Botulinum toxin blocks mast cells and prevents rosacea like inflammation

Abstract

Background: Rosacea is a chronic inflammatory skin condition whose etiology has been linked to mast cells and the antimicrobial peptide cathelicidin LL-37. Individuals with refractory disease have demonstrated clinical benefit with periodic injections of onabotulinum toxin, but the mechanism of action is unknown.

Objectives: To investigate the molecular mechanism by which botulinum toxin improves rosacea lesions.

Methods: Primary human and murine mast cells were pretreated with onabotulinum toxin A or B or control. Mast cell degranulation was evaluated by β-hexosaminidase activity. Expression of botulinum toxin receptor Sv2 was measured by qPCR. The presence of SNAP-25 and VAMP2 was established by immunofluorescence. In vivo rosacea model was established by intradermally injecting LL-37 with or without onabotulinum toxin A pretreatment. Mast cell degranulation was assessed in vivo by histologic counts. Rosacea biomarkers were analyzed by qPCR of mouse skin sections.

Results: Onabotulinum toxin A and B inhibited compound 48/80-induced degranulation of both human and murine mast cells. Expression of Sv2 was established in mouse mast cells. Onabotulinum toxin A and B increased cleaved SNAP-25 and decreased VAMP2 staining in mast cells respectively. In mice, injection of onabotulinum toxin A significantly reduced LL-37-induced skin erythema, mast cell degranulation, and mRNA expression of rosacea biomarkers.

Conclusions: These findings suggest that onabotulinum toxin reduces rosacea-associated skin inflammation by directly inhibiting mast cell degranulation. Periodic applications of onabotulinum toxin may be an effective therapy for refractory rosacea and deserves further study.

Keywords: Botox; Botulinum toxin; Mast cell; Mechanism of action; Rosacea.

Copyright © 2019 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

Figures

Fig 1.. Mast cell degranulation is blocked by onabotulinum toxin in vitro.

(a) Human mast cell (MC) degranulation was assessed by measuring release of β-hexosaminidase. The cells were pretreated with 5 pM botulinum toxin (BoNT) A or BoNT B for 24 h, then treated with compound 48/80 (10 μg/ml) for 30 min; (b-c) Murine MC degranulation was assessed by measuring release of β-hexosaminidase. The cells were pretreated with 0.5-5 pM BoNT A (b) or BoNT B (c) for 24 h, then treated with compound 48/80 (10 μg/ml) for 30 min. ***P<0.001 (n = 3).

Fig 2.. Mast cells express SNAP-25, VAMP2, and SV2.

Immunostaining for cleaved synaptosomal-associated protein-25 (SNAP-25) (a-c) or vesicle-associated membrane protein 2 (VAMP2) (d-f) was performed on mouse MCs. (a) Cells treated with PBS; (b) Cells treated with 0.1 pM BoNT A for 24 h; (d) Cells treated with PBS; (e) Cells treated with 0.1 pM BoNT B for 24 h; (c, f) Corrected total cell fluorescence (CTCF) intensity for cells treated with PBS or BoNT A or B. *P<0.05 (n = 5-6). Expression of the three isoforms of synaptic vesicle glycoprotein 2 (Sv2) in mouse MCs were measured by RT-qPCR; (g) Although all isoforms were detected, Sv2b was expressed at significantly higher levels. ***P<0.001 (n = 3).

Fig 3.. Onabotulinum toxin reduces LL-37-induced skin inflammation in mice.

Cathelicidin LL-37 50 μl of 320 μM was injected intradermally into C57BL/6 mice twice a day for two consecutive days with or without 0.5 unit of BoNT A pretreatment. (a) Control (injected with PBS); (b) Immediately after LL-37 intradermal injection; (c) 12 h after BoNT A pre-treatment and just before LL-37 injection; (d-e) Three days after LL-37 injection without (d) or with (e) BoNT A pretreatment; (f) Collected skin tissues from LL-37 treated mice without and with BoNT A pretreatment; (g) Image J software quantification of area of erythematous skin on LL-37 treated mice without and with BoNT A pretreatment. *P<0.05 (n = 3).

Fig 4.. Mast cell degranulation is blocked by onabotulinum toxin in vivo.

Intact (a) and degranulating (b) dermal MCs were counted in murine skin to assess the effect of BoNT A in vivo. Representative toluidine blue staining of skin tissue taken from mice treated with PBS (d) or cathelicidin LL-37 without (e) or with (f) BoNT A pretreatment; Intact and degranulating mast cells are indicated by red square and red dashed red circle outlines respectively; (c) Quantification of the percentage of dermal mast cells that are degranulating. ***P<0,001 (n = 20 fields of view).

Fig 5.. Onabotulinum toxin inhibits the expression of rosacea biomarkers in LL-37-induced rosacea mouse model.

LL-37 50 μl of 320 μM was injected intradermally into C57BL/6 mice twice a day for two consecutive days with or without 0.5 unit of BoNT A pre-treatment. Control mice were injected with PBS. Cmal (chymase) (a), Klk5 (kallikrein related-peptidase 5) (b), Mmp9 (matrix metallopeptidase 9) (c), and Trpv2 (transient receptor potential cation channel, subfamily V, member 2) (d) mRNA expressions in the skin were measured by RT-qPCR. ***P<0.001 (n = 3).