Type I interferon is induced by hemolysis and drives antibody-mediated erythrophagocytosis in sickle cell disease.

Patients with sickle cell disease (SCD) suffer from intravascular hemolysis-associated vascular injury and tissue damage. Classical monocytes (CMo), which are the most abundant of circulating monocytes, are activated in SCD, but the cause and consequences of activation remain incompletely understood. We found a positive correlation between total plasma heme levels and circulating interferon-α (IFN-α) in patients with SCD along with upregulation of the type I IFN (IFN-I) inducible genes in sort-purified SCD patients' CMo by transcriptome analysis. We demonstrated that hemolysis led to IFN-I expression, predominantly by mouse liver monocyte and macrophages (Mⲫ), primarily through Tank kinase binding 1 (TBK1)/IκB kinase-ε (IKKε) but not TLR4. In response to hemolysis-induced IFN-I, mouse CMo migrated to the liver and differentiated into monocyte-derived Mⲫ, increasing their numbers by sixfold with acute hemin treatment. Hemolysis-driven IFN-I activity also led to the induction of Fc receptor CD64 expression on monocyte and Mⲫ populations, enhancing alloantibody-mediated erythrophagocytosis in SCD both in vivo in mice and in in vitro human cultures. Altogether, these data demonstrate IFN-I response to hemolysis as a novel activation pathway in monocytes and Mⲫ in SCD, opening the possibility for development of IFN-I-based diagnostics and therapeutics against alloantibody-mediated erythrophagocytosis.

Hemolysis inhibits humoral B-cell responses and modulates alloimmunization risk in patients with sickle cell disease.

Red blood cell alloimmunization remains a barrier for safe and effective transfusions in sickle cell disease (SCD), but the associated risk factors remain largely unknown. Intravascular hemolysis, a hallmark of SCD, results in the release of heme with potent immunomodulatory activity, although its effect on SCD humoral response, specifically alloimmunization, remains unclear. Here, we found that cell-free heme suppresses human B-cell plasmablast and plasma cell differentiation by inhibiting the DOCK8/STAT3 signaling pathway, which is critical for B-cell activation, as well as by upregulating heme oxygenase 1 (HO-1) through its enzymatic byproducts, carbon monoxide and biliverdin. Whereas nonalloimmunized SCD B cells were inhibited by exogenous heme, B cells from the alloimmunized group were nonresponsive to heme inhibition and readily differentiated into plasma cells. Consistent with a differential B-cell response to hemolysis, we found elevated B-cell basal levels of DOCK8 and higher HO-1-mediated inhibition of activated B cells in nonalloimmunized compared with alloimmunized SCD patients. To overcome the alloimmunized B-cell heme insensitivity, we screened several heme-binding molecules and identified quinine as a potent inhibitor of B-cell activity, reversing the resistance to heme suppression in alloimmunized patients. B-cell inhibition by quinine occurred only in the presence of heme and through HO-1 induction. Altogether, these data suggest that hemolysis can dampen the humoral B-cell response and that B-cell heme responsiveness maybe a determinant of alloimmunization risk in SCD. By restoring B-cell heme sensitivity, quinine may have therapeutic potential to prevent and inhibit alloimmunization in SCD patients.

Human babesiosis: recent advances and future challenges.

PURPOSE OF REVIEW: As human babesiosis caused by apicomplexan parasites of the Babesia genus is associated with transfusion-transmitted illness and relapsing disease in immunosuppressed populations, it is important to report novel findings relating to parasite biology that may be responsible for such pathology. Blood screening tools recently licensed by the FDA are also described to allow understanding of their impact on keeping the blood supply well tolerated. RECENT FINDINGS: Reports of tick-borne cases within new geographical regions such as the Pacific Northwest of the USA, through Eastern Europe and into China are also on the rise. Novel features of the parasite lifecycle that underlie the basis of parasite persistence have recently been characterized. These merit consideration in deployment of both detection, treatment and mitigation tools such as pathogen inactivation technology. The impact of new blood donor screening tests in reducing transfusion transmitted babesiosis is discussed. SUMMARY: New Babesia species have been identified globally, suggesting that the epidemiology of this disease is rapidly changing, making it clear that human babesiosis is a serious public health concern that requires close monitoring and effective intervention measures. Unlike other erythrocytic parasites, Babesia exploits unconventional lifecycle strategies that permit host cycles of different lengths to ensure survival in hostile environments. With the licensure of new blood screening tests, incidence of transfusion transmission babesiosis has decreased.

Altered parasite life-cycle processes characterize Babesia divergens infection in human sickle cell anemia.

