Babesia microti poses a significant threat to human health, underscoring the need for an improved in vitro culture system to reduce reliance on animal models and support drug and vaccine screening. Key challenges include the tropism of preferential host red blood cells (RBCs), parasite metabolic needs, culture medium formulation, and optimization of the microaerophilic environment (oxygenation).

Abstract Red blood cell (RBC) transfusions remain a life-saving therapy for patients with sickle cell disease (SCD), yet their safety and efficacy are limited by a high incidence of alloimmunization. Emerging evidence suggests that metabolites, including amino acids, can influence humoral immune responses. Here, we found that levels of l-asparagine (Asn), a metabolite previously implicated in immunoregulation, are increased in both plasma and erythroid cells in SCD mice. Reciprocal transfusion indicated that circulating erythroid cells contribute to plasma Asn levels, as transfusion of normal RBCs into SCD mice reduced plasma Asn, whereas transfusion of SCD erythroid cells into wild-type mice increased plasma Asn. We further observed that increased mitochondria-positive (Mito+) erythroid cells in SCD were associated with enhanced Asn synthesis. Functionally, depletion of Asn using asparaginase (ASNase) reduced RBC alloimmunization in SCD mice, albeit more strongly than in control mice, whereas exogenous Asn administration increased alloimmunization in wild-type mice. In SCD mice, treatment with ASNase was associated with inhibition of plasma cell differentiation while sparing resting B cells. In vitro studies revealed that ASNase inhibited, whereas Asn supplementation promoted, human B-cell differentiation, with a more pronounced effect in cells from patients with SCD. ASNase treatment was also associated with reducing Src family kinase (SFK) activation in SCD, suggesting a potential link between Asn and SCD B-cell signaling. Together, these findings point to a potential role for Mito+ erythroid cell Asn-SFK axis in regulating RBC alloimmunization in SCD. Modulation of this pathway may provide a basis for future therapeutic exploration.

Human babesiosis is caused by four Babesia spp., which are intraerythrocytic Apicomplexan zoonotic parasites that, in the last 50 years, have contributed to establishing the parasitic infection as a significant transfusion-transmitted infectious threat and an escalating public-health problem. Of the four species that infect humans, B. divergens was the first to be grown in vitro, via continuous culture in human RBCs. In this chapter, we have comprehensively outlined the basic and necessary tools, protocols, and methodology to successfully grow and study the parasite. Details of in vitro culturing using human serum and Albumax, cryopreservation and thawing, subculturing, maintenance of cultures over extended times, calculation of parasite load using the traditional light microscopy method, and flow cytometry have been elaborated upon. This chapter can serve as standard operating procedures (SOP) for laboratories working on Babesia research and those that want to establish in vitro culturing of B. divergens field isolates, as well as related parasites.

ABSTRACT:Sickle cell nephropathy (SCN) is a major clinical complication in sickle cell disease (SCD), yet its underlying mechanisms remain incompletely defined. Hemolysis, a hallmark of SCD, has been implicated in SCN pathogenesis, but the downstream inflammatory pathways are not fully understood. We previously demonstrated that hemolysis triggers type I interferon (IFN-I) responses, leading to the upregulation of the C-C motif chemokine ligand 2 (CCL2) and recruitment of classical monocytes that differentiate into monocyte-derived macrophages (MoMϕ) within livers in SCD. In this study, we show that IFN-I and CCL2 levels are elevated in the plasma of patients with SCD with abnormal urine albumin-to-creatinine ratio and in the kidneys of the SCD Townes mouse model. Using IFN-I receptor (Ifnar1)-/- and CCL2 receptor (Ccr2)-/- mouse models of SCD, we demonstrate that the loss of IFN-I or CCL2 signaling reduces MoMϕ accumulation, renal inflammation, and renal injury. Mechanistically, we identify that hemin-induced IFN-I production occurs via the Toll-like receptor 3 (TLR3)/TIR-domain-containing adapter-inducing interferon-β (TRIF) signaling axis, independent of MyD88, MAVS, or STING. These findings uncover a previously unrecognized heme-TLR3/TRIF-IFN-I-CCL2 pathway that contributes to renal pathology in SCD and suggest that targeting this axis may offer therapeutic benefit.

