Cell surface RNAs control neutrophil recruitment.

RNAs localizing to the outer cell surface have been recently identified in mammalian cells, including RNAs with glycan modifications known as glycoRNAs. However, the functional significance of cell surface RNAs and their production are poorly known. We report that cell surface RNAs are critical for neutrophil recruitment and that the mammalian homologs of the sid-1 RNA transporter are required for glycoRNA expression. Cell surface RNAs can be readily detected in murine neutrophils, the elimination of which substantially impairs neutrophil recruitment to inflammatory sites in vivo and reduces neutrophils' adhesion to and migration through endothelial cells. Neutrophil glycoRNAs are predominantly on cell surface, important for neutrophil-endothelial interactions, and can be recognized by P-selectin (Selp). Knockdown of the murine Sidt genes abolishes neutrophil glycoRNAs and functionally mimics the loss of cell surface RNAs. Our data demonstrate the biological importance of cell surface glycoRNAs and highlight a noncanonical dimension of RNA-mediated cellular functions.

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.

Dietary iron restriction protects against vaso-occlusion and organ damage in murine sickle cell disease.

Sickle cell disease (SCD) is an inherited disorder resulting from a ß-globin gene mutation, and SCD patients experience erythrocyte sickling, vaso-occlusive episodes (VOE), and progressive organ damage. Chronic hemolysis, inflammation, and repeated red blood cell transfusions in SCD can disrupt iron homeostasis. Patients who receive multiple blood transfusions develop iron overload, and another subpopulation of SCD patients manifest iron deficiency. To elucidate connections between dietary iron, the microbiome, and SCD pathogenesis, we treated SCD mice with an iron-restricted diet (IRD). IRD treatment reduced iron availability and hemolysis, decreased acute VOE, and ameliorated chronic organ damage in SCD mice. Our results extend previous studies indicating that the gut microbiota regulate disease in SCD mice. IRD alters microbiota load and improves gut integrity, together preventing crosstalk between the gut microbiome and inflammatory factors such as aged neutrophils, dampening VOE, and organ damage. These findings provide strong evidence for the therapeutic potential of manipulating iron homeostasis and the gut microbiome to ameliorate SCD pathophysiology. Many treatments, which are under development, focus on lowering the systemic iron concentration to relieve disease complications, and our data suggest that iron-induced changes in microbiota load and gut integrity are related- and novel-therapeutic targets.

Recent advances in "sickle and niche" research - Tribute to Dr. Paul S Frenette.

In this retrospective, we review the two research topics that formed the basis of the outstanding career of Dr. Paul S. Frenette. In the first part, we focus on sickle cell disease (SCD). The defining feature of SCD is polymerization of the deoxygenated mutant hemoglobin, which leads to a vicious cycle of hemolysis and vaso-occlusion. We survey important discoveries in SCD pathophysiology that have led to recent advances in treatment of SCD. The second part focuses on the hematopoietic stem cell (HSC) niche, the complex microenvironment within the bone marrow that controls HSC function and homeostasis. We detail the cells that constitute this niche, and the factors that these cells use to exert control over hematopoiesis. Here, we trace the scientific paths of Dr. Frenette, highlight key aspects of his research, and identify his most important scientific contributions in both fields.

TGF-ß1 Reduces Neutrophil Adhesion and Prevents Acute Vaso-Occlusive Processes in Sickle Cell Disease Mice.

Sickle cell disease (SCD) patients experience chronic inflammation and recurrent vaso-occlusive episodes during their entire lifetime. Inflammation in SCD occurs with the overexpression of several inflammatory mediators, including transforming growth factor beta-1 (TGF-ß1), a major immune regulator. In this study, we aimed to investigate the role played by TGF-ß1 in vascular inflammation and vaso-occlusion in an animal model of SCD. Using intravital microscopy, we found that a daily dose of recombinant TGF-ß1 administration for three consecutive days significantly reduced TNFα-induced leukocyte rolling, adhesion, and extravasation in the microcirculation of SCD mice. In contrast, immunological neutralization of TGF-ß, in the absence of inflammatory stimulus, considerably increased these parameters. Our results indicate, for the first time, that TGF-ß1 may play a significant ameliorative role in vascular SCD pathophysiology, modulating inflammation and vaso-occlusion. The mechanisms by which TGF-ß1 exerts its anti-inflammatory effects in SCD, however, remains unclear. Our in vitro adhesion assays with TNFα-stimulated human neutrophils suggest that TGF-ß1 can reduce the adhesive properties of these cells; however, direct effects of TGF-ß1 on the endothelium cannot be ruled out. Further investigation of the wide range of the complex biology of this cytokine in SCD pathophysiology and its potential therapeutical use is needed.

