Sickle Cell Disease Neutrophils Research Articles

Red cell exchange modulates neutrophil degranulation responses in sickle cell disease.

Neutrophils in sickle cell disease (SCD) are activated, contributing to disease. Red cell exchange (RCE), with the goal of lowering hemoglobin S (HbS), is an important part of therapy for many SCD patients. Whether RCE impacts neutrophil reactivity is unknown. To determine the effect of RCE on neutrophil activation, SCD patients undergoing RCE in steady-state were enrolled. Neutrophil degranulation responses were examined before/after RCE. Kinetic studies were completed to determine the duration of the effect of RCE on neutrophil function. Degranulation results were examined in relation to white blood cell count, neutrophil count, and HbS levels. The effect of RCE on RBC phosphatidylserine (PS) exposure was examined as a possible contributor to modulation of neutrophil function by RCE. Twenty-two patients with SCD, genotype SS, who underwent RCE (average pre-RCE HbS 33 ± 14%) were included for the study. RCE significantly decreased neutrophil degranulation responses. The effect of RCE on neutrophil activation was unrelated to cell count and instead directly correlated with HbS. The effect of RCE on neutrophil activation was sustained over several days post-apheresis. Furthermore, while increased RBC PS exposure results in increased neutrophil degranulation, RCE decreases RBC PS exposure. To our knowledge, this is the first study demonstrating that RCE significantly decreases neutrophil activation in a sustained HbS-dependent manner. Modulation of PS exposure by RCE may be a contributing mechanism by which RCE modulates neutrophil activation. These studies raise the possibility that modulation of neutrophil activation contributes significantly to the therapeutic effect of RCE.

Neutrophils in sickle cell disease: Exploring their potential role as a therapeutic target.

Factors influencing the activation of neutrophils in SCD and the potential neutrophil-mediated ameliorating effects of therapies in SCD.

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.

Resolvins Modulate Netosis and Autoimmunity in Sickle Cell Disease

Sickle cell disease (SCD) is a genetically determined hematological disease which is characterized by anemia, chronic hemolysis, and multi-organ chronic inflammatory vasculopathy with acute vaso-occlusive painful crises and chronic organ damage. The pathophysiology of SCD is complex and involves several other cell types and metabolic pathways than erythrocytes, including neutrophils and soluble factors such as cytokines or alternative complement pathway which affect the function of vascular endothelium. An inflammatory vasculopathy with chronically activated neutrophils releasing Neutrophil Extracellular Traps (NETs) is a key component of the pathophysiology of sickle cell disease (SCD). Chiang et al. have recently shown that resolvins (Rv) reduce NETS and improve NETs clearance in a model of sepsis by Staphylococus aureus, a known trigger of NETosis (Chiang N et al. Blood 139 (8): 1222, 2022). We previously reported that targeting pro-resolution mechanisms with Rv improves SCD vasculopathy and organ damage in humanized sickle mice. Here, we studied 23 adult patients with SCD, followed by the Comprehensive center for hemoglobinopathies and rare anemias, University of Verona and AOUI Verona from January 2019 to December 2021. For each patient the presence/absence of concomitant rheumatological condition such as rheumatoid arthritis, other inflammatory arthropathies, immune-mediated dermatological conditions, Sicca syndrome and Raynaud's phenomenon, were assessed. Eight age, and ethnicity matched healthy controls were analyzed. Blood was collected for hematological and biochemical assay as part of the standard clinical care for these patients. Additional blood was collected for research studies on whole blood and on plasma cytokines. SCD patients display significant increase in tumor- necrosis-factor (TNF)- α (p = 0.011) and interleukin (IL)-10, suggesting both active inflammatory state and an upregulated counterregulatory mechanism to limit inflammation. We observed that antinuclear antibodies (ANA), but not extractable nuclear antibodies (ENA), were significantly increased in SCD patients (provide specific data here). Previous reports have reported the presence of this type of autoantibodies in the absence of autoimmune clinical manifestations in patients with SCD (Toly-Ndour C et al J Rheumatol 38: 302, 2011). ANA+ SCDsubjects (defined by a cell nuclei homogenous pattern, and positive dilution in the 1:80 to 1: 160 range) had a higher inflammatory and hemolytic status, as demonstrated by increased WBC and neutrophil counts and LDH values compared to ANA- subjects. NETosis plays a key role in modification and externalization of autoantigens involved in production of autoantibodies such as ANA. Here, we found basal NETosis was higher, though not significant, in ANA+ SCD subjects compared to ANA-SCD patients. This was associated with lower expression of receptors for RvD1 and D2, namely ALX, GPR32/DRV1, and GPR18/DRV2 on PMN, monocytes, and lymphocytes from ANA+ SCD patients when compared to ANA- SCD subjects, who were also characterized by relatively low LDH compared to ANA+ SCD patients. Exogenous treatment of SCD neutrophils with RvD1 and D4 significantly dampened NETs release and enhanced NETs clearance by macrophages. RvD4 along with other pro-resolving mediators also stimulated ex vivo clearance of P. aeruginosa and S. aureus by SCD neutrophils. Our findings provide a novel link between autoimmunity and neutrophil-mediated vasculopathy in SCD and highlight the importance of Rv and their immune-pro-resolution effects in limiting disease progression in SCD but also to modulate the host response to infections and sepsis, which is altered in SCD.