Babesia divergens is an intra-erythrocytic parasite that causes malaria-like symptoms in infected people. As the erythrocyte provides the parasite with the infra-structure to grow and multiply, any perturbation to the cell should impact parasite viability. Support for this comes from the multitude of studies that have shown that the sickle trait has in fact been selected because of the protection it provides against a related Apicomplexan parasite, Plasmodium, that causes malaria. In this paper, we examine the impact of both the sickle cell anemia and sickle trait red blood cell (RBC) environment on different aspects of the B. divergens life-cycle, and reveal that multiple aspects of parasite biological processes are altered in the mutant sickle anemia RBC. Such processes include parasite population progression, caused potentially by defective merozoite infectivity and/or defective egress from the sickle cell, resulting in severely lowered parasitemia in these cells with sickle cell anemia. In contrast, the sickle trait RBC provide a supportive environment permitting in vitro infection rates comparable to those of wild-type RBC. The elucidation of these naturally occurring RBC resistance mechanisms is needed to shed light on host-parasite interaction, lend evolutionary insights into these related blood-borne parasites, and to provide new insights into the development of therapies against this disease.

Deletion of a flippase subunit Tmem30a in hematopoietic cells impairs mouse fetal liver erythropoiesis.

Transmembrane protein 30A (Tmem30a) is the ß-subunit of P4-ATPases which function as flippase that transports aminophospholipids such as phosphatidylserine from the outer to the inner leaflets of the plasma membrane to maintain asymmetric distribution of phospholipids. It has been documented that deficiency of Tmem30a led to exposure of phosphatidylserine. However, the role of Tmem30a in vivo remains largely unknown. Here we found that Vav-Cre-driven conditional deletion of Tmem30a in hematopoietic cells led to embryonic lethality due to severe anemia by embryonic day 16.5. The numbers of erythroid colonies and erythroid cells were decreased in the Tmem30a deficient fetal liver. This was accompanied by increased apoptosis of erythroid cells. Confocal microscopy analysis revealed an increase of localization of erythropoietin receptor to areas of membrane raft microdomains in response to erythropoietin stimulation in Ter119-erythroid progenitors, which was impaired in Tmem30a deficient cells. Moreover, erythropoietin receptor (EPOR)-mediated activation of the STAT5 pathway was significantly reduced in Tmem30a deficient fetal liver cells. Consistently, knockdown of TMEM30A in human CD34+ cells also impaired erythropoiesis. Our findings demonstrate that Tmem30a plays a critical role in erythropoiesis by regulating the EPOR signaling pathway through the formation of membrane rafts in erythroid cells.

A novel flow cytometric application discriminates among the effects of chemical inhibitors on various phases of Babesia divergens intraerythrocytic cycle.

Human babesiosis is a global emerging infectious disease caused by intraerythrocytic parasites of the genus Babesia. Its biology has remained largely unexplored due to a lack of critical tools and techniques required to define the various stages and phases of the parasite's cycle in its host RBC and the interplay between host and parasite. This article presents a powerful set of tools combining stage synchronization of the parasite with a platform that encompasses both a flow cytometric evaluation of the subpopulation structure of the parasite population together with a morphological assessment of the population parasites using light microscopy of conventional Giemsa stained smears. Together, these yield specific information on the effect of any drug/condition of interest and its targeted biological process, allowing the characterization of the adaptive response of parasites to a particular stressor agent. Three inhibitors were used in this study, each targeting a specific phase of the parasite's lifecycle, neuraminidase for host cell invasion, N-acetyl-L-leucyl-L-leucyl-L-norleucinal for parasite development and EGTA for parasite egress from the host cell. Results presented prove the power of this combination platform in discriminating the specific targets among the life-cycle processes of the parasite-invasion, development/proliferation and egress. This will expand the range of queries that can now be successfully addressed in this parasite, opening avenues for the development of new methods to control babesiosis, either by chemicals (screening for new chemotherapy drugs or defining levels of parasite resistance) or physical methods (light irradiation or heat shock used in pathogen reduction/elimination methods). © 2017 International Society for Advancement of Cytometry.

Babesia divergens builds a complex population structure composed of specific ratios of infected cells to ensure a prompt response to changing environmental conditions.

Babesia parasites cause a malaria-like febrile illness by infection of red blood cells (RBCs). Despite the growing importance of this tick-borne infection, its basic biology has been neglected. Using novel synchronization tools, the sequence of intra-erythrocytic events was followed from invasion through development and differentiation to egress. The dynamics of the parasite population were studied in culture, revealing for the first time, the complete array of morphological forms in a precursor-product relationship. Important chronological constants including Babesia's highly unusual variable intra-erythrocytic life cycle, the life span of each population of infected cells and the time required for the genesis of the different parasite stages were elucidated. Importantly, the maintenance of specific ratios of the infected RBC populations was shown to be responsible for the parasites' choice of developmental pathways, enabling swift responses to changing environmental conditions like availability of RBCs and nutrition. These results could impact the control of parasite proliferation and therefore disease.