Heme induces significant neutrophil adhesion in vitro via an NFκB and reactive oxygen species-dependent pathway.

Intravascular hemolysis, a major manifestation of sickle cell disease (SCD) and other diseases, incurs the release of hemoglobin and heme from red blood cells, in turn triggering inflammatory processes. This study investigated the in vitro effects of heme, a major inflammatory DAMP, on the adhesive properties of isolated human neutrophils. Heme (20 and 50 µM) significantly increased the adhesion of neutrophils to fibronectin and to recombinant ICAM-1, under static conditions, even more efficiently than the potent pro-inflammatory cytokine, tumor necrosis factor-α (TNF); a microfluidic assay confirmed that heme stimulated neutrophil adhesion under conditions of shear stress. Heme-induced neutrophil adhesion was associated with the increased activities, but not expressions, of the Mac-1 and LFA-1 integrin subunits, CD11b and CD11a, on the cell surface. Notably, heme (50 µM) significantly induced NFκB translocation in neutrophils, and inhibition of NFκB activity with the BAY11-7082 molecule abolished heme-induced cell adhesion to fibronectin and significantly decreased CD11a activity. Flow cytometric analysis demonstrated major reactive oxygen species (ROS) generation in neutrophils following heme stimulation that could be inhibited by the antioxidant, α-tocopherol, and by BAY11-7082. Furthermore, co-incubation with α-tocopherol abrogated both heme-stimulated neutrophil adhesion and CD11a/CD11b activation. Thus, our data indicate that heme, at clinically relevant concentrations, is a potent activator of neutrophil adhesion, increasing the ligand affinity of the ß2 integrins via a mechanism that may be partially mediated by an NFkB-dependent pathway and the generation of ROS. Given the fundamental role that the adhesion of neutrophils to the vascular wall plays in SCD vaso-occlusion and other vascular inflammatory processes, our findings provide further evidence that cell-free heme is a major therapeutic target in the hemolytic diseases.

cGMP modulation therapeutics for sickle cell disease.

IMPACT STATEMENT: Sickle cell disease (SCD) is one of the most common inherited diseases and is associated with a reduced life expectancy and acute and chronic complications, including frequent painful vaso-occlusive episodes that often require hospitalization. At present, treatment of SCD is limited to hematopoietic stem cell transplant, transfusion, and limited options for pharmacotherapy, based principally on hydroxyurea therapy. This review highlights the importance of intracellular cGMP-dependent signaling pathways in SCD pathophysiology; modulation of these pathways with soluble guanylate cyclase (sGC) stimulators or phosphodiesterase (PDE) inhibitors could potentially provide vasorelaxation and anti-inflammatory effects, as well as elevate levels of anti-sickling fetal hemoglobin.

Atypical ß-S haplotypes: classification and genetic modulation in patients with sickle cell anemia.

ß-S globin haplotype (ßS haplotype) characterization in sickle cell anemia (SCA) patients is important because it assists individualized treatment. However, the patient with atypical haplotypes do not present detailed studies such as clinical and laboratory data. To understand the phenotypic expression of atypical haplotype patients in relation to typical haplotype ones, it may be necessary to assess the main clinical and laboratorial parameters and investigate transcription factors, as possible genetic modulators that can contribute to the improvement of the SCA patients' clinical condition. The study group was composed of 600 SCA Brazilian patients of both genders ranging in age from 1 to 68 years. The atypical haplotypes were the third most frequent (5.7%) with 11 patterns numerically ranked according to occurrence. We verified that patients with atypical 1 haplotype in combination with Bantu haplotype presented milder clinical outcomes in relation to Bantu/Bantu and Benin/Benin patients, according to improved values of hemoglobin and hematocrit. In clinical severity, we did not observe significant statistical differences between typical and atypical haplotype patients, and this result can be explained with reference to the action of transcription factors in ß-globin cluster. Thus, we presented the atypical haplotype relationship with SCA pathophysiology, reinforcing the hypothesis that individual genetic factors may be responsible for phenotypic diversity of the disease.

Emerging pharmacotherapeutic approaches for the management of sickle cell disease.

