ALBERT

Description: The Journal of Organic Chemistry DOI: 10.1021/jo301572p

Description: Sickle cell disease (SCD) is associated with increased activity of proteases including tryptase and elastase, which can contribute to a wide variety of pathologies including endothelial activation and injury to the neural system. In turn, these effects of proteases contribute to pain, a major comorbidity of SCD (Vincent et al., Blood 2013; Vicuña et al., Nature Med 2015). The enzyme activity of proteases released from granulocytes including neutrophils is regulated by serine protease inhibitors (Serpins). Transcriptomic analysis revealed downregulation of Serpins in the dorsal root ganglion (DRG) of sickle mice (Paul et al., Sci Data 2017). Increased neutrophil activation has been observed in patients with SCD and in mouse models of SCD (Zhang et al., Blood. 2016). Therefore, we hypothesized that increased neutrophil elastase activity contributes to pain in SCD and that a Serpin, a1 anti-trypsin (A1AT) would inhibit elastase activity and reduce pain in SCD. We used transgenic homozygous HbSS-BERK (sickle) and HbAA-BERK (control) mice expressing human sickle hemoglobin and normal human hemoglobin A, respectively, and Prolastin C (Grifols) an A1AT, a Food and Drug Administration (FDA) approved drug, to examine our hypothesis. We observed a significant increase in elastase activity in the plasma, lungs and DRG of sickle mice compared to control mice (p

Description: Sickle cell disease (SCD) is characterized by chronic hemolysis, inflammation, vascular dysfunction, and pain. Earlier we showed that mast cell activation contributes to neuroinflammation and pain and is accompanied by increased toll-like receptor 4 (TLR4) expression on mast cells (Vincent et al., Blood 2013), and that genetic deletion of TLR4 ameliorates neurogenic inflammation and hyperalgesia in HbSS-BERK sickle mice. Several other studies have shown increased TLR4 expression in peripheral system and its involvement in sickle pathobiology. We propose that free heme, due to hemolysis, activates TLR4 in the central nervous system in addition to peripheral activation, which further exacerbates neuroinflammation and hyperalgesia. Spinal cords of HbSS-BERK sickle mice show 3-fold mRNA transcripts for TLR4 and a 2-fold increase in hemin as compared to the spinal cords of HbAA-BERK control mice. Therefore, targeting TLR4 with pharmacological inhibitors may provide a therapeutic approach to attenuate peripheral and central inflammation and hyperalgesia. In the present study we examined the potential of pharmacological inhibition of mast cell activation, neuroinflammation and hyperalgesia in HbSS-BERK sickle mice with TLR4 inhibitor, TAK242. Sickle mice were administrated intravenously with TLR4 inhibitor TAK242 (1 mg/kg body weight/day) for 5 days. Sensory testing was performed at baseline at recruitment and periodically during the 5-day treatment and for another 8 days after concluding the treatment to evaluate mechanical hyperalgesia with von Frey filaments, thermal hyperalgesia in response to heat/cold and grip force for musculoskeletal/deep tissue hyperalgesia. Following the 5-day treatment with TAK242, release of cytokines, tryptase (marker of mast cell activation) and substance P released from skin biopsies and spinal cords were analyzed by ELISA. TAK242 significantly decreased the release of tryptase (TAK242: 5.178 ± 0.7613 pg/ml vs vehicle: 8.801 ± 0.9403 pg/ml, p = 0.0181), substance P (TAK242: 11.56 ± 1.945 pg/ml vs vehicle: 25.51 ± 4.283 pg/ml, p = 0.018), and IL-6 (TAK242: 15.59 ± 0.4541 pg/ml vs vehicle: 29.74 ± 0.8249 pg/ml, p = 0.0045) from skin biopsies, suggesting that TAK242 reduced SCD-induced mast cell activation and inflammation. TAK242 also significantly decreased substance P (TAK242: 0.7198 ± 0.0587 pg/mg vs vehicle: 0.931 ± 0.0676 pg/mg, p = 0.0462) and phosphorylation of p38/MAPK (p = 0.0184) in the spinal cord, as well as dorsal cutaneous blood flow (TAK242: 6.392 ± 0.3857 PU vs vehicle: 12.32 ± 0.5575 PU, p 〈 0.0001), indicating that TAK242 ameliorated SCD-evoked central and peripheral activation of inflammation and nociceptive mechanisms. Furthermore, TAK242 administration gradually reduced the mechanical, deep tissue, and thermal hyperalgesia upto 5-day treatment (p 〈 0.01, vs vehicle HbSS). However, discontinuation of treatment led to a gradual increase in hyperalgesia observed upto day-8 post-treatment. TAK242 also significantly decreased acute pain induced by hypoxia/reoxygenation and accelerated recovery from injury of hypoxia/reoxygenation. These data reveal the significant therapeutic effect of pharmacological inhibition of TLR4 on inflammation and hyperalgesia in sickle mice. Therapies targeting TLR4 inhibition may be potentially beneficial in ameliorating sickle pathobiology and pain. Disclosures No relevant conflicts of interest to declare.

