SS Mice Research Articles

Heme Scavenging by Hemopexin Attenuates Sepsis-Induced Proinflammatory Cytokines and Kidney Insuffciency in a Humanized Mouse Model of Sickle Cell Disease

Sickle cell disease (SCD) is a genetic blood disorder characterized by abnormal hemoglobin production, leading to sickle-shaped red blood cells (RBC). Alteration in RBC shape results in vasoocclusion, chronic hemolysis, and multiple organ damage. One of the severe complications of SCD is an increased susceptibility to infections, including sepsis, which can be life-threatening. Kidney injury, a common and severe complication of sepsis, contributes to the complexity and poor outcomes associated with morbidity and mortality in SCD. However, the mechanisms that underlie septic acute kidney injury (AKI) in SCD are poorly understood. Here, we examined the effects of sepsis in transgenic homozygous SCD (SS; Townes model) mice and their non-sickling (AA) counterparts. We tested the hypothesis that aggravation of inflammation and oxidative stress by free heme released during hemolysis contribute to sepsis-induced kidney insuffciency in SCD. AA and SS mice were subjected to 18 h of sham operation and cecal ligation and puncture (CLP), a pre-clinical model of polymicrobial sepsis, after which kidney function was evaluated by transdermal measurement of glomerular filtration rate (GFR). AKI biomarkers, renal oxidative stress, and cytokines levels were also investigated. We demonstrate that mid- to high-grade CLP (ligation of 60-75% of the cecum and multiple punctures with 21-gauge needles) caused 100% mortality in SS mice. Unlike AA mice (100% survival), SS mice subjected to low-grade CLP (LgCLP; ligation of <45% of the cecum and a single puncture with 21-gauge needles) exhibited 40% survival. LgCLP reduced body weight but did not exacerbate splenomegaly in SS mice. Basal plasma bilirubin, heme, and ferritin were significantly higher in SS than in AA mice. In contrast to AA, LgCLP amplified the increase in plasma bilirubin, heme, and ferritin in SS mice. LgCLP also decreased GFR and increased renal oxidative stress, AKI biomarkers, and kidney-to-body weight ratio in SS mice. Luminex multiplex cytokine analysis indicated that the plasma levels of multiple pro-inflammatory cytokines were elevated in septic SS mice. Heme scavenger hemopexin (HX) diminished LgCLP-induced plasma heme increase. HX also mitigated kidney insuffciency, inflammation, and mortality in the septic SS mice. Our findings suggest that free heme contributes to septic AKI in SCD mice and that HX therapy may attenuate the harmful effect of heme in SCD during sepsis. NHLBI: R01 HL151735. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

CD4 T cell-secreted IFN-γ in Sjögren's syndrome induces salivary gland epithelial cell ferroptosis

BackgroundSjögren's syndrome (SS) is a chronic autoimmune disease that predominantly affects exocrine glands. Previous studies have demonstrated that upregulated interferon-gamma (IFN-γ) in SS triggers ferroptosis in salivary gland epithelial cells (SGECs), resulting in impaired salivary gland secretion. However, the immune cells responsible for secreting IFN-γ remain unclear. Therefore, this study conducted bioinformatics analysis and molecular validation to identify the origin of IFN-γ in SS salivary gland. MethodsThe ‘limma’ package in R software was utilized to identify differentially expressed genes (DEGs) in the human SS dataset. Subsequently, the identified DEGs were compared with the ferroptosis database and screened through Cytoscape to determine candidate genes. The cellular localization and expression patterns of candidate genes were further confirmed in the salivary gland single-cell RNA sequence (scRNA-seq) data set from healthy control and SS mice. Furthermore, in vitro and in vivo studies were performed to analyze the effect of CD4 T-secreted IFN-γ on SGECs' ferroptosis and functions. ResultsUpregulated TLR4, IFNG, and IL33 were screened as candidates ferroptosis ferroptosis-inducing genes in SS salivary glands. The association of IFNG and IL33 with CD4 T cells was established through immune infiltration analysis. The expression of IFN-γ on CD4 T cells was robustly higher compared with that of IL33 as evidenced by scRNA-seq and immunofluorescence co-localization. Subsequent experiments conducted on candidate genes consistently demonstrated the potent ability of IFN-γ to induce SGECs' ferroptosis and inhibit AQP5 expression. ConclusionsOur findings indicate that CD4 T cell-secreted IFN-γ in SS induces SGECs' ferroptosis and inhibits AQP5 expression.

Hemolysis-driven IFNα production impairs erythropoiesis by negatively regulating EPO signaling in sickle cell disease

AbstractDisordered 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 Hbbth3/+ thalassemia mice were increased more than twofold, they were expanded by only ∼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 α (IFNα), 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 IFNα-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-IFNα-CISH axis.

New Insights into Cellular Defects during Haematopoiesis of Sickle Cell Disease Using the Townes Mouse Model

Sickle cell disease (SCD) is an inherited disorder caused by a point mutation in the β globin gene leading to the synthesis of an abnormal haemoglobin (HbS), that under low oxygen tension polymerises driving the sickling of red blood cells and reducing their lifespan. SCD is characterized by anaemia, vaso-occlusion and haemolysis, progressive multiorgan damage and increased mortality. For decades, peripheral haemolysis has been considered as the sole driver of anaemia in SCD. Recently, we have demonstrated the occurrence of ineffective erythropoiesis (IE) in SCD suggesting that anaemia can be impacted by defects of central origin and hinting to abnormalities in the haematopoietic compartment. To gain insight into SCD haematopoiesis, we investigated the haematopoietic compartment of the humanized transgenic Townes mouse model by conducting an in-depth flow cytometry-based analysis in the bone marrow (BM) and spleen, comparing SS mice to control littermates (AA and AS) at 8 and 16 weeks of age (termed 8-w and 16-w, respectively). BM and spleen are two key hematopoietic tissues in adult mice: BM is involved in maintaining homeostatic haematopoiesis and spleen responds to stress haematopoiesis. First, we assessed haematopoietic differentiation in the BM of SS, AA and AS mice. Total number of BM cells and frequency of Lin Neg and Lin NegKit Pos (LK) were identical between SS and control littermates whereas a minor increase in the percentages of Lin NegSca PosKit Pos (LSK) was observed in 16-w SS mice. Within the LSK compartment, we found a 1.81- and 2.5-fold reduction of long-term haematopoietic stem cell (lt-HSC) frequency in 8-w and 16-w SS mice, respectively (Fig1 A). HSCs differentiate into multipotent progenitors (MPPs), which have decreased self-renewal capacity and progressively engage in the megakaryocytic (MPP2), myeloid-biased (MPP3), or lymphoid-biased (MPP4) lineage. The MPP3 compartment showed a 1.6-fold increased frequency in 8-w and 16-w SS mice indicating a skewing toward myeloid cell production and the onset of extramedullary haematopoiesis as early as 8 weeks after birth (Fig1 A). Next, we analysed the downstream lineages of the committed progenitors (lymphoid, granulo-macrophagic, megakaryocytic, and erythroid) and found a 1.4 and 2.3-fold increase of th megakaryocyte-erythroid progenitor cells (MEPs) and pre-CFU-Es in 8-w and 16-w SS mice, respectively (Fig1 A,B). We assessed the potential of SS hematopoietic progenitors (HSPCs) to differentiate into myelomonocytic and erythroid lineages by performing methylcellulose colony forming unit (CFU) assays with LSK and LK cells sorted from 8-w mice. Although no difference was observed in the number and type of colonies formed in vitro from SS and control HSPCs, SS colonies were much smaller than the AA/AS ones. Furthermore, serial replating showed a phenotype of stemness exhaustion in SS mice, with a dramatic decrease in the number of colonies formed by LSK cells (27.5 SS CFUs vs 51.6 AA/AS CFUs), suggesting lower self-renewal capacities of SS HSPCs. In spleen, in addition to the expected splenomegaly of SS mice, we found a trend toward a decreased frequency of the LSK compartment and an increase of the LK compartment. There was a 2.25- and 1.42-fold increase in the percentage of MPP-2 and MPP-3 cells, respectively, and a 2.1-fold increase in the pre-CFU-Es confirming an intense splenic haematopoiesis in SS mice (Fig 1B). HSPCs are known to be retained by the BM stromal cells through the chemotactic interaction of CXCL12 and CXCR4 and the adhesive interaction of VCAM-1 and VLA-4. Measuring HSC-related cytokines in BM supernatants, we found severely diminished levels of CXCL12 in SS mice as compared to control littermates (18 pg.mL -1 in SS vs 84 pg.mL -1 in AA; N=80), suggesting HSC mobilization from the BM to the spleen with disease onset. In addition to the well-established stress erythropoiesis phenotype reported in SCD mice, our study describes cellular abnormalities in the haematopoietic compartment shedding light on potential new mechanisms that contribute to SCD pathology in relation to anaemia and bone marrow stemness.