Identification and Characterization of the Rhoptry Neck Protein 2 in Babesia divergens and B. microti.

Apicomplexan parasites include those of the genera Plasmodium, Cryptosporidium, and Toxoplasma and those of the relatively understudied zoonotic genus Babesia In humans, babesiosis, particularly transfusion-transmitted babesiosis, has been emerging as a major threat to public health. Like malaria, the disease pathology is a consequence of the parasitemia which develops through cyclical replication of Babesia parasites in host erythrocytes. However, there are no exoerythrocytic stages in Babesia, so targeting of the blood stage and associated proteins to directly prevent parasite invasion is the most desirable option for effective disease control. Especially promising among such molecules are the rhoptry neck proteins (RONs), whose homologs have been identified in many apicomplexan parasites. RONs are involved in the formation of the moving junction, along with AMA1, but no RON has been identified and characterized in any Babesia spp. Here we identify the RON2 proteins of Babesia divergens (BdRON2) and B. microti (BmRON2) and show that they are localized apically and that anti-BdRON2 antibodies are significant inhibitors of parasite invasion in vitro Neither protein is immunodominant, as both proteins react only marginally with sera from infected animals. Further characterization of the direct role of both BdRON2 and BmRON2 in parasite invasion is required, but knowledge of the level of conformity of RON2 proteins within the apicomplexan phylum, particularly that of the AMA1-RON2 complex at the moving junction, along with the availability of an animal model for B. microti studies, provides a key to target this complex with a goal of preventing the erythrocytic invasion of these parasites and to further our understanding of the role of these conserved ligands in invasion.

Babesia: impact of cold storage on the survival and the viability of parasites in blood bags.

BACKGROUND: Babesia represents one of the major infectious threats to the blood supply since clinically silent infections in humans are common and these can be life-threatening in certain recipients. It is important to understand the effect of blood storage conditions on the viability of Babesia as this will impact the occurrence and severity of transfusion-transmitted babesiosis. STUDY DESIGN AND METHODS: Babesia divergens was introduced into blood bags containing leukoreduced red blood cells (RBCs) and stored at 4°C for 0 to 31 days. Samples were withdrawn for assessment of the presence, morphology, and viability of parasites. Blood smears were made immediately on removal from blood bags at different time intervals and evaluated by blood film microscopy. RBCs withdrawn from the bags were also cultured for 8 days using conditions optimal for parasite reproduction and growth to allow assessment of parasite viability. RESULTS: After 24 hours of storage at 4°C, there was a substantial reduction of parasitemia in the blood bags, which was maintained throughout storage. This decrease was accompanied by a change in morphology of parasites, with the number of altered parasites increasing through the period of storage. However, viability was maintained through 31 days of cold storage with a lag in achieving exponential growth seen in the parasites subjected to longer periods of refrigeration. CONCLUSION: Refrigeration of B. divergens leads to an alteration of parasite morphology and a decrease in parasite numbers. However, there are sufficient parasites that are robust enough to survive 31 days of storage at 4°C and yield high end-point parasitemia.

Identification of binding domains on red blood cell glycophorins for Babesia divergens.

BACKGROUND: Invasion of red blood cells (RBCs) is one of the critical points in the lifecycle of Babesia. The parasite does not invade other host cells. Earlier work has shown that GPA and GPB function as putative receptors during parasite invasion. The primary focus of this study was the delineation of parasite-binding domains on GPA and GPB. STUDY DESIGN AND METHODS: The assay of choice to validate molecules that participate in invasion is an inhibition of invasion assay, in which changes in parasitemia are assessed relative to a wild-type assay (no inhibitors). Inhibition of invasion can be achieved by modification of different components of the assay or by the addition of competitors of the molecules that participate in invasion. In this study purified antibody fragments to various domains on GPA and GPB were tested for magnitude of inhibition of parasite invasion. Effects on invasion were monitored by assessment of Giemsa-stained smears every 24 hours. RESULTS: Among 10 selected antibodies directed at various epitopes on GPA and GPB, antibodies directed against GPA(M) epitopes had the most severe effect (up to 35%) on inhibition of invasion, followed by antibodies directed against GPB(S) epitope (up to 24%). CONCLUSION: This study confirms the role of RBC glycophorins A and B in Babesia divergens invasion and shows that the GPA(M) and GPB(S) epitopes are likely to play an important role in the entry process.