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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.

Trophoblasts are targets of infection by Brucella spp. but their role in the pathophysiology of pregnancy complications of brucellosis is unknown. Here we show that Brucella abortus invades and replicates in the human trophoblastic cell line Swan-71 and that the intracellular survival of the bacterium depends on a functional virB operon. The infection elicited significant increments of interleukin 8 (IL8), monocyte chemotactic protein 1 (MCP-1), and IL6 secretion, but levels of IL1beta and tumor necrosis factor-alpha (TNF-alpha) did not vary significantly. Such proinflammatory response was not modified by the absence of the Brucella TIR domain-containing proteins BtpA and BtpB. The stimulation of Swan-71 cells with conditioned medium (CM) from B. abortus-infected human monocytes (THP-1 cells) or macrophages induced a significant increase of IL8, MCP-1 and IL6 as compared to stimulation with CM from non-infected cells. Similar results were obtained when stimulation was performed with CM from infected neutrophils. Neutralization studies showed that IL1beta and/or TNF-alpha mediated the stimulating effects of CM from infected phagocytes. Reciprocally, stimulation of monocytes and neutrophils with CM from Brucella-infected trophoblasts increased IL8 and/or IL6 secretion. These results suggest that human trophoblasts may provide a local inflammatory environment during B. abortus infections either through a direct response to the pathogen or through interactions with monocytes/macrophages or neutrophils, potentially contributing to the pregnancy complications of brucellosis., (© 2016 by the Society for the Study of Reproduction, Inc.)

The mechanisms underlying the immune defense by trophoblasts against pathogens remain ill defined. We demonstrated that placental cell death was increased upon in vivo exposure to Listeria monocytogenes. The death of infected cells is an important host innate defense mechanism. Meanwhile, double-stranded DNA (dsDNA) derived from intracellular bacteria or dsDNA viruses is emerging as a potent pathogen-associated molecular pattern recognized by host cells. We sought to characterize trophoblast death in response to cytosolic dsDNA challenge. Our results showed that dsDNA induced caspase-dependent and -independent cell death in human trophoblasts. However, necroptosis, a cell death pathway independent of caspase, could not be induced by dsDNA treatment, even in the presence of exogenously expressed RIPK3. L. monocytogenes-derived genomic DNA triggered a similar cell death pattern. Moreover, the cell death in response to dsDNA was IFI16 dependent. These data suggest that cytosolic dsDNA induces nonnecroptotic cell death in trophoblasts via IFI16, and this could contribute to placental barrier against infection., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Brucellae replicate in a vacuole derived from the endoplasmic reticulum (ER) in epithelial cells, macrophages, and dendritic cells. In animals, trophoblasts are also key cellular targets where brucellae efficiently replicate in association with the ER. Therefore, we investigated the ability of Brucella spp. to infect human trophoblasts using both immortalized and primary trophoblasts. Brucella extensively proliferated within different subpopulations of trophoblasts, suggesting that they constitute an important niche in cases where the fetal-maternal barrier is breached. In extravillous trophoblasts (EVTs), B. abortus and B. suis replicated within single-membrane acidic lysosomal membrane-associated protein 1-positive inclusions, whereas B. melitensis replicated in the ER-derived compartment. Furthermore, B. melitensis but not B. abortus nor B. suis interfered with the invasive capacity of EVT-like cells in vitro. Because EVTs are essential for implantation during early stages of pregnancy, the nature of the replication niche may have a central role during Brucella-associated abortion in infected women.

The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)(2)D), is a potent inducer of the antimicrobial protein cathelicidin, CAMP (LL37). In macrophages this response is dependent on intracrine synthesis of 1,25(OH)(2)D from precursor 25-hydroxyvitamin D (25OHD), catalyzed by the enzyme 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1). In view of the fact that trophoblastic cells also express abundant CYP27B1, we postulated a similar intracrine pathway for induction of CAMP in the placenta. Analysis of placenta explants, primary cultures of human trophoblast, and the 3A trophoblastic cell line treated with 1,25(OH)(2)D (1-100 nM) revealed dose-dependent induction of CAMP similar to that observed with primary cultures of human macrophages. Also consistent with macrophages, induction of trophoblastic CAMP was enhanced via intracrine conversion of 25OHD to 1,25(OH)(2)D. However, in contrast to macrophages, induction of CAMP by vitamin D in trophoblasts was not enhanced by costimulation with Toll-like receptor ligands, such as lipopolysaccharide. Despite this, exposure to vitamin D metabolites significantly enhanced antibacterial responses in trophoblastic cells: 3A cells infected with Escherichia coli showed decreased numbers of bacterial colony-forming units compared with vehicle-treated controls when treated with 25OHD (49.6% +/- 10.9%) or 1,25(OH)(2)D (45.4% +/- 9.2%), both P < 0.001. Treatment with 25OHD (1-100 nM) or 1,25(OH)(2)D (0.1-10 nM) also protected 3A cells against cell death following infection with E. coli (13.6%-26.9% and 22.3%-40.2% protection, respectively). These observations indicate that 1,25(OH)(2)D can function as an intracrine regulator of CAMP in trophoblasts, and may thus provide a novel mechanism for activation of innate immune responses in the placenta.

