RESUMEN
F. nucleatum, involved in carcinogenesis of colon carcinomas, has been described as part of the commensal flora of the female upper reproductive tract. Although its contribution to destructive inflammatory processes is well described, its role as commensal uterine bacteria has not been thoroughly investigated. Since carcinogenesis shares similar mechanisms with early pregnancy development (including proliferation, invasion, blood supply and the induction of tolerance), these mechanisms induced by F. nucleatum could play a role in early pregnancy. Additionally, implantation and placentation require a well-balanced immune activation, which might be suitably managed by the presence of a limited amount of bacteria or bacterial residues. We assessed the effect of inactivated F. nucleatum on macrophage-trophoblast interactions. Monocytic cells (THP-1) were polarized into M1, M2a or M2c macrophages by IFN-γ, IL-4 or TGF-ß, respectively, and subsequently treated with inactivated fusobacteria (bacteria:macrophage ratio of 0.1 and 1). Direct effects on macrophages were assessed by viability assay, flow cytometry (antigen presentation molecules and cytokines), qPCR (cytokine expression), in-cell Western (HIF and P-NF-κB) and ELISA (VEGF secretion). The function of first trimester extravillous trophoblast cells (HTR-8/SVneo) in response to macrophage-conditioned medium was microscopically assessed by migration (scratch assay), invasion (sprouting assay) and tube formation. Underlying molecular changes were investigated by ELISA (VEGF secretion) and qPCR (matrix-degrading factors and regulators). Inflammation-primed macrophages (M1) as well as high bacterial amounts increased pro-inflammatory NF-κB expression and inflammatory responses. Subsequently, trophoblast functions were impaired. In contrast, low bacterial stimulation caused an increased HIF activation and subsequent VEGF-A secretion in M2c macrophages. Accordingly, there was an increase of trophoblast tube formation. Our results suggest that a low-mass endometrial/decidual microbiome can be tolerated and while it supports implantation and further pregnancy processes.
Asunto(s)
Fusobacterium nucleatum , Macrófagos , Trofoblastos , Humanos , Trofoblastos/inmunología , Trofoblastos/microbiología , Trofoblastos/metabolismo , Fusobacterium nucleatum/inmunología , Fusobacterium nucleatum/fisiología , Macrófagos/inmunología , Macrófagos/microbiología , Macrófagos/metabolismo , Femenino , Embarazo , Citocinas/metabolismo , Células THP-1 , FN-kappa B/metabolismo , Infecciones por Fusobacterium/inmunología , Infecciones por Fusobacterium/microbiología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
One of the main causes of human brucellosis is Brucella melitensis infecting small ruminants. To date, Rev1 is the only vaccine successfully used to control ovine and caprine brucellosis. However, it is pathogenic for pregnant animals, resulting in abortions and vaginal and milk shedding, as well as being infectious for humans. Therefore, there is an urgent need to develop an effective vaccine that is safer than Rev1. In efforts to further attenuate Rev1, we recently used wzm inactivation to generate a rough mutant (Rev1Δwzm) that retains a complete antigenic O-polysaccharide in the bacterial cytoplasm. The aim of the present study was to evaluate the placental pathogenicity of Rev1Δwzm in trophoblastic cells, throughout pregnancy in mice, and in ewes inoculated in different trimesters of pregnancy. This mutant was evaluated in comparison with the homologous 16MΔwzm derived from a virulent strain of B. melitensis and the naturally rough sheep pathogen B. ovis. Our results show that both wzm mutants triggered reduced cytotoxic, pro-apoptotic, and pro-inflammatory signaling in Bewo trophoblasts, as well as reduced relative expression of apoptosis genes. In mice, both wzm mutants produced infection but were rapidly cleared from the placenta, in which only Rev1Δwzm induced a low relative expression of pro-apoptotic and pro-inflammatory genes. In the 66 inoculated ewes, Rev1Δwzm was safe and immunogenic, displaying a transient serological interference in standard RBT but not CFT S-LPS tests; this serological response was minimized by conjunctival administration. In conclusion, these results support that B. melitensis Rev1Δwzm is a promising vaccine candidate for use in pregnant ewes and its efficacy against B. melitensis and B. ovis infections in sheep warrants further study.
