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The Role of House Dust Mite Derived Chitin as an Adjuvant in Airway Inflammation Induced by Ovalbumin
난알부민으로 유도한 기도염증에서 집먼지진드기로부터 추출한 키틴의 면역보강제로서의 역할

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dc.contributor.advisor조상헌-
dc.contributor.author이상민-
dc.date.accessioned2017-07-14T01:16:14Z-
dc.date.available2017-07-14T01:16:14Z-
dc.date.issued2014-08-
dc.identifier.other000000020755-
dc.identifier.urihttp://hdl.handle.net/10371/121788-
dc.description학위논문 (박사)-- 서울대학교 대학원 : 협동과정임상약리학전공, 2014. 8. 조상헌.-
dc.description.abstract서론: 집먼지진드기는 가장 흔한 흡입항원으로서 종종 다른 흡입항원에 대한 알레르기 반응을 촉진시키기도 한다. 집먼지진드기 외골격의 주성분은 키틴(chitin)으로서, 자연계에서 2번째로 흔한 다당질(polysaccharide)이다. 최근 키틴은 톨유사수용체2(toll-like receptor 2: TLR2)를 통해 Th1, Th2, Th17 적응면역반응을 일으키는 것으로 알려졌다. 또한 이전 연구에서 종양괴사인자알파(tumor necrosis factor-alpha: TNF-α)와 자연살해T세포(natural killer T cell: NKT cell)는 흡입항원에 대한 Th2 면역반응에서 중요한 역할을 하는 것으로 보고되었다. 이번 연구에서는 집먼지진드기가 다른 흡입항원에 대한 적응면역반응을 촉진시키는 것은 그 구성성분이면서 다양한 면역반응을 일으키는 면역보강제인 키틴 때문이라는 가설을 검증하고자 하였으며, 이를 위해 감작 단계에서 난알부민과 함께 집먼지진드기 혹은 집먼지진드기유래키틴(HDM-derived chitin)을 마우스에게 투여하였다. 또한 톨유사수용체2, 종양괴사인자알파, CD1d가 결여된 마우스를 이용하여 톨유사수용체2, 종양괴사인자알파, 자연살해T세포의 역할을 이러한 마우스 모델에서 각각 평가하고자 하였다.
방법: C57BL/6 배경의 야생형 마우스와 톨유사수용체2, 종양괴사인자알파, CD1d가 각각 결여된 마우스를 난알부민 75 μg과 함께 집먼지진드기 혹은 집먼지진드기유래키틴(HDM-derived chitin) 100 μg으로 감작시킨 후 난알부민 50 μg으로 유발시켰다. 또한, 야생형 마우스에서는 난알부민 75 μg과 함께 키틴분해효소(chitinase)로 처리한 집먼지진드기 혹은 집먼지진드기유래키틴 100 μg으로 감작을 유도하는 실험을 추가적으로 시행하였다.
결과: 집먼지진드기와 집먼지진드기유래키틴은 기도염증과 함께 Th1, Th2, Th17 면역반응의 모든 싸이토카인의 발현과 혈청내 난알부민 특이 IgE, IgG1, IgG2a 항체의 농도를 증가시켰다. TLR2가 결여된 마우스에서는 모든 면역반응과 함께 혈청내 난알부민 특이 IgE, IgG1, IgG2a 항체의 농도가 감소하였다. 종양괴사인자알파가 결여되거나 키틴분해효소를 처리한 경우에는 Th2 싸이토카인의 발현과 혈청내 난알부민 특이 IgE 항체의 농도는 감소하는 반면에 Th1, Th17 싸이토카인의 발현과 혈청내 난알부민 특이 IgG, IgG2a 항체의 농도는 유지되거나 더욱 증가하였다. CD1d가 결여된 마우스에서는 면역조절세포 반응과 관련된 것으로 알려진 IL-10와 난알부민 특이 IgG2a 항체를 제외하고, 모든 면역반응과 함께 혈청내 난알부민 특이 항체의 농도가 감소하였다.
결론: 집먼지진드기가 다른 흡입항원에 대한 적응면역반응을 촉진시키는 것은 그 구성성분이면서 광범위한 면역보강제인 키틴을 통해서 이루어진다. 키틴은 톨유사수용체2, 종양괴사인자알파, 그리고 자연살해T세포를 통해 집먼지진드기와 함께 흡입하는 다른 항원에 대한 Th1, Th2, Th17 면역반응을 모두 일으킨다.
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dc.description.abstractIntroduction: House dust mite (HDM), the most common inhalant allergen, sometimes promotes allergic response to other inhalant allergens. The exoskeleton of HDM mainly consists of chitin which is the second-most abundant polysaccharide in nature. Recently, chitin was reported to be a potent adjuvant in Th1-, Th2- and Th17- type adaptive immune responses through toll-like receptor 2 (TLR2). In previous study, tumor necrosis factor – alpha (TNF-α) and natural killer T (NKT) cell were observed to be important in the development of Th2 cell response to inhaled allergens. The objective of this study is to verify our hypothesis that HDM promotes adaptive immune response to other inhalant allergen through its component, chitin, a multifaceted adjuvant, administrating intranasally ovalbumin (OVA) with HDM or HDM-derived chitin to mouse during sensitization. We also evaluated the role of TLR2, TNF-α and NKT cell in this murine model, using TLR2-, TNF-α-, and CD1d- deficient mice, respectively.
Methods: Wild-type (WT) mice (C57BL/6 background), TLR2-, TNF-α-, and CD1d-deficient mice were sensitized intranasally with 75 μg of OVA and 100 μg of HDM or HDM-derived chitin, then challenged intranasally with 50 μg of OVA. WT mice were also sensitized intranasally with 75 μg of OVA and 100 μg of HDM or HDM-derived chitin in the presence of chitinase.
Results: HDM and HDM-derived chitin promoted airway inflammation and all of Th1, Th2, and Th17 immune response to OVA as well as the serum level of OVA-specific IgE, IgG1, and IgG2a. In the absence of TLR2, all types of immune responses and serum level of OVA-specific IgE, IgG1, and IgG2a were diminished. In the absence of TNF-α or in the presence of chitinase, the expression of Th2 cytokines and the serum level of OVA-specific IgE were alleviated, while the expression of Th1 and Th17 cytokines and the serum level of OVA-specific IgG1 and IgG2a were preserved or more enhanced. In the absence of CD1d, all types of immune responses and serum level of OVA-specific antibodies were diminished, except IL-10 and serum OVA-specific IgG2a which are known to relate to regulator T cell response.
Conclusion HDM promotes adaptive immune response to other inhalant allergen through its component, chitin which is a multifaceted adjuvant. Chitin stimulates all of Th1, Th2, and Th17, but mainly Th2 immune response to concomitantly inhaled other aeroallergen through TLR2, TNF-α and NKT cell.
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dc.description.tableofcontentsI. Introduction ---------------------------------- 1


