Pathogenic Nucleic Acid Of Bacterium Induces Trophoblast Cell Death

Pathogenic Nucleic Acid of Bacterium Induces Trophoblast Cell DeathListeria monocytogenes, an intracellular pathogen, is an important cause ofmaternal-fetal infections. It can spread to the placenta and fetus, serves as a modelorganism to study placental barrier.The death of infected cells is an important host innate defense mechanism. Doublestranded DNA (dsDNA) introduced by intracellular bacteria and dsDNA virus duringinfection, was identified as a potent PAMP. Immune sensing of cytosolic dsDNA hasemerged as a central component of antimicrobial innate defense. While the response oftrophoblast cells to PAMP such as LPS has been extensively studied, little is knownabout the direct effects of cytosolic dsDNA. To analyze the effect of cytosolic foreignDNA delivery on cell survival of trophoblasts cells, poly (dA: dT), a double-strandedhomopolymer with poly dA annealed to poly dT were used. Transfection with poly (dA:dT) on HTR-8/SVneo cell resulted a dose-dependent cell death using MTT and AnnexinV-PI assay. Caspase activation and PARP cleavage is the key event of cell death.Transfection with poly (dA: dT) on HTR-8/SVneo cell induced PARP cleavage.Caspase inhibitor z-VAD-fmk partially inhibited cell death after transfected with poly(dA: dT). Thus, there probably exists a caspase-independent cell death induced by poly(dA: dT) in trophoblast cells.Necroptosis is a new cell death passway which are independent of caspase. Itexhibits a unique signaling pathway that requires the involvement of receptorinteraction protein kinase1and3(RIPK1and RIPK3) and can be specifically inhibitedby necrostatins and/or NSA. The first trimester trophoblast cell line HTR-8/SVneo,BeWo, JAR and JEG-3cells constitutively expressed RIPK1, RIPK3, FADD andcaspase8mRNA, as demonstrated by semi-quantitative RT-PCR. RIPK1and RIPK3were expressed in human first-trimester placenta tissue. Furthermore, the gene andprotein expression of RIPK1and RIPK3were all increased after transfected with poly(dA: dT). Meanwhile, immunofluorescence demonstrated the protein expression and thelocation of RIPK3after transfected with poly (dA: dT). Together these results indicatedthat poly (dA: dT) transfection promoted gene and protein expression of RIPK1andRIPK3, the protein of RIPK3were mostly located in nucleus.In order to measure whether cell death induced by poly (dA: dT) was directly responsible for necroptosis, specific inhibitor of RIPK1and MLKL, a proteindownstream of RIPK3were used. Transfect poly (dA: dT) in the presence or absence ofthe pan-caspase inhibitor z-VAD-fmk, RIPK1inhibitor Nec-1or MLKL inhibitor NSAin trophoblast cells. Cell death was determined using MTT, LDH, Western-Blot andAnnexin V-PI assay. Caspase inhibitor z-VAD-fmk but not Nec-1and NSA may partiallyinhibit caspase activation, PARP cleavage and cell death induced by poly (dA: dT).Moreover, the liberation of LDH induced by poly (dA: dT) were also inhibit byz-VAD-fmk to some extent. Meanwhile, on the condition of inhibit caspase, Nec-1and/or NSA have no effect on cell death induced by poly (dA: dT).For futher determin whether low expression of RIPK3in trophoblast cells give riseto insensitivity of dsDNA induced necroptosis signal. Stable over expression system forRIPK3were used after screening. Unfortunately, necroptosis was not found aftertransfected with poly (dA: dT) in over expression RIPK3trophoblast cells. Caspaseactivation and PARP cleavage were all not increased in over expression RIPK3trophoblast cells. Moreover, Cell death of necroptosis induced by TNF-α andz-VAD-fmk can not be found even if RIPK3is over expression in trophoblast cells.Taken together, these observations suggested that dsDNA may induce the mRNA andprotein of RIPK1and RIPK3expression, while the increased protein of RIPK3may notused for necroptosis.It is often stated that necrosis often provoke inflammation. Morphologically,apoptotic cells initially maintain plasma membrane integrity and don’t rapidly releasetheir intracellular contents. Before apoptotic cells disintegrate, they are usually ingestedby resident phagocytes. Moreover, phagocytosis of apoptotic cells stimulates theproduction of IL-10and TGF-β, which inhibit inflammation. Thus, it is reasonable thatthe invulnerability of trophoblasts to necroptosis induction contributes to placentalbarrier against PAMPs.To investigate the effect of genomic DNA of pathogen on trophoblast cells duringinfection, several concentration of genomic DNA of Listeria monocytogenes were used.Cell death was found after transfection with genomic DNA of Listeria monocytogenesin a dose dependent manner. Furthermore, PARP cleavage was also detected introphoblast cells after transfected with genomic DNA of Listeria monocytogenes. LDHliberation was inhibited by z-VAD-fmk but not RIPK1and MLKL inhibitor.Theseobservations suggested that genomic DNA of Listeria monocytogenes may also induce cell death in trophoblat cells in parallel with poly (dA: dT). Knockdown of AIM2withsmall interfering RNA had no effect on poly (dA: dT)-induced cell death. However, celldeath induced by poly (dA: dT) was inhibited to some extent in the IFI16knockdowntrophoblasts, cell death rate were reduced from17%to10%. Taken together, cytosolicdsDNA induced IFI16-dependent, but AIM2-independent, apoptosis of humantrophoblast cells.