Inflammation In Sickle Cell Disease Research Articles

Mechanisms of Inflammatory Caspase Activation in Sickle Cell Disease

Background: Sickle cell disease (SCD) is an inherited red blood cell disorder that impacts millions of people worldwide. Patients with SCD experience chronic hemolysis and have elevated serum levels of free heme. Free heme activates a variety of inflammatory pathways that contribute to the serious clinical complications of SCD. One of the inflammatory pathways activated by heme is the inflammatory caspase pathway. Our lab previously showed that heme exposure in macrophages leads to the activation of inflammatory caspase proteins caspase-1 and caspase-4. Activation of these proteins by heme leads to IL-1β release and pyroptosis, a form of inflammatory cell death. Patients with SCD have increased circulating IL-1β levels, indicating this pathway is active in patients. In mouse models, inflammatory caspases are required for hemolysis-induced neutrophil recruitment and lethality, indicating these proteins are central to hemolysis-induced inflammation. Despite the key role these proteins play following hemolysis, it remains unknown how heme activates inflammatory caspases. Interestingly, both caspase-1 and caspase-4 are required for heme-induced IL-1β release while caspase-4 alone is required for heme-induced pyroptosis. In fact, pyroptosis likely is required for IL-1β release from the cell. Based on this data, we hypothesized that caspase-4 is the apical step in this pathway and a key druggable target for controlling unchecked inflammation in SCD. Results: We first sought to determine if caspase-4 activation is upstream of caspase-1 activation. To assess this, we used an imaging-based caspase activation reporter assay. Using this system, we found that siRNA knock-down of caspase-4 in primary human macrophages significantly inhibited heme-induced caspase-1 activation (63.8% vs 26.3%, n =4, p = 0.015). This result shows that caspase-4 is upstream of heme-induced caspase-1 activation and confirmed caspase-4 activation is the initial step in this pathway. Next, we sought to determine how caspase-4 activation occurs. Other known activators of caspase-4 induce activation by directly binding to caspase-4 and inducing self-oligomerization. To determine if heme acts in a similar manner, we used a published heme-binding prediction algorithm called HeMoQuest. We found multiple predicted heme-binding sites in caspase-4. The dissociation constants for these binding sites are less than 1 µM, suggesting strong binding affinity. Importantly, there are no predicted heme binding sites in caspase-1, indicating caspase-4 is the only direct heme receptor in this pathway. To validate these binding predictions, we performed molecular docking experiments and confirmed there are multiple heme-binding sites in caspase-4. Specifically, we found two heme-binding regions in the p10 catalytic subunit of caspase-4 comprising 2-4 essential residues. We next confirmed that heme directly interacts with caspase-4 in vitro. Using a pull-down assay with heme conjugated to agarose beads, we observed heme bound to purified caspase-4 and to casapase-4 in macrophage cell-free extracts. Heme did not interact with proteins structurally related to caspase-4 including caspase-1, caspase-7, and caspase-9 and caspase-4 was not pulled down by control agarose. This confirms the specificity of the heme-caspase-4 interaction. To determine if heme binding to caspase-4 results in activation, we performed native PAGE analysis of purified caspase-4 after exposure to heme. Heme induced formation of a caspase-4 oligomer around 200 kDa in size, four-fold larger than the inactive caspase-4 monomer. Other compounds known to induce IL-1β release from cells, like microbial toxin nigericin, did not induce caspase-4 oligomerization, demonstrating the specificity of heme's effect on caspase-4. Conclusions: We have determined that caspase-4 is a direct intracellular heme receptor and that heme exposure leads to caspase-4 activation, which in turn facilitates caspase-1 activation, IL-1β release and cell death. Our future work will elucidate the functional effects of heme binding to caspase-4 in mouse models of sickle cell disease. Caspase-4 is an attractive therapeutic target as it is required for both functional outputs of inflammatory caspase activation, IL-1β release and pyroptosis. Inhibiting heme-caspase-4 interactions would provide a specific way to block the harmful effects of this pathway in patients.

Socioeconomic and Inflammatory Correlates of Plasma Cortisol Among Individuals with Sickle Cell Disease

Introduction: Cortisol plays a critical role in the biological link between stress and health outcomes. It is frequently investigated in the association between socioeconomic stress and morbidity, and the hypothesized biological mechanism by which socioeconomic stress and cortisol impact health is through chronically elevated systemic inflammation. This research is relevant to individuals with sickle cell disease (SCD) for several reasons. First, up to 40% of children and adolescents with SCD are impacted by neighborhood poverty. Furthermore, individuals with SCD are faced with significant disease-related morbidities, such as chronic and acute pain, renal injury, and cerebrovascular disease that are further complicated by elevated inflammatory markers. Finally, cortisol is heavily understudied in this population, with no research investigating socioeconomic or inflammatory correlates. The purpose of this study was to investigate plasma cortisol and its association with neighborhood-level socioeconomic factors (income and poverty) and immunomodulatory cytokines (IL-6, IL-10), among individuals with SCD. Method: Plasma from individuals with SCD within Children's Hospital of Philadelphia (CHOP) and University of Pennsylvania School of Medicine (Penn Medicine) BioBanks were identified. A cortisol radioimmunoassay was used to determine plasma cortisol levels. The Olink immune response panel was used to measure plasma levels of 92 proteins involved in inflammatory pathways. Home address collected from electronic health records was used to determine neighborhood census tract geoid, which was then used to collect data on median neighborhood income and percentage of families living in poverty from the 2020 American Census Survey. Bivariate and multivariate analyses were conducted across 4 subgroups in the total sample: pediatric patients, adult patients, SCA (HbSS/HbSß 0), and other SCD genotypes (HbSC/HbSß +). Control samples (healthy and sickle cell trait; n = 22) without neighborhood information were also available for comparison. Results: Fifty-four independent outpatient samples were identified after excluding duplicates and siblings. Twenty-seven samples were from children and adolescents receiving pediatric care at CHOP (Range age = .8 to 21; Mage= 13.6, SD = 5.3; 51.9% female, 74.1% SCA) and 27 samples were from adults receiving care at Penn Medicine (Range age = 19 to 56; Mage= 35.8, SD = 10.1; 51.9% female, 51.9% SCA). There were no significant differences in cortisol levels across each subgroup ( Mpeds= 16.6, SDpeds = 6.2; Madult= 13.4, SDadult = 6.9; MSCA= 15.1, SDSCA = 7.1; MSCD= 14.9, SDSCD = 6.3). Neighborhood income and poverty did not differ across pediatric and adult patients, and income did not differ across SCD types. However, neighborhood poverty was lower among patients with SCA ( M = 13.9; SD = 11.9) relative to other SCD genotypes ( M = 21.7; SD = 13.5; p = .041). Bivariate Spearman analyses found that neighborhood variables were not correlated with cortisol or cytokines in the total sample or subgroups. Yet, multivariate analyses controlling for sex and time of blood draw showed a positive main effect for patient age ( p = .019) and a significant age by poverty interaction ( p = .041) predicting cortisol among pediatric patients. Cortisol levels were higher with age, yet the association among poverty and cortisol was strongest in younger patients. Analyses also showed a sex*poverty interaction among pediatric patients that approached significance ( p = .081), such that poverty only predicted cortisol in male patients. There were no significant differences for immunomodulatory cytokines across each SCD subgroups; however, IL-6 and IL-10 were elevated in patients with SCA relative to healthy controls ( p IL6 = .021; p IL10 = .003). Bivariate Spearman correlations showed that cortisol was positively related to IL-6 (rho [ρ]= .38, p = .027) and IL-10 (ρ= .34, p = .048) among patients with SCA. There were no additional significant bivariate or multivariate effects. Conclusion: Neighborhood factors likely have downstream effects on the biological stress response and inflammation in SCD. Further research must be conducted to elucidate concurrent and prospective associations among neighborhood socioeconomic factors, stress-related cortisol response, and inflammatory outcomes when extending this work to investigate the impact of stress on SCD morbidity and mortality.