Asunto(s)
Brucella melitensis , Brucelosis , Placenta , Animales , Brucella melitensis/patogenicidad , Brucella melitensis/inmunología , Brucella melitensis/genética , Femenino , Ovinos , Brucelosis/prevención & control , Brucelosis/inmunología , Brucelosis/veterinaria , Embarazo , Placenta/microbiología , Ratones , Enfermedades de las Ovejas/prevención & control , Enfermedades de las Ovejas/inmunología , Enfermedades de las Ovejas/microbiología , Trofoblastos/inmunología , Trofoblastos/microbiología , Vacuna contra la Brucelosis/inmunología , Vacuna contra la Brucelosis/administración & dosificación , Vacuna contra la Brucelosis/genética , Humanos , Vacunas Atenuadas/inmunología , Vacunas Atenuadas/administración & dosificaciónRESUMEN
The placenta is a critical barrier against viral, bacterial, and eukaryotic pathogens. For most teratogenic pathogens, the precise molecular mechanisms of placental resistance are still being unraveled. Given the importance of understanding these mechanisms and challenges in replicating trophoblast-pathogen interactions using in vitro models, we tested an existing stem-cell-derived model of trophoblast development for its relevance to infection with Toxoplasma gondii. We grew human trophoblast stem cells (TSCT) under conditions leading to either syncytiotrophoblast (TSSYN) or cytotrophoblast (TSCYT) and infected them with T. gondii. We evaluated T. gondii proliferation and invasion, cell ultrastructure, as well as for transcriptome changes after infection. TSSYNs cells showed similar ultrastructure compared to primary cells and villous explants when analyzed by transmission electron microscopy and scanning electron microscopy (SEM), a resistance to T. gondii adhesion could be visualized on the SEM level. Furthermore, TSSYNs were highly refractory to parasite adhesion and replication, while TSCYTs were not. RNA-seq data on mock-treated and infected cells identified differences between cell types as well as how they responded to T. gondii infection. We also evaluated if TSSC-derived SYNs and CYTs had distinct resistance profiles to another vertically transmitted facultative intracellular pathogen, Listeria monocytogenes. We demonstrate that TSSYNs are highly resistant to L. monocytogenes, while TSCYTs are not. Like T. gondii, TSSYN resistance to L. monocytogenes was at the level of bacterial adhesion. Altogether, our data indicate that stem-cell-derived trophoblasts recapitulate resistance profiles of primary cells to T. gondii and highlight the critical importance of the placental surface in cell-autonomous resistance to teratogens.IMPORTANCECongenital toxoplasmosis can cause a devastating consequence to the fetus. To reach the fetus's tissues, Toxoplasma gondii must cross the placenta barrier. However, how this parasite crosses the placenta and the precise molecular mechanisms of placental resistance to this parasite are still unknown. In this study, we aimed to characterize a new cellular model of human trophoblast stem cells to determine their resistance, susceptibility, and response to T. gondii. Syncytiotrophoblast derived from trophoblast stem cells recapitulate the resistance profile similarly to placenta cells. We also showed that these cells are highly resistant to Listeria monocytogenes, at the level of bacterial adhesion. Our results suggest that resisting pathogen adhesion/attachment may be a generalized mechanism of syncytiotrophoblast resistance, and trophoblast stem cells represent a promising model to investigate cell-intrinsic mechanisms of resistance to pathogen adhesion and replication.
Asunto(s)
Listeria monocytogenes , Toxoplasma , Trofoblastos , Trofoblastos/microbiología , Trofoblastos/parasitología , Toxoplasma/genética , Toxoplasma/crecimiento & desarrollo , Toxoplasma/fisiología , Toxoplasma/ultraestructura , Humanos , Listeria monocytogenes/crecimiento & desarrollo , Listeria monocytogenes/genética , Listeria monocytogenes/fisiología , Femenino , Embarazo , Adhesión Celular , Placenta/microbiología , Placenta/parasitología , Toxoplasmosis/parasitología , Células MadreRESUMEN
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 Listeria monocytogenes breaches the trophoblast barrier and infects the placental/fetal unit resulting in poor pregnancy outcomes. In this work, we analyzed the L. monocytogenes 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 L. monocytogenes uptake by the SYN rather than inhibition of the bacterial intracellular division or motility. We here report the first transcriptomic analysis of L. monocytogenes-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 L. monocytogenes infection resulting in a pro-inflammatory response, which may contribute to the poor pregnancy outcomes if the pathogen persists.
