Injury In Sickle Cell Disease Research Articles

Effects of Rifaximin on Intestinal Pathophysiologic Changes Associated with Sickle Cell Disease (SCD)

INTRODUCTION Sickle cell disease (SCD) is characterized by episodes of painful vaso-occlusive crises (VOC) and progressive end-organ damage, resulting in poor quality of life and a shortened lifespan. Activated and aged neutrophils are pivotal in pathogenesis of VOC. SCD patients exhibit pathophysiologic changes in the intestine. We previously reported intestinal dysbiosis (Lim et al, Am J Hematol, 2016), and increased intestinal injury and bacterial translocation in SCD patients (Dutta et al, J Transl Med, 2019). These patients also exhibited elevated levels of activated and circulating aged neutrophils (CANs) (Dutta et al, J Transl Med, 2019). Since rifaximin is minimally absorbed into the systemic circulation, we sought to determine how the local effects of rifaximin affects intestinal pathophysiologic changes and circulating neutrophils in SCD patients. We investigated these changes in a cohort of SCD patients accrued to a Phase 2 study of rifaximin for SCD (Clinicaltrials.gov: NCT03719729). METHODS Twelve SCD patients (5 males, 7 females) with a median age 29 years (range 23-56) received 550 mg rifaximin twice daily for 6 months. Blood, urine and stool samples were collected from patients in steady state before and at different intervals during rifaximin treatment. Intestinal microbiome was evaluated by 16S rRNA gene sequence analysis. The following fluorophore-conjugated monoclonal antibodies were used to evaluate CANs: Gr-1-FITC, CD115-PE-Cy7, CXCR4-PE and CD62L-APC. Flow cytometry was performed using the Moflo XDP Sorter and neutrophils gated by Gr-1Hi CD115Lo SSCHi, and CANs were gated by CXCR4Hi CD62LLo within the neutrophil population. Propidium iodide, FSC and SSC were used to exclude dead cells. Urinary 3-indoxyl sulfate (3-IS), and serum soluble CD62L, intestinal fatty acid binding protein (iFABP) and lipopolysaccharide (LPS) were evaluated in triplicates by ELISA using commercially available ELISA kits. RESULTS Rifaximin treatment resulted in a decrease in the intestinal injury, as shown by significant drops in the serum iFABP (p = 0.008) (Figure 1a). The improvement in intestinal integrity was observed as early as two weeks into rifaximin therapy. There were also corresponding decreases in the serum LPS levels (p = 0.01) (Figure 1b) and they also occurred as early as two weeks into rifaximin therapy. Both parameters attained levels comparable to those we previously observed in controls who had similar degree of anemia but without SCD. LPS is one of the pathogen-associated molecular patterns (PAMPs) that trigger toll-like receptors 2/4 (TLRs 2/4) and myeloid differentiation factor 88 (Myd88). Since TLRs2/4 and Myd88 are known to drive neutrophil aging, we next examined changes in CANs. Following rifaximin therapy, CANs decreased significantly in these patients (p = 0.005) (Figure 1c). The decrease was associated with a parallel decline in the number of activated neutrophils, as shown by declines in the serum CD62L (p = 0.0002) (Figure 1d). Unlike CANs, however, the drops in the serum CD62L were not as consistent in some patients and these inconsistencies corresponded with recovery from VOC, highlighting differences in the origins of neutrophil ageing and activation. Not surprisingly, rifaximin alters the profile of the intestinal metabolomics. Rifaximin also resulted in a decrease in urinary 3-IS (p = 0.01) (Figure 1e), being compatible with a narrowing of the intestinal microbial diversity and suggests a reduction in Clostridiales, the major producers of intestinal short-chain fatty acids such as butyrate. CONCLUSION Rifaximin therapy results in improvement in the intestinal pathophysiologic changes observed in SCD. It also reduces the neutrophil subsets pivotal for the pathogenesis of VOC. However, these are achieved in association with a reduction in the intestinal microbial diversity. Further works are needed to delineate the mechanisms rifaximin reduces intestinal injury in SCD. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Rifaximin for sickle cell disease

Impaired Bile Secretion Promotes Chronic Liver Injury in Sickle Cell Disease

Hepatic crisis is an emergent complication affecting sickle cell disease (SCD) patients, however, the molecular mechanism of sickle cell hepatobiliary crisis remains poorly understood. We examined the liver pathophysiology of SCD using a humanized mouse model (townes SCD mice; homozygous for Hbatm1(HBA)Tow, homozygous for Hbbtm2(HBG1,HBB*)Tow). These mice have the major features (irreversibly sickled red cells, splenomegaly, anemia, multiorgan pathology) found in humans with SCD and, as such, represent a useful in vivo system to study hepatobiliary changes in SCD disease. SCD mice manifested progressive hepatomegaly, liver injury and hyperbilirubinemia. RNA-sequence analysis of total RNA from SCD mouse liver compared to control (AS) identified dysregulation of genes encoding proteins responsible for fibrosis, bile acid synthesis, bile transport and cholesterol metabolism. Immunohistochemical analysis confirmed inflammation, fibrosis and increased ductular reaction in SCD mice. To mechanistically address the cholestatic phenotype in SCD mice, we used our recently developed multi-photon-excitation (MPE) enabled in vivo real-time fluorescence microscopy of intact liver in live mice. We used Texas-Red (TXR) dextran to visualize the blood flow through liver sinusoids and carboxyfluorescein (used as a surrogate of bile flow) to visualize bile flow through biliary canaliculi. Real time imaging show that sinusoidal ischemia occurs in the liver of transgenic-humanized SCD mice under basal condition. Intravital imaging also revealed impaired bile secretion into the bile canaliculi, which was associated with loss of apical bile acid transporters and bile acid biosynthetic enzymes, hepatic bile accumulation, and activation of Farnesoid X receptor (FXR) and small heterodimer partner (Shp) in the liver of SCD mice. These findings are the first to identify that impaired bile acid synthesis and misexpression of bile transporters promote intrahepatic bile accumulation and impaired bile secretion, leading to hepatobiliary injury of SCD. Improved understanding of these processes could potentially benefit the development of new therapies to treat sickle cell hepatic crisis. Disclosures Gladwin: Globin Solutions, Inc: Patents & Royalties: Provisional patents for the use of recombinant neuroglobin and heme-based molecules as antidotes for CO poisoning; Bayer Pharmaceuticals: Other: Co-investigator; United Therapeutics: Patents & Royalties: Co-inventor on an NIH government patent for the use of nitrite salts in cardiovascular diseases . Kato:Novartis, Global Blood Therapeutics: Consultancy, Research Funding; Bayer: Research Funding.