Plasma Proteome Profiling of Sickle Cell Patients Reveals Activation of Inflammatory Pathways during Steady State and Vaso-Occlusive Crisis

Background The pathophysiology of Sickle Cell Disease (SCD) is characterized by chronic intravascular hemolysis and ischemia-reperfusion, resulting in oxidative stress and chronic inflammation. Although inflammation seems to play an important role in the development of tissue damage in SCD, little is known about the various cellular and cytokine pathways involved in the pathophysiology of SCD. This study aims to further elucidate the inflammatory pathways in SCD using targeted plasma protein profiling both in steady state and during vaso-occlusive crisis (VOC). Methods In this prospective study, SCD patients with the genotypes HbSS and HbSβ 0-thalassemia and healthy ethnicity matched controls were included. Plasma levels of 92 proteins involved in various inflammatory pathways were measured in EDTA plasma using Olink Proximity Extension Assay (PEA) technology. In brief, pairs of oligonucleotide-labeled antibody probes were added to 20 μL of plasma, resulting in the binding of these antibody probes to their targeted protein. Subsequently DNA polymerase was added, leading to a proximity-dependent DNA polymerization event, generating a unique PCR target sequence. The final assay read-out is presented in Normalized Protein eXpression (NPX) values, which is an arbitrary unit on a log2-scale with an NPX difference (estimate) of 1 meaning a doubling of protein concentration. RStudio version 4.2.1 was used for data analysis and visualization. Differences between subgroups were tested using an unpaired or paired t-test as appropriate. Results were corrected for multiple testing using the Benjamini-Hochberg method. Results with p < 0.05 were considered significant. Furthermore Over-representation Analysis (ORA) was performed to investigate the association of the differentially expressed proteins with specific biological processes. Results Plasma samples were collected from 43 SCD patients in steady state (median age: 25 years (IQR: 20 - 32)) and 13 healthy ethnicity matched controls (median age: 29 (IQR: 28-39)). From 15 SCD patients plasma samples were collected during both steady state and VOC (median age: 27 (IQR: 19 - 34)). Comparison of protein levels between healthy controls and SCD patients in steady state identified 38 proteins (IL-18, ANGPT2, Gal-9, CD4, HGF, HO-1, CCL4, ARG1, ADA, GZMA, PD-L1, CCL23, LAG3, TNFRSF4, CCL3, CXCL11, CRTAM, IL-10, GZMB, CD27, FGF2, IL-8, IL-6, TNF, PGF, NCR1, TNFSF14, CASP-8, IL-7, FASLG, TNFRSF9, LAP TGF-beta-1, PD-L2, CCL19, CD83, GZMH, MCP-4, CD244, TIE2) that were significantly higher in SCD patients as shown in Figure 1. Furthermore soluble CD70 was significantly lower in SCD patients in steady state compared to healthy controls (estimate: -0.34, p < 0.05). Over-representation Analysis revealed a significant upregulation of proteins involved in the regulation of hematopoiesis, the IL-18 signaling pathway, T-cell activation, leukocyte differentiation and leukocyte adhesion (Figure 2). Pairwise comparison of protein levels between steady state and VOC in SCD patients revealed 6 proteins (TWEAK, VEGFR-2, CD244, PDCD1, GZMA, TNFRSF4) which were significantly lower and 12 proteins (IL-6, TNFSF14, ADA, IL-15, HGF, IL-10, IL-8, Gal-9, CSF-1, CCL20, MMP7, VEGFA) which were significantly higher during VOC as compared to steady state. During VOC, pathways involved in the regulation of cell death (IL-6, TNFSF14, ADA, HGF, IL-10, PDCD1, VEGFA) and tissue development (IL-6, ADA, HGF, IL-10, VEGFA) were significantly enriched. Summary/conclusion The results of these proteomic analyses provide new insights into the pathways that regulate inflammation in SCD, both during steady state and VOC. These findings might contribute to a better understanding of SCD pathophysiology and identifying potential new targets for therapeutic interventions in SCD patients. Our results warrant further in-depth investigation of the different activated pathways.

Inhibition of the glycolytic regulator 6-phosphofructokinase/fructose-2,6-bisphosphatase 3 (PFKFB3) in macrophages attenuates heme-induced NF-kB-dependent inflammation in Sickle Cell Disease.

Inhibition of the glycolytic regulator 6-phosphofructokinase/fructose-2,6-bisphosphatase 3 (PFKFB3) in macrophages attenuates heme-induced NF-kB-dependent inflammation in Sickle Cell Disease.

#6368 PLATELET TO LYMPHOCYTE RATIO AND SICKLE CELL NEPHROPATHY

Abstract Background and Aims Platelet-to-lymphocyte ratio (PLR) was introduced as cheap and readily assessed biologic markers of subclinical inflammation in sickle cell disease. Microalbuminuria and hyperfiltration, early markers of Sickle cell nephropathy (SCN) have been reported to be associated with platelet-to-lymphocyte ratio (PLR). This association has not yet been assessed in Congolese Sickle cell disease (SCD) children. Therefore, the aim of the present study was to assess the association between PLR and early markers of kidney disease in SCD children living in the Democratic Republic of Congo (DRC). Method In this cross-sectional study, we have investigated 175 SCD children from four centers (Kinshasa university clinics, CMMASS, CMD Diamand, and Biamba Marie Mutumbo Dikembe) that give a comprehensive care to SCD patients. The PLR were calculated from full blood count. Elevated albuminuria and hyperfiltration, as the main outcomes of the study, were defined by urinary albumin/creatinine ratio (ACR) ≥30 mg/g and estimated glomerular filtration ratio (eGFR) >130 ml/min per 1.73 m2 for females and >140 ml/min per 1.73 m2 for males, respectively. Logistic regression analysis was used to assess the relationship between RPL and early sickle cell nephropathy. Results Of the 175 SCD children enrolled, 41(23.4%) and 67(38.3%) of them presented with abnormal albuminuria and hyperfiltration, respectively. RPL >130 was observed in 90 (51.4%) patients. RPL was strongly, significantly and independently associated with both abnormal albuminuria (aOR 3,381; IC95% 2, 22-1, 63; p = 0,000) and hyperfiltration (aOR 2, 19; 95%IC 2, 98 - 22, 25; p = 0,010). Conclusion The present study has shown that nearly fifty two out of one hundred SCD children bear a high platelet to lymphocyte ratio that is strongly and significantly associated with early markers of kidney disease.

Flurbiprofen inhibits heme induced NLRP3 inflammasome in Berkeley sickle cell disease mice.

