Faculty Bibliography

Macrophages can reprogram their metabolism in response to the surrounding stimuli, which affects their capacity to kill intracellular pathogens. We have investigated the metabolic and immune status of human macrophages after infection with the intracellular trypanosomatid parasites Leishmania donovani, L. amazonensis and T. cruzi and their capacity to respond to a classical polarizing stimulus (LPS and IFN-γ). We found that macrophages infected with Leishmania preferentially upregulate oxidative phosphorylation, which could be contributed by both host cell and parasite, while T. cruzi infection did not significantly increase glycolysis or oxidative phosphorylation. Leishmania and T. cruzi infect macrophages without triggering a strong inflammatory cytokine response, but infection does not prevent a potent response to LPS and IFN-γ. Infection appears to prime macrophages, since the cytokine response to activation with LPS and IFN-γ is more intense in infected macrophages compared to uninfected ones. Metabolic polarization in macrophages can influence infection and immune evasion of these parasites since preventing macrophage cytokine responses would help parasites to establish a persistent infection. However, macrophages remain responsive to classical inflammatory stimuli and could still trigger inflammatory cytokine secretion by macrophages.

BACKGROUND:Tamoxifen is an oestrogen receptor modulator that is widely used for the treatment of early stage breast cancer and reduction of recurrences. Tamoxifen is also used as a powerful research tool for controlling gene expression in the context of the Cre/loxP site-specific recombination system in conditional mutant mice. METHODS:To determine whether the administration of tamoxifen affects Plasmodium growth and/or disease outcome in malaria, in vitro studies assessing the effect of tamoxifen and its active metabolite 4-hydroxytamoxifen on Plasmodium falciparum blood stages were performed. Tamoxifen effects were also evaluated in vivo treating C57/B6 mice infected with Plasmodium berghei (ANKA strain), which is the standard animal model for the study of cerebral malaria. RESULTS:Tamoxifen and its active metabolite, 4-hydroxytamoxifen, show activity in vitro against P. falciparum (16.7 to 5.8 µM IC50, respectively). This activity was also confirmed in tamoxifen-treated mice infected with P. berghei, which show lower levels of parasitaemia and do not develop signs of cerebral malaria, compared to control mice. Mice treated with tamoxifen for 1 week and left untreated for an additional week before infection showed similar parasitaemia levels and signs of cerebral malaria as control untreated mice. CONCLUSIONS:Tamoxifen and its active metabolite, 4-hydroxytamoxifen, have significant activity against the human parasite P. falciparum in vitro and the rodent parasite P. berghei in vivo. This activity may be useful for prevention of malaria in patients taking this drug chronically, but also represents a major problem for scientists using the conditional mutagenic Cre/LoxP system in the setting of rodent malaria. Allowing mice to clear tamoxifen before starting a Plasmodium infection allows the use the Cre/LoxP conditional mutagenic system to investigate gene function in specific tissues.

Anemia is a common complication of malaria that is characterized by the loss of infected and uninfected erythrocytes. In mouse malaria models, clearance of uninfected erythrocytes is promoted by autoimmune anti-phosphatidylserine (PS) antibodies produced by T-bet⁺B-cells, which bind to exposed PS in erythrocytes, but the mechanism in patients is still unclear. In Plasmodium falciparum patients with anemia, we show that atypical memory FcRL5⁺T-bet⁺ B-cells are expanded and associate both with higher levels of anti-PS antibodies in plasma and with the development of anemia in these patients. No association of anti-PS antibodies or anemia with other B-cell subsets and no association of other antibody specificities with FcRL5⁺T-bet⁺ B-cells is observed, revealing high specificity in this response. We also identify FcRL5⁺T-bet⁺ B-cells as producers of anti-PS antibodies in ex vivo cultures of naïve human peripheral blood mononuclear cells (PBMC) stimulated with P.-falciparum-infected erythrocyte lysates. These data define a crucial role for atypical memory B-cells and anti-PS autoantibodies in human malarial anemia.

