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Differential Regulation of IgE and IL-4 Mediated STAT6 Signaling in Allergic Inflammation and Chronic Atopic Dermatitis

알레르기성 염증 및 만성 아토피 피부염에서 IgE와 IL-4 매개 STAT6 신호 전달의 차별적 조절

초록/요약 도움말

Allergic inflammatory diseases, including atopic dermatitis （AD）, are characterized by complex immunological processes involving both early IgE-mediated allergic responses and chronic Th2-dominant inflammation. Although STAT6 signaling has been widely recognized as a key regulator of allergic inflammation, its distinct roles during the early and chronic phases of disease progression remain incompletely understood. Therefore, the present study investigated the stage-specific roles of STAT6 signaling in mast cells and keratinocytes and evaluated the anti-allergic and anti-inflammatory effects of natural product-derived therapeutic candidates targeting these pathways. In Chapter 1, the effects of Paedoksan （PDS）, a traditional herbal prescription, on early allergic immune responses were investigated. PDS did not significantly affect IL-4 production in T cells or innate immune cells, nor did it inhibit IL-4-mediated STAT6 activation in keratinocytes. However, PDS effectively suppressed IgE-mediated mast cell degranulation and attenuated STAT6 phosphorylation under IgE-stimulated conditions in RBL-2H3 cells. Furthermore, PDS ameliorated compound 48/80-induced systemic and local allergic reactions and improved 2,4-Dinitrochlorobenzene （DNCB）-induced AD-like skin lesions in vivo. In addition, PDS reduced serum IgE and IL-4 levels, mast cell infiltration, epidermal hyperplasia, and STAT6 activation in skin tissues. These findings suggest that PDS suppresses early IgE-mediated immune activation and prevents its progression into chronic inflammatory skin diseases. In Chapter 2, dichotomine B （DTB） and dichotomine H （DTH）, diketopiperazine alkaloids derived from Gypsophila oldhamiana, were isolated through bioactivity-guided fractionation and evaluated for their therapeutic effects on chronic atopic dermatitis. DTB and DTH effectively inhibited IL-4-induced phosphorylation of JAK1/3 and STAT6 in keratinocytes and significantly reduced the expression of the STAT6-dependent chemokine CCL26. In addition, both compounds suppressed TSLP expression, eosinophil infiltration, epidermal hyperplasia, and skin barrier dysfunction in a 2,4-Dinitrochlorobenzene （DNCB）-induced AD model. These findings indicate that DTB and DTH attenuate chronic inflammatory responses through suppression of the keratinocyte-associated IL-4–STAT6–CCL26 signaling axis. Taken together, the present study demonstrates that STAT6 signaling serves as a critical regulatory mechanism linking early IgE-mediated allergic responses to chronic atopic dermatitis. Furthermore, stage-specific modulation of STAT6 signaling in mast cells and keratinocytes effectively suppressed both acute and chronic phases of allergic inflammation. These findings provide mechanistic insights into the role of STAT6 in allergic diseases and suggest that targeting STAT6-associated signaling pathways may represent a promising therapeutic strategy for the prevention and treatment of atopic dermatitis and related allergic inflammatory disorders.

