLRs increases susceptibility to an ascending genital tract infection leading to an inflammatory reaction that promotes PPROM and/or PTD. 
Aim I. To characterize TLR expression and function in normal vs. pathological decidua.

Immunohistochemistry, immunofluorescence and microdissection coupled with quantitative RT-PCR will be utilized to quantify the association between altered TLR expression and abruption-associated PTD with and without related CAM. 

Aim II. To determine the mechanisms by which thrombin regulates decidual TLR expression/function.

Studies will dissect out the role of PARs in the expression of TLRs and their downstream signaling intermediates as well as cytokine and NFκB expression. These studies will use agonists, antagonists and small interference RNA (siRNAs) in cell culture. 

Aim III. To determine the functional effects of thrombin on TLR-ligand interactions.

Cultured decidual cells will be treated with thrombin vs. control and then exposed to TLR-2, 3 and 4 ligands. Endpoints of these studies will be assessed by microarray analysis, RT-PCR and immunoassays as well as assessment of the NFκB components by western blotting.

Aim IV. In vivo characterization of thrombin effects on TLR function in pregnant mice.
in coordination with Projects I and III of this PO1, we will utilize a murine model to study the effects of thrombin on TLR expression and function as well as the susceptibility to bacterial infection.


Publications

Krikun G, Lockwood CJ, Abrahams VM, Mor G, Paidas M, Guller S. Expression of toll-like receptors in the human decidua. Histology & Histopathology. 2007;22: 847-854.

Schatz F, Kayisli UA, Vatandaslar E, Ocak N, Guller S, Abrahams VM, Krikun, G, Lockwood, CJ. Toll-like Receptor 4 Expression in Decidual cells and Interstitial Trophoblasts across Human Pregnancy. American Journal of Reproductive Immunology. 2012;68:146-53. 

Krikun G, Trezza J, Shaw J, Rahman M, Guller S, Abrahams VM, Lockwood CJ. LPS appears to activate human endometrial endothelial cells through TLR4-dependent and TLR4-independent mechanisms. American Journal of Reproductive Immunology. 2012 Jun 4 [Epub ahead of print]. 

Kavathas PB, Boeras CM, Mulla MJ, Abrahams VM. Nod1, but not the ASC inflammasome, contributes to induction of IL-1b secretion in human trophoblasts after sensing of Chlamydia trachomatis. Mucosal Immunology. 2012 Jul 4. [Epub ahead of print].