INTRODUCTION: Sickle cell disease (SCD) is an inherited disease with lifelong morbidity, whose complications include frequent acute painful vaso-occlusive episodes (VOEs) that often require hospitalization. The only pharmacotherapy currently in regular use for SCD management is hydroxyurea (hydroxycarbamide). AREAS COVERED: We review recent advances in pharmacotherapy for SCD and summarize promising synthetic agents that are in late-stage development (phase 3) for SCD. EXPERT OPINION: Emerging SCD therapies have been developed to target specific pathophysiological mechanisms of the disease, as either preventative or abortive approaches to VOEs. Continuous-use pharmacotherapeutics in late-phase development for VOE prevention include voxelotor (GBT440), which elevates hemoglobin oxygenation, and prasugrel, a platelet activation inhibitor. However, at least in the near future, it is probable that biological molecules will play a primary role in SCD preventative therapy; in combination with hydroxyurea, crizanlizumab, an anti-P-selectin monoclonal antibody, appears to reduce VOE frequency, while L-glutamine was the first substance licensed by the FDA for use in SCD in 20 years. Synthetic drugs, however, may represent key approaches for the management of individuals upon hospitalization for VOE, a major challenge for SCD. For example, rivipansel (GMI-1070), a pan-selectin inhibitor, has shown encouraging effects on hospitalization time and opioid use.

Impact of genetic polymorphisms in key enzymes of homocysteine metabolism on the pathophysiology of sickle cell anemia.

This work aimed at studying a possible influence of methylenetetrahydrofolate reductase (MTHFR; c. 677C>T) and cystathionine ß-synthase (CBS; 844ins68) polymorphisms on overall oxidative status of sickle cell anemia (SCA) patients and on routine markers, correlating them with hydroxycarbamide (HC) treatment. We evaluated 95 unrelated and diagnosed SCA patients. All patients received a prophylactic treatment with folic acid of 5mg/day, while 41 (43.2%) of them were under hydroxycarbamide (HC) treatment (average dose: 22mg/kg/day). MTHFR and CBS polymorphisms were identified by Polymerase Chain Reaction. Biochemical parameters were measured using spectrophotometric and chromatographic methods. Routine markers were developed by specialized laboratory. We did not find any effect of 677T and "I" allele combination on the biomarkers evaluated. On the other hand, MTHFR 677T mutation was related to a depletion of antioxidant capacity, according to the decreased catalase activity and a reduction about 30% of glutathione levels. Moreover, the presence of the insertion was related to about 23% less biomolecule oxidation levels and lower monocytes count, but about 14% higher lactate dehydrogenase activity. These findings may contribute to highlight that the MTHFR and CBS polymorphisms involvement in SCA pathophysiology is likely to be far more complex than it was explored to date.

Correction: Inflammation in Sickle Cell Disease: Differential and Down-Expressed Plasma Levels of Annexin A1 Protein.

[This corrects the article DOI: 10.1371/journal.pone.0165833.].

Inflammation in Sickle Cell Disease: Differential and Down-Expressed Plasma Levels of Annexin A1 Protein.

Sickle cell disease (SCD) is an inherited hemolytic anemia whose pathophysiology is driven by polymerization of the hemoglobin S (Hb S), leading to hemolysis and vaso-occlusive events. Inflammation is a fundamental component in these processes and a continuous inflammatory stimulus can lead to tissue damages. Thus, pro-resolving pathways emerge in order to restore the homeostasis. For example there is the annexin A1 (ANXA1), an endogenous anti-inflammatory protein involved in reducing neutrophil-endothelial interactions, accelerating neutrophil apoptosis and stimulating macrophage efferocytosis. We investigated the expression of ANXA1 in plasma of SCD patients and its relation with anemic, hemolytic and inflammatory parameters of the disease. Three SCD genotypes were considered: the homozygous inheritance for Hb S (Hb SS) and the association between Hb S and the hemoglobin variants D-Punjab (Hb SD) and C (Hb SC). ANXA1 and proinflammatory cytokines were quantified by ELISA in plasma of SCD patients and control individuals without hemoglobinopathies. Hematological and biochemical parameters were analyzed by flow cytometry and spectrophotometer. The plasma levels of ANXA1 were about three-fold lesser in SCD patients compared to the control group, and within the SCD genotypes the most elevated levels were found in Hb SS individuals (approximately three-fold higher). Proinflammatory cytokines were higher in SCD groups than in the control individuals. Anemic and hemolytic markers were higher in Hb SS and Hb SD genotypes compared to Hb SC patients. White blood cells and platelets count were higher in Hb SS genotype and were positively correlated to ANXA1 levels. We found that ANXA1 is down-regulated and differentially expressed within the SCD genotypes. Its expression seems to depend on the inflammatory, hemolytic and vaso-occlusive characteristics of the diseased. These data may lead to new biological targets for therapeutic intervention in SCD.