We examined if Fc receptor-mediated antibody-dependent enhancement (FcR-ADE) or complement-mediated antibody-dependent enhancement (C'-ADE) of virus infection can contribute to increasing replication of HIV-1 in human syncytiotrophoblast (ST) cells. Here we report that both FcR-ADE and C'-ADE may result in enhanced virus release from HIV-1-infected ST cells. We show that FcR-ADE of HIV-1 infection in ST cells is mediated by FcRIII and other FcR(s) belonging to undetermined Fc classes and does not require CD4 receptors, whereas C'-ADE uses both CD4 and CR2-like receptors. FcR-ADE seems to be more efficient in enhancing HIV-1 replication than C'-ADE. While FcR-ADE leads to increased internalization of HIV-1, C'-ADE does not result in enhanced endocytosis of the virus. In addition, antibodies mediating FcR-ADE are reactive with the gp120 viral envelope antigen, whereas antibodies involved in C'-ADE react with the viral transmembrane glycoprotein gp41. Data suggest that both FcR-ADE and C'-ADE may contribute to the spread of HIV-1 from mother to the fetus.

To characterize the role of the placenta in vertical transmission of human immunodeficiency virus type 1 (HIV-1), the susceptibility of primary human placental cultures and of transformed trophoblast cell lines to infection by several HIV-1 isolates was examined. Placental cultures supported the replication of all strains tested, including lymphocyte-, macrophage-, and amphotropic isolates. All viruses replicated to modest levels, with production of both viral antigen and infectious virus in the culture supernatants. Placental cells demonstrated a pattern of permissiveness for HIV-1 isolates distinct from that seen with lymphocytes, blood-derived macrophages, or T cell lines. Immunofluorescent staining showed that 5%-10% of the cultured placental cells expressed viral antigens, and double labeling revealed that the HIV-positive cells were macrophages not trophoblasts. None of the trophoblast cell line (JEG-3, Jar, BeWo, HP-W1) could be infected by HIV. These results support the hypothesis that infection of the placenta could play a role in maternofetal transmission of HIV-1 and suggest that the placental macrophage is likely to be the primary cell type responsible.

The prevalence of human immunodeficiency virus (HIV) infections among children is increasing in a manner that closely follows the spread of the disease among women. Despite the fact that in utero transmission via the placenta is though to play a major role in the spread of HIV to the pediatric population, little is known about the timing, route(s), and cellular mechanisms by which maternal-fetal transmission occurs. This review attempts to use a developmental and cellular approach to assess the available clinical and laboratory data pertaining to maternal-fetal HIV transmission. While much of this review focuses on the role of the placenta, particularly the placental trophoblast, on the transmission of HIV, potential routes of infection during early development are also discussed. Clinical studies indicate that the placental trophoblast can be infected with HIV but have shed no light on how the virus gains entry to this tissue. While some laboratory studies confirm that trophoblast cells and placental macrophages can be infected with HIV in vitro, many studies are difficult to interpret because of inadequate characterization of the placental cells used. The role of CD4 in the infection of trophoblast remains controversial and clearly warrants a systematic examination. It is also apparent that viral tropism has not received enough attention and more studies using different strains of HIV are required. Thus, several basic questions remain to be answered before strategies to prevent maternal-fetal transmission of HIV can be developed.

The European collaborative study of HIV-infected pregnant women in Europe now indicates a 13% risk of fetal HIV infection (originally thought to be about 30%, and possibly higher in some countries). Several reports suggest trans-placental passage. However, the detailed mechanisms associated with such vertical transmission have not yet been clarified. We have examined the possibility that HIV enters placental tissue from maternal blood via binding to CD4 and Fc receptors (FcR) at the trophoblast level, allowing intraplacental infection. Here we report the detection of several FcR with distinct localization in the placental villus as well as CD4 surface expression on human trophoblast cells. In addition, we show that trophoblastic cells interact specifically with the gp120/gp160 viral envelope protein. By their tissue localization, these receptors could be responsible for the entry of HIV into the fetal placental cells. Furthermore, purified placental cells can be directly infected by HIV in vitro, and the infection is inhibited by soluble CD4. This suggests a crucial role of the CD4 receptor but an additional way of entry cannot be excluded. Such an in vitro model may be suitable for further studies concerning placental HIV transmission and its prevention.

Electron microscopy of near-term placentas of two cottontop marmosets (Saguinus oedipus) revealed, in one placenta, the presence of budding and mature C-type virus particles associated with the basal trophoblast. The particles were morphologically similar to those observed by other investigators in placentas of other primate species.

Type C RNA retroviral particles have been observed budding from the plasma membrane of syncytiotrophoblast during a study of embryo implantation in the common marmoset (Callithrix jacchus). These viral particles appear in profusion at the interface of syncytiotrophoblast with cytotrophoblast. They appear to be a non-infective, endogenous viral component of the host genome, although the precise role of the C-type virus in this context is unclear at present. Such viral particles have been reported previously from studies of human term placenta; however, this is the first report indicating that expression of viral particles occurs at the earliest stages of embryo implantation.