A Landscape of Dendritic Cells Development, Activation and Migration in Mouse Model of Sickle Cell Anemia

Dendritic cells (DCs) are crucial players of innate immunity in initiating, polarizing, and regulating the adaptive immune response. They become activated upon encountering either pathogen- or damage-associated molecular patterns (PAMPs or DAMPs), which are often released due to tissue necrosis or intravascular hemolysis, common events in individuals with sickle cell disease (SCD), one of the most common hemoglobinophaties that affects millions of people worldwide. Upon activation, DCs migrate to peripheral lymph nodes, where they present antigens to T lymphocyte together with co-stimulatory or inhibitory molecules. In addition, they can polarize T cell response by releasing pro- or anti-inflammatory cytokines. We previously showed that SCD patients have higher circulating total and inflammatory DCs, whereas the ratio of type 1 conventional DCs (cDC1) is lower than control individuals. These changes correlated with number of reticulocytes and with the skewed T response towards Th17. Alterations in the development and activation of DC subpopulations, as well as in their function can impair the immune response or tolerance, which may account for the dysfunction in T lymphocyte responses observed in SCD patients. In order to investigate the development, activation and migration of DCs during SCD, we characterized these cells by flow cytometry in lymphoid organs of Townes mice, which have human S hemoglobin (HbS). Our data show that Townes mice (SS) have lower number of total DCs in bone marrow (p=0.057; N=8 each), however, they have higher number of these cells in the spleen (p<0.0001; N=8 each) and lymph nodes (p<0.0021; N=8 each) when compared with the littermate WT mice, suggesting migration of DCs to these peripheral organs. Evaluation of the ratios of DCs subsets shows that the frequency of cDC1 (CD11c +CD8a +CD11b -) among total DCs is higher in the three organs of SS mice compared with WT mice (p=0.004 in spleen; p=0.0015 in bone marrow; and p=0.01 in lymph nodes; N=8 each group). In contrast, the percentage of type 2 conventional DCs, cDC2 (CD11c +CD8a -CD11b +) is similar or lower in SS mice than WT mice (p=0.0005 in spleen; N=8 each). In addition, the frequency of plasmacytoid dendritic cell, pDC (CD11c +B220 +SiglecH +) is higher in lymph nodes (p=0.0006; N=8 each) and lower in bone marrow (p=0.0017; N=8 each) of SS mice when compared with WT mice, indicating that the migration of DCs to peripheral organs is due to cDC1 and pDC. Regarding their maturation and activation phenotype, cDC1 and cDC2 from SS mice have a more immature or inhibitory phenotype than WT mice, as showed by MHC-II (cDC1: p=0.01 in spleen; p=0.0004 in lymph nodes; N=8 each) and CD86 expression (cDC1: p=0.013 in spleen; p=0.0003 in bone marrow; and p= 0.03 in lymph nodes; cDC2: p=0.0067 in bone marrow and p=0.038 in lymph nodes; N=8 each). On the other hand, pDCs from the three organs of SS mice showed a more mature profile than WT mice, as demonstrated by an increase in Sca-1 expression (p=0.0024 in spleen; p=0.037 in bone marrow; and p=0.04 in lymph nodes; N=8 each), indicating that cDCs are being inhibited whereas pDCs are activated during SCD even in steady state. Aimed to understand whether at least part of the alterations on DCs is due to changes in their development, we quantified monocyte and DC progenitor, MDP (Lin -(CD3/CD19/CD56/CD14) CD135 +CD11c -CD115 +CD117 +) and common DC progenitor, CDP (Lin -CD135 +CD11c -CD115 +CD117 -) in bone barrow. Our data show that the ratio of MDP, the earlier progenitor, is increased (p=0.0009; N=8 each), whereas CDP, the progenitor committed to DCs, is diminished (p=0.014; N=8 each) in SS mice when compared with WT mice, indicating a change in the DC compartment in SS mice since their developmental stage. When we performed an in vitro differentiation of DC from bone marrow cells in the presence of flt3l, we did not observe statistical difference in either development and apoptosis of DCs, or in the percentages of DC subsets between SS and WT mice, indicating that the alteration in DC development in SS mice may be due to environmental conditions instead of CDP or DC intrinsic factor. Our findings so far improve our understanding of DCs dynamics in SCD, including the development, activation and migration of the different subsets that may have an important impact on adaptive immune response disfunction in SCD, which may account for the chronic inflammatory state and the susceptibility to infections.

Characterizing Pregnancy Outcomes in a Humanized Mouse Model of Sickle Cell Disease

