Oncostatin M suppresses IL31RA expression in dorsal root ganglia and interleukin-31-induced itching

Abstract

Background: Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by intermittent itchy rash. Type 2 inflammatory cytokines such as interleukin (IL)-4, IL-13, and IL-31 are strongly implicated in AD pathogenesis. Stimulation of IL-31 cognate receptors on C-fiber nerve endings is believed to activate neurons in the dorsal root ganglion (DRG), causing itch. The IL-31 receptor is a heterodimer of OSMRβ and IL31RA subunits, and OSMRβ can also bind oncostatin M (OSM), a pro-inflammatory cytokine released by monocytes/macrophages, dendritic cells, and T lymphocytes. Further, OSM expression is enhanced in the skin lesions of AD and psoriasis vulgaris patients.

Objective: The current study aimed to examine the contributions of OSM to AD pathogenesis and symptom expression.

Methods: The expression levels of the OSM gene (OSM) and various cytokine receptor genes were measured in human patient skin samples, isolated human monocytes, mouse skin samples, and mouse DRG by RT-qPCR. Itching responses to various pruritogens were measured in mice by counting scratching episodes.

Results: We confirmed overexpression of OSM in skin lesions of patients with AD and psoriasis vulgaris. Monocytes isolated from the blood of healthy subjects overexpressed OSM upon stimulation with IL-4 or GM-CSF. Systemic administration of OSM suppressed IL31RA expression in the mouse DRG and IL-31-stimulated scratching behavior. In contrast, systemic administration of OSM increased the expression of IL-4- and IL-13-related receptors in the DRG.

Conclusion: These results suggest that OSM is an important cytokine in the regulation of skin monocytes, promoting the actions of IL-4 and IL-13 in the DRG and suppressing the action of IL-31. It is speculated that OSM released from monocytes in skin modulates the sensitivity of DRG neurons to type 2 inflammatory cytokines and thereby the severity of AD-associated skin itch.

Keywords: IL-31; IL31RA; atopic dermatitis; itch; oncostatin M; pruritus.

Figure 1

Expression of the gene encoding oncostatin M (OSM) is upregulated in atopic dermatitis (AD) lesions and cytokine-stimulated human monocytes. (A) Expression of OSM was undetectable in 14 of 15 healthy human (HH) skin samples but was detected in 8 of 10 AD lesion samples and 5 of 10 psoriasis (PSO) lesion samples by RT-qPCR. (B) OSM protein levels in serum did not differ significantly between healthy humans and patients with AD (n = 20). (C) Stimulation of monocytes isolated from healthy humans with IL-4 (50 ng/mL) or GM-CSF (50 ng/mL) increased OSM expression (n = 6 samples from 6 human volunteers). (D) Stimulation of monocytes from healthy humans with both IL-4 and GM-CSF (50 ng/mL) increased OSM (n = 6 monocyte samples from 6 human volunteers). All results presented as mean ± SD. * p < 0.05, **< 0.01 and **** p < 0.0001 by Tukey’s multiple comparisons test.

Figure 2

Stimulation of isolated mouse dorsal root ganglia (DRG) and cultured human keratinocytes (HEKs) with OSM modulated IL-31 receptor subunit gene expression. (A, B) In isolated mouse DRG, OSM treatment (100 ng/mL) rapidly upregulated Osmr expression, followed by a slow return to baseline, and more slowly downregulated Il31ra expression. (C, D) In HEKs as well, OSM progressively increased OSMR expression (C) and more slowly reduced IL31RA expression (D). (E) In HEKs, CCL2 gene expression increases in an OSM concentration-dependent manner. (F) In lesions of AD and psoriasis vulgaris patients, there was a tendency for higher CCL2 gene expression compared with healthy humans (HH). All results presented as mean ± SD. ** p < 0.01 by unpaired t-test. All experiments except F were performed ≥three times.

Figure 3

High systemic OSM modulates IL-31 receptor subunit gene expression in mouse DRG. (A) Experimental schema. Mice were injected with OSM (200 ng) into the tail vein every 8 h for a total of four doses. At 28 h after the final dose, samples of the dorsal root ganglion (DRG) and dorsal skin were collected. (B, C) Injection of OSM increased DRG expression of Osmr (B) and decreased DRG expression of Il31ra (C) compared to vehicle (Vehi) injection (n = 10 DRG samples from 10 injected mice). (D, E) Alternatively, systemic injection of OSM did not alter IL-31 receptor subunit expression in mouse skin (n = 8 skin samples from 8 mice). (F–I) Systemic injection of OSM also increased DRG expression levels of IL-4 and IL-13 receptor subunits (n = 8 DRG samples from 8 mice). All results are presented as mean ± SD. *p < 0.05, ** p < 0.01, and **** p < 0.0001 by unpaired t-test. ns, non-significant.

Figure 4

Systemic OSM injection suppresses IL-31-induced scratching behavior in mice. (A) Intradermal administration of OSM alone did not induce itching. (n = 12 mice per treatment group). (B) The schedule of OSM and pruritogen administration followed by monitoring of scratching behavior. (C) Intravenous (iv) injection of OSM into the tail vein 12 h prior to subcutaneous (sc) injection of IL-31 reduced scratching behavior compared to iv vehicle (Vehi) injection (n = 12 mice per treatment group). (D, E) Tail vein injection of OSM did not alter scratching behavior induced by sc injection of (D) histamine (His) or (E) serotonin (5-HT) (n = 6 mice per treatment group). All results expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 by Tukey’s multiple comparisons test. n.s, non-significant.

Figure 5

Roles of monocyte and OSM in AD. Monocytes produce OSM in response to stimulation by IL-4 and GM-CSF. OSM triggers the release of CCL2 from keratinocytes, inducing monocytes infiltration into the skin. Under conditions of elevated OSM in skin, IL31RA expression is downregulated while OSMR expression is upregulated in keratinocytes and DRG. Consequently, suppression of IL-31-induced itch is triggered.