Sickle cell disease (SCD) is accompanied by several complications, which emanate from the sickling of erythrocytes due to a point mutation in the β-globin chain of hemoglobin. Sickled erythrocytes are unable to move smoothly through small blood capillaries and therefore, cause vaso occlusion and severe pain. Apart from pain, continuous lysis of fragile sickled erythrocytes leads to the release of heme, which is a strong activator of the NLRP3 inflammasome, thus producing chronic inflammation in sickle cell disease. In this study, we identified flurbiprofen among other COX-2 inhibitors to be a potent inhibitor of heme-induced NLRP3 inflammasome. We found that apart from being a nociceptive agent, flurbiprofen exerts a strong anti-inflammatory effect by suppressing NF-κB signaling, which was evidenced by reduced levels of TNF-α and IL-6 in wild-type and sickle cell disease Berkeley mice models. Our data further demonstrated the protective effect of flurbiprofen on liver, lungs, and spleen in Berkeley mice. The current sickle cell disease pain management regime relies mainly on opiate drugs, which is accompanied by several side effects without modifying the sickle cell disease-related pathology. Considering the potent role of flurbiprofen in inhibiting NLRP3 inflammasome and other inflammatory cytokines in sickle cell disease, our data suggests that it can be explored further for better sickle cell disease pain management along with the possibility of disease modification.

Microbial Metabolites Target Histone Deacetylases to Trigger Vascular Inflammation in Sickle Cell Disease

The gut microbiota is a fundamental regulator of host immune responses. Our group previously demonstrated that the depletion of the gut microbiota by broad-spectrum antibiotics (ABX) prevented neutrophil aging (a type of circadian priming) and vaso-occlusion and prolonged survival in a mouse model for sickle cell disease (SCD) (Zhang, D et al. Nature 2015, 570(7570):528-532); the mechanisms involved, however, are as yet unknown. Diverse metabolites are generated by the microbiota from the fermentation of exogenous undigested dietary components and endogenous substrates secreted by the host. Complex carbohydrates are metabolized into short-chain fatty acids (SCFAs), while cholesterol derivatives from the liver are converted into secondary bile acids by intestinal bacteria. The major SCFA producers are Bacteroidetes (gram-negative) and Firmicutes (gram-positive) divisions, and dysbiosis of these bacterial divisions is found in both SCD humans and mice, (Lim, S et al. Am J Hematol 2018, 93(4):E91-E93). Herein, we investigate whether and how microbiota-derived metabolites modulate the dynamics of neutrophils in vaso-occlusive processes in SCD mice. First, to C57BL/6 (wild-type, WT) mice we administered either: 1) one of three main SCFAs, namely sodium butyrate (Na-B), sodium propionate (Na-P), or sodium acetate (Na-A); or 2) a combination of two main secondary bile acids, lithocholic and deoxycholic acids. The number of circulating neutrophils with an aged phenotype (i.e., Ly6Ghi CD11bhi CD62Llo CXCR4pos, Casanova-Acebes, M et al. Cell 2013, 153(5):1025-1035) was assessed by flow cytometry over time. Numbers started to increase at 2h after Na-B administration and we observed a peak of accumulation of total and aged-like neutrophils at 4-5h, with a return to basal after 9h. Consistent with this finding, administration of Na-B to SCD mice led to augmented counts of aged-like neutrophils and impaired survival of mice subjected to inflammation. Interestingly, when SCD mice receiving prolonged ABX treatment (40-45 days) were also given daily Na-B for the last days of ABX treatment, the percentage of aged-like neutrophils in the circulation was higher than that found in the blood of SCD mice under ABX, but without Na-B (55% vs 24%), showing that Na-B by itself is able to reverse the reduction in neutrophil aging promoted by ABX. To address the impact of Na-B in SCD vaso-occlusion, intravital microscopy of the cremaster muscle of SCD mice, after inflammatory stimulus with TNFa, revealed that Na-B abrogated the beneficial effects of ABX and triggered vaso-occlusive processes. We found a 3-fold rise in the adhesion of leukocytes to the endothelium and a 2-fold slower blood flow rate in the microvasculature of mice who received Na-B (see Figure). These results support our hypothesis that the gut microbiota modulates inflammation and vaso-occlusion in SCD through the generation of SCFAs. Because Na-B is known to be a potent inhibitor of histone deacetylase (HDAC) enzymes, we induced HDAC inhibition in WT mice. Vorinostat, a potent synthetic HDAC inhibitor, led to a 3-fold greater expansion of total and aged-like neutrophils in the blood, suggesting a role for HDACs in neutrophil biology. Consistently, in vitro treatment of WT mouse neutrophils with vorinostat or Na-B accelerated the progressive acquisition of an aged-like phenotype, characterized by loss of L-selectin (CD62L). Proteome of neutrophils from WT mice after induction of circadian aging by anti-P and anti-E-selectin antibodies showed that aged neutrophils are similar to neutrophils from vorinostat-treated mice, with down-regulation of genes involved in mRNA processing, protein synthesis, and proteasome complex formation, suggesting that HDAC inhibition is the underlying mechanism driving neutrophil aging. Importantly, the down- and up-regulated pathways found in these neutrophils were the same as those found in SCD mouse neutrophils, and an opposite proteomic profile was observed in neutrophils from ABX-treated SCD mice. Our findings indicate that the intestinal microbiota drives vaso-occlusion and neutrophil aging in SCD mice through the production of metabolites that inhibit HDAC. They also define the microbiota as instigator and innate immunity as effector of vascular disease, such that targeting their crosstalk could be effective at alleviating the devastating symptoms in SCD patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Colchicine Reduces Inflammation in a Humanized Mouse Model of Sickle Cell Disease

Introduction Inflammation, marked by a "cytokine storm" of circulating proinflammatory cytokines including interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor- α (TNF-α), and C-reactive protein (CRP), and increases in intracellular NLRP3 inflammasomes is an important component of sickle cell disease (SCD). Although current treatments have limited anti-inflammatory effects, targeting inflammation is a promising but unrealized treatment approach. Colchicine, a tubulin inhibitor, is a promising anti-inflammatory agent for SCD. It is useful in gout, familial Mediterranean fever, pericarditis and other inflammatory diseases. In these contexts, it often reduces the same inflammatory proteins and cellular components that are elevated in SCD. We sought preliminary evidence for colchicine as an anti-inflammatory treatment for SCD. Methods Humanized sickle mice (HbSS-BERK) are knockout for murine α and β globins and express human α and βS globins (>99% βS) on a mixed genetic background. HbSS-BERK have severe disease including hyperalgesia. Experiments were performed following protocols approved by the Institutional Animal Care and Use Committee. Female mice of ~ 3.5 months of age received either colchicine 100 µg/kg body weight or vehicle (phosphate-buffered saline) intraperitoneally daily for 14 d. Pain-related behavior testing was performed before treatment and 1 h, 24 h, 7 d, and 14 d after the 1st dose of study agent. Mechanical hyperalgesia was tested by paw withdrawal frequency (PWF) in response to the application of a von Frey monofilament to the mid-plantar surface of the hind paw. Musculoskeletal/deep tissue hyperalgesia was tested by measurement of the peak tensile force exerted by a forelimb upon traction. Cold hyperalgesia was tested by PWF/2 min upon placement of mice on a cold plate maintained at ~4°C. After the 14 d of dosing, mice were euthanized; blood was collected; mice were necropsied; and 4 mm diameter punch biopsies of dorsal skin were harvested and incubated in culture medium for 24 hours to allow for release of proteins into the skin secretagogue Plasma serum amyloid-P (SAP) and IL-6 were analyzed with enzyme-linked immunosorbent assays. Limited blood volumes prevented assay of other circulating proteins. Automated and manual cytometry were used to characterize circulating blood cells. Skin secretagogue was assayed for cytokines IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12, IL-17, monocyte chemoattractant protein 1 (MCP-1), interferon gamma (IFN-γ), TNF-α, macrophage inflammatory protein-1 alpha, granulocyte-macrophage colony-stimulating factor (GM-CSF), and regulated on activation, normal T-cell expressed and secreted protein (RANTES) using Q-PlexTM microarray. Data was analyzed using conventional descriptive statistics. Results As per usual laboratory practice, we considered mouse circulating SAP levels as surrogate for human CRP levels. Following 14 d of dosing, plasma IL-6 levels were ~67% lower (~130 v ~48 µg/g protein; p<0.01), and plasma SAP levels were ~45% lower (~33 v ~18 µg/g protein; p<0.01) in colchicine-treated mice. There were no significant differences in blood cell parameters. Concentrations of IL-3, IL-1, IFN-γ, and GM-CSF were 40% (p<0.05), 20% (p<0.05), 40% (p<0.01), and 40% (p<0.01) lower, respectively, in the skin secretagogue of colchicine-treated mice. Given the chronic nature of inflammation in SCD, we did not anticipate phenotypic changes after only a 14-day exposure to colchicine. Indeed, no significant differences in hyperalgesia or mouse/organ weight were observed. We think that next steps should be dose-finding studies of colchicine in adults living with SCD followed by randomized, placebo-controlled trials with clinically relevant endpoints such as repeated measures of pain interference using a validated instrument. The anticipated lack of colchicine effect upon blood cell parameters suggests that patients would benefit more from a combination of a disease-modifying agent such as hydroxyurea in combination with colchicine.

