Anti-Interleukin-1β Antibody-Based Therapy Ameliorates Endothelial Activation and Inflammation in Sickle Mice
Authors: Kaul Dhananjay K. , Thangaswamy Sangeetha , Suzuka Sandra M. , Fabry Mary E. , Wanderer Alan A. , Gram Hermann 
Published: October 2019

Abstract
Abstract 848
Inflammation is a recognized feature of sickle cell disease (SCD). Reperfusion injury (caused by vaso-occlusive events) and elevated levels of inflammatory cytokines in this disease are implicated in endothelial activation and inflammation. In transgenic sickle mice, exaggerated inflammatory response to hypoxia-reoxygenation is characterized by enhanced endothelial oxidant generation and leukocyte recruitment in venules (Kaul and Hebbel, JCI 2000). While hypoxic phase will enhance intravascular sickling, reperfusion will induce exaggerated inflammatory response and leukocyte recruitment, which will promote flow stasis (Turhan et al. PNAS 2002). Hypoxia also induces endothelial cells and leukocytes to express and secrete proinflammatory cytokines such as interleukin-1 beta (IL-1β). Sickle patients show elevated levels of IL-1β, which may, in part, be attributed to recurring ischemia-reperfusion episodes. IL-1β can up-regulate endothelial adhesion molecules via endothelial oxidant generation and activation of nuclear factor-κB (NF-κB). In SCD, IL-1β is implicated in endothelial activation, cell adhesion and inflammation (Natarajan et al. Blood 1996; Zachlederova and Jarolim, Blood Cells Mol Dis 2006; Wanderer, JPHO 2009). In view of the documented efficacy of anti-IL-1β treatment in ameliorating several human inflammatory syndromes (Dinarello, Blood 2011), we hypothesized that targeting IL-1β will attenuate endothelial activation and exert anti-inflammatory effect in SCD.
To this end, we have examined efficacy of murine anti-IL-1β antibody (Novartis Pharma AG, Basel, Switzerland) in transgenic sickle (NY1DD) and transgenic-knockout sickle (BERK) mice. Whereas BERK mice express exclusively human α- and βS-globins, NY1DD mice express 56% human α and 75% βS on a mouse homozygous βmajor deletional background. For hypoxia-reoxygenation (H/R) experiments, we selected NY1DD mice based on their highly exaggerated response to H/R compared with C57BL mice. NY1DD mice were subjected to 16 hr of hypoxia (8% O2) followed by 3 hr of reoxygenation at ambient air. In NY1DD mice, hypoxia-reoxygenation (H/R) caused almost 8-fold increase in leukocyte adhesion and ∼13-fold increase in transendothelial leukocyte emigration in cremasteric venules as compared with normoxic NY1DD mice (each P<0.0001); leukocyte adhesion often resulted in flow blockage in post-capillary venules. In marked contrast, NY1DD mice, receiving a single i.p. injection of anti-IL-1β antibody (200 μg/mouse) 24 hrs prior to H/R, showed greater than 90% decrease in leukocyte adhesion and emigration compared with NY1 DD mice subjected to H/R alone (P<0.0001); the resulting values were not significantly different from those for normoxic C57BL mice. Also, H/R caused 43% decline in venular wall shear rates in NY1DD mice compared with normoxic NY1DD mice (P<0.0001). In contrast, in NY1DD mice, pretreatment with the antibody followed by H/R almost completely normalized wall shear rates. Importantly, frequent flow stasis observed in NY1DD mice subjected to H/R was completely abolished by the anti-IL-1β antibody. Notably, the decrease in leukocyte adhesion in NY1DD mice receiving the antibody was associated with marked decrease in endothelial oxidant production as measured by dihydrorhodamine (DHR) oxidation intensity (P<0.0001), suggesting alleviation of endothelial activation. The reduced endothelial activation was further evident by 60% decrease in the serum levels of soluble vascular cell adhesion molecule-1 (P<0.016). In experiments with BERK mice, a single dose of anti-IL-1β antibody (300 μg) was injected i.p. under normoxic conditions. We did not subject the severe BERK model to H/R because of their low tolerance to hypoxia. In BERK mice, anti-IL-1β caused 41% decrease in leukocyte adhesion (P<0.036 vs. untreated group). The reduced leukocyte adhesion was accompanied by a pronounced 3.5-fold increase in wall shear rates (P<0.0001), suggesting a generalized improvement in the microvascular flow.
We show for the first time the efficacy of anti-IL-1β therapy in alleviation of reperfusion injury, endothelial activation, inflammation and flow stasis caused by hypoxia-reoxygenation in transgenic sickle mice, as well as its beneficial effect in normoxic BERK mice. Our results provide a strong basis for therapeutic application of this antibody in clinical management of SCD.

Disclosures:
Kaul: NIH: Research Funding; Novartis Pharma AG: Research Funding. Fabry:Novartis Pharma: Collaborator; NIH: Research Funding. Wanderer:Novartis Pharma: Honoraria. Gram:Novartis Pharma AG: Employment.