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Inflamación/etiología , Listeria monocytogenes/fisiología , Trofoblastos/inmunología , Trofoblastos/microbiología , Proteínas Bacterianas/fisiología , Células Cultivadas , Quimiocinas/biosíntesis , Citocinas/biosíntesis , Femenino , Células Gigantes/inmunología , Humanos , Proteínas de la Membrana/fisiología , Embarazo , Resultado del Embarazo , TranscriptomaRESUMEN
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. IMPORTANCE 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.
Asunto(s)
Listeria monocytogenes/inmunología , Macrófagos/inmunología , Macrófagos/microbiología , Placenta/inmunología , Células Cultivadas , Quimiocinas/inmunología , Citocinas/inmunología , Femenino , Humanos , Listeria monocytogenes/patogenicidad , Placenta/citología , Embarazo , Células THP-1 , Trofoblastos/microbiologíaRESUMEN
INTRODUCTION: Recent studies have shown that the endometrium possesses unique microbiomes, including Lactobacillus. However, the roles of these microbes are currently unknown, especially in placentation and the early stage of pregnancy. METHODS: The immortalized human first-trimester trophoblast cell line HTR-8/SVneo was cultured in the presence or absence of Lactobacillus crispatus. Invasive and migrative activities were directly evaluated using an optical microscope and a time-lapse imaging system. Protein levels of the invasion-related protein matrix metalloproteinase (MMP)-1, MMP-2, and MMP-9 were evaluated using ELISA. RESULTS: Matrigel invasion of HTR-8/SVneo cells was significantly increased by L. crispatus, though migration was not affected. The culture supernatant of L. crispatus also promoted invasion. Additionally, levels of the active forms of MMP-1 and MMP-2 in the cell culture medium were upregulated by L. crispatus treatment, but that of MMP-9 was not changed. DISCUSSION: L. crispatus promotes trophoblast invasion with an increase in MMP-1 and MMP-2 activation. Our results might explain why Lactobacillus dominance in the endometrium seems beneficial for implantation. Nevertheless, further research is required to determine whether the promotion of trophoblast invasion by L. cripatus is favorable for successful placentation at the early stage of pregnancy.
Asunto(s)
Movimiento Celular , Lactobacillus crispatus/fisiología , Trofoblastos/fisiología , Línea Celular , Colágeno , Combinación de Medicamentos , Humanos , Laminina , Metaloproteinasas de la Matriz/metabolismo , Proteoglicanos , Trofoblastos/microbiologíaRESUMEN
Bacteria in the genus Brucella are important human and veterinary pathogens. The abortion and infertility they cause in food animals produce economic hardships in areas where the disease has not been controlled, and human brucellosis is one of the world's most common zoonoses. Brucella strains have also been isolated from wildlife, but we know much less about the pathobiology and epidemiology of these infections than we do about brucellosis in domestic animals. The brucellae maintain predominantly an intracellular lifestyle in their mammalian hosts, and their ability to subvert the host immune response and survive and replicate in macrophages and placental trophoblasts underlies their success as pathogens. We are just beginning to understand how these bacteria evolved from a progenitor alphaproteobacterium with an environmental niche and diverged to become highly host-adapted and host-specific pathogens. Two important virulence determinants played critical roles in this evolution: (i) a type IV secretion system that secretes effector molecules into the host cell cytoplasm that direct the intracellular trafficking of the brucellae and modulate host immune responses and (ii) a lipopolysaccharide moiety which poorly stimulates host inflammatory responses. This review highlights what we presently know about how these and other virulence determinants contribute to Brucella pathogenesis. Gaining a better understanding of how the brucellae produce disease will provide us with information that can be used to design better strategies for preventing brucellosis in animals and for preventing and treating this disease in humans.
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Proteínas de la Membrana Bacteriana Externa/metabolismo , Brucella/patogenicidad , Brucelosis/patología , Lipopolisacáridos/inmunología , Sistemas de Secreción Tipo IV/metabolismo , Adaptación Fisiológica , Animales , Proteínas de la Membrana Bacteriana Externa/genética , Brucella/genética , Brucella/inmunología , Femenino , Especificidad del Huésped/genética , Humanos , Macrófagos/microbiología , Embarazo , Complicaciones Infecciosas del Embarazo/microbiología , Complicaciones Infecciosas del Embarazo/patología , Trofoblastos/microbiología , Sistemas de Secreción Tipo IV/genética , Virulencia , Factores de VirulenciaRESUMEN
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-1ß and IL-18, which is blocked by NLRP3 inflammasome inhibitors and occurs without detectable gasdermin D cleavage. We further show that placenta-derived IL-1ß 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.