Circulating Neutrophil Extracellular Traps in the Pathogenesis of Acute Chest Syndrome of Sickle Cell Disease

Introduction: Acute chest syndrome (ACS) is a type of acute lung injury and among the primary reasons for mortality and morbidity among Sickle Cell Disease (SCD) patients. Although epidemiologic evidence suggests that vaso-occlusion in the lung may serve as an antecedent to ACS, the cellular, molecular and biophysical mechanism of ACS is incompletely understood. Our recent findings revealed that the lung vaso-occlusion is enabled by the entrapment of embolic neutrophil-platelet aggregates in the pulmonary arterioles of transgenic humanized SCD mice. Recent evidence also suggests a role for neutrophil extracellular traps (NETs) in ACS. NETs are web-like structures of decondensed nuclear DNA decorated with citrullinated-histones (H3-cit) and neutrophil granule proteins. Interestingly, circulating nucleosomes and NETs fragments are elevated in SCD patient blood and the levels correlate with onset of ACS, however, the molecular mechanism that promotes generation of circulating NETs and the role of circulating NETs in promoting ACS remains poorly understood. Materials and Methods: Townes knock-in humanized SS (hα/hα:βS/βS) and AS (hα/hα:βA/βS) mice were used as SCD and control mice, respectively. SS and AS mice were intravenously (IV) administered 10 µmole/kg Oxy-Hb followed by Sytox orange, FITC-dextran or fluorescent anti-mouse mAbs against Ly6G, CD49b, H3cit, and neutrophil elastase for in vivo visualization of extracellular DNA, blood vessels, neutrophils, platelets and NETs, respectively. Pulmonary microcirculation was monitored using multi-photon-excitation enabled quantitative fluorescence intravital lung microscopy (qFILM). Results and Discussion: IV Oxy-Hb triggered the occlusion of pulmonary arterioles by neutrophil-platelet aggregates leading to loss of pulmonary blood flow in SCD but not control mice. Surprisingly, pulmonary vaso-occlusion in SCD mice was accompanied by the arrival of circulating cell free DNA (CFD) and NETs fragments into the pulmonary circulation. The cell free DNA (CFD) and NETs fragments entered the lung through the arterial circulation suggesting that they originated outside of lung. These cell free DNA (CFD) and NETs fragments contributed to lung vaso-occlusion and injury by promoting neutrophil-platelet aggregation in the lung arterioles. Conclusion: These findings reveal for the first time that circulating cell free DNA (CFD) and NETs fragments originating outside of lung contribute to pathogenesis of ACS. Currently, experiments are underway to identify the innate immune pathways that promote circulating NETs dependent lung injury in SCD. Disclosures Gladwin: Globin Solutions, Inc: Patents & Royalties: Provisional patents for the use of recombinant neuroglobin and heme-based molecules as antidotes for CO poisoning; United Therapeutics: Patents & Royalties: Co-inventor on an NIH government patent for the use of nitrite salts in cardiovascular diseases ; Bayer Pharmaceuticals: Other: Co-investigator.

Proteomic Discovery: Elevated Neurogranin Levels in Children with Sickle Cell Disease

Sickle cell disease (SCD) is an inherited hemoglobinopathy that frequently causes neurological complications, such as stroke, silent cerebral infarct (SCI) and other forms of brain injury, including loss of cognition. SCI is defined as any ischemic lesion visible on T2-weighted magnetic resonance imaging (MRI) that is not associated with a focal neurologic deficit in the same vascular distribution, and is the most commonly recognized form of brain injury in SCD. SCI is associated with decreased neurocognitive function and increased risk for new or enlarging SCI/stroke. The purpose of this study was to use mass spectrometry (MS) based proteomics to discover and validate plasma brain proteins associated with brain injury in children with SCD. The plasma samples used for the proteomics discovery analyses were from two groups: screening samples of participants in the Silent Cerebral Infarct Multi-Center Clinical Trial (SIT Trial) (n=15), and 6 age-matched, healthy non-SCD controls, three of whom had sickle cell trait. Samples from children with SCD were divided into two groups: those with SCI (n=7) and those without SCI (n=8) matched for age, hemoglobin (Hb) and white blood cell counts. To identify circulating markers of SCI, plasma proteomes were analyzed using a sequential separation approach of Hb and top abundant plasma protein depletion, followed by reverse phase separation of intact proteins, trypsin digestion and tandem MS. Neurogranin (NRGN), a small calmodulin-binding synaptic protein (PMID 9886843), was the most abundant brain-enriched protein found in the plasma of children with SCD. We developed a NRGN sandwich immunoassay for verification of MS analysis results in an independent cohort from the SIT Trial longitudinal samples. For this verification study, plasma samples from the SIT Trial and 25 normal controls were tested for NRGN levels at study entry and exit by immunoassay. The majority of the SCD samples were obtained from an ancillary study to collect longitudinal samples that started after the SIT Trial had begun. There was no overlap between the discovery and the verification group samples. The normal control samples were from 25 healthy, age and gender comparable non-SCD pediatric controls unrelated to the SIT Trial, without evidence of acute/chronic illness. From the discovery SCD group, 1172 unambiguous proteins were identified; 639 proteins were identified from normal controls, as shown in Figure 1. Twenty-five percent (289/1172) of these proteins were found solely in the SCI group. Our MS protein identification data were filtered against this list of expressed brain proteins to produce a composite list of brain proteins found in plasma from children with SCD, but not found in plasma from age, gender and race-matched healthy control children. Twenty-five plasma proteins known to be expressed in one or more cell type enriched in the human brain based on bioinformatics searches were more abundant in the SCD vs control groups, as shown in Table 1. Median NRGN levels were higher at study entry in children with SCD (0.44 ng/mL, N=98), in comparison to a healthy, non-SCD control group (0.12 ng/mL, N=25, p < 0.0001), as shown in Figure 2. Using the longitudinal samples from the SIT Trial, there were no differences in NRGN levels between the observation group and the treatment group (p=0.35) over time. There was no significant difference in median NRGN levels between the observation group and the treatment group at the initial visit (0.36 vs 0.19, p=0.069) or final visit (0.39 vs 0.52, p=0.52). Using the initial visit samples from the SIT Trial, there was no significant difference in median NRGN levels between the non-SCI (n=35) and SCI groups (n=61) (p=0.073). There was no significant correlation between NRGN levels and neuropsychological measures of executive function (rho=0.089, p=0.58) and IQ (rho=-0.081, p=0.62). Our non-biased proteomic discovery studies demonstrate that NRGN levels are increased in children with SCD, when compared to normal children. Additional studies will be necessary to determine whether NRGN levels correlate with specific forms of neurologic injury. Further validation studies of larger cohorts with a multi-analyte ELISA of these brain proteins identified in plasma (Table 1) are warranted, and could help establish possible links between brain-specific proteins and neurocognitive outcomes. Disclosures Lance: NIH: Research Funding; KKI: Research Funding. Faulcon:FDA: Employment, Other: Although the author is a FDA/CTP employee, this work was not done as part his/her official duties. This publication reflects the views of the author and should not be construed to reflect the FDA/CTP's views or policies.. Fu:GSK: Employment. Yang:ImmunArray: Patents & Royalties: ImmunArray. Strouse:Global Blood Therapeutics: Consultancy. Barron-Casella:Mast Pharmaceudicals - spouse: Consultancy; ImmunArray - spouse: Patents & Royalties. Van Eck:ImmunArray: Consultancy, Patents & Royalties. Casella:NIH: Research Funding; Mast Pharmaceuticals: Consultancy; Immunoarray Ltd.: Patents & Royalties. Everett:ImmunArray: Consultancy, Patents & Royalties.