Neutrophils as drivers of vascular injury in sickle cell disease.

While neutrophils are the main effectors of protective innate immune responses, they are also key players in inflammatory pathologies. Sickle cell disease (SCD) is a genetic blood disorder in which red blood cells (RBCs) are constantly destroyed in the circulation which generates a highly inflammatory environment that culminates in vascular occlusions. Vaso-occlusion is the hallmark of SCD and a predictor of disease severity. Neutrophils initiate and propagate SCD-related vaso-occlusion through adhesive interactions with the activated and dysfunctional endothelium, sickle RBCs, and platelets, leading to acute and chronic complications that progress to irreversible organ damage and ultimately death. The use of SCD humanized mouse models, in combination with in vivo imaging techniques, has emerged as a fundamental tool to understand the dynamics of neutrophils under complex inflammatory contexts and their contribution to vascular injury in SCD. In this review, we discuss the various mechanisms by which circulating neutrophils sense and respond to the wide range of stimuli present in the blood of SCD patients and mice. We argue that the central role of neutrophils in SCD can be rationalized to develop targets for the management of clinical complications in SCD patients.

Drug Repurposing Patent Applications October-December 2020.

Drug Repurposing Patent Applications October-December 2020.

Targeting the AnxA1/Fpr2/ALX pathway regulates neutrophil function, promoting thromboinflammation resolution in sickle cell disease

Neutrophils play a crucial role in the intertwined processes of thrombosis and inflammation. An altered neutrophil phenotype may contribute to inadequate resolution, which is known to be a major pathophysiological contributor of thromboinflammatory conditions such as sickle cell disease (SCD). The endogenous protein annexin A1 (AnxA1) facilitates inflammation resolution via formyl peptide receptors (FPRs). We sought to comprehensively elucidate the functional significance of targeting the neutrophil-dependent AnxA1/FPR2/ALX pathway in SCD. Administration of AnxA1 mimetic peptide AnxA1Ac2-26 ameliorated cerebral thrombotic responses in Sickle transgenic mice via regulation of the FPR2/ALX (a fundamental receptor involved in resolution) pathway. We found direct evidence that neutrophils with SCD phenotype play a key role in contributing to thromboinflammation. In addition, AnxA1Ac2-26 regulated activated SCD neutrophils through protein kinase B (Akt) and extracellular signal–regulated kinases (ERK1/2) to enable resolution. We present compelling conceptual evidence that targeting the AnxA1/FPR2/ALX pathway may provide new therapeutic possibilities against thromboinflammatory conditions such as SCD.

Sickle cell disease and COVID-19: Atypical presentations and favorable outcomes.

Sickle cell disease and COVID-19: Atypical presentations and favorable outcomes.

