Pain management in inflammatory arthritis (IA) remains challenging. The contribution of multiple mechanistic factors to the initiation and maintenance of pain, and the interplay of sensory, psychological and social factors that modulate perceptual outcomes, requires a multi-faceted approach that does not solely rely on inflammation-targeted therapies. Growing evidence points to the contribution of, and therefore the need to target, centrally mediated pain processes and/or peripheral sensitisation not associated with inflammatory burden. In this review, we examine current evidence on the mechanistic underpinnings of pain in both early and established IA, including novel insights regarding the manifestation of peripherally and centrally mediated pain. Further, we discuss evolving terminology used to describe pain types, including implications for clinical assessment and communication. Finally, we consider how treatment strategies – pharmacological and non-pharmacological – could fit into an individualised approach, exploring barriers to implementation in clinical practice, including limitations in pain assessment and practical challenges in service delivery.

Keloids are scars characterized by excessive collagen deposition and expansion beyond the wound margin, causing aesthetic concerns as well as pain and itching. However, the cellular origins of these sensations remain poorly understood. This study aimed to identify the cellular sources of keloid pain and itch, and explore their potential for in vitro investigation. Single-cell RNA sequencing (scRNAseq) datasets of keloid scars were analysed to identify pain/itch mediators. Expression of key mediators was validated using quantitative polymerase chain reaction (qPCR). The impact of cell passage on keloid and normal-adjacent dermal cells was examined by comparing scRNAseq from primary cells at passage 0 (P0) and passage 4 (P4) from a single patient. Analysis of three scRNAseq datasets identified mesenchymal fibroblasts and ACKR1+ endothelial cells as more abundant in keloid samples, and able to express prurogenic and algesic mediators such as CXCL12, tenascin-C, periostin, endothelin-1, interleukin (IL)-6 and IL-33. However, qPCR validation on expanded primary dermal fibroblasts (passages 5–8) showed discordant results, potentially due to changes during culture. To address this, scRNAseq analysis at P0 and P4 revealed that stromal cell subsets amalgamated and became transcriptionally distinct by P4. Moreover, the differential expression of prurogenic and algesic mediators identified from the literature was decreased or absent in P4 cell subsets, likely attributable to relative proportions of subsets normalizing; this explains the previous discordance between scRNAseq and qPCR observations. These changes demonstrate that prolonged culture alters the representation of stromal cell populations and can impact their expression of pain/itch mediators. This study identified keloid-enriched subsets that express pain and itch-related mediators. However, their expression was lost or reduced through culture in standard conditions. This indicates key biological differences may have been historically missed, and more sophisticated models or fresh tissue analysis may be required to capture certain disease-relevant features.

In vitro models of scarring and fibrosis are essential to improve our understanding of disease mechanisms and ultimately develop much-needed therapeutic strategies. The emerging appreciation of fibroblast heterogeneity leaves a knowledge gap about what is represented in typical fibroblast cultures: are the quantitative differences in fibroblast subtypes observed in pathological tissues represented, and are disease-associated molecular alterations of subtypes maintained? Single-cell (sc) RNA-seq on patient-matched keloid and adjacent non-lesional dermis was compared to sc-and bulk-RNA-seq of fibroblast cultures after 4+ passages. After culture, fibroblast subtypes assimilated, with clustering distinct from the original populations. Pseudo-bulk analysis of non-cultured mesenchymal fibroblasts showed cell-intrinsic keloid-versus-control transcriptional differences consistent with disease understanding; however, only a subset of these persisted in vitro. Cell-cell communication analysis provides insight into potential strategies to maintain cell populations and their in vivo phenotypes. This work provides a greater understanding of, and potential strategies to refine, essential human fibroblast culture models.

