Cathepsin S acts via protease-activated receptor 2 to activate sensory neurons and induce itch-like behaviour

Abstract

Chronic itch is a debilitating condition characterised by excessive scratching and is a symptom frequently reported in skin diseases such as atopic dermatitis. It has been proposed that release of the cysteine protease Cathepsin S (CatS) from skin keratinocytes or immune cells resident in or infiltrating the skin could act as a pruritogen in chronic itch conditions. CatS is known to activate protease-activated receptor 2 (PAR2). We therefore hypothesised that enzymatic activation of neuronally expressed PAR2 by CatS was responsible for activation of sensory neurons and transmission of itch signals. Intradermally-injected human recombinant (hr)-CatS or the PAR2 agonist, SLIGRL-NH₂ behaved as pruritogens by causing scratching behaviour in mice. Hr-CatS-induced scratching behaviour was prevented by CatS inhibitors and PAR2 antagonists and reduced by 50% in TRPV1^-/- mice compared with wild-type mice, whilst no significant reduction in scratching behaviour was observed in TRPA1^-/- mice. Cultured dorsal root ganglion (DRG) cells showed an increase in [Ca²⁺]_i following incubation with hr-CatS, and the percentage of neurons that responded to hr-CatS decreased in the presence of a PAR2 antagonist or in cultures of neurons from TRPV1^-/- mice. Taken together, our results indicate CatS acts as a pruritogen via PAR2 activation in TRPV1-expressing sensory neurons.

Keywords: Itch; Proteases; Sensory neurons.

Fig. 1

Activated recombinant hr-CatS induces itch-like behaviour. A) Itching time (time spent scratching) over the 15 min observation period after intradermal injection in the nape of the neck. B) Number of paw lifts (Itching bouts) over the 15 min observation period. C) Composite scores over the 15 min observation period. D) Total sum behaviour over the 15 min observation period. Data are mean + SEM of 10 mice per group. *P < 0.05, **P < 0.01, Two-way ANOVA followed by Dunnett’s test versus 1 µg in panels A, B and C. One Way ANOVA followed by Dunnett’s test versus 1 µg dose in composite behaviour (panel D).

Fig. 2

SLIGRL-NH2 induces itch-like behaviour. A) Itching time (time spent scratching) over the 15 min observation period after intradermal injection in the nape of the neck. B) Number of paw lifts (Itching bouts) over the 15 min observation period. C) Composite scores over the 15 min observation period. D) Total sum behaviour over the 15 min observation period. Data are mean + SEM of 7 mice per group *P < 0.05, **P < 0.01,***P < 0.001, Two-way ANOVA followed by Dunnett’s test versus 10 µg in panels A,B and C. One-Way ANOVA followed by Dunnett’s test versus 10 µg dose in panel D.

Fig. 3

CatS and SLIGRL-NH2 induce itch-like behaviour in the cheek injection model. A) Total time spent scratching or wiping on the cheek over the 15 min observation period immediately after injection of activated hr-CatS. B) Total time spent scratching or wiping on the cheek immediately after injection of SLIGRL-NH₂. C) Total time spent scratching or wiping on the cheek immediately after injection of chloroquine. D) Total time spent scratching or wiping on the cheek immediately after injection of capsaicin. Data are mean + SEM of 6 mice per group. * P < 0.05,***P < 0.001, Student’s t test.

Fig. 4

CatS inhibitors and PAR2 antagonist prevent activated Cat-S induced itch-like behaviour. A) Total sum behaviour over the 15 min observation period after intradermal injection of activated hr-CatS (20 µg/mouse) and 1 hr pre-treatment with LHVS (30 mg/kg s.c.). B) Total sum behaviour over the 15 min observation period after intradermal injection of activated hr-CatS (20 µg/mouse) and 1 hr pre-treatment with MDV-590 (200 µmol/kg p.o.). C) Total sum behaviour over the 15 min observation period after intradermal injection of activated hr-CatS (20 µg/mouse) and 1 hr pre-treatment FSLLRY (50 µg/ mouse, intradermal). D) Total sum behaviour over the 15 min observation period after intradermal injection of SLIRGL (50 µg/mouse) and 1 hr pre-treatment FSLLRY (50 µg/ mouse, intradermal); *P < 0.05, **P < 0.01 ***P < 0.001, One Way ANOVA followed by Dunnett’s test versus vehicle group. Data are mean + SEM of 8 animals per group.

Fig. 5

Cat-S and SLIGRL-NH2 induced itch-like behaviour in TRPV1^−/− and TRPA1^−/− mice. A) Total sum behaviour over the 15 min observation period after intradermal injection of activated hr-CatS (20 µg/mouse) or SLIRGL-NH₂ (50 µg/mouse). *P < 0.05, **P < 0.01 ***P < 0.001, One Way ANOVA followed by Dunnett’s test versus vehicle group. Data are mean + SEM of 8 animals per group.

Fig. 6

Calcium responses in sensory neurons following application of CatS are reduced by the PAR2 antagonist and in cultures from TRPV1^−/− or TRPA1^−/− mice. A) Representative traces of the calcium response of a cell that responded to application of hr-CatS (400 nM). No changes were observed between 200 and 1000 s and this is reflected in the break of the axis. B) Percentage of sensory neurons in culture that responded to buffer or hr-CatS applied for up to 20 min, from 461 cells, n = 4, 5 from 1 coverslip per mouse. C) Percentage of sensory neurons in culture that responded to application of hr-CatS in the presence of MDV-590 (0.5 µM) or FSLLRY (5 µM), from 663 cells, n = 3, 4 from 1 coverslip per mouse. D) Percentage of sensory neurons cultured from TRPV1^−/− or TRPA1^−/− mice that responded to application of CatS, from 373 cells, n = 3–5 from 1 coverslip per mouse. *P ≤ 0.05, One Way ANOVA followed by Dunnett's test versus vehicle incubation. Data are mean + SEM.