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.

OBJECTIVE:Psoriatic arthritis (PsA) is an HLA class I-associated inflammatory arthritis that develops in up to 30% of people with psoriasis. We tested the hypothesis that skin and joint inflammation in PsA is linked in terms of CD8+ T cell phenotype and clonality. METHODS:Using single-cell RNA sequencing (n = 6 skin samples with n = 5 paired synovial tissue samples and/or n = 5 paired synovial fluid samples) and spatial transcriptomics (n = 1 paired skin and synovial biopsy sample, n = 4 unpaired biopsy samples), we compared the transcriptional signature, T cell receptor repertoire, and cell neighborhoods of T cells from skin and synovial tissue and/or fluid samples from patients with PsA. RESULTS:We identified an enrichment of type 17 CD8+ tissue-resident memory T (Trm) cells in both the skin and joint, with a stronger interleukin-17 signature in the skin than the joint. CD8+ Trm cells resided in distinct cell neighborhoods in the skin and joint but were located adjacent to antigen-presenting cells in both sites. Several T cell clones were shared between the skin and joint. Across the six patients, 155 CD8+ T cell clones were shared between the two sites, comprising 1,071 CD8+ T cells and taking up a median of 13% of the skin and 8% of the joint CD8+ T cell receptor repertoire. CD8+ skin-joint shared clones tended to have a similar phenotype at both sites, characterized by increased expression of genes associated with a cytotoxic, tissue-resident phenotype. CONCLUSION:Our findings support the hypothesis that skin and joint inflammation in PsA is linked in terms of CD8+ T cell clonality and that specific T cells migrate between these compartments to propagate inflammation across both sites.

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.

Objective: Clinical studies suggest that compared to anti-TNF treatment, JAK inhibitors (JAKi) are superior in reducing pain in rheumatoid arthritis (RA). The underlying mechanisms for this observation are still unknown. Sensory neurons transmit noxious signals from inflamed joints to the central nervous system, where a pain percept is generated. We investigated whether JAKi exert direct effects on sensory neurons. Methods: In-house and public RNA sequencing datasets of sensory neurons were analysed for relevant JAK/STAT and cytokine-receptor gene expression. Human induced pluripotent stem cell (IPSC)-derived sensory neurons were stimulated with serum and synovial fluid (SF) from individuals with RA, or with selected cytokines that were found in RA SF by Luminex. Phosphorylation of STAT3 (pSTAT3) was assessed by Western blot. Sensory neuron activation was examined by recording neuronal firing using multi-electrode array and measuring expression levels of pain-relevant genes with STAT3-binding sites. Results: Cell-free RA synovial fluid induced pSTAT3 in IPSC-derived sensory neurons, an effect which was completely blocked by the JAKi tofacitinib. Compared to paired serum, RA SF was enriched for the JAK/STAT cytokines IL-6, IL-11, LIF, IFN-alpha and IFN-beta, with their requisite receptors present on sensory neurons. Stimulation of IPSC-derived sensory neurons with these recombinant cytokines recapitulated pSTAT3 induction in these cells. Furthermore, IL-6+sIL-6R or LIF upregulated expression of pain-relevant genes which was blocked by tofacitinib. Finally, we provided evidence that LIF can induce neuronal sensitisation. Conclusion: Our data indicate that JAKi can act directly on sensory neurons, providing a potential mechanistic explanation for their suggested superior analgesic properties. ### Competing Interest Statement LST and FD have received consultancy fees and/or research funding from AbbVie, CESAS Medical, GSK, Sanofi A/S, Ono Pharmaceuticals, UCB outside this work. OB is a cofounder, CEO and shareholder of LIFE & BRAIN GmbH. The remaining authors do not declare any conflicts of interest.

We hypothesised that skin and joint inflammation in psoriatic arthritis (PsA) is linked in terms of CD8+ T-cell phenotype and clonality. We employed scRNAseq to directly compare the transcriptional signature and T-cell receptor repertoire of memory T-cells from paired skin and synovial tissue and/or fluid from patients with PsA. We identified an enrichment of type-17 CD8+ tissue-resident memory (TRM) T-cells in both skin and joint, with a stronger IL-17 signature in the skin than the joint. Several T-cell clones were shared between the skin and joint and these shared clones tended to have the same signature at both sites, characterised by increased expression of genes associated with a cytotoxic, tissue-resident phenotype. Our findings support the hypothesis that skin and joint inflammation in PsA is linked in terms of T-cell clonality and raises the possibility that specific T-cells migrate between these compartments to propagate inflammation across both sites.### Competing Interest StatementBWK has received speaker or consultancy fees and/or research support from AbbVie, Eli Lilly, Galapagos, Janssen, Novartis, Pfizer and UCB, outside of this work. LST has received speaker or consultancy fees and/or research support from AbbVie, GSK, IMID forum, Sanofi and UCB, outside of this work.

