theLiTE™: A Screening Platform to Identify Compounds that Reinforce Tight Junctions.

Tight junctions (TJ) are formed by transmembrane and intracellular proteins that seal the intercellular space and control selective permeability of epithelia. Integrity of the epithelial barrier is central to tissue homeostasis and barrier dysfunction has been linked to many pathological conditions. TJ support the maintenance of cell polarity through interactions with the Par complex (Cdc42-Par-6-Par-3-aPKC) in which Par-6 is an adaptor and links the proteins of the complex together. Studies have shown that Par-6 overexpression delays the assembly of TJ proteins suggesting that Par-6 negatively regulates TJ assembly. Because restoring barrier integrity is of key therapeutic and prophylactic value, we focus on finding compounds that have epithelial barrier reinforcement properties; we developed a screening platform (theLiTE™) to identify compounds that modulate Par-6 expression in follicular epithelial cells from Par-6-GFP Drosophila melanogaster egg chambers. Hits identified were then tested whether they improve epithelial barrier function, using measurements of transepithelial electrical resistance (TEER) or dye efflux to evaluate paracellular permeability. We tested 2,400 compounds, found in total 10 hits. Here we present data on six of them: the first four hits allowed us to sequentially build confidence in theLiTE™ and two compounds that were shortlisted for further development (myricetin and quercetin). We selected quercetin due to its clinical and scientific validation as a compound that regulates TJ; food supplement formulated on the basis of this discovery is currently undergoing clinical evaluation in gastroesophageal reflux disease (GERD) sufferers.

Bradykinin Sequestration by Plasmodium berghei Infected Erythrocytes Conditions B2R Signaling and Parasite Uptake by Fetal Trophoblasts.

Plasmodium infection during pregnancy causes placental malfunction reducing fetus sustainability and leading to abortions, stillbirths, low birth weight or premature delivery. Accumulation of infected erythrocytes (IE) in the placenta is a key factor in placental malaria pathogenesis but the role played by fetal trophoblast that contact maternal blood has been neglected. Here we explore the hypothesis that interactions between Plasmodium-IE and fetal trophoblast cells impact on vasoactive alterations underlying placental dysfunction. We screened gene expression of key mediators in vasoactive pathways. We found that mRNA of bradykinin receptor 2 (B2R) and nitric oxide synthase (eNOS), as well as levels of bradykinin (BK), were decreased in late gestation placentas of pregnant Plasmodium berghei-infected mice. Co-culturing mouse trophoblasts with IE down-regulated B2R transcription and interleukin (IL)-6 secretion in a B2R-signaling dependent manner. IE showed increased levels of surface B2R and enhanced capacity to bind BK. We propose that down-regulation of B2R signaling in the course of IE-trophoblast interactions is due to BK sequestration by IE. In corroboration, levels of BK were lower in infected placentas and the positive correlation of B2R gene expression and fetal weight was disrupted by infection. This indicates that deregulation of the BK-B2R pathway is associated to placental dysfunction provoked by malaria infection. We further found that upon inhibition of B2R signaling, trophoblasts engulf IE to a lesser extent and show reduced production of IL-6. Our data suggest that BK sequestration by P. berghei represents a strategy for the parasite to ameliorate the risk of phagocytic capture by down modulating B2R activation.

HGF Secreted by Activated Kupffer Cells Induces Apoptosis of Plasmodium-Infected Hepatocytes.

Malaria liver stage infection is an obligatory parasite development step and represents a population bottleneck in Plasmodium infections, providing an advantageous target for blocking parasite cycle progression. Parasite development inside hepatocytes implies a gross cellular insult evoking innate host responses to counteract intra-hepatocytic infection. Using primary hepatocyte cultures, we investigated the role of Kupffer cell-derived hepatocyte growth factor (HGF) in malaria liver stage infection. We found that Kupffer cells from Plasmodium-infected livers produced high levels of HGF, which trigger apoptosis of infected hepatocytes through a mitochondrial-independent apoptosis pathway. HGF action in infected hepatocyte primary cultures results in a potent reduction of parasite yield by specifically sensitizing hepatocytes carrying established parasite exo-erythrocytic forms to undergo apoptosis. This apoptosis mechanism is distinct from cell death that is spontaneously induced in infected cultures and is governed by Fas signaling modulation through a mitochondrial-dependent apoptosis pathway. This work indicates that HGF and Fas signaling pathways are part of an orchestrated host apoptosis response that occurs during malaria liver stage infection, decreasing the success of infection of individual hepatocytes. Our results raise the hypothesis that paracrine signals derived from Kupffer cell activation are implicated in directing death of hepatocytes infected with the malaria parasite.