Babesiosis in sickle cell disease (SCD) is marked by severe anemia but the underlying red blood cell (RBC) rheological parameters remain largely undefined. Here, we describe altered RBC deformability from both primary (host RBC sickle hemoglobin mediated) and secondary changes (Babesia parasite infection mediated) to the RBC membrane using wild type AA, sickle trait AS and sickle SS RBCs. Our ektacytometry (LORRCA) analysis demonstrates that the changes in the host RBC bio-mechanical properties, pre- and post- Babesia infection, reside on a spectrum of severity, with wild type infected AA cells, despite showing a significant reduction of deformability under both shear and osmolarity gradients, exhibiting only a mild phenotype; compared to infected AS RBCs which show median changes in deformability and infected SS RBCs which exhibit the most dramatic impact of infection on cellular rheology, including an increase in Point of Sickling values. Further, using Image stream cytometric technology to quantify changes in cellular shape and area along with a tunable resistive pulse sensor to measure release of extra-cellular vesicles (EV) from these host RBCs, before and after infection, we offer a potential mechanistic basis for this extreme SS RBC rheologic profile, which include enhanced sickling rates and osmotic fragility, loss of RBC surface area and hypervesiculation in infected SS host RBCs. These results underly the importance of understanding the impact of intra-erythrocytic parasitic infections of SCD RBCs, especially on their cellular membranes and studying the mechanisms that lead to hyper hemolysis and extreme anemia in the SCD patient population.

Purpose of review Malaria and babesiosis are important transfusion-transmitted diseases, therefore, it is important to report novel insights into the complex interactions the causative parasites share with their common host RBCs. Metabolomics is an important tool that can be used to reveal an in-depth analysis of parasite infections in the context of the host. Similarities and differences in the biochemical fingerprints between malaria and babesia infected RBCs are reviewed with potential reasons for these differences and implications for the host. Recent findings Recent results from Babesia-infected RBCs offer an opportunity to develop comparative models of pathogenesis for both infections. Perturbation in the levels of key biomolecules including sugars, amino-acids and lipids, along with redox homeostasis, and heme utilization, are hallmarks of both diseases. Key similarities include enhanced glycolytic rate in both infected RBCs together with lipid scavenging from RBC membranes. Differences relate to hemoglobin breakdown and the use of resultant amino acids for propagation. Summary Altered metabolic profiles reflect the unique lifecycles of Plasmodium and Babesia, pointing to how they carve out a niche for successful proliferation. A comprehensive understanding of the metabolic similarities and differences between the two parasites will aid in identifying new biomarkers as well as specific, effective targeted therapies.

Newly produced platelets acquire a low activation state, but whether the megakaryocyte plays a role in this outcome has not been fully uncovered. Mesenchymal stem cells (MSCs) were previously shown to promote platelet production and lower platelet activation. We found that healthy megakaryocytes transfer mitochondria to MSCs, which is mediated by connexin 43 (Cx43) gap junctions on MSCs and leads to platelets at a low energetic state with increased LYN activation, characteristic of resting platelets with increased LYN activation, characteristic of resting platelets. On the contrary, MSCs have a limited ability to transfer mitochondria to megakaryocytes. Sickle cell disease (SCD) is characterized by hemolytic anemia and results in heightened platelet activation, contributing to numerous disease complications. Platelets in SCD mice and human samples had a heightened energetic state with increased glycolysis. MSC exposure to heme in SCD led to decreased Cx43 expression and a reduced ability to uptake mitochondria from megakaryocytes. This prevented LYN activation in platelets and contributed to increased platelet activation at steady state. Altogether, our findings demonstrate an effect of hemolysis in the microenvironment leading to increased platelet activation in SCD. These findings have the potential to inspire new therapeutic targets to relieve thrombosis-related complications of SCD and other hemolytic conditions.