HbS promotes TLR4-mediated monocyte activation and proinflammatory cytokine production in sickle cell disease

AbstractMonocytes are considered crucial actors of inflammation in sickle cell disease (SCD), being responsible for an increased production of proinflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and IL-6. Although a role of free heme released by intravascular hemolysis has been suspected, the mechanisms underlying monocyte activation in patients with SCD remain unknown. Using purified human hemoglobin (Hb), we demonstrate herein, that cell-free HbS, unlike HbA or heme, is responsible for a major enhancement in the expression of proinflammatory cytokines by human monocytes. This effect was found mediated by direct interaction with the Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD-2) complex, resulting in the activation of both the nuclear factor-κB (NF-κB) and type I interferon pathways. In Townes SCD mice, injection of HbS, unlike HbA, was responsible for an increased production of proinflammatory cytokines, which was prevented by the TLR4 inhibitor, TAK-242. Our results reveal a novel mechanism of monocyte activation and systemic inflammation in SCD, which opens new promising therapeutic perspectives targeting the HbS-TLR4 interaction.

TGF-β1 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-β1), a major immune regulator. In this study, we aimed to investigate the role played by TGF-β1 in vascular inflammation and vaso-occlusion in an animal model of SCD. Using intravital microscopy, we found that a daily dose of recombinant TGF-β1 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-β, in the absence of inflammatory stimulus, considerably increased these parameters. Our results indicate, for the first time, that TGF-β1 may play a significant ameliorative role in vascular SCD pathophysiology, modulating inflammation and vaso-occlusion. The mechanisms by which TGF-β1 exerts its anti-inflammatory effects in SCD, however, remains unclear. Our in vitro adhesion assays with TNFα-stimulated human neutrophils suggest that TGF-β1 can reduce the adhesive properties of these cells; however, direct effects of TGF-β1 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.

Individuals with Sickle Cell Disease Have a Higher Burden of Mitochondrial DNA Heteroplasmy

Background: The simple point mutation that causes sickle cell disease (SCD) belies the extensive systemic damage it can cause. While the sickle pathology is initiated by polymerization of HbS, the multiple end-organ damage is inflicted by years of on-going inflammation and vasculopathy. An emerging marker of inflammation is the accumulation of acquired heteroplasmy mutations in mitochondrial DNA (mtDNA). Given the underlying chronic inflammation in SCD, we hypothesized that SCD patients display increased rates of mtDNA mutations, and previously confirmed (1). Here, we further performed indepth analyses in an ethnically matched normal (HbAA) as well as sickle trait (HbAS) subjects from another independent cohort, the Jackson Heart Study (JHS).Methods: We analyzed and compared whole genome sequencing (WGS) data from the from NIH cohort of 676 SCD patients of African ancestry with that of 621 ethnic-matched indviduals from the 1000 Genomes Project (1KG), and 3,580 individuals from the JHS cohort. The NIH SCD cohort included 561 HbSS & HbSβ 0thalassemia (combined), 90 HbSC, and 25 HbSβ + thalassemia genotypes, the 1KG cohort - 516 HbAA and 105 HbAS and JHS cohort - 3,200 HbAA, 89 HbAC (hemoglobin C trait), and 291 HbAS. Additionally, to further understand any potential sequencing depth bias, as well as to compare between two patient cohorts (NIH SCD & JHS cohorts) with underlying conditons that may influence the heteroplasmy bias, we downsampled 300 NIH cohort HbSS samples to a sequencing depth similar to JHS cohort, and compared their heteroplasmy burden. Mitochondrial sequences extracted from the cleaned WGS data of these 3 cohorts were analyzed for heteroplasmic and homoplasmic variants using mitoCaller from the package mitoAnalyzer.Results: The average depth per locus was ~6,671X for the NIH SCD cohort , ~2,879X for the 1KG cohort, and ~2169X for JHS cohort. We compared the quantity of heteroplasmic variants across the different NIH SCD genotype with 1KG (HbAA & HbAS), and JHS (HbAA, HbAC and HbAS) genotypic groups. The median number of heteroplasmic variants per individual increased progressively from HbAA, HbAS, HbSβ +thalassemia, and HbSC with the highest median number of 118 in HbSS & HbSβ 0 (Fig 1A) in NIH SCD cohort. It is noteworthy that the median mtDNA heteroplasmy in HbAA individuals in 1KG cohort was significantly lower than those in JHS cohort (Table insert in Fig 1A) which may be related to the underlying cardiovascular disease in the JHS cohort; whereas similar heteroplasmy burden in HbAS individuals between these 2 cohorts may underscore the genotype (HbAS) as the driver of heteroplasmy in these cohorts. We compared the heteroplasmy burden of a downsampled subset (n=300) NIH HbSS with that of JHS HbAA, HbAC and HbAS genotypes (Fig 1B). Although, the 70% reduction in sequencing depth resulted in the slight reduction in heteroplasmy burden, we noticed higher heteroplasmic variability (standard deviation) in this subset of NIH HbSS patients. This variability may be attributable to extreme variation in SCD phenotypic severity. We then applied cumulative distribution function to this downsampled subset and compared with JHS genotypes. We found the NIH HbSS patients have disproportionately higher proportion of heteroplasmy variants (Fig 1D) when compared to the JHS genotypes (HbAA, HbAC, and HbAS).Conclusion: We conclude that there is an increased prevalence of heteroplasmic mtDNA variants in SCD compared to ethnic-matched normal (HbAA) populations. Normal individuals with HbAA in JHS cohort have significantly higher heteroplasmic burden compared to those in 1KG cohort, suggesting an underlying cardiovascular disease in JHS cohort as a driving factor. Within each 1KG and JHS cohorts, individuals with sickle cell trait (HbAS) have similar heteroplasmy burden and also higher than those with HbAA, highlighting the potential significance of this genotype. Reducing the sequencing depth by > 70% (downsampling) led to the filtering out of heteroplasmy variants that would have been discovered with the original deeper sequencing depth of ~7300X. Nonetheless, downsampled HbSS samples still retained disproportionately higher heteroplasmy burden compared to non-SCD subjects. We are currently investigating if there is any correlation between mtDNA heteroplasmy burden and severity of clinical phenotypes among the SCD patients.1. Ahmad, MM et al, Blood 136 (1):11-11 (2020) [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Imaging Blood-Brain Barrier Permeability Through MRI in Pediatric Sickle Cell Disease: A Feasibility Study.