Description: Sickle cell disease (SCD) is associated with pain, which remains a major challenge to treat. Earlier, we showed that peripheral mechanisms including mast cell activation in the skin contribute to pain in sickle mice (Vincent et al., Blood 2013). Mast cell activation in sickle mice was accompanied by a significant increase in toll-like receptor 4 (TLR4) as compared to mast cells from control mice. Since peripheral as well as central mechanisms are involved in nociception, we examined the central mechanisms underlying pain in SCD. TLR4 signaling is involved in inflammatory and neuropathic pain (Wang et al., FASEB 2013 and Hutchinson et al., Eur J Neurosci 2008). Microglial cells, the “macrophages” of the central nervous system in the spinal cord are critically involved in the development and maintenance of pain. Binding of an endogenous ligand to TLR4 is an important step in the regulation of microglial activity in pain facilitation. We hypothesized that heme, released during hemolysis in SCD, is a ligand for TLR4 expressed on spinal microglia. Methods. We isolated microglial cells from the spinal cords of HbSS-BERK (sickle) and HbAA-BERK (control) mice. To assess mitochondrial activity, we analyzed reactive oxygen species (ROS) and ATP, since increased ROS and decreased ATP are suggestive of mitochondrial dysfunction, which in turn is influenced by endoplasmic reticulum (ER) stress. ROS in the microglial cells was determined by utilizing the cell permeable reagent 2’,7’-dichlorofluorescein, which is oxidized by ROS to form a fluorescent compound, with the max excitation and emission spectra of 495 nm and 529 nm, respectively. ATP production was measured by a luminescence based assay from PerkinElmer (ATPlite). Results. Stimulation of microglia from control and sickle mice with hemin in vitro led to a several-fold increase in TLR4 gene transcripts in a time-dependent manner. Additionally, hemin induced the production of proinflammatory cytokines, TNF-α and IL-6, and ROS compared to vehicle-treated microglial cells from both sickle and control mice (p

Description: Mast cell activation in the skin contributes to inflammation, neurogenic inflammation and hyperalgesia in sickle mice (Vincent et al., Blood 2013). However, the underlying cause of mast cell activation and challenges in inhibiting mast cell activation remain unclear. Hemolysis in sickle cell disease leads to excess free heme, which contributes to endothelial activation and neutrophil extracellular trap (NET) formation. We therefore hypothesized that free heme activates mast cells and extracellular trap formation in a sickle microenvironment. We examined hemin-induced activation in mast cells from the skin of HbAA-BERK control and HbSS-BERK sickle mice. Mast cells in culture were incubated with vehicle, or I ng/ml TNF-α for 4 hours, or 40 μM hemin for 2 hours, or with hemin for 2 hours after 2 hours of priming with TNF-α to create an inflammatory sickle microenvironment. Cells were stained with cell impermeable DNA dye SYTOX orange and cell permeable dye SYTO13 to visualize the extracellular DNA fibers. Incubation with TNF-α or hemin did not show significant eruption of DNA from the cell body. However, sickle mast cells incubated with both TNF-α and hemin showed distinct DNA containing fibers exploding from the cell body with the appearance of spider web like formation similar to NETs. The length of majority of individual fibers was more than 50 μm stretching to more than 500 μM, indicative of TRAP formation. This response was blunted in mast cells from control mice. Thus, mast cells in a sickle microenvironment are primed and activated, and further priming with cytokines such as TNF-α leads to hemin-induced mast cell extracellular trap (MET) formation. Sickle mast cells express significantly higher TLR4 and FcεRI as compared to control mice (Vincent et al., Blood 2013). Therefore, we examined if silencing of FcεRI and/or inhibition of TLR4 attenuated TNF-α primed/hemin-induced MET formation. TAK242 at a dose of 1 μM but not at 0.5 μM inhibited TNF-α primed/hemin-induced MET formation, but silencing of FcεRI had no effect. Since cannabinoids, imatinib, palmitoylethanolamide (PEA) and cromolyn are known mast cell inhibitors, we examined their effect on TNF-α/hemin-induced METs. Imatinib (30 - 100 μM), a known mast cell inhibitor, showed no significant effect, while cromolyn (100 μM) led to a modest decrease in MET formation. Cannabinoid CP 55,940 and PEA at a relatively low dose of 30 μM completely blocked MET formation. Functionally, imatinib, PEA and CP 55,940 reduced the release of cytokines TNF-α and RANTES from mast cells incubated with TNF-α/hemin for 24 hours. Hemin in the presence of TNF-α stimulated sustained activation of NLRP3 inflammasome signaling. Thus, mast cells in a sickle microenvironment are activated due to a rich inflammatory cytokine milieu, via activation of the inflammasome signaling upon stimulation with hemin. The unique phenomenon of MET formation is orchestrated by a combined action of inflammatory milieu and hemin, imparting resistance to common mast cell inhibitors such as cromolyn and imatinib. This new phenomenon of MET formation identified by us may in part contribute to challenges of treating sickle cell disease and other mast cell associated pathological conditions. Disclosures No relevant conflicts of interest to declare.