Phenotypic Diversity of Sickle Cell Disease in Patients with a Double Heterozygosity for Hb S and Hb D-Punjab.

Phenotypic heterogeneity for sickle cell disease is associated to several genetic factors such as genotype for sickle cell disease, ß-globin gene cluster haplotypes and Hb F levels. The coinheritance of Hb S (HBB: c.20A > T) and Hb D-Punjab (HBB: c.364G > C) results in a double heterozygosity, which constitutes one of the genotypic causes of sickle cell disease. This study aimed to assess the phenotypic diversity of sickle cell disease presented by carriers of the Hb S/Hb D-Punjab genotype and the Bantu [- + - - - -] haplotype. We evaluated medical records from 12 patients with sickle cell disease whose Hb S/Hb D-Punjab genotype and Bantu haplotype were confirmed by molecular analysis. Hb S and Hb D-Punjab levels were quantified by chromatographic analysis. Mean concentrations of Hb S and Hb D-Punjab were 44.8 ± 2.3% and 43.3 ± 1.8%, respectively. Painful crises were present in eight (66.7%) patients evaluated, representing the most common clinical event. Acute chest syndrome (ACS) was the second most prevalent manifestation, occurring in two individuals (16.7%). Three patients were asymptomatic, while another two exhibited greater diversity of severe clinical manifestations. Medical records here analyzed reported a significant clinical diversity in sickle cell disease ranging from the absence of symptoms to wide phenotypic variety. The sickle cell disease genotype, Bantu haplotype and hemoglobin (Hb) levels did not influence the clinical diversity. Thus, we concluded that the phenotypic variation in sickle cell disease was present within a specific genotype for disease regardless of the ß-globin gene cluster haplotypes.

Inheritance of the Bantu/Benin haplotype causes less severe hemolytic and oxidative stress in sickle cell anemia patients treated with hydroxycarbamide.

Beta S-globin gene cluster haplotypes (ß(S)-haplotypes) can modulate the response to hydroxycarbamide (HC) treatment in sickle cell anemia (SCA) patients. In Brazil, the most common haplotypes are Bantu and Benin, and both confer a poor prognosis for patients when untreated with HC. We evaluated oxidative and hemolytic biomarkers in 48 SCA patients undergoing HC treatment separated in three subgroups: Bantu/Bantu, Bantu/Benin and Benin/Benin haplotype. On the basis of reduced haptoglobin (HP) levels, patients with Bantu/Bantu haplotypes had 3.0% higher hemolysis degree when compared with those with Bantu/Benin haplotypes (P=0.01). The Benin/Benin patients had 53.6% greater lipid peroxidation index than the Bantu/Bantu patients (P=0.01) because of evaluated thiobarbituric acid reactive species levels. The Bantu/Benin subgroup had intermediate levels of hemolytic and oxidative stress markers compared with the homozygous subgroups. Through strict inclusion criteria adopted, as well as consolidated and well-described hemolytic and the oxidative parameters evaluated, we suggest a haplotype-interaction response to HC treatment mediated by a 'balance' between the genetic factors of each haplotype studied.

Plasma levels of TGF-ß1 in homeostasis of the inflammation in sickle cell disease.

Sickle cell disease (SCD) represents a chronic inflammatory condition with complications triggered by the polymerization of hemoglobin S (Hb S), resulting in a series of cellular interactions mediated by inflammatory cytokines, as the transforming growth factor beta (TGF-ß), which plays an important role in inflammation resolution. This study assessed the relation between SCD inflammation and the plasma concentration of TGF-ß1, and also checked the influence of the presence of -509C/T polymorphism in TGFB1 gene on TGF-ß1 plasma values. The plasma levels of TGF-ß1 were quantified by ELISA in 115 patients with SCD (genotypes SS, SD-Los Angeles, Sß-thalassemia and SC) and in 58 individuals with no hemoglobinopathies (Hb AA), as the control group. The -509C/T polymorphism in TGFB1 gene was screened by PCR-RFLP. The correlation between TGF-ß1 plasma levels and the inflammation was based on its association with the count of platelets, total white blood cells (WBC) and neutrophils in the peripheral blood. Patients with SCD showed plasma levels of TGF-ß1 higher than the control group, especially the Hb SS genotype, followed by the group with Hb SD. Polymorphism investigation showed no interference in the values obtained for the cytokine in the groups evaluated. All SCD groups showed TGF-ß1 levels positively correlated to the platelets and WBC counts. The original data obtained in this study for SCD support the involvement of TGF-ß1 in regulating of the inflammatory response and suggest that this marker possibly may become a potential therapeutic target in the treatment of the disease.

