John M. Dawes

The role of the immune system in the generation of neuropathic pain

Persistent pain is a sequela of several neurological conditions with a primary immune basis, such as Guillain-Barré syndrome and multiple sclerosis. Additionally, diverse forms of injury to the peripheral or the central nervous systems--whether traumatic, metabolic, or toxic--result in substantial recruitment and activation of immune cells. This response involves the innate immune system, but evidence also exists of T-lymphocyte recruitment, and in some patient cohorts antibodies to neuronal antigens have been reported. Mediators released by immune cells, such as cytokines, sensitise nociceptive signalling in the peripheral and central nervous systems. Preclinical data suggest an immune pathogenesis of neuropathic pain, but clinical evidence of a central role of the immune system is less clear. An important challenge for the future is to establish to what extent this immune response initiates or maintains neuropathic pain in patients and thus whether it is amenable to therapy.

CXCL5 mediates UVB irradiation-induced pain.

The cytokine CXCL5 is a peripheral mediator of pain induced by UVB irradiation to the skin. Pinpointing the Cause of Sunburns Pain As any summer sunbather knows, when pain persists after the immediate cause is removed, it can be debilitating and can cause delayed problems such as cancer. To better understand this undesirable form of pain, Dawes et al. examine sunburned skin, a good example of inflammatory pain. Pain caused by ultraviolet B (UVB) light–induced DNA damage, these investigators found, is caused by the cytokine CXCL5. They made sure that their results from ras apply to human skin by carefully comparing how both human and rodent skin react to UVB irradiation. UV irradiation of rat foot and human forearm skin causes increased blood flow and painful hypersensitivity to mechanical and heat-induced stimuli 40 hours later. By using a large array that detected expression of many cytokines and chemokines, the authors saw an expected up-regulation of interleukin-1β (IL-1β) and cyclooxygenase-2 (COX-2) and found that the most markedly enhanced chemokine was CXCL5—in both species. Not only was CXCL5 most elevated at the time of maximum pain, it was directly shown to be an important contributor to UVB-induced pain: Its injection alone into rat skin caused mechanical (but not thermal) hypersensitivity. CXCL5 attracted neutrophils and macrophages to the inflamed area. The final proof that CXCL5 is the key regulator for UVB-induced pain is that a neutralizing antibody to the chemokine protected against the pain and infiltration of immune cells. This fits with the known ability of CXCL5 to attract neutrophils (and as shown here macrophages) by regulating their chemotaxis. Pain is not always controlled in rodent models the same way that it is in humans. This has often prevented efficient translation of results in animal models to humans. Here, the authors guarded against this problem by showing a clear correlation of the UVB response in rat and in human skin, giving them confidence that their rat results would apply in humans. At least for inflammatory pain caused by the UVB rays of the sun, CXCL5 is an attractive target for therapeutic agents. Many persistent pain states (pain lasting for hours, days, or longer) are poorly treated because of the limitations of existing therapies. Analgesics such as nonsteroidal anti-inflammatory drugs and opioids often provide incomplete pain relief and prolonged use results in the development of severe side effects. Identification of the key mediators of various types of pain could improve such therapies. Here, we tested the hypothesis that hitherto unrecognized cytokines and chemokines might act as mediators in inflammatory pain. We used ultraviolet B (UVB) irradiation to induce persistent, abnormal sensitivity to pain in humans and rats. The expression of more than 90 different inflammatory mediators was measured in treated skin at the peak of UVB-induced hypersensitivity with custom-made polymerase chain reaction arrays. There was a significant positive correlation in the overall expression profiles between the two species. The expression of several genes [interleukin-1β (IL-1β), IL-6, and cyclooxygenase-2 (COX-2)], previously shown to contribute to pain hypersensitivity, was significantly increased after UVB exposure, and there was dysregulation of several chemokines (CCL2, CCL3, CCL4, CCL7, CCL11, CXCL1, CXCL2, CXCL4, CXCL7, and CXCL8). Among the genes measured, CXCL5 was induced to the greatest extent by UVB treatment in human skin; when injected into the skin of rats, CXCL5 recapitulated the mechanical hypersensitivity caused by UVB irradiation. This hypersensitivity was associated with the infiltration of neutrophils and macrophages into the dermis, and neutralizing the effects of CXCL5 attenuated the abnormal pain-like behavior. Our findings demonstrate that the chemokine CXCL5 is a peripheral mediator of UVB-induced inflammatory pain, likely in humans as well as rats.

