Airway Hyper-Responsiveness Associated With Sickle Cell Disease Is Mediated By Placenta Growth Factor

Airway hyper-responsiveness (AHR) affects about 55-77% of children with sickle cell disease (SCD) and is a risk factor for asthma, increasing the risk of vaso-occlusive events and acute chest syndrome in this patient population. Although in the non-SCD pediatric population AHR is associated with an active allergic response, the molecular mechanisms underlying the high incidence of AHR in a red blood cell disorder remain unclear. Our laboratory has reported that placenta growth factor (PlGF), an erythroid cell secreted-growth factor belonging to the vascular endothelial growth factor family is found at high levels in patients with SCD, mediates increased 5-lipoxygenase (5LO) expression in vitro (Patel et al, 2009). 5LO is the key enzyme in the leukotriene synthetic pathway (LT) and LT levels have been reported to be elevated in SCD (Setty et al, 2002; Jennings et al, 2008). We hypothesized that PlGF contributes to AHR in SCD and investigated the direct contribution of PlGF to AHR in mice with genetically imposed deficiency of PlGF and in the well characterized Berkeley sickle mouse model (HbS mice). We have previously reported that imposing PlGF deficit in HbS mice resulted in extremely small litters and the rare HbS/PlGF-/- mice born were infertile (Patel et al, 2010). Hence the PlGF deficient and HbS mice were studied separately. We utilized a well-established murine model of allergen induced AHR using the highly AHR susceptible mouse strain (BALB/c), that either had normal levels of PlGF (PlGF^WT) or had a genetically imposed deficit of PlGF (PlGF^-/- mice). Results were validated in HbS mice by using pharmacological blockade of this mechanistic pathway. Following allergen treatment with either IL-13 or house dust mites, mice were subjected to a cholinergic challenge and AHR was assessed using either APTI or airway resistance, using an established AHR protocol. Upon allergen challenge, PlGF^-/- BALB/c mice showed significantly blunted AHR compared to PlGF^WT BALB/c mice: the airway resistance was 683 ± 93.5 vs 1283 ± 229 cm²xs; p≤0.04. PlGF neutralization with a blocking antibody (kindly provided by Thrombogenics, Inc.) reduced the severity of AHR in PlGF^WT mice to levels similar to PlGF^-/- mice. Consistent with the importance of eosinophils in driving AHR development, bronchoalveolar lavage fluid (BALF) from PlGF^-/- mice revealed reduced eosinophils (9.3 ± 6.4 x10⁴ vs. 27.5 ± 4.8 x 10⁴ cell/ml P≤0.05), and decreased leukocyte infiltration of lungs by histology. Mechanistically, PlGF deficiency was associated with decreased 5LO expression in lungs both at the mRNA and protein level. We also found that in addition to 5LO, genetically imposed deficiency of PlGF or its pharmacological inhibition were associated with a 2.5-5 fold decrease in IL17A expression in CD4+ lymphocytes from BALF. AHR susceptibility across mouse strains varies tremendously, and has been reported to be mediated via IL-13 and IL-17A (Lajoie and Wills Karp, Nature Immunology, 2010). The HbS mice were originally outbred and have been crossed to C57Bl/6 mice for six generations. To study AHR in HbS mice while eliminating confounding variables attributable to genetic background difference, we transplanted sickle hematopoietic stem cells from HbS donors into Bl/6 (BoyJ) mice, and derived HbS/Bl/6 chimeric mice that had donor derived sickle hematopoiesis replaced the normal recipient hematopoiesis. C57Bl/6 mice transplanted into congenic BoyJ (Bl/6) mice served as controls. Four months after transplant when the mice were totally chimeric, allergen challenge revealed significant increased AHR in HbS/Bl/6 mice compared to C57/Bl/6 mice. Interestingly, IL17A levels were similar between HbS/Bl/6 and C57/Bl/6mice; whereas they were significantly lower in HbS mice compared to control C57/Bl6 mice prior transplant. Consistent with the role of PlGF in AHR in SCD, PlGF neutralizing antibody reduced the AHR severity in chimeric HbS/Bl/6 mice; and pharmacological blockade of 5-LO using zileuton, a 5-LO inhibitor significantly decreased the severity of AHR in HbS/Bl/6 mice in comparison to vehicle treated HbS/Bl/6 mice. Overall, our results indicate that PlGF expression exacerbates AHR via regulating the leukotriene and IL-17A pathways. Leukotriene antagonists are licensed for asthma and PlGF antibody is in clinical trials for cancer. These can be used to reduce the pulmonary morbidity in susceptible patients with moderate to severe SCD.