Blood-brain barrier (BBB) disruption may lead to endothelium dysfunction and inflammation in sickle cell disease (SCD). However, abnormalities of BBB in SCD, especially in pediatric patients for whom contrast agent administration less than optimal, have not been fully characterized. To examine BBB permeability to water in a group of pediatric SCD participants using a non-invasive magnetic resonance imaging technique. We hypothesized that SCD participants will have increased BBB permeability. Prospective cross-sectional. Twenty-six pediatric participants (10 ± 1 years, 15F/11M) were enrolled, including 21 SCD participants and 5 sickle cell trait (SCT) participants, who were siblings of SCD patients. 3 T. Water extraction with phase-contrast arterial spin tagging with echo-planer imaging, phase-contrast and T1 -weighted magnetization-prepared rapid acquisition of gradient echo. Water extraction fraction (E), BBB permeability-surface area product (PS), cerebral blood flow, hematological measures (hemoglobin, hematocrit, hemoglobin S), neuropsychological scores (including domains of intellectual ability, attention and executive function, academic achievement and adaptive function, and a composite score). Regions of interest were drawn by Z.L. (6 years of experience). Wilcoxon rank sum test and chi-square test for group comparison of demographics. Multiple linear regression analysis of PS with diagnostic category (SCD or SCT), hematological measures, and neuropsychological scores. A two-tailed P value of 0.05 or less was considered statistically significant. Compared with SCT participants, SCD participants had a significantly higher BBB permeability to water (SCD: 207.0 ± 33.3 mL/100 g/minute, SCT: 171.2 ± 27.2 mL/100 g/minute). SCD participants with typically more severe phenotypes also had a significantly leakier BBB than those with typically milder phenotypes (severe: 217.3 ± 31.7 mL/100 g/minute, mild: 193.3 ± 31.8 mL/100 g/minute). Furthermore, more severe BBB disruption was associated with worse hematological symptoms, including lower hemoglobin concentrations (β=-8.84, 95% confidence interval [CI] [-14.69, -3.00]), lower hematocrits (β=-2.96, 95% CI [-4.84, -1.08]), and higher hemoglobin S fraction (β=0.77, 95% CI [0.014, 1.53]). These findings support a potential role for BBB dysfunction in SCD pathogenesis of ischemic injury. 2 TECHNICAL EFFICACY: Stage 2.

Potential Implications of a Type 1 Interferon Gene Signature on COVID-19 Severity and Chronic Inflammation in Sickle Cell Disease.

At the onset of the corona virus disease 19 (COVID-19) pandemic, there were concerns that patients with sickle cell disease (SCD) might be especially vulnerable to severe sequelae of SARS-CoV-2 infection. While two reports support this conclusion, multiple studies have reported unexpectedly favorable outcomes in patients with SCD. However, mechanisms explaining these disparate conclusions are lacking. Here, we review recent studies indicating that the majority of patients with SCD express elevated levels of anti-viral type 1 interferons (IFNα/β) and interferon stimulated genes, independent of COVID-19, during their baseline state of health. We also present our data from the pre-COVID-19 era, illustrating elevated expression of a well-characterized interferon stimulated gene in a cohort of patients with SCD, compared to race-matched controls. These type 1 interferons and interferon stimulated genes have the potential to contribute to the variable progression of COVID-19 and other viral infections in patients with SCD. While the majority of evidence supports a protective role, the role of IFNα/β in COVID-19 severity in the general population remains an area of current investigation. We conclude that type 1 interferon responses in patients with SCD may contribute to the variable COVID-19 responses reported in prior studies. Additional studies investigating the mechanisms underlying IFNα/β production and other clinical consequences of IFNα/β-mediated inflammation in SCD disease are warranted.

Chronic Hemolysis, Oxidative Stress and Inflammation Contribute to Dysfunctional HDL in Sickle Cell Disease

IntroductionHypocholesterolemia was observed in sickle cell disease (SCD) patients according to various reports in the literature. It was previously linked to anemia and inflammation by others and to oxidative stress by our group. In this study we investigated the relationship between cholesterol metabolism, hemolysis, inflammation and oxidative stress in SCD.MethodsThe study consisted of pediatric SCD patients (N=34) and healthy controls (N=19). Patients were crisis-free and did not receive transfusion for the last 3 months.Hemolysis was assessed with the measurement of haptoglobin (Hpg), bilirubin and LDH. Hemin values were accepted as a representation of oxidative stress; while serum amyloid A (SAA) and myeloperoxidase (MPO) levels were measured to determine the level of inflammation.Hemin, SAA and MPO levels were measured by commercial kits while all other parameters including the lipid panel and liver function tests were measured by autoanalyzers in the clinical chemistry lab. Statistical analyses were performed with IBM SPSS v23.0. The study was approved by the Institutional Review Board of Mersin University.ResultsMean age of the patients and controls were 13.71±3.79 and 13.47±3.49 years respectively. Patient and control groups were similar in terms of age and sex. All values are given as patients vs. controls and mean±SD.Among the values representing hemolysis, statistically significant differences between groups were found for Hpg (3.84±16.18 mg/dL vs. 69.98±37.66 mg/dL, p<0.001), total and direct bilirubin (1.93±1.17 mg/dL vs. 0.27±0.14 mg/dL and 0.40±0.21 mg/dL vs. 0.06±0.04 mg/dL, p<0.001), and LDH (540.29±217.32 U/L vs. 202.79±49.46 U/L, p<0.001).Hemin levels, as an index of oxidative stress, were significantly higher in the patient group (9.52±8.75 mg/L vs. 0.27±0.01 mg/L, p<0.001).SAA and MPO levels which were accepted to determine the level of chronic inflammation were both significantly higher in the patient group (1.19±0.60 mg/L vs. 0.68±0.52 mg/L, p=0.013 and 76.53±31.03 μg/L vs. 44.56±8.29 μg/L, p=0.001; respectively).In the lipid panel, significant differences between groups were found in total cholesterol (116.97±17.22 mg/dL vs. 169.84±33.70 mg/dL, p<0.001), HDL (28.31±5.21 mg/dL vs. 50.95±12.95 mg/dL, p<0.001), LDL (70.80±11.96 mg/dL vs. 99.89±22.95 mg/dL, p<0.001), and apolipoprotein AI (149.61±54.88 mg/dL vs. 199.32±33.48 mg/dL, p=0.001); whereas no difference was found in apolipoprotein B (77.65±25.52 mg/dL vs. 81.57±13.96 mg/dL, p=0.541) levels.ConclusionPediatric SCD patients in this study had hypocholesterolemia reflected by low LDL and HDL levels. Apolipoprotein AI levels were also lower in patients suggesting either low production or increased catabolism of the HDL particle.The drastic decrease in Hpg levels and the increase in bilirubin and LDH values show the overwhelming chronic hemolysis in SCD. Hpg protects vascular endothelium by binding to hemoglobin. Hpg was also shown to protect Apo AI from oxidative damage. However, the consumption of Hpg in our patients may possibly render Apo AI open to the damaging effects of free radicals formed during hemolysis.Hemin is produced by the oxidation of free heme to the ferric state. Increased hemin levels in SCD patients, compared to the almost-undetectable levels in controls, may cause oxidative damage to plasma proteins and lipids as well as activating neutrophils with its oxidative effect. Apolipoproteins and lipids in the structure of HDL and LDL particles are targets for oxidative damage.The increase in SAA and MPO levels indicate the chronic inflammation in SCD. SAA is an important acute phase protein secreted by the liver during inflammation. It translocates Apo AI by incorporating into HDL and slows down reverse cholesterol transport by inhibiting cholesterol incorporation into HDL. A previous study reports SAA as a marker of crisis in SCD. Increased MPO release by the leukocytes was shown to oxidize Apo AI and disrupt its function in patients with cardiovascular disease. Therefore, increased MPO levels in our patients may contribute to the oxidative damage on the Apo AI of the HDL particle.Thus, the cumulative effect of chronic hemolysis, oxidative stress and inflammation in SCD may result with a pathological structural alteration in HDL and contribute to HDL dysfunction. The slowing down of reverse cholesterol transport may be an important cause of hypocholesterolemia in SCD. DisclosuresNo relevant conflicts of interest to declare.