II. Materials and Methods ------------------------- 4
2.1 Mice --------------------------------------------- 4
2.2 Reagents ----------------------------------------- 4
2.3 Purification of chitin from HDM and FTIR analysis --------------------------------------------------------- 4
2.4 Generation of murine model ----------------------- 5
2.5 Cellularity in bronchoalveolar lavage fluid ------ 6
2.6 Macrophage preparation from peritoneum and alveolus ------------------------------------------------------- 6
2.7 Single-cell preparation from lung and lung-draining lymph nodes ------------------------------------------- 6
2.8 Lung tissue histology ---------------------------- 7
2.9 Immune response in the lung and lung-draining lymph nodes ------------------------------------------------- 7
2.10 Cytokine measurement ---------------------------- 8
2.11 OVA specific antibody level evaluation ---------- 8
2.12 Fluorescence-activated cell sorting analyses ---- 8
2.13 Statistical analyses ---------------------------- 9

III. Results ----------------------------------------- 10
3.1 House dust mites promotes innate and adaptive immune response to other inhalant allergens through their component, chitin, a multifaceted adjuvant ----------- 10
3.2 The isolation of chitin from house dust mite and the determination of adequate chitin’s dose for inducing airway inflammation ---------------------------------- 12
3.3 House dust mite derived (HDM-derived) chitin induces airway inflammation and innate immune responses to inhalant allergen ------------------------------------ 13
3.4 House dust mite derived (HDM-derived) chitin induces Th1, Th2, and Th17 adaptive immune responses to inhalant allergen, which was reversed by chitinase ------------ 15
3.5 House dust mite derived (HDM-derived) chitin induces Th1, Th2, and Th17 adaptive immune responses to inhalant allergen via toll-like receptor 2 (TLR2)---------------17
3.6 The main adaptive immune response to inhalant allergen induced by house dust mite derived (HDM-derived) chitin is Th2 rather than Th1 or Th17, and it is mediated by TNF-α signaling ---------------------------------- 19
3.7 House dust mite derived (HDM-derived) chitin induces Th1, Th2, and Th17 adaptive immune responses to inhalant allergen though natural killer T (NKT) cell ---------- 21

IV. Discussion --------------------------------------- 24

V. References ---------------------------------------- 31

VI. Figure legends ----------------------------------- 40

VII. Figure ------------------------------------------ 49

VIII. Korean abstract -------------------------------- 62
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dc.formatapplication/pdf-
dc.format.extent1895656 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjecthouse dust mite-
dc.subjectchitin-
dc.subjectadjuvant-
dc.subjecttoll-like receptor 2-
dc.subjecttumor necrosis factor–alpha-
dc.subjectnatural killer T cell-
dc.subject집먼지진드기-
dc.subject키틴-
dc.subject면역보강제-
dc.subject톨유사수용체2-
dc.subject종양괴사인자알파-
dc.subject자연살해T세포-
dc.subject.ddc615-
dc.titleThe Role of House Dust Mite Derived Chitin as an Adjuvant in Airway Inflammation Induced by Ovalbumin-
dc.title.alternative난알부민으로 유도한 기도염증에서 집먼지진드기로부터 추출한 키틴의 면역보강제로서의 역할-
dc.typeThesis-
dc.contributor.AlternativeAuthorSang Min Lee-
dc.description.degreeDoctor-
dc.citation.pagesxi, 64-
dc.contributor.affiliation의과대학 협동과정임상약리학전공-
dc.date.awarded2014-08-
Appears in Collections:
College of Medicine/School of Medicine (의과대학/대학원)Program in Clinical Pharmacology (협동과정-임상약리학전공)Theses (Ph.D. / Sc.D._협동과정-임상약리학전공)
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