Activation of the erythroid K-Cl cotransporter Kcc1 enhances sickle cell disease pathology in a humanized mouse model

Key Points A missense mutation in the cytoplasmic tail of Kcc1 activates K-Cl cotransporter activity by impairing phosphorylation of nearby threonines. In vivo evidence shows that activation of Kcc1 directly contributes to the pathogenesis of sickle cell disease.

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Oral Monomethyl Fumarate Therapy Ameliorates Retinopathy in a Humanized Mouse Model of Sickle Cell Disease

Antioxidants and Redox Signaling ◽

10.1089/ars.2016.6638 ◽

2016 ◽

Vol 25 (17) ◽

pp. 921-935 ◽

Cited By ~ 11

Author(s):

Wanwisa Promsote ◽

Folami Lamoke Powell ◽

Satyam Veean ◽

Menaka Thounaojam ◽

Shanu Markand ◽

...

Keyword(s):

Sickle Cell Disease ◽

Mouse Model ◽

Sickle Cell ◽

Cell Disease ◽

Humanized Mouse ◽

Humanized Mouse Model ◽

Monomethyl Fumarate

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Pomalidomide Augments Erythropoiesis and Fetal Hemoglobin Production in a Humanized Mouse Model of Sickle Cell Disease

Blood ◽

10.1182/blood.v112.11.536.536 ◽

2008 ◽

Vol 112 (11) ◽

pp. 536-536 ◽

Cited By ~ 2

Author(s):

Steffen E. Meiler ◽

Marlene Wade ◽

Zhong Chen ◽

Preetha Ramalingam ◽

Laure A Moutouhde Parseval ◽

...

Keyword(s):