Background: Sickle cell disease (SCD) is a group of hereditary disorders commonly associated with the distortion of red blood cells (RBC) at low oxygen levels. It is characterized by complications such as vaso-occlusion, anemia, and hemolysis. These complications are lifelong and become extremely relevant during periods of increased physiological stress such as pregnancy. Pregnancy in sickle cell disease (SCD) is associated with an increased risk of complications including, pre-term delivery, deep vein thrombosis, intra-uterine growth restriction, pre-eclampsia, and intra-uterine death. Recent meta-analyses have shown that women with SCD are at 26x greater risk for maternal mortality than their healthy counterparts; a figure which, despite the adoption of improved care plans, has shown little to no significant improvement over the last two decades. While impaired vascular maintenance underlies multiple complications of pregnancy outside of sickle cell disease, it is especially relevant to, and characteristic of sickle cell-related pathologies at baseline and is highly likely to be exacerbated as pregnancy proceeds. Through this study we aimed to characterize the effects of SCD-related vasculopathy on pregnancy outcomes and overall placental health using the Townes humanized mouse model of SCD. Methods: SS (sickle hemoglobin) and AA (normal hemoglobin) mice underwent timed breeding to produce pregnant dams. These dams were housed under normal conditions until 17.5 days post conception (dpc), at which point they began undergoing procedures for analysis including 24-hour urine collection, high-resolution ultrasound analysis (18.5dpc), and post-mortem analysis of pregnancy outcomes and tissue collection (18.5dpc). Included in this report are gross pregnancy outcomes, histological assessments of placental tissue, and reports from ultrasound analysis including 3-D volume measures, placental vascularization renders, and laser-Doppler assessment of uterine and umbilical arteries. Continuing research efforts will further utilize tissues from this study to evaluate the biochemical interactions influencing poor pregnancy outcomes related to SCD. Results: These studies show that the SCD mice recapitulate many similarly poor outcomes as women. We observed: reduced litter sizes (AA 6.9 ± 1.5 embryos vs SS 5.2 ± 1.2 embryos**), fetal weight (AA 0.49 ± 0.14g vs SS 0.38 ± 0.16g*), viability of embryos (AA 100.0% IQR 0.0 vs SS 20.0% IQR 33.3****), and maternal mortality (AA 0.0% vs SS 8.7%, OR = 4.3 ns). In assessing placental histology, we identified evidence of vasculopathy common to end organ damage in SCD such as increased presence of avascular areas, reduced relative labyrinth area (AA 55.6 ± 8.6% vs SS 44.7 ± 6.0%*) and reduced vascular density (AA 38.9 ± 6.2% vs SS 34.4 ± 7.0%*) which are likely responsible for worsening outcomes. Additional pathologies include increased occurrence of placental ectasia, calcification of the labyrinth, vascular thrombi, and perivascular fibrosis when compared to controls. Further, ultrasound analysis revealed several measures of placental insufficiency amongst SS mice that were significantly impaired including uterine blood flow and an overall reduction in placental vascularization and oxygenation. Conclusions: SCD mice demonstrate many similar pregnancy outcomes as humans, allowing for increased translational application. We've shown these outcomes to be severe and largely driven by vascular impairment specifically within the placenta, the main maternal-fetal interface. This study serves as the groundwork for future studies aimed at investigating the underlying mechanisms of these outcomes while also establishing baseline measures and novel methods of approach towards the broader goal of improving obstetric disparities faced by women with SCD.

The Role of P-Selectin in Microvascular Hemodynamics and Cerebral Brain Volumes in Aging Sickle Cell Mice

Cerebral vasculopathy has been linked to the development of cognitive impairment in sickle cell disease (SCD). We have previously demonstrated that 13-month-old sickle cell mice recapitulate features of cerebral vasculopathy such as tortuous vessels, microthrombi, and hypoperfusion seen in adults with SCD. Histologically, we also showed these mice have increased cerebral microvascular deposition of P-selectin and more frequent and larger microinfarcts compared to matched controls. We further showed that these mice developed evidence of cognitive deficit compared to matched controls. Based on these observations, we hypothesized P-selectin may play a critical role in development of cerebral micro vasculopathy and downstream pathologic sequalae such as microinfarcts by enhancing leukocyte-endothelial interactions. Furthermore, abundant evidence from non-sickle mice shows that microinfarcts can exacerbate small vessel changes including gray matter volume loss. Building on our prior work, in this study, we examined the effect of P-selectin deficiency on cerebral microvascular hemodynamics and leukocyte-endothelial interaction and well as changes in cortical and subcortical gray matter volume in aged sickle mice. Male, P-selectin knockout (Psel KO) or intact (Psel WT) chimeric mice were generated by transplanting control (AA) or sickle (SS) bone marrow into P-selectin knockout (Psel -/-) or C57BL/6 (B6) recipients. To assess the impact of vasculopathy and brain gray matter volume changes longitudinally, male mice were assessed at 12-, 14- or 16-month post-transplant. Hemodynamics (mean velocity, red blood cell (RBC) flux, white blood cell stalls in capillaries as well as venules) were obtained from line-scan images acquired using in-vivo two-photon microscopy, using custom and validated MATLAB scripts. Gray matter volumes in 26 cortical and subcortical brain regions with relevance to cognitive function, were derived from parcellation of T2-weighted magnetic resonance imaging after non-linear registration to Allen brain atlas T1 anatomical space. Parcellation was accomplished by registering the Paxinos atlas to the mouse T2 now in Allen space. Brain volume analyses was accomplished using Analyze 12 software. After the completion of in-vivo studies, brains were extracted for immunohistochemistry analysis. Statistical analysis was performed using two-way ANOVA. The breakdown of the mice included in this study are; 14 B6 AA, 12 B6 SS, 12 Psel KO AA and 13 Psel KO SS. Given the high attrition rate in older mice, data from 12-, 14- and 16-month-old cohorts were combined. Mean age of B6 AA cohort was 13.1 months, 12.8 months for B6 SS, 13. 9 months for Psel KO AA and 13.6 months for Psel KO SS mice and were not statistically different. Psel KO sickle mice had a significantly lower mean capillary RBC velocity (1.733 mm/s vs 4.717 mm/s, p < 0.01) compared to Psel WT SS mice. In addition, Psel KO SS mice had relatively smaller (more normal) capillary diameter compared to Psel WT SS mice (6.248 um vs 8.613 um, p < 0.001). Interestingly, there was no statistically significant difference in RBC flux between Psel KO SS and Psel WT SS mice, however Psel KO SS mice show a lower red blood cell flux similar to that seen in Psel WT AA mice ( Figure 1). Of the 26 brain regions that were assessed, Psel KO SS mice had larger measured gray matter volume in the frontal association cortex (3.01 ± 0.77 vs 3.66± 0.33 mm 3, p= 0.0083), prelimbic cortex (1.72 ± 0.27 vs 1.95 ± 0.17 mm 3, p=0.0079), hippocampus (17.67 ± 1.56 vs 16.21± 1.97 mm 3, p=0.01)( Figure 1) including other regions such as lateral (2.12 ± 0.19 vs 1.88 ± 0.31 vs mm 3 p=0.0096) medial (1.84± 0.16 vs 1.64 ± 0.26 mm 3, p=0.01), and ventral orbital cortex( 1.95± 0.17 vs1.72 ± 0.27 mm 3, p=0.01) and dorsolateral olfactory tract (1.46 6± 0.13 vs 1.31 ± 0.20 mm 3, p=0.023 compared to the Psel WT SS mice (data not shown). In conclusion, non-hematopoetic P-selectin deficiency improves cerebral vasculopathy as demonstrated by normalization of hemodynamics in sickle mice. We also show that Psel KO SS mice had larger brain gray matter volume in regions involved in cognitive function. Additional studies are needed to identify the underlying mechanism of improvement in brain gray matter volume as well as whether P-selectin could be potential target for reducing SCD associated neurological complications.

