This is Part 2 of a two-part report on selected talks from the 33rd Annual Scientific Meeting of the American Pain Society (APS), held April 30-May 3, 2014 in Tampa, Florida, US. See also Part 1.
Inflammatory pain: novel mechanisms and mediators
Yuriy Usachev, University of Iowa, Iowa City, US, is examining the role of the complement system, a key component of innate immunity, in inflammatory pain. The idea that the complement system could be important in pain signaling derives support from microarray gene expression studies of pain in rodents in which several complement genes surfaced as statistically significant “hits” (Lacroix-Fralish et al., 2011). A notable recent study implicated C1q, a key molecule in the complement cascade, in persistent inflammatory pain signaling in mice (see PRF related news story).
Usachev is interested in another complement molecule, the proinflammatory complement fragment C5a. Previous research showed that blocking the C5a receptor with an antagonist reduced mechanical allodynia, edema, and skin cytokine levels in the mouse hindpaw incision model (Clark et al., 2006). At the meeting, Usachev reported unpublished data showing that complete Freund’s adjuvant (CFA)-induced thermal hyperalgesia was significantly reduced in C5a receptor knockout mice. Administration of C5a receptor antagonists also decreased CFA-induced hyperalgesia. Meanwhile, intraplantar injection of C5a itself resulted in thermal and mechanical hyperalgesia that could be reversed by a TRPV1 antagonist or by TRPV1 deletion, suggesting that the ion channel may mediate the effects of C5a on pain.
To more precisely delineate the signaling mechanisms by which C5a contributes to inflammatory pain, Usachev has turned his focus to macrophages. Macrophages in skin tissue express the C5a receptor, and macrophage cell lines respond to C5a with calcium transients. Thermal and mechanical hyperalgesia induced by C5a were eliminated in transgenic mice lacking macrophages in the periphery, further implicating these white blood cells in C5a-induced pain hypersensitivity.
Could macrophages communicate with neurons via C5a signaling to cause pain, and if so, how? Here Usachev pointed to nerve growth factor (NGF), which macrophages express. NGF levels in the skin are elevated after C5a injection, and Usachev’s group has found that an NGF-neutralizing antibody reduced C5a-induced thermal hyperalgesia, as did an NGF receptor (Trk) inhibitor.
Based on these preliminary findings, Usachev is proposing a model in which C5a binding to its receptor on macrophages increases NGF release from macrophages, which then sensitizes TRPV1 on neurons, leading to pain sensitivity. Usachev’s work thus provides further evidence that the role of immune system components such as complement factors extends far beyond the immune system into pain signaling, too.
David Clark, Stanford University School of Medicine, Stanford, US, presented his group’s work on the role of epigenetic mechanisms and mediators in inflammatory pain. This research has important clinical relevance for postoperative pain, a significant problem for many patients, especially for those on chronic opioid treatment.
Enzymatic modification of histones, the proteins that package DNA, by histone acetyltransferases (HATs) and histone deacetylases (HDACs) is one key epigenetic mechanism that may contribute to pain states (for a review, see Crow et al., 2013). Clark previously reported that treatment of mice with the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) worsened mechanical hypersensitivity in that model, but did not affect thermal sensitivity (Sun et al., 2013). Conversely, administration of the HAT inhibitor anacardic acid ameliorated mechanical hypersensitivity, also without impacting thermal sensitivity. Using an incision model of hyperalgesic priming, Clark also found that treatment with the HAT inhibitor around the time of incision partially blocked hyperalgesia when the animals were challenged two weeks later with prostaglandin E2, whereas the HDAC inhibitor had no effect.
The results suggested that incision increased histone acetylation levels to cause pain, and further experiments looking at spinal cord tissue after incision indeed revealed increased acetylation of an important histone subtype, H3K9, in dorsal horn neurons; SAHA treatment further increased levels of acetylated H3K9. Clark and colleagues further found that incision increased chemokine signaling involving CXC chemokine receptor 2 (CXCR2) and its ligand, CXCL1, a pathway known to play a role in pain and inflammation; SAHA treatment further increased that signaling. Together, the findings suggested that epigenetic processes affecting CXCL1/CXCR2 signaling could be a target for postoperative pain.