Asunto(s)
Vellosidades Coriónicas/inmunología , Inflamasomas/inmunología , Listeria monocytogenes/inmunología , Listeriosis/inmunología , Transducción de Señal/inmunología , Trofoblastos/inmunología , Células CACO-2 , Vellosidades Coriónicas/microbiología , Vellosidades Coriónicas/patología , Femenino , Humanos , Interleucina-18/inmunología , Interleucina-1beta/inmunología , Listeriosis/microbiología , Listeriosis/patología , Monocitos/inmunología , Monocitos/microbiología , Monocitos/patología , Células THP-1 , Trofoblastos/microbiología , Trofoblastos/patologíaRESUMEN
Porphyromonas gingivalis is a periodontal pathogen implicated in a range of pregnancy disorders that involve impaired spiral artery remodeling (ISAR) with or without fetal growth restriction (FGR). Using a rodent periodontitis model, we assessed the ability of P. gingivalis to produce ISAR and FGR in Sprague Dawley (SD) and Wistar (WIS) rats. Both infected SD and WIS rats developed ISAR, but only WIS rats developed FGR despite both rat strains having equivalent microbial loads within the placenta. Neither maternal systemic inflammation nor placental (fetal) inflammation was a feature of FGR in WIS rats. Unique to infected WIS rats, was loss of trophoblast cell density within the junctional zone of the placenta that was not present in SD tissues. In addition, infected WIS rats had a higher proportion of junctional zone trophoblast cells positive for cytoplasmic high temperature requirement A1 (Htra1), a marker of cellular oxidative stress. Our results show a novel phenomenon present in P. gingivalis-induced FGR, with relevance to human disease since dysregulation of placental Htra1 and placental oxidative stress are features of preeclamptic placentas and preeclampsia with FGR.
Asunto(s)
Arterias/patología , Infecciones por Bacteroidaceae/complicaciones , Retardo del Crecimiento Fetal/etiología , Porphyromonas gingivalis/patogenicidad , Trofoblastos/patología , Remodelación Vascular , Animales , Arterias/microbiología , Infecciones por Bacteroidaceae/microbiología , Femenino , Retardo del Crecimiento Fetal/patología , Masculino , Embarazo , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Trofoblastos/microbiologíaRESUMEN
Maternal decidual NK (dNK) cells promote placentation, but how they protect against placental infection while maintaining fetal tolerance is unclear. Here we show that human dNK cells highly express the antimicrobial peptide granulysin (GNLY) and selectively transfer it via nanotubes to extravillous trophoblasts to kill intracellular Listeria monocytogenes (Lm) without killing the trophoblast. Transfer of GNLY, but not other cell death-inducing cytotoxic granule proteins, strongly inhibits Lm in human placental cultures and in mouse and human trophoblast cell lines. Placental and fetal Lm loads are lower and pregnancy success is greatly improved in pregnant Lm-infected GNLY-transgenic mice than in wild-type mice that lack GNLY. This immune defense is not restricted to pregnancy; peripheral NK (pNK) cells also transfer GNLY to kill bacteria in macrophages and dendritic cells without killing the host cell. Nanotube transfer of GNLY allows dNK to protect against infection while leaving the maternal-fetal barrier intact.
