PROJECT SUMMARY/ABSTRACT Chronic pain and the addictive effects of opioids used to control pain are major health problems affecting millions of Americans. Validation of novel targets for the safe treatment of chronic pain is urgently needed. We identified peptidase inhibitor 16 (PI16) as a novel regulator of chronic pain in an unbiased RNA seq screen. PI16 is a putative peptidase inhibitor that has not been studied in the context of pain. We showed that male and female Pi16-/- mice are protected against mechanical allodynia in the spared nerve injury (SNI) and paclitaxel models of neuropathic pain. Along the neuraxis, PI16 is only detectable in fibroblasts around peripheral nerves (perineurium), and in the meninges of dorsal root ganglia (DRG), spinal cord, and brain, but not in neurons, glia or leukocytes. . PI16 levels in perineurial and DRG meningeal fibroblasts increase during neuropathic pain. The overall objective of this project is to validate PI16 as a novel target for the treatment of chronic pain using mouse models and human tissues of neuropathy patients and controls and to identify the underlying mechanisms. Our central hypothesis is that increased PI16 secretion by DRG meningeal and perineurial fibroblasts promotes chronic pain by increasing blood nerve barrier (BNB) permeability and leukocyte trafficking into nerve and DRG. The significance is in the validation of PI16 as a novel, potentially druggable, regulator of chronic pain and the discovery of fibroblasts as key regulators of chronic pain. We propose the following three specific aims: 1. Validate the key role of PI16 in chronic pain. In two independent laboratories, we will test the hypothesis that genetic deletion of Pi16 in male and female mice protects against chronic pain in well-established models of chronic pain. 2. Investigate expression and regulation of PI16 in fibroblasts around mouse DRG and mouse and human peripheral nerves. The hypothesis is that PI16 production by fibroblasts in DRG meninges and perineurium is increased via a TGFβ− and Epac1-mediated pathway to promote chronic pain. We will use tissues from the neuraxis in mouse pain model and nerves from humans with neuropathic pain. 3. Determine the contribution of PI16 to blood nerve barrier integrity in mouse pain models and in a human in vitro model. Using a model of the human BNB, we will test the hypothesis that PI16 increases BNB permeability and leukocyte transmigration. Using co-immuno-precipitation and mass spectrometry we will identify novel PI16 targets. This proposal is conceptually innovative because successful completion will validate PI16 as a novel key regulator of neuropathic and inflammatory pain and establish a novel role of perineurial fibroblasts in regulating BNB function with major consequences for chronic pain. The expected findings are significant because they will advance our fundamental understanding of cellular and molecular mechanisms that contribute to chronic pain and will identif...