Hairy Platelet-Derived Extracellular Vesicles Promote Lung Vaso-Occlusion in Sickle Cell Disease

Abstract Background: Acute chest syndrome (ACS) is a type of acute lung injury and the leading cause of mortality in Sickle Cell Disease (SCD). Current treatments for ACS are primarily supportive, and there is a critical need for rescue therapies. ACS is often a sequela of acute systemic vaso-occlusive crisis and preceded by thrombocytopenia. However, the role of platelets in the pathogenesis of ACS remains largely unknown. Methods: We used our validated model of vaso-occlusive crisis in transgenic, humanized SCD mice, which is triggered by intravenous challenge with nanogram levels of the TLR4 ligand, lipopolysaccharide (LPS). Platelet-neutrophil aggregates and blood flow in the lung microcirculation was visualized in real time in vivo, using multi-photon-excitation microscopy of intact lung in live SCD mice. SCD or control human blood was perfused through microfluidic channels in vitro and neutrophil-platelet aggregation was visualized using fluorescence microscopy. Platelet derived extracellular vesicles were characterized using nanoparticle tracking and biochemical approaches. Results: We have made a novel finding that the arrest of blood flow and injury in the lung is secondary to blockade of pulmonary arterioles by platelet-neutrophil aggregates. Using in vitro microfluidic studies, we confirmed that platelet-neutrophil aggregation is higher in LPS-treated SCD patient blood compared with healthy controls, and this correlates with increased numbers of platelet-derived extracellular vesicles (EVs) that express IL-1β. Our studies also reveal that platelet-neutrophil aggregation in pulmonary arterioles of SCD mice is associated with an increase in peripheral blood levels of platelet-derived EVs containing IL-1β. Remarkably, inhibition of TLR4 or TLR4/NLRP3-inflammasome activated caspase-1, or inhibition of IL-1β signaling, attenuated release of platelet EVs and platelet-neutrophil aggregation in the lung arterioles of SCD mice in vivo and SCD human blood in vitro . Conclusions: TLR4 and NLRP3-inflammasome-mediated caspase-1 activation in platelets during vaso-occlusive crisis leads to release of IL-1β-containing EVs into the circulation. These circulating platelet EVs promote platelet-neutrophil aggregation in pulmonary arterioles, which results in arrest of blood flow in the lung, leading to ACS. Therapeutic inhibition of TLR4/NLRP3-caspase-1 signaling in platelets or IL-1β signaling is a potential therapy for ACS in SCD patients. Acknowledgments: This study was supported by 1R01HL128297-01 (P.S.) and VMI startup funds (P.S.). Disclosures No relevant conflicts of interest to declare.

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The Essential Oil of Artemisia argyi H.Lév. and Vaniot Attenuates NLRP3 Inflammasome Activation in THP-1 Cells

Frontiers in Pharmacology ◽

10.3389/fphar.2021.712907 ◽

2021 ◽

Vol 12 ◽

Author(s):

Pengxiao Chen ◽

Qi Bai ◽

Yanting Wu ◽

Qiongzhen Zeng ◽

Xiaowei Song ◽

...

Keyword(s):

Essential Oil ◽

Nlrp3 Inflammasome ◽

Therapeutic Potential ◽

Inflammasome Activation ◽

Caspase 1 ◽

Artemisia Argyi ◽

Nlrp3 Inflammasome Activation ◽

Long Time

Artemisia argyi H. Lév. and Vaniot is a traditional medical herb that has been used for a long time in China and other Asian counties. Essential oil is the main active fraction of Artemisia argyi H. Lév. and Vaniot, and its anti-inflammatory potential has been observed in vitro and in vivo. Here, we found that the essential oil of Artemisia argyi H. Lév. and Vaniot (EOAA) inhibited monosodium urate (MSU)- and nigericin-induced NLRP3 inflammasome activation. EOAA suppressed caspase-1 and IL-1β processing and pyroptosis. NF-κB p65 phosphorylation and translocation were also inhibited. In addition, EOAA suppressed nigericin-induced NLRP3 inflammasome activation without blocking ASC oligomerization, suggesting that it may inhibit NLRP3 inflammasome activation by preventing caspase-1 processing. Our study thus indicates that EOAA inhibits NLRP3 inflammasome activation and has therapeutic potential against NLRP3-driven diseases.