Sickle Cell Disease ◽

Mouse Model ◽

Sickle Cell ◽

Fetal Hemoglobin ◽

Spleen Weight ◽

Cell Disease ◽

Humanized Mouse ◽

Humanized Mouse Model ◽

Cd34 Cells

Abstract Introduction: Pomalidomide (PL) is an IMiD™ immunomodulatory compound. Thalidomide and lenalidomide have previously been shown to mitigate blood transfusion dependency in patients with thalassemia major and myelodysplastic syndromes, respectively.1,2 In a recent in vitro study, PL, which is currently being evaluated for the treatment of hematological cancers, stimulated erythropoiesis, F-cell production, total hemoglobin (Hb) and fetal hemoglobin (HbF) synthesis in human CD34+ cells.3 Furthermore, combined treatment of CD34+ cells with PL and Hydroxyurea (HU) synergistically upregulated HbF expression. These results suggest a promising role for PL in the treatment of β-hemoglobinopathies. To evaluate the in vivo efficacy of PL in sickle cell disease (SCD), we conducted an eight week study in a relevant knockout-transgenic (KT) mouse model. 4 Methods: Animals. Six week old KT homozygous sickle mice were treated daily (Mon–Fri; i.p. injections) for eight weeks with the following compounds: Grp 1. Vehicle (n=8); Grp 2. PL (10 mg/kg; n=9); Grp 3. HU (100 mg/kg; n=7); Grp 4. PL+ HU (n=8). Mice were maintained in an accredited pathogen-free animal facility according to NIH and institutional guidelines. Mice were anesthetized with Ketamine/Xylazine and blood collected by intracardiac puncture into 0.5 ml vacutainer EDTA tubes. Complete blood count (CBC) was analyzed with the CBC-Diff™ Veterinary Hematology System (Heska Inc., Loveland, CO). Reticulocyte counts were determined by supravital staining with methylene blue. HbF analysis of mouse hemolysates was done by HPLC using a weak cation-exchange column SynChropak CM-300 (Elchrom INC, Darien, IL). Organ analysis (liver, spleen, and femur marrow) included organ weights (%bw) and histology of H&E paraffin sections. Statistical analysis. One-Way ANOVA followed by Student-Newman-Kuels (Sigma Stat). Data are reported as the mean ± SE. A P-value of < 0.05 was considered significant. Results: Activity level and social behavior were unaffected by the treatment groups. There were no physical signs of drug toxicity and weight gains were comparable for all groups. PL significantly augmented HbF expression comparable to HU (HbF [peak%]: Veh: 6.24±0.35; PL: 9.51±0.83, P<0.01; HU: 10.54±0.77, P<0.01/total Hb [gm/dl]: Veh: 7.16±0.38; PL: 7.67±0.33; HU: 6.09±0.79). Surprisingly, PL-induced HbF production returned to control values after combinatory therapy with HU (HbF [peak%]: Veh: 6.24±0.35; PL+HU: 7.14±0.32). In contrast to HU, PL enhanced erythropoiesis as evidenced by increases in total RBCs, reticulocytes, and spleen weight (RBCs [×106/μl]: Veh: 4.84±0.14; PL: 5.49±0.19, P<0.05; HU: 4.47±0.51/Retics [%]: Veh: 36.81±5.78; PL: 40.47±4.73; HU: 16.41±3.94, P=0.01/Spleen weight [%bw]: Veh: 4.07±0.27; PL: 4.92±0.2, P=0.02; HU: 2.75±0.25, P<0.01). The total WBC count was largely unaffected by PL, but decreased significantly with HU (WBC [×103/μl]: Veh: 14.43±2.74; PL: 11.62±2.62; HU: 6.85±0.82, P<0.05). The PL group showed a trend toward increased marrow hyperplasia. A detailed analysis of cell density, erythroid and myeloid cells is underway. Liver histology revealed decreased tissue inflammation and focal necrosis in ~50% of PL-treated animals. Summary & Conclusions: 1. PL caused a robust induction of HbF in sickle mice. These results extend earlier in vitro findings of PL’s bioactivity in human erythroid progenitor cells. The HbF response to PL was similar to the established HbF-inducer HU, but surprisingly was lost in combination with HU. This inhibitory effect suggests that PL and HU stimulate HbF by distinct mechanisms. 2. Additionally, PL augmented erythropoiesis as demonstrated by increases in total RBCs, reticulocytes, spleen weight, and marrow hyperplasia. In contrast, HU showed the expected findings of generalized myelosuppression. 3. PL reduced liver injury and inflammation in ~50% of treated animals. Liver preservation tended to be associated with higher HbF values, though PL’s immunomodulatory properties may have contributed to this outcome. 4. In summary, PL exhibits a highly favorable hematological profile in a humanized mouse model of SCD. These results warrant further investigation in a Phase I trial of patients with SCD.

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Calpain-1 regulates platelet function in a humanized mouse model of sickle cell disease

Thrombosis Research ◽

10.1016/j.thromres.2017.10.018 ◽

2017 ◽

Vol 160 ◽

pp. 58-65 ◽

Cited By ~ 3

Author(s):

Jennifer O. Nwankwo ◽

Thomas Gremmel ◽

Anja J. Gerrits ◽

Farha J. Mithila ◽

Rod R. Warburton ◽

...

Keyword(s):

Sickle Cell Disease ◽

Mouse Model ◽

Sickle Cell ◽

Platelet Function ◽

Cell Disease ◽

Humanized Mouse ◽

Humanized Mouse Model

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Genetic inactivation of calpain-1 attenuates pain sensitivity in a humanized mouse model of sickle cell disease

Haematologica ◽

10.3324/haematol.2016.148106 ◽

2016 ◽

Vol 101 (10) ◽

pp. e397-e400 ◽

Cited By ~ 8

Author(s):

J. O. Nwankwo ◽

J. Lei ◽

J. Xu ◽

A. Rivera ◽

K. Gupta ◽

...