Description: Background: Inflammation, neurogenic inflammation and pain remain challenging to treat in sickle cell disease (SCD). Alternative therapies including acupuncture have been used for centuries to reduce pain and ameliorate underlying pathobiology of many disorders. We examined the mechanisms underlying acupuncture therapy in sickle mice. To prevent the influence of anesthetics and constraint on the pathobiology we developed electroacupuncture (EA) treatment for awake/conscious freely moving mice to simulate treatment conditions in patients, and then examined the peripheral and central mechanisms of neuroinflammation and nociception. Methods: HbSS-BERK sickle and HbAA-BERK control mice were treated with four EA treatments (every 3rd day, frequency: 4 or 10 Hz, pulse width: 100 microsecond, duration: 30 min) at acupoint GB30. Untreated and sham-EA treated (acupuncture without electrical stimulation) were used as controls. Hyperalgesia was evaluated daily by determining mechanical threshold, deep tissue hyperalgesia and thermal hyperalgesia using von Frey filaments, grip force, and cold plate, respectively. Blood and tissues were collected for analysis after four sessions of treatment. Skin biopsies were incubated overnight and culture medium was analyzed for mast cell activation marker tryptase, and neuromodulatory marker substance P. Results: Varied analgesic response to EA treatment was observed in sickle mice. About 86% treated mice equally showed positive (〉50% pain relief) or moderate (20-30% pain relief) response and 14% were non-responsive (

Description: Patients experiencing chronic and/or recurrent pain show a correlative decline in health and lifespan. In turn, declining health and sensory effects may contribute to persistent pain and poor response to analgesia. We therefore hypothesized that dietary alterations to improve health and stress reduction by mating may reduce chronic pain in male sickle mice. Methods. We used male HbSS-BERK (sickle) mice, which show hyperalgesia as compared to and HbAA-BERK (control) mice (Kohli et al., Blood 2010). Breeders and pups up to 12 wks of age were fed Sickle Mouse Diet (59M3, TestDiet) and 18% Protein Rodent Diet (2018, Harlan), thereafter, called ‘sickle’ and ‘rodent’ diet. Sickle diet contained 26.4% Protein, 11.1% fat and 27.5% and 26% kcal/g of each, respectively. Rodent diet contained 18.6% protein and 6.2% fat, and 24% and 18% kcal/g of each, respectively. Vitamins were about 2-fold higher in sickle as compared to rodent diet. Sickle mice showing hyperalgesia were recruited and treated as follows: [A] Rodent diet without mating (RD/M-); [B] Sickle diet with mating (SD/M+); [C] Rodent diet with mating (RD/M+); [D] Sickle diet without mating (SD/M-) and [E] mice on SD and mating for 4 weeks, were deprived of SD and mating and fed RD. Control mice which do not show hyperalgesia were fed RD without mating. Sensory testing was performed at baseline (BL) at recruitment and weekly, to evaluate mechanical hyperalgesia with von Frey filaments, thermal hyperalgesia in response to heat/cold and grip force for musculoskeletal/deep tissue hyperalgesia. Following the treatments, release of cytokines from skin biopsies was analyzed by cytokine arrays as described by us earlier (Vincent et al., Blood 2013), and spinal cords were analyzed for nociceptive signaling. Results. We did not observe difference in the body weight of mice between different groups at any time. White blood cell counts and spleen weight were significantly increased in group E following the withdrawal of SD and mating as compared to group C on SD/M+ (p