Identification of a Heme Activation Site on the MD-2/TLR4 Complex

Lipopolysaccharide (LPS), the first-identified TLR4 agonist, binds myeloid differentiation factor-2 (MD-2) in association with TLR4 to initiate TLR4 signaling. LPS binds to a large hydrophobic pocket in MD-2 and directly bridges the MD-2/TLR4 heterodimer. The MD-2/TLR4 complex also recognizes a diverse number of endogenous molecules released from injured cells called damage-associated molecular patterns or DAMPs. One such DAMP is heme. Large amounts of heme can be released intravascularly by trauma, sepsis, malaria and red blood cell disorders such as sickle cell disease (SCD). Recent studies underscore the importance of heme-mediated MD-2/TLR4 activation in inflammation, vessel occlusion, lethality and pulmonary injury in SCD. Therefore, we examined human MD-2 for potential heme activation sites. Recombinant MD-2 (rMD-2) was produced by transfecting Chinese hamster ovary (CHO) cells with human MD-2 plasmids. After 72 hours, Western blots of the CHO-conditioned media demonstrated soluble rMD-2 was present. Heme was shown to bind rMD-2 using pull-down assays utilizing heme-agarose or biotin-heme with streptavidin-agarose coupled with MD-2 Western blots of the pellet. These pull-down assays of rMD2 were inhibited by excess heme, indicating specific binding of heme to rMD-2. UV/visible scanning spectroscopy (250 - 550 nm) of purified rMD-2 in the presence or absence of heme, confirmed specific rMD-2-heme binding. In silico analyses combining both structure and sequence-based methods, identified two potential heme docking sites on MD-2 near conserved amino acids W23/S33/Y34 and Y36/C37/I44 (Figure 1). To determine whether MD-2 mutations at these two sites affect heme-MD-2/TLR4 signaling, HEK293 cells were transfected with plasmids encoding human MD-2, TLR4, CD14 and an NF-κB luciferase reporter. After 24 hours, transfected cells were stimulated with heme (10 μM) or LPS (10 ng/ml) for 6 hours and NF-κB luciferase reporter activity was measured. Heme or LPS treatment elicited robust luciferase activity. The addition of both heme and LPS had an additive effect on NF-κB luciferase activity. Absence of an MD-2, TLR4 or CD14 plasmid abolished NF-κB luciferase reporter responses to heme and/or LPS. When plasmids encoding MD-2 point mutants W23A or Y34A were introduced into MD-2, heme-induced NF-κB luciferase activity was inhibited 91-92% compared to WT-MD-2. The S33A MD-2 mutant stimulated NF-κB luciferase activity by 40%. NF-κB activation by LPS was marginally affected by the same mutants. Biotin-heme/streptavidin-agarose pulled down 68% less W23A mutant MD-2 and 80% less W23A/S33A/Y34A mutant MD-2 than WT-MD-2. In contrast, at the other potential heme binding site, heme-induced NF-κB luciferase activity was increased in mutants Y36A (120%), C37A (121%) and I44A (230%) compared to WT-MD-2. These data suggest that amino acids W23 and Y34 on MD-2 are specific for heme binding and TLR4 signaling. This heme activation site was targeted for potential inhibitors using virtual screening. The virtual screen identified 60 potential inhibitors for screening in heme-stimulated primary human umbilical vein endothelial cells (HUVEC) and a human U-937 monocyte cell line. Four of these molecules inhibited Weibel-Palade body P-selectin and von Willebrand factor expression in HUVEC and IL-8 secretion by U-937 cells stimulated with heme. We conclude that heme activates MD-2/TLR4 signaling at residues W23 and Y34 on MD-2, which might be a drugable target in SCD and other hemolytic diseases. Disclosures Belcher: Mitobridge, an Astellas Company: Consultancy, Research Funding. Vercellotti:Mitobridge, an Astellas Company: Consultancy, Research Funding.

Acute hydroxyurea treatment reduces tubular damage following bilateral ischemia-reperfusion injury in a mouse model of sickle cell disease

Ischemic injury is a primary contributor to the initiation of renal tubular epithelial cell damage in sickle cell disease (SCD). In this study, we investigated the effects of bilateral ischemia-reperfusion injury, which is a common type of acute kidney injury (AKI), in male and female genetic mouse model of SCD. Bilateral occlusion of both renal hila for 21 min led to a significantly higher detection of established serum markers of AKI (creatinine, KIM-1 and NGAL) compared to sham-operated male SCD mice. Severe damage to the outer medullary tubules was determined in the ischemia-reperfision injury (IRI)-treated SCD male mice. In female SCD mice with a longer ischemic time (23 min), the serum markers of AKI were not as highly elevated compared to their male counterparts, and the extent of outer medullary tubular injury was less severe. To assess the potential benefit in the use of hydroxyurea (50 mg/kg IP) following bilateral renal IRI, we observed that the serum markers of AKI and the outer medullary tubular damage were markedly improved compared to male SCD mice that were not treated with hydroxyurea. In this study, we confirmed that male SCD mice were more susceptible to increased tubular damage and a loss in renal function compared to female SCD mice, and that hydroxyurea may partially prevent the extent of tubular injury following severe ischemia-reperfusion injury in SCD.

Depletion of haptoglobin and hemopexin promote hemoglobin-mediated lipoprotein oxidation in sickle cell disease.