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목차 도움말

1. Chapter 1 3
1.1. Introduction 4
1.2. Materials and Methods 10
1.2.1 Paedoksan preparation 10
1.2.2 Cell cultures 11
1.2.3 Animals 12
1.2.4 Quantitative real-time PCR （qPCR） 14
1.2.5 Measurement of IL-4 mRNA expression during T helper2differentiation 16
1.2.6 Measurement of IL-4 mRNA expression induced by the T helper2 immune response 17
1.2.7 Western blot 18
1.2.8 IL-4-mediated STAT6 phosphorylation and CCL26 mRNA expression 19
1.2.9 β-hexosaminidase release 20
1.2.10 Anaphylaxis test 21
1.2.11 Scratching behavior in a localized allergic reaction test 21
1.2.12 IgE-mediated STAT6 phosphorylation 22
1.2.13 Measurement of TEWL 22
1.2.14 Histological analysis 23
1.2.15 Analysis of total IL-4 and IgE in serum 23
1.2.16 Statistical analysis 24
1.3. Results 25
1.3.1 Effect of PDS on IL-4 cytokine production in activation-dependent cytokine-producing T Cells 25
1.3.2 Effect of PDS on Th2 cytokine IL-4 mRNA expression in RBL-2H3 Cells 27
1.3.3 Effect of PDS on IL-4-mediated STAT6 phosphorylation in keratinocytes. 29
1.3.4 Effect of PDS on STAT6-dependent CCL26 expression 31
1.3.5 Effect of PDS on degranulation in RBL-2H3 cells 33
1.3.6 Effect of PDS on anaphylaxis as an early allergic response 35
1.3.7 Effect of PDS compound 48/80-induced local allergic responses 37
1.3.8 Effect of PDS on IgE-mediated STAT6 phosphorylation in RBL-2H3 39
1.3.9 Effect of PDS on DNCB-induced atopic dermatitis in mouse model 42
1.3.10 Effect of PDS on histological change in a DNCB-induced mousemodel 45
1.3.11 Effect of PDS on serum total IgE and IL-4 levels in a DNCB-induced atopic dermatitis model 48
1.3.12 Effect of PDS on STAT6 Phosphorylation in skin tissues of a DNCB-induced atopic dermatitis model 50
1.4. Discussion 52
2. Chapter 2 55
2.1. Introduction 56
2.2. Materials and Methods 60
2.2.1 Plant material and extraction of GRE 60
2.2.2 Fractionation and isolation of DTB and DTH 61
2.2.3 HPLC analysis of GRE 62
2.2.4 Cell cultures 63
2.2.5 Measurement of IL-4 mRNA expression during T helper2differentiation 64
2.2.6 Measurement of IL-4 mRNA expression induced by the T helper2 immune response 65
2.2.7 β-hexosaminidase ＆ Histamine release 66
2.2.8 Western blot 67
2.2.9 IL-4-mediated STAT6 phosphorylation and CCL26 mRNA expression 68
2.2.10 STAT6 reporter gene activity assay 69
2.2.11 Animals 70
2.2.12 Measurement of TEWL and skin hydration 72
2.2.13 Histological analysis 72
2.2.14 Analysis of total IL-4 and IgE in serum 74
2.2.15 Statistical analysis 74
2.3. Results 75
2.3.1 HPLC profiling of GRE evaluation of the effects of isolated compound on IL-4 expression 75
2.3.2 Effects of DTB and DTH on IL-4 cytokine production in activation-dependent cytokine-producing T cells 79
2.3.3 Effects of DTB and DTH on Th2 inflammatory cytokine IL-4 mRNA expression in RBL-2H3 cells 81
2.3.4 Effects of DTB and DTH on degranulation and histamine release in RBL-2H3 cells 83
2.3.5 Effects of DTB and DTH on IL-4-mediated JAK1/3-STAT6 signaling pathway 86
2.3.6 Effects of DTB and DTH on STAT6 activity determined by a STAT6 reporter gene assay 90
2.3.7 Effects of DTB and DTH on CCL26 expression as a downstream target of the IL-4-JAK1/3-STAT6 axis 92
2.3.8 Effects of DTB and DTH on DNCB-induced atopic dermatitis in mice 94
2.3.9 Histological analysis of epidermal hyperkeratosis by H＆E staining in a DNCB-induced mouse model treated with DTB and DTH 98
2.3.10 Histological analysis of immune cell infiltration by toluidine blue and chromotrope 2R staining in a DNCB-induced mouse model treated with DTB and DTH 100
2.3.11 Immunohistochemical analysis of Th2 inflammatory cytokine TSLP following DTB and DTH treatment 103
2.3.12 Effect of DTB and DTH on serum inflammatory cytokines and eosinophil levels in a DNCB-induced mouse model 105
2.4 Discussion 107
3. Summary 111
4. Reference 114
5. Acknowledgement 125

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