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Astragaloside IV Suppresses High Glucose-Induced NLRP3 Inflammasome Activation by Inhibiting TLR4/NF-κB and CaSR

Mediators of Inflammation ◽

10.1155/2019/1082497 ◽

2019 ◽

Vol 2019 ◽

pp. 1-16 ◽

Cited By ~ 10

Author(s):

Bin Leng ◽

Yingjie Zhang ◽

Xinran Liu ◽

Zhen Zhang ◽

Yang Liu ◽

...

Keyword(s):

Nlrp3 Inflammasome ◽

High Glucose ◽

Inflammasome Activation ◽

Astragaloside Iv ◽

Caspase 1 ◽

Endothelial Inflammation ◽

Nlrp3 Inflammasome Activation ◽

Anti Inflammatory

Long-term exposure to high glucose induces vascular endothelial inflammation that can result in cardiovascular disease. Astragaloside IV (As-IV) is widely used for anti-inflammatory treatment of cardiovascular diseases. However, its mechanism of action is still not fully understood. In this study, we investigated the effect of As-IV on high glucose-induced endothelial inflammation and explored its possible mechanisms. In vivo, As-IV (40 and 80 mg/kg/d) was orally administered to rats for 8 weeks after a single intraperitoneal injection of streptozotocin (STZ, 65 mg/kg). In vitro, human umbilical vein endothelial cells (HUVECs) were treated with high glucose (33 mM glucose) in the presence or absence of As-IV, NPS2143 (CaSR inhibitor), BAY 11-7082 (NF-κB p65 inhibitor), and INF39 (NLRP3 inhibitor), and overexpression of CaSR was induced by infection of CaSR-overexpressing lentiviral vectors to further discuss the anti-inflammatory property of As-IV. The results showed that high glucose increased the expression of interleukin-18 (IL-18), interleukin-1β (IL-1β), NLRP3, caspase-1, and ASC, as well as the protein level of TLR4, nucleus p65, and CaSR. As-IV can reverse these changes in vivo and in vitro. Meanwhile, NPS2143, BAY 11-7082, and INF39 could significantly abolish the high glucose-enhanced NLRP3, ASC, caspase-1, IL-18, and IL-1β expression in vitro. In addition, both NPS2143 and BAY 11-7082 attenuated high glucose-induced upregulation of NLRP3, ASC, caspase-1, IL-18, and IL-1β expression. In conclusion, this study suggested that As-IV could inhibit high glucose-induced NLRP3 inflammasome activation and subsequent secretion of proinflammatory cytokines via inhibiting TLR4/NF-κB signaling pathway and CaSR, which provides new insights into the anti-inflammatory activity of As-IV.

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In vivo survival studies of 51Cr-labeled methyl acetimidate treated erythrocytes in patients with sickle cell disease

Blood ◽

10.1182/blood.v56.6.1041.1041 ◽

1980 ◽

Vol 56 (6) ◽

pp. 1041-1047 ◽

Cited By ~ 5

Author(s):

TG Gabuzda ◽

TL Chao ◽

MR Berenfeld ◽

T Gelbart

Keyword(s):

Sickle Cell Disease ◽

Survival Time ◽

Sickle Cell ◽

Clinical Testing ◽

Cell Disease ◽

Show Evidence ◽

Survival Studies ◽

Circulating Antibody

Abstract Studies of the survival time of 51Cr labeled erythrocytes treated in vitro with methyl acetimidate (MAI) were conducted in 13 patients with sickle cell disease in order to assess the suitability of this antisickling agent for more extensive clinical testing. In comparison with previously measured control values (average t1/2 8.4 +/- 1.1 days a), the survival time of the treated erythrocytes in 10 of the patients who were not transfused was initially prolonged (average t1/2 24.4 +/- 4.6 days). However, 5 of the 13 patients studied developed circulating antibody against the MAI treated erythrocytes, markedly reducing the survival time of MAI treated erythrocytes in subsequent studies. Two patients, each challenged 3 times with infused MAI treated erythrocytes, failed to show evidence of antibody production, suggesting that not all subjects become immunized even after repeated exposure. In spite of many other promising properties of MAI as an antisickling agent of potential value, consideration of its use in further clinical testing must depend on successful avoidance of immunization in patients receiving infusions of treated erythrocytes.

