Biology subjects

Denk, F.

Publications and source records attributed to Denk, F..

16 recordsLinked to original sources

bioRxiv2026

LIF is a pro-algesic mediator in human and mouse models of arthritis-related pain

Purpose: Emerging clinical and experimental data suggest that JAK inhibitors (JAKi) are effective in reducing pain in Rheumatoid Arthritis (RA). We recently showed that JAK/STAT signalling cytokines, specifically IL-6, LIF and IL-11, are present in the RA joint and can act directly on human induced pluripotent stem cell (iPSC) derived sensory neurons by increasing their STAT3 phosphorylation (pSTAT3) levels and expression of select pain-relevant genes. In this study, we determined that LIF is the most consequential cytokine out of the three, altering neuronal signalling, outgrowth and pain-like behaviours in in vitro and in vivo models of arthritis. Methods: Human iPSC-derived sensory neurons were stimulated with RA synovial fluid (SF) or with cytokine-treated RA synovial fibroblast conditioned media (CM) in the absence or presence of anti-cytokine antibodies. pSTAT3 and pSTAT1 levels, and gene expression patterns, were assessed by Western blot and RNA-sequencing (RNA-seq). The immortalised dorsal root ganglion (DRG)-derived cell line (HD 10.6) was assessed for LIF-induced JAK/STAT signalling in addition to neurite outgrowth assays as a functional readout of the sensory neurons. Lastly, mouse models were used to assess pain-related behaviours in vivo. Results: LIF was found to be consistently present in the SF from painful joints from individuals with inflammatory arthritis. Western blotting revealed that the addition of RA SF to iPSC-derived sensory neurons led to LIF-dependent induction of pSTAT3 and pSTAT1, while RNA-seq demonstrated that LIF alters the transcriptome of sensory neurons more prominently than IL-6 and IL-11, up-regulating signature genes related to peripheral neuron sensitization, such as TRPV1, NTRK1, BDNF, HRH1 and SCN10A. Similarly, cytokine-treated fibroblast conditioned media was shown to induce neuronal pSTAT3 and pSTAT1 and upregulate expression of BDNF, HRH1 and VGF in a LIF-dependent manner. Additionally, the immortalised dorsal root ganglion (DRG)-derived cell line (HD 10.6), which also exhibited LIF-induced pSTAT1 and pSTAT3, showed increased neurite outgrowth after axotomy when treated with LIF. Finally, in a mouse model of inflammatory arthritis, LIF was shown to be significantly increased in the affected joint. Furthermore, intraarticular injection of LIF into a healthy mouse knee resulted in increased responsiveness to painful pressure stimuli in that joint. Conclusions: Our data indicate that LIF may be a pro-algesic mediator and a possible therapeutic target to improve pain management in individuals living with inflammatory arthritis.

neuroscience↗

bioRxiv2025

Generation of parasympathetic neurons from hiPSC that reproduce the electrophysiological properties of native neurons and modulate the activity of hiPSC-atrial cardiomyocytes

Peripheral parasympathetic ganglia lie adjacent to their target organs; their dysfunction contributes to diseases like atrial fibrillation. Due to their challenging accessibility for primary culture, we provide a step-by-step protocol to generate parasympathetic neurons from human induced pluripotent stem cells (hiPSC). They reproduce numerous native physiological features: cholinergic and autonomic markers, functional nicotinic receptors and electrophysiological properties. We also describe a co-culture system with hiPSC-atrial cardiomyocytes allowing neuromodulation of cardiomyocyte activity and offering a platform for drug discovery and disease modelling. HighlightsO_LISteps to generate parasympathetic neurons from human induced pluripotent stem cells C_LIO_LIGuidance of the functional assessment of the neurons (whole-cell patch clamp and Ca2+ imaging) C_LIO_LIInstructions on how to co-culture these neurons with hiPSC-derived atrial cardiomyocytes C_LIO_LIGuidance of the functional assessment of the neuronal modulation of cardiomyocyte activity C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/684157v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1e77cfborg.highwire.dtl.DTLVardef@be6cbforg.highwire.dtl.DTLVardef@1d58aa6org.highwire.dtl.DTLVardef@81ba0e_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

Single-cell RNA sequencing clarifies dermal fibroblast subset representation in vitro and reveals variable persistence of keloid disease-associated features

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.

cell biology↗

bioRxiv2025

Macrophages amplify the spontaneous activity of damaged sensory neurons in a human co-culture model of 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.

neuroscience↗

bioRxiv2024

A role for fibroblast and mural cell subsets in models of neuropathic pain.