Mitochondrial DNA Variation in Individuals with Sickle Cell Disease

Background: Sickle cell disease (SCD) is a complex multi-system disorder that predominantly affects individuals of African heritage. While the sickle pathology is initiated by polymerization of HbS, multiple end-organ damage is inflicted by years of on-going inflammation and vasculopathy. An emerging marker of inflammation is the accumulation of mutations in mitochondrial DNA (mtDNA), the phenotypic effect of which will depend on the nature of the gene that harbors the mutation, the mutant allele fraction, and the pathogenicity of the mutant allele. A mutation in mtDNA is heteroplasmic when it is present in only a proportion of mtDNA, and homoplasmic, when it is present in all mtDNA molecules. Mitochondrial heteroplasmy can also occur at different tissue or cell levels, even within the same individual. Given the underlying chronic inflammation in SCD, we hypothesize that SCD patients display increased rates of mtDNA mutations. Methods: We analyzed and compared whole genome sequencing (WGS) data from the cohort of 683 SCD patients (SCD cohort) of African ancestry with that of 621 individuals of African ancestry from the 1000 Genomes Project (1KG). The SCD cohort included 561 HbSS &amp; HbSβ0 thalassemia (combined), 90 HbSC, and 25 HbSβ+ thalassemia. The 1KG cohort included 516 HbAA and 105 sickle carriers (HbAS). mtDNA sequences of SCD and 1KG cohorts were initially aligned to the revised Cambridge Reference Sequence (rCRS NC_012920), and subsequently base recalibrated and deduplicated. Mitochondrial sequences extracted from the cleaned WGS data of both cohorts were analyzed for heteroplasmic and homoplasmic variants using mitoCaller from the package mitoAnalyzer. Results: The average depth per locus is ~6,828X for the SCD cohort and ~2,879X for the 1KG cohort. We performed a locus by locus comparison between the mtDNA sequences of both cohorts. No homoplasmic variants unique to the SCD cohort were found when compared to the 1KG cohort. In contrast, there were several "hotspots" of heteroplasmic variants that were unique to the SCD cohort, and largely shared amongst the SCD patient population (Figure 1A). To identify these unique variants, we used MITOMASTER and Ensembl VEP to annotate the heteroplasmic variants that had above 40% population frequency within the SCD cohort and below 10% population frequency in the 1KG cohort. Several heteroplasmic variants were non-synonymous and the selected variants originated from the Control Region (D-loop), RNR1, RNR2, ND1, ND4, and ND5. One of the heteroplasmic variants, 2623 A&amp;gt;G, was found to be age linked for HbSS &amp; HbSβ0 (Figure 1B), with its minor allele frequency (MAF) increasing with age. Further analysis needs to be done in order to determine if more variants unique to the SCD cohort are age-linked. We then compared the quantity of heteroplasmic variants across the different SCD genotypic groups with the 1KG HbAA and the 1KG HbAS groups. The median number of heteroplasmic variants per individual increased progressively from HbAA, HbAS, HbSβ+ thalassemia, and HbSC with the highest median number of 119 in HbSS &amp; HbSβ0 (Figure 1C). Mitochondrial heteroplasmy for 1KG HbAA and 1KG HbAS were statistically significant when tested against each other and against every SCD sub-group; however, the difference was not statistically significant between the different SCD genotypes (Table insert in Fig 1C). It is important to note that we did not apply a MAF threshold, thus many of the heteroplasmic variants may be present at very low levels. Conclusion: Our findings suggest that there is an increased prevalence of heteroplasmic variants in SCD compared to ethnic-matched healthy populations. Within the SCD genotypes, the heteroplasmic burden increased progressively (HbAS &amp;lt; HbSβ+ thalassemia &amp;lt; HbSC &amp;lt; HbSS &amp; HbSβ0) with genotypic groups that are associated with increasing phenotypic severity. mtDNA heteroplasmic burden for one variant also increased with age in HbSS &amp; HbSβ0 individuals, but further studies are needed to explore if mtDNA heteroplasmic burden correlates with the degree of organ damage and disease severity within the same genotypic group. Although it is not clear if the variants are a cause or effect of the sickle inflammatory pathology, our data suggest that mtDNA heteroplasmic burden is a potential biomarker of SCD severity. Disclosures No relevant conflicts of interest to declare.

Biased PAR-1 Signaling Regulates Thrombo-Inflammation in a Mouse Model of Sickle Cell Disease