ReadqPCR and NormqPCR: R packages for the reading, quality checking and normalisation of RT-qPCR quantification cycle (Cq) data.

BackgroundMeasuring gene transcription using real-time reverse transcription polymerase chain reaction (RT-qPCR) technology is a mainstay of molecular biology. Technologies now exist to measure the abundance of many transcripts in parallel. The selection of the optimal reference gene for the normalisation of this data is a recurring problem, and several algorithms have been developed in order to solve it. So far nothing in R exists to unite these methods, together with other functions to read in and normalise the data using the chosen reference gene(s).ResultsWe have developed two R/Bioconductor packages, ReadqPCR and NormqPCR, intended for a user with some experience with high-throughput data analysis using R, who wishes to use R to analyse RT-qPCR data. We illustrate their potential use in a workflow analysing a generic RT-qPCR experiment, and apply this to a real dataset. Packages are available from http://www.bioconductor.org/packages/release/bioc/html/ReadqPCR.htmland http://www.bioconductor.org/packages/release/bioc/html/NormqPCR.htmlConclusionsThese packages increase the repetoire of RT-qPCR analysis tools available to the R user and allow them to (amongst other things) read their data into R, hold it in an ExpressionSet compatible R object, choose appropriate reference genes, normalise the data and look for differential expression between samples.

Neuregulin-1 controls an endogenous repair mechanism after spinal cord injury

Spontaneous remyelination after spinal cord injury is mediated largely by Schwann cells of unknown origin. Bartus et al. show that neuregulin-1 promotes differentiation of spinal cord-resident precursor cells into PNS-like Schwann cells, which remyelinate central axons and promote functional recovery. Targeting the neuregulin-1 system could enhance endogenous regenerative processes.

Synthesis of lipid mediators during UVB-induced inflammatory hyperalgesia in rats and mice.

Peripheral sensitization during inflammatory pain is mediated by a variety of endogenous proalgesic mediators including a number of oxidized lipids, some of which serve endogenous modulators of sensory TRP-channels. These lipids are eicosanoids of the arachidonic acid and linoleic acid pathway, as well as lysophophatidic acids (LPAs). However, their regulation pattern during inflammatory pain and their contribution to peripheral sensitization is still unclear. Here, we used the UVB-model for inflammatory pain to investigate alterations of lipid concentrations at the site of inflammation, the dorsal root ganglia (DRGs) as well as the spinal dorsal horn and quantified 21 lipid species from five different lipid families at the peak of inflammation 48 hours post irradiation. We found that known proinflammatory lipids as well as lipids with unknown roles in inflammatory pain to be strongly increased in the skin, whereas surprisingly little changes of lipid levels were seen in DRGs or the dorsal horn. Importantly, although there are profound differences between the number of cytochrome (CYP) genes between mice and rats, CYP-derived lipids were regulated similarly in both species. Since TRPV1 agonists such as LPA 18∶1, 9- and 13-HODE, 5- and 12-HETE were elevated in the skin, they may contribute to thermal hyperalgesia and mechanical allodynia during UVB-induced inflammatory pain. These results may explain why some studies show relatively weak analgesic effects of cyclooxygenase inhibitors in UVB-induced skin inflammation, as they do not inhibit synthesis of other proalgesic lipids such as LPA 18∶1, 9-and 13-HODE and HETEs.

Chemokines as peripheral pain mediators

Multiple lines of evidence support the notion that much if not most chronic pain is dependent on on-going peripheral activity in nociceptors. This is not to say that central changes are unimportant, only that much of the central change is supported by a peripheral drive. This begs the question of what causes this peripheral drive. In some instances, particularly in association with peripheral nerve injury, nociceptors may become spontaneously active because of alterations in ion channel function or expression. But in most cases nociceptor activity arises because of the actions of peripheral mediators released by injured or damaged tissue. Some of these mediators are well known, such as the prostanoids. Others have more recently been identified, such as nerve growth factor (NGF). However, the limited efficacy of existing analgesic therapies strongly suggests that other important pain mediators exist. Here we discuss the evidence that a family of secreted proteins, the chemokines - well known for their actions in regulating immune cell migration - also play an important role in sustaining abnormal nociceptor activity in persistent pain states.

Genome-wide transcriptional profiling of skin and dorsal root ganglia after ultraviolet-B-induced inflammation.

