PolyI:C Induces Necroptosis In Mouse Decidual Stromal Cells

Pathogenic infections during pregnancy are important causes resulting in fetalloss, preterm delivery, stillbirth and other pregnancy diseases. Pathogenic infectionsdampen not only the survival of the fetus, but also threaten the maternal health andviability. It is well known that variety of viruses such as cytomegalovirus, rubellavirus, adenovirus, herpes simplex virus, etc could impair normal pregnancy progress.However, the mechanism that viral infections resulting in pregnancy failure is stillunclear. Their self-components or secretory products of pathogenic microorganismsare important pathogenic factors, the molecular structure of these components isconstant and evolutionarily conserved, which can be shared by varieties of pathogenicmicro-objects, called pathogen-associated molecular pattern (PAMP). These PAMPscan be respectively recognized by pattern recognition receptors (PRRs), and thusinitiated host immune responses. Pathogen-derived PAMPs usually induceoverwhelming inflammatory responses, which promoting pathogenic infection andpathogenicity and resulting in host damage or death. Nucleic acids, one type of PAMP,function as an important pathogenic factor of virus. There are several forms of viralnucleic acids, including single-stranded or double-stranded RNA and DNA. Variousdouble-stranded RNA viruses cause pregnancy infection. Fully understanding of theeffect and mechanism of double-stranded RNA on pregnancy outcome is theprerequisite and basis of preventing viral infection during pregnancy.The mouse dicudual stromal cells (DSCs) constituted the primary cells of thematernal-fetal interface, and its functioning properly played an important role forpregnancy maintenance. Placenta and fetus got infected by pathogen through twomajor routes: ascending from the uterus or through the mother’s blood circulation.Pathogenic microorganisms from two routes infect DSCs directly and damage theirnormal physiological function, thereby terminate the pregnancy process. Except for releasing cytokine, the cells endow another important immune response to stimulatesor infections, namely cell death. Pathogens could damage the structural integrity ofcells and enhance infectivity and virulence of infected cells by inducing excessive celldeath. Necroptosis is one of the frontiers in the field of mechanisms of cell death inrecent years. Necroptosis is a kind of regulated cellular necrosis, which depends onactivities of RIPK1/RIPK3kinase, an increasing number of studies showed thatnecroptosis plays an important role in various diseases. In this study, based on theabove theories the following hypothesis were posed, the mechanisms of viral infectionresulting to adverse pregnancy outcomes may be that with the placenta infected byvirus, PAMP such as pathogen derived-dsRNA promoted cell death and inflammatorycytokines production of decidual stromal cells and destroyed the structural integrity ofthe placenta and pregnancy immune balance, therefore terminating the pregnancyprocess.The methods of isolating and culturing mouse decidual stromal cells in vitrowere established. Our results showed that exogenous polyI:C transfection in DSCpromoted cell death in a significant dose-dependent manner by MTT and FACSanalysis. Moreover, polyI:C transfection could promote the cleavage of PARP andCaspase-3proteins, and thus it confirmed that polyI:C transfection induced cell death.In order to disclose the mode of cell death, the combination of polyI:C transfectionwith RIPK1inhibitor necrostatin-1(nec-1), MLKL inhibitors Necrosulfonamide(NSA) and caspase pan inhibitor z-VAD-Fmk was adopted, and the results showedthat polyI:C transfection could induce necroptosis in DSC, while necroptoticinduction was independant of caspase inhibition. Morphological analysis using SEMfurther confirmed that polyI:C transfection induced necroptosis in DSC. In order toanalyze signaling mechanism of polyI:C-induced cell death, RNAi technologies andknockout mice are used, the results showed that polyI:C transfection-inducednecroptosis was dependent on MDA5-Mavs signaling pathway, and independent ofTLR3-TRIF signaling pathway. PolyI:C delivery activated cytoplasmic MDA5-Mavssignaling. TLR3-TRIF signaling is mainly located within endosome. Furthermore, this study have tried to understand the effect of exogenous polyI:C on cell death in anon-transfected manner. Experimental results showed that there is no obvious celldeath after polyI:C challengement under non-transfection condition. In this situation,it only induced slightly cell death at a higher concentration. However, lowconcentrations of polyI:C could significantly induce cell death in coorporation withz-VAD-Fmk, and this induction could be reversed by necrostatin-1, a RIPK1inhibitor,otherwise MLKL inhibitor NSA, which suggested that non-transfectedpolyI:C-induced necroptosis in DSC depended mainly on RIPK1signaling and didnot rely on MLKL signaling. Analysis of TRIF KO DSC showed that thenon-transfected polyI:C-induced necroptosis depended on the TLR3-TRIF signaling..In summary, this study demonstrated polyI:C can induce necroptosis in DSC. Tosome extent, it revealed the underlying molecular mechanisms that double-strandedRNA or viral infection affected the outcome of pregnancy, so as to provide atheoretical basis to prevent and treat infections during pregnancy. This study alsoprovided an important theoretical basis for clarifying the involvement and regulationof necroptosis in double-stranded RNA-induced diseases in pregnancy.