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Interleukin-1 receptor in sickle cell disease
Company, Arlington, TX, USA), according to the manufacturer's instructions and expressed as a percentage of total erythrocytes.
Stroke model
Sixteen weeks after BMT, middle cerebral artery (MCA) occlu- sion was induced by photochemical injury as previously described,41,42 n=3-5. On day 3 following MCA occlusion, mice were anesthetized with pentobarbital, and blood drawn via car- diac puncture. Mouse bodies were perfused with PBS, then brains were excised and sliced into 2 mm segments before staining for 20 minutes with 2, 3, 5-triphenyltetrazolium chloride at room tem- perature while protected from light. The brain sections were imaged with a Nikon SMZ-2T microscope and Spot Idea camera model 29.2-13MP using Spot 5.1 software, and stroke size was then calculated as done previously.41 Brain macrophages were stained with an anti-mouse MAC3 antibody (1:200; #550292, BD Biosciences, San Jose, CA), n=3-5. Fibrin(ogen) was stained with an anti-mouse fibrin(ogen) antibody (1:4,000; ab189490, Abcam, Cambridge, MA) and ICAM-1 was stained with anti-mouse ICAM-1 antibody (2 ug/mL; #14-0542-85, ThermoFisher Scientific, Waltham, MA), followed by a biotin-conjugated sec- ondary IgG (1:100), n=3-5. A Nikon Microphot-SA Epi-FL3 micro- scope, Nikon Ds Fi3 camera, and NIS Elements software were used to capture images. Quantification of fibrin(ogen) stained area was performed with Image J software. Quantification of MAC3- positive cells was attained for each mouse by manually counting stained cells in 20 fields of view at 10x. For each field, the number of MAC3-positive cells was divided by the area of brain imaged in each field.
Circulating E-selectin and IL-6 measurements
Soluble E-selectin (sEsel), and IL-6 enzyme-linked immunosor- bent assays (ELISA) were performed according to the manufactur- er’s instructions (R&D Systems, Inc.; Minneapolis MN, USA; Cat#: MES00 & MPS00), n=3-5. Blood for ELISA was collected via cardiac puncture at the time of sacrifice and plasma prepared by centrifugation at 8,500 rpm for 10 min.
Drug treatment
Anakinra (Swedish Orphan Biovitrum AB, Stockholm, Sweden) (100 mg/kg via one intraperitoneal injection [i.p.]) or vehicle con- trol (phosphate buffered saline) was administered 1 hour follow- ing induction of stroke (n=4 per group).
Statistical analysis
Data are presented as mean ± standard deviation. Analysis was carried out using GraphPad Prism and tests for normality were performed using the Shapiro-Wilk test. Differences between groups were then analyzed using a one way ANOVA or an unpaired t-test for comparison between groups or Mann Whitney U test. Probability values of P < 0.05 were considered statistically significant.
Data sharing
For original data, please contact deitzman@umich.edu. Results
Effect of IL-1R and IL-6 status on hematological data in SCD mice
In order to determine whether signaling through the IL- 1R or IL-6 in non-hematopoietic tissues would impact ane- mia in SCD mice, whole blood was analyzed for cell counts, platelets, and reticulocytes 15 weeks following
BMT. Compared to WT mice transplanted with WT bone marrow (Wt,WTbmt), WT recipients of SCD bone marrow (Wt,SCDbmt) were more anemic with elevated leukocyte and reticulocyte counts (Figure 1). IL-1R-/- and IL-6-/- recipi- ents of SCD marrow (IL1R-/-,SCDbmt and IL6-/-, SCDbmt) dis- played similar anemia and reticulocyte counts compared to Wt,SCDbmt mice (Figure 1).
Effect of IL-1R and IL-6 status on circulating levels of IL- 6 in SCD mice
Plasma levels of IL-6 were detectable in Wt,SCDbmt mice (4.877±3.62 pg/mL) but undetectable in both IL-1R-/-,SCDbmt and IL6-/-,SCDbmt mice, consistent with a non-hematopoietic source for circulating IL-6 in SCD and a critical role for non-hematopoietic IL-1 receptor signaling towards IL-6 levels in SCD.
Effect of IL-1R and IL-6 status on stroke size in sickle cell disease following middle cerebral artery occlusion
SCD mice have been shown to experience larger strokes following MCA occlusion due to vasocclusion by sickled erythrocytes in the penumbra.42 In order to determine whether IL1R-/-,SCDbmt mice would be protected from the increased stroke size associated with SCD, photochemical- mediated thrombosis was induced in the MCA in Wt,SCDbmt mice and IL-1R-/-,SCDbmt mice. Three days later, the stroke area was quantitated and IL-1R-/-,SCDbmt mice were found to have a similar stroke areas to Wt,WTbmt mice, both of which had reduced areas when compared to Wt,SCDbmt mice (Figure 2A to C). In contrast, stroke size in IL6-/-,SCDbmt mice was not reduced compared to Wt,SCDbmt mice (Figure 2D). Thus, although non-hematopoietic IL-1R signaling pathways regulate circulating IL-6 levels, this pathway does not account for the effects of non- hematopoietic IL-1R signaling on stroke size in SCD.
Reduction in stroke size in IL-1R-/-,SCDbmt mice was also associated with reduced peri-infarct infiltration of macrophages, as denoted by staining of MAC3 (Figure 3). Since endothelial IL-1R signaling regulates expression of endothelial adhesion molecules40,44 which could affect the stroke phenotype, levels of sEsel were measured given its endothelial specificity. Plasma levels of sEsel were found to be significantly reduced in IL-1R-/-,SCDbmt compared to Wt,SCDbmt mice (32.12±2.08 ng/mL vs. 50.10±2.31 ng/mL; P=0.03). Circulating values of sEsel in IL6-/-,SCDbmt were not significantly different compared to Wt,SCDbmt (54.03±10.10 ng/mL, P=0.37). Fixed brain sections were also stained for ICAM-1. Expression of ICAM-1 was significantly decreased IL-1R-/-,SCDbmt compared to Wt,SCDbmt mice (0.008±0.002% area vs. 0.029±0.007% area, P<0.05).
Increased infiltration of leukocytes may diminish blood brain barrier integrity, leading to leakage of fibrin(ogen)- containing plasma into the brain from the vasculature.45-47 Fibrin(ogen) immunopositivity was significantly decreased in IL-1R-/-,SCDbmt compared to Wt,SCDbmt mice, whereas IL6-/-,SCDbmt were not significantly different than Wt,SCDbmt mice (Figure 4).
Effect of single dose anakinra on stroke size given post middle cerebral artery occlusion
From a practical therapeutic standpoint, treatment with antagonists of IL-1β or IL-1 receptor signaling pathways may not be feasible in SCD patients prior to the onset of stroke, however therapies could be administered following
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