Critical Role of Protein Arginine Deiminase 4 in Mast Cell Extracellular Trap Formation Leading to Neuropathic Pain in Sickle Mice

Extracellular trap (ET) formation primarily meant to ensnare the pathogens, is also involved in sterile inflammation, platelet activation, impaired wound healing, oxidative stress, organ damage, etc (Jorch and Kubes, reviewed in Nat Med 2017). We have identified a novel role of mast cell ETs (MCETs) in contributing to neuropathic pain via Protein Arginine Deiminase 4 (PAD4), an enzyme that catalyzes chromatin decondensation in the nucleus by converting arginine to citrulline on histones. We used homozygous BERK sickle (HbSS) mice expressing human sickle hemoglobin (Hb) and control BERK (HbAA) mice expressing normal human HbA, and mast cells isolated from their dorsal skin. MCETs were visualized by laser scanning confocal microscopy in 100 μm thick dorsal skin sections and in mast cells in vitro with SYTO13 and SYTOX Orange (Invitrogen) and citrullinated histone antibodies (Abcam); and co-localized in the skin using mast cell-specific c-kit, FcεR1 and tryptase, nerve-fiber-specific anti-NF200 and anti-PGP9.5 and anti-CD31 for vasculature.

We observed vibrant clusters of trap-shooting mast cells surrounding the vasculature and nerve fibers in the skin of sickle mice. The exuberant spread of MCETs and tryptase permeated the vessel lumen and may contribute to observed increase in circulating tryptase, DNA and endothelial activation in sickle mice. Notably, large aggregates of MCETs and mast cells appeared occluding blood vessels, and tightly surrounded the axonal nerve fibers demonstrating signs of injury and axonal sprouting, features underlying neuropathic pain. We stimulated mast cells in vitro with 40µM hemin for 2h after 2-hour priming with 1ng/ml TNFα to create a sickle microenvironment in vitro . SYTO13 and citrullinated histone staining showed that nearly all the cells from sickle but fewer cells from control mice showed MCET formation with hemin after priming with TNFα. Tubulin staining showed contraction and extension into long fibers in hemin/TNFα treated sickle mast cells, suggesting that ongoing hemolysis releasing free-heme in an inflammatory mileau in SCD may contribute to MCETosis observed in the skin of sickle mice. Transcripts of TLR4 and FcεR1 are significantly elevated in sickle compared to control mast cells (Vincent et al., Blood 2013). Silencing of TLR4 and/or FcεR1 did not inhibit hemin/TNFα-induced MCET formation completely. However, inhibition of PAD4 with 10µM GSK484 (Cayman) completely abrogated hemin/TNFα-induced MCET formation. Several signaling pathways including PKB/Akt, MAPK/ERK, p38MAPK and Syk were constitutively activated in sickle mast cells. Collectively, these in vitro observations suggest that multiple receptors and pathways converge at the downstream activation of PAD4 leading to MCETosis.

Since MCETs incited direct peripheral nerve injury, we examined if inhibition of MCETs in vivo using PAD4 inhibitor would attenuate pain. Sickle and control mice were treated subcutaneously with PAD4 inhibitor GSK484 (20 mg/kg/day) or 0.9% saline for 30 days. GSK484 significantly reduced cold and heat hyperalgesia after 48h (p=.0066) and 72h (p=.0002), respectively, but reduced mechanical hyperalgesia significantly after 1 week (p=.0036) of treatment Vs baseline before treatment. Attenuation of hyperalgesia remained effective until 30 days, last day of treatment and observation, suggesting that mice do not develop analgesic tolerance. Mast cell degranulation was decreased by 90% in the skin (p=.0004); white blood cells and serum GM-CSF were significantly reduced (p= .010 and <.0001, respectively), but the hematocrit was significantly increased (p=.035) in sickle mice treated with GSK484 Vs saline. Skin secretagogue of GSK484 treated sickle mice showed about 50% reduction in inflammatory cytokines CCL2/MCP-1, TNFα and RANTES (p=.0229, .0154 and .0290, respectively Vs saline treated mice).

Together, these data demonstrate an anti-inflammatory effect of GSK484 in the periphery and attenuation of neuropathic pain. Thus, mast cell degranulation and MCETosis contribute to inflammation and neurovascular injury leading to inflammatory as well as neuropathic pain. We speculate that MCETosis may also contribute to vascular dysfunction and organ damage in SCD. Thus, PAD4 inhibition provides a potential therapeutic target to attenuate the onset and progression of pain and perhaps other pathological consequences of SCD.