Intravascular sickling and lysis of red blood cells, a hallmark feature of sickle cell disease (SCD), releases hemoglobin (Hb) into the circulation. Increased cell-free Hb has been linked to vasculopathy and in vitro lipid oxidation. Scavenger plasma proteins haptoglobin (Hp) and hemopexin (Hpx) can attenuate cell-free Hb and total plasma heme lipid-oxidative capacity but are depleted in SCD. Here, we isolated lipids from BERK-SS mice, guinea pigs (GP) infused with heme-albumin, and patients with SCD undergoing regular exchange transfusion therapy and evaluated the level of lipid oxidation. Malondialdehyde formation, an end product of lipid peroxidation, was increased in BERK-SS mice, purified lipid fractions of the heme-albumin infused GP, and patients with SCD compared with controls. In humans, the extent of lipid oxidation was associated with the absence of Hp as well as decreased Hpx in plasma samples. Postmortem pulmonary tissue obtained from patients with SCD demonstrated oxidized LDL deposition in the pulmonary artery. The relationship between no Hp and low Hpx levels with greater LDL and HDL oxidation demonstrates the loss of protection against cell-free Hb and total plasma heme-mediated lipid oxidation and tissue injury in SCD. Strategies to protect against plasma lipid oxidation by cell-free Hb and total plasma heme (e.g., therapeutic Hp and Hpx replacement) may diminish the deleterious effects of cell-free Hb and total plasma heme toward the vascular system in SCD.

Elevated Proangiogenic Markers are Associated with Vascular Complications within Ghanaian Sickle Cell Disease Patients.

Sickle cell disease (SCD) is an inherited blood disorder that can result in vasculopathy and end organ damage. Angiogenesis has been implicated as a key contributing factor to vascular mediated tissue injury in SCD. The relative plasma levels of angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), and vascular endothelial growth factor (VEGF) greatly influence angiogenesis. Dysregulation of these growth factors, leading to a pro-angiogenic state in SCD patients, has been documented in the developed world but there is very little data in Africa. There is the need, therefore, for studies in Ghanaian SCD patients. The aim of this study was to assess plasma levels of Ang-1, Ang-2, and VEGF in homozygous (HbSS) SCD patients with or without complications and healthy controls (HbAA) in Ghana. The study was a case-control study involving 544 participants: 396 HbSS SCD patients and 148 HbAA healthy controls. The study was conducted at the Center for Clinical Genetics (Sickle Cell Clinic) and Accra Area Blood Centre for National Blood transfusion at the Korle-Bu Teaching Hospital, Accra, Ghana. The plasma levels of Ang-1, Ang-2, and VEGF of study participants were measured with a double sandwich enzyme-linked immunosorbent assay (ELISA) technique. Complete blood count (CBC) was measured with an autoanalyser. The mean plasma Ang-1, Ang-2, and VEGF were significantly higher in HbSS SCD patients with or without complications than healthy controls (p < 0.001). The Ang-2/Ang-1 ratio was significantly lower in the controls than the HbSS patients (p < 0.001). The Ang-2/Ang-1 ratio was higher in the HbSS patients with leg ulcers as compared with patients with other complications and healthy controls (p < 0.001). There were higher leucocyte counts in HbSS patients than healthy controls. Overall, there was elevated plasma levels of Ang-1, Ang-2, and VEGF in SCD patients. The higher Ang-2/Ang-1 plasma levels in patients with leg ulcers suggests a possible ongoing angiogenesis and response to inflammatory stimuli. The study provides a first report on plasma levels of angiopoietin-1, angiopoietin-2, and vascular endothelial growth factors in homozygous sickle cell disease patients in Ghana.

GBT440 reverses sickling of sickled red blood cells under hypoxic conditions in vitro.

Sickle cell disease is characterized by hemolytic anemia, vasoocclusion and early mortality. Polymerization of hemoglobin S followed by red blood cell sickling and subsequent vascular injury are key events in the pathogenesis of sickle cell disease. Sickled red blood cells are major contributors to the abnormal blood rheology, poor microvascular blood flow and endothelial injury in sickle cell disease. Therefore, an agent that can prevent and or reverse sickling of red blood cells, may provide therapeutic benefit for the treatment of sickle cell disease. We report here that GBT440, an anti-polymerization agent being developed for the chronic treatment of sickle cell disease, increases hemoglobin oxygen affinity and reverses in vitro sickling of previously sickled red blood cells under hypoxic conditions. Our results suggest that besides preventing sickling of red blood cells, GBT440 may mitigate vasoocclusion and microvascular dysfunction by reversing sickling of circulating sickled red blood cells in vivo.

Apolipoprotein L1 Genetic Variants Are Associated with Evidence of Early Kidney Injury in Sickle Cell Disease

▪Background and Objectives: Apolipoprotein L1 (APOL1) renal risk variants prevalent in African-ancestry populations are associated with chronic kidney disease (CKD). CKD is a major cause of morbidity and mortality in sickle cell disease (SCD) in adults; yet the prevalence and significance of APOL1 renal-risk variants in this population remains unknown. Our objective was to determine expression of biomarkers of early kidney disease in African American youth with SCD, based on their APOL1 genotype.Methods:We enrolled65 African American subjects between 5 to 21 years of age with SCD (Hb SS or Hb S β0 thalassemia). Blood and concurrent urine samples were collected. Allenrolled subjects underwent APOL1 genotyping by PCR analysis of DNA extracted from whole blood. Presence of two renal-risk variants qualified for APOL1 High Risk (HR) genotype, while presence of zero or one copy of renal-risk variants qualified for APOL1 Low Risk (LR) genotype.Results: APOL1 HR genotype was expressed by 22% of our subjects; this is comparable to 23% prevalence of APOL1 HR genotype in African American adults with CKD (AASK study). Both groups were similar with respect to distribution of age, BMI z-scores, renal function and urine osmolality. Hyperfiltration was noted in both groups based on low serum creatinine for age, and elevated eGFR based on creatinine clearance. However, median urine albumin excretion rate was significantly higher in HR group vs. LR group.Conclusions: Preliminary data in this cohort of 65 children and adolescents with SCD detected greater urine albumin excretion rate in the presence of APOL1 HR genotype. Screening SCD youth for heightened CKD risk may be an avenue to initiate targeted preventive interventions to preserve renal function and reduce progression of kidney disease.Acknowledgment: Research reported in this publication was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number P20GM109021; National Kidney Foundation Young Investigator Grant and DE-CTR-ACCEL grant number U54-GM104941 (PI: Binder-Macleod). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Graph Lasso-Based Test for Evaluating Functional Brain Connectivity in Sickle Cell Disease.

