Synovial fibroblast gene expression is associated with sensory nerve growth and pain in rheumatoid arthritis.

It has been presumed that rheumatoid arthritis (RA) joint pain is related to inflammation in the synovium; however, recent studies reveal that pain scores in patients do not correlate with synovial inflammation. We developed a machine-learning approach (graph-based gene expression module identification or GbGMI) to identify an 815-gene expression module associated with pain in synovial biopsy samples from patients with established RA who had limited synovial inflammation at arthroplasty. We then validated this finding in an independent cohort of synovial biopsy samples from patients who had early untreated RA with little inflammation. Single-cell RNA sequencing analyses indicated that most of these 815 genes were most robustly expressed by lining layer synovial fibroblasts. Receptor-ligand interaction analysis predicted cross-talk between human lining layer fibroblasts and human dorsal root ganglion neurons expressing calcitonin gene-related peptide (CGRP+). Both RA synovial fibroblast culture supernatant and netrin-4, which is abundantly expressed by lining fibroblasts and was within the GbGMI-identified pain-associated gene module, increased the branching of pain-sensitive murine CGRP+ dorsal root ganglion neurons in vitro. Imaging of solvent-cleared synovial tissue with little inflammation from humans with RA revealed CGRP+ pain-sensing neurons encasing blood vessels growing into synovial hypertrophic papilla. Together, these findings support a model whereby synovial lining fibroblasts express genes associated with pain that enhance the growth of pain-sensing neurons into regions of synovial hypertrophy in RA.

Machine Learning Reveals Synovial Fibroblast Genes Associated with Pain Affect Sensory Nerve Growth in Rheumatoid Arthritis.

It has been presumed that rheumatoid arthritis (RA) joint pain is related to inflammation in the synovium; however, recent studies reveal that pain scores in patients do not correlate with synovial inflammation. We identified a module of 815 genes associated with pain, using a novel machine learning approach, Graph-based Gene expression Module Identification (GbGMI), in samples from patients with longstanding RA, but limited synovial inflammation at arthroplasty, and validated this finding in an independent cohort of synovial biopsy samples from early, untreated RA patients. Single-cell RNA-seq analyses indicated these genes were most robustly expressed by lining layer fibroblasts and receptor-ligand interaction analysis predicted robust lining layer fibroblast crosstalk with pain sensitive CGRP+ dorsal root ganglion sensory neurons. Netrin-4, which is abundantly expressed by lining fibroblasts and associated with pain, significantly increased the branching of pain-sensitive CGRP+ neurons in vitro . We conclude GbGMI is a useful method for identifying a module of genes that associate with a clinical feature of interest. Using this approach, we find that Netrin-4 is produced by synovial fibroblasts in the absence of inflammation and can enhance the outgrowth of CGRP+ pain sensitive nerve fibers. One Sentence Summary: Machine Learning reveals synovial fibroblast genes related to pain affect sensory nerve growth in Rheumatoid Arthritis addresses unmet clinical need.

Chemogenetic Silencing of NaV1.8-Positive Sensory Neurons Reverses Chronic Neuropathic and Bone Cancer Pain in FLEx PSAM4-GlyR Mice.

Drive from peripheral neurons is essential in almost all pain states, but pharmacological silencing of these neurons to effect analgesia has proved problematic. Reversible gene therapy using long-lived chemogenetic approaches is an appealing option. We used the genetically activated chloride channel PSAM4-GlyR to examine pain pathways in mice. Using recombinant AAV9-based delivery to sensory neurons, we found a reversal of acute pain behavior and diminished neuronal activity using in vitro and in vivo GCaMP imaging on activation of PSAM4-GlyR with varenicline. A significant reduction in inflammatory heat hyperalgesia and oxaliplatin-induced cold allodynia was also observed. Importantly, there was no impairment of motor coordination, but innocuous von Frey sensation was inhibited. We generated a transgenic mouse that expresses a CAG-driven FLExed PSAM4-GlyR downstream of the Rosa26 locus that requires Cre recombinase to enable the expression of PSAM4-GlyR and tdTomato. We used NaV1.8 Cre to examine the role of predominantly nociceptive NaV1.8+ neurons in cancer-induced bone pain (CIBP) and neuropathic pain caused by chronic constriction injury (CCI). Varenicline activation of PSAM4-GlyR in NaV1.8-positive neurons reversed CCI-driven mechanical, thermal, and cold sensitivity. Additionally, varenicline treatment of mice with CIBP expressing PSAM4-GlyR in NaV1.8+ sensory neurons reversed cancer pain as assessed by weight-bearing. Moreover, when these mice were subjected to acute pain assays, an elevation in withdrawal thresholds to noxious mechanical and thermal stimuli was detected, but innocuous mechanical sensations remained unaffected. These studies confirm the utility of PSAM4-GlyR chemogenetic silencing in chronic pain states for mechanistic analysis and potential future therapeutic use.