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Antígenos de Diferenciación de Linfocitos T/inmunología , Bacterias/inmunología , Movimiento Celular/inmunología , Células Asesinas Naturales/inmunología , Trofoblastos/inmunología , Animales , Línea Celular , Línea Celular Tumoral , Células Dendríticas/inmunología , Femenino , Células HeLa , Humanos , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Placenta/inmunología , Placenta/microbiología , Embarazo , Ratas , Células THP-1 , Trofoblastos/microbiologíaRESUMEN
Extravillous trophoblasts (EVT) migration into the decidua is critical for establishing placental perfusion and when dysregulated, may lead to pre-eclampsia (PE) and intrauterine growth restriction (IUGR). The breast cancer resistance protein (BCRP; encoded by ABCG2) regulates the fusion of cytotrophoblasts into syncytiotrophoblasts and protects the fetus from maternally derived xenobiotics. Information about BCRP function in EVTs is limited, however placental exposure to bacterial/viral infection leads to BCRP downregulation in syncitiotrophoblasts. We hypothesized that BCRP is involved in the regulation of EVT function and is modulated by infection/inflammation. We report that besides syncitiotrophoblasts and cytotrophoblasts, BCRP is also expressed in EVTs. BCRP inhibits EVT cell migration in HTR8/SVneo (human EVT-like) cells and in human EVT explant cultures, while not affecting cell proliferation. We have also shown that bacterial-lipopolysaccharide (LPS)-and viral antigens-single stranded RNA (ssRNA)-have a profound effect in downregulating ABCG2 and BCRP levels, whilst simultaneously increasing the migration potential of EVT-like cells. Our study reports a novel function of BCRP in early placentation and suggests that exposure of EVTs to maternal infection/inflammation could disrupt their migration potential via the downregulation of BCRP. This could negatively influence placental development/function, contribute to existing obstetric pathologies, and negatively impact pregnancy outcomes and maternal/neonatal health.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Lipopolisacáridos/efectos adversos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , ARN Viral/efectos adversos , Trofoblastos/citología , Línea Celular , Movimiento Celular , Proliferación Celular , Regulación hacia Abajo , Femenino , Humanos , Placentación , Embarazo , Trofoblastos/metabolismo , Trofoblastos/microbiología , Trofoblastos/virologíaRESUMEN
INTRODUCTION: Salmonella species are gram-negative facultative intracellular bacteria that are common causes of foodborne illness in North America. Infections by Salmonella during pregnancy are a significant cause of fetal loss in domestic livestock, and fetal and maternal mortality in mice. Furthermore, Salmonella infection is associated with miscarriage, stillbirth and preterm birth in pregnant women. Despite these collective associations, the extent to which Salmonella can infect the human placenta has not been investigated. METHODS: Human placental villous explants from several gestational ages were exposed to Salmonella enterica serovar Typhimurium (STm) ex vivo. Infection was assessed by colony forming unit assay and whole mount immunofluorescence (WMIF). RESULTS: Viable bacteria were recovered from placental villous explants of all gestational ages tested, but the bacterial burden was highest in 1st trimester explants. Bacterial numbers did not change appreciably with time post-infection in explants from any gestational age examined, suggesting that STm does not proliferate in placental villi. Exposure of villous explants to STm strains defective for the type III secretion systems revealed that Salmonella pathogenicity island 1 is essential for optimal invasion. In contrast to placental explants, STm infected and proliferated within villous cytotrophoblast cells isolated from term placentas. WMIF demonstrated that STm was restricted primarily to the syncytiotrophoblast layer in infected placentas. DISCUSSION: Our study demonstrates that STm can invade into the syncytiotrophoblast but does not subsequently proliferate. Thus, the syncytiotrophoblast may function as a barrier to STm infection of the fetus.
Asunto(s)
Enfermedades Placentarias/microbiología , Placenta/microbiología , Complicaciones Infecciosas del Embarazo/microbiología , Infecciones por Salmonella/complicaciones , Salmonella typhimurium/patogenicidad , Carga Bacteriana , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Vellosidades Coriónicas/microbiología , Femenino , Edad Gestacional , Humanos , Técnicas In Vitro , Microscopía Fluorescente , Embarazo , Infecciones por Salmonella/microbiología , Salmonella typhimurium/genética , Salmonella typhimurium/fisiología , Trofoblastos/microbiología , Sistemas de Secreción Tipo III/deficiencia , Sistemas de Secreción Tipo III/genética , Sistemas de Secreción Tipo III/fisiología , Virulencia/fisiologíaRESUMEN