Prognosis of patients with sickle cell disease and COVID-19: a French experience

Prognosis of patients with sickle cell disease and COVID-19: a French experience

Neutrophil Extracellular Traps Are an Intrinsic Feature in Sickle Cell Disease

Polymerisation of deoxygenated-HbS in sickle cell disease (SCD) leads to alteration of the shape and function of the red blood cells (RBC), resulting in hemolytic anemia and recurrent episodes of micro-vascular occlusion, triggering a cascade of downstream events and inflammation that underlies the progressive multi-systemic organ damage. Neutrophils have a critical role in promoting sickle pathophysiology; patients with higher leukocyte count, particularly neutrophils, tend to have a higher risk for more severe manifestations as well as earlier mortality. SCD neutrophils display a specific overreactive phenotype, including increased adhesive properties and production of reactive oxygen species. Activated neutrophils can also produce extracellular traps (NETs) in response to pathogens or other inflammatory stimuli. Studies have shown that prolonged exposure of healthy neutrophils to SCD plasma leads to formation of mature string-like NETs. These strings of decondensed chromatin covered with elastase (NE) or myeloperoxidase (MPO) might contribute to SCD pathophysiology. Here, we investigated if SCD neutrophils had enhanced intrinsic potential for NETs formation (i.e. could form NETs in the absence of a stimulus).We evaluated NETosis both indirectly, in which neutrophils isolated from healthy subjects were incubated with plasma from HbSS patients and directly, in which neutrophils isolated from HbSS patients were compared with those isolated from healthy donors. 17 HbSS patients in steady-state (all on hydroxyurea) and 11 race and sex matched healthy donors were included in the direct assays; the NETosis-related events were observed over a period of 7 minutes to 4 hours. For the indirect assay, we used plasma from 19 HbSS patients (14 on hydroxyurea, 5 off hydroxyurea and/or transfused), all of whom provided samples in steady-state and acute pain crisis (paired samples); the healthy neutrophils were treated with SCD plasma for up to 7 hours. A pain crisis was defined as an episode of acute pain that has no evident cause other than SCD, resulting in hospitalization and treatment with parenteral opioids. Steady state was defined as the period from at any time 8 weeks prior to or after a crisis. Patients were excluded when they were <18 or >80 years of age, pregnant or had a history of blood transfusion in the previous 8 weeks. The IRB approved the studies under NIH protocol numbers NCT00081523 (SCD subjects) and 03-H-0015 (healthy controls and SCD subjects). The NETosis features (histone citrullination H4cit3, nuclear decondensation, DNA-NE-MPO strings) were assessed by imaging flow cytometry and fluorescence microscopy.In the indirect assays, healthy neutrophils made more string NETs when treated with the SCD plasma (from both steady state and crisis) compared to plasma from healthy donors (Figure 1A), and NETs response was further increased with plasma from acute compared to their paired steady state sample (N=19, p=0.0114) (Figure 1B). Plasma from patients on or off hydroxyurea induced comparable NETosis responses. This confirmed a higher ability for the crisis milieu to modulate NETosis.Directly, neutrophils isolated from SCD patients as compared to those from healthy controls, showed increased number of decondensed H4cit3 positive nuclei, after only 7 min of incubation in RPMI (N=7, p=0.0688). By comparison the response to high concentration (20 uM) of Hemin - a regular product of hemolysis, was similar in the SCD and the healthy neutrophils (p=0.6871) (Figure 1C). This data suggested that the SCD environment might activate neutrophils to produce NETs in the absence of a specific stimulus. Fluorescence microscopy confirmed that neutrophils isolated from the patients did produce significantly more DNA-NE strings without any stimulation compared to healthy neutrophils (N=9, p=0.0079). After Hemin treatment, NETs response in SCD neutrophils was also higher than that of the healthy controls, but not statistically significant (Figure 1D).Our data provide the first direct in vitro evidence that neutrophils in SCD are innately prone to NETosis but whether and how these might contribute to the initiation or progress of SCD vaso-occlusive crises is yet to be determined. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Elevated Levels of CD64 MFI on Monocyte Subsets Are Associated with a History of Stroke in Sickle Cell Disease

Background: Ischemic stroke is a cause of morbidity and mortality in children and adults with sickle cell disease (SCD). Recent studies showed that non-classical monocytes may be protective against vaso-occlusive crisis. Monocyte subsets have been examined in non-SCD stroke, but little is known about the role of monocyte or their subsets in ischemic stroke in SCD. CD64 (FCγRI), a high affinity receptor for IgG, is a marker of endothelial adherence on immature neutrophils in SCD, however it is more classically exhibited on activated pro-inflammatory monocytes. We hypothesized that higher levels of CD64 mean fluorescence index (MFI) on monocyte subsets may be associated with ischemic stroke due to increased adherence.Methods: Adults (age ≥18) who presented for regularly scheduled clinic visits were enrolled. Blood samples were taken when subjects were at their healthy baseline and sent for complete blood count with differential. Monocytes were analyzed by flow cytometry and subsequently sub-classified by their CD14 and CD16 expression into classical (CD14high, CD16 low), intermediate (CD14high, CD16high), and non-classical (CD14low, CD16high). The CD64 MFI was determined for each monocyte subset. History of ischemic stroke was confirmed by neurologist or neuroimaging documentation in the medical record. Laboratory results between those with a history of stroke and those without history of stroke were compared using Student's T Tests. We modeled log-odds of stroke history for increasing levels of each monocyte subtype and CD64 MFI using both marginal and age-adjusted multivariate logistic regression.Results: We enrolled 49 adults, of whom 17 (35%) had a history of ischemic stroke. There was no difference in age, gender, or genotype between the two groups (Table 1). Patients with a history of stroke had higher total neutrophils, but there was otherwise no difference in complete blood counts (Table 1). When monocyte subsets were examined, patients with a history of stroke had a higher percent of intermediate monocytes and a lower percent of classical monocytes (Table 1). Patients with history of stroke also had a higher MFI (mean fluorescence index) both overall and on each monocyte subset. (Table 1). In univariate models increased levels of classical monocytes were associated with decreased odds of stroke history and increased levels of intermediate monocytes, CD64 MFI on each monocyte subtype, total monocyte CD64 MFI, and total neutrophil count were all associated with increased odds of stroke history (Table 2). However, in multivariate models adjusted for age and monocyte subtype percent only total monocyte CD64 MFI and total neutrophil counts remained significant and both had increased odds ratios for ischemic stroke history, (OR 3.8 per 1000 CD64 95% CI 1.2 - 11.4, p= 0.02, OR 1.2 per 1x10^9/L 95% CI 1.0 - 1.4, p=0.05 respectively, R-squared 0.45).Conclusion: Levels of monocyte subsets have been shown to correlate with clinical outcomes in non-SCD stroke, but to our knowledge this is the first study to examine their roles in ischemic stroke in SCD. The pathophysiology of stroke in SCD is unique and the role of monocytes in it deserves separate study from the role of monocytes in non-SCD stroke. We saw that while monocyte subsets were associated with a history of ischemic stroke, CD64 MFI on all monocyte subsets showed a strong association. We wonder if this may be due to CD64 MFI being a marker for cells which are more adherent to endothelium as has been shown in previous studies. This creates a need for future studies. In particular it would be valuable to know if increased CD64 MFI precedes stroke and could be used as a marker of future stroke risk. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Aged Neutrophils in Sickle Cell Disease Impede Ischemic Repair

