Molecular Mechanism Of Listeria Monocytogenes-induced Abortion In Mice Based On Inflammasome Activation

Listeria Monocytogenes (LM), a gram-positive bacteria, parasitizing inside or outsidethe host cells, is an important foodborne zoonotic pathogen likely causing the reproductivedisorders of human and animals, especially ruminant animals (cattle, sheep et al.), resultingin abortion, stillbirths or even fetal infection. LM-induced animal reproductive failureresults in great economic loss to the animal producers. In recent years, many investigationson the pathogenic mechanism of LM-induced reproductive disorder were extensivelyconducted and showed that LM-induced reproductive disorder were largely associated withLM-induced immune imbalance and cell apoptosis/death, however, its intrinsic mechanismremains to be elucidated. It is known that there are many immune cells such asmacrophages and natural killer cells exists in the maternal-fetal interface of pregnantanimals, where the cells can interact with trophoblasts to maintain the embryodevelopment, regulate the local immune balance and resist the exogenous pathogeninvasion. Recently the inflammasome has been identified to play an important role inmediating local inflammatory reaction and defending foreign pathogen invasion. It wasproved that many pathogens can activate the inflammasome of monocyte/macrophages inthe host, and the inflammasome can mediate the non-specific immune response which ischaracterized by inflammatory response. However excessive inflammatory response canlead to tissue damage and related diseases. Studies proved that LM can activate theinflammasome of macrophages, however, whether the inflammasome activiation can leadto the immune imbalance of maternal-fetal interface and affect the function of trophoblasts,and then cause the reproductive disorders of the animals are still unclear.To explore the molecular mechanism of LM-induced reproductive disorder of animals,embryo survival rate and cytokine production of pregnant mice infected by LM wereanalyzed. The results showed that LM greatly reduced embryo survival rates and increasedthe expression of Th1(IL-2, INF-γ) cytokines and the release of IL-1β in the placenta.Increased Th1cytokines contributed to the imbalance of Th1/Th2cytokines, and increasedIL-1βmediated inflammatory response，which were considered the important factorsleading to pregnancy failure. LM induced-IL-1β maturation and release suggests that LMlikely activate the inflammasome in the maternal-fetal interface。Therefore, one of theinflammasome component, caspase1in the placental tissue was detected. The resultsshowed that LM promoted the caspase1muturation in placenta of pregnant mice. Listeriolysin O (LLO）is a major virulence factor of LM. In order to determine whetherLLO plays an important role in LM induced reproductive disorders, the pregnant micewere infected with LLO-deficient LM (LM/LLO-/-). The result showed that the LM/LLO-/-infection did not significantly affect the mouse embryo survival, cytokine expressions andthe caspase1muturation in the placental tissue of pregnant mice compared with uninfectedmice, indicating that LLO plays an important role in LM-induced reproductive disorder inpregnant mice by activating inflammasome.To investigate LLO function in inflammasome activation, a certain amount ofrecombinant LLO are needed. So we constructed LLO prokaryotic secretory expressionvector using molecular genetic techniques and expressed the recombinant LLO in E. coli.Hemolysis assay showed that the LLO prepared in this study had the significant hemolyticactivity, indicating that the LLO has the biological activity.In order to confirm LM can activate inflammasomes, the mouse macrophages wereinfected by LM. The result demonstrated that LM could promote the IL-1β maturation andrelease, stimulate caspase1muturation, and induce apoptosis and pyroptosis of themacrophages. All these results indicate that LM is able to activate the inflammasome ofmacrophages. To further explore the role and molecular mechanism of LLO ininflammasome activation, the mouse B6macrophages and NLRP3-KO B6macrophageswere infected by wild-type LM (LM/WT), LM/LLO-/-, and treated with recombinant LLOin vitro, respectively. The results showed that:(1) LM/WT, not LM/LLO-/-, could promoteIL-1β release and caspase1p10(mature caspase1) generation, and increase the expressionsof pro-IL-1β, TNF and IL-6in macrophage. However, the LM/WT did not significantlypromote IL-1β release and caspase1p10generation in NLRP3-KO B6macrophage.(2)The similar results of increasing IL-1β release and caspase1-P1genaration were obtainedfrom LLO-treated LPS-primed B6cells. The above results demonstrate that LM activatesNLRP3inflammasome and induce IL-1β maturation and release by its LLO. In order toclarify the mechanism of LLO-induced NLRP3inflammasome activation, the cathepsin Bdistribution in the different compartments of B6macrophages was detected with DQavalbumin staining and observed under the confocal microscopy, the K+efflux and Ca2+influx induced by recombinant LLO were also analyzed simultaneously. The resultsconfirmed that the intracellular LLO produced by infected LM disruptedphagosome/lysosomal membrane resulting in the release of cathepsin B into cytosol, andthe extracellar recombinant LLO promoted the K+efflux and Ca2+influx by incresedLLO-induced membrane permeability. The cathepsin B release and the ion flux wereconsidered important stimulators for NLRP3inflammasome activation..To investigate the regulatory role of macrophages based on inflammasome activationon the trophoblasts, the mouse trophoblasts were treated either with the culture medium ofLM-infected macrophages or with recombinant IL-1β in vitro to analyze the effects of the inflammasome activation on trophoblast invasion and inflammatory immune response. Theresults showed that both the culture medium of LM-infected macrophages and the IL-1βcould significantly stimulate the trophoblast invasion by upregulating matrixmetalloproteinases (MMPs) expression. This result suggests that the moderateinflammatory response mediated by IL-1β is beneficial for the trophoblast invasion andembryo implantation. However, the excessive inflammatory response can causeinflammatory damage and stimulate excessive trophoblast invasion, which likely affect thestructure of the decidual tissue and the implantation and development of the embryo. Theresults also showed that the IL-1β induced the inflammatory cytokine expressions(including IL-6, IL-8, TNFα) and monocyte chemotactic protein-1(MCP-1) expression introphoblasts. The former can mediate the inflammatory response in maternal-fetal interface,and the latter can promote infiltration of inflammatory cells, thereby affecting the normalembryo development and stability, which might be another important factor contributing tothe abortion induced by LM.In summary, the present study in vivo and in vitro proved that LM infection can causeimbalance of Th1/Th2cytokine in the maternal-fetal interface of pregnant mice. LLO canactivate NLRP3inflammasome of macrophage by its pore-forming toxic effect.LM-induced inflammasome activation of mouse B6macrophages leads tocaspase1-mediated apoptosis and pyroptosis, promote IL-1β release and IL-1-mediatedlocal inflammatory response. The results also showed that LM-induced macrophageinflammasome activation could stimulate the invasion of trophoblasts and inflammatoryimmune responses. These results not only provide important experimental basis to studythe molecular mechanism of reproductive disorders in humans and animals caused by LM,but also provide a potential target for the prevention and treatment of the LM-inducedreproductive disease.