Malaria is a highly inflammatory disease caused by the protozoan parasite Plasmodium During the blood stage of infection, patients exhibit fever with high levels of inflammatory cytokines in their blood. However, when cells of the immune system are incubated with the parasite in vitro, their cytokine response is low. In particular, human primary dendritic cells (DCs) respond to Plasmodium falciparum-infected erythrocytes by upregulating maturation markers and chemokines but lack a substantial cytokine response. Because oxidative stress is a trigger of inflammatory cytokines in malaria and synergizes with P. falciparum to induce IL-1β secretion by macrophages, we assessed whether oxidative stress has an impact on DC maturation and function in response to P. falciparum Using xanthine oxidase, a reactive oxygen species- (ROS) producing enzyme that is increased during malaria, we observed that exposure to extracellular ROS potentiated DC maturation in response to the parasite. Xanthine oxidase-derived ROS increased parasite-induced cytokine secretion and CD80 surface expression in DCs. This enhanced maturation phenotype boosted the DCs' ability to prime autologous naive CD4⁺ T cells, resulting in higher T cell proliferation in vitro. Xanthine oxidase-derived ROS did not have an effect on the cytokines produced by primed T cells. We propose that oxidative stress during malaria contributes to the inflammatory response by enhancing the magnitude of DC and CD4⁺ T cell responses without changing the quality.

Autoantibodies targeting host antigens contribute to autoimmune disorders, frequently occur during and after infections and have been proposed to contribute to malaria-induced anemia. We measured anti-phosphatidylserine (PS) and anti-DNA antibody levels in 382 Ugandan children prospectively recruited in a study of severe malaria (SM). High antibody levels were defined as antibody levels greater than the mean plus 3 standard deviations of community children (CC). We observed increases in median levels of anti-PS and anti-DNA antibodies in children with SM compared to CC (p < 0.0001 for both). Children with severe malarial anemia were more likely to have high anti-PS antibodies than children with cerebral malaria (16.4% vs. 7.4%), p = 0.02. Increases in anti-PS and anti-DNA antibodies were associated with decreased hemoglobin (p < 0.05). A one-unit increase in anti-DNA antibodies was associated with a 2.99 (95% CI, 1.68, 5.31) increase odds of acute kidney injury (AKI) (p < 0.0001). Elevated anti-PS and anti-DNA antibodies were associated with post-discharge mortality (p = 0.031 and p = 0.042, respectively). Children with high anti-PS antibodies were more likely to have multiple hospital readmissions compared to children with normal anti-PS antibody levels (p < 0.05). SM is associated with increased autoantibodies against PS and DNA. Autoantibodies were associated with anemia, AKI, post-discharge mortality, and hospital readmission.

Zika virus infection is associated with the development of Guillain-Barré syndrome (GBS), a neurological autoimmune disorder caused by immune recognition of gangliosides and other components at nerve membranes. Using a high-throughput ELISA, we have analyzed the anti-glycolipid antibody profile, including gangliosides, of plasma samples from patients with Zika infections associated or not with GBS in Salvador, Brazil. We have observed that Zika patients that develop GBS present higher levels of anti-ganglioside antibodies when compared to Zika patients without GBS. We also observed that a broad repertoire of gangliosides was targeted by both IgM and IgG anti-self antibodies in these patients. Since Zika virus infects neurons, which contain membrane gangliosides, antigen presentation of these infected cells may trigger the observed autoimmune anti-ganglioside antibodies suggesting direct infection-induced autoantibodies as a cause leading to GBS development. Collectively, our results establish a link between anti-ganglioside antibodies and Zika-associated GBS in patients.