20-HETE As an Emerging Target to Improve Renal Health in Sickle Cell Disease

The kidney is one of the most severely affected organs in sickle cell disease (SCD), with glomerular hyperfiltration and microalbuminuria beginning in childhood, and progressing to chronic kidney disease (CKD) in majority of adults. Sickle individuals also suffer from acute kidney injury (AKI) often during characteristic vaso-occlusive crisis. Renal pathology in sickle cell nephropathy includes extensive microvascular congestions and thrombotic microangiopathy. The hypercoagulable and vasoconstrictive environment of SCD, in conjunction with medullary hypoperfusion and cortical hyperperfusion contributes to vaso-occlusive injury and deregulates intrarenal hemostasis leading to CKD development. Ischemic injury increases arachidonic acid hydroxylation producing 20-hydroxyeicosatetraenoic acid (20-HETE), which in turn triggers thrombosis and promotes vasoconstriction in the kidney. However, the role of 20-HETE in regulating renal hemostasis in SCD nephropathy has never been reported. We found that plasma 20-HETE is significantly elevated in SCD patients (n=9-11; p<0.01) compared to normal individuals. Similarly, transgenic SCD mice (SS), homozygous for human hemoglobin S, have increased plasma 20-HETE compared to control mice (AA), homozygous for human hemoglobin A (n=6; p<0.01). We have previously shown that elevated circulating heme mimicking hemolytic crisis triggers clinically relevant acute kidney injury (AKI) in SS mice (Blood (2020) 135(13): 1044-1048). We postulated that hemolytic stress may exacerbate 20-HETE production which in turn aggravates renal microvascular occlusion and vasoconstriction, leading to renal damage in SCD. Immunohistochemistry and biochemical experiments showed that the expression of CYP4A12a, a cytochrome 450-dependent enzyme responsible 20-HETE production in mice, is substantially elevated in the kidneys of SS mice compared to AA mice, associated with increased vascular congestion and renal thrombogenesis. Renal CYP4A12a and plasma 20-HETE were increased substantially following heme-induced AKI in SS mice, associated with a heightened expression of renal angiotensin II receptor 1 (ATIIR1), thus indicating induced vasoconstrictive activity. In vitro, we found that 20-HETE induced ATIIR1 expression in human renal glomerular endothelial cells (hRGEC), which was further exacerbated by low-concentration of heme challenge (5 mM for 24 hour). Nitro-oleic acid (NO 2-OA) is a nitrated fatty acid that binds ATIIR1 and inhibits angiotensin II induced vasoconstriction. Pretreatment with NO 2-OA reduced 20-HETE-dependent ATIIR1 expression in hRGEC, while NO 2-OA prophylaxis during hemin-induced AKI inhibited albuminuria, normalized plasma creatinine levels, and lowered the expression of ATIIR1 in SS mice kidneys. Overall, our data show that elevated 20-HETE levels during hemolytic stress may aggravate renal injury in SCD which can be prevented by NO 2-OA prophylaxis. Ongoing experiments will determine the direct effect of elevated 20-HETE in renal hemostasis and CKD development in SCD.

Unravelling the Mechanism behind Ineffective Erythropoiesis in Sickle Cell Disease: The Role of GATA-1

Background & Objectives: Ineffective erythropoiesis (IE) is an important factor in many types of anemias, including beta-thalassemia. Recently, a study by El Hoss et al provided compelling evidence for IE being an important part of the pathophysiology of sickle cell anemia (SCA) (PMID: 32855279). Furthermore, we developed a clinical index to measure IE and showed that patients with the SS genotype, but not SC, had increased IE compared to healthy donors (HD) (PMID: 34670360). However, the molecular mechanisms of IE in SCA, its relationship to disease heterogeneity and its potential as a therapeutic target, remain unknown. Importantly, GATA1, an essential erythroid transcription factor known to be downregulated in haematological conditions presenting with IE, has not been previously investigated in SCA. Thus, the aim of this project is to investigate the molecular basis of IE in SCA by focusing on GATA1 functions in erythropoiesis. Methods: HUDEP-2 cells, gene-edited sickle HUDEP-2 (SS HUDEP-2) (PMID: 32405513) cells and CD34+ cells isolated from individuals with HbSS and healthy donors, were differentiated ex vivo using a two-phase culture and (i) erythroid differentiation kinetics, (ii) markers of oxidative stress, (iii) GATA1 expression and (iv) antioxidant response factors NRF2, FOXO3 and heme-oxygenase 1 (HO-1) were assayed every other day during phase 2. IE was also studied in SS Berkeley transgenic mouse (PMID: 16166585). Both the spleen and the bone marrow were investigated in AA, AS and SS mice. Results: No significant differences were observed in the differentiation kinetics and in levels of oxidative stress in in vitro differentiated HUDEP-2 and SS HUDEP-2 cells. Moreover, GATA1 levels were similar in both cases. Importantly, ex vivo differentiation of human SS CD34+ cells showed a striking delay accompanied by high oxidative stress and a decrease in GATA1 protein levels (Figure 1) compared to normal controls. We also found a significant decrease in FOXO3, NRF2 and HO-1 levels in SS erythroblasts compared to healthy donor erythroblasts. IE was evident in SS mice compared to AA and AS controls. SS mice presented with higher levels of reactive oxygen species (ROS) in both the bone marrow and the spleen tissue. Histological studies showed that the SS Berkley mouse has a very congested bone marrow with a disorganized architecture. Moreover, we showed a significant decrease in GATA1 protein in SS spleen and bone marrow erythroid cells. Conclusion: Using ex vivo differentiation of primary SS cells and an in vivo mouse model for SCA, we show for the first time that aberrant GATA-1 levels play a key role during IE in SCA. During SS erythropoiesis, a significant reduction in the protein levels of the GATA1 erythroid master transcription factor and of FOXO3 and NF-E2, important transcriptional regulators of the oxidative stress response occur. These same changes were not seen in SS HUDEP-2 cells, suggesting that abnormalities of the bone marrow niche are central to abnormal erythropoiesis in SCA. Modification of the bone marrow niche and manipulation of GATA-1 levels may be important therapeutic tools in SCA.

Predictive Biomarker Analysis from the GBT021601 Survival Study in Townes Sickle Mice

Introduction : Sickle cell disease (SCD) is caused by a single nucleotide mutation in the β-globin gene resulting in sickle hemoglobin (HbS). Red blood cell (RBC) deoxygenation leads to HbS polymerization, RBC sickling and subsequent chronic hemolysis, anemia, vascular endothelial cell activation, vaso-occlusion, and ischemia. SCD is associated with extensive morbidity and markedly reduced survival. GBT021601 is an investigational, second-generation HbS polymerization inhibitor that reduces RBC sickling and anemia, and improves both O 2 delivery and consumption in chronically treated Townes Sickle Cell mice that harbor 2 HbS alleles (SS). Objectives of this 2-part study were to (1) characterize the effects of GBT021601 administered ad libitum on compound exposure and pharmacodynamics (PD), (2) examine the impact of long-term (>24 weeks) treatment with GBT021601 on % reticulocytes and overall survival (OS) of Townes SS mice, and (3) assess the utility of early changes in % reticulocytes as a predictive biomarker of survival in Townes SS mice treated with GBT021601. Methods: In Part 1, 24 male Townes SS mice 8-10 weeks old were split into 3 cohorts (n=8/group) and for 3 weeks were fed standard chow containing 0% (control), 0.05% (low dose), or 0.2% (high dose) of GBT021601. Body weight and food consumption were measured weekly. Mice were bled daily on days 10-21 to determine RBC half-life and on days 10 and 21 for analysis of compound exposure. On day 22 mice were euthanized. Whole blood was collected for analyses of compound exposure, % reticulocytes, Hb levels, RBC numbers, RBC half-life, and RBC deformability. In Part 2, 5-week-old Townes mice (AA and SS genotype; 50% male) were fed either standard chow (AA [n=57] and SS mice [n=54]) or chow containing 0.05% or 0.2% of GBT021601 (SS mice only; n=55 each). Body weight and food consumption were measured weekly. Blood was collected at 1, 14, 31, 43, 55, and 67 weeks of treatment for compound exposure analysis and % reticulocytes. Mice were euthanized when the humane end-point criteria were met or after 72 weeks of treatment, and whole blood was collected for compound exposure and % reticulocyte analyses. Results: GBT021601 in chow was well tolerated during both study parts; food consumption and body weight were unaffected. Outcomes in Part 1 confirmed the same compound exposure/PD trends as mice orally gavaged with GBT021601 as observed by Dufu et al. (Br J Haematol 2023); Hb levels, RBC numbers, and RBC half-life increased, RBC deformability improved, and % reticulocytes decreased. Long-term dosing of GBT021601 in the chow of Townes SS mice revealed similar compound exposure and % reticulocytes as our 3-week dosing study (Part 1) and Dufu et al. In Part 2, Townes SS mice fed GBT021601 chow, achieving compound exposures after 1 week of dosing as depicted in Figure 1, had a longer median survival vs control Townes SS mice (60 vs 56 weeks). In control Townes SS mice, % reticulocytes increased during the first 14 weeks of the study and then plateaued, whereas in Townes SS mice fed GBT021601 there was a dose-dependent sustained reduction in % reticulocytes compared with control Townes SS mice. Reductions in % reticulocytes after 1 week of dosing were greater in Townes SS mice achieving a higher compound exposure (Figure 1), a trend sustained after 14 and 31 weeks of treatment. OS increased in Townes SS mice of the same sex achieving a ≤30% reduction in % reticulocytes vs Townes SS mice with % reticulocytes >30% after 1 week of treatment; P=0.00048 (Figure 2). Multivariate survival analysis, including % reticulocytes at 1 week (categorical variable of ≤30% or >30%) and sex as predictors confirmed significant association of >30% reticulocytes with reduced survival (hazard ratio=1.48, P=0.027 using Cox proportional hazard). Conclusions:Longer-term GBT021601 treatment as part of chow was well tolerated by Townes SS mice, and compound exposure was comparable with studies of a shorter duration of treatment. Early compound exposure was predictive of a long-term survival benefit in Townes SS mice. Early reductions in % reticulocytes were more likely in Townes SS mice with a high GBT021601 exposure and were predictive of a long-term survival benefit. Overall, our findings support the use of GBT021601 to reduce % reticulocytes and improve OS in Townes SS mice and suggest the clinical relevance of early reductions in % reticulocytes as a biomarker to predict long-term survival.