Sickle cell disease (SCD) is a vascular disorder that is often associated with recurrent ischemia-reperfusion injury, anemia, vasculopathy, and strokes. These cerebral injuries are associated with neurological dysfunction, limiting the full developing potential of the patient. However, recent large studies of SCD have demonstrated that cognitive impairment occurs even in the absence of brain abnormalities on conventional magnetic resonance imaging (MRI). These observations support an emerging consensus that brain injury in SCD is diffuse and that conventional neuroimaging often underestimates the extent of injury. In this article, we postulated that alterations in the cerebral connectivity may constitute a sensitive biomarker of SCD severity. Using functional MRI, a connectivity study analyzing the SCD patients individually was performed. First, a robust learning scheme based on graphical lasso model and Fréchet mean was used for estimating a consistent descriptor of healthy brain connectivity. Then, we tested a statistical method that provides an individual index of similarity between this healthy connectivity model and each SCD patient's connectivity matrix. Our results demonstrated that the reference connectivity model was not appropriate to model connectivity for only 4 out of 27 patients. After controlling for the gender, two separate predictors of this individual similarity index were the anemia (p = 0.02) and white matter hyperintensities (WMH) (silent stroke) (p = 0.03), so that patients with low hemoglobin level or with WMH have the least similarity to the reference connectivity model. Further studies are required to determine whether the resting-state connectivity changes reflect pathological changes or compensatory responses to chronic anemia.

Cytoprotective Chaperone Proteins Are Novel Anti-Inflammatory Targets in Sickle Cell Disease

Sickle cell disease (SCD) is a pro-inflammatory condition. Levels of TNF-α, IL-6, IL-8, and IL-10 are elevated. There is clear evidence of endothelial cells (EC) dysfunction, and increased leukocyte, and erythrocyte adhesion in patients even in the non-crisis "steady state" condition. Additional insult, either via infection or vaso-occlusive ischemia, induce a dramatic increase in inflammation and EC dysfunction in SCD. Furthermore, there is a kindling of coagulation activation in patients with SCD. We, and others, have reported elevated levels of thrombin and monocyte tissue factor (TF) expression in patients. Both thrombin and monocyte TF expression increase during acute clinical events. In addition to the chronic impairment of lung function, acute chest syndrome (ACS) adds further insult to lung and cardiovascular impairment. In fact, ACS is the leading cause of sudden death in patients with SCD. Although there are multiple etiologies for ACS, infection/sepsis and the dramatic innate immune and coagulation response to it remain a major contributor to morbidity and mortality during ACS. Novel methods to reduce the inflammatory response during infection are needed as are methods that normalize the chronic pro-inflammatory state. Chaperone proteins, namely HSP90 and HSP70, are known agents that participate in inflammation and thus have significant potential to influence the inflammatory, pro-coagulant burden. Therefore, in this study, we wanted to evaluate the novel anti-inflammatory, anti-coagulatory properties of the chaperone proteins in SCD. We had previously determined that inhibition of HSP90 using the drug AUY-922 could block the bacterial toxin lipopolysaccharide (LPS) - induced TF expression and pro-inflammatory cytokine release from monocytes. Therefore, we used the Townes mouse model of SCD to evaluate AUY-922 in a pre-clinical study. Townes mice with SCD or without were administered AUY-922 intraperitoneal (IP) for 4 days prior to a 6 hour LPS-mediated induction of the inflammatory response and coagulation activation. Notably, the dose of LPS failed to induce any pro-inflammatory response in the AA mice (n=24). However, LPS-induced an exaggerated response in the SS mice. Levels of TNF-α, IL-6, IL-8, and IL-10 were elevated up to 40,000 fold over control treated SS mice. Pre-treatment with AUY-922 either completely ablated, or significantly attenuated the inflammatory cytokine response and normalized EC function. Furthermore, the treatment with AUY-922 doubled the amount of the anti-inflammatory chaperone molecule HSP70 in the livers of the SS mice. This particular result suggested that the function of HSP90 could be spared, and the induction of HSP70 was potentially sufficient to protect against the LPS-induced insult.Of note, the main function of HSP70 is cytoprotection in response to oxidative and febrile stress. Therefore, we next sought to determine, in a proof of principle in vitro study, whether induction of HSP70 alone was sufficient to block LPS-induced cytokine release and coagulation activation. We treated human monocytes with the HSP70 inducer, celastrol for 24h, followed by treatment with LPS (1µg/ml). We observed a significant release of the cytokines IL-6 and TNF-α with LPS treatment. However, induction of HSP70 via celastrol was sufficient to block this inflammatory response. Furthermore, we observed that celastrol blocked the LPS-induced, TF-specific clotting of plasma in vitro. Interestingly, we also observed that conditioned media from celastrol treated monocytes could block LPS-induced IL-6 release in an HSP70 dependent manner. Thus, secreted HSP70 was an active participant in cellular protection from LPS-induced insult. Initial studies suggest that secreted HSP70 levels may be lower in patients with SCD than in unaffected individuals. Therefore, replacement of this chaperone may be of significant benefit as therapeutic. Thus, taken together, our data demonstrate in both a pre-clinical and an in vitro proof of principle study, that the chaperone proteins HSP90 and HSP70 are attractive targets at reducing the inflammatory burden and associated acute lung injury in SCD. DisclosuresNo relevant conflicts of interest to declare.

Endothelin-1 contributes to the progression of renal injury in sickle cell disease via reactive oxygen species.

Endothelin-1 (ET-1) is increased in patients with sickle cell disease and may contribute to the development of sickle cell nephropathy. The current study was designed to determine whether ET-1 acting via the ETA receptor contributes to renal injury in a mouse model of sickle cell disease. Adult, humanized HbSS (homozygous for sickle Hb) mice had increased ET-1 mRNA expression in both the cortex and the glomeruli compared with mice heterozygous for sickle and Hb A (HbAS controls). In the renal cortex, ETA receptor mRNA expression was also elevated in HbSS (sickle) mice although ETB receptor mRNA expression was unchanged. Ligand binding assays confirmed that sickle mice had increased ETA receptors in the renal vascular tissue when compared with control mice. In response to PKC stimulation, reactive oxygen species production by isolated glomeruli from HbSS sickle mice was increased compared with that from HbSA controls, an effect that was prevented by 1 week in vivo treatment with the selective ETA antagonist, ABT-627. Protein and nephrin excretion were both elevated in sickle mice, effects that were also significantly attenuated by ABT-627. Finally, ETA receptor antagonism caused a significant reduction in mRNA expression of NADPH oxidase subunits, which may contribute to nephropathy in sickle cell disease. These data support a novel role for ET-1 in the progression of sickle nephropathy, specifically via the ETA receptor, and suggest a potential role for ETA receptor antagonism in a treatment strategy.

