关键词: 皮炎, 特应性, 香叶基香叶基转移酶Ⅱ, B-淋巴细胞, 细胞凋亡, 浆细胞, 免疫球蛋白E

Abstract: 【Abstract】 Objective To investigate roles and action mechanisms of geranylgeranyl transferase Ⅱ （GGTase Ⅱ） in regulating B-cell function and the pathological progression of atopic dermatitis （AD）. Methods Cd19creRabggtafl/fl （cKO） mice were established, and the knockout efficiency of the Rabggta gene in B cells was validated by real-time quantitative reverse transcription PCR （RT-qPCR）. Wild-type （WT） mice and cKO mice were assigned to 3 groups: （1） vehicle control group （n = 4）, in which WT mice received daily application of an equal volume of anhydrous ethanol to the left ear; （2） WT model group （n = 6）, in which WT mice received daily application of calcipotriol to the left ear to induce AD-like dermatitis; （3） cKO model group （n = 6）, in which cKO mice received daily application of calcipotriol to the left ear to induce AD-like dermatitis. Modeling was performed for 12 consecutive days, and ear thickness was measured every 3 days. On day 13, the mice were sacrificed, and ear tissue samples were collected. Hematoxylin and eosin staining was used to evaluate skin inflammation and pathological changes, and immunofluorescence staining was performed to assess B-cell infiltration in the skin lesions. Flow cytometry was performed to determine the number of B cells, the expression of the apoptosis marker cleaved caspase-3, and the proportion of CD138?IgE? plasmacytoid cells in the draining lymph nodes of the mouse ears. Serum levels of IgE, interleukin （IL）-4, and IL-13 were measured by enzyme-linked immunosorbent assay （ELISA）. Primary splenic B cells were isolated from WT and cKO mice and divided into 4 groups: control costimulation group and?Rabggta-knockout costimulation group （cells from WT and cKO mice, respectively, treated with 5 μg/ml anti-CD40 antibody, 20 ng/ml IL-4, and 20 ng/ml IL-5 for 72 hours）, and control unstimulated group and?Rabggta-knockout unstimulated group （cells from WT and cKO mice, respectively, cultured in RPMI 1640 complete medium containing 10% fetal bovine serum for 72 hours）. Flow cytometry was performed to assess the relative cell viability, mean fluorescence intensity of cleaved caspase-3, and the proportion of CD138?IgE? plasmacytoid cells. After extraction of membrane protein fractions, Western blot analysis was performed to determine the expression of key RAB proteins involved in vesicular transport （RAB5A, RAB8A, and RAB11A）. Statistical analyses were performed using the two-independent-samples t test, one-way analysis of variance, repeated measures analysis of variance, and factorial analysis of variance. Results Compared with WT mice, cKO mice showed significantly lower Rabggta mRNA expression in B cells （t = 8.59, P = 0.001）. At the end of calcipotriol-induced modeling, mice in the WT model group developed typical AD-like skin lesions on the ears, characterized by marked erythema, scaling, and ear thickening; compared with the WT model group, the cKO model group showed significantly reduced ear thickness （P = 0.009）, decreased epidermal thickness and attenuated inflammatory cell infiltration in histopathological sections, and fewer scratching episodes （P < 0.001）. Immunofluorescence staining showed reduced B-cell infiltration in the skin lesions in the cKO model group than in the WT model group. Compared with the WT model group, the cKO model group exhibited significantly decreased total cell counts in the ear-draining lymph nodes （P < 0.001）, reduced proportion and absolute number of B220? B cells （both P < 0.001）, increased expression of cleaved caspase-3 （P < 0.001）, and reduced proportion and absolute number of CD138?IgE? plasmacytoid cells （P = 0.001, < 0.001, respectively）. ELISA revealed that serum levels of total IgE, IL-4, and IL-13 were significantly lower in the cKO model group than in the WT model group （all P < 0.05）. In vitro experiments further confirmed that, compared with the control costimulation group, the Rabggta-knockout costimulation group exhibited significantly reduced relative cell viability （P < 0.001）, increased mean fluorescence intensity of cleaved caspase-3 （P = 0.019）, and a decreased proportion of CD138?IgE? plasmacytoid cells （P < 0.001）. Moreover, compared with the control costimulation group, the Rabggta-knockout costimulation group showed significantly lower protein expression levels of RAB5A, RAB8A, and RAB11A （all P < 0.001）. Conclusions B cell-specific knockout of Rabggta promoted B-cell apoptosis, accompanied by reduced formation of CD138?IgE? plasmacytoid cells and decreased IgE levels, and impaired RAB protein-mediated vesicular secretory pathways, thereby attenuating calcipotriol-induced AD-like skin lesions in mice. Therefore, targeting GGTase Ⅱ may represent a potential novel therapeutic strategy for AD.

Key words: Dermatitis, atopic, Geranylgeranyl transferaseⅡ, B-lymphocytes, Apoptosis, Plasma cells, Immunoglobulin E