Interleukin‐1 receptor antagonism leads to improved anaemia in a murine model of sickle cell disease and is associated with reduced ex vivo platelet‐mediated erythrocyte sickling

Background: Sickle cell disease (SCD) is associated with chronic hemolytic anemia and a heightened inflammatory state. Multiple inflammatory pathways have been shown to be activated in SCD, however the causal role of these pathways in mediating organ damage and contributing to stroke is unclear. Methods: Bone marrow transplantation (bmt) from SCD (Townes) to wild-type (WT) recipient mice was used to generate SCD mice (WTSCD). To generate mice with non-hematopoietic deficiency of the interleukin-1 receptor (IL1R), SCD marrow was transplanted into IL1R deficient recipients (IL1R-/- SCD). Other WTSCD mice were given the IL1R inhibitor, anakinra, or PBS for 2 or 4 weeks before sacrifice. Iron and collagen staining were performed in multiple formalin-fixed tissues with Prussian blue and Masson's Trichrome, respectively. Complete blood counts and reticulocyte percentages were analyzed 15 weeks following BMT. Plasma levels of soluble E-selectin (sE-sel) and soluble P-selectin (sP-sel) were measured 14 weeks following bmt with ELISA. Ischemic stroke was induced by middle cerebral artery (MCA) photothrombosis at 16 weeks of age. WTSCD mice were given anakinra following stroke induction. Seventy-two hours after MCA occlusion, stroke volume was assessed by staining brain sections with 2, 3, 5-triphenyltetrazolium chloride. Formalin-fixed brain sections were also stained for macrophages. Results: All SCD mice were anemic and the severity of anemia was not different between WTSCD and IL1R-/- SCD mice. Increased circulating erythrocytes (9.01 ± 0.78 vs 6.77 ± 0.18 M/uL; p<0.05) and decreased reticulocytes (25.31 ± 1.79 vs 32.45 ± 2.74%; p<0.05) were observed in WTSCD mice treated with anakinra for 4 weeks. Two week anakinra treatment was sufficient to decrease organ iron deposition and lung collagen deposition. In vivo treatment with anakinra also decreased erythrocyte sickling in a whole blood ex vivo sickling assay (15.14 ± 0.94 vs 24.18 ± 1.95 % sickled; p<0.01). Further, in vitro treatment of WTSCD whole blood with IL-1β increased sickling (26.70 ± 0.39 vs 22.34 ± 0.95; p<0.05), and this was ameliorated with in vitro pretreatment with anakinra. Alterations to ex vivo sickling were not observed when conditions were repeated on erythrocytes alone. Stroke volume was significantly reduced in IL1R-/- SCD mice compared to WTSCD mice 3 days following MCA occlusion (8.08 ± 0.91 vs 11.10 ± 1.16 % hemisphere; p<0.05). Post-stroke treatment of WTSCD mice with anakinra also decreased stroke size compared to vehicle-treated mice (14.45 ± 1.44 vs 9.7 ± 3.23 % hemisphere; p=0.05). Non-hematopoietic deficiency of IL1R decreased sE-sel (vs 50.10 ± 2.31 vs 32.12 ± 2.08 pg/mL; p<0.05) and sP-sel (155.98 ± 24.44 vs 327.23 ± 18.31 ng/mL; p<0.05) levels in post-stroke animals and decreased the number of macrophages observed in the peri-stroke area (0.26 ± 0.05 vs 0.722 ± 0.15 % hemisphere; p<0.05). Conclusions: Deficiency of non-hematopoietic IL1R or treatment with an IL1R antagonist was appears beneficial towards several phenotypes in a mouse model of SCD. Given the safety of IL1R inhibitors in various patient populations, this study reveals a potential therapeutic intervention for treatment of sickle cell patients. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Non-hematopoietic deficiency of proprotein convertase subtilisin/kexin type 9 deficiency leads to more severe anemia in a murine model of sickle cell disease

Scientific Reports ◽

10.1038/s41598-020-73463-9 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

J. Venugopal ◽

J. Wang ◽

C. Guo ◽

H. Lu ◽

Y. E. Chen ◽

...

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Ex Vivo ◽

Vascular Complications ◽

Lipid Lowering ◽

Cell Disease ◽

Proprotein Convertase ◽

Beneficial Effects ◽

Cholesterol Levels ◽

Erythrocyte Sickling

Abstract Proprotein convertase subtilisin/kexin type 9 (PCSK9) deficiency leads to lower cholesterol and is associated with reduced vascular complications in the general population. Cholesterol lowering may also have beneficial effects in sickle cell disease (SCD). The objective of this study was to determine effects of PCSK9 deficiency in a mouse model of SCD. Bone marrow transplantation (BMT) was performed from donor SCD mice to wild-type, PCSK9-deficient, and LDLR-deficient recipients to generate SCD controls (Pcsk9+/+, SCDbmt) with preserved PCSK9 status, SCD mice with deficiency of PCSK9 (Pcsk9−/−, SCDbmt), and SCD mice with deficiency of LDLR (Ldlr−/−, SCDbmt). Although cholesterol levels were lower in Pcsk9−/−, SCDbmt mice compared to Pcsk9+/+, SCDbmt mice, anemia was more severe in Pcsk9−/−, SCDbmt mice. Increased reticulocytosis, enhanced ex vivo erythrocyte sickling, and increased erythrocyte phosphatidylserine exposure was also observed. Livers, spleens, and kidneys contained increased iron in Pcsk9−/−, SCDbmt mice compared to Pcsk9+/+, SCDbmt mice consistent with greater hemolysis. SCD mice with deficiency of LDLR (Ldlr−/−, SCDbmt mice) had similar anemia as Ldlr+/+, SCDbmt mice despite higher serum cholesterol. In conclusion, deficiency of PCSK9 is associated with worsened anemia in SCD mice due to increased hemolysis. These findings may have implications for lipid-lowering strategies in patients with SCD, as well as for potential novel modifiers of anemia severity.

