Immune Mechanisms in Inflammatory Anemia.

Maintaining the correct number of healthy red blood cells (RBCs) is critical for proper oxygenation of tissues throughout the body. Therefore, RBC homeostasis is a tightly controlled balance between RBC production and RBC clearance, through the processes of erythropoiesis and macrophage hemophagocytosis, respectively. However, during the inflammation associated with infectious, autoimmune, or inflammatory diseases this homeostatic process is often dysregulated, leading to acute or chronic anemia. In each disease setting, multiple mechanisms typically contribute to the development of inflammatory anemia, impinging on both sides of the RBC production and RBC clearance equation. These mechanisms include both direct and indirect effects of inflammatory cytokines and innate sensing. Here, we focus on common innate and adaptive immune mechanisms that contribute to inflammatory anemias using examples from several diseases, including hemophagocytic lymphohistiocytosis/macrophage activation syndrome, severe malarial anemia during Plasmodium infection, and systemic lupus erythematosus, among others.

The Gut Microbiome Regulates Psychological-Stress-Induced Inflammation.

Psychological stress has adverse effects on various human diseases, including those of the cardiovascular system. However, the mechanisms by which stress influences disease activity remain unclear. Here, using vaso-occlusive episodes (VOEs) of sickle cell disease as a vascular disease model, we show that stress promotes VOEs by eliciting a glucocorticoid hormonal response that augments gut permeability, leading to microbiota-dependent interleukin-17A (IL-17A) secretion from T helper 17 (Th17) cells of the lamina propria, followed by the expansion of the circulating pool of aged neutrophils that trigger VOEs. We identify segmented filamentous bacteria as the commensal essential for the stress-induced expansion of aged neutrophils that enhance VOEs in mice. Importantly, the inhibition of glucocorticoids synthesis, blockade of IL-17A, or depletion of the Th17 cell-inducing gut microbiota markedly reduces stress-induced VOEs. These results offer potential therapeutic targets to limit the impact of psychological stress on acute vascular occlusion.

ZNF410 Uniquely Activates the NuRD Component CHD4 to Silence Fetal Hemoglobin Expression.

Metazoan transcription factors typically regulate large numbers of genes. Here we identify via a CRISPR-Cas9 genetic screen ZNF410, a pentadactyl DNA-binding protein that in human erythroid cells directly activates only a single gene, the NuRD component CHD4. Specificity is conveyed by two highly evolutionarily conserved clusters of ZNF410 binding sites near the CHD4 gene with no counterparts elsewhere in the genome. Loss of ZNF410 in adult-type human erythroid cell culture systems and xenotransplantation settings diminishes CHD4 levels and derepresses the fetal hemoglobin genes. While previously known to be silenced by CHD4, the fetal globin genes are exposed here as among the most sensitive to reduced CHD4 levels.. In vitro DNA binding assays and crystallographic studies reveal the ZNF410-DNA binding mode. ZNF410 is a remarkably selective transcriptional activator in erythroid cells, and its perturbation might offer new opportunities for treatment of hemoglobinopathies.

Iron scavenging and myeloid cell polarization.

Myeloid cells that populate all human organs and blood are a versatile class of innate immune cells. They are crucial for sensing and regulating processes as diverse as tissue homeostasis and inflammation and are frequently characterized by their roles in either regulating or promoting inflammation. Recent studies in cultured cells and mouse models highlight the role of iron in skewing the functional properties of myeloid cells in tissue damage and repair. Here, we review certain emerging concepts on how iron influences and determines myeloid cell polarization in the context of its uptake, storage, and metabolism, including in conditions such as multiple sclerosis (MS), sickle cell disease, and tumors.

The genetic dissection of fetal haemoglobin persistence in sickle cell disease in Nigeria.

