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Mast cells are key mediators of cathelicidin-initiated skin inflammation in rosacea.

Muto Y, Wang Z, Vanderberghe M, Two A, Gallo RL, Di Nardo A - J. Invest. Dermatol. (2014)

Bottom Line: MC proteases not only recruit other immune cells, which amplify the inflammatory response, but also cause vasodilation and angiogenesis.Here, we demonstrate that MCs are key mediators of cathelicidin-initiated skin inflammation.Our data were confirmed on erythematotelangiectatic rosacea subjects who showed a decrease in matrix metalloproteinase activity (P<0.05), after 8 weeks of topical cromolyn treatment.

View Article: PubMed Central - PubMed

Affiliation: Division of Dermatology, Department of Medicine, University of California, San Diego, San Diego, California, USA.

ABSTRACT
Rosacea is a chronic inflammatory skin disease whose pathophysiological mechanism is still unclear. However, it is known that mast cell (MC) numbers are increased in the dermis of rosacea patients. MC proteases not only recruit other immune cells, which amplify the inflammatory response, but also cause vasodilation and angiogenesis. MCs are also one of the primary sources of cathelicidin LL-37 (Cath LL-37), an antimicrobial peptide that has been shown to be an enabler of rosacea pathogenesis. Here, we demonstrate that MCs are key mediators of cathelicidin-initiated skin inflammation. After Cath LL-37 injection into the dermis, MC-deficient B6.Cg-Kit(W-sh)/HNihrJaeBsmJ (KitW-sh) mice did not develop rosacea-like features. Conversely, chymase (P<0.001), tryptase, and Mmp9 (P<0.01) mRNA levels were significantly higher in C57BL/6 wild-type (WT) mice. Treating WT mice with an MC stabilizer significantly decreased the expressions of Mmp9 and Cxcl2 (P<0.01). Our data were confirmed on erythematotelangiectatic rosacea subjects who showed a decrease in matrix metalloproteinase activity (P<0.05), after 8 weeks of topical cromolyn treatment. We conclude that MCs have a central role in the development of inflammation subsequent to Cath LL-37 activation and that downregulation of activated MCs may be a therapy for rosacea treatment.

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(a-c) Cromolyn treatment of the rosacea-mouse modelMice were treated with Cromolyn (C) or Cromolyn and LL-37 (C+LL-37). (a) Skin mRNA expression of mouse metalloprotease 9 (Mmp9) and pro-inflammatory cytokine (Cxcl2). (b) Skin Metalloproteases total activity (MMP activity). (c) Mouse skin immunohistochemistry of mMC-MMP9 contents after LL-37 challenge (left image, LL-37) and cromolyn pre-treatment LL-37-challenge (right image, C+LL-37). MMP-9 (green), FcεRI (Red: MC marker) and DAPI (Blue: cell nuclei). Yellow –orange hue indicates MMP9 and mMC co-localization. Yellow orange color is not present in the right image. Scale bar = 50.0μm. (d-e) MC degranulation increases protease activity in NHEK. (d) MMP and KLK protease activities in NHEK after addition of the product of huMC degranulation in saline buffer (+Sup), or saline buffer (+Saline). (e) RT-PCR Cath LL-37 mRNA (CAMP) expression in NHEK cells treated with the product of huMC degranulation in saline buffer and 1,25(OH)2VD3 (Sup+VD), or saline buffer and 1,25(OH)2VD3 (Saline+VD) and saline alone (Saline); (f-h) Cromolyn effect on protease activity of rosacea patients. (f) MMP activity on the skin of patient treated with placebo (n=5) or cromolyn (n=5) (g) KLK activity. (h) LL-37 peptide level. Statistics: ***p<0.001, **p<0.01, *p<0.05
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Figure 3: (a-c) Cromolyn treatment of the rosacea-mouse modelMice were treated with Cromolyn (C) or Cromolyn and LL-37 (C+LL-37). (a) Skin mRNA expression of mouse metalloprotease 9 (Mmp9) and pro-inflammatory cytokine (Cxcl2). (b) Skin Metalloproteases total activity (MMP activity). (c) Mouse skin immunohistochemistry of mMC-MMP9 contents after LL-37 challenge (left image, LL-37) and cromolyn pre-treatment LL-37-challenge (right image, C+LL-37). MMP-9 (green), FcεRI (Red: MC marker) and DAPI (Blue: cell nuclei). Yellow –orange hue indicates MMP9 and mMC co-localization. Yellow orange color is not present in the right image. Scale bar = 50.0μm. (d-e) MC degranulation increases protease activity in NHEK. (d) MMP and KLK protease activities in NHEK after addition of the product of huMC degranulation in saline buffer (+Sup), or saline buffer (+Saline). (e) RT-PCR Cath LL-37 mRNA (CAMP) expression in NHEK cells treated with the product of huMC degranulation in saline buffer and 1,25(OH)2VD3 (Sup+VD), or saline buffer and 1,25(OH)2VD3 (Saline+VD) and saline alone (Saline); (f-h) Cromolyn effect on protease activity of rosacea patients. (f) MMP activity on the skin of patient treated with placebo (n=5) or cromolyn (n=5) (g) KLK activity. (h) LL-37 peptide level. Statistics: ***p<0.001, **p<0.01, *p<0.05

