Regulation of Elastase By SerpinA3N Contributes to Pain in Sickle Cell Disease

Abstract

Abstract SerpinA3N, a serine protease inhibitor, has been shown to ameliorate neuropathic pain by inhibiting leukocyte elastase activity (Vicuña et al., Nature Med 2015, 21:518-523). Patients with sickle cell disease (SCD) have activated leukocytes and experience neuropathic pain. We observed that SerpinA3N protein expression is significantly reduced in the dorsal root ganglion (DRG) of 5 month old homozygous HbSS-BERK sickle mice, compared to age-matched HbAA-BERK control mice expressing normal human hemoglobin A (p < 0.05). Since HbSS-BERK sickle mice demonstrate tonic hyperalgesia (Kohli et al., Blood 2010, 116:456-465), we hypothesize that decreased expression of SerpinA3N contributes to hyperalgesia by increasing elastase activity in these mice. We examined our hypothesis by using both genders of HbSS-BERK sickle and HbAA-BERK control mice, treated intravenously with and without sivelestat, a small molecule inhibitor of elastase. Mechanical, thermal and musculoskeletal/deep tissue hyperalgesia were analyzed before (considered baseline, BL) and after treatments. Elastase activity was determined using a fluorescent elastase substrate MeOSuc-AAPV-AMC (EMD Milipore). We observed that elastase activity is significantly increased in the DRG and lungs of male and female sickle mice compared to gender and age-matched control mice (p < 0.05 and < 0.005 for female and male DRG, respectively; p < 0.001 and < 0.05 for female and male lungs, respectively). Thus, decreased SerpinA3N expression perhaps contributes to increased elastase activity in sickle mice. Treatment of sickle mice with a single dose of 2 mg/Kg sivelestat led to a significant decrease in mechanical, heat and cold hyperalgesia for up to 24 hours compared to BL (p < 0.05), but no effect was noted in deep tissue hyperalgesia. Increasing the single dose to 4.5 mg/Kg led to a significant decrease in mechanical and thermal as well as deep tissue hyperalgesia for up to 24 hours in both genders compared to BL or saline treated mice (p < 0.05). Continuation of treatment with sivelestat 4.5 mg/Kg/day for 3 days led to significantly reduced hyperalgesia for up to 6 days in both genders of mice. These findings suggest that elastase activity contributes to nociceptive mechanisms, since hyperalgesia remained reduced for up to 3 days even after discontinuation of sivelestat. Reduced hyperalgesia following 3 days of sivelestat (4.5 mg/Kg/day) treatment was accompanied by a significant decrease in elastase activity in the DRG (p < 0.005) and lungs (p < 0.05) of both genders of sickle mice compared to saline treatment, but no histopathological changes were observed in lungs, kidney, liver, spleen and heart. Activating transcription factor 3 (ATF3), a marker of neuropathic pain, is elevated in the DRG of sickle as compared to control mice (Vincent et al., Blood 2013, 122:1853-1862). Sivelestat treatment significantly reduced ATF3-immunoreactivity compared to saline treatment in the DRG of sickle mice (p < 0.05), suggesting a decrease in neuronal injury which underlies neuropathic pain. None of the treatments had any effect on control mice. Sivelestat treatment showed no significant effect on serum tryptase or cutaneous mast cell degranulation in sickle or control mice. Thus, sivelestat specifically inhibits elastase activity. However, both elastase and tryptase activate protease-activated receptor-2 (PAR2) in the peripheral nervous system, stimulating the release of pro-nociceptive neuropeptides and activation of transient receptor potential vanilloid (TRPV) channels, leading to hyperalgesia. Since PAR-2 and TRPV channels are activated in sickle mice, it is likely that sivelestat treatment has an inhibitory effect on these mechanisms. It has been suggested that leukocytes, including activated neutrophils, contribute to acute lung injury (a common complication of SCD) and the pathogenesis of SCD (Zhang et al., Blood 2016, 127:801-809). Our observations that decreased SerpinA3N protein expression leads to increased elastase activity in DRG and lung in sickle mice suggest that this mechanism may contribute to sickle pathobiology and pain. In Japan, sivelestat is already approved for treatment of acute lung injury. Therefore, pharmacological inhibition of elastase may offer the advantage of treating chronic pain while concomitantly ameliorating one of the pathogenic mechanisms of SCD. Disclosures No relevant conflicts of interest to declare.