Menthoxypropanediol inhibits nerve growth factor‐induced nerve fibre sprouting in coculture models of sensory neurons and skin cells

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Abstract

Atopic dermatitis (AD) is a chronic, relapsing and highly pruritic inflammatory skin disease affecting 10–20% of children and 2–5% of adults in developed countries 1, 2. In AD, inflammation is related to an elevated skin cell-derived release of nerve growth factor (NGF), leading to an increased density of cutaneous C-fibres 3, 4 that might be linked to pruritus 5. Cooling agents, such as menthol or icilin, suppress itch by activating transient receptor potential ion channel M8 (TRPM8) on cutaneous Aδ fibres 6, 7 and have therefore been used as an antipruritic treatment for many years 8. Additionally, TRPM8 activation has recently been shown to attenuate inflammatory responses in a mouse model of colitis 9. A potential anti-inflammatory effect of cooling agents and its impact on itch-associated hyperinnervation in AD had not been studied. In this study, we investigate the role of menthoxypropanediol (MPD) in modulating inflammatory NGF upregulation and itch-associated hyperinnervation in coculture models of sensory neurons and skin cells. Methods are presented in supplementary material. In the pathogenesis of AD, TNF-α acts synergistically with Th2 cytokines via NFκB to promote inflammatory processes [s1-s3]. In a screening, we identified the menthol derivative MPD (Fig. 1a,b) as a potent inhibitor of TNF-α-induced NFκB activation (Fig. 1d). This effect might be TRPM8 independent as menthol activated TRPM8 to the same extent as MPD (Fig. 1c), but did not modulate NFκB activation (Fig. 1d). In accordance with our findings, Ramachandran et al. observed a potent anti-inflammatory response with TRPM8 agonist icilin, but not with menthol 9. These findings indicate different mechanisms of cooling agents to modulate anti-inflammatory signalling that requires further investigation. In AD, pruritus is associated with elevated cutaneous levels of NGF and an increased intra-epidermal nerve fibre density [s4, s5]. Because of the fact that NGF is regulated by NFκB [s6], we hypothesized that MPD modulates NGF expression in skin cells. As TNF-α is known to upregulate NGF in an NFκB-dependent manner [s7] and is reported as an aggravation factor in atopic skin [s1], we treated dermal fibroblasts with TNFα and MPD. In fact, MPD reduced TNF-α-induced upregulation of ngf mRNA (Fig. 1e) and NGF protein (Fig. 1f), suggesting an impact of MPD on the regulation of cutaneous innervation. The TRPM8 antagonist AMTB did not abrogate the effect of MPD on ngf regulation (Fig. S1) pointing to a TRPM8-independent mechanism. To investigate whether the effect of MPD on NGF expression influences neurite outgrowth in vitro, we used our compartmented coculture model of porcine DRG neurons and dermal fibroblasts (Fig. 2). The model enables a culture of dermal fibroblasts with nerve endings spatially compartmented from DRG somata (Figs S2 and 2e). To mimic itch-associated hyperinnervation, we treated fibroblasts with histamine, inducing an increased outgrowth of nerve fibres into side compartments (Fig. 2a,c,f). As histamine induces NGF via NFκB [s8, s9], MPD limited the increase in histamine-induced ngf mRNA concentration in dermal fibroblasts (Fig. S3). Consequently, MPD inhibited increased histamine-induced nerve fibre sprouting in coculture models (Fig. 2c–f). Apart from its effect on NGF expression in fibroblasts, MPD might also modulate neurite outgrowth directly via activation of TRPM8 on nerve endings. Formerly, we showed that atopic skin cells mediate increased neurite outgrowth in air-exposed skin equivalents due to elevated NGF release [s10], pointing to the important role of skin cells in the generation of itch-associated hyperinnervation. In this study, we provide first evidence that MPD inhibits neurite outgrowth in innervated skin models (Fig. S5d) comprising atopic dermal fibroblasts and normal keratinocytes (Fig. S5a–c). The analysis of skin model epidermis suggests that MPD reduces ngf mRNA expression in keratinocytes as well (Fig. S5d), confirming the effect of MPD on TNF-α- and histamine-induced NGF regulation, thus partially explaining the reduced neurite density. Nerve fibres in innervated skin models released a basal level of the neuropeptide calcitonin gene-related peptide (CGRP) that plays a major role in neurogenic inflammation [s11, s12]. In line with the MPD-induced reduction in nerve fibre density, CGRP levels decreased by trend as well (Fig. S5e). Ramachandran et al. showed that TRPM8 agonist icilin inhibited pro-inflammatory CGRP release from distal colon tissue 9. As TRPM8 is coexpressed with TRPV1 in a subset of sensory neurons and suppresses TRPV1 activity [s13], we investigated whether MPD modulates the response to TRPV1 agonist capsaicin in sensory neurons. In fact, MPD reduced capsaicin-induced CGRP release of DRG neurons in vitro (Fig. S5f), pointing to the role of MPD in modulating not only nerve cell–skin cell interaction, but also activation of peptidergic, TRPV1-positive nerve fibres. Neuropeptides such as CGRP and substance P are known to induce NGF expression in skin cells [s14]. Based on our results, we suggest that MPD modulates NGF expression in atopic skin and consequently cutaneous nerve fibre density by (i) inhibition of NFκB activation in skin cells and (ii) reduction of inflammation-induced neuronal CGRP release. In this study, we report an inhibitory effect of MPD on NFkB activation, counteracting TNF-α-induced NGF upregulation in dermal fibroblasts. This modulation of NGF expression was functionally analysed in complex coculture models of porcine DRG neurons and human skin cells. MPD inhibited neurite outgrowth of histamine-treated dermal fibroblasts as well as nerve fibre sprouting in innervated skin models, comprising atopic fibroblasts and normal keratinocytes, by reducing NGF expression. As elevated NGF levels and increased cutaneous nerve fibre density are associated with itch 3-5, these observations point to an antipruritic effect of MPD that is based not only on the activation of cold-sensing nerve fibres 6, 7, but also on a modified crosstalk between nerve endings and skin cells. We thank Elmar Forsch, Ursula Wensorra and Alisa Gruschka for technical assistance. D.R. planned all experiments, was responsible for data interpretation and wrote the manuscript. A-C.W. and M.S. performed all experiments and were responsible for data interpretation. H.W. and F.S. supervised this project and were involved in data interpretation. G.N. supervised the study and wrote the manuscript. The authors are employed at Beiersdorf AG, Hamburg. Figure S1. TRPM8 antagonist AMTB did not abrogate MPD-induced ngf mRNA regulation. Figure S2. Compartmented culture model. Figure S3. Histamine-induced ngf mRNA upregulation is reduced by MPD. Figure S4. Cultivation of the innervated skin model. Figure S5. Menthoxypropanediol (MPD) inhibits nerve fiber sprouting and CGRP release in innervated skin models. Data S1. Material and Methods. Data S2. References Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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