The role of danger associated molecular patterns in human fetal membrane weakening - SUMMARY
The rate of prematurity in the US is currently 1 in 10 births, resulting in large numbers of infants that are
vulnerable to both short-term and long-term health problems. Preterm Premature Rupture of Membranes
(pPROM) causes thirty percent of preterm birth, with almost half of these cases due to infection. Although the
infectious agent can often be identified, little can be done to halt its progression or identify those who are at risk.
The central premise of this work is that understanding the normal mechanisms of membrane rupture will help
to understand how they weaken in normal term pregnancies and how this may be altered in pPROM. This
proposal tests the hypothesis that Danger Associated Molecular Pattern (DAMP) ligands for the Toll-like
receptors (TLR) have a key role in the initiation of inflammation and the resultant weakening of the amnion in
normal rupture of the membranes. This drives the predictions that (1) DAMPs are produced in the amnion in
response to cell stress, and also (2) that DAMPs and the TLR infection ligands, Pathogen Associated
Molecular Patterns (PAMPs), activate TLR on the mesenchymal cells (AMC) of the amnion causing its
weakening and rupture through different inflammatory signatures.
Specific Aim 1: Ascertain if and how DAMPs are produced in mesenchymal amnion cells.
Hypothesis: The amnion produces DAMP signals that act locally to initiate fetal membrane weakening.
Approach: Normal term human amnion samples will be utilized to detect the production of various TLR
activating DAMPs by immunocytochemistry, western blotting and ELISA. Human AMC will be grown in culture
and subjected to stretch or oxidative stress and the resultant DAMP production measured. Outcomes: The
data will link the known term causes of cell stress with the DAMP/TLR pathway, supporting them as the key
initiators of sterile inflammation that results in rupture at term.
Specific Aim 2: Determine the mechanism(s) by which DAMPs weaken the human amnion via TLRs and
compare that to the response seen to PAMPs. Hypothesis: TLR on AMC are activated by DAMPs and
PAMPs to drive extracellular matrix degradation and apoptosis through the initiation of different inflammatory
signatures. Approach: AMC will be cultured in 3D AlvetexTM to measure the effect of known DAMPs on the
enzymatic control of the extracellular matrix, cell viability and inflammation. The inflammatory signature for
TLR2/6 after activation by DAMPs and PAMPs will be compared by cytokine secretion and the temporal
analysis of miRNA expression. Outcome: This data will establish the mechanisms of TLR amnion weakening
by sterile inflammation and help to understand why the clinical outcomes seen during normal membrane
rupture and infection driven rupture are different.