The clinical severity of sickle cell disease (SCD) is strongly influenced by the level of fetal haemoglobin (HbF) persistent in each patient. Three major HbF loci (BCL11A, HBS1L-MYB, and Xmn1-HBG2) have been reported, but a considerable hidden heritability remains. We conducted a genome-wide association study for HbF levels in 1006 Nigerian patients with SCD (HbSS/HbSß0), followed by a replication and meta-analysis exercise in four independent SCD cohorts (3,582 patients). To dissect association signals at the major loci, we performed stepwise conditional and haplotype association analyses and included public functional annotation datasets. Association signals were detected for BCL11A (lead SNP rs6706648, ß = -0.39, P = 4.96 × 10-34) and HBS1L-MYB (lead SNP rs61028892, ß = 0.73, P = 1.18 × 10-9), whereas the variant allele for Xmn1-HBG2 was found to be very rare. In addition, we detected three putative new trait-associated regions. Genetically, dissecting the two major loci BCL11A and HBS1L-MYB, we defined trait-increasing haplotypes (P < 0.0001) containing so far unidentified causal variants. At BCL11A, in addition to a haplotype harbouring the putative functional variant rs1427407-'T', we identified a second haplotype, tagged by the rs7565301-'A' allele, where a yet-to-be-discovered causal DNA variant may reside. Similarly, at HBS1L-MYB, one HbF-increasing haplotype contains the likely functional small indel rs66650371, and a second tagged by rs61028892-'C' is likely to harbour a presently unknown functional allele. Together, variants at BCL11A and HBS1L-MYB SNPs explained 24.1% of the trait variance. Our findings provide a path for further investigation of the causes of variable fetal haemoglobin persistence in sickle cell disease.

Sickle Cell Disease: From Genetics to Curative Approaches.

Sickle cell disease (SCD) is a monogenic blood disease caused by a point mutation in the gene coding for ß-globin. The abnormal hemoglobin [sickle hemoglobin (HbS)] polymerizes under low-oxygen conditions and causes red blood cells to sickle. The clinical presentation varies from very severe (with acute pain, chronic pain, and early mortality) to normal (few complications and a normal life span). The variability of SCD might be due (in part) to various genetic modulators. First, we review the main genetic factors, polymorphisms, and modifier genes that influence the expression of globin or otherwise modulate the severity of SCD. Considering SCD as a complex, multifactorial disorder is important for the development of appropriate pharmacological and genetic treatments. Second, we review the characteristics, advantages, and disadvantages of the latest advances in gene therapy for SCD, from lentiviral-vector-based approaches to gene-editing strategies.

High-throughput quantification of red blood cell deformability and oxygen saturation to probe mechanisms of sickle cell disease.

The complex, systemic pathology of sickle cell disease is driven by multiple mechanisms including red blood cells (RBCs) stiffened by polymerized fibers of deoxygenated sickle hemoglobin. A critical step toward understanding the pathologic role of polymer-containing RBCs is quantifying the biophysical changes in these cells in physiologically relevant oxygen environments. We have developed a microfluidic platform capable of simultaneously measuring single RBC deformability and oxygen saturation under controlled oxygen and shear stress. We found that RBCs with detectable amounts of polymer have decreased oxygen affinity and decreased deformability. Surprisingly, the deformability of the polymer-containing cells is oxygen-independent, while the fraction of these cells increases as oxygen decreases. We also find that some fraction of these cells is present at most physiologic oxygen tensions, suggesting a role for these cells in the systemic pathologies. Additionally, the ability to measure these pathological cells should provide clearer targets for evaluating therapies.

SMARCB1 regulates the hypoxic stress response in sickle cell trait.