Neutrophils infiltrate sensory ganglia and mediate chronic widespread pain in fibromyalgia.

Fibromyalgia is a debilitating widespread chronic pain syndrome that occurs in 2 to 4% of the population. The prevailing view that fibromyalgia results from central nervous system dysfunction has recently been challenged with data showing changes in peripheral nervous system activity. Using a mouse model of chronic widespread pain through hyperalgesic priming of muscle, we show that neutrophils invade sensory ganglia and confer mechanical hypersensitivity on recipient mice, while adoptive transfer of immunoglobulin, serum, lymphocytes, or monocytes has no effect on pain behavior. Neutrophil depletion abolishes the establishment of chronic widespread pain in mice. Neutrophils from patients with fibromyalgia also confer pain on mice. A link between neutrophil-derived mediators and peripheral nerve sensitization is already established. Our observations suggest approaches for targeting fibromyalgia pain via mechanisms that cause altered neutrophil activity and interactions with sensory neurons.

Disease modification and symptom relief in osteoarthritis using a mutated GCP-2/CXCL6 chemokine.

We showed that the chemokine receptor C-X-C Motif Chemokine Receptor 2 (CXCR2) is essential for cartilage homeostasis. Here, we reveal that the CXCR2 ligand granulocyte chemotactic protein 2 (GCP-2) was expressed, during embryonic development, within the prospective permanent articular cartilage, but not in the epiphyseal cartilage destined to be replaced by bone. GCP-2 expression was retained in adult articular cartilage. GCP-2 loss-of-function inhibited extracellular matrix production. GCP-2 treatment promoted chondrogenesis in vitro and in human cartilage organoids implanted in nude mice in vivo. To exploit the chondrogenic activity of GCP-2, we disrupted its chemotactic activity, by mutagenizing a glycosaminoglycan binding sequence, which we hypothesized to be required for the formation of a GCP-2 haptotactic gradient on endothelia. This mutated version (GCP-2-T) had reduced capacity to induce transendothelial migration in vitro and in vivo, without affecting downstream receptor signaling through AKT, and chondrogenic activity. Intra-articular adenoviral overexpression of GCP-2-T, but not wild-type GCP-2, reduced pain and cartilage loss in instability-induced osteoarthritis in mice. We suggest that GCP-2-T may be used for disease modification in osteoarthritis.

Mechanisms of cancer pain.

Personalised and targeted interventions have revolutionised cancer treatment and dramatically improved survival rates in recent decades. Nonetheless, effective pain management remains a problem for patients diagnosed with cancer, who continue to suffer from the painful side effects of cancer itself, as well as treatments for the disease. This problem of cancer pain will continue to grow with an ageing population and the rapid advent of more effective therapeutics to treat the disease. Current pain management guidelines from the World Health Organisation are generalised for different pain severities, but fail to address the heterogeneity of mechanisms in patients with varying cancer types, stages of disease and treatment plans. Pain is the most common complaint leading to emergency unit visits by patients with cancer and over one-third of patients that have been diagnosed with cancer will experience under-treated pain. This review summarises preclinical models of cancer pain states, with a particular focus on cancer-induced bone pain and chemotherapy-associated pain. We provide an overview of how preclinical models can recapitulate aspects of pain and sensory dysfunction that is observed in patients with persistent cancer-induced bone pain or neuropathic pain following chemotherapy. Peripheral and central nervous system mechanisms of cancer pain are discussed, along with key cellular and molecular mediators that have been highlighted in animal models of cancer pain. These include interactions between neuronal cells, cancer cells and non-neuronal cells in the tumour microenvironment. Therapeutic targets beyond opioid-based management are reviewed for the treatment of cancer pain.

