Study of Type I interferon responses during Orientia tsutsugamushi infection
쯔쯔가무시균 감염에 의한 제 1형 인터페론 반응에 대한 연구

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dc.description학위논문 (박사)-- 서울대학교 대학원 : 의과학과, 2016. 8. 조남혁.-
dc.description.abstractType I Interferons (TI IFNs) induced by microbial infections play a pivotal role in protective immune responses. Recently, various bacterial pathogens have been shown to cause TI IFN production. However, the effect of TI IFNs induced by bacteria on immune responses is quite controversial. Orientia tsutsugamushi, an obligate intracellular bacterium that causes scrub typhus, can also induce TI IFNs during mammalian infection, but the effect of TI IFNs on immune responses against O. tsutsugamushi remains elusive. Here, I studied the molecular details of the induction of TI IFNs during O. tsutsugamushi infection in vitro and the role of TI IFNs in generating adaptive immune responses against the bacterial pathogen in vivo. Induction of TI IFNs during O. tsutsugamushi infection was consistently observed in mouse embryonic fibroblasts (MEF) and bone marrow-derived macrophages (BMDM). Using various MEF and BMDM derived from specific transgenic knockout (KO) mice, I searched for signaling molecules required for induction of TI IFNs by O. tsutsugamushi infection. Studies showed that IKK-, RIG-I, cGAS, MAVS, and STING are required for the induction of TI IFNs, but signaling adaptors involved in TLR and NOD signaling are dispensable. In vitro replication of O. tsutsugamushi in MEF or BMDM deficient in a gene encoding the TI IFN receptor (IFNAR) did not significantly change when compared to wild type cells. In addition, the survival rate of TI IFNAR KO mice lethally challenged with O. tsutsugamushi was similar to that of wild type mice, indicating that T1 IFN responses do not play a significant role in protective immunity during primary infection of O. tsutsugamushi. Nevertheless, it is interesting to note that early antibody responses against 56 kD type specific antigen (TSA56), a major outer membrane protein, were significantly delayed in IFNAR KO mice when compared to wild type. These findings correlate with a delayed alteration of follicular architecture in the spleen as well as a delay in active differentiation of follicular helper T (TFH) cells in the spleen of IFNAR KO mice infected with O. tsutsugamushi, suggesting that T1 IFNs may affect the generation of specific antibody responses via promoting the differentiation of naïve CD4+ T cells into TFH cells in secondary lymphoid organs. Also, CD8+ memory T cell responses against O. tsutsugamushi antigen are significantly reduced in IFNAR KO mice whereas CD4+ memory T cell responses remain intact. Therefore, T1 IFNs produced by O. tsutsugamushi infection do not significantly affect protective immunity during primary infection, but they might promote the generation of specific adaptive immune responses by supporting the differentiation of TFH cells as well as enhancing the generation of CD8+ memory T cell responses.-
dc.description.tableofcontentsIntroduction 1

Materials and Methods 9
1. Ethics statement 9
2. Mice 9
3. Cell Culture 9
4. Preparation of mouse embryonic fibroblasts 10
5. Generation of bone marrow derived macrophages 10
6. Preparation of O. tsutsugamushi 11
7. Immunofluorescence assay 12
8. siRNA knockdown 12
9. Immunoblot analysis 13
10. RNA purification and RT-qPCR 13
11. Purification of Recombinant antigen 15
12. Enzyme-linked immunosorbent assay 15
13. Type I IFN bioassay and luciferase reporter assay 16
14. Flow cytometry 16
15. Fluorescent Immunohistochemisty 17
16. Statistical analysis 18

Results 19
Induction of T1 IFNs by O. tsutsugamushi infection 19
Induction of T1 IFN by O. tsutsugamushi is mediated by IKKα-dependent pathway 21
MyD88, TRIF, and RIP2 are dispensable for T1 IFN induction by O. tsutsugamushi in BMDM 23
Induction of T1 IFN by O. tsutsugamushi is mediated by MAVS or STING-mediated pathways 25
RIG-I and cGAS are the potential sensors for the induction of T1 IFNs in host cells infected with O. tsutsugamushi 27
Replication of O. tsutsugamushi is not significantly affected in IFNAR KO cells 29
The mortality and morbidity of mice infected with O. tsutsugamushi are not significantly changed in the absence of IFNAR 31
Lymphocyte responses in IFNAR KO mice infected with O. tsutsugamushi 33
Antibody responses in IFNAR KO mice infected with O. tsutsugamushi 38
Memory T cell responses in IFNAR KO mice infected with O. tsutsugamushi 42

Discussion 45

References 53

국문 초록 70
dc.format.extent1210766 bytes-
dc.publisher서울대학교 대학원-
dc.subjectOrieintia tsutsugamushi-
dc.subjectType I interferon-
dc.titleStudy of Type I interferon responses during Orientia tsutsugamushi infection-
dc.title.alternative쯔쯔가무시균 감염에 의한 제 1형 인터페론 반응에 대한 연구-
dc.contributor.AlternativeAuthorMin Chan-ki-
dc.contributor.affiliation의과대학 의과학과-
Appears in Collections:
College of Medicine/School of Medicine (의과대학/대학원)Dept. of Biomedical Sciences (대학원 의과학과)Theses (Ph.D. / Sc.D._의과학과)
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