Mechanisms of 8-aminoquinoline induced haemolytic toxicity in a G6PDd humanized mouse model.

Primaquine (PQ) and Tafenoquine (TQ) are clinically important 8-aminoquinolines (8-AQ) used for radical cure treatment of P. vivax infection, known to target hepatic hypnozoites. 8-AQs can trigger haemolytic anaemia in individuals with glucose-6-phosphate dehydrogenase deficiency (G6PDd), yet the mechanisms of haemolytic toxicity remain unknown. To address this issue, we used a humanized mouse model known to predict haemolytic toxicity responses in G6PDd human red blood cells (huRBCs). To evaluate the markers of eryptosis, huRBCs were isolated from mice 24-48 h post-treatment and analysed for effects on phosphatidylserine (PS), intracellular reactive oxygen species (ROS) and autofluorescence. Urinalysis was performed to evaluate the occurrence of intravascular and extravascular haemolysis. Spleen and liver tissue harvested at 24 h and 5-7 days post-treatment were stained for the presence of CD169+ macrophages, F4/80+ macrophages, Ter119+ mouse RBCs, glycophorin A+ huRBCs and murine reticulocytes (muRetics). G6PDd-huRBCs from PQ/TQ treated mice showed increased markers for eryptosis as early as 24 h post-treatment. This coincided with an early rise in levels of muRetics. Urinalysis revealed concurrent intravascular and extravascular haemolysis in response to PQ/TQ. Splenic CD169+ macrophages, present in all groups at day 1 post-dosing were eliminated by days 5-7 in PQ/TQ treated mice only, while liver F4/80 macrophages and iron deposits increased. Collectively, our data suggest 8-AQ treated G6PDd-huRBCs have early physiological responses to treatment, including increased markers for eryptosis indicative of oxidative stress, resulting in extramedullary haematopoiesis and loss of splenic CD169+ macrophages, prompting the liver to act as the primary site of clearance.

Validation of the hen's egg test for micronucleus induction (HET-MN): detailed protocol including scoring atlas, historical control data and statistical analysis.

A validation exercise of the hen's egg test for micronucleus induction was finalised with a very good predictivity based on the analysis of micronuclei in peripheral erythrocytes of fertilised chicken eggs (Reisinger et al. The hen's egg test for micronucleus-induction (HET-MN): validation data set. Mutagenesis, this issue). For transparency reasons this complementary publication provides further details on the assay especially as it was the first validation study in the field of genotoxicity testing involving the use of chicken eggs. Thus, the experimental protocol is described in detail and is complemented by a scoring atlas for microscopic analysis in blood cells. In addition, general characteristics of the test system, which is able to mirror the systemic availability of test compounds, are delineated: the test compound passes the egg membrane and is taken up by the blood vessels of the underlying chorioallantoic membrane. Subsequently, it is distributed by the circulating blood, metabolised by the developing liver and the yolk sac membrane and finally excreted into the allantois, a bladder equivalent. In specific, the suitability of the test system for genotoxicity testing is shown by, inter alia, a low background DNA damage in a comprehensive historical control database. In addition, the state-of-the-art statistical method used to evaluate obtained data is delineated. It combines laboratory-specific effect threshold with the Umbrella-Williams test, a statistical model also of interest for other genotoxicity test methods.

The hen's egg test for micronucleus induction (HET-MN): validation data set.

The classical in vitro genotoxicity test battery is known to be sensitive for indicating genotoxicity. However, a high rate of 'misleading positives' was reported when three assays were combined as required by several legislations. Despite the recent optimisations of the standard in vitro tests, two gaps could hardly be addressed with assays based on 2D monolayer cell cultures: the route of exposure and a relevant intrinsic metabolic capacity to transform pro-mutagens into reactive metabolites. Following these considerations, fertilised chicken eggs have been introduced into genotoxicity testing and were combined with a classical read-out parameter, the micronucleus frequency in circulating erythrocytes, to develop the hen's egg test for micronucleus induction (HET-MN). As a major advantage, the test mirrors the systemic availability of compounds after oral exposure by reflecting certain steps of Absorption, Distribution, Metabolism, Excretion (ADME) without being considered as an animal experiment. The assay is supposed to add to a toolbox of assays to follow up on positive findings from initial testing with classical in vitro assays. We here report on a validation exercise, in which >30 chemicals were tested double-blinded in three laboratories. The specificity and sensitivity of the HET-MN were calculated to be 98 and 84%, respectively, corresponding to an overall accuracy of 91%. A detailed protocol, which includes a picture atlas detailing the cell and micronuclei analysis, is published in parallel (Maul et al. Validation of the hen's egg test for micronucleus induction (HET-MN): detailed protocol including scoring atlas, historical control data and statistical analysis).

