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Development of Immune Modulatory Adjuvants for Antigen Presenting Cells and Immunological Role of CD30 in Adaptive Immunity

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Author(s)
김미은
Issued Date
2014
Abstract
The immune response is a host defense mechanism against foreign pathogens and tumors. Immunity is composed of two major responses, the "innate immune response" and “adaptive immune response". The innate immune response is non-specific. For example, inflammation is an innate immunological response induced by macrophages, which is characterized by fever, vasodilatation, and inflammatory cytokine production. In addition, the innate immune response plays a role in presenting antigenic information to the adaptive arm of the immune response, which induces the antigen-specific immune response. Dendritic cells (DCs) are powerful antigen presenting cells (APCs) that present antigen-specific peptides to T cells. In contrast to the innate immune response, the adaptive immune response is antigen-specific, and is divided into cell-mediated and humoral immunity. Many studies have shown that these immune responses are tightly associated with health; however, aberrant immune responses are associated with many types of disease including diabetes, sepsis, cardiovascular disease, allergy, and cancer. It has been suggested that these immune-related diseases might be cured or prevented through modulation of the immune response. Therefore, many researchers have attempted to understand the regulation of immunological functions in various aspects. The two major ways to regulate the immunological functions are via chemical modulation and genetic modulation. The present study identified new immunomodulatory chemicals and genetic regulators for innate immunity and adaptive immunity.
Sargassum horneri (Turner) C. Agardh is a brown algae and galangin is a member of the flavonols found in Alpinia officinarum. In the present study, ethanolic extracts of S. horneri (Turner) C. Agardh (ESH) and galangin exhibited anti-inflammatory effects on RAW 264.7 macrophages in vitro. Treatment with ESH and galangin resulted in reduced nitric oxide (NO) production as well as reduced expression of pro-inflammatory genes such as interleukins, inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-stimulated macrophages. These anti-inflammatory activities were mediated by the mitogen-activated protein kinase (MAPK) and nuclear factor-B (NF-B) pathways.
Baicalin is a flavonoid found in Scutellaria baicalensis, and 18β-glycyrrhetic acid (β-GA) is a triterpenoid compound derived from Licorice root. The present study investigated whether baicalin and β-GA could influence surface molecule expression, antigen uptake capacity, and cytokine production on DCs, and induce subsequent T cell differentiation. Baicalin and β-GA significantly suppressed the expression of surface molecules, CD80, CD86, major histocompatibility complex (MHC) class I, and MHC class II and reduced the levels of IL-12 production in LPS-stimulated DCs. Moreover, baicalin or β-GA-treated DCs showed an impaired induction of the T helper type 1 (Th1) immune response. These findings provided clues to important immunopharmacological functions of baicalin and β-GA, which have ramifications for the development of therapeutic adjuvants for the treatment of DC-related acute and chronic diseases.
CD30, also called tumor necrosis factor receptor superfamily (TNFRSF) 8, is a co-stimulatory molecule of immune cells. In a previous study, CD30 was shown to be expressed on lymphoid cells such as lymphoma cells, and activated T cells and B cells. Although CD30 is a marker for several diseases including lymphoma and allergy, it is also related to helper type 2 (Th2) responses in adaptive immunity. The present study investigated the immunological role of CD30 in CD4+ T cells and tumor immunity using CD30-/- mice. CD30 deficiency did not affect immune cell populations or DC maturation. However, the lack of CD30 caused CD4+ T cell proliferation and activation, and decreased survival of the MC-38 murine colon cancer tumor metastasis model. These data indicate that CD30 is involved inCD4+ T cell function and tumor immunity.