INTRODUCTIONRepeated bout of tissue ischemia is a hallmark of sickle cell disease (SCD). Neutrophils are critical first responder cells that initiate host response to sterile injury, including ischemia. Of the numerous neutrophils in circulation, a small proportion undergo physiologic changes (termed aging) which directs their homing to the bone marrow for clearance. How neutrophil homeostasis is regulated in sickle cell disease (SCD) to influence ischemic repair is unknown. HYPOTHESISWe hypothesized that SCD may alter the homeostasis and function of aged neutrophil. METHODSAged neutrophils from the Townes humanized sickle cell (SS) mice were compared to aged neutrophils from MerTK deficient mice (MerKO), which age in the circulation owing to defective efferocytosis. Hind limb ischemia (HLI) was used as model of vascular injury. LASER Doppler perfusion imaging (LDPI) measured perfusion recovery after HLI. RESULTSCompared to wild-type mice (7 ± 1.2%), the proportion of CXCR4HiCD62Llo aged neutrophils in circulation was markedly elevated in the SS (34 ± 4%) and MerKO mice (38 ± 4%). However, in contrast with aged neutrophils from MerKO mice, those from SS mice showed impaired phagocytosis, increased capacity to produce reactive oxygen species, and pronounced release of extracellular traps (NETs). In response to HLI, perfusion recovery in the MerKO and wild type mice were similar, but this was dramatically impaired in SS mice. Importantly, the phenotype of SS mice was reversed by treatment with the anti-oxidant N-acetyl cysteine (NAC), but NAC therapy had no impact on MerKO mice. CONCLUSIONSAlthough SS and MerKO mice demonstrated elevated numbers of aged neutrophils, our data suggest that oxidant stress causes aged CXCR4HiCD62Llo neutrophils in SS to impede vascular repair after ischemia. This specialized subset of neutrophils may be targeted to improve vascular health in SCD. DisclosuresNo relevant conflicts of interest to declare.

Heterogeneous Hypoxia-Mediated Neutrophil and Red Blood Cell Adhesion to E-Selectin in Microscale Flow