Neuropathic pain is a devastating type of pain that significantly reduces the quality of life of affected people. Traditionally considered as mechanistically distinct from pain induced by classical inflammatory states, studies continue to reveal more commonalities than differences, with a whole host of pathological changes in the environment of local peripheral nerves accompanying chronic neuropathic pain conditions. This narrative review provides an overview of the cellular and molecular drivers of neuropathic pain, highlighting some of the seminal publications from past and present. We discuss both neuronal and non-neuronal mechanisms contributing to neuropathic pain (eg, immune and stromal cell dysregulation). Particular attention is given to studies involving human cohorts which, until recently, have been less common in the field, due to the difficulties in accessing relevant tissues, like nervous system samples. The consequences of recent findings for analgesic drug development are also discussed, both in the context of neuropathic and non-neuropathic pain.

Neuropathic pain is a highly prevalent condition for which treatments are hampered by low efficacy and dose-limiting side-effects. Injury to the somatosensory nervous system causes maladaptive plasticity that initiates and maintains chronic pain. Emerging evidence suggests that inflammatory cells of the innate immune system shape the response of the injured nervous system and thereby contribute to the pathogenesis of pain. Data from preclinical models and human patient biopsies have specifically implicated peripheral macrophage populations for a pro-algesic role, yet how these cell types influence damaged sensory neurons and whether they directly contribute to neuronal hyperexcitability is unclear. Here, we have developed an iPSC co-culture system to study the interactions of macrophages and sensory neurons in a fully humanised experimental model. We found that analogous to endogenous counterparts, iPSC-derived macrophages (iMacs) display a dynamic molecular and functional profile that is highly dependent on neuronal state. Co-culture with injured iPSC-derived sensory neurons (iSNs) induces morphological, gene expression, and secretory profile changes in iMacs that are consistent with the response of macrophages to nerve injury in vivo. iMacs in turn amplify spontaneous firing in damaged sensory neurons, implicating macrophages in this cardinal feature of neuropathic pain. These results illustrate the utility of an iPSC-based model to study signalling between these two cell types; they support a role for macrophages in directly amplifying damaged sensory neuron activity and highlight disrupting pathological signalling between these cell types as a promising strategy for future analgesic drug development.

Keloids are a type of scar characterized by abnormal collagen deposition and expansion beyond the wound boundary. Besides the unwanted aesthetics, keloids can also produce severe pruritus, leading to patients experiencing decreased health-related quality of life. Unfortunately, a lack of research means the source of this sensation is unknown. Therefore, this study aims to explore itch-specific targets for therapeutic purposes. People living with keloids were asked to complete a questionnaire regarding their keloid itch status. A list of putative itch mediators was assembled using a literature search. Using three published single-cell RNA sequencing datasets of keloid scars, the expression of itch mediators will be explored and cell subset sources ascertained. The future work will include growing the specific cell populations that differentially express the identified itch genes and explore the interference with the nervous system. Survey results (n = 96) highlighted that itch is prevalent across the keloid population and can be moderate to severe in severity. Standard intralesional steroid injections lead to no improvement or worsening of symptoms in the majority of cases. These results highlighted the need to address the specific source(s) of keloid itch in order to develop targeted treatments. The next steps will be to analyse three published single-cell RNA sequencing datasets of keloid scars, and cross-reference cell subset expression with a list of literature-sourced putative itch mediators to identify potential sources of keloid-specific itch. We have confirmed that itch is a significant issue for people living with keloids. Next steps will be to identify cells expressing putative itch mediators by single-cell RNA sequencing. This will be followed by validation studies using ex vivo labelling of the identified molecular targets in keloid tissue samples.