Mentions: According to the obtained results, MC proteases and MMP-9, which are released from MCs upon stimulation by Cath LL-37, are central in promoting rosacea–like skin inflammation. Therefore, we anticipated that blocking MC degranulation would also prevent the formation of rosacea-like inflammation in the skin. To test this hypothesis, cromolyn sodium, a very well known MC stabilizer, was injected intra peritoneal (I.P.) into WT mice (10mg/kg body weight, per day) for 4 days before Cath LL-37 challenge. 24 hours after the last cromolyn sodium injection, mice were injected with 50 μl of 320 μm Cath LL-37 or PBS, twice a day for 2 days. As expected, skin inflammation did not develop in the mice pretreated with cromolyn. Mmp9 and Cxcl2 expressions were significantly decreased in the cromolyn treated mice (p<0.01) (Figure 3a). Consistent with Mmp9 mRNA expression, MMP activity in the tissue was also dramatically decreased by cromolyn pretreatment (p<0.01) (Figure 3b). In addition, frozen skin sections were stained with anti-MMP-9 and anti-FcεRI antibodies for the detection of MCs. The immunostaining images showed that numerous MMP-9 positive MCs were observed in Cath LL-37 treated mice, but not in cromolyn pretreated Cath LL-37 mice (Figure 3c left panel versus right panel).


Mast cells are key mediators of cathelicidin-initiated skin inflammation in rosacea.

Muto Y, Wang Z, Vanderberghe M, Two A, Gallo RL, Di Nardo A - J. Invest. Dermatol. (2014)