Renal medullary carcinoma (RMC) is an aggressive kidney cancer that almost exclusively develops in individuals with sickle cell trait (SCT) and is always characterized by loss of the tumor suppressor SMARCB1. Because renal ischemia induced by red blood cell sickling exacerbates chronic renal medullary hypoxia in vivo, we investigated whether the loss of SMARCB1 confers a survival advantage under the setting of SCT. Hypoxic stress, which naturally occurs within the renal medulla, is elevated under the setting of SCT. Our findings showed that hypoxia-induced SMARCB1 degradation protected renal cells from hypoxic stress. SMARCB1 wild-type renal tumors exhibited lower levels of SMARCB1 and more aggressive growth in mice harboring the SCT mutation in human hemoglobin A (HbA) than in control mice harboring wild-type human HbA. Consistent with established clinical observations, SMARCB1-null renal tumors were refractory to hypoxia-inducing therapeutic inhibition of angiogenesis. Further, reconstitution of SMARCB1 restored renal tumor sensitivity to hypoxic stress in vitro and in vivo. Together, our results demonstrate a physiological role for SMARCB1 degradation in response to hypoxic stress, connect the renal medullary hypoxia induced by SCT with an increased risk of SMARCB1-negative RMC, and shed light into the mechanisms mediating the resistance of SMARCB1-null renal tumors against angiogenesis inhibition therapies.

Effective therapies for sickle cell disease: are we there yet?

Sickle cell disease (SCD) is a common genetic blood disorder associated with acute and chronic pain, progressive multiorgan damage, and early mortality. Recent advances in technologies to manipulate the human genome, a century of research and the development of techniques enabling the isolation, efficient genetic modification, and reimplantation of autologous patient hematopoietic stem cells (HSCs), mean that curing most patients with SCD could soon be a reality in wealthy countries. In parallel, ongoing research is pursuing more facile treatments, such as in-vivo-delivered genetic therapies and new drugs that can eventually be administered in low- and middle-income countries where most SCD patients reside.

Diverse Approaches to Gene Therapy of Sickle Cell Disease.

Sickle cell disease (SCD) results from a single base pair change in the sixth codon of the ß-globin chain of hemoglobin, which promotes aggregation of deoxyhemoglobin, increasing rigidity of red blood cells and causing vaso-occlusive and hemolytic complications. Allogeneic transplant of hematopoietic stem cells (HSCs) can eliminate SCD manifestations but is limited by absence of well-matched donors and immune complications. Gene therapy with transplantation of autologous HSCs that are gene-modified may provide similar benefits without the immune complications. Much progress has been made, and patients are realizing significant clinical improvements in multiple trials using different approaches with lentiviral vector-mediated gene addition to inhibit hemoglobin aggregation. Gene editing approaches are under development to provide additional therapeutic opportunities. Gene therapy for SCD has advanced from an attractive concept to clinical reality.

Accurate long-read sequencing allows assembly of the duplicated RHD and RHCE genes harboring variants relevant to blood transfusion.

Next-generation sequencing (NGS) technologies have transformed medical genetics. However, short-read lengths pose a limitation on identification of structural variants, sequencing repetitive regions, phasing of distant nucleotide changes, and distinguishing highly homologous genomic regions. Long-read sequencing technologies may offer improvements in the characterization of genes that are currently difficult to assess. We used a combination of targeted DNA capture, long-read sequencing, and a customized bioinformatics pipeline to fully assemble the RH region, which harbors variation relevant to red cell donor-recipient mismatch, particularly among patients with sickle cell disease. RHD and RHCE are a pair of duplicated genes located within an ∼175 kb region on human chromosome 1 that have high sequence similarity and frequent structural variations. To achieve the assembly, we utilized palindrome repeats in PacBio SMRT reads to obtain consensus sequences of 2.1 to 2.9 kb average length with over 99% accuracy. We used these long consensus sequences to identify 771 assembly markers and to phase the RHD-RHCE region with high confidence. The dataset enabled direct linkage between coding and intronic variants, phasing of distant SNPs to determine RHD-RHCE haplotypes, and identification of known and novel structural variations along with the breakpoints. A limiting factor in phasing is the frequency of heterozygous assembly markers and therefore was most successful in samples from African Black individuals with increased heterogeneity at the RH locus. Overall, this approach allows RH genotyping and de novo assembly in an unbiased and comprehensive manner that is necessary to expand application of NGS technology to high-resolution RH typing.