Lysophosphatidic acid receptor 5 inhibits B cell antigen receptor signaling and antibody response.

Lysophospholipids have emerged as biologically important chemoattractants capable of directing lymphocyte development, trafficking, and localization. Lysophosphatidic acid (LPA) is a major lysophospholipid found systemically, and its levels are elevated in certain pathological settings, such as cancer and infections. In this study, we demonstrate that BCR signal transduction by mature murine B cells is inhibited upon LPA engagement of the LPA5 (GPR92) receptor via a Gα12/13-Arhgef1 pathway. The inhibition of BCR signaling by LPA5 manifests by impaired intracellular calcium store release and most likely by interfering with inositol 1,4,5-triphosphate receptor activity. We further show that LPA5 also limits Ag-specific induction of CD69 and CD86 expression and that LPA5-deficient B cells display enhanced Ab responses. Thus, these data show that LPA5 negatively regulates BCR signaling, B cell activation, and immune response. Our findings extend the influence of lysophospholipids on immune function and suggest that alterations in LPA levels likely influence adaptive humoral immunity.

Development of an ectopic huLiver model for Plasmodium liver stage infection.

Early Plasmodium falciparum and P. vivax infection requires parasite replication within host hepatocytes, referred to as liver stage (LS). However, limited understanding of infection dynamics in human LS exists due to species-specificity challenges. Reported here is a reproducible, easy-to-manipulate, and moderate-cost in vivo model to study human Plasmodium LS in mice; the ectopic huLiver model. Ectopic huLiver tumors were generated through subcutaneous injection of the HC-04 cell line and shown to be infectible by both freshly dissected sporozoites and through the bite of infected mosquitoes. Evidence for complete LS development was supported by the transition to blood-stage infection in mice engrafted with human erythrocytes. Additionally, this model was successfully evaluated for its utility in testing antimalarial therapeutics, as supported by primaquine acting as a causal prophylactic against P. falciparum. Presented here is a new platform for the study of human Plasmodium infection with the potential to aid in drug discovery.

Liver-targeted polymeric prodrugs of 8-aminoquinolines for malaria radical cure.

Primaquine and tafenoquine are the two 8-aminoquinoline (8-AQ) antimalarial drugs approved for malarial radical cure - the elimination of liver stage hypnozoites after infection with Plasmodium vivax. A single oral dose of tafenoquine leads to high efficacy against intra-hepatocyte hypnozoites after efficient first pass liver uptake and metabolism. Unfortunately, both drugs cause hemolytic anemia in G6PD-deficient humans. This toxicity prevents their mass administration without G6PD testing given the approximately 400 million G6PD deficient people across malarial endemic regions of the world. We hypothesized that liver-targeted delivery of 8-AQ prodrugs could maximize liver exposure and minimize erythrocyte exposure to increase their therapeutic window. Primaquine and tafenoquine were first synthesized as prodrug vinyl monomers with self-immolative hydrolytic linkers or cathepsin-cleavable valine-citrulline peptide linkers. RAFT polymerization was exploited to copolymerize these prodrug monomers with hepatocyte-targeting GalNAc monomers. Pharmacokinetic studies of released drugs after intravenous administration showed that the liver-to-plasma AUC ratios could be significantly improved, compared to parent drug administered orally. Single doses of the liver-targeted, enzyme-cleavable tafenoquine polymer were found to be as efficacious as an equivalent dose of the oral parent drug in the P. berghei causal prophylaxis model. They also elicited significantly milder hemotoxicity in the humanized NOD/SCID mouse model engrafted with red blood cells from G6PD deficient donors. The clinical application is envisioned as a single subcutaneous administration, and the lead tafenoquine polymer also showed excellent bioavailability and liver-to-blood ratios exceeding the IV administered polymer. The liver-targeted tafenoquine polymers warrant further development as a single-dose therapeutic via the subcutaneous route with the potential for broader patient administration without a requirement for G6PD diagnosis.

