Project description:Mitochondria are essential and highly dynamic organelles, constantly undergoing fusion and fission. We analyzed mitochondrial dynamics during infection with the human bacterial pathogen Listeria monocytogenes and show that this infection profoundly alters mitochondrial dynamics by causing transient mitochondrial network fragmentation. Mitochondrial fragmentation is specific to pathogenic Listeria monocytogenes, and it is not observed with the nonpathogenic Listeria innocua species or several other intracellular pathogens. Strikingly, the efficiency of Listeria infection is affected in cells where either mitochondrial fusion or fission has been altered by siRNA treatment, highlighting the relevance of mitochondrial dynamics for Listeria infection. We identified the secreted pore-forming toxin listeriolysin O as the bacterial factor mainly responsible for mitochondrial network disruption and mitochondrial function modulation. Together, our results suggest that the transient shutdown of mitochondrial function and dynamics represents a strategy used by Listeria at the onset of infection to interfere with cellular physiology.
Project description:Introduction: Placental infection and inflammation are risk factors for adverse pregnancy outcomes, including preterm labor. However, the mechanisms underlying these outcomes are poorly understood. Methods: To study this response, we have employed a pregnant mouse model of placental infection caused by the bacterial pathogen Listeria monocyogenes, which infects the human placenta. Through in vivo bioluminescence imaging, we confirm the presence of placental infection and quantify relative infection levels. Infected and control placentas were collected on embryonic day 18 for RNA sequencing to evaluate gene expression signatures associated with infection by Listeria. Results: We identified an enrichment of genes associated with eicosanoid biosynthesis, suggesting an increase in eicosanoid production in infected tissues. Because of the known importance of eicosanoids in inflammation and timing of labor, we quantified eicosanoid levels in infected and uninfected placentas using semi-targeted mass spectrometry. We found a significant increase in the concentrations of several key eicosanoids: leukotriene B4, lipoxin A4, prostaglandin A2, prostaglandin D2, and eicosatrienoic acid. Discussion: Our study provides a likely explanation for dysregulation of the timing of labor following placental infection. Further, our results suggest potential biomarkers of placental pathology and targets for clinical intervention. Overall design: Mouse placental samples. 20 infected replicates (infected with L. monocytogenes) and four uninfected controls.
Project description:The human placenta is a dynamic organ that modulates physiological adaptations to pregnancy. To define the immunological signature of the human placenta, we performed unbiased profiling of secreted immune factors from human chorionic villi isolated from placentas at mid and late stages of pregnancy. We show that placental trophoblasts constitutively secrete the inflammasome-associated cytokines IL-1β and IL-18, which is blocked by NLRP3 inflammasome inhibitors and occurs without detectable gasdermin D cleavage. We further show that placenta-derived IL-1β primes monocytes for inflammasome induction to protect against Listeria monocytogenes infection. Last, we show that the human placenta responds to L. monocytogenes infection through additional inflammasome activation and that inhibition of this pathway sensitizes villi to infection. Our results thus identify the inflammasome as an important mechanism by which the human placenta regulates systemic and local immunity during pregnancy to defend against L. monocytogenes infection.
Project description:Listeria monocytogenes is an important cause of maternal-fetal infections and serves as a model organism to study these important but poorly understood events. L. monocytogenes can infect non-phagocytic cells by two means: direct invasion and cell-to-cell spread. The relative contribution of each method to placental infection is controversial, as is the anatomical site of invasion. Here, we report for the first time the use of first trimester placental organ cultures to quantitatively analyze L. monocytogenes infection of the human placenta. Contrary to previous reports, we found that the syncytiotrophoblast, which constitutes most of the placental surface and is bathed in maternal blood, was highly resistant to L. monocytogenes infection by either internalin-mediated invasion or cell-to-cell spread. Instead, extravillous cytotrophoblasts-which anchor the placenta in the decidua (uterine lining) and abundantly express E-cadherin-served as the primary portal of entry for L. monocytogenes from both extracellular and intracellular compartments. Subsequent bacterial dissemination to the villous stroma, where fetal capillaries are found, was hampered by further cellular and histological barriers. Our study suggests the placenta has evolved multiple mechanisms to resist pathogen infection, especially from maternal blood. These findings provide a novel explanation why almost all placental pathogens have intracellular life cycles: they may need maternal cells to reach the decidua and infect the placenta.