ABSTRACT:Focal nerve injuries are often associated with neuropathic pain. Preclinical research suggests altered neuroimmune signalling underlies such neuropathic pain; however, its cause remains poorly understood in humans. In this multicentre cohort study, we describe the local cellular and molecular signature of neuropathic pain at the lesion site, using Morton's neuroma as a human model system of neuropathic pain (n = 22; 18 women) compared with nerves from participants without nerve injury (n = 11; 4 women). Immunofluorescent staining revealed demyelination and chronic infiltration of immune cells in Morton's neuroma. RNA bulk sequencing identified 3349 differentially expressed genes between Morton's neuroma and controls. Gene ontology enrichment analysis and weighted gene co-expression network analyses revealed modules specific for host defence and neurogenesis. Deconvolution analysis confirmed higher densities of macrophages and B cells in Morton's neuroma than control samples. Modules associated with defence response, neurogenesis, and muscle system development as well as macrophage cell populations identified by deconvolution correlated with patients' paroxysmal or evoked pain. Of note, we identified a consistently differentially expressed gene signature ( MARCO, CD163, STAB1 ) , indicating the presence of a specific M(GC) subset of macrophages. MARCO gene expression correlated with paroxysmal pain. Targeted immunofluorescent analyses confirmed higher densities of intraneural CD163 + MARCO + macrophage subsets in Morton's neuroma. Our findings provide detailed insight into the local molecular signature in the context of human focal nerve injury. There is clear evidence for an ongoing role of the immune system in chronic peripheral neuropathic pain in humans, with macrophages and specifically the M(GC) MARCO + subset implicated.

Peripheral sensitization of nociceptors is believed to be a key driver of chronic pain states. Here, we sought to study the effects of a modified version of inflammatory soup on the excitability of human stem cell-derived sensory neurons. For this, we used a preexisting and a novel stem cell line, modified to stably express the calcium sensor GCamP6f. Upon treatment with inflammatory soup, we observed no changes in neuronal transcription or functional responses upon calcium imaging and only a very minor increase in resting membrane potential (RMP) via whole cell patch clamping: control RMP (-71.31 +/- 1.1 mV) vs inflammatory soup RMP (-67.74 +/- 1.29 mV), uncorrected 2-tailed independent samples t test, P = 0.0383. Similarly, small changes were observed when treating mouse primary sensory neurons with inflammatory soup. A semi-systematic reexamination of past literature further indicated that observed effects of inflammatory mediators on dissociated sensory neuron cultures are generally small. We conclude that modelling inflammation-induced peripheral sensitization in vitro is nontrivial and will require careful selection of mediators and/or more complex, longitudinal multicellular setups. Especially in the latter, our novel GCamP6f-induced pluripotent stem cell line may be of value.

JAK inhibitors (JAKi) are widely used antiinflammatory drugs. Recent data suggest that JAKi have superior effects on pain reduction in rheumatoid arthritis (RA). However, the underlying mechanisms for this observation are not fully understood. We investigated whether JAKi can act directly on human sensory neurons. We analyzed RNA-seq datasets of sensory neurons and found that they expressed JAK1 and STAT3. Addition of cell-free RA synovial fluid to human induced pluripotent stem cell-derived (iPSC-derived) sensory neurons led to phosphorylation of STAT3 (pSTAT3), which was completely blocked by the JAKi tofacitinib. Compared with paired serum, RA synovial fluid was enriched for the STAT3 signalling cytokines IL-6, IL-11, LIF, IFN-α, and IFN-β, with their requisite receptors present in peripheral nerves postmortem. Accordingly, these recombinant cytokines induced pSTAT3 in iPSC-derived sensory neurons. Furthermore, IL-6 + sIL-6R and LIF upregulated expression of pain-relevant genes with STAT3-binding sites, an effect that was blocked by tofacitinib. LIF also induced neuronal sensitization, highlighting this molecule as a putative pain mediator. Finally, over time, tofacitinib reduced the firing rate of sensory neurons stimulated with RA synovial fluid. Together, these data indicate that JAKi can act directly on human sensory neurons, providing a potential mechanistic explanation for their suggested superior analgesic properties.