Quantitative MRI of Hemodynamic Compromise in Children with Sickle Cell Disease: New Insights into Pathophysiology

Introduction:Sickle cell disease (SCD) is the leading cause of overt stroke in children, resulting in increased morbidity and mortality (Prengler et al. 2002). Neurologic injury in SCD is caused by a cascade of complex physiological processes that may manifest as abnormal cerebral blood flow (CBF), abnormal oxygen extraction fraction (OEF), and ultimately lead to oxygen deprivation of the brain tissue (Adams et al. 2007., Cheung et al. 2002), however, the underlying pathophysiology is not well understood. While much research has focused on the role of vasculopathy in neurologic injury in SCD, over one third of children with sickle cell disease who suffer an overt ischemic event do not present with arterial stenosis (Hulbert et al. 2011). Extending the work of Nur et al. (2009), we have recently shown in children without stenosis in SCD that elevated cerebral blood flow and diminished cerebrovascular reserve is associated with the severity of anemia (Kosinski et al. 2015). However, to date, little is known about the relative contributions of non-vasculopathic hemodynamic compromise that may occur in children with SCD. Therefore, a comprehensive model of pathophysiology in this population will help stratify stroke risk and guide therapeutic strategies.Aim:To noninvasively quantify both CBF and OEF in children with SCD using magnetic resonance imaging.Methods:11 patients (7M/4F; age 14 ± 2.7 years (mean ± SD)) with SCD and no history of stroke nor arterial stenosis were imaged on a 3T MRI system. 9 patients were on hydroxyurea (average dose: 19.7 ± 3 mg/kg/day). Hematological parameters were acquired at a clinic visit no more than one month prior to the MRI scan (Table 1). Pulsed arterial spin labelling MRI was used to noninvasively assess grey matter cerebral blood flow according to a standard kinetic model. A T2*-weighted flow-compensated image provided phase information, with the phase signal difference between the sagittal sinus and surrounding tissue used to quantify venous oxygenation (SvO2) (Driver et al. 2014). Arterial oxygen saturation (SaO2) was measured via peripheral pulse oximetry and OEF was determined by (SaO2 - SvO2/SaO2) according to Fick's principle of arteriovenous difference.Results:A significant positive association was observed between CBF and OEF (p = 0.82, p = 0.02, pearson product moment correlation coefficient), as shown in Figure 1. Mean values (± SD) were 0.75 ± 0.08, 0.23 ± 0.08 and 54.1 ± 11 ml/min/100g for SvO2, OEF and CBF respectively.Discussion:The observed correlation between CBF and OEF suggests that children with SCD who do not present with vasculopathy, utilize both the perfusion and metabolic reserve in order to meet the cerebral demand for O2. This allows CBF and OEF to increase simultaneously rather than sequentially as proposed by Powers et al. (1991). This non-sequential hemodynamic compromise is in agreement with previous literature in cerebrovascular disease (Kanno et al. 1988) and may serve to minimise increases in cardiac output (Varat et al. 1972). This data suggests that elevated OEF may serve as an early risk marker for overt stroke in addition to elevated blood flow velocity and thus may help better screen for those who will most benefit from transfusion therapy.Table 1SCD PatientsSample Size (N)11Gender (M/F)7/4Age (y)13.7 ± 2.7BMI (Kg/m2)18.8 ± 3.0Hematocrit0.282 ± 0.03Hemoglobin (g/L)100 ± 10.3SaO20.99 ± 0.02SvO20.75 ± 0.08Absolute Reticulocyte (K/uL)205 ± 72Neutrophil Count (K/uL)5.3 ± 3.7Clinical parameters acquired within 30 days of the MRI session [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

MiRNA-146a, miRNA-203a, and miRNA-223 Modulate Inflammatory Response in LPS- Acute Lung Injury in Sickle Cell Transgenic Mice

Introduction: Mouse models have allowed a wider investigation of the inflammatory process in Sickle cell disease (SCD). Previous studies have demonstrated that neutrophil recruitment inresponse to LPS is upregulated in Berkeley sickle mice and the development of acute LPS-induced airway inflammation is associated with enhanced expression of cytokines, chemokines and MMPs expression in lungs. MicroRNAs (miRNAs) belong to a regulatory class of RNAs with great importance in several biological processes, including the development and fate of immune cells, and in both innate and adaptive immune responses, whereby strong evidence links miRNAs expression to signalling pathways and receptors. Some miRNAs are positive regulators of inflammation and others are involved in the negative-feedback regulation of inflammatory processes. A growing amount of evidence indicates that these molecules are involved in various lung diseases. However, the expression profile of miRNAs in lung injury in SCD have not been investigated. Since pulmonary complications are an important cause of morbidity and mortality in SCD, the aim of this study was to determine whether miRNAs implicated in LPS-mediated inflammation are involved in neutrophil recruitment and inflammation in lung injury in SCD using the sickle mice model.Methods: Acute lung inflammation and injury was induced in wild- type (WT, C57BL/6, n=5) and Berkeley SCD mice (n=5) by intranasal administration of LPS (50 ml of 250 mg/ml). The vehicle group received a similar volume of sterile PBS. BAL was performed 4h after LPS challenge. qPCR analysis was used to examine gene expression and ELISA was used to determine protein production. For miRNAs expression analysis, extraction of lung RNA was performed using mirVanaTM miRNA Isolation kit and TaqManTM MicroRNA Reverse Transcription.The expression level of miRNAs was determined using the Taqman miRNA expression assay by qPCR with U6 snRNA as an endogenous control.Results: Levels of miR-15a-5p, miR-16-5p, miR-26a-5p and miR-98-5p in lung tissue increased almost twofold at 4h after LPS challenge in SS compared with SS PBS group (2.9, 3.7; 2.3; 2.9-fold change, respectively). The expressions of miR-106a-5p, miR-146a-5p, miR-146b-5p, and miR-181a-5p were not altered (2.0; 1.2; 1.55; 1.4-fold change, respectively). However, the expressions of miR-203a-3p and miR-223-3p were significantly up-regulated compared to animals from the PBS group (4.3 and 8.8 fold change, respectively, p<0.05). These increases correlated with the increased expressions of Tnf- α, Il-1 β, Il-6, Cxcl1 and Mmp-9 mRNA in the lungs (LPS: 0.27±0.05; 0.24±0.04; 0.18±0.007; 0.44±0.09, 0.46±0.09 vs PBS: 0.02±0.001; 0.03±0.02; 0.007±0.18; 0.052±0.03; 0.07±0.03, respectively) and increased BAL levels of Tnf-α, Cxcl1 and Mmp-9 (4250±636.4; 2491±454.3; 187.5±16.7 vs PBS: 20.8±8.9; 186.6±35.4, 5.4±2.9, pg/ml, respectively). LPS challenge also led to a higher expression of miRNAs in the lungs of WT animals; however only expression levels of miR-146a-5p and miR-223-3p were significantly up-regulated (2.6 and 3.2-fold change, respectively, p<0.05).Conclusion: We have shown that theLPS-induced inflammatory response in SCD transgenic mice is associated with increases in miR-203a-3p and miR-223-3p expression in the mouse lung; suggesting a role of the miRNAs in the regulation of inflammatory mediator production, since these augmentations are associated with the increases in cytokines and chemokines. Furthermore, miR-146a-5p, a negative regulator of inflammatory gene expression, is not upregulated in SS mice and could contribute to the exaggerated inflammation observed in these animals. These data indicate that alterations in the miRNA expression may be associated with the inflammatory state in SCD. Financial Support: FAPESP DisclosuresNo relevant conflicts of interest to declare.