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Hemoglobin Blocks Von Willebrand Factor Proteolysis by ADAMTS-13: A Mechanism Associated with Acquired ADAMTS-13 Deficiency in Patients with Sickle Cell Disease

Blood ◽

10.1182/blood.v112.11.3919.3919 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3919-3919

Author(s):

Zhou Zhou ◽

Han Hyojeong ◽

Miguel A. Cruz ◽

Jose A. Lopez ◽

Jing-fei Dong ◽

...

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Von Willebrand Factor ◽

Elevated Level ◽

Dependent Manner ◽

Cell Disease ◽

Von Willebrand ◽

Willebrand Factor

Abstract One of the hallmark events of sickle cell disease (SCD) is vasoocclusion and episodic pain crisis. Although the mechanism of vascular occlusion is very complicated, processes like thrombosis and thromboembolism have been recognized to play an important role in the development of such clinical manifestation in SCD. Studies have shown that the von Willebrand factor (VWF), especially the ultra-large (UL) multimers play a major role in vasoocclusion, which clearly indicates a possible impairment of the VWF-cleaving metalloproteae ADAMTS-13 in these patients with SCD. In a recent work, indeed we have mentioned that the plasma ADAMTS-13 in patients with SCD having normal antigen level showed 35% less protease activity than the normal. There may be several plasma factors responsible for the acquired deficiency of ADAMTS-13 in SCD. Since, the increasing evidences suggest that the elevated level of extracellular hemoglobin (Hb) in plasma parallely associated with the pathogenesis of SCD, we investigated the effects of extracellular Hb on VWF proteolysis by ADAMTS-13. We observed that purified Hb dose-dependently inhibited the ADAMTS-13 cleavage of recombinant(r) VWF and endothelial ULVWF multimers under static and flow conditions. Hb bound to VWF multimers in a saturation-dependent manner and more potently to the rVWFA2 domain (affinity Kd~24nM), which contains the cleavage site for ADAMTS-13. Hb bound also to the ADAMTS-13 (Kd~65nM), with 2.7 times less affinity than to VWFA2. The bindings were neither calcium-dependent nor affected by haptoglobin. However, it is the Hb-binding to VWF that prevented the substrate from being cleaved by ADAMTS-13. These in vitro findings are consistent with the in vivo observations in patients with SCD. An elevated level of extracellular Hb in plasma was inversely correlated (linear regression, r2 =0.6354) with the low activity of ADAMTS-13 in a cohort of ten adult patients with SCD (mean±SE, Hb 346±138 mg/l; activity 33.3±30%) compared to age and gender-matched normal individuals (n=10; Hb 24±8 mg/l; activity 76.2±16%). The data together suggest that patients with SCD suffer from acquired ADAMTS-13 deficiency, primarily because Hb competitively binds and inhibits the proteolysis of VWF multimers, leading to ULVWF accumulation on vascular endothelium and in circulation. The Hb-VWF interaction may therefore be considered as a therapeutic target for reducing thrombotic and vasoocclusive complications in patients with severe hemolysis such as those with SCD.

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Platelet Activation and Inhibition in Sickle Cell Disease (PAINS) Study

Blood ◽

10.1182/blood.v120.21.5147.5147 ◽

2012 ◽

Vol 120 (21) ◽

pp. 5147-5147

Author(s):

Andrew L. Frelinger ◽

Joseph A. Jakubowski ◽

Julie K. Brooks ◽

Sabrina L. Zayas ◽

Michelle A. Berny-Lang ◽

...

Keyword(s):

