Rosacea is a common inflammatory skin disease that affects 3% of the population aged >30 years, and is characterized by facial erythema, papulopustules, and telangiectasia (Straus, 1991
; Kelly, 1996
; Crawford et al., 2004
). Disease severity in rosacea is exacerbated by a variety of external stimuli, and involves aberrant expression of KLK5 and cathelicidin (Yamasaki et al., 2007
). In this study we asked why patients with rosacea are abnormally reactive to the environment and respond with excessive KLK5 expression. We found that keratinocytes in rosacea skin have higher TLR2 expression compared with normal skin. Subsequent analysis of the functional consequences of this observation revealed that the expression of TLR2 in keratinocytes, or exposure to TLR2 ligands, enhances KLK5 production and protease activity. These data provide the first molecular explanation for the enhanced sensitivity of facial skin of patients with this disease, and provide further insight into systems that trigger innate inflammatory responses.
The findings of this study suggest that the capacity of TLR2 to increase release of KLK5 from keratinocytes is strongly dependent on calcium. Previous work has shown that calcium influences keratinocyte differentiation (Menon et al., 1985
; Mauro et al., 1998
). Direct observations of extracellular calcium concentrations in whole skin show that they are low in the basal epidermis, increased in mid epidermis, and highest in stratum granulosum. KLK5 is normally detectable only in differentiated keratinocytes at the layer of granular and cornified cells of the epidermis in vivo
, the region of highest calcium concentrations. Thus, a potential role for calcium in control of KLK5 production is suggested by the colocalization of these molecules. The data here confirmed that calcium is a stimulus for increased KLK5 transcription, and are in agreement with a recent previous publication from our group (Morizane et al., 2010
), but go on to make the important new observation that the functional release of KLK5 and total serine protease activity in the epidermis is controlled by the action of TLR2. Supporting this conclusion, we show for the first time to our knowledge that TLR2 ligands induce enhanced calcium flux in epidermal keratinocytes and that the increase in KLK5 release occurs in a calcium-dependent manner. This response reveals a less-known function for TLR2, as the vast majority of previous observations following activation of TLR2 have focused on de novo
mRNA transcription after encountering pathogen-associated molecules. The implications of these observations extend beyond understanding a disease process such as rosacea, and suggest a role for the skin microbiome in influencing normal keratinocyte function during epidermal barrier development. Such an observation would complement recent observations of a role for the microbiome and TLR2 in controlling epidermal inflammation following injury (Lai et al., 2009
), and evidence that signaling through TLR2 enhances bronchial epithelial cell growth and epithelial wound repair (Shaykhiev et al., 2008
We observed elevated TLR2 expression in rosacea skin and in keratinocytes cultured in high calcium media (). This increase in TLR2 expression by calcium may explain reason why keratinocytes in high calcium media showed better responses to TLR2/1 ligands. From a functional perspective, it is also logical that increased TLR2 in suprabasal epidermis would be beneficial to detect microbes at the surface. However, some reports have shown increased TLR2 expression in basal cells of normal human epidermis (Baker et al., 2003
; Pivarcsi et al., 2003
; Begon et al., 2007
). Three possibilities could explain the reason for these diverse observations: body site from where skin specimens were taken, antibodies used, and methods of immunodetection. Our samples were taken from the faces of normal and rosacea individuals, a significant difference from previous studies. Regarding the antibodies, we used monoclonal antibodies against TLR2. A close examination of previous publications using this antibody shows that TLR2 localized in the suprabasal area of normal epidermis and in hair follicle, similar to our results (Pivarcsi et al., 2003
). In contrast, Baker et al. (2003)
used polyclonal antibodies when detecting TLR2 at the basal layer. Finally, in our work we used an immunofluorescence staining technique and set the exposure time such that signals were not saturated. Under these conditions, the abundance of TLR2 in normal skin is barely detected, and this is in upper epidermis. With longer exposure times, one can see staining in the entire epidermis of normal skin as previously reported (Begon et al., 2007
). Thus, our findings may be consistent with the distribution of TLR2 previously reported, but now more specifically shows relative expression in facial skin.
Although microbes have long been thought to have a role in the pathology of rosacea, studies have shown contradictory results regarding microbial colonization and the associated incidence of rosacea (Buechner, 2005
; Yamasaki and Gallo, 2009
). We showed here that TLR2 overexpression by adenovirus vector system in normal human keratinocytes increased TLR2 expression 2- to 3-fold. Similarly, stimulation with the TLR2/1L Pam3CSK4 enhanced KLK5 expression, and elevated TLR2 2.0±1.2-fold more than normal skin. In addition, P. acnes
, the most abundant of the facial skin commensal microbes (Roth and James, 1988
), could induce KLK5 and cathelicidin in a TLR2-dependent manner. Therefore, elevated TLR2 expression could be a cause of aberrant expression of both KLK5 and cathelicidin, both of which are important in rosacea (Yamasaki et al., 2007
). Also, although it is known that KLK5, as well as cathelicidin, are localized and transported in the lamellar granule system in keratinocytes (Braff et al., 2005
; Ishida-Yamamoto et al., 2005
), the mechanisms that are involved in lamellar granule formation and release are not fully understood. Our data explain why rosacea skin is potentially susceptible to a wide variety of microbial molecules because of the relatively promiscuous nature of TLR2 recognition. Therefore, rosacea patients might respond more than normals, although bacterial diversity and quantities are similar between rosacea and normal skin. Thus, our findings suggest a molecular basis for increased microbial sensitivity. These results also show how many different microbes could contribute to the disease in different individuals and may explain previous contradictory studies attempting to isolate a single microbe as associated with the disease.
In summary, in this study we show that increased TLR2 expression and a subsequent broad increased susceptibility to innate immune stimuli may explain the aberrant expression of cathelicidin and KLK in rosacea. A proper balance between pattern recognition receptors and the external and internal ligands are necessary to maintain homeostasis of healthy skin and the cutaneous barrier (Yamasaki and Gallo, 2008
; Lai and Gallo, 2009
). In the case of this disease, the recognition of a specific role for TLR2 therefore offers greater insight toward an appropriate therapy.