Oncostatin M enhances the lengthening of sensory nerves and skin hypersensitivity

Abstract

Background: Oncostatin M (OSM) is a cytokine that mediates inflammatory processes and is overexpressed in skin lesions of atopic dermatitis (AD). By amplifying neural responses to chemicals such as histamine, OSM increases sensitivity to pruritus. However, the morphological effects of OSM on peripheral sensory nerves and their subsequent impact on pruritus remain unclear. This study investigated OSM-induced peripheral nerve elongation, which may contribute to skin hypersensitivity.

Methods: We assessed neurite outgrowth using primary mouse dorsal root ganglion (DRG) cells treated with OSM, IL-31, or nerve growth factor. Next, we pre-treated the cells with inhibitors of downstream signaling pathways of OSM, including extracellular signal-regulated kinase (ERK), signal transducers and activator of transcription (STAT) 3, c-Jun N-terminal kinase (JNK), and p38, followed by OSM administration to measure neurite outgrowth. Furthermore, OSM receptor β-overexpressing cell lines were established by gene transfer into the DRG cell line, and nerve elongation was measured after OSM administration. In vivo studies involved OSM administration in mouse skin models. Immunofluorescence staining was used to evaluate nerve elongation. We examined whether OSM-infused mice had increased hypersensitivity to mechanical stimuli-induced pruritus. Various cytokine stimuli were applied to CD4+ T cells isolated from healthy humans to examine the conditions under which OSM production increases.

Results: OSM significantly induced neurite outgrowth in DRG cells and the effect of OSM surpassed the effects of IL-31 and nerve growth factor. The neurite outgrowth effect of OSM involved the JAK/STAT3, MEK/ERK, and p38/MAPK pathways. Compared to control cells, DRG cell lines that overexpressed OSM receptor β showed significantly enhanced neurite outgrowth upon OSM treatment. In vivo, OSM treatment increased nerve elongation in the mouse dermis. Behavioral assays in mice showed that OSM administration increased sensitivity to mechanical stimuli. IL-4 and TNFα increased OSM production in CD4+ T cells.

Conclusion: OSM induces neurite elongation and may contribute to skin hypersensitivity. This suggests the potential utilization of OSM as a therapeutic target for inflammatory skin diseases such as AD.

Keywords: atopic dermatitis; itch; nerve elongation; oncostatin M; pruritus; sensory nerve.

Figure 1

Representative microscopic images of primary DRG cells cultured with the following stimulatory conditions: (A) control, (B) OSM, (C) IL-31, and (D) NGF. The neurite length measurements and statistical analyses are presented in (E). The neurite lengths of OSM-stimulated primary DRG cells pretreated with the inhibitors were measured and statistically evaluated (F). For each group, 9–11 random fields were imaged, and the five longest neurites per field were measured. The experiment was repeated in triplicate, and the data were combined for analysis. Data are presented as the mean ± standard error (SE), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, not significant; DRG, dorsal root ganglion; OSM, oncostatin M; NGF, nerve growth factor.

Figure 2

Representative microscopic images of cells cultured with OSM stimulation in Control, Mock, and OSMRβ-overexpressing cell lines. (A) Control/OSM-, (B) Control/OSM+, (C) Mock/OSM-, (D) Mock/OSM+, (E) OSMRβ overexpressing/OSM-, (F) OSMRβ overexpressing/OSM+. Results of neurite length measurements and statistical evaluation after OSM stimulation of Control, Mock, and OSMRβ-overexpressing cell lines (G). Data are presented as the mean ± standard error (SE), *p<0.05, ****p<0.0001. For each condition, 9–11 random fields were imaged, and the five longest neurites per field were measured. The experiment was repeated in triplicate, and the data were combined for analysis. OSM, oncostatin M; OSMR, oncostatin M receptor.

Figure 3

Mice were subcutaneously injected with either saline or OSM in the back of the neck for two months, followed by fluorescent immunostaining. (A) Saline and (B) OSM. Arrows indicate stained nerve fibers. Statistical evaluation of neurite elongation and enlargement is shown in (C). Eight mice were used in each group, and 2–3 skin sections per mouse were analyzed for quantification. Mice were subcutaneously injected with either saline or OSM in the external ear for two months, followed by fluorescent immunostaining: (D) saline and (E) OSM. Statistical evaluation of neurite elongation and enlargement is presented in (F). Both ears of each mouse were used for analysis, and two peripheral areas were randomly imaged per ear (i.e., four areas per mouse). A total of eight mice were analyzed per group. Data are presented as the mean ± standard deviation (SD), **p<0.01. OSM, oncostatin M.

Figure 4

Mice were subcutaneously injected with either saline or OSM in the back of the neck for one month. Scratching behavior in response to von Frey filament stimulation was recorded in terms of (A) number of occurrences and (B) duration. Those measured without stimulation by von Frey filaments are (C) number of occurrences and (D) duration. A total of seven mice were used per group (N=7). Data are presented as the mean ± standard error (SE), *p<0.05. OSM, oncostatin M. ns, not significant.

Figure 5

CD4+ T cells isolated from healthy humans were stimulated with (A) IL-4 (10 ng/mL), (B) IL-13 (10 ng/mL), (C) IL-31 (10 ng/mL), (D) TNFa (10 ng/mL), and (E) TSLP (10 ng/mL). N=8 samples from 8 human volunteers. Data are presented as the mean ± standard deviation (SD), **p<0.01, ns, not significant.