Sickle Cell Disease ◽

Antiplatelet Therapy ◽

Platelet Activation ◽

Healthy Subjects ◽

Research Funding ◽

Cell Disease ◽

Activation Markers ◽

Circulating Levels

Abstract Abstract 5147 Platelet activation/aggregation in sickle cell disease (SCD) may promote tissue ischemia, suggesting antiplatelet therapy may be useful. However, assessing platelet function and the effect of antiplatelet therapy in blood from SCD patients may be confounded by hemolysis with release of ADP. Here we evaluate levels of platelet activation markers in SCD adolescents vs. normal controls and compare, by multiple methods, the effect of in vitro blockade of the platelet ADP receptor P2Y12 by prasugrel's active metabolite, R-138727. Platelet activation markers in blood from SCD adolescents (n=15) and healthy adults (n=10), and the effect of R-138727 (0. 1 – 10 μM) added in vitro, were evaluated with and without ADP stimulation. Circulating levels of platelet-monocyte and platelet-neutrophil aggregates were significantly higher (p <0. 01) in SCD patients than in healthy controls. R-138727, in a concentration-dependent manner, inhibited platelet function in both SCD patients and healthy subjects as judged by ADP-stimulated light transmission aggregation, VerifyNow P2Y12 assay, multiple electrode aggregometry, and flow cytometric analysis of platelet vasodilator-stimulated phosphoprotein, activated GPIIb-IIIa and P-selectin. The R-138727 IC50s for each assay were not significantly different in SCD vs. healthy subjects. In summary: 1) The high circulating levels of platelet-monocyte and platelet-neutrophil aggregates demonstrate in vivo platelet activation in SCD and may be useful as markers of the in vivo pharmacodynamic efficacy of antiplatelet therapy in SCD. 2) The similar in vitro R-138727 IC50s in SCD and healthy subjects suggest that the prasugrel dose-dependence for platelet inhibition in SCD patients will be similar to that previously observed in healthy subjects. Disclosures: Frelinger: Eli Lilly: Consultancy, Research Funding; Daiichi Sankyo: Research Funding; GLSynthesis: Research Funding. Jakubowski:Eli Lilly: Employment. Heeney:Novartis: Consultancy, Research Funding; Eli Lilly and Company: Research Funding; Pfizer: Consultancy. Michelson:Eli Lilly: Data monitoring committee and idependent external monitor of clinical trials, Research Funding; Takeda: Research Funding; Oxygen Biotherapeutics: Research Funding; Alexion: Research Funding; Omthera: Research Funding.

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Regulation of Na+/Mg2+ Exchange in Sickle Erythrocytes By Endothelin-1

Blood ◽

10.1182/blood.v124.21.4064.4064 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4064-4064

Author(s):

Pablo A. Rivera ◽

Yaritza Inostroza ◽

Jose R. Romero ◽

Alicia Rivera

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Ex Vivo ◽

Red Cells ◽

Cell Disease ◽

Magnesium Homeostasis ◽

Increased Activity ◽

Exchange Activity

Abstract Excess levels of endothelin-1 (ET-1), erythrocyte sickling and chronic inflammation have been proposed as important contributors to the pathophysiology of sickle cell disease (SCD). We have shown that ET-1 receptor antagonists improve hematological parameters by reducing Gardos channel activity in two transgenic mouse models of SCD while reducing oxidant stress by decreasing circulating levels of protein disulfide isomerase. Magnesium (Mg2+) deficiency, mediated in part via increased erythrocyte Na+/Mg2+ exchanger activity, has been demonstrated to contribute to erythrocyte dehydration, K+ loss and sickling in SCD. However, the relationship between ET-1 and the Na+/Mg2+ exchanger in SCD remains unclear. We measured Na+/Mg2+ exchange activity in ex vivo red cells and observed increased activity following in vitro incubation of human (2.2 ± 0.2 to 3.2 ± 0.1 mmol/1013 cell x h, P<0.03, n=5) and mouse red blood cells with ET-1 (P<0.001, n=5); events that were significantly blocked by pre-incubation of cells with 1 μM BQ788, a selective inhibitor of ET-1 type B receptors. In addition, in vitro deoxygenation of sickle red cells led to increased exchanger activity that was inhibited by impramine, a Na+/Mg2+ exchange inhibitor, and associated with reduced deoxygenation-stimulated sickle cell dehydration. These results suggest an important role for ET-1 and cellular magnesium homeostasis in sickle cell disease. To this end, we studied Na+/Mg2+ exchange activity in ex vivo erythrocytes from three transgenic sickle mouse models and observed increased activity in these cells when compared to red cells from either Hb A transgenic or C57BL/J6 wild-type mice (P<0.03, n=4). We then tested the in vivo effects of ET-1 receptor antagonists on erythrocyte Na+/Mg2+ exchange activity in the BERK mouse, a transgenic model of SCD. We blocked ET-1 receptors type A and B by in vivo treatment with BQ-788 and BQ-123 (360mg/Kg/Day) for 14 days and observed lower erythrocyte exchanger activity when compared to cells from vehicle treated BERK mice (P<0.02, n=6). Thus our results suggest that ET-1 receptor blockade represents an important therapeutic approach to control erythrocyte volume and magnesium homeostasis that may lead to improved inflammatory and vascular complications observed in SCD. Supported by NIH R01HL090632 to AR. Disclosures No relevant conflicts of interest to declare.