Impaired Hemoglobin Clearance By Liver Sinusoidal Endothelium Promotes Liver Damage in Sickle Cell Disease

Sickle cell disease (SCD) is a monogenic disorder that affects 100,000 African Americans and millions of people worldwide. Intra-erythrocytic polymerization of sickle hemoglobin (HbS) promotes erythrocyte sickling, impaired rheology, ischemia and hemolysis, leading to the development of progressive liver injury in SCD. Liver resident macrophages and monocytes are known to enable the clearance of HbS, however, the role of liver sinusoidal endothelial cells (LSECs) in HbS clearance and liver injury in SCD remains unknown. Using real-time intravital ( in vivo) imaging of the liver as well as primary culture of human and mice LSECs, we show for the first time that liver injury is associated with accumulation of HbS and iron in the LSECs, leading to the development of senescence in LSECs. Hepatic monocytes were observed to attenuate LSEC-senescence by accelerating HbS clearance in the liver of SS mice, however, this protection was impaired in P-selectin-deficient SCD mice secondary to reduced monocyte recruitment in the liver. These findings are the first to suggest that LSECs contribute to HbS clearance and LSEC-senescence promotes progressive liver injury in SS mice. Our results reveal a novel insight into the pathogenesis of hemolysis induced chronic liver injury in SCD caused by LSEC senescence. Identifying the regulators of LSEC mediated HbS clearance may lead to new therapies to prevent the progression of liver and other organ injuries in SCD.

Deciphering Metabolomic Signatures Associated with Sickle Cell Disease Using Mouse Models

Introduction: Sickle cell disease (SCD) is debilitating and affects millions of people worldwide. Although SCD is caused by a single-point mutation of the β-globin chain of hemoglobin, the clinical manifestations are heterogeneous, and there is a lack of well-characterized predictive biomarkers of disease progression. Metabolomics is a promising tool to interrogate disease pathophysiology and underlying molecular mechanisms. Although prior metabolomic studies have been performed on human and murine red blood cells (RBCs) in the context of SCD, no systematic investigation across commonly used mouse models of SCD has been published. In the present study, we examined the blood cell metabolome of 2 widely used SCD mouse models to study and compare the metabolomic changes in disease models. Methods: Blood samples were collected from 5 groups of mice (10-14 weeks old, n=6-7 per group): wild-type (WT) C57BL/6J mice; Townes AA, AS, and SS mice; and Berkeley SCD mice. Direct injection high-resolution mass spectrometry was used to generate untargeted metabolomics data. Three matrices (RBC, plasma, and whole blood) were isolated from each mouse group. The RBC fraction, containing peripheral blood mononuclear cells, was separated from plasma via centrifugation of whole blood. The current study focuses on the RBC dataset.Raw profile data were centroided, merged, and recalibrated using methods previously described (Fuhrer et al. Anal Chem 2011). Putative annotations were generated based on compounds contained in the Human Metabolome Database, KEGG, and ChEBI databases using accurate mass per charge (tolerance 0.001 m/z) and isotopic correlation patterns. Z-score normalization was applied across all samples for unsupervised clustering using principal component analysis (PCA) and to identify metabolites with significant changes between groups. Metabolites were mapped to central RBC metabolic pathways using MetaboAnalyst (Xia et al. Nat Protoc 2011) and manual curation. Results: Unsupervised PCA that used all detectable RBC metabolites resulted in clear separation of mouse groups by disease status (Figure 1), suggesting robust metabolomic changes in mouse models of SCD. Focusing on the top divergent metabolites across the mouse groups, several enriched pathways were identified, such as amino acid metabolism, pyrimidine and purine metabolism, and aminoacyl-tRNA biosynthesis, all potentially attributable to the buildup of RBC precursors. Metabolites characteristic of immature RBCs were further examined, with elevated hexokinase activity (hexose:hexose-6-phosphate ratio), ATP levels, and mitochondrial metabolites found in RBCs from SCD, in line with the significantly elevated reticulocyte content in SCD mice. Metabolites were curated from additional relevant RBC metabolic pathways; some showed significant differences among mouse groups, and others were significantly elevated in sickle mice (Townes SS and Berkeley SCD) compared with non-SCD controls (WT and Townes AA), including some glycolytic and pentose phosphate pathway (PPP) intermediates (Figure 2). The product-to-substrate ratios of metabolite levels from glycolytic and oxidative PPP (oxPPP) steps suggested glycolysis was favored and the later stage of the oxPPP was reduced in mouse models of SCD. There was also a higher metabolic turnover of glutathione, which is characteristic of SCD in humans, but we observed divergent metabolomic signatures between the disease models in our study (Figure 2). Conclusions: To our knowledge, this is the most comprehensive metabolomics study in mouse models of SCD. It provides a foundation to assess disease biology and response to treatment in future studies. Untargeted RBC metabolomics revealed metabolomic signatures that are consistent with human disease pathology in both murine SCD models, including reticulocytosis, hemolysis, and mitochondria retention in RBCs, and signatures that are unique to the murine models. Of the 2 main RBC metabolic pathways, glycolysis was favored over the oxPPP in SCD mice. This is the first step towards understanding the impact of SCD on RBC carbohydrate metabolism.