Safety and efficacy studies of CRISPR-Cas9 treatment of sickle cell disease highlights disease-specific responses.

Fetal hemoglobin (HbF) reactivation expression through CRISPR-Cas9 is a promising strategy for the treatment of sickle cell disease (SCD). Here, we describe a genome editing strategy leading to reactivation of HbF expression by targeting the binding sites (BSs) for the lymphoma-related factor (LRF) repressor in the γ-globin promoters. CRISPR-Cas9 treatment in healthy donor (HD) and patient-derived HSPCs resulted in a high frequency of LRF BS disruption and potent HbF synthesis in their erythroid progeny. LRF BS disruption did not impair HSPC engraftment and differentiation but was more efficient in SCD than in HD cells. However, SCD HSPCs showed a reduced engraftment and a myeloid bias compared with HD cells. We detected off-target activity and chromosomal rearrangements, particularly in SCD samples (likely because of the higher overall editing efficiency) but did not impact the target gene expression and HSPC engraftment and differentiation. Transcriptomic analyses showed that the editing procedure results in the up-regulation of genes involved in DNA damage and inflammatory responses, which was more evident in SCD HSPCs. This study provides evidence of efficacy and safety for an editing strategy based on HbF reactivation and highlights the need of performing safety studies in clinically relevant conditions, i.e., in patient-derived HSPCs.

Autologous gene therapy for hemoglobinopathies: From bench to patient's bedside.

In recent years, a growing number of clinical trials have been initiated to evaluate gene therapy approaches for the treatment of patients with transfusion-dependent ß-thalassemia and sickle cell disease (SCD). Therapeutic modalities being assessed in these trials utilize different molecular techniques, including lentiviral vectors to add functional copies of the gene encoding the hemoglobin ß subunit in defective cells and CRISPR-Cas9, transcription activator-like effector protein nuclease, and zinc finger nuclease gene editing strategies to either directly address the underlying genetic cause of disease or induce fetal hemoglobin production by gene disruption. Here, we review the mechanisms of action of these various gene addition and gene editing approaches and describe the status of clinical trials designed to evaluate the potentially for these approaches to provide one-time functional cures to patients with transfusion-dependent ß-thalassemia and SCD.

Development and IND-enabling studies of a novel Cas9 genome-edited autologous CD34+ cell therapy to induce fetal hemoglobin for sickle cell disease.

Sickle cell disease (SCD) is a common, severe genetic blood disorder. Current pharmacotherapies are partially effective and allogeneic hematopoietic stem cell transplantation is associated with immune toxicities. Genome editing of patient hematopoietic stem cells (HSCs) to reactivate fetal hemoglobin (HbF) in erythroid progeny offers an alternative potentially curative approach to treat SCD. Although the FDA released guidelines for evaluating genome editing risks, it remains unclear how best to approach pre-clinical assessment of genome-edited cell products. Here, we describe rigorous pre-clinical development of a therapeutic γ-globin gene promoter editing strategy that supported an investigational new drug application cleared by the FDA. We compared γ-globin promoter and BCL11A enhancer targets, identified a potent HbF-inducing lead candidate, and tested our approach in mobilized CD34+ hematopoietic stem progenitor cells (HSPCs) from SCD patients. We observed efficient editing, HbF induction to predicted therapeutic levels, and reduced sickling. With single-cell analyses, we defined the heterogeneity of HbF induction and HBG1/HBG2 transcription. With CHANGE-seq for sensitive and unbiased off-target discovery followed by targeted sequencing, we did not detect off-target activity in edited HSPCs. Our study provides a blueprint for translating new ex vivo HSC genome editing strategies toward clinical trials for treating SCD and other blood disorders.

Endothelial VWF is critical for the pathogenesis of vaso-occlusive episode in a mouse model of sickle cell disease.