Physiologic osteoclasts are not sufficient to induce skeletal pain in mice.

BACKGROUND: Increased bone resorption is driven by augmented osteoclast activity in pathological states of the bone, including osteoporosis, fracture and metastatic bone cancer. Pain is a frequent co-morbidity in bone pathologies and adequate pain management is necessary for symptomatic relief. Bone cancer is associated with severe skeletal pain and dysregulated bone remodelling, while increased osteoclast activity and bone pain are also observed in osteoporosis and during fracture repair. However, the effects of altered osteoclast activity and bone resorption on nociceptive processing of bone afferents remain unclear. METHODS: This study investigates whether physiologic osteoclasts and resulting changes in bone resorption can induce skeletal pain. We first assessed correlation between changes in bone microarchitecture (through µCT) and skeletal pain using standardized behavioural phenotyping assays in a mouse model of metastatic bone cancer. We then investigated whether increased activity of physiologic osteoclasts, and the associated bone resorption, is sufficient to induce skeletal pain using mouse models of localized and widespread bone resorption following administration of exogenous receptor activator of nuclear factor kappa-B ligand (RANKL). RESULTS: Our data demonstrates that mice with bone cancer exhibit progressive pain behaviours that correlate with increased bone resorption at the tumour site. Systemic RANKL injections enhance osteoclast activity and associated bone resorption, without producing any changes in motor function or pain behaviours at both early and late timepoints. CONCLUSION: These findings suggest that activation of homeostatic osteoclasts alone is not sufficient to induce skeletal pain in mice. SIGNIFICANCE STATEMENT: The role of osteoclasts in peripheral sensitization of sensory neurones is not fully understood. This study reports on the direct link between oestrogen-independent osteoclast activation and skeletal pain. Administration of exogenous receptor activator of nuclear factor kappa-B ligand (RANKL) increases bone resorption, but does not produce pro-nociceptive changes in behavioural pain thresholds. Our data demonstrates that physiologic osteoclasts are not essential for skeletal pain behaviours.

Sensitization of Cutaneous Primary Afferents in Bone Cancer Revealed by In Vivo Calcium Imaging.

Cancer-induced bone pain (CIBP) is a complex condition, comprising components of inflammatory and neuropathic processes, but changes in the physiological response profiles of bone-innervating and cutaneous afferents remain poorly understood. We used a combination of retrograde labelling and in vivo calcium imaging of bone marrow-innervating dorsal root ganglia (DRG) neurons to determine the contribution of these cells in the maintenance of CIBP. We found a majority of femoral bone afferent cell bodies in L3 dorsal root ganglia (DRG) that also express the sodium channel subtype Nav1.8-a marker of nociceptive neurons-and lack expression of parvalbumin-a marker for proprioceptive primary afferents. Surprisingly, the response properties of bone marrow afferents to both increased intraosseous pressure and acid were unchanged by the presence of cancer. On the other hand, we found increased excitability and polymodality of cutaneous afferents innervating the ipsilateral paw in cancer bearing animals, as well as a behavioural phenotype that suggests changes at the level of the DRG contribute to secondary hypersensitivity. This study demonstrates that cutaneous afferents at distant sites from the tumour bearing tissue contribute to mechanical hypersensitivity, highlighting these cells as targets for analgesia.

Spread the Word: There Are Two Opioid Crises!

Pain is associated with emotional and physical suffering that severely impacts quality of life. Many guidelines for the treatment of moderate to severe cancer pain indicate the use of opioids. For a small proportion of the global population, opioids are readily accessible, but are consequently also subject to risk of overuse and misuse. On the other hand, many regions provide limited access to licensed opioid therapeutics and patients struggle for better pain management. The use of prescription opioids for treatment of severe cancer and acute pain is well established, but opioid use in chronic non-cancer pain is controversial and not supported by the literature. The opioid crisis and the increasing overdose fatalities in some countries have resulted in a resurgence of opiophobia in these countries, but even worse, amplified opiophobia in countries with lower opioid consumption. In this narrative review, we highlight how the opioid crisis of overuse in some countries can negatively impact appropriate access to opioids elsewhere. The availability of opioids for clinical and recreational use differs between countries worldwide-this is an important factor in determining the occurrence of a 'crisis of recreational use of opioids' or a 'crisis of under-prescription of opioids' for pain management.