Lsc activity is controlled by oligomerization and regulates integrin adhesion.

Lsc is a hematopoietic-restricted protein that functions as an effector of G alpha(12/13)-associated G-protein coupled receptors that activates RhoA. In the absence of Lsc leukocytes exhibit impaired migration and B lymphocytes inefficiently resolve integrin-mediated adhesion. Here, we demonstrate that Lsc exists physiologically in primary B lymphocytes as a large molecular weight complex resembling a homo-tetramer. Interfering with the assembly of this large molecular weight Lsc oligomer results in the activation of both Lsc functional activities and leads to cell rounding and inhibition of integrin-mediated adhesion. During cell migration on integrin ligands we find Lsc localizes predominantly toward the rear of migrating cells where we suggest it activates RhoA to resolve integin-mediated adhesion. Together these data demonstrate that Lsc regulates integrin-mediated adhesive events at the trailing edge of migrating cells.

Active PI3K abrogates central tolerance in high-avidity autoreactive B cells.

Autoreactive B cells that bind self-antigen with high avidity in the bone marrow undergo mechanisms of central tolerance that prevent their entry into the peripheral B cell population. These mechanisms are breached in many autoimmune patients, increasing their risk of B cell-mediated autoimmune diseases. Resolving the molecular pathways that can break central B cell tolerance could therefore provide avenues to diminish autoimmunity. Here, we show that B cell-intrinsic expression of a constitutively active form of PI3K-P110α by high-avidity autoreactive B cells of mice completely abrogates central B cell tolerance and further promotes these cells to escape from the bone marrow, differentiate in peripheral tissue, and undergo activation in response to self-antigen. Upon stimulation with T cell help factors, these B cells secrete antibodies in vitro but remain unable to secrete autoantibodies in vivo. Overall, our data demonstrate that activation of the PI3K pathway leads high-avidity autoreactive B cells to breach central, but not late, stages of peripheral tolerance.

LPA5 Is an Inhibitory Receptor That Suppresses CD8 T-Cell Cytotoxic Function via Disruption of Early TCR Signaling.

Persistent T cell antigen receptor (TCR) signaling by CD8 T cells is a feature of cancer and chronic infections and results in the sustained expression of, and signaling by, inhibitory receptors, which ultimately impair cytotoxic activity via poorly characterized mechanisms. We have previously determined that the LPA5 GPCR expressed by CD8 T cells, upon engaging the lysophosphatidic acid (LPA) bioactive serum lipid, functions as an inhibitory receptor able to negatively regulate TCR signaling. Notably, the levels of LPA and autotaxin (ATX), the phospholipase D enzyme that produces LPA, are often increased in chronic inflammatory disorders such as chronic infections, autoimmune diseases, obesity, and cancer. In this report, we demonstrate that LPA engagement selectively by LPA5 on human and mouse CD8 T cells leads to the inhibition of several early TCR signaling events including intracellular calcium mobilization and ERK activation. We further show that, as a consequence of LPA5 suppression of TCR signaling, the exocytosis of perforin-containing granules is significantly impaired and reflected by repressed in vitro and in vivo CD8 T cell cytolytic activity. Thus, these data not only document LPA5 as a novel inhibitory receptor but also determine the molecular and biochemical mechanisms by which a naturally occurring serum lipid that is elevated under settings of chronic inflammation signals to suppress CD8 T cell killing activity in both human and murine cells. As diverse tumors have repeatedly been shown to aberrantly produce LPA that acts in an autocrine manner to promote tumorigenesis, our findings further implicate LPA in activating a novel inhibitory receptor whose signaling may be therapeutically silenced to promote CD8 T cell immunity.