Urinary Transforming Growth Factor β-1 as a Marker of Renal Dysfunction in Sickle Cell Disease

Sickle cell disease (SCD) is a genetic disorder that results in deformity of red blood cells. Renal dysfunction affects 5-18% of patients with SCD. To date, few studies have described urinary levels of transforming growth factor β-1 (TGF-β1), which is a marker of fibrosis, as a biomarker in identifying patients at risk of developing renal disease in SCD. The aim of this study is to determine prevalence of sickle cell nephropathy in Egyptian SCD patients. We aimed also to evaluate the association of urinary TGF-β1 with other conventional biomarkers of renal damage in SCD patients to identify a novel renal biomarker for early diagnosis of sickle nephropathy. We examined 40 SCD patients, 21 with sickle cell anemia, 16 sickle thalassemia, and three with sickle trait. We compared them to 20 control children with matched age and sex. The study was held in the time period between May 2013 and December 2013 in the Hematology Clinic, New Cairo University Children Hospital, Cairo, Egypt. Urinary excretion of TGF-β1 was 7.07±1.91ng/mL in SCD patients versus 2.23±0.76ng/mL in control children (p < 0.001). SCD patients had elevated estimated glomerular filtration rate (177.44±35.6mL/min/1.73m(2)), denoting a state of glomerular hyperfiltration. 47.5% of SCD patients had microalbuminuria. Urinary TGF-β1 correlated positively with microalbuminuria and estimated glomerular filtration rate (p=0.001 and p=0.018, respectively). We concluded that urinary TGF-β1 may serve as a marker of early renal injury in SCD.

Cognitive and behavior deficits in sickle cell mice are associated with profound neuropathologic changes in hippocampus and cerebellum

Strokes are perhaps the most serious complications of sickle cell disease (SCD) and by the fifth decade occur in approximately 25% of patients. While most patients do not develop strokes, mounting evidence indicates that even without brain abnormalities on imaging studies, SCD patients can present profound neurocognitive dysfunction. We sought to evaluate the neurocognitive behavior profile of humanized SCD mice (Townes, BERK) and to identify hematologic and neuropathologic abnormalities associated with the behavioral alterations observed in these mice. Heterozygous and homozygous Townes mice displayed severe cognitive deficits shown by significant delays in spatial learning compared to controls. Homozygous Townes also had increased depression- and anxiety-like behaviors as well as reduced performance on voluntary wheel running compared to controls. Behavior deficits observed in Townes were also seen in BERKs. Interestingly, most deficits in homozygotes were observed in older mice and were associated with worsening anemia. Further, neuropathologic abnormalities including the presence of large bands of dark/pyknotic (shrunken) neurons in CA1 and CA3 fields of hippocampus and evidence of neuronal dropout in cerebellum were present in homozygotes but not control Townes. These observations suggest that cognitive and behavioral deficits in SCD mice mirror those described in SCD patients and that aging, anemia, and profound neuropathologic changes in hippocampus and cerebellum are possible biologic correlates of those deficits. These findings support using SCD mice for studies of cognitive deficits in SCD and point to vulnerable brain areas with susceptibility to neuronal injury in SCD and to mechanisms that potentially underlie those deficits.

Quantitation of Iron in Brain Structures of Children with Sickle Cell Disease and Transfusion Hemosiderosis

Introduction:Children with sickle cell disease (SCD) and abnormal cerebral blood flow often receive cyclic blood transfusions (CTx) to reduce risk of stroke. While effective for stroke prevention, CTx causes systemic iron accumulation that may damage multiple organs (Harmatz P., Blood 2000). Iron toxicity from CTx is poorly described for the brain, in comparison to the liver and heart. Quantifying iron in the subcortical structures and the choroid plexus is of renewed clinical importance, due to recent findings that iron accumulation in these regions may predict CNS injury in neurodegenerative disorders. In this study, a new imaging protocol is applied to quantify iron levels and measure structural volumes for the subcortical structures and choroid plexus of children with SCD on CTx, using healthy children for comparison.Methods:SCD patients were eligible if at least 12 consecutive months of CTx were received for abnormal transcranial doppler. Patients with history of CVA were excluded, due to potential confounding effects of prior ischemic damage.Healthysiblings and relatives were used as ethnic- and age-matched controls.MRI scans of the liver and brain, using a Siemens 3T Trio scanner, was performed without sedation for all subjects. A modified pulse sequence (R2*) for the 3T scanner was used to determine liver iron concentration. T1-weighted imaging sequence with MPRAGE was used for anatomical identification and volume measurements of brain regions (Kirk G., Cerebral Cortex 2009). A 3D multi-echo gradient-echo sequence (FOV=256x192mm, Matrix=256x192, Slice thickness = 2mm, 72 slices, TR = 50 ms, first TE = 3.7ms, 12 echoes, echo spacing = 3.8ms) was used for the quantitative susceptibility mapping (QSM), a MRI method that is sensitive to tissue iron (Shmueli K., Magn Reson Med 2009). Iron levels were generated with a post-processing protocol developed for this application (modification of Qiu D., AJNR 2014). To compare iron levels between the two groups a general linear model (GLM) was used that incorporated the ages of the subjects into the comparison. For the patients, an additional GLM analysis was performed to determine if iron level when in each region when corrected for age was associated duration of transfusions, liver R2* and average serum ferritins.Results:MRI scans were evaluable for 25 SCD patients on CTx and 23 controls. There were no significant differences between patients and healthy controls with respect to age, sex or ethnicity. The average duration of CTx was 6.0 (1.5-12.6) yrs and the average serum ferritin and liver iron R2* were 2540 ng/ml and 819 Hz (~14.8mg/g dry weight), respectively. As anticipated, iron level in the choroid plexus was significantly elevated in patients compared to controls (p=1.4x10-7), and levels correlated well with serum ferritin (p=0.0003) and liver R2* (p=0.001) (panel a). While there were no significant differences detected between the groups with respect to iron levels in the subcortical structures, there was a trend to increasing differences between the groups with age in the Substanita Nigra, Red Nucleus and Dentate Nucleus (panels b,c,d). In the GLM analysis, age was significantly associated with iron levels in the subcortical structures, not in the choroid plexus. Although iron levels in some of the subcortical structures were associated with duration of CTx, ferritin and liver R2*, the significance was predominately lost when corrected for age. These observations suggest additional studies with older subjects may reveal increased differences. Volumetric analysis was performed to assess potential injury to deep gray matter. The measured volumes of the subcortical structures were not statistically different between the groups, with a noted exception of the caudate nucleus (p=0.021), a region of known predisposition to injury in SCD (Steen R., Ann Neurol 1999).Conclusions: The MRI protocol successfully quantified iron in brain structures, associated with neurodegenerative disorders. Iron accumulation in the choroid plexus was significant in SCD patients on CTx, and correlated with other markers of iron overload. While iron was detectable in subcortical structures, the levels were not significant different from controls after adjusting for age. These finding should be helpful in planning for a longitudinal study that includes a broader age range. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Activated Human iNKT Cells in Pediatric Sickle Cell Disease Patients and in Culture Upregulate Ectonucleotidase CD39 and Adenosine a2A Receptor