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Therapeutic Efficacy of Semisynthetic Supra Perfusion Resuscitation Fluids, EAF Peg Alb and EAF Peg Hb, Are Differentiated By Their Cerebral Effects in Animal Models of Sickle Cell Disease

Blood ◽

10.1182/blood.v126.23.773.773 ◽

2015 ◽

Vol 126 (23) ◽

pp. 773-773

Author(s):

Craig A Branch ◽

Min-Hui Cui ◽

Sangeetha Thangaswamy ◽

Nicholas Branch ◽

Seetharama Acharya

Keyword(s):

Blood Flow ◽

Sickle Cell Disease ◽

Oxygen Delivery ◽

Therapeutic Efficacy ◽

Systemic Circulation ◽

Cerebral Oxygen ◽

Cremaster Muscle ◽

Cell Disease ◽

The Brain

Abstract Background: Extension Arm Facilitated (EAF) PEG Alb and EAF PEG Hb are low viscosity semisynthetic hybrid biopolymers which are isoviscous with conventional colloidal plasma expanders but are distinguished from them because they are supra perfusion resuscitation fluids (SPF's). These SPF's have longer half-life, are pseudoplastic and facilitate the production of NO in vivo by increasing shear thinning of RBC's. We recently tested two SPF's, EAF-P5K6 Alb and P3K6 Hb in WT mice, and in two Tg models of Sickle Cell Disease (SCD): the Berkley mouse (BERK), which is a severe anemic model exhibiting a high impairment of systemic blood flow, and in the NY1DD mouse which only exhibits extensive blood flow impairment when challenged with hypoxia followed by reoxygenation. Here we present a comparison of the systemic and cerebral effects of the EAF PEGgylated SPF's. Methods: A single intraperitoneal 10% top-load dose of either drug was given to WT, NY1DD or BERK mice. In NY1DD mice SPF's were administered after hypoxia at the beginning of reoxygenation (8% for 18 hours), while SPF's were given to WT or BERK mice under normoxia conditions. Three hours after the administration of drug, in vivo intra-vital microscopic observation of post-capillary venules in cremaster muscle was performed. In a separate group of WT and BERK animals, we employed MRI to examine the therapeutic efficacy of a single dose of the same SPF's by measuring cerebral blood flow (CBF) and sufficiency of cerebral oxygen delivery (B OLD MRI R esponse to a brief period of H yperO xia, BRHO) serially following treatment. Results: In NY1DD mice, EAF P5K6 Alb significantly attenuated hopoxia reoxygenation induced impairment of cremaster blood flow and associated vaso-occlusion, while EAF P3K6 Hb completely neutralized the experimentally induced sickle crisis. In BERK mice, both SPF's had comparable effects: the chronic state of vaso-occluison as observed in the cremaster muscle was eliminated completely by EAF P3K6-Hb. In MRI experiments in WT mice, both drug candidates resulted in increases in CBF, which resolved over 1 week. The increased CBF was accompanied by decreased BRHO consistent with a pseudo 'luxury perfusion' afforded by the accentuated delivery of oxygen. On the other hand, when BERK mice were treated with EAF P5K6 Alb or EAF P3K6 Hb, CBF trended lower, but with the Alb SPF, BRHO increased, and the Hb SPF, BHRO was unchanged, suggesting that the slightly reduced CBF led to increased O2 deficiency with the PEG-Alb, but not with the PEG-Hb. Conclusion : In WT mice, SPF's increase CBF in the brain where the facility to modify NO production is intact, resulting in over delivery of oxygen as confirmed by reductions in deoxy-Hb levels by BROH imaging, confirming supraperfusionary properties of the SPF's. In SCD animals, both SPF's attenuate muscle vaso-occlusion and restore blood flow. In addition, in experimentally induced sickle crisis (NY1DD), EAF P3K6 Hb maintained O2 level in the plasma and attenuate depolymerization of deoxyHb. In the severely anemic BERK mouse, EAF P5K6-Alb slightly attenuated CBF, likely due to reduced cerebral perfusion pressure (CPP), while O2 extraction increased suggesting that reduced CBF was detrimental to cerebral oxygen delivery. This effect was remediated when EAF P3K6-Hb is administered, which afforded additional oxygen to offset the losses due to reduced CBF. EAF P3K6 Hb led to slightly reduced CBF in NY1DD and BERK mice to levels approaching that obtained after administering EAF P5K6 Alb, but without inducing further oxygen debt. EAF P3K6 Hb appears to be the choice agent as this SPF facilitates increased delivery of O2 to hypoxic tissues thereby neutralizing painful crisis, and protects the brain from further ischemic insults. The influence of SCD on CBF by MRI is opposite to the decrease in blood flow observed in the systemic circulation. The infusion of SFA's increased flow in the systemic circulation, but reduced CBF in a disease dependent fashion. These divergent responses suggest the need for oxygen supplementation when developing SCD therapeutics. In particular, these studies suggest that high oxygen affinity PEG-Hb may have increased the therapeutic efficacy of this SPF by preventing the complete deoxygenation of HbS in the RBC. An antioxidant conjugated to the SFP, such as quercetin, could attenuate the hypoxia reoxygenation induced acute crisis and improve the efficacy of SCD therapeutics. Disclosures No relevant conflicts of interest to declare.