Introduction Pain in patients with rheumatoid arthritis (RA) is an unmet clinical need. Targeting joint inflammation with disease-modifying antirheumatic drugs has not resulted in the anticipated reduction in pain for many patients. This can partly be explained by the concept of central sensitisation whereby spinal and supraspinal pathways have a lower threshold of activation, leading to increased perception of pain. Synovial stromal cells, such as fibroblasts, are also thought to play a role through peripheral sensitisation of nerves in the joint. Synovial fibroblasts are known to produce pro-algesic mediators such as interleukin 6 and nerve growth factor at the messenger RNA level. These pro-algesic mediators could activate sensory nerve fibres that send signals from the joint to the spinal cord, thereby driving persistent pain in RA. The purpose of this study is to evaluate which pro-algesic mediators are produced by lining versus sub-lining fibroblasts and whether the level of these mediators correlates with clinical measures of pain in patients with RA.Methods and analysis FiND-Pain RA is a multicentre observational study which will recruit 50 patients with seropositive RA who attend the rheumatology department of Guy’s and St Thomas’ Hospital, London, and the Nuffield Orthopaedic Centre, Oxford. Clinical examination, pain-focused patient-reported outcome measures, ultrasound examination and ultrasound-guided synovial biopsy of the knee will be performed. The levels of known and putative pro-algesic mediators will be measured in fibroblasts from the lining and sub-lining layer of the synovium. The location and spatial morphology of sensory nerve fibres and their proximity to lining and sub-lining fibroblasts will be characterised. The primary outcome will be to determine whether the knee pain scores of participants correlate with the level of leukaemia inhibitory factor, a novel putative pain-mediator expressed in sub-lining fibroblasts. The secondary outcomes will be to determine whether other pro-algesic mediators produced by lining or sub-lining fibroblasts correlate with clinical measures of pain and to assess the location and proximity of sensory nerve fibres to lining versus sub-lining fibroblasts.Ethics and dissemination The study is a sub-study of the PUMIA (Pain Phenotypes and their Underlying Mechanisms in Inflammatory Arthritis) study, which has been approved by the Bromley Research Ethics Committee (REC: 21/LO/0712). The findings of this study will be disseminated through open-access publications, as well as scientific and clinical conferences.

In vitro models of scarring and fibrosis are essential to improve our understanding of disease mechanisms and ultimately develop much-needed effective therapeutic strategies. This is particularly true for keloids, the example of pathological scarring exploited in this study, as there is no animal model. Our emerging appreciation of fibroblast heterogeneity from single cell RNA sequencing (scRNA-seq) information leaves a knowledge gap about what is represented in typical fibroblast cultures. Specifically, it is important to know whether quantitative differences in fibroblast subtypes observed in pathological tissues are represented and/or whether disease-associated molecular alterations of subtypes are maintained. This study performed scRNA-seq on patient-matched keloid and normal adjacent dermis immediately following surgical removal, which was compared to sc- and bulk-RNA-seq on primary dermal fibroblast cultures from the same samples after 4+ passages. Freshly dissociated tissue showed anticipated differences in cell proportions in keloid versus normal skin; however, comparably for both tissue types, there was an assimilation of fibroblast subtypes after culture. Cultured cells clustered conspicuously from the original populations, with evidence of only minor heterogeneity persisting. Cells displayed, to varying degrees, elements of each of the original subset signatures, with FAP+/SFRP2+ mesenchymal features the strongest. Pseudo-bulk analysis of mesenchymal subpopulations ex vivo showed cell-intrinsic keloid versus normal skin transcriptional differences consistent with current disease understanding; however, only a subset of these persisted in vitro . Cell-cell communication analysis provides potential strategies to maintain specific cell populations and their in vivo phenotypes. As an example, we report that culture with ascorbic acid (stimulating cell-derived extracellular matrix) enriched the mesenchymal signature. The data presented herein provide resources supporting greater understanding of, and strategies to refine, essential human fibroblast culture models. ### Competing Interest Statement The authors have declared no competing interest. * scRNA-seq : Single-cell RNA sequencing P0 : non-passaged (not cultured) P4 : 4th passage SMC : Smooth muscle cell Medical Research Council, https://ror.org/03x94j517, MR/N013700/1, MR/X502923/1 Wellcome Trust, https://ror.org/029chgv08, 107859/Z/15/Z, 108874/Z/15/Z