Background: Sickle cell disease (SCD) is a chronic degenerative disease punctuated by periodic painful vaso-occlusive crises (pVOC). When sickle hemoglobin polymerizes, it causes erythrocytes to become rigid and sticky and produces multi-cellular aggregates that can occlude venules. We have shown that invariant natural killer T (iNKT) cells are pivotal in causing inflammation and injury in sickle cell disease (Wallace KL, Marshall MA, et al. Blood. 2009; 114(3):667-76). Activation of iNKT cells causes an induction of anti-inflammatory adenosine A2A receptors (A2ARs) that have a negative feedback role to limit inflammation. We found that an A2AR activation inhibits iNKT cell activation and protects SCD mice from tissue injury (Wallace KL, Linden J. Blood 2010; 116(23):5010-20). We have also seen in adult SCD patients that the expression of the A2AR is significantly higher in iNKT cells during pVOC than at steady state (Field JJ, Lin G, et al. Blood 2013; 121(17):3329-42). In addition, CD39 and CD73, ectonucleotidases that generate adenosine from ATP, reduce inflammation by activating the A2AR (Crikis S, Lu B, et al. Am J Transplant 2010; 10(12):2586-95).Hypothesis and Aims: We hypothesized that pediatric SCD patients will have elevated A2AR and CD39 expression on iNKT cells during pVOC compared to steady state. We also hypothesized that the elevated A2AR immunoreactivity on activated iNKT cells is associated with enhanced A2AR signaling and reduced iNKT cell activation and inflammation. Our aims were: 1) to measure A2ARs and CD39 on iNKT cells of pediatric SCD patients at steady state and during pVOC, and also on cultured human iNKT cells in vitro with and without activation; and 2) to measure immunoreactive A2AR protein on activated cultured iNKT cells and correlate it with functional A2AR signaling.Methods: We collected blood from pediatric SCD patients at steady state and during pVOC. We used fluorescence-activated cell sorting (FACS) assays to evaluate the level of A2AR and CD39 immunofluorescence. For cultured human iNKT cell studies, human iNKT cells were prepared from healthy donor whole blood and expanded in culture. iNKT cells were activated through engagement of TCRs (with anti-CD3 and anti-CD28 Abs), cytokines (IL-12 and IL-18), or a combination of TCR engagement and cytokines. We treated the activated iNKT cells with vehicle or the A2AR agonist, regadenoson. iNKT cells were analyzed at 48 hours by FACS for A2AR and CD39. To determine if induced A2ARs are functional we utilized cAMP assays. A2AR is a Gs coupled receptor, which when activated, activates adenylyl cyclase to generate cAMP.Results: iNKT cells from pediatric sickle cell patients have higher A2AR and CD39 immunofluorescence during pVOC than at steady state. In cultured iNKT cells we found both A2AR and CD39 immunofluorescence are higher in activated iNKT cells than in non-activated cells. We discovered that iNKT cell activation through TCRs and cytokines is significantly stronger than either activation method alone. We also found both in vivo and in vitro that high A2AR expression on iNKT cells is associated with high CD39 expression. The A2ARs induced on activated iNKT cells are functional by showing significantly higher cAMP from activated iNKT cells exposed to Regadenoson.Conclusions: Our findings indicate that CD39 and the A2AR function together to provide a mechanism to gradually turn off activated iNKT cells and modulate the inflammatory response. Understanding this negative feedback system will help us to better understand the control of inflammation in SCD during vaso-occlusive crises and to identify novel therapies to treat and prevent the complications of SCD. Further studies may include determining if A2AR activation decreases iNKT cell activation, and if blockade of CD39 enhances iNKT cell activation. DisclosuresNo relevant conflicts of interest to declare.

Abstract 18: Circulating Extracellular DNA Mediates Vaso-Occlusions in Sickle Cell Disease

Rationale: It was recently reported that circulating extracellular DNA (cirDNA) is increased in sickle cell disease (SCD). CirDNA apears to be associated to intravascular hemolysis and cell-free heme in plasma. But nothing is known of the physiopathological impact of cirDNA in SCD. Here, we aimed to define the role of cirDNA and hypothesized that it may favor red blood cell (RBC) aggregation, vaso-occlusive crises (VOC) and ischemic events. Material and Methods: A blood collection from SCD patients and matched healthy volunteers was built. RBCs and neutrophils were separated from plasma by centrifugation. We cultured human neutrophils and endothelial cells in vitro. We quantified extracellular DNA with a fluorescent intercalant probe. We also used our model of renal vaso-occlusions induced by infusion of heme-loaded RBC microparticles in SAD transgenic mice. Results: In SCD patients at steady state, cirDNA was increased by 300% over health, and a further 100% during VOC. Next, we treated cultured neutrophils and endothelial cells with purified heme. Heme induced neutrophil extracellular traps (NETs) and endothelial apoptosis, with release of genomic DNA in supernatants. We purified neutrophil DNA, added it to whole blood and studied RBC aggregation by laser-assisted optical red blood cell aggregometry. DNA induced RBC aggregation in SCD blood, but not healthy blood. Conversely, DNase-1 added to SCD blood collected during VOC reduced RBC aggregation back to steady state levels. Next, we triggered renal vaso-occlusions in SAD transgenic mice, and cirDNA levels were increased by 200% within 30 minutes. Moreover, one intravenous bolus of DNase-1, administered after inducing the occlusions, accelerated renal reperfusion back to normal levels in under 20 minutes. In contrast, spontaneous reperfusion with vehicle injection took over 90 minutes. Conclusion: Our data reveal a novel mechanism of vascular occlusion in SCD: CirDNA can bind RBCs in blood, promote aggregation and small vessel occlusions. CirDNA and NETs may thus bridge intravascular hemolysis, inflammation and ischemic tissue injury in SCD. Interestingly, DNase digestion of plasma DNA and NETs may prove an unexpected but powerful novel pharmaceutical approach to treat vaso-occlusive events.