Ultraviolet-B (UVB)-induced inflammation produces a dose-dependent mechanical and thermal hyperalgesia in both humans and rats, most likely via inflammatory mediators acting at the site of injury. Previous work has shown that the gene expression of cytokines and chemokines is positively correlated between species and that these factors can contribute to UVB-induced pain. In order to investigate other potential pain mediators in this model we used RNA-seq to perform genome-wide transcriptional profiling in both human and rat skin at the peak of hyperalgesia. In addition we have also measured transcriptional changes in the L4 and L5 DRG of the rat model. Our data show that UVB irradiation produces a large number of transcriptional changes in the skin: 2186 and 3888 genes are significantly dysregulated in human and rat skin, respectively. The most highly up-regulated genes in human skin feature those encoding cytokines (IL6 and IL24), chemokines (CCL3, CCL20, CXCL1, CXCL2, CXCL3 and CXCL5), the prostanoid synthesising enzyme COX-2 and members of the keratin gene family. Overall there was a strong positive and significant correlation in gene expression between the human and rat (Ru200a=u200a0.8022). In contrast to the skin, only 39 genes were significantly dysregulated in the rat L4 and L5 DRGs, the majority of which had small fold change values. Amongst the most up-regulated genes in DRG were REG3B, CCL2 and VGF. Overall, our data shows that numerous genes were up-regulated in UVB irradiated skin at the peak of hyperalgesia in both human and rats. Many of the top up-regulated genes were cytokines and chemokines, highlighting again their potential as pain mediators. However many other genes were also up-regulated and might play a role in UVB-induced hyperalgesia. In addition, the strong gene expression correlation between species re-emphasises the value of the UVB model as translational tool to study inflammatory pain.

The Role of G-Protein Receptor 84 in Experimental Neuropathic Pain

G-protein receptor 84 (GPR84) is an orphan receptor that is induced markedly in monocytes/macrophages and microglia during inflammation, but its pathophysiological function is unknown. Here, we investigate the role of GPR84 in a murine model of traumatic nerve injury. Naive GPR84 knock-out (KO) mice exhibited normal behavioral responses to acute noxious stimuli, but subsequent to partial sciatic nerve ligation (PNL), KOs did not develop mechanical or thermal hypersensitivity, in contrast to wild-type (WT) littermates. Nerve injury increased ionized calcium binding adapter molecule 1 (Iba1) and phosphorylated p38 MAPK immunoreactivity in the dorsal horn and Iba1 and cluster of differentiation 45 expression in the sciatic nerve, with no difference between genotypes. PCR array analysis revealed that Gpr84 expression was upregulated in the spinal cord and sciatic nerve of WT mice. In addition, the expression of arginase-1, a marker for anti-inflammatory macrophages, was upregulated in KO sciatic nerve. Based on this evidence, we investigated whether peripheral macrophages behave differently in the absence of GPR84. We found that lipopolysaccharide-stimulated KO macrophages exhibited attenuated expression of several proinflammatory mediators, including IL-1β, IL-6, and TNF-α. Forskolin-stimulated KO macrophages also showed greater cAMP induction, a second messenger associated with immunosuppression. In summary, our results demonstrate that GPR84 is a proinflammatory receptor that contributes to nociceptive signaling via the modulation of macrophages, whereas in its absence the response of these cells to an inflammatory insult is impaired.

Genomics of pain in osteoarthritis

Summary Osteoarthritis (OA) accounts for the majority of the disease burden for musculoskeletal disorders and is one of the leading causes of disability worldwide. This disability is the result not of the cartilage loss that defines OA radiographically, but of the chronic pain whose presence defines symptomatic OA. It is becoming clear that many genes, each with a small effect size, contribute to the risk of developing OA. However, the genetics of OA pain are only just starting to be explored. This review will describe the first genes to have been identified in genomic studies of OA pain, as well as the possible dual roles of genes previously identified in genomic studies of OA in the context of pain. Difficulties associated with attempting to characterise the genetics of OA pain will be discussed and promising future avenues of research into genetic and epigenetic factors affecting OA pain described.

Lentiviral vectors encoding short hairpin RNAs efficiently transduce and knockdown LINGO-1 but induce an interferon response and cytotoxicity in central nervous system neurones

Knocking down neuronal LINGO‐1 using short hairpin RNAs (shRNAs) might enhance axon regeneration in the central nervous system (CNS). Integration‐deficient lentiviral vectors have great potential as a therapeutic delivery system for CNS injuries. However, recent studies have revealed that shRNAs can induce an interferon response resulting in off‐target effects and cytotoxicity.