ABSTRACT:The pathophysiology of sickle cell disease (SCD) is characterized by hemolytic anemia and vaso-occlusion, although its impact on the adaptive immune responses remains incompletely understood. To comprehensibly profile the humoral immune responses, we immunized SCD mice with T-cell-independent (TI) and T-cell-dependent (TD) antigens (Ags). Our study showed that SCD mice have significantly enhanced type 2 TI (TI-2) immune responses in a manner dependent on the level of type I interferons (IFN-I), while maintaining similar or decreased TD immune responses depending on the route of Ag administration. Consistent with the enhanced TI-2 immune responses in SCD mice, the frequencies of B-1b cells (B-1 cells in humans), a major cell type responding to TI-2 Ags, were significantly increased in both the peritoneal cavity and spleens of SCD mice and in the blood of patients with SCD. In support of expanded B-1 cells, elevated levels of anti-red blood cell (anti-RBC) autoantibodies were detected in both SCD mice and patients. Both the levels of TI-2 immune responses and anti-RBC autoantibodies were significantly reduced after IFN-I receptor (IFNAR) antibody blockades and in IFNAR1-deficient SCD mice. Moreover, the alterations of B-1 cell subsets were reversed in IFNAR1-deficient SCD mice, uncovering a critical role for IFN-I in the enhanced TI-2 immune responses and the increased production of anti-RBC autoantibodies by modulating the innate B-1 cell subsets in SCD. Overall, our study provides experimental evidence that the modulation of B-1 cells and IFN-I can regulate TI immune responses and the levels of anti-RBC autoantibodies in SCD.

Free hemin released during intravascular hemolysis induces inflammatory responses and plays a pivotal role in the pathogenesis of various hemolytic disorders, including sickle cell disease (SCD). Several strategies including the use of hemopexin have been developed to eliminate or inactivate the pro-inflammatory effects of free hemin. Compared to biologic drugs, small molecule drugs are more affordable, which is critical for the SCD patient cohort. Several hemin binding small molecule drugs, including quinine derivatives, have been developed and approved by FDA as malaria therapies, but it is unclear whether they can alleviate the detrimental effects of free heme and be used as a therapeutic for SCD. These approved drugs have been extensively studied for safety, pharmacokinetics and targets, which will substantially reduce the effort required for anti-hemolysis drug development. Because in a recent publication (Pal et al, Blood, 2021), quinine demonstrated immunologic bioactivity against B cells after binding with free heme, we selected quinine as the first candidate. Using human primary monocytes, we found that quinine can inhibit hemin-mediated inflammatory cytokine production. Specifically, we measured the levels of IL-1β, IL-6, and TNF-α produced by human monocytes treated with medium, LPS alone, LPS plus hemin (25µM) or quinine alone (25µM), and LPS plus hemin (25µM) mixed with 2.5-25µM quinine for 4 hours. We found increased IL-1β (~3.6 fold) and TNF-α (~1.5 fold) production in LPS plus hemin treatment compared to LPS alone treatment but no change on IL-6 production (quinine alone treatment had no effect). However, adding quinine to hemin led to a dose-dependent inhibition of all three cytokines, such that TNF-α was reversed to the level to the LPS alone group while IL-1β and IL-6 levels were inhibited below the level of the LPS alone group (IL-1β:15% of LPS alone, IL-6: 36% of LPS alone), indicating a novel anti-inflammatory activity of the quinine hemin compound (QHC). To test the potential of quinine binding free heme in vivo and exhibiting anti-inflammatory activities in hemolytic diseases, we treated SCD mice and control mice with quinine (I.P., 2 weeks) using a dose (5mg/kg BW) lower than normally used (>20mg/kg BW). We found quinine treatment (I.P., 2 weeks) decreased plasma levels of IL-1β, IFN-β, IL-6, and TNF-α, and reduced liver tissue damage in SCD mice, while it had no effect on control mice. These results demonstrated the anti-inflammatory activity of quinine in hemolytic conditions, and the mechanism needed further studies to elucidate. Our data suggest that QHC can inhibit the production of multiple inflammatory cytokines. We decided to focus on the mechanism of QHC inhibiting IL-1β production because it is specifically controlled by the inflammasome pathway. Using ATP, an NLRP3 inflammasome agonist, treatment on human monocytes as the model, we found that QHC (2.5µM), but not hemin alone or quinine alone, reduced >80% IL-1β production and ~50% cell death induced by ATP through inhibiting Caspase 1 and GASDMD activation. Similar results were obtained using other NLRP3 agonists, NLRC4 agonist, AIM2 agonist, and non-canonical inflammasome agonist. These results indicate that QHC is a broad-spectrum inhibitor of inflammasome activation. In addition to quinine, we tested the effect of combination hemin with chloroquine, amodiaquine, and dihydroartemisinin on IL-1β, and no effect was identified at the concentration of 2.5 µM. The results indicates that specific molecular space structure of QHC is required for the activity. In vivo, QHC (1.25mg/kg BW, I.P.), but not hemin or quinine alone, inhibited ~70% of IL-1β production and ~50% of neutrophil infiltration in peritoneal lavage in a classic alum crystal I.P. injection inflammasome activation mouse model. alum crystal I.P. injection can cause ~70% lethality in SCD mice. Pre-treating SCD mice with QHC (1.25mg/kg BW, I.P.) protected 100% of the mice from alum-induced lethality, while the same dose of hemin alone or quinine alone showed no effects. Our data support that QHC inhibits inflammasome activation both in vitro and in vivo. In summary, our data support the use of hemin-binding small molecules as potential therapeutics for SCD and other inflammasome activated diseases.