Keyword(s):

Sickle Cell Disease ◽

Mouse Model ◽

Sickle Cell ◽

Pain Sensitivity ◽

Cell Disease ◽

Humanized Mouse ◽

Humanized Mouse Model ◽

Genetic Inactivation

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Reconstruction of Sickle Cell Disease with Circulating Sickling Red Blood Cells in Novel Humanized Cytokines and Liver Mistrg Mice

Blood ◽

10.1182/blood-2020-141603 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 29-30

Author(s):

Yuanbin Song ◽

Rana Gbyli ◽

Liang Shan ◽

Wei Liu ◽

Yimeng Gao ◽

...

Keyword(s):

Sickle Cell Disease ◽

Mouse Model ◽

Red Blood Cells ◽

Sickle Cell ◽

Blood Cells ◽

Red Cell ◽

Cell Disease ◽

Ineffective Erythropoiesis ◽

Cd34 Cells

In vivo models of human erythropoiesis with generation of circulating mature human red blood cells (huRBC) have remained elusive, limiting studies of primary human red cell disorders. In our prior study, we have generated the first combined cytokine-liver humanized immunodeficient mouse model (huHepMISTRG-Fah) with fully mature, circulating huRBC when engrafted with human CD34+ hematopoietic stem and progenitor cells (HSPCs)1. Here we present for the first time a humanized mouse model of human sickle cell disease (SCD) which replicates the hallmark pathophysiologic finding of vaso-occlusion in mice engrafted with primary patient-derived SCD HSPCs. SCD is an inherited blood disorder caused by a single point mutation in the beta-globin gene. Murine models of SCD exclusively express human globins in mouse red blood cells in the background of murine globin knockouts2 which exclusively contain murine erythropoiesis and red cells and thus fail to capture the heterogeneity encountered in patients. To determine whether enhanced erythropoiesis and most importantly circulating huRBC in engrafted huHepMISTRG-Fah mice would be sufficient to replicate the pathophysiology of SCD, we engrafted it with adult SCD BM CD34+ cells as well as age-matched control BM CD34+ cells. Overall huCD45+ and erythroid engraftment in BM (Fig. a, b) and PB (Fig. c, d) were similar between control or SCD. Using multispectral imaging flow cytometry, we observed sickling huRBCs (7-11 sickling huRBCs/ 100 huRBCs) in the PB of SCD (Fig. e) but not in control CD34+ (Fig. f) engrafted mice. To determine whether circulating huRBC would result in vaso-occlusion and associated findings in SCD engrafted huHepMISTRG-Fah mice, we evaluated histological sections of lung, liver, spleen, and kidney from control and SCD CD34+ engrafted mice. SCD CD34+ engrafted mice lungs showed an increase in alveolar macrophages (arrowheads) associated with alveolar hemorrhage and thrombosis (arrows) but not observed control engrafted mice (Fig. g). Spleens of SCD engrafted mice showed erythroid precursor expansion, sickled erythrocytes in the sinusoids (arrowheads), and vascular occlusion and thrombosis (arrows) (Fig. h). Liver architecture was disrupted in SCD engrafted mice with RBCs in sinusoids and microvascular thromboses (Fig. i). Congestion of capillary loops and peritubular capillaries and glomeruli engorged with sickled RBCs was evident in kidneys (Fig. j) of SCD but not control CD34+ engrafted mice. SCD is characterized by ineffective erythropoiesis due to structural abnormalities in erythroid precursors3. As a functional structural unit, erythroblastic islands (EBIs) represent a specialized niche for erythropoiesis, where a central macrophage is surrounded by developing erythroblasts of varying differentiation states4. In our study, both SCD (Fig. k) and control (Fig. l) CD34+ engrafted mice exhibited EBIs with huCD169+ huCD14+ central macrophages surrounded by varying stages of huCD235a+ erythroid progenitors, including enucleated huRBCs (arrows). This implies that huHepMISTRG-Fah mice have the capability to generate human EBIs in vivo and thus represent a valuable tool to not only study the effects of mature RBC but also to elucidate mechanisms of ineffective erythropoiesis in SCD and other red cell disorders. In conclusion, we successfully engrafted adult SCD patient BM derived CD34+ cells in huHepMISTRG-Fah mice and detected circulating, sickling huRBCs in the mouse PB. We observed pathological changes in the lung, spleen, liver and kidney, which are comparable to what is seen in the established SCD mouse models and in patients. In addition, huHepMISTRG-Fah mice offer the opportunity to study the role of the central macrophage in human erythropoiesis in health and disease in an immunologically advantageous context. This novel mouse model could therefore serve to open novel avenues for therapeutic advances in SCD. Reference 1. Song Y, Shan L, Gybli R, et. al. In Vivo reconstruction of Human Erythropoiesis with Circulating Mature Human RBCs in Humanized Liver Mistrg Mice. Blood. 2019;134:338. 2. Ryan TM, Ciavatta DJ, Townes TM. Knockout-transgenic mouse model of sickle cell disease. Science. 1997;278(5339):873-876. 3. Blouin MJ, De Paepe ME, Trudel M. Altered hematopoiesis in murine sickle cell disease. Blood. 1999;94(4):1451-1459. 4. Manwani D, Bieker JJ. The erythroblastic island. Curr Top Dev Biol. 2008;82:23-53. Disclosures Xu: Seattle Genetics: Membership on an entity's Board of Directors or advisory committees. Flavell:Zai labs: Consultancy; GSK: Consultancy.