Severity of Brazilian sickle cell disease patients: severity scores and feasibility of the Bayesian network model use.

The integration of the several clinical and laboratory dimensions and the influence of each parameter on the sickle cell disease (SCD)-related mortality is useful for predicting the phenotype of an individual. This study evaluated the feasibility of the SCD severity calculator use to measure disease severity in Brazilian patients. The study group was composed of 500 SCD patients (440 HbSS and 60 HbSC) diagnosed by molecular biology. We observed a decrease in severity scores in 72 SCD patients assessed before and after the hydroxyurea (HU) use. Furthermore, the HU influenced the increase of mean corpuscular volume (MCV) and HbF concentration, and the decrease of leukocytes and total bilirubin. We found 180 (36.0%) patients with intermediate phenotype, 170 (34.0%) mild phenotype and 150 (30.0%) with severe phenotype. Patients with ages >40 years had higher mean score (0.778±0.177) than patients between 18 and 40 years (0.562±0.152) and patients between 5 and 17 years (0.322±0.145). We observe that there is a tendency of individuals with leg ulcers, avascular necrosis and cardiac complications with increasing age. Correlation analysis showed relations between severity scores with leukocytes, reticulocytes, bilirubin, lactate dehydrogenase, HbS, hemoglobin and hematocrit (p<0.05). Several comparisons involving age groups, SCD genotype and phenotypic classification had satisfactory results and this classification will be used for future studies involving genetic polymorphisms, response to treatment with HU and oxidative stress markers in SCD.

Hemoglobin D-Punjab: origin, distribution and laboratory diagnosis

This review discusses hemoglobin D-Punjab, also known as hemoglobin D-Los Angeles, one of the most common hemoglobin variants worldwide. It is derived from a point mutation in the beta-globin gene (HBB: c.364G>C; rs33946267) prevalent in the Punjab region, North-western Indian. Hemoglobin D-Punjab can be inherited in heterozygosis with hemoglobin A causing no clinical or hematological alterations, or in homozygosis, the rarest form of inheritance, a condition that is commonly not related to clinical symptomatology. Moreover, this variant can exist in association with other hemoglobinopathies, such as thalassemias; the most noticeable clinical alterations occur when hemoglobin D-Punjab is associated to hemoglobin S. The clinical manifestations of this association can be similar to homozygosis for hemoglobin S. Although hemoglobin D-Punjab is a common variant globally with clinical importance especially in cases of double heterozygosis, hemoglobin S/D-Punjab is still understudied. In Brazil, for example, hemoglobin D-Punjab is the third most common hemoglobin variant. Thus, this paper summarizes information about the origin, geographic distribution, characterization and occurrence of hemoglobin D-Punjab haplotypes to try to improve our knowledge of this variant. Moreover, a list of the main techniques used in its identification is provided emphasizing the importance of complementary molecular analysis for accurate diagnosis.

Hemoglobin D-Punjab: origin, distribution and laboratory diagnosis.

This review discusses hemoglobin D-Punjab, also known as hemoglobin D-Los Angeles, one of the most common hemoglobin variants worldwide. It is derived from a point mutation in the beta-globin gene (HBB: c.364G>C; rs33946267) prevalent in the Punjab region, Northwestern Indian. Hemoglobin D-Punjab can be inherited in heterozygosis with hemoglobin A causing no clinical or hematological alterations, or in homozygosis, the rarest form of inheritance, a condition that is commonly not related to clinical symptomatology. Moreover, this variant can exist in association with other hemoglobinopathies, such as thalassemias; the most noticeable clinical alterations occur when hemoglobin D-Punjab is associated to hemoglobin S. The clinical manifestations of this association can be similar to homozygosis for hemoglobin S. Although hemoglobin D-Punjab is a common variant globally with clinical importance especially in cases of double heterozygosis, hemoglobin S/D-Punjab is still understudied. In Brazil, for example, hemoglobin D-Punjab is the third most common hemoglobin variant. Thus, this paper summarizes information about the origin, geographic distribution, characterization and occurrence of hemoglobin D-Punjab haplotypes to try to improve our knowledge of this variant. Moreover, a list of the main techniques used in its identification is provided emphasizing the importance of complementary molecular analysis for accurate diagnosis.