Ineffective Erythropoiesis Negatively Impacts the Erythroblastic Island in Sickle Cell Disease

Sickle cell disease (SCD) is a genetic recessive disorder, characterized by painful episodes of vaso-occlusion, chronic hemolytic anemia, and progressive organ failure. It is one of the most common and severe monogenic diseases worldwide. SCD is caused by a point mutation in the β-globin gene, leading to the expression of an abnormal hemoglobin (HbS) that polymerizes under hypoxic conditions, driving red blood cells (RBC) sickling. Peripheral RBCs are extensively studied in SCD, as hemolysis is the major contributor to anemia, but little is known about erythroid differentiation in this pathology. We recently demonstrated the occurrence of ineffective erythropoiesis in the bone marrow (BM) of SCD patients, characterized by the death of a significant proportion of erythroblasts between the polychromatic and the orthochromatic stages. We hypothesize that this increased cell death could negatively impact the central macrophages of the erythroblastic islands (EBIs) as they would engulf high numbers of hemoglobin-rich apoptotic erythroblasts and undergo ferroptosis because of excess iron. To investigate our hypothesis, we used an in vitro system whereby CD14 + monocytes were isolated from peripheral blood and cultured in media supplemented with M-CSF and dexamethasone. This led them to differentiate towards anti-inflammatory tissue macrophages, with an “EBI-like” phenotype, expressing markers such as CD16, CD163, CD169, CD206. Phagocytosis of high numbers of RBCs by “EBI-like” macrophages led to increased cell death, reactive oxygen species, and lipid peroxidation, and to the upregulation of genes involved in ferroptosis pathways such as HO-1 and SLC7A11. This was circumvented by the ferroptosis inhibitors ferrostatin-1 and liproxtatin-1. Furthermore, phagocytosis of apoptotic in vitro-generated SCD erythroblasts also induced ferroptosis in EBI-like macrophages, with increased cell death and lipid peroxidation in a time-dependent manner. Interestingly, while this was the case with hemoglobin-rich, terminally differentiating erythroblasts, phagocytosis of cells without hemoglobin, such as K562 or GPA - progenitor cells did not affect cell viability, indicating that the observed ferroptosis was due to the iron content of the internalized cells. We investigated the effect of increased RBC phagocytosis on the phenotype of surviving macrophages and found an M2 to M1 shift indicating a switch to a pro-inflammatory phenotype under the condition of elevated levels of intracellular iron and oxidative stress. We challenged these findings in vivo using the Townes humanized SCD mouse model. Data on 4-month-old mice showed the expected switch in erythroid production from steady-state erythropoiesis in the BM to a stress erythropoiesis in the spleen. This was associated with a decrease in the number of EBI macrophages (F4/80 HighVCAM+CD169+Ly6G-) in the BM of SS mice when compared to AS and AA mice, and an expansion of red pulp macrophages (RPMs) F4/80 HighCD169+VCAM+. Using a flow cytometry-based assay we found high lipid peroxidation levels in BM EBI macrophages of SS mice as compared to AA, suggesting occurrence of ferroptosis in these cells. Our study suggests that IE in SCD may contribute to anemia, directly by cell death of differentiating erythroblasts, and indirectly by disrupting the erythroblastic island or by inducing its collapse following ferroptosis of the central macrophage. Our work with the Townes mouse model and BM samples from SCD patients will help to elucidate this hypothesis.

Enhanced Expression of Heme Oxygenase-1 (HO-1) Among Children with Sickle Cell Disease: Results of the Sickle Cell Disease Genomics of Africa (SickleGenAfrica) Study

Sickle cell disease (SCD) is characterized by intense and chronic intravascular hemolysis that generates multiple erythroid danger associated molecular pattern (eDAMP) molecules in the extracellular space. Extracellular heme is a prototypical eDAMP that causes tissue injury and is associated with multiple complications of SCD including vaso-occlusive crisis, acute kidney injury and acute chest syndrome (ACS). Heme-oxygenase-1 (HO-1) is the rate-limiting enzyme that degrades heme to neutralize its deleterious effects on organ function. Raised extracellular heme is associated with 2.5 higher odds ratio of developing ACS, while HO-1 gene promoter allelic variants that drive robust HO-1 expression are associated with lower ACS-related hospitalization rates among children. Young transgenic homozygous SCD (SS) mice have higher survival rates than adult SS mice from a heme-induced ACS model due to their ability to rapidly degrade excess extracellular heme. While these data suggest that HO-1 may be involved in the well-established higher ACS survival among children, and potentially other ameliorating features of SCD in early development, no study had previously studied HO-1 expression in this patient population. The SickleGenAfrica Network enrolled over 2,000 children and adults with SCD from three sites in Ghana from 2018 to 2021, with consent for use of samples in future studies. The SCD status of participants was confirmed using automated capillary hemoglobin electrophoresis, and serum HO-1 level measured using ELISA. Patients were grouped based on age, using five-year intervals. The median HO-1 level comparisons across groups were performed using the pairwise Wilcoxon test, with Bonferroni correction for multiple comparisons. All analyses were performed using R version 4.3.1. The study included 1,971 patients with SCD, with a median age of 13 years (range 0 - 78), of which 1,067 participants (55%) were female. The majority of patients (1149 ;58%) had HbSS, 589 (30%) had HbSC, 171 (9%) sickle-hereditary persistence of fetal hemoglobin, and 62 (3%) HbS-beta(+). We observed an approximately 3-fold drop-off in median HO-1 levels between the 11-15-year-old (73.9 ng/ml) and 16-20-year-old (25.1 ng/ml) age groups (adj. p<0.001). Furthermore, males had higher average HO-1 levels (85.5 ng/ml) relative to females (63.1 ng/ml) (p<0.001). Our data show for the first time that children with SCD have significantly elevated HO-1 levels relative to adults, with a sharp decline in HO-1 levels during adolescence. HO-1 may serve as a protective SCD factor, contributing to enhanced ACS survival rates in children versus adults. To the best of our knowledge, this is the first study of HO-1 levels in patients with SCD at different ages. Our future work aims to establish whether baseline blood HO-1 levels can predict ACS incidence and severity, in addition to further investigating genetic and epigenetic mechanisms controlling HO-1 expression.

8-Oxoguanine DNA Glycosylase 1 Inhibition Suppresses Inflammatory Responses in Sickle Cell Disease