(a-c) Cromolyn treatment of the rosacea-mouse modelMice were treated with Cromolyn (C) or Cromolyn and LL-37 (C+LL-37). (a) Skin mRNA expression of mouse metalloprotease 9 (Mmp9) and pro-inflammatory cytokine (Cxcl2). (b) Skin Metalloproteases total activity (MMP activity). (c) Mouse skin immunohistochemistry of mMC-MMP9 contents after LL-37 challenge (left image, LL-37) and cromolyn pre-treatment LL-37-challenge (right image, C+LL-37). MMP-9 (green), FcεRI (Red: MC marker) and DAPI (Blue: cell nuclei). Yellow –orange hue indicates MMP9 and mMC co-localization. Yellow orange color is not present in the right image. Scale bar = 50.0μm. (d-e) MC degranulation increases protease activity in NHEK. (d) MMP and KLK protease activities in NHEK after addition of the product of huMC degranulation in saline buffer (+Sup), or saline buffer (+Saline). (e) RT-PCR Cath LL-37 mRNA (CAMP) expression in NHEK cells treated with the product of huMC degranulation in saline buffer and 1,25(OH)2VD3 (Sup+VD), or saline buffer and 1,25(OH)2VD3 (Saline+VD) and saline alone (Saline); (f-h) Cromolyn effect on protease activity of rosacea patients. (f) MMP activity on the skin of patient treated with placebo (n=5) or cromolyn (n=5) (g) KLK activity. (h) LL-37 peptide level. Statistics: ***p<0.001, **p<0.01, *p<0.05
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Figure 3: (a-c) Cromolyn treatment of the rosacea-mouse modelMice were treated with Cromolyn (C) or Cromolyn and LL-37 (C+LL-37). (a) Skin mRNA expression of mouse metalloprotease 9 (Mmp9) and pro-inflammatory cytokine (Cxcl2). (b) Skin Metalloproteases total activity (MMP activity). (c) Mouse skin immunohistochemistry of mMC-MMP9 contents after LL-37 challenge (left image, LL-37) and cromolyn pre-treatment LL-37-challenge (right image, C+LL-37). MMP-9 (green), FcεRI (Red: MC marker) and DAPI (Blue: cell nuclei). Yellow –orange hue indicates MMP9 and mMC co-localization. Yellow orange color is not present in the right image. Scale bar = 50.0μm. (d-e) MC degranulation increases protease activity in NHEK. (d) MMP and KLK protease activities in NHEK after addition of the product of huMC degranulation in saline buffer (+Sup), or saline buffer (+Saline). (e) RT-PCR Cath LL-37 mRNA (CAMP) expression in NHEK cells treated with the product of huMC degranulation in saline buffer and 1,25(OH)2VD3 (Sup+VD), or saline buffer and 1,25(OH)2VD3 (Saline+VD) and saline alone (Saline); (f-h) Cromolyn effect on protease activity of rosacea patients. (f) MMP activity on the skin of patient treated with placebo (n=5) or cromolyn (n=5) (g) KLK activity. (h) LL-37 peptide level. Statistics: ***p<0.001, **p<0.01, *p<0.05
Mentions: According to the obtained results, MC proteases and MMP-9, which are released from MCs upon stimulation by Cath LL-37, are central in promoting rosacea–like skin inflammation. Therefore, we anticipated that blocking MC degranulation would also prevent the formation of rosacea-like inflammation in the skin. To test this hypothesis, cromolyn sodium, a very well known MC stabilizer, was injected intra peritoneal (I.P.) into WT mice (10mg/kg body weight, per day) for 4 days before Cath LL-37 challenge. 24 hours after the last cromolyn sodium injection, mice were injected with 50 μl of 320 μm Cath LL-37 or PBS, twice a day for 2 days. As expected, skin inflammation did not develop in the mice pretreated with cromolyn. Mmp9 and Cxcl2 expressions were significantly decreased in the cromolyn treated mice (p<0.01) (Figure 3a). Consistent with Mmp9 mRNA expression, MMP activity in the tissue was also dramatically decreased by cromolyn pretreatment (p<0.01) (Figure 3b). In addition, frozen skin sections were stained with anti-MMP-9 and anti-FcεRI antibodies for the detection of MCs. The immunostaining images showed that numerous MMP-9 positive MCs were observed in Cath LL-37 treated mice, but not in cromolyn pretreated Cath LL-37 mice (Figure 3c left panel versus right panel).

Bottom Line: MC proteases not only recruit other immune cells, which amplify the inflammatory response, but also cause vasodilation and angiogenesis.Here, we demonstrate that MCs are key mediators of cathelicidin-initiated skin inflammation.Our data were confirmed on erythematotelangiectatic rosacea subjects who showed a decrease in matrix metalloproteinase activity (P<0.05), after 8 weeks of topical cromolyn treatment.

View Article: PubMed Central - PubMed

Affiliation: Division of Dermatology, Department of Medicine, University of California, San Diego, San Diego, California, USA.

ABSTRACT
Rosacea is a chronic inflammatory skin disease whose pathophysiological mechanism is still unclear. However, it is known that mast cell (MC) numbers are increased in the dermis of rosacea patients. MC proteases not only recruit other immune cells, which amplify the inflammatory response, but also cause vasodilation and angiogenesis. MCs are also one of the primary sources of cathelicidin LL-37 (Cath LL-37), an antimicrobial peptide that has been shown to be an enabler of rosacea pathogenesis. Here, we demonstrate that MCs are key mediators of cathelicidin-initiated skin inflammation. After Cath LL-37 injection into the dermis, MC-deficient B6.Cg-Kit(W-sh)/HNihrJaeBsmJ (KitW-sh) mice did not develop rosacea-like features. Conversely, chymase (P<0.001), tryptase, and Mmp9 (P<0.01) mRNA levels were significantly higher in C57BL/6 wild-type (WT) mice. Treating WT mice with an MC stabilizer significantly decreased the expressions of Mmp9 and Cxcl2 (P<0.01). Our data were confirmed on erythematotelangiectatic rosacea subjects who showed a decrease in matrix metalloproteinase activity (P<0.05), after 8 weeks of topical cromolyn treatment. We conclude that MCs have a central role in the development of inflammation subsequent to Cath LL-37 activation and that downregulation of activated MCs may be a therapy for rosacea treatment.

Show MeSH
Related in: MedlinePlus