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Differential contribution of FXa and thrombin to vascular inflammation in a mouse model of sickle cell disease

Blood ◽

10.1182/blood-2013-08-523936 ◽

2014 ◽

Vol 123 (11) ◽

pp. 1747-1756 ◽

Cited By ~ 71

Author(s):

Erica M. Sparkenbaugh ◽

Pichika Chantrathammachart ◽

Jacqueline Mickelson ◽

Joanne van Ryn ◽

Robert P. Hebbel ◽

...

Keyword(s):

Sickle Cell Disease ◽

Mouse Model ◽

Sickle Cell ◽

Vascular Inflammation ◽

Vascular Pathology ◽

Cell Disease ◽

Potential Target ◽

Key Points ◽

Thrombotic Complications ◽

Differential Contribution

Key Points Inhibition of FXa or thrombin might be considered to reduce thrombotic complications and vascular inflammation in sickle cell patients. PAR-2 could be a potential target to inhibit vascular pathology associated with sickle cell disease.

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Contributions of Immune Cells to Vascular Occlusion in Sickle Cell Disease.

Blood ◽

10.1182/blood.v112.11.sci-44.sci-44 ◽

2008 ◽

Vol 112 (11) ◽

pp. sci-44-sci-44

Author(s):

Paul S. Frenette ◽

Andres Hidalgo ◽

Jungshan Chang ◽

Anna Peired

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Blood Cells ◽

High Speed ◽

Globin Gene ◽

Clinical Manifestations ◽

Leading Edge ◽

Cell Disease ◽

Humanized Mouse Model

Abstract Sickle cell disease (SCD) results from a single missense mutation in the β-globin gene, making the gene product susceptible to polymerize in conditions of low O2 tension. The resulting polymers can disrupt the normal architecture of sickle red blood cells (sRBC), altering their membrane and promoting adherence. sRBC adhesion events within the vasculature produces the painful vaso-occlusive episodes that account for most of the morbidity and mortality of this disease.1 The mechanisms mediating vaso-occlusion (VOC) in SCD are not well understood. Several studies have shown that sRBC, most notably the immature reticulocytes, can bind to endothelial cells (EC) though multiple overlapping adhesion mechanisms. However, whether these interactions are directly involved in VOC is controversial. sRBC can clearly interact with other blood cells inside the vasculature. For example, in a humanized mouse model of SCD, sRBCs interact prominently with leukocytes that are adherent in inflamed venules.2 A role for leukocytes in VOC is consistent with clinical data that have linked leukocyte counts, but not reticulocyte counts, with clinical manifestations of SCD. High-speed multichannel fluorescence intravital microscopy (MFIM) using low doses of lineage-specific antibodies has shown that sRBCs interact specifically with adherent neutrophils.3 Most adherent neutrophils are not immobile but rather crawl along inflamed venules, and virtually all of them exhibit a polarized appearance in vivo with clustered L-selectin at the uropod. Inhibition of or deficiency in the endothelial selectins (E- and P-selectins) protects SCD mice from VOC. Recent studies into the individual role of