Vaso-occlusive episode (VOE) is a common and critical complication of sickle cell disease (SCD). Its pathogenesis is incompletely understood. von Willebrand factor (VWF), a multimeric plasma hemostatic protein synthesized and secreted by endothelial cells and platelets, is increased during a VOE. However, whether and how VWF contributes to the pathogenesis of VOE is not fully understood. In this study, we found increased VWF levels during tumor necrosis factor (TNF)-induced VOE in a humanized mouse model of SCD. Deletion of endothelial VWF decreased hemolysis, vascular occlusion, and organ damage caused by TNF-induced VOE in SCD mice. Moreover, administering ADAMTS13, the VWF-cleaving plasma protease, reduced plasma VWF levels, decreased inflammation and vaso-occlusion, and alleviated organ damage during VOE. These data suggest that promoting VWF cleavage via ADAMTS13 may be an effective treatment for reducing hemolysis, inflammation, and vaso-occlusion during VOE.

Phenotypic screening of the ReFRAME drug repurposing library to discover new drugs for treating sickle cell disease.

Stem cell transplantation and genetic therapies offer potential cures for patients with sickle cell disease (SCD), but these options require advanced medical facilities and are expensive. Consequently, these treatments will not be available for many years to the majority of patients suffering from this disease. What is urgently needed now is an inexpensive oral drug in addition to hydroxyurea, the only drug approved by the FDA that inhibits sickle-hemoglobin polymerization. Here, we report the results of the first phase of our phenotypic screen of the 12,657 compounds of the Scripps ReFRAME drug repurposing library using a recently developed high-throughput assay to measure sickling times following deoxygenation to 0% oxygen of red cells from sickle trait individuals. The ReFRAME library is a very important collection because the compounds are either FDA-approved drugs or have been tested in clinical trials. From dose-response measurements, 106 of the 12,657 compounds exhibit statistically significant antisickling at concentrations ranging from 31 nM to 10 µM. Compounds that inhibit sickling of trait cells are also effective with SCD cells. As many as 21 of the 106 antisickling compounds emerge as potential drugs. This estimate is based on a comparison of inhibitory concentrations with free concentrations of oral drugs in human serum. Moreover, the expected therapeutic potential for each level of inhibition can be predicted from measurements of sickling times for cells from individuals with sickle syndromes of varying severity. Our results should motivate others to develop one or more of these 106 compounds into drugs for treating SCD.

Profiling the Plasmodium falciparum Erythrocyte Membrane Protein 1-Specific Immununoglobulin G Response Among Ghanaian Children With Hemoglobin S and C.

Members of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family are important targets for protective immunity. Abnormal display of PfEMP1 on the surfaces of infected erythrocytes (IEs) and reduced cytoadhesion have been demonstrated in hemoglobin (Hb) AS and HbAC, inherited blood disorders associated with protection against severe P. falciparum malaria. We found that Ghanaian children with HbAS had lower levels of immunoglobulin G against several PfEMP1 variants and that this reactivity increased more slowly with age than in their HbAA counterparts. Moreover, children with HbAS have lower total parasite biomass than those with HbAA at comparable peripheral parasitemias, suggesting impaired cytoadhesion of HbAS IEs in vivo and likely explaining the slower acquisition of PfEMP1-specific immunoglobulin G in this group. In contrast, the function of acquired antibodies was comparable among Hb groups and appears to be intact and sufficient to control parasitemia via opsonization and phagocytosis of IEs.

Hemoglobin scavenger receptor CD163 as a potential biomarker of hemolysis-induced hepatobiliary injury in sickle cell disease.