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 mesenchymal lineage 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 mesenchymal cell abnormalities should be considered when developing novel strategies to tackle neuropathic pain. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/627455v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@186492corg.highwire.dtl.DTLVardef@12deb90org.highwire.dtl.DTLVardef@1c43b8aorg.highwire.dtl.DTLVardef@211952_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

Using in vivo calcium imaging and home cage behavioural analysis to study pain in mouse models of rheumatoid- and osteo-arthritis

BackgroundStudying pain in rodent models of arthritis is challenging. For example, assessing functional changes in joints 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 osteoarthritis: 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. MethodsAntigen 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. ResultsAntigen induced arthritis produced knee joint swelling and PMX caused degeneration of articular cartilage in the knee. In the first 46 hours 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. ConclusionGroup-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.

neuroscience↗

bioRxiv2024

Blockade of rheumatoid arthritis synovial fluid-induced sensory neuron activation by JAK inhibitors

ObjectiveClinical 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. MethodsIn-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. ResultsCell-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. ConclusionOur data indicate that JAKi can act directly on sensory neurons, providing a potential mechanistic explanation for their suggested superior analgesic properties.

immunology↗

bioRxiv2024

Modelling inflammation-induced peripheral sensitization in a dish - more complex than expected?

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 pre-existing 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 via whole cell patch clamping. Similarly small changes were observed when treating mouse primary sensory neurons with inflammatory soup. A semi-systematic re-examination of past literature further indicated that observed effects of inflammatory mediators on dissociated sensory neuron cultures are generally very small. We conclude that modelling inflammation-induced peripheral sensitization in vitro is non-trivial and will require careful selection of mediators and/or more complex, longitudinal multi-cellular setups. Especially in the latter, our novel GCamP6f induced-pluripotent stem cell line may be of value.

neuroscience↗

bioRxiv2023

Deep sequencing of Phox2a nuclei reveals five classes of anterolateral system neurons

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 3 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 & 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. Significance StatementThe 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 the development of new 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 these cells by 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 novel 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.

neuroscience↗

bioRxiv2023

Spontaneous activity in peripheral sensory nerves - A systematic review

In the peripheral nervous system, spontaneous activity in sensory neurons is considered to be one of the two main drivers of chronic pain states, alongside neuronal sensitization. Despite this, the precise nature and timing of this spontaneous activity in neuropathic pain is not well-established. Here, we have carried out a systematic search and data extraction of existing electrophysiological literature to shed light on which fibre types have been shown to maintain spontaneous activity and over what time frame. We examined both in vivo recordings of pre-clinical models of neuropathic pain, as well as microneurography recordings in humans. Our analyses reveal that there is broad agreement on the presence of spontaneous activity in neuropathic pain conditions, even months after injury or years after onset of neuropathic symptoms in humans. However, due to the highly specialised nature of the electrophysiological methods used to measure spontaneous activity, there is also a high degree of variability and uncertainty around these results. Specifically, there are very few directly controlled experiments, with little directly comparable data between human and animals. Given that spontaneous peripheral neuron activity is considered to be a key mechanistic feature of chronic pain conditions, it may be beneficial to conduct further experiments in this space.

neuroscience↗

bioRxiv2023

Assessing spontaneous sensory neuron activity using in vivo calcium imaging

Heightened spontaneous activity in sensory neurons is often reported in individuals living with chronic pain. It is possible to study this activity in rodents using electrophysiology, but these experiments require great skill and can be prone to bias. Here, we have examined whether in vivo calcium imaging with GCaMP6s can be used as an alternative approach. We show that spontaneously active calcium transients can be visualised in the fourth lumbar dorsal root ganglion (L4 DRG) via in vivo imaging in a mouse model of pain. Application of lidocaine to the nerve, between the inflamed site and the DRG, silenced spontaneous firing and revealed the true baseline level of calcium for spontaneously active neurons. We used this data to train a machine leaning algorithm to predict when a neuron is spontaneously active. We show that our algorithm is accurate in two different models of pain: intraplantar Complete Freunds Adjuvant and antigen-induced arthritis, with accuracies of 90.0% +/-1.2 and 85.9 % +/-2.1, respectively, assessed against visual inspection by an experienced observer. The algorithm can also detect neuronal activity in imaging experiments generated in a different lab using a different microscope configuration (Accuracy = 94.0 % +/2.2). We provide a Google Colaboratory Notebook to allow anyone easy access to this novel tool, for assessment of peripheral neuron activity in their own calcium imaging setups.

neuroscience↗

bioRxiv2022

In vivo calcium imaging shows that satellite glial cells have increased activity in painful states

Satellite glial cells (SGCs) are important for proper neuronal function of primary sensory neurons to whom they provide homeostatic support. Most research of SGC function has been performed with in vitro studies, but recent advances in calcium imaging and transgenic mouse models have enabled this first in vivo study of single cell SGC function in mouse models of inflammation and neuropathic pain. We found that in naive condition SGCs do not respond in a time-locked fashion to neuronal firing. In painful inflammatory and neuropathic states we detected time-locked signals in a subset of SGCs, but only with suprathreshold stimulation of the sciatic nerve. Surprisingly therefore, we conclude that most calcium signals in SGCs seem to develop at arbitrary intervals not directly linked to neuronal activity patterns. More in line with expectations, our experiments also revealed that the number of active SGCs was increased under conditions of inflammation or nerve injury. This could reflect the increased requirement for homeostatic support across dorsal root ganglion neuron populations, which are more active during such painful states.

