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J. Venugopal et al.
risk of stroke in SCD patients, with those with hemoglo- bin (Hb)SS having the highest risk and those with sickle β+-thalassemia having the lowest risk.2 In general, the most prevalent subtype of stroke associated with SCD patients is ischemic stroke, however between the ages of 20 and 29 hemorrhagic strokes are more prevalent.2 Stroke prevention in SCD patients is primarily accomplished through chronic blood transfusions4 and hydroxyurea treatment.5-7
Although erythrocyte sickling in response to stressors constitutes the primary underlying defect of SCD, subse- quent inflammatory responses to vascular occlusive events contribute to organ damage and further vascular dysfunction.8-11 This heightened inflammatory milieu is characterized by leukocytosis and elevated levels of cytokines in SCD.12-14 Therapies shown to be beneficial in SCD such as hydroxyurea and anti-selectin antibodies may exert their beneficial effects, in part, via dampening of leukocyte-mediated inflammatory responses.15,16
Hemolysis in SCD may result in the activation of leuko- cytes via Toll-like receptors (TLR) and NOD-like receptor 3 (NLRP3) by free heme.17 TLR and NLRP3 inflammasome expression levels, including interleukin-1β (IL-1β ), are increased in peripheral blood monocytes from SCD patients.18,19 IL-1β is a particularly important mediator of acute and chronic inflammatory disease processes, as ther- apeutic targeting of IL-1β has proven beneficial in several inflammatory diseases.20-22 Additionally, some genetic polymorphisms of IL-1β have been shown to affect IL-1β transcription and are associated with arthritis, cardiovas- cular disease, and complications of SCD.9,23,24 While these studies suggest IL-1β signaling pathways are involved in some manifestations of SCD, the causal role of these path- ways remains unclear.
IL-1β may represent a particularly important modulator of stroke outcomes.25,26 IL-1β is rapidly upregulated during ischemic stroke and may contribute to ischemic injury.26 In a meta-analysis of 16 non-SCD animal studies, admin- istration of the IL-1R antagonist, anakinra, produced a 36% reduction of infarct volume.27 IL-1β may promote neuronal death indirectly, via effects on astrocytes and endothelial cells.25 The binding of IL-1β to astrocyte IL-1R activates signaling cascades resulting in the production of IL-6, TNF-α, and other chemokines which influence cen- tral nervous system (CNS) inflammation28 and neurotoxi- city.28 Neurotoxicity mediated by IL-1β may also occur through endothelial interleukin-1 receptor (IL-1R)-mediat- ed activation of cerebral endothelial cells,30 leading to leukocyte infiltration31 and the loss of blood brain barrier integrity.32 The recruitment of peripheral leukocytes by IL-1β can sustain neuroinflammation,33 further promoting neurotoxicity,34 and blood brain barrier (BBB) disruption.35 IL-1β may also induce permeability of the BBB directly through endothelial cell signaling pathways.36
Mouse models of SCD have been developed that mimic the predominant features of SCD in humans.37-39 In gener- al, these mice exhibit hemolysis, anemia, splenomegaly, and multi-organ infarcts.37-39 SCD mice have thus been a useful aid to identify mechanisms involved in vaso-occlu- sion and to test potential therapeutic interventions. Because of reduced fertility and complex genetics, gener- ating SCD mice with complete deficiency of a disease- modifying candidate gene through intercrosses is cumber- some, as is generation of suitable littermate controls. However, bone marrow transplantation (BMT) is an effi-
cient means to generate SCD mice, and if a candidate gene of interest exerts its effects via non-bone marrow-derived cellular pools, then informative SCD mice can be readily generated by transplanting SCD marrow to recipient mice with deficiency of the candidate gene. In order to modify IL-1β signaling pathways using this strategy, transplanta- tion of SCD marrow to mice lacking the receptor for IL-1β, (IL-1R), leads to lack of IL-1 signaling in non- hematopoietic IL-1R cellular pools. The endothelial IL-1R pool is responsible for mediating the upregulation of endothelial adhesion molecules and leukocyte-endothelial interactions in response to IL-1β stimulation,40 which might be particularly relevant to SCD pathogenesis. Therefore, to study IL-1β signaling pathways in SCD, mice were generated by transplanting SCD marrow into recipients with IL-1R deficiency and compared to control wild-type (WT) recipients on the same C57BL6/J strain background. The role of IL-1R signaling was then ana- lyzed with regards to anemia and stroke in SCD mice. The effect of an IL-1R pharmacologic antagonist was also assessed.
Methods
Animals
Male C57BL/6J wild-type (WT), homozygous SCD ( SCD, Stock No:013071 Townes model), IL1R null mice (IL1R-/-, Stock No: 003245), interleukin-6 null mice (IL6-/-, Stock No: 002650) were purchased from Jackson Laboratory (Bar Harbor, Maine, USA). SCD and control experimental mice were then generated by BMT from SCD mice into WT, IL1R-/-, and IL6-/- recipients. Additional controls were generated by transplantation of WT marrow into WT recipients. Mice were housed under specific pathogen-free conditions in static microisolator cages with tap water ad libitum in a temperature-controlled room with a 12:12-hour light/dark cycle. Mice were fed a standard laboratory rodent diet (No. 5001, TestDiet, Richmond, IN, USA). All animal use protocols complied with the Principle of Laboratory and Animal Care established by the National Society for Medical Research and were approved by the University of Michigan Committee on Use and Care of Animals.
Bone marrow transplantation and blood parameter analysis
SCD mice were generated by BMT as previously described.41,42 Briefly, 8 week-old male WT, IL1R-/- and IL6-/- mice were used as recipients that received bone marrow from 8 week-old SCD male donors. Bone marrow was harvested from the donor mice by flushing their femurs and tibias with RPMI medium (Gibco/Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (Gibco/Invitrogen, Carlsbad, CA, USA). Cells were then centrifuged at 300g and resuspended in phosphate-buffered saline before injection. Each recipient mouse was irradiated (2×650 rad [0.02×6.5 Gy]) and injected with 4×106 bone marrow cells via the tail vein in a 200 μL bone marrow suspension in phos- phate-buffered saline. Acid water (6 mM HCl, pH=2.5) was pro- vided to animals beginning 4 days before BMT to 4 weeks follow- ing BMT. Transplant efficiency was determined by hemoglobin electrophoresis, as done previously.41,43,15 weeks following BMT, blood parameter analyses were performed with a Hemavet (Drew Scientific, Inc) on whole blood collected in EDTA-lined tubes via retro-orbital sampling from isofluorane-anesthetized mice (n=5 per group). Reticulocyte percentages were quantified by new methylene blue staining (n=5 per group) (Ricca Chemical
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haematologica | 2021; 106(9)