Background: The continuous production of reactive oxygen species (ROS) and oxidative stress contribute to ongoing inflammation and the pathology of sickle cell disease (SCD). ROS are known to mediate DNA damage through oxidation of nucleotide bases with guanine being the primary target. Although guanine base lesions vary according to the nature of the oxidants, 7,8-dihydro-8-oxoguanine (8-oxoG) is the most frequent oxidation product of DNA, which is repaired by 8-Oxoguanine-DNA glycosylase-1 (OGG1) via the DNA base excision repair pathway (OGG1-BER). The release of the 8-oxoG base from the genome by OGG1-BER is a prerequisite for increasing expression of pro-inflammatory cytokines and immune response. OGG1 inhibition has been shown to attenuate proinflammatory responses via the mitochondrial DNA-cGAS-STING axis in mice (Qin et al, 2020), a pathway also involved in the activation of neutrophils in SCD (Tumburu et al, 2021). Here, we evaluate the effect of OGG1 inhibition on expression of pro-inflammatory cytokines and immune response in SCD. Methods: Blood samples were obtained from ethnic-matched healthy controls (HbAA) and patients with SCD (HbSS) enrolled under protocols NCT03049475 and NCT00047996. CD14+ monocytes were isolated from PBMC of HbAA and HbSS subjects using EasySep™ Human Monocyte Isolation Kit (STEMCELL Technologies). 8-oxoG was measured by staining CD14+ monocytes with anti-8-oxoG antibody; OGG1 and pro-inflammatory cytokine levels were evaluated by qRT-PCR and western blotting. After differentiation, macrophages derived from CD14+ monocytes were treated with or without 10 µM SU0268 (OGG1 inhibitor) for 1 hour and then incubated with plasma from healthy controls or HbSS patients in steady or crisis status, followed by qRT-PCR quantitation of IL-1ꞵ, IL-6, TNF-α, and OGG1 mRNA levels. These measurements were repeated after OGG1 knockdown using OGG1 siRNA in macrophages derived from CD14+ monocytes. We repeated the study on peritoneal macrophages (PMs) from Townes HbAA and HbSS mice that were collected 4 days after a 3% Brewer's thioglycolate medium intraperitoneal injection. The level of 8-oxoG was measured by staining PMs with anti-8-oxoG antibody. The mRNA and protein level of OGG1 and pro-inflammatory cytokines were evaluated by qRT-PCR and western blotting. PMs from HbAA mice were treated with 10 µM SU0268 (OGG1 inhibitor) or vehicle for 1 hour, before incubation with plasma from HbAA or HbSS mice, the mRNA levels of IL-1ꞵ, IL-6, TNF-α, and OGG1 were measured by qRT-PCR. Results: CD14+ monocytes isolated from human HbSS PBMC showed increased levels of 8-oxoG nuclear staining when compared with human HbAA CD14+ monocytes, consistent with an increased oxidative burden in SCD patients (Fig 1A). The mRNA and protein levels of OGG1 were also increased in HbSS monocytes, suggestive of a potential therapeutic target of OGG1. Consistent with previous studies, mRNA levels of pro-inflammatory cytokines IL-1ꞵ, IL-6, and TNF-α were increased in SS compared with AA monocytes. To investigate the role of OGG1 in regulating cytokine expression, we treated the macrophages derived from CD14+ monocytes with SU0268, a selective OGG1 inhibitor (Tahara et al, 2018). Macrophages treated with plasma from HbSS patients (steady and crisis status) showed increased expression of OGG1, IL-1ꞵ, IL-6, and TNF-α compared with cells treated with normal plasma, and the increase was largely blocked by SU0268 pretreatment (Fig1B). Further, siRNA knockdown of OGG1 decreased the expression of IL-1ꞵ and TNF-α, confirming the role of OGG1 in regulating pro-inflammatory cytokine expression. We then evaluated the effect of OGG1 in regulating cytokines expression in cells form SCD mice. The levels of 8-oxoG and OGG1, as well as cytokines (IL-1ꞵ, IL-6, and TNF-α), were increased in PMs obtained from HbSS mice compared with macrophages obtained from HbAA mice. Similar to the results obtained from human macrophages, murine cells treated with plasma from SS mice showed increased expression of IL-1ꞵ, IL-6, TNF-α and OGG1 compared with cells treated with plasma from HbAA mice, and pretreatment with SU0268 largely blocked their increased expression. Conclusion: OGG1 has a regulatory role in the expression of pro-inflammatory cytokines in SCD. OGG1 inhibition could be a potential therapeutic approach to control sickle cell inflammation.

Exploring Mitochondrial DNA Heteroplasmy in a Mouse Model of Sickle Cell Disease

BACKGROUND: Mitochondria are important in the pathology of sickle cell disease (SCD). They are abnormally retained in sickle red blood cells (RBCs) and the likely source of cell-free mitochondrial DNA (mtDNA), an erythrocytic-DAMP that triggered formation of neutrophil extracellular traps (Tumburu et al, 2022). Acquired mutations in the mitochondrial genome, referred to mtDNA heteroplasmy, are emerging markers of aging and inflammation. Using whole genome sequence data from peripheral blood DNA, we previously established that patients with SCD not only have increased mtDNA heteroplasmy, but the burden also varied with the sickle genotype [Ahmad et. al, 2021]. The burden of mtDNA heteroplasmy is known to vary across different organs and tissues. Here, we utilized the Townes mouse model of SCD to explore differential mtDNA heteroplasmy burden across the different tissues and sickle genotypes in SCD. METHODS: Genomic DNA was extracted from 9 different organs (heart, brain, muscle, kidney, blood, bone marrow, liver, lungs, spleen) of littermate mice (3 male and 3 female) of three genotypes (HbAA, HbAS, HbSS) in three replica sets (total number of mice = 18, 9 different tissue samples per mouse, total sample number = 162). Mitochondria DNA was enriched from genomic DNA by long range PCR in 2 amplicons of 9kb and 7kb sizes (Misa Hirose et al, 2018). The enriched mtDNA PCR amplicons were mixed together and subjected to library preparation for deep sequencing using Illumina platform. Sequence reads aligned to mtDNA were mapped and called for variants using LoFreq variant calling tool. RESULTS: Of the 162 samples, 1 sample (HbAA, lung) was excluded from analysis due to low coverage. Using LoFreq variant caller tool (VAF cut-off 4% & Coverage Filter 3500X), and after removing the repeating variants, 11 unique variants spanning 16kb mouse mitochondrial genome, were found in 106 of the 161 (65.8%) samples. The variants were differentially distributed across the tissues and genotypes with the variant at mito-position 9820 (MT: 9820) being most frequent, present in 19, 16 and 18 samples in AA, AS and SS mice, respectively (Fig. 1). Two variants at MT: 11866 and MT: 6881 were shared between AA and AS mice. Four variants were unique to SS mice: MT: 6753 in COX1 gene causing Phe476-Leu substitution, MT: 6794 in intergenic region upstream to COX2 gene, MT: 1220 in intergenic region upstream to ND1 gene, and MT425 in regulatory/D-Loop region (Table -1). The SS-unique variants were present in all tissues except for Cox1 Phe476-Leu (MT: 6753), a single variant that was found only in the spleen. Overall, HbSS had the highest concentration of variants distributed among 41/54 (75.9%) followed by AS 38 /54 (70.3 %) and AA 27/53 (50.9%). Female mice had higher mtDNA heteroplasmy burden compared to male mice in all genotypes. Across all genotypes and tissues, spleen had the highest number of variants (16.03%) and liver, the lowest (9.4%). CONCLUSION: In keeping with data in patients with SCD, HbSS mice had the highest mtDNA heteroplasmic burden followed by HbAS and HbAA genotypes. Four mito-variants were unique to HbSS. We also found differentially higher mtDNA heteroplasmic burden in splenic tissues and speculate that this could arise from metabolic stress conditions in the spleen.

Red Blood Cell Pyruvate Kinase Properties in Sickle Cell Disease - of Mice and Men