Intravital placenta imaging reveals microcirculatory dynamics impact on sequestration and phagocytosis of Plasmodium-infected erythrocytes.

Malaria in pregnancy is exquisitely aggressive, causing a range of adverse maternal and fetal outcomes prominently linked to Plasmodium-infected erythrocyte cytoadherence to fetal trophoblast. To elucidate the physiopathology of infected erythrocytes (IE) sequestration in the placenta we devised an experimental system for intravital placental examination of P. berghei-infected mice. BALB/c females were mated to C57Bl/6 CFP+ male mice and infected with GFP+ P. berghei IE, and at gestational day 18, placentas were exposed for time-lapse imaging acquisition under two-photon microscopy. Real-time images and quantitative measurements revealed that trophoblast conformational changes transiently restrain blood flow in the mouse placental labyrinth. The complex dynamics of placental microcirculation promotes IE accumulation in maternal blood spaces with low blood flow and allows the establishment of stable IE-trophoblast contacts. Further, we show that the fate of sequestered IE includes engulfment by both macrophagic and trophoblastic fetal-derived cells. These findings reinforce the current paradigm that IE interact with the trophoblast and provide definitive evidence on two novel pathogenesis mechanisms: (1) trophoblast layer controls placental microcirculation promoting IE sequestration; and (2) fetal-derived placental cells engulf sequestered IE.

Distinct placental malaria pathology caused by different Plasmodium berghei lines that fail to induce cerebral malaria in the C57BL/6 mouse.

BACKGROUND: Placental malaria (PM) is one major feature of malaria during pregnancy. A murine model of experimental PM using BALB/c mice infected with Plasmodium berghei ANKA was recently established, but there is need for additional PM models with different parasite/host combinations that allow to interrogate the involvement of specific host genetic factors in the placental inflammatory response to Plasmodium infection. METHODS: A mid-term infection protocol was used to test PM induction by three P. berghei parasite lines, derived from the K173, NK65 and ANKA strains of P. berghei that fail to induce experimental cerebral malaria (ECM) in the susceptible C57BL/6 mice. Parasitaemia course, pregnancy outcome and placenta pathology induced by the three parasite lines were compared. RESULTS: The three P. berghei lines were able to evoke severe PM pathology and poor pregnancy outcome features. The results indicate that parasite components required to induce PM are distinct from ECM. Nevertheless, infection with parasites of the ANKAΔpm4 line, which lack expression of plasmepsin 4, displayed milder disease phenotypes associated with a strong innate immune response as compared to infections with NK65 and K173 parasites. CONCLUSIONS: Infection of pregnant C57BL/6 females with K173, NK65 and ANKAΔpm4 P. berghei parasites provide experimental systems to identify host molecular components involved in PM pathogenesis mechanisms.

Early IL-10 production is essential for syngeneic graft acceptance.

We performed a comparative study and evaluated cellular infiltrates and anti-inflammatory cytokine production at different time-points after syngeneic or allogeneic skin transplantation. We observed an early IL-10 production in syngeneic grafts compared with allografts. This observation prompted us to investigate the role of IL-10 in isograft acceptance. For this, we used IL-10 KO and WT mice to perform syngeneic transplantation, where IL-10 was absent in the graft or in the recipient. The majority of syngeneic grafts derived from IL-10 KO donors did not engraft or was only partially accepted, whereas IL-10 KO mice transplanted with skin from WT donors accepted the graft. We evaluated IL-10 producers in the transplanted skin and observed that epithelial cells were the major source. Taken together, our data show that production of IL-10 by donor cells, but not by the recipient, is determinant for graft acceptance and strongly suggest that production of this cytokine by keratinocytes immediately upon transplantation is necessary for isograft survival.

Expansion of CD4+ CD25+ Foxp3+ T cells by bone marrow-derived dendritic cells.

