Association of Mu Opioid Receptor with Neurogenic Inflammation in Sickle Mic
Authors: Luk Kathryn , Nguyen Julia , Vang Derek , Taiwo Oludare B , Kohli Divyanshoo , Ericson Marna , Ansonoff Michael A , Pintar John E , Hebbel Robert P , Gupta Kalpna 
Published: October 2019

Abstract
Abstract 895
Sickle cell disease (SCD) is accompanied by chronic inflammation and severe pain for which available therapies are unsatisfactory. We found that pain was accompanied by increased peripheral expression of pro-inflammatory and vasoactive neuropeptides, substance P (SP) and calcitonin gene related peptide (CGRP), and decreased expression of mu opioid receptor (MOR) in the skin of BERK mice expressing sickle hemoglobin (HbSS) compared to control HbA-BERK mice expressing normal human hemoglobin (Kohli et al., Blood 2010). Neuropeptide modulations are key features of neurogenic inflammation in chronic pain states. Tryptase released from mast cells is known to activate the release of neuropeptides from peripheral nerve endings, which in turn act on target cells including mast cells, inflammatory cells, endothelial cells and smooth muscle cells, resulting in increased vascular flow, inflammation and plasma extravasation collectively termed neurogenic inflammation. MOR influences neuropeptide release and perhaps modulates neurogenic inflammation. We hypothesize that neurogenic inflammation contributes to chronic pain and inflammation in SCD. We first examined the quantitative expression of MOR-, SP-, CGRP- and tryptase-immunoreactivity (ir) using laser scanning confocal microscopy (LSCM) in thick skin sections of BERK sickle and HbA-BERK control mice. Significantly decreased MOR-ir (p<0.001), but increased SP-, CGRP- (p<0.01 for both) and tryptase-ir (p<0.001) was observed in the skin of BERK as compared to HbA-BERK. Decreased MOR-ir co-localized with epidermal and dermal layers in the skin of BERK, but blood vessels showed robust MOR-ir in BERK similar to HbA-BERK. Tryptase-ir was more pronounced in the epidermal layer, indicative of its proximity to peripheral nerve endings. Increased tryptase co-localized with CD117/c-kit, confirming the mast cell origin. Next, we analyzed if MOR downregulation contributes to these alterations, by using mice with deletion of mu-, delta- and kappa-opioid receptors (MOR, DOR and KOR, respectively) and wild type (WT), 129S6 mice. SP-, CGRP- and tryptase-ir were increased about 2-fold in the skin of MOR, DOR and KOR knockout as compared to WT mice, suggesting that downregulation of MOR in sickle mice may contribute to increased neuropeptide and tryptase expression. Consistent with an increase in cutaneous neuropeptides, BERK and MOR knockout mice showed ∼2.5- and ∼1.5-fold increase in dorsal skin blood flow using Laser Doppler blood perfusion monitor, as compared to HBA-BERK and WT control mice, respectively. Thermography measurements revealed a correlative increase in temperature in dorsal skin of BERK as compared to HbA-BERK (p<0.05), indicative of increased inflammation. These neurochemical and physiological alterations in the skin were accompanied by activation of dorsal root ganglion (DRG) neuron and glial cells. LSCM of whole DRG revealed increased-ir for activating transcription factor-3 (ATF3), a marker of neuropathic and inflammatory insult; and increased glial fibrillary acidic protein (GFAP)-ir, a marker of inflammation, in BERK relative to HbA-BERK mice. It is likely that chronic pain and ischemia-reperfusion injury in SCD activates ATF3 in DRG leading to the release of mast cell tryptase followed by release of neuropeptides in the DRG and their ‘antidromic' release from the nociceptive C-fibers in the skin. In addition, recurrent/chronic pain and inflammation in SCD may cause peripheral nociceptor sensitization mediated by mast cell-tryptase, and neuropeptide release from nerve endings. Nociceptor sensitization together with decrease in MOR, in turn may activate mast cell tryptase, promoting a vicious cycle between components of central nervous system and inflammation that may exacerbate neurogenic inflammation in SCD. It is likely that the pro-inflammatory neuropeptide-enhanced microenvironment has a compounding effect on inflammation and pain in SCD. We speculate that therapies targeting neuropeptides and their release mechanism(s) may potentiate the effect of analgesics and anti-inflammatory therapies for treating pain in SCD.

Disclosures:
No relevant conflicts of interest to declare.