The effectors of the type IV secretion system (T4SS) of bacteria play important roles in mediating bacterial intracellular proliferation and manipulating host-related pathway responses to bacterial infection. Brucella Spp. inhibit the apoptosis of host cells to benefit their own intracellular proliferation. However, the underlying mechanisms between T4SS effectors and Brucella-inhibited apoptosis in goat trophoblast cells remain unclear. Here, based on Brucella suis vaccine strain 2, the VceC was deleted by allelic exchange. We show that ΔVceC was able to infect and proliferate to high titers in goat trophoblast cells (GTCs) and increase C/EBP-homologous protein (CHOP)-mediated apoptosis. GRP78 expression decreased upon ΔVceC infection. In addition, we discovered that the inositolrequiring enzyme 1 (IRE1) pathway was inhibited in this process. Changing endoplasmic reticulum (ER) stress affected Brucella intracellular replication in GTCs. The replication of ΔVceC was more sensitive under the different ERstress conditions in the GTC line after treatment with ER stress inhibitors 4 phenyl butyric acid (4-PBA) or ER stress activator Tm. Together, our findings show that VceC has a protective effect on the intracellular persistence of Brucella infection, and inhibits ER stress-induced apoptosis in the CHOP pathway. The present work provides new insights for understanding the mechanism of VceC in the establishment of chronic Brucella infection.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Brucella/fisiología , Brucelosis/veterinaria , Proteínas Serina-Treonina Quinasas/metabolismo , Trofoblastos/metabolismo , Trofoblastos/microbiología , Secuencia de Aminoácidos , Animales , Apoptosis , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/genética , Cabras , Interacciones Huésped-Patógeno , Humanos , Viabilidad Microbiana , Mutación , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Ovinos , Enfermedades de las Ovejas/metabolismo , Enfermedades de las Ovejas/microbiología , Transducción de SeñalRESUMEN
Subversion of endoplasmic reticulum (ER) function is a feature shared by multiple intracellular bacteria and viruses, and in many cases this disruption of cellular function activates pathways of the unfolded protein response (UPR). In the case of infection with Brucella abortus, the etiologic agent of brucellosis, the unfolded protein response in the infected placenta contributes to placentitis and abortion, leading to pathogen transmission. Here we show that B. abortus infection of pregnant mice led to death of infected placental trophoblasts in a manner that depended on the VirB type IV secretion system (T4SS) and its effector VceC. The trophoblast death program required the ER stress-induced transcription factor CHOP. While NOD1/NOD2 expression in macrophages contributed to ER stress-induced inflammation, these receptors did not play a role in trophoblast death. Both placentitis and abortion were independent of apoptosis-associated Speck-like protein containing a caspase activation and recruitment domain (ASC). These studies show that B. abortus uses its T4SS to induce cell-type-specific responses to ER stress in trophoblasts that trigger placental inflammation and abortion. Our results suggest further that in B. abortus the T4SS and its effectors are under selection as bacterial transmission factors.IMPORTANCEBrucella abortus infects the placenta of pregnant cows, where it replicates to high levels and triggers abortion of the calf. The aborted material is highly infectious and transmits infection to both cows and humans, but very little is known about how B. abortus causes abortion. By studying this infection in pregnant mice, we discovered that B. abortus kills trophoblasts, which are important cells for maintaining pregnancy. This killing required an injected bacterial protein (VceC) that triggered an endoplasmic reticulum (ER) stress response in the trophoblast. By inhibiting ER stress or infecting mice that lack CHOP, a protein induced by ER stress, we could prevent death of trophoblasts, reduce inflammation, and increase the viability of the pups. Our results suggest that B. abortus injects VceC into placental trophoblasts to promote its transmission by abortion.
Asunto(s)
Brucella abortus/patogenicidad , Muerte Celular , Estrés del Retículo Endoplásmico , Placenta/microbiología , Trofoblastos/microbiología , Sistemas de Secreción Tipo IV/metabolismo , Animales , Femenino , Ratones , Ratones Endogámicos C57BL , Proteína Adaptadora de Señalización NOD1/genética , Proteína Adaptadora de Señalización NOD2/genética , Placenta/citología , Embarazo , Factor de Transcripción CHOP/genética , Trofoblastos/patología , Respuesta de Proteína DesplegadaRESUMEN
Brucella melitensis infection causes acute necrotizing inflammation in pregnant animals; however, the pathophysiological mechanisms leading to placentitis are unknown. Here, we demonstrate that high-mobility group box 1 (HMGB1) acts as a mediator of placenta inflammation in B. melitensis-infected pregnant mice model. HMGB1 levels were increased in trophoblasts or placental explant during B. melitensis infection. Inhibition of HMGB1 activity with neutralising antibody significantly reduced the secretion of inflammatory cytokines in B. melitensis-infected trophoblasts or placenta, whereas administration of recombinant HMGB1 (rHMGB1) increased the inflammatory response. Mechanistically, this decreased inflammatory response results from inhibition of HMGB1 activity, which cause the suppression of both mitogen-activated protein kinases and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation. Moreover, neutralising antibody to HMGB1 prevented B. melitensis infection-induced activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in trophoblasts. In contrast, in vitro stimulation of trophoblasts with rHMGB1 caused activation of NADPH oxidase and increased the production of ROS, which contributes to high bacterial burden within trophoblasts or placenta. In vivo, treatment with anti-HMGB1 antibody increases the number of Brucella survival within placenta in B. melitensis-infected pregnant mice but successfully reduced the severity of placentitis and abortion.
