Cytoprotective Chaperone Proteins Are Novel Anti-Inflammatory Targets in Sickle Cell Disease

Sickle cell disease (SCD) is a pro-inflammatory condition. Levels of TNF-α, IL-6, IL-8, and IL-10 are elevated. There is clear evidence of endothelial cells (EC) dysfunction, and increased leukocyte, and erythrocyte adhesion in patients even in the non-crisis "steady state" condition. Additional insult, either via infection or vaso-occlusive ischemia, induce a dramatic increase in inflammation and EC dysfunction in SCD. Furthermore, there is a kindling of coagulation activation in patients with SCD. We, and others, have reported elevated levels of thrombin and monocyte tissue factor (TF) expression in patients. Both thrombin and monocyte TF expression increase during acute clinical events. In addition to the chronic impairment of lung function, acute chest syndrome (ACS) adds further insult to lung and cardiovascular impairment. In fact, ACS is the leading cause of sudden death in patients with SCD. Although there are multiple etiologies for ACS, infection/sepsis and the dramatic innate immune and coagulation response to it remain a major contributor to morbidity and mortality during ACS. Novel methods to reduce the inflammatory response during infection are needed as are methods that normalize the chronic pro-inflammatory state. Chaperone proteins, namely HSP90 and HSP70, are known agents that participate in inflammation and thus have significant potential to influence the inflammatory, pro-coagulant burden. Therefore, in this study, we wanted to evaluate the novel anti-inflammatory, anti-coagulatory properties of the chaperone proteins in SCD. We had previously determined that inhibition of HSP90 using the drug AUY-922 could block the bacterial toxin lipopolysaccharide (LPS) - induced TF expression and pro-inflammatory cytokine release from monocytes. Therefore, we used the Townes mouse model of SCD to evaluate AUY-922 in a pre-clinical study. Townes mice with SCD or without were administered AUY-922 intraperitoneal (IP) for 4 days prior to a 6 hour LPS-mediated induction of the inflammatory response and coagulation activation. Notably, the dose of LPS failed to induce any pro-inflammatory response in the AA mice (n=24). However, LPS-induced an exaggerated response in the SS mice. Levels of TNF-α, IL-6, IL-8, and IL-10 were elevated up to 40,000 fold over control treated SS mice. Pre-treatment with AUY-922 either completely ablated, or significantly attenuated the inflammatory cytokine response and normalized EC function. Furthermore, the treatment with AUY-922 doubled the amount of the anti-inflammatory chaperone molecule HSP70 in the livers of the SS mice. This particular result suggested that the function of HSP90 could be spared, and the induction of HSP70 was potentially sufficient to protect against the LPS-induced insult.

Of note, the main function of HSP70 is cytoprotection in response to oxidative and febrile stress. Therefore, we next sought to determine, in a proof of principle in vitro study, whether induction of HSP70 alone was sufficient to block LPS-induced cytokine release and coagulation activation. We treated human monocytes with the HSP70 inducer, celastrol for 24h, followed by treatment with LPS (1µg/ml). We observed a significant release of the cytokines IL-6 and TNF-α with LPS treatment. However, induction of HSP70 via celastrol was sufficient to block this inflammatory response. Furthermore, we observed that celastrol blocked the LPS-induced, TF-specific clotting of plasma in vitro. Interestingly, we also observed that conditioned media from celastrol treated monocytes could block LPS-induced IL-6 release in an HSP70 dependent manner. Thus, secreted HSP70 was an active participant in cellular protection from LPS-induced insult. Initial studies suggest that secreted HSP70 levels may be lower in patients with SCD than in unaffected individuals. Therefore, replacement of this chaperone may be of significant benefit as therapeutic. Thus, taken together, our data demonstrate in both a pre-clinical and an in vitro proof of principle study, that the chaperone proteins HSP90 and HSP70 are attractive targets at reducing the inflammatory burden and associated acute lung injury in SCD.