Sickle cell disease (SCD) is a hereditary hemoglobinopathy characterized by painful vaso-occlusive crises (VOC) and chronic hemolysis. The mononuclear phagocyte system is pivotal to SCD pathophysiology, but the mechanisms governing monocyte/macrophage differentiation remain unknown. This study examined the influence of hemolysis on circulating monocyte trajectories in SCD. We discovered that hemolysis stimulated CSF-1 production, partly by endothelial cells via Nrf2, promoting classical monocyte (CMo) differentiation into blood patrolling monocytes (PMo) in SCD mice. However, hemolysis also upregulated CCL-2 through IFN-I, inducing CMo transmigration and differentiation into tissue monocyte-derived macrophages. Blocking CMo transmigration by anti-P selectin antibody in SCD mice increased circulating PMo, corroborating that CMo-to-tissue macrophage differentiation occurs at the expense of CMo-to-blood PMo differentiation. We observed a positive correlation between plasma CSF-1/CCL-2 ratios and blood PMo levels in patients with SCD, underscoring the clinical significance of these two opposing factors in monocyte differentiation. Combined treatment with CSF-1 and anti-P selectin antibody more effectively increased PMo numbers and reduced stasis compared with single-agent therapies in SCD mice. Altogether, these data indicate that monocyte fates are regulated by the balance between two heme pathways, Nrf2/CSF-1 and IFN-I/CCL-2, and suggest that the CSF-1/CCL-2 ratio may present a diagnostic and therapeutic target in SCD.

Disordered erythropoiesis is a feature of many hematologic diseases, including sickle cell disease (SCD). However, very little is known about erythropoiesis in SCD. Here, we show that although bone marrow (BM) erythroid progenitors and erythroblasts in Hbb(th3/+) thalassemia mice were increased more than twofold, they were expanded by only similar to 40% in Townes sickle mice (SS). We further show that the colony-forming ability of SS erythroid progenitors was decreased and erythropoietin (EPO)/EPO receptor (EPOR) signaling was impaired in SS erythroid cells. Furthermore, SS mice exhibited reduced responses to EPO. Injection of mice with red cell lysates or hemin, mimicking hemolysis in SCD, led to suppression of erythropoiesis and reduced EPO/EPOR signaling, indicating hemolysis, a hallmark of SCD, and could contribute to the impaired erythropoiesis in SCD. In vitro hemin treatment did not affect Stat5 phosphorylation, suggesting that hemin-induced erythropoiesis suppression in vivo is via an indirect mechanism. Treatment with interferon alpha (IFN alpha), which is upregulated by hemolysis and elevated in SCD, led to suppression of mouse BM erythropoiesis in vivo and human erythropoiesis in vitro, along with inhibition of Stat5 phosphorylation. Notably, in sickle erythroid cells, IFN-1 signaling was activated and the expression of cytokine inducible SH2-containing protein (CISH), a negative regulator of EPO/EPOR signaling, was increased. CISH deletion in human erythroblasts partially rescued IFN alpha-mediated impairment of cell growth and EPOR signaling. Knocking out Ifnar1 in SS mice rescued the defective BM erythropoiesis and improved EPO/EPOR signaling. Our findings identify an unexpected role of hemolysis on the impaired erythropoiesis in SCD through inhibition of EPO/EPOR signaling via a heme-IFN alpha-CISH axis.