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Brucella melitensis/fisiología , Brucelosis/inmunología , Proteína HMGB1/metabolismo , Placenta/inmunología , Trofoblastos/metabolismo , Trofoblastos/microbiología , Aborto Espontáneo/genética , Aborto Espontáneo/metabolismo , Aborto Espontáneo/microbiología , Animales , Brucella melitensis/genética , Brucella melitensis/metabolismo , Brucella melitensis/patogenicidad , Brucelosis/genética , Brucelosis/metabolismo , Citocinas/metabolismo , Replicación del ADN/inmunología , Femenino , Proteína HMGB1/administración & dosificación , Proteína HMGB1/antagonistas & inhibidores , Proteína HMGB1/genética , Inflamación/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , NADPH Oxidasas/química , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Fosforilación , Placenta/microbiología , Placenta/patología , Embarazo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Trofoblastos/enzimologíaRESUMEN
BACKGROUND: Group B streptococcus (GBS) is the main bacteria that infects pregnant women and can cause abortion and chorioamnionitis. The impact of GBS effects on human trophoblast cells remains largely elusive, and actions toward anti-inflammatory strategies in pregnancy are needed. A potent anti-inflammatory molecule, uvaol is a triterpene from olive oil and its functions in trophoblasts are unknown. We aimed to analyze biomechanical and functional effects of inactivated GBS in trophoblast cells, with the addition of uvaol to test potential benefits. METHODS: HTR-8/SVneo cells were treated with uvaol and incubated with inactivated GBS. Cell viability and death were analyzed. Cellular elasticity and topography were accessed by atomic force microscopy. Nitrite production was evaluated by Griess reaction. Nuclear translocation of NFkB p65 was detected by immunofluorescence and Th1/Th2 cytokines by bead-based multiplex assay. RESULTS: GBS at 108â¯CFU increased cell death, which was partially prevented by uvaol. Cell stiffness, cytoskeleton organization and morphology were changed by GBS, and uvaol partially restored these alterations. Nuclear translocation of NFkB p65 began 15â¯min after GBS incubation and uvaol inhibited this process. GBS decreased IL-4 secretion and increased IL-1ß, IFN-γ and IL-2, whereas uvaol reverted this. CONCLUSIONS: The increased inflammation and cell death caused by GBS correlated with biomechanical and cytoskeleton changes found in trophoblast cells, while uvaol was effective its protective role. GENERAL SIGNIFICANCE: Uvaol is a natural anti-inflammatory product efficient against GBS-induced inflammation and it has potential to be acquired through diet in order to prevent GBS deleterious effects in pregnancy.
Asunto(s)
Streptococcus agalactiae/patogenicidad , Triterpenos/farmacología , Trofoblastos/efectos de los fármacos , Trofoblastos/microbiología , Animales , Transporte Biológico , Fenómenos Biomecánicos , Muerte Celular , Línea Celular , Núcleo Celular/metabolismo , Supervivencia Celular , Chlorocebus aethiops , Citocinas/metabolismo , Femenino , Humanos , FN-kappa B/metabolismo , Nitritos/metabolismo , Embarazo , Células TH1/metabolismo , Trofoblastos/metabolismo , Células VeroRESUMEN
Brucellosis is a zoonosis caused by bacteria of the Brucella genus. In ruminants, brucellosis causes abortion, followed by chronic infection and secretion of bacteria in milk. In humans, it usually presents as flu-like symptoms, with serious complications if untreated. Epidemiological studies have only recently established that brucellosis can also cause pregnancy complications in women, but the pathogenic mechanisms are unknown. Pioneering studies in ruminants showed that Brucella infect trophoblasts and then colonise the placenta where they grow to high density. A recent study showed that the main zoonotic Brucella species can infect human cytotrophoblasts (CTB) and extravillous trophoblasts (EVT). In this work, we show that Brucella papionis (associated with stillbirth in primates) also infects human trophoblasts. However, it replicates actively in CTB, whereas its replication is very restricted within EVT. We also observed alteration of several trophoblastic functions upon infection by B. papionis or Brucella melitensis (the most prevalent species in human brucellosis). Infection altered the production of hormones, the ability of CTB to form syncytiotrophoblasts, and the invasion capacity of EVT. We also found that infection can spread between different types of trophoblasts. These findings constitute a new step in understanding how Brucella infection causes adverse pregnancy outcomes.