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Mycobacterium aviumComplex Infection in a Patient with Sickle Cell Disease and Severe Iron Overload

Case Reports in Infectious Diseases ◽

10.1155/2014/405323 ◽

2014 ◽

Vol 2014 ◽

pp. 1-5 ◽

Cited By ~ 1

Author(s):

Kamal Shemisa ◽

Nasima Jafferjee ◽

David Thomas ◽

Gretta Jacobs ◽

Howard J. Meyerson

Keyword(s):

Sickle Cell Disease ◽

Iron Overload ◽

Sickle Cell ◽

Unknown Origin ◽

Mycobacterial Infection ◽

In Vivo Studies ◽

Recurrent Pain ◽

Cell Disease

A 34-year-old female with sickle cell anemia (hemoglobin SS disease) and severe iron overload presented to our institution with the subacute presentation of recurrent pain crisis, fever of unknown origin, pancytopenia, and weight loss. A CT scan demonstrated both lung and liver nodules concerning for granulomatous disease. Subsequent biopsies of the liver and bone marrow confirmed the presence of noncaseating granulomas and blood cultures isolatedMycobacterium aviumcomplex MAC. Disseminated MAC is considered an opportunistic infection typically diagnosed in the immunocompromised and rarely in immunocompetent patients. An appreciable number of mycobacterial infection cases have been reported in sickle cell disease patients without immune dysfunction. It has been reported that iron overload is known to increase the risk for mycobacterial infection in vitro and in vivo studies. While iron overload is primarily known to cause end organ dysfunction, the clinical relationship with sickle cell disease and disseminated MAC infection has not been reported. Clinical iron overload is a common condition diagnosed in the sub-Saharan African population. High dietary iron, genetic defects in iron trafficking, as well as hemoglobinopathy are believed to be the etiologies for iron overload in this region. Patients with iron overload in this region were 17-fold more likely to die fromMycobacterium tuberculosis. Both experimental and clinical evidence suggest a possible link to iron overload and mycobacterial infections; however larger observational studies are necessary to determine true causality.

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OLT1177, a β-sulfonyl nitrile compound, safe in humans, inhibits the NLRP3 inflammasome and reverses the metabolic cost of inflammation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1716095115 ◽

2018 ◽

Vol 115 (7) ◽

pp. E1530-E1539 ◽

Cited By ~ 93

Author(s):

Carlo Marchetti ◽

Benjamin Swartzwelter ◽

Fabia Gamboni ◽

Charles P. Neff ◽

Katrin Richter ◽

...