Human babesiosis is caused by apicomplexan parasites of the Babesia genus and is associated with transfusion-transmitted illness and relapsing disease in immunosuppressed populations. Through its continuous cycles of invasion, proliferation, and egress, B. divergens radically changes the metabolic environment of the host red blood cell, allowing us opportunities to study potential chemical vulnerabilities that can be targeted by drugs. ABSTRACT Babesia divergens is an apicomplexan parasite that infects human red blood cells (RBCs), initiating cycles of invasion, replication, and egress, resulting in extensive metabolic modification of the host cells. Babesia is an auxotroph for most of the nutrients required to sustain these cycles. There are currently limited studies on the biochemical pathways that support these critical processes, necessitating the high-resolution global metabolomics approach described here to uncover the metabolic interactions between parasite and host RBC. Our results reveal an extensive parasite-mediated modulation of RBC metabolite levels of all classes, including lipids, amino acids, carbohydrates, and nucleotides, with numerous metabolic species varying in proportion to the level of infection. Many of these molecules are scavenged from the host RBCs. This is in accord with the needs of a rapidly proliferating parasite with limited biosynthetic capabilities. Probing these pathways in depth, we used growth inhibition assays to quantitate parasite susceptibility to drugs targeting these pathways and stimulated emission depletion (STED) microscopy to obtain high-resolution images of drug-treated parasites to correlate changes in morphology with specific metabolic blocks in order to validate the data generated by the untargeted metabolomics platform. Thus, interruption of cholesterol scavenging from the host cell led to premature parasite egress, while chemical targeting of the hydrolysis of acyl glycerides led to the buildup of malformed parasites that could not successfully egress. This is the first report detailing the global metabolomic profile of the B. divergens-infected RBC. Besides deciphering diverse aspects of the host-parasite relationship, our results can be exploited by others to uncover further drug targets in the host-parasite biochemical network. IMPORTANCE Human babesiosis is caused by apicomplexan parasites of the Babesia genus and is associated with transfusion-transmitted illness and relapsing disease in immunosuppressed populations. Through its continuous cycles of invasion, proliferation, and egress, B. divergens radically changes the metabolic environment of the host red blood cell, allowing us opportunities to study potential chemical vulnerabilities that can be targeted by drugs. This is the first global metabolomic profiling of Babesia-infected human red blood cells, and our analysis revealed perturbation in all biomolecular classes at levels proportional to the level of infection. In particular, lipids and energy flux pathways in the host cell were altered by infection. We validated the changes in key metabolic pathways by performing inhibition assays accompanied by high-resolution microscopy. Overall, this global metabolomics analysis of Babesia-infected red blood cells has helped to uncover novel aspects of parasite biology and identified potential biochemical pathways that can be targeted for chemotherapeutic intervention.

Sickle red blood cells (RBCs) represent a naturally existing host-cell resistance mechanism to hemoparasite infections. We investigate the basis of this resistance using Babesia divergens grown in sickle (SS) and sickle trait (AS) cells. We found that oxygenation and its corresponding effect on RBC sickling, frequency of fetal hemoglobin positive (HbF+) cells, cellular redox environment, and parasite proliferation dynamics, all played a role in supporting or inhibiting Babesia proliferation. To identify cellular determinants that supported infection, an image flow cytometric tool was developed that could identify sickled cells and constituent Hb. We showed that hypoxic conditions impaired parasite growth in both SS and AS cells. Furthermore, cell sickling was alleviated by oxygenation (hyperoxic conditions), which decreased inhibition of parasite proliferation in SS cells. Interestingly, our tool identified HbF+-SS as host-cells of choice under both hypoxic and hyperoxic conditions, which was confirmed using cord RBCs containing high amounts of HbF+ cells. Uninfected SS cells showed a higher reactive oxygen species-containing environment, than AA or AS cells, which was further perturbed on infection. In hostile SS cells we found that Babesia alters its subpopulation structure, with 1N dominance under hypoxic conditions yielding to equivalent ratios of all parasite forms at hyperoxic conditions, favorable for growth. Multiple factors, including oxygenation and its impact on cell shape, HbF positivity, redox status, and parasite pleiotropy allow Babesia propagation in sickle RBCs. Our studies provide a cellular and molecular basis of natural resistance to Babesia, which will aid in defining novel therapies against human babesiosis.