Background: Townes and Berkeley mouse models of sickle cell disease (SCD) are commonly used to study SCD pathophysiology and to develop novel therapies. Although the pathophysiology in these models is similar to the human disease, there are distinct differences. For example, Townes SS mice have higher ATP and lower 2,3-diphosphyglycerate (2,3-DPG) levels compared to Townes AA mice. This contrasts with SCD patients, who show lower ATP and higher 2,3-DPG levels, suggesting potential differences between human and murine red blood cell (RBC) metabolism. Pyruvate kinase (PK), a key ATP-generating enzyme in glycolysis, is a target for novel SCD therapies. While PK activators have been shown to increase hemoglobin in clinical trials, such treatments do not consistently lead to the same hematological change in mouse models, further indicating that PK properties may differ between SCD mouse models and human patients. A detailed comparative characterization of PK and glycolytic metabolites in murine SCD models and SCD patients is therefore important. Aim: To study PK properties in murine and human SCD RBCs. Methods: Wild-type (C57BL/6J), Townes (AA and SS) and Berkeley mice (non-sickling and sickling) were used. Human blood was obtained from healthy controls (HbAA) and untreated SCD patients (HbSS). Hematological parameters were measured using the Sysmex hematology analyzer. PK (Vmax, 5mM substrate) and hexokinase (HK) activity, and PK thermostability (53°C) were measured in purified RBCs. Levels of ATP, 2,3-DPG, as well as reduced glutathione (GSH) were measured by targeted LC-MS/MS and spectrophotometry, respectively. P50 (oxygen tension at which 50% of hemoglobin is saturated with oxygen) was measured with the Hemox analyzer (TCS). Statistical analysis was performed in Graphpad Prism 9.4.1. by Welch's t-test. Results: Samples from 17 wild-type mice, 21 Townes mice (10 AA, 11 SS), 15 Berkeley mice (7 non-sickling, 8 sickling) and 12 human subjects (6 HbAA, 6 HbSS) were included. Both Townes and Berkeley SCD mice showed increased PK activity (Table 1; p<0.0001). This may be attributed to their high reticulocyte counts (Townes SS vs. AA: 43% vs. 4%; Berkeley sickling vs. non-sickling: 30% vs. 6%), because when normalizing PK activity to that of HK, another RBC age-dependent enzyme, no genotype differences were found in these mouse models. This lack of PK/HK difference was in sharp contrast with our finding in human controls vs. SCD patients (Table 1). Notably, both Berkeley sickling and non-sickling mice had lower PK/HK ratio than their Townes counterparts (p<0.0001). Mouse PK thermostability was markedly reduced compared to that of human, and PK from the two sickling mice was less stable than their non-sickling controls (Fig. 1). Moreover, both sickling mice showed higher ATP/2,3-DPG ratio (Table 1; p<0.01). This was again in direct contrast with our observation using human blood, where SCD patients had lower ATP/2,3-DPG ratio than healthy controls (p<0.05). Even though there was no difference in ATP/2,3-DPG ratio between the two SCD models, Townes SS mice had significantly lower 2,3-DPG and higher ATP levels than Townes AA mice, whereas Berkeley sickling mice only had significantly higher ATP level compared to their controls. Furthermore, sickling mice had lower P50 value compared to non-sickling mice, reflecting a higher oxygen affinity. This also contrasted with human SCD patients, who had higher P50 value than healthy controls (Table 1). GSH levels were increased in both SCD patients and sickling mice, although the elevation was more pronounced in sickling mice (Table 1). Conclusions: This study reveals important distinctions between mouse models and humans: no differences in PK/HK ratio between sickling and non-sickling mice, and an overall lower PK thermostability in mice. Furthermore, this study shows significant changes in 2,3-DPG content (important for oxygen affinity), energy level (ATP), oxygen affinity (P50) and antioxidant level (GSH) when comparing sickling to non-sickling mice. Sickling mice also have higher ATP/2,3-DPG ratio, which contrasts with SCD patients. These differences should be considered when studying PK activators in SCD mouse models and extrapolating results from SCD mouse models to humans.

Rhabdomyolysis aggravates renal iron accumulation and acute kidney injury in a humanized mouse model of sickle cell disease

Individuals with sickle cell disease (SCD) are at greater risk of rhabdomyolysis, a potentially life-threatening condition resulting from the breakdown of skeletal muscle fibers. Acute kidney injury (AKI) is one of the most severe complications of rhabdomyolysis. Chronic kidney and cardiovascular disease, which account for SCD mortality, are long-term consequences of AKI. Although SCD elevates the risks of rhabdomyolysis-induced sudden death, the mechanisms that underlie rhabdomyolysis-induced AKI in SCD are unclear. In the present study, we show that, unlike their control non-sickling (AA) counterparts, transgenic homozygous SCD (SS; Townes model) mice exhibited 100% mortality 8–24 h after intramuscular glycerol injection. Five hours after glycerol injection, SS mice showed a more significant increase in myoglobinuria and plasma creatine kinase levels than AA mice. Basal plasma heme and kidney tissue iron levels were significantly higher in SS than in AA mice. In contrast to AA, glycerol-induced rhabdomyolysis aggravated these parameters in SS mice. Rhabdomyolysis also amplified oxidative stress in SS compared to AA mice. Glycerol-treated SS mice exhibited worse renal function, exemplified by a reduction in GFR with a corresponding increase in plasma and urinary biomarkers of early AKI and renal tubular damage. The free radical scavenger and Fenton chemistry inhibitor, TEMPOL, ameliorated rhabdomyolysis-induced AKI in the SS mice. These findings demonstrate that oxidative stress driven by renal iron accumulation amplifies rhabdomyolysis-induced AKI in SCD mice.

Impaired post-stroke collateral circulation in sickle cell anemia mice.

Patients with sickle cell anemia (SCA) have a high incidence of ischemic stroke, but are usually excluded from thrombolytic therapy due to concerns for cerebral hemorrhage. Maladaptation to cerebral ischemia may also contribute to the stroke propensity in SCA. Here we compared post-stroke cortical collateral circulation in transgenic sickle (SS) mice, bone marrow grafting-derived SS-chimera, and wildtype (AA) controls, because collateral circulation is a critical factor for cell survival within the ischemic penumbra. Further, it has been shown that SS mice develop poorer neo-collateral perfusion after limb ischemia. We used the middle cerebral artery (MCA)-targeted photothrombosis model in this study, since it is better tolerated by SS mice and creates a clear infarct core versus peri-infarct area. Compared to AA mice, SS mice showed enlarged infarction and lesser endothelial proliferation after photothrombosis. SS-chimera showed anemia, hypoxia-induced erythrocyte sickling, and attenuated recovery of blood flow in the ipsilateral cortex after photothrombosis. In AA chimera, cerebral blood flow in the border area between MCA and the anterior cerebral artery (ACA) and posterior cerebral artery (PCA) trees improved from 44% of contralateral level after stroke to 78% at 7 d recovery. In contrast, blood flow in the MCA-ACA and MCA-PCA border areas only increased from 35 to 43% at 7 d post-stroke in SS chimera. These findings suggest deficits of post-stroke collateral circulation in SCA. Better understanding of the underpinnings may suggest novel stroke therapies for SCA patients.

A mass spectrometry assay for detection of endogenous and lentiviral engineered hemoglobin in cultured cells and sickle cell disease mice.

Sickle cell disease (SCD) results from a sequence defect in the β-globin chain of adult hemoglobin (HbA) leading to expression of sickle hemoglobin (HbS). It is traditionally diagnosed by cellulose-acetate hemoglobin electrophoresis or high-performance liquid chromatography. While clinically useful, these methods have both sensitivity and specificity limitations. We developed a novel mass spectrometry (MS) method for the rapid, sensitive and highly quantitative detection of endogenous human β-globin and sickle hβ-globin, as well as lentiviral-encoded therapeutic hβAS3-globin in cultured cells and small quantities of mouse peripheral blood. The MS methods were used to phenotype homozygous HbA (AA), heterozygous HbA-HbS (AS) and homozygous HbS (SS) Townes SCD mice and detect lentiviral vector-encoded hβAS3-globin in transduced mouse erythroid cell cultures and transduced human CD34+ cells after erythroid differentiation. hβAS3-globin was also detected in peripheral blood 6 weeks post-transplant of transduced Townes SS bone marrow cells into syngeneic Townes SS mice and persisted for over 20 weeks post-transplant. As several genome-editing and gene therapy approaches for severe hemoglobin disorders are currently in clinical trials, this MS method will be useful for patient assessment before treatment and during follow-up.