Keyword(s):

Human Blood ◽

Nlrp3 Inflammasome ◽

Inflammatory Diseases ◽

Plasma Concentrations ◽

Metabolic Cost ◽

Inflammasome Activation ◽

Potassium Efflux ◽

Caspase 1 ◽

Healthy Humans

Activation of the NLRP3 inflammasome induces maturation of IL-1β and IL-18, both validated targets for treating acute and chronic inflammatory diseases. Here, we demonstrate that OLT1177, an orally active β-sulfonyl nitrile molecule, inhibits activation of the NLRP3 inflammasome. In vitro, nanomolar concentrations of OLT1177 reduced IL-1β and IL-18 release following canonical and noncanonical NLRP3 inflammasome activation. The molecule showed no effect on the NLRC4 and AIM2 inflammasomes, suggesting specificity for NLRP3. In LPS-stimulated human blood-derived macrophages, OLT1177 decreased IL-1β levels by 60% and IL-18 by 70% at concentrations 100-fold lower in vitro than plasma concentrations safely reached in humans. OLT1177 also reduced IL-1β release and caspase-1 activity in freshly obtained human blood neutrophils. In monocytes isolated from patients with cryopyrin-associated periodic syndrome (CAPS), OLT1177 inhibited LPS-induced IL-1β release by 84% and 36%. Immunoprecipitation and FRET analysis demonstrated that OLT1177 prevented NLRP3-ASC, as well as NLRP3-caspase-1 interaction, thus inhibiting NLRP3 inflammasome oligomerization. In a cell-free assay, OLT1177 reduced ATPase activity of recombinant NLRP3, suggesting direct targeting of NLRP3. Mechanistically, OLT1177 did not affect potassium efflux, gene expression, or synthesis of the IL-1β precursor. Steady-state levels of phosphorylated NF-κB and IkB kinase were significantly lowered in spleen cells from OLT1177-treated mice. We observed reduced IL-1β content in tissue homogenates, limited oxidative stress, and increased muscle oxidative metabolism in OLT1177-treated mice challenged with LPS. Healthy humans receiving 1,000 mg of OLT1177 daily for 8 d exhibited neither adverse effects nor biochemical or hematological changes.

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Evaluation of persistence and fate of ex vivo edited-HSC modified with donor template and Its role in correcting Sickle Cell Disease

10.1101/2021.06.30.450644 ◽

2021 ◽

Author(s):

Sowmya Pattabhi ◽

Samantha N Lotti ◽

Mason P Berger ◽

David J Rawlings

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Ex Vivo ◽

Cell Disease ◽

Gene Correction ◽

Healthy Human ◽

Peripheral Blood Stem Cells ◽

Donor Template

Sickle cell disease (SCD) is caused by a single nucleotide transversion in exon 1 of the HBB gene that changes the hydrophobicity of adult globin (βA), leading to substantial morbidity and reduced lifespan. Ex vivo autologous gene editing utilizing co-delivery of a designer nuclease along with a DNA donor template allows for precise homology-directed repair (HDR). These gene corrected cells when engrafted into the bone marrow (BM) can prove to be therapeutic and serves as an alternative to HLA-matched BM transplantation. In the current study, we extensively explored the role of single stranded oligonucleotide (ssODN) and recombinant adeno-associated 6 (rAAV6) donor template delivery to introduce a codon-optimized change (E6optE) or a sickle mutation (E6V) change following Crispr/Cas9-mediated cleavage of HBB in healthy human mobilized peripheral blood stem cells (mPBSCs). We achieved efficient HDR in vitro in edited cells and observed robust human CD45+ engraftment in the BM of NBSGW mice at 16-17 weeks. Notably, recipients of ssODN-modified HSC exhibited a significantly higher proportion of HDR-modified cells within individual BM, CD34+ and CD235+ compartments of both E6optE and E6V cohorts. We further assessed key functional outcomes including RNA transcripts analysis and globin sub-type expression. Our combined findings demonstrate the capacity to achieve clinically relevant HDR in vitro and in vivo using both donor template delivery method. The use of ssODN donor template-delivery is consistently associated with higher levels of gene correction in vivo as demonstrated by sustained engraftment of HDR-modified HSC and erythroid progeny. Finally, the HDR-based globin protein expression was significantly higher in the E6V ssODN-modified animals compared to the rAAV6-modified animals confirming that the ssODN donor template delivery outperforms rAAV6-donor template delivery.

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