Distinct transcriptional responses of mouse sensory neurons in models of human chronic pain conditions.

Background: Sensory neurons play an essential role in almost all pain conditions, and have recently been classified into distinct subsets on the basis of their transcriptomes. Here we have analysed alterations in dorsal root ganglia (DRG) gene expression using microarrays in mouse models related to human chronic pain. Methods: Six different pain models were studied in male C57BL/6J mice: (1) bone cancer pain using cancer cell injection in the intramedullary space of the femur; (2) neuropathic pain using partial sciatic nerve ligation; (3) osteoarthritis pain using mechanical joint loading; (4) chemotherapy-induced pain with oxaliplatin; (5) chronic muscle pain using hyperalgesic priming; and (6) inflammatory pain using intraplantar complete Freund's adjuvant. Microarray analyses were performed using RNA isolated from dorsal root ganglia and compared to sham/vehicle treated controls. Results: Differentially expressed genes (DEGs) were identified. Known and previously unreported genes were found to be dysregulated in each pain model. The transcriptomic profiles for each model were compared and expression profiles of DEGs within subsets of DRG neuronal populations were analysed to determine whether specific neuronal subsets could be linked to each of the pain models.  Conclusions: Each pain model exhibits a unique set of altered transcripts implying distinct cellular responses to different painful stimuli. No simple direct link between genetically distinct sets of neurons and particular pain models could be discerned.

Effects of intraplantar botulinum toxin-B on carrageenan-induced changes in nociception and spinal phosphorylation of GluA1 and Akt.

Increasing evidence suggests that botulinum neurotoxins (BoNTs) delivered into the skin and muscle in certain human and animal pain states may exert antinociceptive efficacy though their uptake and transport to central afferent terminals. Cleavage of soluble N-methylaleimide-sensitive attachment protein receptor by BoNTs can impede vesicular mediated neurotransmitter release as well as transport/insertion of channel/receptor subunits into plasma membranes, an effect that can reduce activity-evoked facilitation. Here, we explored the effects of intraplantar botulinum toxin- B (BoNT-B) on peripheral inflammation and spinal nociceptive processing in an inflammatory model of pain. C57BL/6 mice (male) received unilateral intraplantar BoNT (1 U, 30 µL) or saline prior to intraplantar carrageenan (20 µL, 2%) or intrathecal N-methyl-D-aspartate (NMDA), substance P or saline (5 µL). Intraplantar carrageenan resulted in edema and mechanical allodynia in the injected paw and increased phosphorylation of a glutamate subunit (pGluA1ser845) and a serine/threonine-specific protein kinase (pAktser473) in spinal dorsal horn along with an increased incidence of spinal c-Fos positive cells. Pre-treatment with intraplantar BoNT-B reduced carrageenan evoked: (i) allodynia, but not edema; (ii) pGluA1 and pAkt and (iii) c-Fos expression. Further, intrathecal NMDA and substance P each increased dorsal horn levels of pGluA1 and pAkt. Intraplantar BoNT-B inhibited NMDA, but not substance P evoked phosphorylation of GluA1 and Akt. These results suggest that intraplantar toxin is transported centrally to block spinal activation and prevent phosphorylation of a glutamate receptor subunit and a kinase, which otherwise contribute to facilitated states.

Genes, molecules and patients--emerging topics to guide clinical pain research.

This review selectively explores some areas of pain research that, until recently, have been poorly understood. We have chosen four topics that relate to clinical pain and we discuss the underlying mechanisms and related pathophysiologies contributing to these pain states. A key issue in pain medicine involves crucial events and mediators that contribute to normal and abnormal pain signaling, but remain unseen without genetic, biomarker or imaging analysis. Here we consider how the altered genetic make-up of familial pains reveals the human importance of channels discovered by preclinical research, followed by the contribution of receptors as stimulus transducers in cold sensing and cold pain. Finally we review recent data on the neuro-immune interactions in chronic pain and the potential targets for treatment in cancer-induced bone pain.