Keloids are a severe form of scarring for which the underlying mechanisms are poorly understood, and treatment options are limited or inconsistent. Although biomechanical forces are potential drivers of keloid scarring, the direct cellular responses to mechanical cues have yet to be defined. The aim of this study was to examine the distinct responses of normal dermal fibroblasts and keloid-derived fibroblasts (KDFs) to changes in extracellular matrix stiffness. When cultured on hydrogels mimicking the elasticity of normal or scarred skin, KDFs displayed greater stiffness-dependent increases in cell spreading, F-actin stress fiber formation, and focal adhesion assembly. Elevated actomyosin contractility in KDFs disrupted the normal mechanical regulation of extracellular matrix deposition and conferred resistance on myosin inhibitors. Transcriptional profiling identified mechanically regulated pathways in normal dermal fibroblasts and KDFs, including the actin cytoskeleton, Hippo signaling, and autophagy. Further analysis of the autophagy pathway revealed that autophagic flux was intact in both fibroblast populations and depended on actomyosin contractility. However, KDFs displayed marked changes in lysosome organization and an increase in lysosomal exocytosis, which was mediated by actomyosin contractility. Together, these findings demonstrate that KDFs possess an intrinsic increase in cytoskeletal tension, which heightens the response to extracellular matrix mechanics and promotes lysosomal exocytosis.

Keloids are a severe form of scarring for which the underlying mechanisms are poorly understood, and treatment options are limited or inconsistent. While biomechanical forces are potential drivers of keloid scarring, the direct cellular responses to mechanical cues have yet to be defined. The aim of this study was to examine the distinct responses of normal dermal fibroblasts (NDFs) and keloid-derived fibroblasts (KDFs) to changes in extracellular matrix (ECM) stiffness. When cultured on hydrogels mimicking the elasticity of normal or scarred skin, KDFs displayed greater stiffness-dependent increases in cell spreading, F-actin stress fibre formation, and focal adhesion assembly. Elevated acto-myosin contractility in KDFs disrupted the normal mechanical regulation of ECM remodelling, leading to constitutive collagen and fibronectin deposition. Transcriptional profiling identified mechanically-regulated pathways in NDFs and KDFs, including the actin cytoskeleton, Hippo signalling, and autophagy. Further analysis of the autophagy pathway revealed that autophagic flux was intact in both fibroblast populations and depended on acto-myosin contractility. However, KDFs displayed marked changes in lysosome organisation and an increase in lysosomal exocytosis, which was mediated by acto-myosin contractility. Together, these findings demonstrate that KDFs possess an intrinsic increase in cytoskeletal tension, which heightens the response to ECM mechanics and promotes lysosomal exocytosis.

Fibrotic scarring is prevalent in a range of collagenous tissue disorders. Understanding the role of matrix biophysics in contributing to fibrotic progression is important to develop therapies, as well as to elucidate biological mechanisms. Here, we demonstrate how microfocus small-angle X-ray scattering (SAXS), with in situ mechanics and correlative imaging, can provide quantitative and position-resolved information on the fibrotic matrix nanostructure and its mechanical properties. We use as an example the case of keloid scarring in skin. SAXS mapping reveals heterogeneous gradients in collagen fibrillar concentration, fibril pre-strain (variations in D-period) and a new interfibrillar component likely linked to proteoglycans, indicating evidence of a complex 3D structure at the nanoscale. Furthermore, we demonstrate a proof-of-principle for a diffraction-contrast correlative imaging technique, incorporating, for the first time, DIC and SAXS, and providing an initial estimate for measuring spatially resolved fibrillar-level strain and reorientation in such heterogeneous tissues. By application of the method, we quantify (at the microscale) fibrillar reorientations, increases in fibrillar D-period variance, and increases in mean D-period under macroscopic tissue strains of ~20%. Our results open the opportunity of using synchrotron X-ray nanomechanical imaging as a quantitative tool to probe structure–function relations in keloid and other fibrotic disorders in situ.

The keratin network of intermediate filaments provides keratinocytes with essential mechanical strength and resilience, but the contribution to mechanosensing remains poorly understood. Here, we investigated the role of the keratin cytoskeleton in the response to altered matrix rigidity. We found that keratinocytes adapted to increasing matrix stiffness by forming a rigid, interconnected network of keratin bundles, in conjunction with F-actin stress fiber formation and increased cell stiffness. Disruption of keratin stability by overexpression of the dominant keratin 14 mutation R416P inhibited the normal mechanical response to substrate rigidity, reducing F-actin stress fibers and cell stiffness. The R416P mutation also impaired mechanotransduction to the nuclear lamina, which mediated stiffness-dependent chromatin remodeling. By contrast, depletion of the cytolinker plectin had the opposite effect and promoted increased mechanoresponsiveness and up-regulation of lamin A/C. Together, these results demonstrate that the keratin cytoskeleton plays a key role in matrix rigidity sensing and downstream signal transduction.