Inhibition of Phosphodiesterase 9A (PDE9A) Significantly Reduces Cytokine-Stimulated Adhesion of Neutrophils From Sickle Cell Disease Individuals, in Vitro, but Not Red Cell Adhesion.
Authors: Miguel Lediana I , Almeida Camila B. , Franco-Penteado Carla F , Bezerra Marcos André C , Traina Fabiola , Dominical Venina M , Araujo Aderson S , Saad Sara T.O. , Costa Fernando F. , Conran Nicola 
Published: October 2019

Abstract
Abstract 1520
Poster Board I-543
The adhesion of both red and white cells to the vessel walls of the microcirculation initiates vaso-occlusion in sickle cell disease (SCD). The chronic inflammatory nature of SCD leads to elevation of circulating cytokines in patients, which may contribute significantly to the activation of blood cells and their consequent adhesion. Nitric oxide (NO) has important inhibitory effects on cellular adhesive properties and drugs that enhance NO bioavailability or NO-cGMP-dependent signaling may hold potential for treatment of various aspects of SCD. This study aimed to observe the effect of cytokines, often found augmented in the plasma of SCD individuals, on the in vitro adhesive properties of neutrophils (neu) and red blood cells (RBC) from healthy control (CON) and steady-state SCD (SCD) individuals. Furthermore, the effects of BAY 73-6691, an inhibitor of the cGMP-hydrolyzing enzyme, phosphodiesterase 9A (PDE9A) and BAY 41-2271, a guanylate cylase activator, on non-stimulated and cytokine-stimulated cell adhesion were determined. Neus and RBCs were isolated from peripheral blood of CON and SCD individuals. Cell adhesion (5×106neu/ml in RPMI or 2×108RBC/ml in HBSS) to immobilized fibronectin (FN;20μg/ml) was assessed using static adhesion assays (30min, 37°C, 5%CO2) in the presence or absence of cytokines IL-8 (100-500ng/ml), TNF-alpha (0.1-1μg/ml) and GM-CSF (1-100ng/ml) and/or in the presence/absence of BAY 73-6691 (10-60μM), BAY 41-2271 (60-150nM) or DMSO vehicle (0.02%v/v). As previously demonstrated, SCDneu have a greater capacity to adhere to FN than CONneu (see Table). Stimulation of cells in vitro with all three cytokines significantly augmented both CONneu adhesion to FN and further increased SCDneu adhesion (Table). Co-incubation of both CONneu and SCDneu with BAY 73-6691, but not BAY 41-2271, significantly reduced their adhesions to FN (Table). Furthermore, BAY 73-6691, but essentially not BAY 41-2271, significantly inhibited CONneu and SCDneu adhesion stimulated by IL-8, TNF-alpha and GM-CSF (Table). DMSO vehicle had no significant effect upon neu adhesion (data not shown). As previously reported, SCD RBC have a greater capacity to adhere to FN, in vitro, compared to CON RBC (12.8±1.7% comp. 7.8±1.0%, n=7, p<0.05). However, in contrast to neutrophils, cytokines IL-8 (10-500ng/ml) and TNF-alpha (0.1-1μg/ml) did not alter the capacities of neither CON RBC nor SCD RBC to adhere to FN (200ng/ml IL-8: CON RBC, 6.8±0.8%; SCD RBC, 13.5±1.8%, n≥6, p>0.05) (1μg/ml TNF-alpha: CON RBC, 7.0±1.0%; SCD RBC, 11.4±1.9%, n=6, p>0.05). Furthermore, BAY 73-6691 and BAY 41-2271 did not affect either basal CON RBC (data not shown) or SCD RBC adhesion (SCD RBC basal; 14.8±1.0%; 60μM BAY 73-6691, 16.4±1.3%; 150nM BAY 41-2271, 15.7±1.2%, n=6). Key SCD inflammatory mediators were found, at physiologically relevant concentrations, to augment the adhesive properties of neutrophils, but not RBC, from control and SCD individuals. Circulating inflammatory cytokines may play a role in the induction of leukocyte adhesive properties in SCD; in contrast factors other than inflammatory stimuli may be more important for induction of SCD RBC adhesion. Data suggest that elevation of intracellular cGMP may be an important approach for reducing SCD leukocyte, but not SCD RBC, adhesive properties, even in an inflammatory environment. PDE9A is highly expressed in hematopoietic cells and inhibition of this enzyme, with consequent augmentation of cGMP, may represent a tissue/cell-specific therapeutic drug target worthy of further in vitro and in vivo studies as a therapy for SCD.
Table Effect of cytokine stimulation and cGMP-elevating agents upon adhesive properties of neutrophils from healthy control and steady-state SCD individuals % Neutrophil adhesion to Fibronectin (mean±SEM) Controls SCD Basal N=18 IL-8 (200ng/ml) N≥7 TNF-alpha (500ng/ml) N≥7 GM-CSF (100ng/ml) N≥7 Basal N=18 IL-8 (200ng/ml) N≥7 TNF-alpha (500ng/ml) N≥7 GM-CSF (100ng/ml) N≥7 Basal 16.1±1.5 26.6±2.8 27.4±5.5 21.4±1.6 21.4±1.9 32.5±3.5 29.2±3.0 25.2±2.2 * p<0.05 * p<0.05 * p<0.05 & p<0.05 * p<0.05 * p<0.05 * p<0.05 BAY 13.5±2.2 14.5±2.9 17.4±4.6 9.42±2.3 15.5±1.2 10.5±3.5 23.2±3.1 15.4±1.9 736691 (60μM) # p<0.01 # p<0.001 # p<0.01 # p<0.01 # p<0.001 # p<0.001 # p<0.05 # p<0.05 BAY 412271 (60 nM) 17.5±1.9 23.6±3.8 25.8±5.0 18.2±2.1 19.2±1.2 20.4±3.5 # p<0.05 27.4±2.7 20.0±1.4 &, comp. to control neutrophils (Mann-Whitney); *, comp. to non-cytokine stimulated adhesion; #, comp. to basal adhesion without co-incubation with BAY-736691/412271(Repeated measures ANOVA, Dunn post test)

Disclosures
No relevant conflicts of interest to declare.