Background & Aims
Peripheral neuropathy is a common neurological complication of diabetes, and about half of the patients develop pain 1. Research and available therapies for neuropathic pain, with prominent peripheral pathologic mechanisms, have until now mostly been directed towards the central nervous system, which induce many undesirable side effects and only provide partial relief in a subpopulation of patients 2.
We propose that targeting the peripheral source of inflammatory mediators of pain in the skin may promote a local homeostatic metabolic milieu that disrupts the inflammatory cascade and relieve pain. Inflammation has also previously been linked to metabolism and mitochondrial function 3-5.
Skin fibroblasts are in intensive interaction with nerves and nociceptors6. Human and experimental data show that skin fibroblasts during disease become dysregulated in their release of pain-related mediators 7-9, making skin fibroblasts a unique cell model to study peripheral pain sensitization.
Methods
We have established a biobank of skin fibroblasts from 30 skin biopsies from deeply phenotyped patients divided into four groups: patients with diabetes 1) without neuropathy, 2) with pain-free neuropathy, 3) with painful neuropathy, and 4) non-diabetic controls. All groups were matched for age and sex, while the diabetic groups were matched for glycemic control as defined by HbA1C and diabetes duration.
We have measured the excretion of IL-1?, IL-1?, IL-2, IL-4, IL-6, IL-8, IL-10, and TNF-? from skin fibroblasts into the growth media using Luminex Multiplex Technology and ELISA at either baseline or TNF-?/LPS stimulation for a subset of 16 skin fibroblast samples. We are currently expanding the assays to include all 30 skin fibroblast lines in our biobank.
Additionally, we are currently assessing the growth rate, the mitochondrial membrane potential, and mitochondrial superoxide levels of our fibroblasts to assess a potential change in mitochondrial function.
Results
Our initial findings demonstrate that skin fibroblasts from our diabetic groups release IL-6 and IL-8 into the growth media under baseline conditions. Notably, fibroblasts from patients, but not controls, exhibit this behavior, with those suffering from painful neuropathy showing a heightened tendency to release IL-6. Other cytokines were not detected in the growth media.
Additionally, our results show that when stimulated with the pro-inflammatory mediator TNF-?, skin fibroblasts have the capacity to increase the release of IL-6 compared to baseline.
Preliminary data also suggest an elevation in mitochondrial superoxide levels (redox stress) in fibroblasts from patients with painful neuropathy compared to control fibroblasts, though these findings require further validation on a larger cohort that includes all four groups for potential group-specific phenotype detection.
Conclusions
Together, our preliminary data suggest a potential connection between skin fibroblast behavior and painful neuropathy in patients with diabetes. Since mitochondrial metabolism and redox signaling are interconnected with inflammatory responses 3-5, this connection could be through a mitochondrial-stress-mediated production and release of pro-inflammatory cytokines such as IL-6 into the local environment of the skin. This, however, needs further validation on the full fibroblast cohort and additional skin-nerve co-culture experiments to validate and strengthen the hypothesis.
References
1Hagedorn, J. M. et al. An overview of painful diabetic peripheral neuropathy: Diagnosis and treatment advancements. Diabetes Res Clin Pract 188, 109928 (2022). https://doi.org:10.1016/j.diabres.2022.109928
2Bort, A. et al. Effects of JWH015 in cytokine secretion in primary human keratinocytes and fibroblasts and its suitability for topical/transdermal delivery. Mol Pain 13, 1744806916688220 (2017). https://doi.org:10.1177/1744806916688220
3McGettrick, A. F. & O’Neill, L. A. How metabolism generates signals during innate immunity and inflammation. J Biol Chem 288, 22893-22898 (2013). https://doi.org:10.1074/jbc.R113.486464
4Mehta, M. M., Weinberg, S. E. & Chandel, N. S. Mitochondrial control of immunity: beyond ATP. Nat Rev Immunol 17, 608-620 (2017). https://doi.org:10.1038/nri.2017.66
5O’Neill, L. A., Kishton, R. J. & Rathmell, J. A guide to immunometabolism for immunologists. Nat Rev Immunol 16, 553-565 (2016). https://doi.org:10.1038/nri.2016.70
6Karl, F. et al. Patient-derived in vitro skin models for investigation of small fiber pathology. Ann Clin Transl Neurol 6, 1797-1806 (2019). https://doi.org:10.1002/acn3.50871
7Kreß, L. et al. Differential impact of keratinocytes and fibroblasts on nociceptor degeneration and sensitization in small fiber neuropathy. Pain 162, 1262-1272 (2021). https://doi.org:10.1097/j.pain.0000000000002122
8Lakomá, J., Donadio, V., Liguori, R. & Caprini, M. Characterization of Human Dermal Fibroblasts in Fabry Disease. J Cell Physiol 231, 192-203 (2016). https://doi.org:10.1002/jcp.25072
9Paish, H. L. et al. Fibroblasts Promote Inflammation and Pain via IL-1? Induction of the Monocyte Chemoattractant Chemokine (C-C Motif) Ligand 2. Am J Pathol 188, 696-714 (2018). https://doi.org:10.1016/j.ajpath.2017.11.007
10Gylfadottir, S. S. et al. Diagnosis and prevalence of diabetic polyneuropathy: a cross-sectional study of Danish patients with type 2 diabetes. Eur J Neurol 27, 2575-2585 (2020). https://doi.org:10.1111/ene.14469
11Gylfadottir, S. S. et al. The characteristics of pain and dysesthesia in patients with diabetic polyneuropathy. PLoS One 17, e0263831 (2022). https://doi.org:10.1371/journal.pone.0263831
Presenting Author
Zahra Nochi
Poster Authors
Zahra Nochi
PhD
Danish Pain Research Center, Aarhus University, Denmark
Lead Author
Julie Mie Bentzen
BSc
Research Unit for Molecular Medicine, Department of Clinical Medicine, Aarhus University
Lead Author
Peter Kolind Brask-Thomsen
MD
Danish Pain Research Center, Department of Clinical Medicine, Aarhus University
Lead Author
Sif Gylfadottir
ORG-100009397
Lead Author
Páll Karlsson
MSc
Danish Pain Research Center, Department of Clinical Medicine, Aarhus University
Lead Author
Nanna Finnerup
The Danish Pain Research Center - Department of Clinical Medicine, Aarhus University
Lead Author
Rikke Olsen
MSc
Research Unit for Molecular Medicine, Department of Clinical Medicine, Aarhus University
Lead Author
Zahra Nochi
MSc
Danish Pain Research Center, Department of Clinical Medicine, Aarhus University
Lead Author
Topics
- Mechanisms: Biological-Molecular and Cell Biology