Pain and inflammation following tissue damage serve as powerful protective measures to promote rest and enable healing. While researchers have a good grasp of what causes pain and inflammation in the first place after injury, little is known about how these problems are resolved over time.
But now, a new study led by Ru-Rong Ji, Duke University, Durham, US, shows that GPR37, a G protein-coupled receptor (GPCR) expressed on macrophages but not microglia, contributes to the resolution of inflammation and inflammatory pain in mice. When activated, GPR37 stimulates phagocytosis of proinflammatory particles, boosts levels of anti-inflammatory cytokines, and suppresses levels of proinflammatory cytokines. Further, mice lacking GPR37 show deficits in inflammatory pain resolution and macrophage phagocytic activity.
“I really like this work, which comes from a group that is consistently producing novel and exciting data. The identification of a GPCR on macrophages involved in the resolution of inflammation and inflammatory pain is timely and provides some much-needed emphasis on this area,” said Marzia Malcangio, King’s College London, UK, who was not involved in the study.
The paper was published online July 16 in the Journal of Clinical Investigation, along with an accompanying commentary by Lintao Qu and Michael Caterina, Johns Hopkins School of Medicine, Baltimore, US.
Identifying a receptor
The process of inflammation, which includes sensitization of nociceptors, recruitment of immune cells, and the generation of edema, has been extensively studied, but the resolution of inflammation and pain that accompanies tissue healing after injury is relatively less well understood. Specialized proresolving mediators (SPMs), including so-called resolvins, are produced during the resolution phase and serve to dampen inflammation and inflammatory pain (Serhan et al., 2002). Resolvins have potential as analgesics since they suppress inflammatory pain at doses much lower than those needed for pain relief from morphine (Xu et al., 2010).
“One particular family of SPMs known as protectins or neuroprotectins produce potent inhibition of inflammatory pain by acting on multiple cells including neurons, immune cells, and glial cells. All of the known receptors for SPMs are G protein-coupled receptors. However, the receptor for one neuroprotectin in particular, called neuroprotectin D1, or NPD1, was unknown,” explained Ji. “Another important function of resolvins is to promote phagocytosis by macrophages, so we were interested to investigate which receptor mediates this particular effect of neuroprotectin.”
One specific receptor, GPR37, caught the researchers’ interest, as its activation produces analgesic and neuroprotective effects in rodents, but the effects of GPR37 activation on immune cells, if any, were unknown. So first author Sangsu Bang and colleagues began by examining the hind paw skin of wild-type mice for expression of GPR37. Using immunofluorescence staining, they observed strong GPR37 expression that was co-localized with CD68, a macrophage marker.
Expression of GPR37 was high, with 30 percent expression in macrophages in the hind paw skin and 60 percent expression in macrophages in the peritoneum, a thin layer of tissue that forms the inside lining of the abdomen. Interestingly, no GPR37 expression was observed in microglia, which are macrophage-like cells that patrol the central nervous system to regulate neuroinflammation and chronic pain.
Next, by overexpressing GPR37 in culture using a commonly used cell line (HEK293 cells) and measuring intracellular calcium signaling (a surrogate marker of cellular activity) in response to a variety of SPMs, the team found that NPD1 robustly increased calcium levels in the cells. NPD1 also produced increases in calcium signaling in peritoneal macrophages, an effect that was abolished in mice lacking the GPR37 receptor. This provided even stronger evidence that GPR37 serves as the receptor for NPD1.
“Identifying the receptor for NPD1 is an important contribution to the field of inflammation and pain resolution. Several receptors for SPMs had previously been identified and, along with this new one, will allow us to further exploit this field,” according to Malcangio.
GPR37 triggers phagocytosis
Macrophages respond to infections by surveilling and engulfing foreign particles or pathogens, and by regulating the local inflammatory environment via the release of pro- or anti-inflammatory cytokines. In the current study, in vitro, NPD1 stimulated macrophage phagocytosis of particles of zymosan, which is derived from yeast, an effect that was diminished in GPR37 knockout mice. This again provided evidence for a critical role of this receptor in macrophage function.