Project description:Pregnant women are highly susceptible to infection by the bacterial pathogen Listeria monocytogenes, leading to miscarriage, premature birth, and neonatal infection. L. monocytogenes is thought to breach the placental barrier by infecting trophoblasts at the maternal/fetal interface. However, the fate of L. monocytogenes within chorionic villi and how infection reaches the fetus are unsettled. Hofbauer cells (HBCs) are fetal placental macrophages and the only leukocytes residing in healthy chorionic villi, forming a last immune barrier protecting fetal blood from infection. Little is known about the HBCs' antimicrobial responses to pathogens. Here, we studied L. monocytogenes interaction with human primary HBCs. Remarkably, despite their M2 anti-inflammatory phenotype at basal state, HBCs phagocytose and kill non-pathogenic bacteria like Listeria innocua and display low susceptibility to infection by L. monocytogenes. However, L. monocytogenes can exploit HBCs to spread to surrounding placental cells. Transcriptomic analyses by RNA sequencing revealed that HBCs undergo pro-inflammatory reprogramming upon L. monocytogenes infection, similarly to macrophages stimulated by the potent M1-polarizing agents lipopolysaccharide (LPS)/interferon gamma (IFN-γ). Infected HBCs also express pro-inflammatory chemokines known to promote placental infiltration by maternal leukocytes. However, HBCs maintain the expression of a collection of tolerogenic genes and secretion of tolerogenic cytokines, consistent with their tissue homeostatic role in prevention of fetal rejection. In conclusion, we propose a previously unrecognized model in which HBCs promote the spreading of L. monocytogenes among placental cells and transition to a pro-inflammatory state likely to favor innate immune responses, while maintaining the expression of tolerogenic factors known to prevent maternal anti-fetal adaptive immunity. <b>IMPORTANCE</b> Infection of the placental/fetal unit by the facultative intracellular pathogen Listeria monocytogenes results in severe pregnancy complications. Hofbauer cells (HBCs) are fetal macrophages that play homeostatic anti-inflammatory functions in healthy placentas. HBCs are located in chorionic villi between the two cell barriers that protect fetal blood from infection: trophoblast cells at the maternal interface (in contact with maternal blood), and fetal endothelial cells at the fetal interface (in contact with fetal blood). As the only leukocytes residing in chorionic villi, HBCs form a critical immune barrier protecting the fetus from infection. Here, we show that although HBCs display low susceptibility to L. monocytogenes, the bacterium still replicates intracellularly and can spread to other placental and fetal cells. We propose that HBCs are permissive to L. monocytogenes transplacental propagation and can repolarize toward a pro-inflammatory phenotype upon infection. However, consistent with their placental homeostatic functions, repolarized HBCs maintain the expression of tolerogenic factors known to prevent maternal anti-fetal adaptive immunity, at least at early stages of infection.
Project description:Immunity and metabolism are intimately linked; manipulating metabolism, either through diet or genetics, has the power to alter survival during infection. However, despite metabolism's powerful ability to alter the course of infections, little is known about what being "sick" means metabolically. Here we describe the metabolic changes occurring in a model system when Listeria monocytogenes causes a lethal infection in Drosophila melanogaster. L. monocytogenes infection alters energy metabolism; the flies gradually lose both of their energy stores, triglycerides and glycogen, and show decreases in both intermediate metabolites and enzyme message for the two main energy pathways, beta-oxidation and glycolysis. L. monocytogenes infection also causes enzymatic reduction in the anti-oxidant uric acid, and knocking out the enzyme uric oxidase has a complicated effect on immunity. Free amino acid levels also change during infection, including a drop in tyrosine levels which may be due to robust L. monocytogenes induced melanization.
Project description:Mammalian pregnancy requires protection against immunological rejection of the developing fetus bearing discordant paternal antigens. Immune evasion in this developmental context entails silenced expression of chemoattractant proteins (chemokines), thereby preventing harmful immune cells from penetrating the maternal-fetal interface. Here, we demonstrate that fetal wastage triggered by prenatal Listeria monocytogenes infection is driven by placental recruitment of CXCL9-producing inflammatory neutrophils and macrophages that promote infiltration of fetal-specific T cells into the decidua. Maternal CD8+ T cells with fetal specificity upregulated expression of the chemokine receptor CXCR3 and, together with neutrophils and macrophages, were essential for L. monocytogenes-induced fetal resorption. Conversely, decidual accumulation of maternal T cells with fetal specificity and fetal wastage were extinguished by CXCR3 blockade or in CXCR3-deficient mice. Remarkably, protection against fetal wastage and in utero L. monocytogenes invasion was maintained even when CXCR3 neutralization was initiated after infection, and this protective effect extended to fetal resorption triggered by partial ablation of immune-suppressive maternal Tregs, which expand during pregnancy to sustain fetal tolerance. Together, our results indicate that functionally overriding chemokine silencing at the maternal-fetal interface promotes the pathogenesis of prenatal infection and suggest that therapeutically reinforcing this pathway represents a universal approach for mitigating immune-mediated pregnancy complications.