Thrombin and activated protein C (APC) are serine proteases involved in coagulation and inflammatory responses. Protease activated receptor-1 (PAR-1) is a G-protein coupled receptor (GPCR) activated by proteolytic cleavage of the extracellular N-terminus. PAR-1 cleavage at R41 by thrombin or at R46 by APC creates unique tethered ligands that activate different signaling pathways. On endothelial cells, thrombin employs PAR-1 to promote endothelial barrier disruption, vascular leakage, and inflammation. In contrast, APC employs PAR-1 to stabilize endothelial barrier and provides anti-inflammatory and anti-apoptotic activities. These opposing effects of PAR-1 activation are a classic example of "GPCR biased signaling". We have previously demonstrated that PAR-1 deficiency in all hematopoietic cells reduces heme-induced vascular stasis in sickle mice. In contrast it does not affect the elevated plasma levels of IL-6 or thrombin-antithrombin (TAT) complexes observed in sickle cell disease (SCD) mice compared to non-SCD controls during steady state. In this study, we investigated the effects of thrombin- and APC-induced PAR-1 activation on thromboinflammation in the well-characterized Townes mouse model of SCD. Procoagulant and proinflammatory responses were analyzed in control (AA) and sickle (SS) mice at steady state (saline injection) and 5 hours after TNFα (2 µg/kg, i.p.) to mimic the pro-inflammatory milieu that occurs during painful crisis in SCD patients. As previously described, SS mice had elevated plasma levels of TAT compared to AA mice (8.6± 2.4 vs 3.5±0.7ng/mL; p&amp;lt;0.05), that were further enhanced in SS mice by TNFα injection (21.5 ± 4.2 ng/mL, p&amp;lt;0.001). A similar pattern was observed for plasma levels of IL-6 (AA: 2.4±2 vs SS: 18±7.8 ng/mL, p&amp;lt;0.05; SS+TNFα: 231 ± 32 ng/mL, p&amp;lt;0.0001 vs SS). Using these experimental conditions, we first investigated the role of endogenous APC in SCD. We used a rat anti-mouse protein C mAb (SPC-54), which inhibits the active site of APC, thereby blocking all its enzymatic activities, including PAR-1-dependent signaling and anticoagulant actions. SS mice were injected with anti-APC SPC54 (10 mg/kg) or control IgG antibodies. Eighteen hours later both groups of mice were injected with either saline or TNFα and plasma samples were collected 5 hours later. We TNFαSSdata suggest that endogenous APC has anticoagulant and anti-inflammatory roles for both the steady state and after TNFα challenge. To address the individual roles of thrombin and APC signaling on vascular inflammation in SCD, we used mice with point mutations in PAR-1 at R41 or R46. Mutation of R41 to Glu (R41Q) renders PAR-1 insensitive to thrombin while preserving APC-mediated cleavage at R46. Mutation of R46 to Glu (R46Q) ablates APC's cytoprotective effects. Bone marrow (BM) from SS mice was transplanted into PAR-1WT, PAR-1R41Q, or PAR-1R46Q mice to generate SSBM mice with either normal PAR-1 (SSBM/PAR-1WT) or the mutated form of this receptor on all non-hematopoietic cells (SSBM/PAR-1R41Q and SSBM/PAR-1R46Q). Four months after BM transplantation, plasma levels of TAT, IL-6, and HMGB1 were analyzed in all 3 groups at steady state or 5 hours after TNFα challenge. Neither the PAR-1R41Q nor PAR-1R46Q mutation on non-hematopoietic cells affected plasma levels of TAT, IL-6, or HMGB1 in SS mice at steady state. Interestingly, plasma levels of IL-6 were significantly elevated in SSBM/PAR-1R46Q mice and reduced in SSBM/PAR-1R41Q mice compared to SSBM/PAR-1WT mice after TNFα challenge, with a statistically significant difference between the two PAR-1 mutant groups. Furthermore plasma levels of TAT and HMGB1 were significantly reduced in SSBM/PAR-1R41Q but not changed in SSBM/PAR-1R46Q mice after TNFα challenge (Figure 2A-C). These data suggest that PAR-1 activation at R41 regulates TAT, IL-6, and HMGB1, whereas activation of R46 negatively regulates IL-6 in sickle mice during TNFα challenge. Our data imply that pharmacological approaches aiming to eliminate thrombin-dependent, while preserving APC-dependent PAR-1 activation, may be effective in attenuating thrombo-inflamatory complications associated with SCD. Studies evaluating the effects of R41Q and R46Q mutations on microvascular stasis in SS mice are currently ongoing. Disclosures No relevant conflicts of interest to declare.

Plasma-Based Inflammatory Signatures in Patients with Sickle Cell Disease during Baseline Health and Acute Pain

INTRODUCTION: Pain, the most common complication of sickle cell disease (SCD), presents as both sudden acute pain and chronic daily pain. However, there is wide variability in frequency and presentation of pain despite inheritance of the same monogenic gene defect. SCD has long been recognized as a chronic inflammatory condition. The ongoing effect of repeated vaso-occlusion, ischemia-reperfusion injury and hemolysis contribute to further SCD inflammation and likely pain. Regulation of the immune response can potentially modulate the inflammatory impact on pain. The collective balance of these inflammatory mediators in union in SCD patients and how this balance may change during baseline health and acute pain is unknown. The objective of this work was to determine the balance between patients' inflammatory and immune regulatory response and examine whether this balance changes during acute pain in patients with SCD. METHODS: We conducted a cross sectional analysis involving 3 cohorts: patients with SCD who were in their baseline health state, patients with SCD who had an acute pain episode and healthy African American controls. We used a novel bioassay originally developed for use in type 1 diabetes and applied to cystic fibrosis, inflammatory bowel disease and influenza to determine the inflammatory/immune regulatory response. This response was calculated as a composite Inflammatory Index (I.I.com) from these 3 patient cohorts. Patient plasma was co-cultured with cryopreserved PBMCs from a healthy donor to induce transcription (Figure 1). We identified informative transcripts that differentiate SCD patients from healthy controls thereby defining the disease-specific plasma-induced signature and retained ones differentially expressed between patients with SCD and controls that exhibit a fold change &amp;gt;1.4, ANOVA p-value of &amp;lt;0.05 and an FDR &amp;lt;10%. The data were subjected to ontological analyses for quantitative interpretation with Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity Pathway Analysis (IPA). Our scoring strategy used the degree of induction of genes in inflammatory and regulatory ontological classes. The composite I.I.com was calculated using the average ratio between the mean log intensity of the genes classified as being "inflammatory" versus "regulatory". Independent samples Student's t-test was used to compare the mean I.I.com between 1) SCD baseline health cohort and controls and 2) SCD baseline health cohort and SCD acute pain cohort. RESULTS: Plasma from 16 patients with SCD in baseline health, 27 patients with SCD with an acute pain episode, and 45 African American controls were collected and analyzed. The average age of the study population was 12.6 (SD=3.6) years old and 52.3% were female. Quantitative scoring of plasma-induced signatures showed SCD patients had significantly higher mean I.I.com during baseline health compared to controls (0.713 vs. -1.235-12, p=5.4625-11). In addition, patients with SCD during acute pain episodes had significantly higher I.I.com than patients in baseline health (1.282 vs. 0.713, p=5.2051-8) (Figure 2). Heat map in Figure 3 shows differential gene expression between the cohorts; green and red colors in heat maps represent lower or higher relative expression respectively. CONCLUSION: Our findings show distinct immune signatures in SCD patients compared to controls and distinct signatures in SCD patients during acute pain episodes as compared to baseline health. The novel assay used to assess the inflammatory and immune regulatory gene expression in the three cohorts studied allowed for the determination of the balance between the two immune states. The imbalance between inflammation and immune regulation shown in our results in SCD patients of SCD pain. Further investigation into the specific inflammatory pathways that contribute to altered immune response could lead to novel targets for pain treatment. Disclosures Mucalo: NIH/NINDS: Research Funding; NIH/NHLBI: Research Funding. Jia:NIH/NHBLI: Research Funding; NIH/NINDS: Research Funding. Panepinto:NINDS: Research Funding; HRSA: Research Funding; NINDS: Research Funding; NHLBI: Research Funding. Roethle:NIH/NHLBI: Research Funding; NIH/NINDS: Research Funding. Hessner:NIH/NHLBI: Research Funding; NIH/NINDS: Research Funding. Brandow:NIH / NHLBI: Research Funding; Greater Milwaukee Foundation: Research Funding.