Neuropathic pain is a particularly intractable type of chronic pain that can result from physical nerve damage due to surgery or entrapment. Here, we present data which suggest that a particular subclass of fibroblast and mural cells may be implicated in the sensory neuron dysfunction that is characteristic of this pain state. In a mouse model of traumatic painful neuropathy, we used cell sorting, nerve tissue clearing and RNA sequencing to study stromal cells. With cell sorting (n = 4 mouse nerves) and tissue clearing (n = 5), we show that fibroblasts and mural cells positive for the platelet-derived growth factor receptor beta (Pdgfrb) gene are increased in number for at least two months post-nerve damage. Moreover, single cell RNA sequencing data (n = 4) from our own lab and those of three other laboratories reveal that Pdgfrb+ cells express high levels of known and putative pro-algesic mediators. Bulk sequencing of sorted Pdgfrb+ fibroblasts (n = 10) and Pdgfrb+/Cd146+ mural cells (n = 11) further indicate that many of these mediators are upregulated in neuropathy. We go on to demonstrate that a human nerve pericyte line releases a selection of these pro-algesic mediators at protein level. Moreover, conditioned media from stimulated human pericytes induces intra-cellular changes in human induced pluripotent stem cell derived sensory neurons (n = 5 independent differentiations); these changes (phosphorylation of the transcription factor signal transducer and activator of transcription 3, STAT3) have been previously linked to sensory neuron activation. In summary, our data indicate that stromal cell abnormalities should be considered when developing novel strategies to tackle neuropathic pain.

Studying pain in rodent models of arthritis is challenging. For example, assessing functional changes in joint neurons is challenging due to their relative scarcity amongst all sensory neurons. Additionally, studying pain behaviors in rodent models of arthritis poses its own set of difficulties. Commonly used tests, such as static weight-bearing, often require restraint, which can induce stress and consequently alter nociception. The aim of this study was to evaluate two emerging techniques for investigating joint pain in mouse models of rheumatoid- and osteo-arthritis: In vivo calcium imaging to monitor joint afferent activity and group-housed home cage monitoring to assess pain-like behaviors. Specifically, we examined whether there was increased spontaneous activity in joint afferents and reduced locomotor activity following induction of arthritis. Antigen induced arthritis (AIA) was used to model rheumatoid arthritis and partial medial meniscectomy (PMX) was used to model osteoarthritis. Group-housed home cage monitoring was used to assess locomotor behavior in all mice, and weight bearing was assessed in PMX mice. In vivo calcium imaging with GCaMP6s was used to monitor spontaneous activity in L4 ganglion joint neurons retrogradely labelled with fast blue 2 days following AIA and 13–15 weeks following PMX model induction. Cartilage degradation was assessed in knee joint sections stained with Safranin O and fast green in PMX mice. Antigen induced arthritis produced knee joint swelling and PMX caused degeneration of articular cartilage in the knee. In the first 46 h following AIA, mice travelled less distance and were less mobile compared to their control cage mates. In contrast, no such differences were found between PMX and sham mice when measured between 4–12 weeks post-surgery. A larger fraction of joint neurons showed spontaneous activity in AIA but not PMX mice. Spontaneous activity was mostly displayed by medium-sized neurons in AIA mice and was not correlated with any of the home cage behaviors. Group-housed home cage monitoring revealed locomotor changes in AIA mice, but not PMX mice (with n = 10/group). In vivo calcium imaging can be used to assess activity in multiple retrogradely labelled joint afferents and revealed increased spontaneous activity in AIA but not PMX mice.

Pain research continues to face the challenge of poor translatability of pre-clinical studies. In this short primer, we are summarizing the possible causes, with an emphasis on practical and constructive solutions. In particular, we stress the importance of increased heterogeneity in animal studies; formal or informal pre-registration to combat publication bias; and increased statistical training in order to help pre-clinical scientists appreciate the usefulness of available experimental design and reporting guidelines.