Project description:The placenta controls the growth of the fetus and ensures its immune protection. Key to these functions, the syncytiotrophoblast (SYN) is a syncytium formed by fusion of underlying mononuclear trophoblasts. The SYN covers the placental surface and is bathed in maternal blood to mediate nutritional and waste exchanges between the mother and fetus. The bacterial pathogen <i>Listeria monocytogenes</i> breaches the trophoblast barrier and infects the placental/fetal unit resulting in poor pregnancy outcomes. In this work, we analyzed the <i>L. monocytogenes</i> intracellular lifecycle in primary human trophoblasts. In accordance with previous studies, we found that the SYN is 20-fold more resistant to infection compared to mononuclear trophoblasts, forming a protective barrier to infection at the maternal interface. We show for the first time that this is due to a significant reduction in <i>L. monocytogenes</i> uptake by the SYN rather than inhibition of the bacterial intracellular division or motility. We here report the first transcriptomic analysis of <i>L. monocytogenes</i>-infected trophoblasts (RNA sequencing). Pathway analysis showed that infection upregulated TLR2, NOD-like, and cytosolic DNA sensing pathways, as well as downstream pro-inflammatory circuitry (NF-κB, AP-1, IRF4, IRF7) leading to the production of mediators known to elicit the recruitment and activation of maternal leukocytes (IL8, IL6, TNFα, MIP-1). Signature genes associated with poor pregnancy outcomes were also upregulated upon infection. Measuring the release of 54 inflammatory mediators confirmed the transcriptomic data and revealed sustained production of tolerogenic factors (IL-27, IL-10, IL-1RA, TSLP) despite infection. Both the SYN and mononuclear trophoblasts produced cytokines, but surprisingly, some cytokines were predominantly produced by the SYN (IL-8, IL-6) or by non-fused trophoblasts (TNFα). Collectively, our data support that trophoblasts act as placental gatekeepers that limit and detect <i>L. monocytogenes</i> infection resulting in a pro-inflammatory response, which may contribute to the poor pregnancy outcomes if the pathogen persists.
Project description:The peroxisomal proliferator-activated receptor ? (PPAR?) is a nuclear receptor that controls inflammation and immunity. Innate immune defense against bacterial infection appears to be compromised by PPAR?. The relevance of PPAR? in myeloid cells, that organize anti-bacterial immunity, for the outcome of immune responses against intracellular bacteria such as Listeria monocytogenes in vivo is unknown. We found that Listeria monocytogenes infection of macrophages rapidly led to increased expression of PPAR?. This prompted us to investigate whether PPAR? in myeloid cells influences innate immunity against Listeria monocytogenes infection by using transgenic mice with myeloid-cell specific ablation of PPAR? (LysMCre×PPAR?(flox/flox)). Loss of PPAR? in myeloid cells results in enhanced innate immune defense against Listeria monocytogenes infection both, in vitro and in vivo. This increased resistance against infection was characterized by augmented levels of bactericidal factors and inflammatory cytokines: ROS, NO, IFN? TNF IL-6 and IL-12. Moreover, myeloid cell-specific loss of PPAR? enhanced chemokine and adhesion molecule expression leading to improved recruitment of inflammatory Ly6C(hi) monocytes to sites of infection. Importantly, increased resistance against Listeria infection in the absence of PPAR? was not accompanied by enhanced immunopathology. Our results elucidate a yet unknown regulatory network in myeloid cells that is governed by PPAR? and restrains both listeriocidal activity and recruitment of inflammatory monocytes during Listeria infection, which may contribute to bacterial immune escape. Pharmacological interference with PPAR? activity in myeloid cells might represent a novel strategy to overcome intracellular bacterial infection.