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Unlabelled: Chronic pain is a major clinical problem that is difficult to treat and requires novel therapies. Although most pain therapies primarily target neurons, neuroinflammatory processes characterized by spinal cord and dorsal root ganglion production of proinflammatory cytokines play an important role in persistent pain states and represent potential therapeutic targets. Anti-inflammatory cytokines are attractive candidates to regulate aberrant neuroinflammatory processes, but the therapeutic potential of these cytokines as stand-alone drugs is limited. Their optimal function requires concerted actions with other regulatory cytokines, and their relatively small size causes rapid clearance. To overcome these limitations, we developed a fusion protein of the anti-inflammatory cytokines interleukin 4 (IL4) and IL10. The IL4-10 fusion protein is a 70 kDa glycosylated dimeric protein that retains the functional activity of both cytokine moieties. Intrathecal administration of IL4-10 dose-dependently inhibited persistent inflammatory pain in mice: three IL4-10 injections induced full resolution of inflammatory pain in two different mouse models of persistent inflammatory pain. Both cytokine moieties were required for optimal effects. The IL4-10 fusion protein was more effective than the individual cytokines or IL4 plus IL10 combination therapy and also inhibited allodynia in a mouse model of neuropathic pain. Mechanistically, IL4-10 inhibited the activity of glial cells and reduced spinal cord and dorsal root ganglion cytokine levels without affecting paw inflammation. In conclusion, we developed a novel fusion protein with improved efficacy to treat pain, compared with wild-type anti-inflammatory cytokines. The IL4-10 fusion protein has potential as a treatment for persistent inflammatory pain., Significance Statement: The treatment of chronic pain is a major clinical and societal challenge. Current therapies to treat persistent pain states are limited and often cause major side effects. Therefore, novel analgesic treatments are urgently needed. In search of a novel drug to treat chronic pain, we developed a fusion protein consisting of two prototypic regulatory cytokines, interleukin 4 (IL4) and IL10. The work presented in this manuscript shows that this IL4-10 fusion protein overcomes some major therapeutic limitations of pain treatment with individual cytokines. The IL4-10 fusion protein induces full resolution of persistent inflammatory pain in two different mouse models. These novel findings are significant, as they highlight the IL4-10 fusion protein as a long-needed potential new drug to stop persistent pain states., (Copyright © 2016 the authors 0270-6474/16/367353-11$15.00/0.)

Hyperexcitability of peripheral nociceptive pathways is often associated with inflammation and is an important mechanism underlying inflammatory pain. Here we describe a completely novel mechanism via which nociceptor G-protein-coupled receptor kinase 2 (GRK2) contributes to regulation of inflammatory hyperalgesia. We show that nociceptor GRK2 is downregulated during inflammation. In addition, we show for the first time that prostaglandin E2 (PGE2)-induced hyperalgesia is prolonged from <6 h in wild-type (WT) mice to 3 d in mice with low GRK2 in Nav1.8+ nociceptors (SNS-GRK2+/- mice). This prolongation of PGE2 hyperalgesia in SNS-GRK2+/- mice does not depend on changes in the sensitivity of the prostaglandin receptors because prolonged hyperalgesia also developed in response to 8-Br-cAMP. PGE2 or cAMP-induced hyperalgesia in WT mice is PKA dependent. However, PKA activity is not required for hyperalgesia in SNS-GRK2+/- mice. SNS-GRK2+/- mice developed prolonged hyperalgesia in response to the Exchange proteins directly activated by cAMP (Epac) activator 8-pCPT-2'-O-Me-cAMP (8-pCPT). Coimmunoprecipitation experiments showed that GRK2 binds to Epac1. In vitro, GRK2 deficiency increased 8-pCPT-induced activation of the downstream effector of Epac, Rap1, and extracellular signal-regulated kinase (ERK). In vivo, inhibition of MEK1 or PKCε prevented prolonged PGE2, 8-Br-cAMP, and 8-pCPT hyperalgesia in SNS-GRK2+/- mice. In conclusion, we discovered GRK2 as a novel Epac1-interacting protein. A reduction in the cellular level of GRK2 enhances activation of the Epac-Rap1 pathway. In vivo, low nociceptor GRK2 leads to prolonged inflammatory hyperalgesia via biased cAMP signaling from PKA to Epac-Rap1, ERK/PKCε pathways.

Chronic pain associated with inflammation is a common clinical problem, and the underlying mechanisms have only begun to be unraveled. GRK2 regulates cellular signaling by promoting G-protein-coupled receptor (GPCR) desensitization and direct interaction with downstream kinases including p38. The aim of this study was to determine the contribution of GRK2 to regulation of inflammatory pain and to unravel the underlying mechanism. GRK2(+/-) mice with an approximately 50% reduction in GRK2 developed increased and markedly prolonged thermal hyperalgesia and mechanical allodynia after carrageenan-induced paw inflammation or after intraplantar injection of the GPCR-binding chemokine CCL3. The effect of reduced GRK2 in specific cells was investigated using Cre-Lox technology. Carrageenan- or CCL3-induced hyperalgesia was increased but not prolonged in mice with decreased GRK2 only in Na(v)1.8 nociceptors. In vitro, reduced neuronal GRK2 enhanced CCL3-induced TRPV1 sensitization. In vivo, CCL3-induced acute hyperalgesia in GRK2(+/-) mice was mediated via TRPV1. Reduced GRK2 in microglia/monocytes only was required and sufficient to transform acute carrageenan- or CCL3-induced hyperalgesia into chronic hyperalgesia. Chronic hyperalgesia in GRK2(+/-) mice was associated with ongoing microglial activation and increased phospho-p38 and tumor necrosis factor alpha (TNF-alpha) in the spinal cord. Inhibition of spinal cord microglial, p38, or TNF-alpha activity by intrathecal administration of specific inhibitors reversed ongoing hyperalgesia in GRK2(+/-) mice. Microglia/macrophage GRK2 expression was reduced in the lumbar ipsilateral spinal cord during neuropathic pain, underlining the pathophysiological relevance of microglial GRK2. Thus, we identified completely novel cell-specific roles of GRK2 in regulating acute and chronic inflammatory hyperalgesia.