Sickle cell disease (SCD)-associated chronic hemolysis promotes oxidative stress, inflammation, and thrombosis leading to organ damage, including liver damage. Hemoglobin scavenger receptor CD163 plays a protective role in SCD by scavenging both hemoglobin-haptoglobin complexes and cell-free hemoglobin. A limited number of studies in the past have shown a positive correlation of CD163 expression with poor disease outcomes in patients with SCD. However, the role and regulation of CD163 in SCD-related hepatobiliary injury have not been fully elucidated yet. Here we show that chronic liver injury in SCD patients is associated with elevated levels of hepatic membrane-bound CD163. Hemolysis and increase in hepatic heme, hemoglobin, and iron levels elevate CD163 expression in the SCD mouse liver. Mechanistically we show that heme oxygenase-1 (HO-1) positively regulates membrane-bound CD163 expression independent of nuclear factor erythroid 2-related factor 2 (NRF2) signaling in SCD liver. We further demonstrate that the interaction between CD163 and HO-1 is not dependent on CD163-hemoglobin binding. These findings indicate that CD163 is a potential biomarker of SCD-associated hepatobiliary injury. Understanding the role of HO-1 in membrane-bound CD163 regulation may help identify novel therapeutic targets for hemolysis-induced chronic liver injury.

Nontargeted Plasma Proteomic Analysis of Renal Disease and Pulmonary Hypertension in Patients with Sickle Cell Disease.

Sickle cell disease (SCD) is characterized by red blood cell sickling, vaso-occlusion, hemolytic anemia, damage to multiple organ systems, and, as a result, shortened life expectancy. Sickle cell disease nephropathy (SCDN) and pulmonary hypertension (pHTN) are common and frequently co-occurring complications of SCD; both are associated with markedly accelerated mortality. To identify candidate circulating biomarkers of SCDN and pHTN, we used mass spectrometry to quantify the relative abundance of >1000 proteins in plasma samples from 189 adults with SCD from the Outcome Modifying Genes in SCD (OMG-SCD) cohort (ProteomeXchange identifier PXD048716). Forty-four proteins were differentially abundant in SCDN, most significantly cystatin-C and collagen α-1(XVIII) chain (COIA1), and 55 proteins were dysregulated in patients with SCDN and pHTN, most significantly insulin-like growth factor-binding protein 6 (IBP6). Network analysis identified a module of 133 coregulated proteins significantly associated with SCDN, that was enriched for extracellular matrix proteins, insulin-like growth factor binding proteins, cell adhesion proteins, EGF-like calcium binding proteins, and several cadherin family members. Collectively, these data provide a comprehensive understanding of plasma protein changes in SCDN and pHTN which validate numerous studies of chronic kidney disease and suggest shared profiles of protein disruption in kidney dysfunction and pHTN among SCD patients.

Urine concentration impairment in sickle cell anemia: genuine nephrogenic diabetes insipidus or osmotic diuresis?

The urine concentration impairment responsible for hyposthenuria in sickle cell nephropathy is currently thought to be a consequence of renal medulla lesions, which lead to nephrogenic diabetes insipidus. The objective of the present study was to investigate the mechanism of hyposthenuria in patients with sickle cell anemia. We performed an observational study of patients with homozygous SS sickle cell anemia and data available on the fasting plasma antidiuretic hormone (ADH) concentration. A total of 55 patients were analyzed. The fasting plasma ADH values ranged from 1.2 to 15.4 pg/mL, and 82% of the patients had elevated ADH values and low fasting urine osmolality (<505 mosmol/kgH2O). Plasma ADH was positively associated with plasma tonicity and natremia (P < 0.001). None of the patients experienced polyuria and fasting free water clearance was negative in all cases, thus, ruling out nephrogenic diabetes insipidus. The tertile groups did not differ with regard to fasting urine osmolality, plasma renin level, mGFR, or several hemolysis biomarkers. The negative fasting free water clearance in all cases and the strong association between 24-h osmolal clearance and 24-h diuresis favors the diagnosis of osmotic diuresis due to an impaired medullary gradient, rather than lesions to collecting tubule.NEW & NOTEWORTHY The urine concentration impairment in sickle cell anemia is an osmotic diuresis related to an impaired renal medullary gradient leading to an ADH plateau effect. The fasting plasma ADH was high in the context of a basic state of close-to-maximal urine concentration probably driven by short nephrons maintaining a cortex-outer medullary gradient (about 400 milliosmoles). The patients had a low daily osmoles intake without evidence of thirst dysregulation so no one experienced polyuria.