Hypoxia promotes red blood cell (RBC) sickling, oxidative stress, systemic endothelium activation, vascular inflammation, and activation of coagulation in sickle cell disease (SCD), which results in a malicious cycle contributing to the progression of vaso-occlusion and other consequent clinical manifestations, such as acute chest syndrome and ischemic injuries. In the multistep and multicellular paradigm of vaso-occlusion, recruitment of neutrophils and adhesion of sickle RBCs to activated endothelial cells are critical in initiating this cascade of events. To better understand the role of hypoxia in this pathophysiological process, we assessed the adhesion profiles of RBCs and neutrophils to immobilized e-selectin utilizing blood samples from a clinically diverse patient population with SCD.Blood samples were collected from 11 subjects with homozygous SCD (HbSS) and 5 normal subjects (HbAA). Prior to the experiments, whole blood samples were mixed with Hank's balanced salt buffer solution modified with calcium and magnesium (1:1 v/v). A total volume of 25 µl blood sample was perfused through each e-selectin immobilized microchannel under both normoxic and hypoxic (7.5% oxygen level) conditions using SCD Biochip microfluidic adhesion assay [1, 2]. Blood perfusion was followed by a rinse with Hank's buffer solution at 1 dyne/cm2 corresponding to the typical shear stress levels observed in post-capillary venules. Thereafter, neutrophil adhesion, neutrophil rolling, neutrophil-platelet aggregation, and RBC adhesion data were obtained and analyzed.E-selectin functionalized microchannels supported neutrophil adhesion as well as neutrophil rolling when flowing normal blood samples, where we observed higher adhesion and rolling rates in hypoxia (Fig. 1A). SCD subjects were categorized into two distinct groups based on their adhesion profiles: Group 1: hypoxia-enhanced neutrophil adhesion without significant RBC adhesion (N=7), and Group 2: hypoxia-reduced RBC adhesion with marginal neutrophil adhesion (N=4) (Fig. 1B). We find that both normal and SCD neutrophil adhesion to e-selectin is significantly enhanced under hypoxic conditions (Fig. 1C, p<0.05, paired t-test). Moreover, we observed significantly increased neutrophil-platelet aggregates and an increase in the percentage of adhered neutrophils involved in neutrophil-platelet aggregation induced by hypoxia (Fig. 1D&E), suggesting that hypoxia is strongly associated with neutrophil-platelet aggregation driven vaso-occlusive events in Group 1. Furthermore, rolling velocity of 20 neutrophils from each SCD subjects under shear stress was measured, and hypoxia-mediated neutrophil rolling behavior was determined (Fig. 1F). A unique adhesion profile was observed in Group 2, in which the number of adhered RBCs was significantly reduced in response to hypoxia (Fig. 1G, p<0.05, Mann-Whitney non-parametric analysis).Here, we report two different adhesion profiles among SCD sub-populations using an e-selectin functionalized microfluidic model. We observed elevated numbers of adherent neutrophils, decreased neutrophil rolling velocities, and enhanced neutrophil-platelet aggregation induced by hypoxia in one group, while lowered number of adhered RBCs mediated by hypoxia in the other group. We speculate that there may be two distinct mechanisms that initiate vaso-occlusive events in SCD: in Group 1, neutrophils are responsive to endothelial activation. In this group, neutrophil recruitment and the resulting neutrophil-platelet aggregates and other complexes may precipitate vaso-ooclusion, which is strongly susceptible to hypoxia. In Group 2, in whom neutrophil recruitment is less effective, vaso-occlusion may be induced by vascular RBC adhesion, in which hypoxia may be a less proximate trigger.

35 - Neutrophils in sickle cell disease: the missing candidate – myeloperoxidase

Sickle cell disease (SCD) is caused by a single point mutation of the β-globin gene that increases the polymerization of hemoglobin under hypoxia and subsequent sickling of red blood cells (RBCs). Sickled RBCs occlude capillaries, inducing ischemic attacks in the lungs of affected individuals. Increased hemolysis and repeated bouts of ischemia-reperfusion injury lead to chronic states of oxidative stress and inflammation. Previously, we reported that sickle disease increases neutrophil activation and release of myeloperoxidase (MPO), which generates toxic oxidants that impair vascular function in SCD mice. In the present study, we determine if N-acetyl lysyltyrosylcysteine amide (KYC), a tripeptide inhibitor of MPO prevents toxic oxidant production and oxidative damage to sickle RBCs in SCD mice. KYC treatment of SCD mice reduced NO2-Tyr and Cl-Tyr, both markers of MPO-mediated damage, on sickle RBC membranes. Interestingly, the number of sickled RBCs in the lungs in KYC-treated SCD mice were reduced, which correlated with a reduction in the number of occluded blood vessels in the lungs of perfused SCD mice. KYC treatment also reduced MPO deposition, Cl-Tyr formation, and IgG deposition in the interstitium of the lungs of SCD mice. These data indicate KYC reduces oxidative damage to sickle RBCs which decreases sickling, vaso-occlusion, and ischemic injury of the lungs in sickle animals. KYC-dependent reductions in MPO-dependent oxidative damage to the lungs correlated with decreased vascular leakage in the lungs of SCD mice and reductions in SCD-related lung pathology. In conclusion, inhibitors that target MPO, such as KYC, may be an effective strategy for reducing vaso-occlusive events in SCD.

