A sensory neuron-expressed IL-31 receptor mediates T helper cell-dependent itch: Involvement of TRPV1 and TRPA1

Abstract

Background: Although the cytokine IL-31 has been implicated in inflammatory and lymphoma-associated itch, the cellular basis for its pruritic action is yet unclear.

Objective: We sought to determine whether immune cell-derived IL-31 directly stimulates sensory neurons and to identify the molecular basis of IL-31-induced itch.

Methods: We used immunohistochemistry and quantitative real-time PCR to determine IL-31 expression levels in mice and human subjects. Immunohistochemistry, immunofluorescence, quantitative real-time PCR, in vivo pharmacology, Western blotting, single-cell calcium imaging, and electrophysiology were used to examine the distribution, functionality, and cellular basis of the neuronal IL-31 receptor α in mice and human subjects.

Results: Among all immune and resident skin cells examined, IL-31 was predominantly produced by TH2 and, to a significantly lesser extent, mature dendritic cells. Cutaneous and intrathecal injections of IL-31 evoked intense itch, and its concentrations increased significantly in murine atopy-like dermatitis skin. Both human and mouse dorsal root ganglia neurons express IL-31RA, largely in neurons that coexpress transient receptor potential cation channel vanilloid subtype 1 (TRPV1). IL-31-induced itch was significantly reduced in TRPV1-deficient and transient receptor channel potential cation channel ankyrin subtype 1 (TRPA1)-deficient mice but not in c-kit or proteinase-activated receptor 2 mice. In cultured primary sensory neurons IL-31 triggered Ca(2+) release and extracellular signal-regulated kinase 1/2 phosphorylation, inhibition of which blocked IL-31 signaling in vitro and reduced IL-31-induced scratching in vivo.

Conclusion: IL-31RA is a functional receptor expressed by a small subpopulation of IL-31RA(+)/TRPV1(+)/TRPA1(+) neurons and is a critical neuroimmune link between TH2 cells and sensory nerves for the generation of T cell-mediated itch. Thus targeting neuronal IL-31RA might be effective in the management of TH2-mediated itch, including atopic dermatitis and cutaneous T-cell lymphoma.

Keywords: AD; AITC; Allyl isothiocyanate; Atopic dermatitis; Cytokine; DRG; Dorsal root ganglia; ERK; Extracellular signal-regulated kinase; GRPR; Gastrin-releasing peptide receptor; HBSS; Hanks balanced salt solution; High-power field; IB4; Isolectin B4; KO; Knockout; MEK; Mas-related G protein–coupled receptor; Mitogen-activated protein kinase enzyme; Mrgpr; NPR-A; Natriuretic peptide receptor A; OSMRβ; OVA; Oncostatin M receptor β; Ovalbumin; PAR-2; Proteinase-activated receptor 2; Quantitative real-time PCR; SC; SEB; Spinal cord; Staphylococcal enterotoxin B; TG; TRPA1; TRPV1; Transient receptor channel potential cation channel ankyrin subtype 1; Transient receptor potential cation channel vanilloid subtype 1; Trigeminal ganglion; atopic dermatitis; hpf; qPCR; sensory nerve; skin; transient receptor potential channel.

Figure 1. IL-31 derives from human T_H2 cells, and IL-31RA is expressed on human DRG neurons

(a) QPCR of IL-31, IL-31RAlong, IL-31RAshort, and OSMR-β. (b) Co-localization of CLA (red) and IL-31 (green) in AD skin (scale bar = 100 μm). (c) Human T_H2 cells express IL-31 mRNA. (d) Immature and mature dendritic cells (iDC/mDC) express IL-31 mRNA. (e) IL-31RA immunostaining in human DRG neurons (scale bar = 50 μm, (f) control). *P<0.05, **P<0.01, ***P<0.001, Mann-Whitney U test.

Figure 2. Superantigen-induced upregulation of IL-31 in AD-like mouse model

(a) Treatment regimen. (b) HE-staining of vehicle (PBS)- and SEB-treated skin (scale bar = 200 μm). Number of CD3⁺ T cells (c) and eosinophils (d) in vehicle- vs. SEB-treated skin. (e-f) qPCR from skin samples reveal increased mRNA levels for IL-31 (e) and IL-4 (f) in SEB-treated skin. N=8 mice/group. **p<0.01, ***p<0.001, Student’s t-test, error bars indicated as SEM.

Figure 3. In vivo effects of IL-31 in mice

(a) Injection of IL-31 into the nape of neck induced profound scratching. (b) Intraplantar IL-31 significantly increased paw-licking. (c) Cheek injection of IL-31 only produced scratching but no wiping (d). (e) Intrathecal injection of IL-31 induced significant dose-dependent scratching compared to vehicle. N=8 mice/group. **p<0.01, ***p<0.001, Student’s t-test, error bars indicated as SEM.

Figure 4. Localization of IL-31RA in murine DRG and spinal cord

(a) IL-31RA⁺ (red) and TRPV1⁺ (green) neurons partly co-localize. (b) Minimal overlap of IL-31RA (red) and IB4⁺ (green) subset of non-peptidergic nociceptors. (c) No overlap of IL-31RA⁺ (red) and N52⁺ unmyelinated neurons (green). (d) IL-31RA⁺/TRPV1⁺ in nerve terminals of the superficial dorsal horn. I.t. capsaicin (e), but not vehicle (d), ablated TRPV1⁺ (green) and IL-31RA (red) immunoreactivity. Scale bars = 100 μm.

Figure 5. Neuronal requirement of IL-31-induced itch

(a) Depletion of TRPV1⁺ neurons by i.t. capsaicin significantly decreased i.t. IL-31 induced scratching. (b) TRPV1KO and (c) TRPA1KO mice show reduction of IL-31 induced scratching compared to WT littermates. (d) c-kit mutant mice and (e) PAR-2 KO mice showed equal scratching to WT mice after IL-31 injection. N=8 mice/group. **p<0.01, ***p<0.001, Student’s t-test, error bars indicated as SEM.

Figure 6. IL-31-induced calcium mobilization, and characterization of IL-31-responsive DRG neurons

(a) Neurons responding to IL-31 only (blue), histamine only (green), IL-31 and histamine (black), neither IL-31 nor histamine (red). (b) Percentages of IL-31-responsive neurons which also respond to other compounds. (c-d) Venn diagrams for DRG neurons in percentages. (e) Percentages of IL-31-responsive neurons in different KO mice. N=193-981 cells/group. For quantification, 10–30 dishes/group were used, and 20–50 cells/dish were counted. *p<0.05, **p<0.01, unpaired t-test, error bars indicated as SEM.

Figure 7. ERK1/2 phosphorylation in DRG is critical for IL-31-induced itch

(a) Western blot and (b) densitometry analysis of murine cultured DRG for pERK1/2 illustrate peak activation of ERK1/2 after 5 min. (c) Pre-treatment with the ERK1/2 inhibitor U0126 blocked IL-31-induced ERK1/2 activation. (d) IL-31 stimulation does not lead to p38-phosphorylation in cultured DRG. (e) I.p. injection of U0126 prior to IL-31 blocked IL-31-evoked scratching. N = 8 mice/group. p<0.001, Student’s t-test, error bars indicated as SEM.