This protocol describes how to use the home cage analyser developed by Actual Analytics to measure activity in group-housed mice.

The study of pain mechanisms has advanced significantly with the development of innovative in vitro models. This chapter explores those already used in or potentially useful for neuropathic pain research, emphasizing the complementary roles of animal and human cellular models to enhance translational success. Traditional animal models have provided foundational insights into the neurobiology of pain and remain invaluable for understanding complex pain pathways. However, integrating human cellular models addresses the need for better replication of human nociceptors. The chapter details methodologies for culturing rodent and human primary sensory neurons, including isolation and culture techniques, advantages, and limitations. It highlights the application of these models in neuropathic pain research, such as identifying pain-associated receptors and ion channels. Recent advancements in using induced pluripotent stem cell (iPSC)-derived sensory neurons are also discussed. Finally, the chapter explores advanced in vitro models, including 2D co-cultures and 3D organoids, and their implications for studying neuropathic pain. These models offer significant advantages for drug screening and ethical research practices, providing a more accurate representation of human pain pathways and paving the way for innovative therapeutic strategies. Despite challenges such as limited access to viable human tissue and variability between samples, these in vitro models, alongside traditional animal models, are indispensable for advancing our understanding of neuropathic pain and developing effective treatments.

Significance The anterolateral system (ALS) is a major ascending pathway from the spinal cord that underlies perception of pain, itch, and skin temperature. It is therefore an important target for development of treatments for chronic pain. Our understanding of this system has been hampered by the considerable diversity of its constituent cells. Here, we dissect the complex heterogeneity of a major subset of these cells, using high-resolution RNA sequencing. We reveal five distinct types of ALS neurons, which are differentially distributed within the spinal cord and probably represent functional populations. Our data provide insights into the molecular architecture of the ALS and will be important for future studies to define the roles of different ALS cell types in sensory processing.

Abstract Neuropathic pain remains difficult to treat, with drug development hampered by an incomplete understanding of the pathogenesis of the condition, as well as a lack of biomarkers. The problem is compounded by the scarcity of relevant human peripheral tissues, including skin, nerves, and dorsal root ganglia. Efforts to obtain such samples are accelerating, increasing the need for standardisation across laboratories. In this white paper, we report on a consensus meeting attended by neuropathic pain experts, designed to accelerate protocol alignment and harmonization of studies involving relevant peripheral tissues. The meeting was held in London in March 2024 and attended by 28 networking partners, including industry and patient representatives. We achieved consensus on minimal recommended phenotyping, harmonised wet laboratory protocols, statistical design, reporting, and data sharing. Here, we also share a variety of relevant standard operating procedures as supplementary protocols. We envision that our recommendations will help unify human tissue research in the field and accelerate our understanding of how abnormal interactions between sensory neurons and their local peripheral environment contribute towards neuropathic pain.

The anterolateral system (ALS) is a major ascending pathway from the spinal cord that projects to multiple brain areas and underlies the perception of pain, itch, and skin temperature. Despite its importance, our understanding of this system has been hampered by the considerable functional and molecular diversity of its constituent cells. Here, we use fluorescence-activated cell sorting to isolate ALS neurons belonging to the Phox2a-lineage for single-nucleus RNA sequencing. We reveal five distinct clusters of ALS neurons (ALS1-5) and document their laminar distribution in the spinal cord using in situ hybridization. We identify three clusters of neurons located predominantly in laminae I-III of the dorsal horn (ALS1-3) and two clusters with cell bodies located in deeper laminae (ALS4 and ALS5). Our findings reveal the transcriptional logic that underlies ALS neuronal diversity in the adult mouse and uncover the molecular identity of two previously identified classes of projection neurons. We also show that these molecular signatures can be used to target groups of ALS neurons using retrograde viral tracing. Overall, our findings provide a valuable resource for studying somatosensory biology and targeting subclasses of ALS neurons.