Neutrophil Activation In Sickle Cell Disease: Biochemical and Functional Changes At Baseline and During Acute Vaso-Occlusive Crises

Neutrophilia is a well-recognized complication of sickle cell disease (SCD) and is a risk factor for increased disease severity and early death. Increased neutrophil activation, adhesion and oxidant production have been reported in SCD. While changes in neutrophil surface marker expression, degranulation, migration, adhesion and intracellular oxidative stress have been reported in SCD, there is little mechanistic underpinning for these observed functional changes. Moreover, how neutrophils specifically contribute to SCD pathology, particularly during acute vaso-occlusive crises, remains unknown. We hypothesize that chronic biochemical and functional changes of neutrophils in SCD contribute to disease pathology and are amplified during vaso-occlusive crisis. To define biochemical differences between neutrophils isolated from individuals with SCD and those isolated from healthy controls, we performed global gene expression analysis using the Affymetrix HG133 plus 2 GeneChip. Highly distinct transcriptional profiles were observed in neutrophils isolated from controls versus individuals with SCD at baseline and SCD in crisis. These were sufficiently distinct as to be separable by disease group in unsupervised hierarchical clustering analyses. Ontological analyses identified enriched transcriptional networks in SCD neutrophils related to elevated reactive oxygen species (ROS) production (p<6.3E-4) as well as potentiated respiratory burst (p<0.008). Using extracellular flux technology, we were able to measure oxygen consumption rates (OCR) of intact cells and observed that neutrophils from individuals with SCD have an elevated basal OCR over control cells. The elevated basal OCR could be due to increased NADPH oxidase 2 activity or increased mitochondrial respiration. While mitochondrial activity in healthy neutrophils is minimal, these organelles are essential to drive apoptosis, which is required for adequate resolution of inflammatory processes. In turn, neutrophil apoptosis is suppressed in multiple inflammatory diseases, including SCD. The transcriptional studies identified an upregulation of genes that promote mitochondrial stability (2.1- to 3.5-fold increase, p<0.02), a potential mechanism for both increased neutrophil lifespan and perhaps ROS production. Normally, neutrophils maintain ample antioxidant capacity to prevent intracellular damage from ROS generation. Vitamin E is a particularly important antioxidant in neutrophil biology as it attenuates multiple pro-inflammatory activities of these cells. We find that SCD neutrophils have markedly diminished vitamin E levels (p=0.031). Thus, loss of this important antioxidant may promote a pro-inflammatory phenotype of neutrophils in SCD. Next, we examined the potential contribution of neutrophils to the SCD pathobiology in vivo particularly during acute crisis. Berkeley sickle mice (SS mice) and controls (AA mice) were exposed to normoxia (baseline) or hypoxia (3 hrs 10% FIO2) to induce acute sickling followed by reoxygenation (2 hrs room air, H/R). Liver histopathology of SS mice displayed areas of leukocyte infiltration at baseline, with a potentiation in the number of these inflammatory sites after acute sickling induced by H/R. Importantly, examination of these sites at higher magnification reveals significant neutrophil infiltration near congested vessels, suggesting that H/R may potentiate neutrophil recruitment to injured tissues in SCD. To further assess neutrophil activation post H/R, plasma levels of myeloperoxidase (MPO) and DNA, evidence of neutrophil extracellular trap (NET) formation, were measured. SS mice have elevated plasma MPO levels compared to AA mice at baseline (p=0.007), with a trend towards a further increase in SS mice post H/R (no change in plasma MPO in AA mice post H/R). Plasma DNA levels were also elevated in SS mice compared to AA mice at baseline (p=0.01) and exposure of SS mice to H/R significantly increased plasma DNA levels (p=0.009), consistent with an acute increase in NET formation. In summary, chronic neutrophil activation in SS mice at baseline is exacerbated by acute sickling. Taken together, our data supports a role for neutrophils in SCD pathology which is amplified during vaso-occlusive crisis. Thus, targeting neutrophils may offer a novel therapeutic strategy for the prevention and treatment of vaso-occlusive complications in SCD.▪ Disclosures:Wandersee:Bayer: Consultancy. Hillery:Bayer: Consultancy; Biogen Idec: Consultancy.

Alterations in cell maturity and serum survival factors may modulate neutrophil numbers in sickle cell disease

Leukocytes are known to exacerbate inflammatory and vaso-occlusive processes in sickle cell disease (SCD). The aim of this study was to determine whether alterations in neutrophil maturity and/or cell-death modulating factors in the circulation contribute to the increased leukocyte counts and leukocyte survival observed in SCD. The maturity of circulating neutrophils from healthy control individuals (CON), SCD and SCD patients on hydroxyurea therapy (SCDHU) was determined immunophenotypically. Serum factors affecting neutrophil apoptosis (determined by annexin V-binding) were analyzed by culturing control neutrophils (CON neutrophils) with pooled serum from CON, SCD and SCDHU individuals. Immunophenotypic characterization of neutrophils suggested a slight, but significant, increase in the circulation of immature neutrophils in SCD. While SCD neutrophils cultured in the presence of CON serum presented delayed apoptosis, unexpectedly, the culture of CON neutrophils with SCD serum significantly augmented apoptosis and caspase-9 activity. Inhibition of the activity of serum interleukin-8, a neutrophil-apoptosis-inhibiting cytokine, significantly increased SCD serum-induced CON neutrophil apoptosis, indicating that SCD serum may have both apoptotic and antiapoptotic properties. The decreased maturity of SCD neutrophils observed is suggestive of an accelerated immigration of leukocytes from the bone marrow to the circulating pool that may contribute to an increase in cell survival, subject to modulation by a complex balance of both anti- and proapoptotic factors contained in the circulation of SCD individuals.