Babesiosis is a zoonosis and an important blood-borne human parasitic infection that has gained attention because of its growing infection rate in humans by transfer from animal reservoirs. Babesia represents a potential threat to the blood supply because asymptomatic infections in man are common, and blood from such donors can cause severe disease in certain recipients. Extracellular vesicles (EVs) are vesicles released by cells that contain a complex mixture of proteins, lipids, glycans, and genetic information that have been shown to play important roles in disease pathogenesis and susceptibility, as well as cell–cell communication and immune responses. In this article, we report on the identification and characterization of EVs released from red blood cells (RBCs) infected by two major human Babesia species—Babesia divergens from in vitro culture and those from an in vivo B. microti mouse infection. Using nanoparticle tracking analysis, we show that there is a range of vesicle sizes from 30 to 1,000 nm, emanating from the Babesia-infected RBC. The study of these EVs in the context of hemoparasite infection is complicated by the fact that both the parasite and the host RBC make and release vesicles into the extracellular environment. However, the EV frequency is 2- to 10-fold higher in Babesia-infected RBCs than uninfected RBCs, depending on levels of parasitemia. Using parasite-specific markers, we were able to show that ~50%–60% of all EVs contained parasite-specific markers on their surface and thus may represent the specific proportion of EVs released by infected RBCs within the EV population. Western blot analysis on purified EVs from both in vivo and in vitro infections revealed several parasite proteins that were targets of the host immune response. In addition, microRNA analysis showed that infected RBC EVs have different microRNA signature from uninfected RBC EVs, indicating a potential role as disease biomarkers. Finally, EVs were internalized by other RBCs in culture, implicating a potential role for these vesicles in cellular communication. Overall, our study points to the multiple functional implications of EVs in Babesia–host interactions and support the potential that EVs have as agents in disease pathogenesis.

Red blood cells (RBCs) generated ex vivo have the potential to be used for transfusion. Human embryonic stem cells (ES) and induced pluripotent stem cells (iPS) possess unlimited self-renewal capacity and are the preferred cell sources to be used for ex vivo RBC generation. However, their applications are hindered by the facts that the expansion of ES/iPS-derived erythroid cells is limited and the enucleation of ES/iPS-derived erythroblasts is low compared to that derived from cord blood (CB) or peripheral blood (PB). To address this, we sought to investigate the underlying mechanisms by comparing the in vitro erythropoiesis profiles of CB CD34 + and ES CD34 + cells. We found that the limited expansion of ES CD34 + cell-derived erythroid cells was associated with defective cell cycle of erythroid progenitors. In exploring the cellular and molecular mechanisms for the impaired enucleation of ES CD34 + cell-derived orthochromatic erythroblasts (ES-ortho), we found the chromatin of ES-ortho was less condensed than that of CB CD34 + cell-derived orthochromatic erythroblasts (CB-ortho). At the molecular level, both RNA-seq and ATAC-seq analyses revealed that pathways involved in chromatin modification were down-regulated in ES-ortho. Additionally, the expression levels of molecules known to play important role in chromatin condensation or/and enucleation were significantly lower in ES-ortho compared to that in CB-ortho. Together, our findings have uncovered mechanisms for the limited expansion and impaired enucleation of ES CD34 + cell-derived erythroid cells and may help to improve ex vivo RBC production from stem cells.

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.

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.

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.

Babesia divergens is an obligate intracellular protozoan parasite that causes zoonotic disease. Central to its pathogenesis is the ability of the parasite to invade host red blood cells of diverse species, and, once in the host blood stream, to manipulate the composition of its population to allow it to endure unfavorable conditions. Here we will review key in vitro studies relating to the survival strategies that B. divergens adopts during its intraerythrocytic development to persist and how proliferation is restored in the parasite population once optimum conditions return.