Clark’s latest work, published shortly after the meeting, shows that hyperalgesia induced by chronic opioid use also involves epigenetic alterations of CXCL1/CXCR2 signaling (Sun et al., 2014). Again using the mouse hindpaw incision model, Clark showed that chronic morphine administration, which produced mechanical allodynia and thermal sensitivity pre-incision, and worsened incision-induced mechanical allodynia, also increased expression of CXCL1/CXCR2 in the wound area of the skin (dermal layer), though not in the spinal cord; neutrophils in the wound area were the likely source of CXCL1. Chronic morphine further increased the acetylation of H3K9 observed after incision in dermal neutrophils infiltrating the wound area, and acetylation of the CXCL1 promoter was also elevated. In addition, SAHA had effects on CXCL1 levels in the skin similar to those seen with chronic morphine administration.
The recent results suggest that opioids may regulate CXCL1 expression and function via epigenetic processes in injured tissues. Compounds targeting those processes could have value to dampen postoperative pain, especially for patients taking chronic opioids.
Rheumatoid arthritis: a spinal player in post-inflammatory pain
Historically, pain in rheumatoid arthritis has been attributed to inflammation, but many patients with the condition continue to experience pain despite treatment with disease-modifying antirheumatic drugs (DMARDs) that effectively control inflammation. In a plenary lecture, Camilla Svensson, Karolinska Institute, Stockholm, Sweden, focused on mechanisms that may drive persistent pain in rheumatoid arthritis not only during inflammation but also after inflammation has subsided. A particular highlight was discussion of her work, published shortly after the meeting, on the role of extracellular high mobility group box-1 (HMGB1) protein in arthritis pain (Agalave et al., 2014).
HMGB1 is a damage-associated molecular pattern (DAMP) molecule that helps mediate the response to tissue damage and inflammation (Harris et al., 2012) and has been linked to neuropathic pain, low back pain, bone cancer-induced pain, diabetes-induced pain, and migraine in experimental models (e.g., see Shibasaki et al., 2010). Svensson’s path to HMGB1 stemmed from earlier work in which she and her colleagues used a serum transfer arthritis model in mice where mechanical hypersensitivity persists for several weeks after joint inflammation resolves (so-called “late phase” hypersensitivity; Christianson et al., 2011). The investigators found that spinal deficiency of the innate immune receptor toll-like receptor 4 (TLR4), as well as intrathecal administration of TLR4 antagonists, prevented the development of persistent mechanical hypersensitivity in the animals. Because HMGB1 is an endogenous TLR4 ligand, it seemed plausible that HMGB1 could play a part in TLR4-mediated arthritis pain.
In her recently published research, Svensson used a collagen antibody-induced arthritis (CAIA) model in mice to study HMGB1 function. Immunohistochemistry findings revealed the expression of HMGB1 in spinal cord dorsal horn neurons and glia of naïve animals, and that HMGB1 levels were significantly increased in the spinal cord in CAIA animals. Furthermore, blocking HMGB1 with an intrathecal neutralizing monoclonal antibody or with a recombinant peptide known to block extracellular HMGB1 activities reversed mechanical hypersensitivity during both the inflammatory and late phase in the CAIA mice. The investigators also discovered that only a particular, partially oxidized isoform of HMGB1 (disulfide HMGB1) caused mechanical hypersensitivity when administered intrathecally to naïve mice; it is only the disulfide form that activates TLR4. Furthermore, while intrathecal disulfide HMGB1 caused mechanical hypersensitivity in wild-type mice, as well as in animals missing immune receptors other than TLR4, TLR4 knockout mice did not show the same response, indicating that TLR4 mediated HMGB1-induced hypersensitivity. Finally, as activation of TLR4 is associated with activation of glial cells and production of cytokines, the researchers examined the effects of HMGB1 on glial and cytokine gene expression. Only the disulfide form of HMGB1 induced expression of cytokine and glial genes, further underscoring the importance of that particular isoform in rheumatoid arthritis pain signaling.
Collectively, the results show that disulfide HMGB1 expressed in spinal neurons and glial cells acts through TLR4 to enhance glial activity and drive rheumatoid arthritis-induced pain, even in the absence of inflammation, and that compounds that interfere with this pathway could have salutary effects on pain. Svensson is now investigating whether levels of HMGB1 (either alone or in complex with other factors) are altered in cerebrospinal fluid from patients with rheumatoid arthritis.
Next year’s APS Annual Scientific Meeting will take place in Palm Springs, California. For more information, see the APS website.
See Part 1.
Image credit: American Pain Society