each selectin have revealed, unexpectedly, a major function for E-selectin. Whereas E-selectin inhibition or deficiency does not significantly affect neutrophil adhesion in inflamed venules, it dramatically blocks the interactions of RBC with adherent neutrophils, suggesting that E-selectin signaling into an adherent leukocyte may induce RBC capture. We have found that RBCs carrying normal hemoglobin (nRBCs) also interact with adherent neutrophils in inflamed venules of wildtype mice. In the mouse, three glycoproteins, P-selectin glycoprotein ligand-1 (PSGL-1, encoded by the Selplg gene), CD44 (encoded by Cd44), and E-selectin ligand-1 (ESL-1, encoded by Glg1) mediate all E-selectin binding activity of leukocytes.4 Detailed analyses of Selplg−/− mice, Cd44−/− mice, or chimeric mice, in which Glg1 is knocked-down by RNA interference, revealed that E-selectin-induced signals are mediated specifically by Glg1. Using MFIM analyses, we have mapped the location of RBC captures on the leading edge of polarized neutrophils. Further investigations have revealed that RBC capture was mediated by the β2 integrin Mac-1 (αMβ2). We have developed a new method to assay Mac-1 activation on adherent leukocytes in live mice. These analyses have shown that Glg1-mediated signals activate αMβ2 regionally at the leading edge, allowing RBC capture. These results suggest a new pathway for the development of targeted therapies for VOC. The fact that genetically normal RBCs are captured by neutrophils through mechanisms similar to sRBCs also suggests broad functions for this paradigm in other thrombo-inflammatory diseases.

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A functional DC cross talk promotes human ILC homeostasis in humanized mice

Blood Advances ◽

10.1182/bloodadvances.2017004358 ◽

2017 ◽

Vol 1 (10) ◽

pp. 601-614 ◽

Cited By ~ 15

Author(s):

Silvia Lopez-Lastra ◽

Guillemette Masse-Ranson ◽

Oriane Fiquet ◽

Sylvie Darche ◽

Nicolas Serafini ◽

...

Keyword(s):

Mouse Model ◽

Cross Talk ◽

Humanized Mice ◽

Humanized Mouse ◽

Humanized Mouse Model ◽

Key Points

Key Points A novel humanized mouse model to study human ILC biology. Human DC cross talk with ILCs in vivo.

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Soluble GARP has potent antiinflammatory and immunomodulatory impact on human CD4+ T cells

Blood ◽

10.1182/blood-2012-12-474478 ◽

2013 ◽

Vol 122 (7) ◽

pp. 1182-1191 ◽

Cited By ~ 33

Author(s):

Susanne A. Hahn ◽

Heiko F. Stahl ◽

Christian Becker ◽

Anita Correll ◽

Franz-Joseph Schneider ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Mouse Model ◽

Cd4 T Cells ◽

Humanized Mouse ◽

Humanized Mouse Model ◽

Key Points ◽

Destructive Inflammation

Key Points GARP efficiently represses proliferation of naïve and resting CD4+ T cells and is involved in the induction of adaptive regulatory T cells. In vivo, GARP prevents T cell–mediated destructive inflammation in a preclinical humanized mouse model of GVHD.

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