Correlation between pyrrolidone carboxylic acid in corneocytes and the severity of clinical symptom or lesional inflammation in atopic dermatitis patients

Abstract

Atopic dermatitis (AD) has been known as a multifactorial disease affected by genetic factors and environmental factors in pathogenesis. It is most persuasively accepted that when filaggrin protein – a structural protein – decreases or becomes deficient by genetic mutation, exogeneous antigens easily penetrate the skin. After antigens penetrate the stratum corneum (SC), they are phagocytized by antigen presenting cells such as Langerhans cell and presented to naive T helper cell (Th0). Th0 cell polarizes to type 2 T helper cell (Th2) under increased thymic stromal lymphopoietin (TSLP) environment. Therefore, we measured the quantity of pyrrolidone carboxylic acid (PCA), which is a final product of filaggrin protein and works as a representative natural moisturizing factor (NMF) and is maintained in a high level of concentration in horny cells. In this study, 75 subjects participated. Among 42 AD patients, 21 subjects were mild AD patients with a EASI score under 15. 13 subjects were moderate AD patients with a EASI score between 15 and 29, and 8 of them were severe AD patients with a EASI score beyond 30. For normal healthy control group, 18 non-atopic healthy volunteers were recruited after sex and age matching. In addition, 13 recessive X-linked ichthyosis (RXLI) patients were included as a negative control of filaggrin gene (FLG) mutation. Fluorescence in situ hybridization (FISH) analysis was employed to identify the deletion of steroid sulfatase gene (STS) for exact diagnosis of RXLI. For skin barrier function of the AD patients, stratum corneum (SC) hydration, basal transepidermal water lose (TEWL), and skin surface pH were measured. And then D-squame® disc was stripped horny cells from the lesional and non-lesional skin to evaluate PCA. PCA was extracted from the horny cells and quantified by liquid chromatography tandem mass spectrometry (LC/MS-MS) analysis. To identify FLG mutation, we collected blood from the AD patients to extract their DNA. Two types of mutation (pK4022X and c3321delA), which are most frequently found in Korean AD patients, were gone through polymerase chain reaction (PCR) analysis and sequence analysis to identify FLG mutation. As a result, it was found that only 2 of 42 AD patients had pK4022X mutation. The quantification of PCA in corneocytes decreased in the lesional skin of the AD patients. It was decreased even in the non-lesional skin of the moderate to severe AD patients. PCA quantity between the healthy control subjects and RXLI patients wasn’t found to be significant. In addition, as basal TEWL and the pH of skin surface increased the SC hydration decreased, PCA quantity decreased. Furthermore, when PCA quantity was compared with serum immunoglobulin E (IgE) level, there was a inverse relation has been seen. The expression level of caspase-14 and PCA quantity were significantly decreased in lesion than non-lesional skin of AD pateints and in was found to be statiscally significant, compared with healthy control. In addition, RXLI patients showed a similar expression level of caspase-14 to healthy control. Moreover, like PCA quantity, the moderate to severe AD patients showed a significant decrease in the expression level even in the non-lesional skin. The correlation between PCA quantity and caspase-14 expression level on lesional skin showed a proportionate relation. These results explain that the decreased expression of caspase-14 in corneocyte triggers the degradation defect of filaggrin monomer, and leads to the decrease in the PCA quantity. Such degradation defect of filaggrin monomer deters the generation of free amino acid including PCA, causing the insufficiency of NMF and finally xerosis.