Participation of Mac-1, LFA-1 and VLA-4 integrins in the in vitro adhesion of sickle cell disease neutrophils to endothelial layers, and reversal of adhesion by simvastatin

Pharmacological approaches to inhibit increased leukocyte adhesive interactions in sickle cell disease may represent important strategies for the prevention of vaso-occlusion in patients with this disorder. We investigated, in vitro, the adhesion molecules involved in endothelial-sickle cell disease neutrophil interactions and the effect of simvastatin on sickle cell disease neutrophil adhesion to tumor necrosis factor-α-activated endothelial monolayers (human umbilical vein endothelial cells), and neutrophil chemotaxis. Sickle cell disease patients in steady state and not on hydroxyurea were included in the study. Endothelial cells treated, or not, with tumor necrosis factor-α and simvastatin were used for neutrophil adhesion assays. Neutrophils treated with simvastatin were submitted to interleukin 8-stimulated chemotaxis assays. Sickle cell disease neutrophils showed greater adhesion to endothelial cells than control neutrophils. Adhesion of control neutrophils to endothelial cells was mediated by Mac-1 under basal conditions and by the Mac-1 and LFA-1 integrins under inflammatory conditions. In contrast, adhesion of sickle cell disease neutrophils to endothelium, under both basal and tumor necrosis factor-α-stimulated conditions, was mediated by Mac-1 and LFA-1 integrins and also by VLA-4. Under stimulated inflammatory conditions, simvastatin significantly reduced sickle cell disease neutrophil adhesion, and this effect was reversed by inhibition of nitric oxide synthase. Furthermore, intercellular adhesion molecule-1 expression was significantly abrogated on tumor necrosis factor-α-stimulated endothelium incubated with simvastatin, and statin treatment inhibited the interleukin-8-stimulated migration of both control and sickle cell disease neutrophils. The integrins Mac-1, LFA-1 and, interestingly, VLA-4 mediate the adhesion of sickle cell disease leukocytes to activated endothelial cell layers, in vitro. Our data indicate that simvastatin may be able to reduce endothelial activation and consequent leukocyte adhesion in this in vitro model; future experiments and clinical trials may determine whether simvastatin therapy could be employed in patients with sickle cell disease, with beneficial effects on vaso-occlusion.

Simvastatin Reduces the in Vitro Adhesion of Sickle Cell Disease Neutrophils to Endothelial Layers

Leukocytes may have a propagating and, possibly, initiating role in sickle cell disease (SCD) vaso-occlusion. Endothelial dysfunction contributes to the vaso-occlusion process and leads to inflammation, leukocyte and red cell adhesion. Markers of neutrophil activation are also increased in SCD, in association with increased levels of circulating cytokines and increased leukocyte adhesion. In animal models, vaso-occlusion causes hypoxia/reperfusion, leading to vascular endothelium damage and an inflammatory response. We postulate that anti-inflammatory agents may reduce the participation of activated endothelium in the vaso-occlusive process. Statins are commonly used to treat arteriosclerosis and have anti-inflammatory effects that include a regulatory action on endothelial function, reduced oxidative stress and inflammation. The objective of this study was to investigate the in vitro effect of simvastatin on the adhesion of sickle neutrophils to activated endothelial cell layers (HUVEC). Neutrophils (Neu) were isolated from the peripheral blood of healthy controls (ConNeu) and SCD (SCDNeu) individuals in steady state over ficoll-paque gradients. Cell adhesion (2×106 cell/ml in Ham's F12 K) to cultured human umbilical vein endothelial cells (HUVEC) grown to confluence was assessed using static adhesion assays. HUVEC cells were treated with or without 1 μg/ml simvastatin for 6 hours in the absence or presence of a 10nM TNF-α activating stimulus (3 hours) before allowing adhesion of Neu to the cell layers (30 min, 37°C, 5%CO2). Neu from SCD patients demonstrated a significantly greater adhesion to HUVEC than ConNeu (20.5 ± 1.9% compared to 13.8 ± 1.7 %; n=15; p<0.02; Mann Whitney test). Subsequently, Neu from patients and controls were allowed to adhere to endothelial layers previously treated with simvastatin; adhesion was not significantly different to the adhesion of Neu to nonsimvastatin treated HUVEC (16.7 ± 3.2% for ConNeu; n=8, p>0.05 and 19.8 ±2.7% for SCDNeu; n=11, p>0.05, paired t test). Pre-treatment of HUVEC with the cytokine TNF-α increased the adhesion of SCD and Con Neu to HUVEC (40.9 ± 5.4%; 28.9 ± 5.0%, respect, N>8, P<0.01 compared to adhesion to non-activated HUVEC). Interestingly, when the endothelium layer was protected with simvastatin and then stimulated with TNF-α, SCDNeu adhesion was significantly diminished (reduced to 31.3% ± 3.6%; n=11, p<0.005 comp. to adhesion to non-simvastatin-treated HUVEC); in contrast, no difference in the adhesion of ConNeu to HUVEC treated with TNF-α and simvastatin was observed (31.9 ± 5.8%, n=8, p>0.05 for ConNeu). In conclusion, data indicate that under in vitro inflammatory conditions, simvastatin appears to protect endothelium layers and reduces SCD leukocyte adhesion. We speculate that statins may have anti-inflammatory properties and, as such, may be useful for diminishing endothelial activation and, in turn, preventing the adhesion of leukocytes adhesion to the vascular wall in SCD, a mechanism that is essential to the vaso-occlusive process.