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Brucella melitensis/patogenicidad , Brucella/patogenicidad , Brucelosis/microbiología , Trofoblastos/microbiología , Brucelosis/patología , Femenino , Humanos , Embarazo , Trofoblastos/patologíaRESUMEN
A successful pregnancy depends on not only the tolerance of the fetal immune system by the mother but also resistance against the threat of hazardous microorganisms. Infection with pathogenic microorganisms during pregnancy may lead to premature delivery, miscarriage, growth restriction, neonatal morbidity, and other adverse outcomes. Moreover, the host also has an intact immune system to avoid these adverse outcomes. It is important to note the presence of normal bacteria in the maternal reproductive tract and the principal role of the maternal-placental-fetal interaction in antimicrobial immunity. Previous studies mainly focused on maternal infection during pregnancy. However, this review summarizes the new views on the study of the maternal microbiome and expounds the innate immune defense mechanism of the maternal vagina and decidua as well as how cytotrophoblasts and syncytiotrophoblasts recognize and kill bacteria in the placenta. Fetal immune systems, thought to be weak, also exhibit an immune defense function that is indispensable for maintaining the safety of the fetus. The skin, lungs, and intestines of the fetus during pregnancy constitute the main immune barriers. These findings will provide a new understanding of the effects of normal microbial flora and how the host resists harmful microbes during pregnancy. We believe that it may also contribute to the reference on the clinical prevention and treatment of gestational infection to avoid adverse pregnancy outcomes.
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Inmunidad Innata , Intercambio Materno-Fetal/inmunología , Microbiota/inmunología , Decidua/inmunología , Decidua/microbiología , Femenino , Humanos , Embarazo , Complicaciones del Embarazo/inmunología , Complicaciones del Embarazo/microbiología , Trofoblastos/inmunología , Trofoblastos/microbiología , Vagina/inmunología , Vagina/microbiologíaRESUMEN
Epidemiological studies suggested that periodontitis is a risk factor for pregnancy complications including preterm birth. Porphyromonas gingivalis, a vital periodontal pathogen found in amniotic fluid and intact membranes of women who deliver preterm low birth weight infants, is thought to contribute to preterm labor. However, molecular and cellular interactions between P. gingivalis and placental cells are not clear. In this study, we investigated the effect of P. gingivalis on human extravillous trophoblasts and observed that it triggered apoptosis and inflammation and that Akt was activated in this process. In addition, when Akt activation was inhibited, apoptosis and inflammation was significantly increased. Thus, P. gingivalis infection contributes to preterm low birth weight infants by triggering excessive inflammation and increasing apoptosis in trophoblasts and that the Phosphatidylinositol 3-Kinase/Akt signaling pathway is involved in the regulation of Pg-induced apoptosis and inflammation.
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Apoptosis/inmunología , Infecciones por Bacteroidaceae/inmunología , Fosfatidilinositol 3-Quinasas/inmunología , Porphyromonas gingivalis/inmunología , Proteínas Proto-Oncogénicas c-akt/inmunología , Transducción de Señal/inmunología , Trofoblastos/inmunología , Infecciones por Bacteroidaceae/patología , Línea Celular , Humanos , Inflamación/inmunología , Inflamación/microbiología , Inflamación/patología , Trofoblastos/microbiología , Trofoblastos/patologíaRESUMEN
Since pathogenic Brucella survive and replicate inside phagocytes, cellular models of infection constitute important tools in brucellosis research. We describe the behavior of B. ovis PA (which causes a type of ovine brucellosis mainly affecting the male reproductive tract) and representative attenuated mutants in two commercially available cell lines of non-professional phagocytes related to Brucella tissue preference: OA3.Ts ovine testis cells and JEG-3 human trophoblasts. In comparison with J774.A1 macrophages and HeLa cells, intracellular bacteria were enumerated at several post-infection time points and visualized by confocal microscopy. Replication of B. ovis in OA3.Ts and JEG-3 cells was equivalent to that observed in J774.A1 macrophages-despite the more efficient internalization in the latter-and better than in HeLa cells. Multiplication and/or survival in all phagocytes was dependent on virB2 and vjbR but independent of cgs, despite the attenuation in mice of the Δcgs mutant. However, Omp25c was required for B. ovis internalization only in HeLa cells, and removal of Omp31 increased bacterial internalization in human HeLa and JEG-3 cells. The results presented here demonstrate variability in the interaction of B. ovis with different host cells and provide advantageous models of non-professional phagocytes to study the intracellular behavior of B. ovis.