Immunogenicity of Structurally Perturbed Hen Egg Lysozyme Adsorbed to Silicone Oil Microdroplets in Wild-Type and Transgenic Mouse Models.

Silicone oil microdroplets may act as adjuvants, promoting unwanted immune responses against both foreign and self-proteins. Proteins often unfold upon adsorption to silicone oil microdroplets, but it is unclear how such unfolding might affect the immune response. In this study, we found that hen egg lysozyme (HEL) readily adsorbed to silicone oil microdroplets and perturbed the conformation of HEL. We compared the immune response to injections of HEL formulated in the presence and absence of silicone oil microdroplets in both wild-type mice and transgenic littermates that express a soluble form of HEL (sHEL), thus rendering them immunologically tolerant to this nominal self-antigen. Following 2 subcutaneous injections of a HEL formulation containing silicone oil microdroplets, wild-type mice exhibited a stronger IgG1 antibody response against HEL compared to the response in wild-type mice that administered an oil-free HEL formulation. However, when HEL was subcutaneously administered to sHEL-transgenic mice, immunological tolerance to sHEL was not broken in the presence of silicone oil microdroplets. Thus, although structural perturbations in proteins adsorbed to silicone oil microdroplets may augment the immune response, in the case of endogenously expressed proteins, such structural perturbations may not be sufficient to result in a breach of immunological tolerance.

Breaching peripheral tolerance promotes the production of HIV-1-neutralizing antibodies.

A subset of characterized HIV-1 broadly neutralizing antibodies (bnAbs) are polyreactive with additional specificities for self-antigens and it has been proposed immunological tolerance may present a barrier to their participation in protective humoral immunity. We address this hypothesis by immunizing autoimmune-prone mice with HIV-1 Envelope (Env) and characterizing the primary antibody response for HIV-1 neutralization. We find autoimmune mice generate neutralizing antibody responses to tier 2 HIV-1 strains with alum treatment alone in the absence of Env. Importantly, experimentally breaching immunological tolerance in wild-type mice also leads to the production of tier 2 HIV-1-neutralizing antibodies, which increase in breadth and potency following Env immunization. In both genetically prone and experimentally induced mouse models of autoimmunity, increased serum levels of IgM anti-histone H2A autoantibodies significantly correlated with tier 2 HIV-1 neutralization, and anti-H2A antibody clones were found to neutralize HIV-1. These data demonstrate that breaching peripheral tolerance permits a cross-reactive HIV-1 autoantibody response able to neutralize HIV-1.

Receptor editing and genetic variability in human autoreactive B cells.

The mechanisms by which B cells undergo tolerance, such as receptor editing, clonal deletion, and anergy, have been established in mice. However, corroborating these mechanisms in humans remains challenging. To study how autoreactive human B cells undergo tolerance, we developed a novel humanized mouse model. Mice expressing an anti-human Igκ membrane protein to serve as a ubiquitous neo self-antigen (Ag) were transplanted with a human immune system. By following the fate of self-reactive human κ(+) B cells relative to nonautoreactive λ(+) cells, we show that tolerance of human B cells occurs at the first site of self-Ag encounter, the bone marrow, via a combination of receptor editing and clonal deletion. Moreover, the amount of available self-Ag and the genetics of the cord blood donor dictate the levels of central tolerance and autoreactive B cells in the periphery. Thus, this model can be useful for studying specific mechanisms of human B cell tolerance and to reveal differences in the extent of this process among human populations.

Lysophosphatidic acid inhibits CD8 T cell activation and control of tumor progression.