Delving a little deeper, the team examined the intracellular signaling processes underlying phagocytosis. They discovered that phagocytosis was inhibited by a known blocker of intracellular calcium signaling called BAPTA-AM. And, ionomycin, which boosts calcium levels in cells, markedly increased phagocytosis in both wild-type and GPR37 knockout mice, suggesting that increasing calcium signaling may be sufficient to trigger phagocytosis.
Next, using a model in which zymosan produces inflammation upon injection into the hind paw of mice, the researchers mapped the time course of edema (swelling), infiltration of immune cells, and phagocytosis of zymosan particles. Edema was apparent within two hours of injection, peaked at four hours, and began to decline after that. Immunohistochemistry showed that neutrophils were the first immune cells to enter the inflamed hind paw skin, with peak levels seen at 24 hours after zymosan injection, and a decline at 48 hours. Macrophage infiltration began slightly later, on day one, peaked on day five and began to decline on day eight.
Interestingly, the time course of phagocytosis of the zymosan particles and expression of GPR37 closely resembled that of macrophage infiltration at the site of inflammation. Staining of phagocytized zymosan particles showed that they were predominantly present in macrophages that also expressed GPR37. Further, the phagocytized zymosan particles were also significantly reduced in mice that lacked GPR37. Together, these findings point to a role for GPR37 specifically on macrophages in the resolution of inflammation.
“It’s likely that GPR37 is not the only receptor that, when activated, can trigger phagocytosis by macrophages. Other receptors, such as ChemR23 for resolvin E1, should all have this function of phagocytosis but perhaps through a different signaling mechanism that may be calcium dependent or independent,” said Ji.
Resolution of inflammatory pain
GPR37 on macrophages also contributed to the resolution of inflammatory pain. Following zymosan injection, wild-type mice developed heat and mechanical sensitivity that resolved on days two and five, respectively; this behavior followed a similar time course to that of immune cell infiltration. However, both heat and mechanical sensitivity were prolonged in mice lacking GPR37.
Notably, there was no difference between wild-type and GPR37 knockout mice in the early onset of inflammatory pain in the first several hours after zymosan. This indicated that this receptor regulates the resolution but not the induction of inflammatory pain.
To better understand the specific contribution of macrophages to inflammatory pain caused by zymosan, Ji and colleagues then transplanted macrophages into either wild-type or GPR37-deficient mice (adoptive transfer). Wild-type macrophages rescued the deficits in pain resolution previously observed in the GPR37 knockouts. But adoptive transfer of macrophages lacking GPR37 into knockout mice did not do so. This showed that macrophage GPR37 was playing a critical role in pain resolution.
Finally, the group also saw that zymosan caused both pro- and anti-inflammatory cytokine expression in wild-type mice, but the increased expression of anti-inflammatory cytokines such as interleukin-10 (IL-10) was abolished in the GPR37 knockouts. Lastly, blocking IL-10 signaling with an antibody in the inflamed hind paw skin of wild-type mice impaired the resolution of inflammation and heat as well as mechanical pain sensitivity.
Overall, the results show that GPR37 in macrophages promotes the resolution of inflammation and inflammatory pain in part by promoting the release of IL-10. And, the receptor could be a good drug target, according to Ji.
“We definitely see some potentially viable therapeutic targets in this whole signaling pathway, be it GPR37 or SPMs, perhaps, and exploring these further is a direction in which we want to go,” he told PRF.
Another potential avenue for future exploration, says Malcangio, is to better understand why pain remains even after inflammation has gone down. “Does neuroprotectin D1 or GPR37 play a role in models where pain persists after inflammation has been resolved, such as in rheumatoid arthritis models?”
Dara Bree is a postdoctoral fellow at Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, US.
Image credit: Qu and Caterina, 2018