Taken together, it was concluded that various natural compounds could regulate the immunological functions of APCs such as macrophages and DCs through modulation of immune response-related gene expression, expression of co-stimulatory molecules, and pro-inflammatory cytokine production. Furthermore, the regulation of DCs resulted in T cell polarization, which would influence the fate of the adaptive immune response. In addition, CD30 deficiency was shown to suppress T cell activation and anti-tumor immunity. Our findings suggest that ESH, galangin, baicalin, and β-GA have potential as immunological therapeutic adjuvants, while CD30 might be a genetic modulator of adaptive immunity.|면역반응은 외부로부터 침입한 항원 또는 종양으로부터 신체를 보호하기 위한 숙주의 방어기작으로, 선천면역반응과 적응면역반응 두 가지로 구분된다. 선천면역반응은 비특이적 면역반응으로, 대식세포에 의해 유발되는 염증반응은 대표적인 면역학적 징후이며 열, 혈관확장, 염증성 사이토카인의 생성을 동반한다. 또한, 선천면역반응에서는 항원 특이적인 면역반응을 위하여 적응면역반응에 항원의 정보를 제공한다. 수지상세포는 강력한 항원제시세포이며 T 세포에 항원특이성에 대한 정보를 제공한다. 선천면역반응과 달리 적응면역반응은 항원 특이적 면역반응으로 세포매개성 면역반응과 체액성 면역반응으로 나뉜다. 현재까지, 수많은 연구를 통해서 이러한 면역반응들이 인체의 건강과 당뇨, 패혈증, 심혈관질환, 알러지, 종양 등 다양한 질병과 밀접한 연관이 있다고 알려져 왔으며, 이러한 질병은 면역반응을 조절함으로써 완화될 수 있다고 보고 있다. 따라서 많은 사람들은 다양한 측면에서 면역반응을 조절할 수 있는 방법을 찾고자 하였으며, 크게 화학적 조절방법과 유전적 조절방법으로 볼 수 있다. 본 연구에서는 새로운 선천면역반응과 적응면역반응에 대한 새로운 화학적 조절인자와 유전적 조절인자를 찾고자 하였다.
괭생이모자반은 갈조류에 속하는 해조류이며 galangin은 양강에서 유래된 flavonol의 일종이다. 본 연구에서는 쥐의 대식세포인 RAW 264.7 세포주를 이용하여 괭생이모자반의 에탄올 추출물(ESH)와 galangin의 항염증 효과를 확인하였다. ESH와 galangin은 각각 200 μg/ml 과 50 μM까지 대식세포에 독성을 나타내지 않았으며 지질다당체 (LPS)가 처리된 대식세포에서 염증의 지표인 일산화질소뿐만 아니라 인터루킨, inducible nitric oxygenase (iNOS), cyclooxygenase (COX-2)의 생성을 저해하였다. 이러한 항염증성 활성은 mitogen-activated protein kinase (MAPK) 신호전달 경로 억제를 통해서 나타남을 확인하였다.
Baicalin은 황금에서 유래되는 flavonoid의 일종이며 18β-Glycyrrhetic acid는 감초에서 유래되는 triterpenoid의 일종이다. 본 연구에서는 bacalin과 β-GA가 수지상세포의 세표표면분자 발현, 항원포식능, 사이토카인 생성능과 T 세포 분화 유도능에 영향을 미칠 수 있는지 확인하였다. Baicalin과 β-GA는 LPS에 의해 자극된 수지상세포에서 세포표면 분자인 CD80, CD86, MHC class I, MHC class II의 발현뿐만 아니라 IL-12의 생성능을 억제하였다. 또한 baicalin과 β-GA는 수지상세포의 Th1 면역반응을 유도할 수 있는 능력을 저해하였다. 이러한 연구 결과는 baicalin과 β-GA가 수지상세포에 의해 매개되는 과민성 면역질환에 대하여 치료용 보조약물로 개발될 수 있는 가능성을 제시하였다.
CD30은 TNRFSF8이라고도 알려져 있으며 면역세포 표면에 발현되는 공동자극인자로 알려져 있다. 기존에 보고된 바에 의하면, CD30은 주로 림프종, 활성화된 T 세포, B 세포 등 림프계 세포에 주로 발현되는 것으로 알려져 있다. CD30이 림프종과 다양한 질환의 지표로 알려져 있음에도 불구하고 Th2 면역반응에도 밀접한 연관이 있다고 알려져 있다. 본 연구에서는 CD30결핍 마우스를 통해 CD4+ T 세포와 종양면역에 대한 CD30의 면역반응 기능에 대하여 확인하였다. CD30의 결핍은 비장에 존재하는 면역세포들의 수와 LPS를 통한 수지상세포의 활성에는 영향을 미치지 않았다. 그러나 CD30의 결핍은 CD4+ T 세포의 증식과 활성을 저해하는 효과를 나타내었고 쥐의 대장암세포인 MC-38을 주입한 마우스에서 정상 쥐에 비하여 생존율이 낮아지는 효과를 나타내었다. 이러한 결과는 CD30이 CD4+ T 세포의 활성과 종양면역에 관련이 있으며 CD30이 결핍될 경우, CD4+ T cell의 활성이 억제됨을 통해서 종양세포에 더 취약해짐을 알 수 있다.
결론적으로, 다양한 천연물은 면역관련 유전자, 공동자극인자, 사이토카인 생성 등의 조절을 통해서 대식세포와 수지상세포와 같은 항원제시세포의 면역학적 기능을 조절할 수 있다. 더욱이 수지상세포 조절은 T 세포의 극성을 결정하는 능력을 가지며, 이는 적응면역반응의 방향을 결정하는데 중요한 요소이다. 또한, 공동자극인자인 CD30의 결핍은 T 세포의 활성과 항종양 면역반응을 저해하는 효과를 가짐을 관찰 할 수 있다. 이러한 연구 결과들은 ESH, galangin, baicalin과 β-GA와 같은 천연물이 면역학적 치료보조제로써의 가능성을 가지며 CD30 분자가 적응면역반응에서 유전적 조절인자가 될 수 있음을 제시한다.
Alternative Title
항원제시세포의 면역조절 보조제 발굴과 적응면역반응에서 CD30의 면역학적 역할 규명 연구
Alternative Author(s)
Kim, Mi Eun
Department
일반대학원 생명과학
Advisor
이준식
Awarded Date
2015-02
Table Of Contents
I. INTRODUCTION..................................................1
1. Immunity...........................................................1
2. Innate immunity (Inflammation)............................3
3. Antigen Presenting Cells.....................................7
(1) Macrophage.....................................................7
(2) Dendritic cell.....................................................8
4. Adaptive Immunity..............................................10
(1) T cell..............................................................12
(2) APC - T cell interaction......................................12
II. MATERIALS AND METHODS...................................15
(1) Reagents..........................................................15
(2) Extraction of Sargassum horneri (Turner) C. Agardh...15
(3) Cell culture.....................................................15
(4) EHS treatment....................................................16
(5) Galangin treatment............................................16
(6) Cell viability assay...............................................16
(7) NO assay.............................................................17
(8) Reverse transcription (RT)-PCR....................17
(9) Quantitative real-time PCR................................18
(10) Western blot analysis..........................20
(11) Enzyme-linked immunosorbent assay (ELISA)........22
(12) Animals and chemicals.......................................22
(13) Reagents and Abs............................................22
(14) Generation and culture of DCs...............................23
(15) Stimulation of DC by β-GA.....................................24
(16) Stimulation of DC by baicalin...........................24
(17) Flow cytometric analysis....................................24
(18) Cytokines assay..........................................25
(19) Mixed Lymphocyte Reaction.................................26
(20) Experimental lung metastasis model and survival study..26
(21) T cell stimulation and intracellular cytokine staining...27
(22) Statistical analysis...................................27
III. RESULTS.........................................................28
Part I. Immune modulatory adjuvant for antigen presenting cells

