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Distinct Roles of ERAP1 and Tartaric Acid in Immune and Inflammatory Responses in a DSS- Induced Colitis Model

초록/요약

Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by continuous mucosal inflammation of the colon. Its pathogenesis involves genetic, environmental, immune, and microbial factors. Despite the availability of therapeutic intervention, many patients remain unresponsive to treatment, indicating unresolved mechanisms. This study explores the distinct roles of endoplasmic reticulum aminopeptidase 1 (ERAP1) and tartaric acid (TA) in UC pathogenesis using a dextran sulfate sodium (DSS)-induced colitis mouse model. ERAP1, a key enzyme in antigen processing, was examined in wild- type (WT) and ERAP1-deficient (ERAP1+/−) mice. Following DSS induction and treatment with the UC drug sulfasalazine, ERAP1⁺/⁻ mice exhibited mildly increased susceptibility to DSS-induced colitis, with altered immune cell infiltration compared to WT mice. RNA sequencing identified 428 differentially expressed genes between ERAP1⁺/⁻ and WT mice. Among these, 28 genes were associated with colitis or colorectal cancer, of which 11 were upregulated and 17 downregulated in ERAP1⁺/⁻ mice. RT-qPCR confirmed significantly elevated expression of Anxa9, Atp2a1, and Hepacam2 in ERAP1⁺/⁻ mice after sulfasalazine treatment, indicating a differential therapeutic response. TA, a naturally occurring organic acid, elicited dose-dependent eosinophil activation in both healthy and colitis mice. In normal mice, TA administration led to a progressive increase in eosinophil markers (CD11b⁺, SiglecF⁺, and CCR3⁺), accompanied by elevated Th2 cytokines (IL-4, IL-13, IL-31) and IL-17. Moreover, eosinophil-associated genes such as CCL11, IL- 5Rα, and TLR4 were significantly upregulated at higher TA doses. In DSS- induced colitis mice, TA further enhanced IL-13 and IL-5Rα expression, potentially exacerbating eosinophilic inflammation. Collectively, ERAP1 deficiency and high-dose TA enhance susceptibility to DSS-induced colitis via distinct mechanisms: ERAP1 deficiency disrupts immune regulation, inflammation-associated genes, and reduces sulfasalazine efficacy, while TA enhances eosinophilic and Th2- mediated inflammation. These findings identify ERAP1 and TA act as immunological modulators influencing mucosal inflammation and may represent potential targets for understanding or managing UC.

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목차

1 INTRODUCTION 1
1.1 Ulcerative colitis: Overview and challenges 1
1.2 Dextran sulfate sodium- induced colitis: A widely used model 2
1.3 Immune cell dynamics in UC 3
1.4 Eosinophils and their regulatory molecules in colitis 5
1.5 Endoplasmic reticulum aminopeptidase 1 and sulfasalazine: Intersecting roles in immune regulation and colitis therapy 6
1.6 Immunological implications of tartaric acid in inflammation 8
1.7 Study objectives and significance 9
2 MATERIALS AND METHODS 10
2.1 Mice 10
2.2 Generation of ERAP1⁺/⁻ mice 11
2.3 Study design 12
2.4 Sample collection 15
2.5 Flow cytometry analysis 16
2.6 RNA extraction and real-time quantitative PCR 19
2.7 Colonic histopathology 21
2.8 RNA-sequencing (RNA-seq) 21
2.9 Differentially expressed gene (DEGs) analysis 23
2.10 Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses 23
2.11 Morphological observation under transmission electron microscopy (TEM) 24
2.12 Statistical analysis 25
3 RESULTS 26
3.1 ERAP1 regulates inflammatory responses in DSS-induced colitis 26
3.1.1 Impact of ERAP1 on colitis severity and sulfasalazine efficacy in DSS-treated mice 26
3.1.2 Changes in the frequencies of Ly6G+ CD11b+ neutrophils in ERAP1 WT and ERAP1⁺/⁻ mice during sulfasalazine treatment in colitis model 30
3.1.3 ERAP1 modulates the co-stimulatory molecules of DCs in ERAP1WT and ERAP1⁺/⁻ mice under colitis and sulfasalazine treatment 34
3.1.4 Alteration of T cell and NK cell frequencies in ERAP1 WT and ERAP1⁺/⁻ mice under colitis and sulfasalazine treatment 38
3.1.5 DEGs under ERAP1 haploinsufficiency in the intestine tissue 41
3.1.6 Functional enrichment analysis 44
3.1.7 Validation of RNA-Seq results with RT-qPCR 47
3.2 TA enhances eosinophilic and Th2-mediated responses in normal and DSS-induced colitis mice 51
3.2.1 Expression levels of eosinophil activation markers in PBLs of normal mice treated with TA 51
3.2.2 Increased frequencies of SiglecF+ cells in LN of TA-treated mice 54
3.2.3 Upregulation of SiglecF+ and SiglecF+CCR3+ expressions in the spleens of TA-treated mice 57
3.2.4 Increased Th2 cytokine expression in TA-treated mice 60
3.2.5 Changes in IL-5, IL-5Rα, and CCL11 mRNA expression after TA administration to mice 64
3.2.6 Eosinophil identification in LN of TA-treated mice by TEM 68
3.2.7 Effects of TA on DSS-induced colitis in mice 70
3.2.8 mRNA expression of eosinophil-related regulatory molecules after TA administration to mice with colitis 73
3.2.9 Th2 cytokine mRNA expression by TA administration in colitis mice 77
4 DISCUSSION 80
5 CONCLUSION 99
REFERENCES 101
국문초록 118

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