Download Full-text

3057 – MATURATION OF EX VIVO-CULTURED HUMAN ERYTHROCYTES AND SICKLE CELL DISEASE MODELLING USING CRISPR-CAS9

Experimental Hematology ◽

10.1016/j.exphem.2020.09.074 ◽

2020 ◽

Vol 88 ◽

pp. S55

Author(s):

Yelena Boccacci ◽

Guillaume Margaillan ◽

Nellie Dumont ◽

Mathieu Drouin ◽

Yannick Doyon ◽

...

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Ex Vivo ◽

Human Erythrocytes ◽

Disease Modelling ◽

Cell Disease

Download Full-text

Interferon Induction By HbS, SERINC5 Upregulation and Reduction of HIV-1 Nef and AP2 Contribute to HIV-1 Restriction in Sickle Cell Disease

Blood ◽

10.1182/blood-2020-142699 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 5-6

Author(s):

Namita Kumari ◽

Marina Jerebtsova ◽

Songping Wang ◽

Sharmin Diaz ◽

Sergei Nekhai

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Interferon Response ◽

Cell Disease ◽

Restriction Factors ◽

Peripheral Blood Mononuclear ◽

Hiv 1

Concerted action of numerous positively acting cellular factors is essential for Human immunodeficiency virus type 1 (HIV-1) replication but in turn is challenged by anti-viral restriction factors. Previously we showed that ex vivo one round HIV-1 replication and replication of fully competent T-tropic HIV-1(IIIB) is significantly reduced in peripheral blood mononuclear cells (PBMCs) obtained from patients with Sickle Cell Disease (SCD). Further, we identified and confirmed CDKN1A (p21) and CH25H as host restriction factors expressed in SCD PBMCs that may contribute to the HIV-1 inhibition, in addition to the previously reported SAMHD1 and IKBα. Since CH25H is an interferon stimulated gene (ISG), we analyzed IRFs and interferon expression in SCD PBMCs. Higher levels of IRF7 and IFNβ mRNA were observed in SCD PBMCs compared to controls. We probed further to ascertain if hemin or sickle Hb was responsible for interferon response. We found upregulation of IFNβ in THP-1 - derived macrophages treated with lysates of HbSS RBCs or purified HbS as compared to untreated or HbA treated controls. HbSS RBCs lysates and purified HbS inhibited HIV-1 gag mRNA expression in monocyte-derived macrophages infected with HIV-1(Ba-L). Recent clinical study showed increased levels of CD4 in HIV-1 infected SCD patients in Africa. Thus we analyzed CD4 levels in HIV-1 IIIB infected SCD PBMCs, and found them to be higher compared to controls. Levels of HIV-1 nef mRNA, that controls CD4 expression was lower in HIV-1 IIIB infected SCD PBMCs. As Nef counteracts SERINC3/5 restriction factor, we analyzed its expression as well as the expression of AP2 clathrin adaptor that is required for Nef mediated internalization of CD4. AP2 expression was lower and SERINC5 expression was higher in SCD PBMCs. CONCLUSIONS: SCD PBMCs could resist HIV-1 infection because of the increased IFNβ production by macrophages exposed to HbSS or sickle cell RBCs. SCD PBMC have increased levels of SERNIC5 and lower levels of HIV-1 Nef and host AP2 expression that, culumlatively, can increased CD4 levels and lead to the overall improved immunological health of SCD patients. ACKNOWLEDGMENTS: This work was supported by NIH Research Grants (1P50HL118006, 1R01HL125005, 1SC1HL150685, 5U54MD007597, 1UM1AI26617 and P30AI087714). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Successful Engraftment of Umbilical Cord Blood (UCB) Cells after Co-Transplantation of Nicord® (Ex Vivo Expanded UCB Progenitor Cells with Nicotinamide) and an Unmanipulated UCB Unit after Myeloablative Chemotherapy in Severe Sickle Cell Disease

Biology of Blood and Marrow Transplantation ◽

10.1016/j.bbmt.2016.12.291 ◽

2017 ◽

Vol 23 (3) ◽

pp. S175-S176 ◽

Cited By ~ 1

Author(s):

Suhag Parikh ◽

Joel Brochstein ◽

Paul L. Martin ◽

Mitchell E. Horwitz ◽

Einat Galamidi ◽

...

Keyword(s):

Sickle Cell Disease ◽

Cord Blood ◽

Progenitor Cells ◽

Umbilical Cord ◽

Sickle Cell ◽

Umbilical Cord Blood ◽

Ex Vivo ◽

Cell Disease ◽

Myeloablative Chemotherapy

Download Full-text

248. Evidence of Globin Lentiviral Vector-Induced Genotoxicity in a Murine Model of Sickle Cell Disease

Molecular Therapy ◽

10.1016/s1525-0016(16)44454-0 ◽

2007 ◽

Vol 15 ◽

pp. S95

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Murine Model ◽

Lentiviral Vector ◽

Cell Disease

Download Full-text

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.

Download Full-text