CD8 T lymphocytes are able to eliminate nascent tumor cells through a process referred to as immune surveillance. However, multiple inhibitory mechanisms within the tumor microenvironment have been described that impede tumor rejection by CD8 T cells, including increased signaling by inhibitory receptors. Lysophosphatidic acid (LPA) is a bioactive lysophospholipid that has been shown repeatedly to promote diverse cellular processes benefiting tumorigenesis. Accordingly, the increased expression of LPA and LPA receptors is a common feature of diverse tumor cell lineages and can result in elevated systemic LPA levels. LPA is recognized by at least 6 distinct G-protein-coupled receptors and several of which are expressed by T cells, although the precise role of LPA signaling in CD8 T cell activation and function has not been defined. Here, we demonstrate that LPA signaling via the LPA5 receptor expressed by CD8 T cells suppresses antigen receptor signaling, cell activation and proliferation in vitro and in vivo. Importantly, in a mouse melanoma model tumor-specific CD8 T cells that are LPA5-deficient are able to control tumor growth significantly better than wild-type tumor-specific CD8 T cells. Together, these data suggest that the production of LPA by tumors serves not only in an autocrine manner to promote tumorigenesis but also as a mechanism to suppress adaptive immunity and highlights a potential novel target for cancer treatment.

Type I IFN enhances follicular B cell contribution to the T cell-independent antibody response.

Humoral immunity to viruses and encapsulated bacteria is comprised of T cell-independent type 2 (TI-2) antibody responses that are characterized by rapid antibody production by marginal zone and B1 B cells. We demonstrate that toll-like receptor (TLR) ligands influence the TI-2 antibody response not only by enhancing the overall magnitude but also by skewing this response to one that is dominated by IgG isotypes. Importantly, TLR ligands facilitate this response by inducing type I interferon (IFN), which in turn elicits rapid and significant amounts of antigen-specific IgG2c predominantly from FO (follicular) B cells. Furthermore, we show that although the IgG2c antibody response requires B cell-autonomous IFN-alpha receptor signaling, it is independent of B cell-intrinsic TLR signaling. Thus, innate signals have the capacity to enhance TI-2 antibody responses by promoting participation of FO B cells, which then elaborate effective IgG anti-pathogen antibodies.

TLR agonists promote marginal zone B cell activation and facilitate T-dependent IgM responses.

Although IgM serves as a first barrier to Ag spreading, the cellular and molecular mechanisms following B lymphocyte activation that lead to IgM secretion are not fully understood. By virtue of their anatomical location, marginal zone (MZ) B cells rapidly generate Ag-specific IgM in response to blood-borne pathogens and play an important role in the protection against these potentially harmful Ags. In this study, we have explored the contribution of TLR agonists to MZ B cell activation and mobilization as well as their ability to promote primary IgM responses in a mouse model. We demonstrate that diverse TLR agonists stimulate MZ B cells to become activated and leave the MZ through pathways that are differentially dependent on MyD88 and IFN-alphabeta receptor signaling. Furthermore, in vivo stimulation of MZ B cells with TLR agonists led to a reduction in the expression of the sphingosine-1-phosphate (S1P) receptors expressed by MZ B cells and/or increased CD69 cell surface levels. Importantly, as adjuvants for a T cell-dependent protein Ag, TLR agonists were found to accelerate the kinetics but not magnitude of the Ag-specific IgM response. Together, these data demonstrate that in vivo TLR agonist treatment enhances the early production of Ag-specific IgM and activates MZ B cells to promote their relocation.

Lsc regulates marginal-zone B cell migration and adhesion and is required for the IgM T-dependent antibody response.

The humoral immune response to protein antigens is composed of a rapid low-affinity IgM antibody response followed by an IgG response exhibiting higher affinity. Here, we demonstrate that Lsc, a protein that regulates G protein-coupled-receptor signaling and RhoA activation, is required by B lymphocytes for the antigen-specific IgM antibody response to a protein antigen. We further show that in lsc(-/-) mice, MZB cells are selectively affected such that naive and in vivo-activated MZB cells migrate toward sphingosine-1-phosphate at increased proportions but release inefficiently from integrin ligands. Consequently, lsc(-/-) MZB cells do not traffick appropriately in an immune response and do not contribute to the TD antibody response. These data demonstrate that Lsc regulates the migration and adhesion of MZB cells, and this regulation appears to be required for these cells to contribute to the antibody response to TD antigens.