neuroscience↗

bioRxiv2021

Comparative transcriptional analysis of the satellite glial cell injury response

Satellite glial cells (SGCs) tightly surround and support primary sensory neurons in the peripheral nervous system and are increasingly recognized for their involvement in the development of neuropathic pain following nerve injury. The SGCs are difficult to investigate due to their flattened shape and tight physical connection to neurons in vivo and their rapid changes in phenotype and protein expression when cultured in vitro. Consequently, several aspects of SGC function under normal conditions as well as after a nerve injury remain to be explored. The recent advance in single cell RNAseq technologies has enabled a new approach to investigate SGCs. Here we publish a dataset from mice subjected to sciatic nerve injury as well as a dataset from dorsal root ganglia cells after 3 days in culture. We use a meta-analysis approach to compare the injury response with that in other published datasets and conclude that SGCs share a common signature following sciatic nerve crush and sciatic ligation, involving transcriptional regulation of cholesterol biosynthesis. We also observed a considerable transcriptional change when culturing SGCs, suggesting that some differentiate into a specialised in vitro state, while others start resembling Schwann cell-like precursors. The datasets are available via the Broad Institute Single Cell Portal.

neuroscience↗

bioRxiv2021

Fibroblasts - the neglected cell type in peripheral sensitization and chronic pain? - A systematic view on the current state of the literature.

Chronic pain and its underlying biological mechanisms have been studied for many decades, with a myriad of molecules, receptors and cell types known to contribute to abnormal pain sensations. We now know that besides an obvious role for neuronal populations in the peripheral and central nervous system, immune cells like microglia, macrophages and T cells are also important drivers of persistent pain. While neuroinflammation has therefore been widely studied in pain research, there is one cell-type that appears to be rather neglected in this context: the humble fibroblast. Fibroblasts may seem unassuming, but actually play a major part in regulating immune cell function and driving chronic inflammation. What is known about them in the context chronic pain? Here we set out to analyze the literature on this topic - using systematic screening and data extraction methods to obtain a balanced view on what has been published. We found that there has been surprisingly little research in this area: 134 articles met our inclusion criteria, only a tiny minority of which directly investigated interactions between fibroblasts and peripheral neurons. We categorized the articles we included - stratifying them according to what was investigated, the estimated quality of results, and any common conclusions. Fibroblasts are a ubiquitous cell type and a prominent source of many pro-algesic mediators in a wide variety of tissues. We think that they deserve a more central role in pain research and propose a new, testable model of how fibroblasts might drive peripheral neuron sensitization.

neuroscience↗

bioRxiv2019

A transcriptional toolbox for exploring peripheral neuro-immune interactions.

Correct communication between immune cells and peripheral neurons is crucial for the protection of our bodies. Its breakdown is observed in many common, often painful conditions, including arthritis, neuropathies and inflammatory bowel or bladder disease. Here, we have characterised the immune response in a mouse model of neuropathic pain using flow cytometry and cell-type specific RNA sequencing (RNA-seq). We found few striking sex differences, but a very persistent inflammatory response, with increased numbers of monocytes and macrophages up to 3[1/2] months after the initial injury. This raises the question of whether the commonly used categorisation of pain into \"inflammatory\" and \"neuropathic\" is one that is mechanistically appropriate. Finally, we collated our data with other published RNA-seq datasets on neurons, macrophages and Schwann cells in naive and nerve injury states. The result is a practical web-based tool for the transcriptional data-mining of peripheral neuroimmune interactions.\n\nhttp://rna-seq-browser.herokuapp.com/

neuroscience↗

bioRxiv2018

Novel mechanisms of bone cancer pain revealed with in vivo GCaMP6s imaging

Skeletal metastases are frequently accompanied by chronic pain that is mechanoceptive in nature and not easily managed by available therapies. The peripheral sensory profile of primary afferents responsible for transmitting the pain-related messages from cancerous bone to central sites is investigated here. We imaged thousands of primary sensory dorsal root ganglion neurons in vivo in healthy (sham-operated) and cancer-induced bone pain (CIBP) rats in order to analyse and compare their function. Utilising Markov Cluster Analysis we identified distinct clusters of primary afferent responses to limb compression and position. In CIBP rats, three times as many sensory afferents responded to knee compression in the leg ipsilateral to the tumour compared to sham-operated rats. We present evidence that the observed increase in sensory afferent response was not due to increased individual afferent activity but rather represents activation of silent nociceptors, whose origin we propose is largely from outside of the bone.

neuroscience↗