Malaria is a highly inflammatory disease caused by Plasmodium infection of host erythrocytes. However, the parasite does not induce inflammatory cytokine responses in macrophages in vitro and the source of inflammation in patients remains unclear. Here, we identify oxidative stress, which is common in malaria, as an effective trigger of the inflammatory activation of macrophages. We observed that extracellular reactive oxygen species (ROS) produced by xanthine oxidase (XO), an enzyme upregulated during malaria, induce a strong inflammatory cytokine response in primary human monocyte-derived macrophages. In malaria patients, elevated plasma XO activity correlates with high levels of inflammatory cytokines and with the development of cerebral malaria. We found that incubation of macrophages with plasma from these patients can induce a XO-dependent inflammatory cytokine response, identifying a host factor as a trigger for inflammation in malaria. XO-produced ROS also increase the synthesis of pro-IL-1β, while the parasite activates caspase-1, providing the two necessary signals for the activation of the NLRP3 inflammasome. We propose that XO-produced ROS are a key factor for the trigger of inflammation during malaria.

A high-throughput screening (HTS) campaign was carried out for Trypanosoma cruzi glucokinase (TcGlcK), a potential drug-target of the pathogenic protozoan parasite. Glycolysis and the pentose phosphate pathway (PPP) are important metabolic pathways for T. cruzi and the inhibition of the glucose kinases (i.e. glucokinase and hexokinase) may be a strategic approach for drug discovery. Glucose kinases phosphorylate d-glucose with co-substrate ATP to yield G6P, and moreover, the produced G6P enters both pathways for catabolism. The TcGlcK - HTS campaign revealed 25 novel enzyme inhibitors that were distributed in nine chemical classes and were discovered from a primary screen of 13,040 compounds. Thirteen of these compounds were found to have low micromolar IC₅₀ enzyme - inhibition values; strikingly, four of those compounds exhibited low toxicity towards NIH-3T3 murine host cells and notable in vitro trypanocidal activity. These compounds were of three chemical classes: (a) the 3-nitro-2-phenyl-2H-chromene scaffold, (b) the N-phenyl-benzenesulfonamide scaffold, and (c) the gossypol scaffold. Two compounds from the 3-nitro-2-phenyl-2H-chromene scaffold were determined to be hit-to-lead candidates that can proceed further down the early-stage drug discovery process.

Lapatinib, an approved epidermal growth factor receptor inhibitor, was explored as a starting point for the synthesis of new hits against Trypanosoma brucei, the causative agent of human African trypanosomiasis (HAT). Previous work culminated in 1 (NEU-1953), which was part of a series typically associated with poor aqueous solubility. In this report, we present various medicinal chemistry strategies that were used to increase the aqueous solubility and improve the physicochemical profile without sacrificing antitrypanosomal potency. To rank trypanocidal hits, a new assay (summarized in a cytocidal effective concentration (CEC₅₀)) was established, as part of the lead selection process. Increasing the sp³ carbon content of 1 resulted in 10e (0.19 μM EC₅₀ against T. brucei and 990 μM aqueous solubility). Further chemical exploration of 10e yielded 22a, a trypanocidal quinolinimine (EC₅₀: 0.013 μM; aqueous solubility: 880 μM; and CEC₅₀: 0.18 μM). Compound 22a reduced parasitemia 10⁹ fold in trypanosome-infected mice; it is an advanced lead for HAT drug development.

Cerebral malaria (CM) is a clinical syndrome involving irreversible and lethal signs of brain injury associated to infection by parasites of the genus Plasmodium. The pathogenesis of CM derives from infection-induced proinflammatory cytokines associated with cytoadherence of parasitized red blood cells to brain microvasculature. Glycoconjugates are very abundant in the surface of Plasmodium spp., and are critical mediators of parasite virulence in host-pathogen interactions. Herein, we show that 6-Diazo-5-oxo-L-norleucine (DON) therapeutically used for blocking hexosamine biosynthetic pathway leads to recovery in experimental murine cerebral malaria. DON-induced protection was associated with decreased parasitism, which severely reduced Plasmodium transmission to mosquitoes. These findings point to a potential use of DON in combination therapies against malaria.