학위논문 (박사)-- 서울대학교 대학원 : 의과대학 의학과, 2018. 2. 김미금.Purpose: Extracellular high mobility group box 1 (HMGB1) acts as a damage associated molecular pattern molecule through the Toll-like receptor (TLR) to promote autoreactive B cell activation, which may be involved in the pathogenesis of Sjӧgrens syndrome. Moreover, extracellularly secreted HMGB1 is reported to be involved in the activation of T helper (Th) 17 cells during inflammatory disease, and may also be involved in interleukin (IL)-17 or IL-22 secretion in innate lymphoid cells (ILCs) which have important functions in innate and adaptive immunity. Considering that TLR9 abundantly expressed on B cells and plasmacytoid dendritic cells and main role of reactive B cells and plasma cells that secrete autoantibodies in the pathogenesis of Sjӧgrens syndrome together, we hypothesized that chronic epithelial damage to the cornea or lacrimal gland may contribute to trigger the vicious cycle of inflammation by secreting HMGB1 as a danger signal. Therefore, this study was aimed to investigate the effect of subconjunctival administration of anti-HMGB1 antibodies on dry eye in a mouse model of Sjӧgrens syndrome via evaluation of changes in clinical manifestations of dry eye and immunologic responses.
Methods: NOD.B10.H2b mice were used as primary Sjӧgrens syndrome model, and to investigate inhibitory effect of triggering by HMGB1 in early stage of inflammatory cascades, ten weeks-old mice were chosen based on previous preliminary data. Twenty-four mice were divided into 4 groups and anti-HMGB1 antibodies (0.02, 0.2 and 2 µg/0.1 cc) or phosphate buffered saline (PBS) containing chicken immunoglobulin Y (0.2 µg/0.1 cc) were injected subconjunctivally twice a week for two consecutive weeks.
To evaluate changes in clinical manifestations, tear volume and corneal staining scores were measured and compared between before- and after-treatment. Goblet cell density was counted in periodic acid Schiff (PAS) stained forniceal conjunctiva.
For the evaluation of changes in immunologic responses, flow cytometry was performed to evaluate the changes in BrdU+ cells, IL-17-, IL-10-, or IFNγ-secreting cells, functional B cells, and IL-22 secreting group 3 ILCs in cervical lymph nodes. The level of anti-SSA in plasma and IL-22 in intraorbital glands were also measured using enzyme-linked immunosorbent assay. Inflammatory foci score (>50 cells/focus) was measured in extraorbital glands after CD3 and B220 immunohistochemical staining.
Results: Injection of 2 µg or 0.02 µg anti-HMGB1 antibodies significantly attenuated corneal epithelial erosions (p<0.05). Tear volume significantly increased in groups treated with 2 µg or 0.02 µg anti-HMGB1 antibodies (p<0.05). Although statistical significance was failed to be shown, clinical manifestation of corneal epithelial disruption and tear secretion tended to be improved in the 0.2 µg anti-HMGB1 antibodies treated group. Goblet cell density was increased in 0.2 µg and 2 µg anti-HMGB1-treated-mice with marginal significance.
The percentage and number of BrdU+CD3+T and BrdU+B220+B cells in the draining lymph nodes of treated mice were not significantly altered compared to that in controls. For effector T cell responses, the percentage of Th17 cells and cytotoxic T cells that secret IL-17 were not different and IFNγ-secreting cells were not changed following treatment. For functional B cell responses, no significant changes were found in the percentage of plasma cells or regulatory B cells in the draining lymph nodes. Level of anti-SSA in plasma was not significantly changed. The infiltrating focus of CD3+T cells was very similar to that of B220+B cells in extraorbital gland and scores of inflammatory foci were not different among the groups. Unexpectedly, the percentage of group 3 ILCs was significantly increased in the draining lymph nodes (p<0.05), and the expression of IL-22 was significantly increased in the intraorbital glands (p<0.05) after administration of 2 µg anti-HMGB1.
Conclusion: Subconjunctival administration of anti-HMGB1 improved clinical manifestations of dry eye in NOD.B10.H2b mice. The improvement of dry eye may involve an increase of group 3 ILCs, rather than modulation of B cells or plasma cells, as shown in the present study using a mouse model of Sjӧgrens syndrome.Chapter 1. Introduction 1
1.1. Study Background 1
1.2. Purpose of Research 6
Chapter 2. Materials and Methods 7
2.1. Animals 7
2.2. Mouse model of Sjӧgrens syndrome 7
2.3. Anti-HMGB1 treatment 8
2.4. BrdU proliferation analysis 8
2.5. Phenol red thread test for tear volume measurement 8
2.6. Corneal dye staining 9
2.7. Periodic acid Schiff (PAS) staining for identification of goblet cells 9
2.8. Histopathology 10
2.9. Flow cytometry 10
2.10. Enzyme-linked immunosorbent assay 12
2.11. Statistical analysis 12
Chapter 3. Results 13
3.1. Effect of anti-HMGB1 treatment on the clinical manifestation of dry eye 13
3.2. Changes in immunologic responses in the extraorbital glands, plasma, and draining lymph nodes 15
3.3. Changes in innate lymphoid cell type 3 in the draining lymph nodes 19
Chapter 4. Discussion 21
Chapter 5. Conclusion 26
Bibliography 27
Supporting Information 31
Abstract in Korean 35Docto