Modulation of redox pathways in neutrophils from sickle cell disease patients

Interaction of nitric oxide (NO) with enzymatic sources of reactive species exerts modulatory actions on inflammatory signaling mechanisms. NADPH oxidase, total peroxidase, cyclooxygenase (COX) activity, and NO consumption were measured in neutrophils isolated from sickle cell disease (SCD) patients and healthy controls. Glutathione (GSH) levels and expression of inducible NO synthase (NOS-2) were also analyzed to assess intracellular redox state and NO production, respectively. Functional assay of NADPH oxidase was performed by measuring superoxide release, which was similar in control and SCD, both at basal conditions and in response to N-formyl-methionyl-leucyl-phenylalanine stimulation. Peroxidase activity, assessed spectrophotometrically, was not significantly different in SCD neutrophils compared to controls. Total COX activity, measured via an assay kit, was significantly increased in SCD neutrophils. The increase in total COX activity observed in SCD was due to enhanced activity of COX-2, differentiated by using the isoform-specific inhibitors DuP-697 and SC-560. Western blot analysis of COX-2 protein in SCD and control neutrophils confirmed increased enzyme activity in the diseased group. Western blot analysis of neutrophil lysates from SCD patients showed significantly increased NOS-2 protein content, compared to controls. Spectrophotometric measurement of GSH and nitrate/nitrite levels showed a decrease in GSH and an increase in nitrate/nitrite content in SCD neutrophils. Electrochemical measurement of NO consumption both under basal conditions and after N-formyl-methionyl-leucyl-phenylalanine stimulation revealed a significant decrease in SCD neutrophils compared to controls. Depletion of GSH in SCD neutrophils may impact on rates of NO consumption and reflects increased oxidative stress associated with neutrophil activation.

High expression of the cGMP‐specific phosphodiesterase, PDE9A, in sickle cell disease (SCD) and the effects of its inhibition in erythroid cells and SCD neutrophils

Modulation of intracellular cyclic guanosine monophosphate (cGMP) may characterize a therapeutic target for sickle cell disease (SCD); cGMP-dependent signalling may be important for erythroid foetal haemoglobin induction and exert anti-inflammatory functions in leucocytes. As the inhibition of phosphodiesterases (PDEs), which regulate intracellular cGMP, can result in tissue-specific elevation of cGMP, we studied the gene expressions of cGMP-specific PDEs (-1A, -5A and -9A) in the reticulocytes and neutrophils of healthy controls, steady-state SCD patients and SCD patients on hydroxycarbamide therapy (SCDHC). PDE9A gene expression was found in numerous cell types; however, high expression was found in neutrophils, reticulocytes, CD34(+)-derived erythroid cells and K562 erythroleukaemic cells, indicating a high haematopoietic cell expression. PDE9A gene expression was, however, significantly higher in the reticulocytes and neutrophils of SCD individuals, compared to control cells; Western blotting confirmed the production of PDE9A protein in SCD neutrophils and K562 cells. Inhibition of PDE9A enzyme with the specific inhibitor, BAY73-6691, significantly increased production of the gamma-globin gene (HBG) in K562 cells and reversed the increased adhesive properties of SCD neutrophils. Since elevation of haematopoietic intracellular cGMP may be beneficial in SCD, the relatively limited tissue distribution of PDE9A suggests that it could represent a novel drug target worthy of further study.