Dendritic cells (DCs) are the most important antigen-presenting cells of the immune system and have a crucial role in T-lymphocyte activation and adaptive immunity initiation. However, DCs have also been implicated in maintaining immunological tolerance. In this study, we evaluated changes in the CD4(+) CD25(+) Foxp3(+) T-cell population after co-culture of lymph node cells from BALB/c mice with syngeneic bone marrow-derived DCs. Our results showed an increase in CD4(+) CD25(+) Foxp3(+) T cells after co-culture which occurred regardless of the activation state of DCs and the presence of allogeneic apoptotic cells; however, it was greater when DCs were immature and were pulsed with the alloantigen. Interestingly, syngeneic apoptotic thymocytes were not as efficient as allogeneic apoptotic cells in expanding the CD4(+) CD25(+) Foxp3(+) T-cell population. In all experimental settings, DCs produced high amounts of transforming growth factor (TGF)-beta. The presence of allogeneic apoptotic cells induced interleukin (IL)-2 production in immature and mature DC cultures. This cytokine was also detected in the supernatants under all experimental conditions and enhanced when immature DCs were pulsed with the alloantigen. CD4(+) CD25(+) Foxp3(+) T-cell expansion during co-culture of lymph node cells with DCs strongly suggested that the presence of alloantigen enhanced the number of regulatory T cells (Tregs) in vitro. Our data also suggest a role for both TGF-beta and IL-2 in the augmentation of the CD4(+) CD25(+) Foxp3(+) population.

Supplementation of CXCL12 (CXCL12) induces homing of CD11c+ dendritic cells to the spleen and enhances control of Plasmodium berghei malaria in BALB/c mice.

In malaria, parasitaemia is controlled in the spleen, a multicomponent organ that undergoes changes in its cellular constituents to control the parasite. During this process, dendritic cells (DCs) orchestrate the positioning of effector cells in a timely manner for optimal parasite clearance. We have recently demonstrated that CXCL12 [stromal cell-derived factor-1 (CXCL12)] supplementation partially restores the ability to control parasitaemia in Plasmodium berghei-infected mice. In the present study, we investigated the nature of the DCs involved by flow cytometry and immunohistochemistry of CD11c(+) cells. Flow cytometry of bone marrow cells showed that infection with P. berghei did not alter the proportion of CD11c(+) cells present in this haematopoietic compartment, while CXCL12 supplementation of naïve uninfected mice induced only minor increases in the population of CD11c(+) cells. In the spleen, P. berghei infection alone resulted in an increase in CD11c(+) cells as compared with naïve animals. Exogenously administered CXCL12 in the absence of infection resulted in a significant expansion of the splenic CD11c(+) population, and this effect was even more pronounced in infected and supplemented mice. Immunohistochemistry revealed that CD11c(+) cells infiltrated the perivascular areas and marginal zone of the spleen in infected animals treated with CXCL12, suggesting that this chemokine induces homing of CD11c(+) dendritic cells to the splenic compartment. Our results show that small amounts of CXCL12 supplementation are effective in recruiting DCs to the spleens of both uninfected and infected mice, suggesting the participation of CXCL12 and CD11c(+) cells in the establishment of an adequate environment in the spleen for malaria control.

Administration of a peptide inhibitor of alpha4-integrin inhibits the development of experimental autoimmune uveitis.

PURPOSE: Recruitment of lymphocytes into the retina and to the vitreous during the development of experimental autoimmune uveitis (EAU) is governed by factors such as the state of activation of inflammatory cells and the repertoire of adhesion molecules expressed by the local vascular endothelia. alpha4 Integrins and their receptors play an important role during homing of cells to the inflammatory site. In the present study, the effect of alpha4-integrin inhibitor on the development of EAU was investigated. METHODS: EAU was induced either by immunizing B10.RIII mice with the 161-180 peptide or by adoptive transfer of interphotoreceptor retinoid-binding protein (IRBP)-specific uveitogenic T cells. Animals were treated with an active peptide inhibitor (alpha4-api) or a peptide control at different time points after induction of disease. EAU was evaluated by histology 21 to 49 days after immunization. Antigen-specific cell proliferation was evaluated by thymidine incorporation. Cytokine synthesis in culture supernatants and anti-IRBP-specific serum IgG1 and IgG2a were evaluated by ELISA. Delayed-type hypersensitivity was evaluated by ear challenge 2 days before the termination of the experiment. RESULTS: Treatment with alpha4-api had a significant ameliorating effect on EAU. The anti-IRBP antibody response and cellular proliferation were not affected by the treatment, whereas delayed-type hypersensitivity was significantly diminished. Cytokine synthesis was not changed by treatment, except for a decrease in IL-10 levels. CONCLUSIONS: The results show that small-molecule inhibitors of alpha4-integrins can act therapeutically in EAU, possibly by interfering with cell adhesion events involved in the development of the disease.