1. Anti-inflammatory effects of ethanolic extract from Sargassum horneri (Turner) C. Agardh on lipopolysaccharide-stimulated macrophage activation via NF-κB pathway regulation....................28
a. Introduction...........................................................28
b. Results................................................31
(1) ESH inhibits NO production in LPS-stimulated macrophages.31
(2) ESH suppresses the expression of pro-inflammatory genes..34
(3) ESH inhibits LPS-induced activation of ERK.............37
(4) ESH attenuates LPS-stimulated NF-κB activation......37
c. Discussion..........................................................41

2. Anti-inflammatory effects of galangin on lipopolysaccharide-activated macrophages via ERK and NF-κB pathway regulation...................................44
a. Introduction.................................................44
b. Results........................................................47
(1) Galangin inhibits NO production in LPS-stimulated macrophages......................................................47
(2) Galangin suppresses the expression of pro-inflammatory factors(3) Galangin inhibits LPS-induced ERK and NF-κB-p65 phosphorylation..............................51
(4) Galangin attenuates IL-1β mRNA expression levels...59
c. Discussion......................................................63

3. 18β-Glycyrrhetinic acid from licorice root impairs dendritic cells maturation and Th1 immune responses.....65
a. Introduction..........................................................65
b. Results.............................................................68
(1) β-GA inhibits LPS-stimulated DCs maturation........................68
(2) β-GA impairs IL-12 secretion, and does not influence IL-10 production during LPS-stimulated DCs maturation..................71
(3) β-GA suppresses endocytic activity of DCs.............................74
(4) β-GA inhibits the capacity of DCs to induce Th1 response.....77
c. Discussion.....................................................80

4. Baicalin from Scutellaria baicalensis impairs Th1 polarization through inhibition of dendritic cell maturation....................................................83
a. Introduction................................................83
b. Results........................................................86
(1) Baicalin inhibits LPS-induced DCs maturation......86
(2) Baicalin impairs the secretion of IL-12 during LPS-induced DCs maturation................................................89
(3) Baicalin enhances the immature state of DCs with high endocytic capacity..................................................92
(4) Baicalin impairs DC-mediated induction of Th1 polarization...95
c. Discussion.........................................98

Part II. Genetical regulation of adaptive immunity
1. CD30 deficiency impairs anti-tumor activity through CD4+ T cell response..................................................102
a. Introduction..................................................102
b. Results..............................................................104
(1) CD30 deficiency has normal immune cells populations..........104
(2) CD30 deficiency has no effect on DCs maturation..................109
(3) CD30 deficiency impairs CD4+ T cell proliferation and activation..................................................................111
(4) CD30 deficiency is vulnerable to cancer metastasis..115
c. Discussion..........................................................117

IV. CONCLUSION................................................119

V. REFERENCES.................................................123
Degree
Doctor
Publisher
조선대학교
Citation
김미은. (2014). Development of Immune Modulatory Adjuvants for Antigen Presenting Cells and Immunological Role of CD30 in Adaptive Immunity.
Type
Dissertation
URI
https://oak.chosun.ac.kr/handle/2020.oak/12346
http://chosun.dcollection.net/common/orgView/200000264557
Appears in Collections:
General Graduate School > 4. Theses(Ph.D)
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