NFkB mediated sustained inflammation promotes hepatobiliary injury in Sickle Cell Disease

Abstract Hepatic crisis is an emergent complication affecting sickle cell disease (SCD) patients, however, the molecular mechanism of sickle cell hepatobiliary injury remains poorly understood. Using the knock-in humanized mouse model of SCD and SCD patient blood, we sought to mechanistically characterize SCD-associated hepatopathophysiology applying our recently developed quantitative liver intravital imaging, RNA sequence analysis, and biochemical approaches. SCD mice manifested sinusoidal ischemia, progressive hepatomegaly, progressive inflammation, hyperbilirubinemia, and increased ductular reaction under basal condition. NFkB activation in the SCD mice liver inhibited FXR signaling and its downstream targets, leading to loss of canallicular bile transport, sustained inflammation and altered bile acid pool. Intravital imaging revealed impaired bile secretion into the bile canaliculi, which was secondary to loss of canallicular bile transport and bile acid metabolism, leading to intrahepatic bile accumulation in SCD mice liver. These findings are the first to identify that NFkB-FXR dependent impaired bile secretion promotes intrahepatic bile accumulation, which contributes to hepatobiliary injury and liver inflammation in SCD. Improved understanding of these processes could potentially benefit the development of new therapies to treat sickle cell hepatic crisis.

Effect of Hydroxyurea Treatment on the Inflammatory Markers Among Children With Sickle Cell Disease.

Background:Neutrophil-to-lymphocyte ratio (NLR) was introduced as a potential inflammatory marker in sickle cell disease (SCD). This study aimed to evaluate the impact of hydroxyurea (HU) treatment on the value of NLR and some inflammatory mediators in SCD.Methods:The hematological parameters and clinical events were analyzed in 35 children with SCD under HU treatment and followed up for 1 year and in 20 healthy controls. Enzyme-linked immunosorbent assay was performed for the evaluation of proinflammatory cytokines, including interleukin (IL) 6, IL-8, high-sensitivity C-reactive protein (hs-CRP), and tumor necrosis factor α (TNF-α).Results:Hydroxyurea significantly improves most of the hematological parameters in children with SCD. The percentages of hemoglobin fraction S, serum levels of TNF-α and IL-6 were significantly decreased when compared to baseline value but did not reach the value of the healthy control. The HU treatment led to a significant decrease in NLR compared to the baseline values and reached healthy control values. Neutrophil-to-lymphocyte ratio was positively correlated with hs-CRP, TNF-α, and IL-8 serum levels and negatively correlated with percentage of fetal hemoglobin and hematocrit values. The cutoff value of NLR to expect a response to HU among SCD was 3.0, with 76% specificity and 85% sensitivity (area under the curve: 0.85, P < .0001). In conclusion, hydroxyurea induced a decrease in NLR and inflammatory cytokines, which represent a biomarker of inflammation in SCD. The calculation of NLR is a straightforward and cheap method for SCD outcome prediction in young children.

The Gut Microbiome Regulates Psychological Stress-Induced Inflammation in Sickle Cell Disease

Psychological stress (PS) is reported to precipitate vaso-occlusive episides (VOE) in sickle cell disease (SCD), but the mechanisms by which it does, are largely unknown. We subjected humanized (Berkeley) SCD mice to restraint stress and analyzed VOE using intravital microscopy. We found that stress exacerbated VOE as shown by reduced venular blood flow rates (control: 494 ± 30 × 103 pL/s; stress: 380 ± 20 × 103 pL/s; n = 25 - 43; p = 0.009) and reduced survival time (median survival time in control and stress groups: 394 min and 175 min, respectively; p = 0.02, Log-rank test). As previous studies show that adherent polymorphonuclear neutrophils (PMNs), especially aged PMNs marked by low CD62L and high CXCR4 expression, contribute to VOEs, we evaluated the behavior of leukocytes in live mice and observed a robust PMN response manifested by significantly increased adhesion and interactions with sickle RBC in stressed SCD animals. Flow cytometry analysis revealed a striking increase in aged PMNs in stressed SCD mice (control: 0.7 ± 0.1 × 106/ml; stress: 2.9 ± 0.3 × 106/ml; n = 3 - 4; p = 0.001), suggesting that the aged PMN expansion induced by stress may lead to the exacerbation of VOE in stressed SCD mice. Since aged PMN numbers are regulated by microbiota under steady state, we investigated whether response to PS also required the presence of the microbiota. Depletion of microbiota with broad-spectrum antibiotics (ABX) in SCD mice significantly prevented stress-induced aged PMN expansion (control: 4.8 ± 1.2 × 106/ml; ABX: 1.2 ± 0.3× 106/ml; n = 4 - 5; p = 0.0006), which was further confirmed using C57BL/6 germ-free (GF) mice (control: 1.1 ± 0.3 × 106/ml; GF: 0.2 ± 0.1 × 106/ml; n = 4; p = 0.003). Interestingly, analysis of inflammatory cytokines revealed a microbiota-dependent augmentation of IL-17A in the stressed mice. To investigate the role of IL-17A in the stress response, we neutralized IL-17A using an antibody (Ab) in C57BL/6 mice and found that IL-17A inhibition significantly dampened the aged PMN expansion induced by stress (isotype control Ab: 0.6 ± 0.1 × 106/ml; anti-IL-17A: 0.1 ± 0.0 × 106/ml; n = 4 - 5; p = 0.0004), which was also confirmed using IL-17A-/- mice. Further analyses revealed that Th17 lymphocytes were the major IL-17A-producing cells contributing to the stress response. In addition, depletion of microbiota with ABX or neutralization of IL-17A significantly alleviated stress-exacerbated VOE as measured by reduced PMN activation, improved blood flow rate and prolonged SCD mouse survival. As segmented filamentous bacteria (SFB) commensals were previously found to induce a Th17 response, we next tested the hypothesis that SFB was the driving bacteria in the stress response. Indeed, we found that PS-induced aged PMN expansion was greater in SFB+ mice (from Taconic) than in SFB- mice (from Jackson lab) (Taconic: 0.64 ± 0.11× 106/ml; Jax: 0.13 ± 0.02 × 106/ml; n = 4 - 5; p = 0.005). Depletion of SFB using vancomycin, a drug that eliminates Gram+ bacteria (including SFB), significantly reduced stress-induced aged PMN expansion. Furthermore, colonization of GF mice with SFB augmented aged PMN expansion after stress compared to stress control GF mice (control GF: 0.08 ± 0.02 × 106/ml; GF + SFB: 0.34 ± 0.10× 106/ml; n = 4 - 5; p = 0.04), suggesting that SFB was the key bacteria promoting aged PMN expansion during stress. The stress mechanisms involved the hypothalamic-pituitary-adrenal (HPA) axis since the blockade of glucocorticoid (GC) production using metyrapone or following adrenalectomy significantly prevented aged PMN expansion in stressed mice (PBS: 0.5 ± 0.1 × 106/ml; metyrapone: 0.1 ± 0.1× 106/ml; n = 5; p = 0.02. sham: 1.2 ± 0.2 × 106/ml; adrenalectomy: 0.4 ± 0.2 × 106/ml; n = 4 - 5; p = 0.01). Moreover, PS and dexamethasone greatly increased the gut permeability. Blockade of GC synthesis using metyrapone significantly alleviated stress-induced exacerbation of VOE manifested by reduced inflammation, improved the blood flow rate and prolonged SCD mice survival (median survival time in control and metyrapone groups: 155 min and 200 min, respectively; p = 0.02, Log-rank test). Overall, our results suggest that stress activation of the HPA axis increases the intestinal permeability, thereby promoting exposure to commensals that stimulate Th17 cells to produce IL-17A, which enables the expansion of aged polymorphonuclear neutrophils driving vaso-occlusion in SCD. Disclosures Frenette: Albert Einstein College of Medicine, Inc: Patents &amp; Royalties; Ironwood Pharmaceuticals: Research Funding; Cygnal Therapeutics: Equity Ownership; Pfizer: Consultancy.