||The Role of Leukocyte Signal Transduction on the Pathogenesis of Secondary Immunodeficiency in Obesity
||Institute of Basic Medical Sciences
肥胖可能導致第二型糖尿病以及心血管疾病等併發症，為世界上重要的公衛健康問題之一。免疫細胞的活化以及全身性的慢性發炎，已知是肥胖引起併發症的重要致病機轉。但是同時，研究指出肥胖患者有免疫力低下並且較容易受到感染的現象。此現象與肥胖患者體內免疫系統較為活化的現象相牴觸。因此，本研究想要藉由研究肥胖患者的白血球在受到 Toll-like receptor (TLR) 刺激之後產生細胞激素的反應，來了解造成肥胖患者免疫力低下的致病機轉。我們發現到肥胖患者的全血細胞在經由 TLR 刺激劑刺激之後所產生的促發炎細胞激素包括 gamma型干擾素、介白素 (interleukin, IL)-6 以及腫瘤壞死因子明顯少於健康對照組，而抑制性細胞激素 IL-10 的產量則有顯著的提升。肥胖患者周邊血單核球 (peripheral blood mononuclear cells, PBMCs) 中的肝醣合成酶激酶-3β (glycogen synthase kinase-3β, GSK-3β) 有較高的磷酸化程度。同時，胰島素或是脂多糖 (lipopolysaccharide, LPS) 的刺激無法促進肥胖患者 PBMC 中 GSK-3β 進一步的磷酸化。另外，我們發現到 LPS 刺激肥胖患者 PBMC所引起的 IκB 降解也較健康受試者低下。為了瞭解這些現象的分子機轉，我們抑制人類單核球細胞株 THP-1 中 GSK-3β 的表現，或是將細胞培養於高胰島素以及高游離脂肪酸的環境，研究細胞在模擬肥胖環境中受到 LPS 刺激後促進發炎的轉錄因子 NF-κB 以及抑制發炎的的轉錄因子 cAMP response element-binding protein (CREB) 的反應。我們發現處於模擬肥胖環境之 THP-1 細胞受到 LPS 刺激後，NF-κB 活化的程度有明顯下降。同時CREB 的活性有明顯的增強。在後續追蹤實驗中，我們發現肥胖患者在經過減重手術後，上述細胞激素分泌能力缺損的現象有明顯的改善。我們另外發現到可能由代謝不正常的肥胖所引起之第二型糖尿病病患也有類似於肥胖病患的不正常先天性免疫反應。經由本研究，我們得知肥胖引起的不正常代謝在第二型糖尿病發病之前，會因為GSK-3β 的過度磷酸化而影響免疫細胞在感染的情況下細胞激素之產生，造成自然免疫反應的繼發型缺損。
Obesity is a severe health problem worldwide which leads to multiple comorbidities including type 2 diabetes mellitus (DM) and cardiovascular diseases. Inflammation has been found to be an important characteristic of adipose tissue in obese subjects. However, obesity is also associated with compromised immune responses to infections and the impact of obesity on immune function has not been fully understood. To clarify the role of obesity in the immune responses, I investigated the Toll-like receptor (TLR)-induced cytokine secretion by leukocytes from obese and lean subjects. The relationship between insulin-induced intracellular signaling and cytokine production using peripheral blood mononuclear cells (PBMC) and a monocytic cell line THP-1 was also investigated. I found decreased TLR-induced interferon-gamma, interleukin-6 and tumor necrosis factor-alpha secretions and elevated IL-10 secretion by leukocytes from obese subjects when compared with those in lean controls. PBMCs from obese subjects showed enhanced basal Akt and glycogen synthase kinase 3β (GSK-3β) phosphorylation which did not further increased with insulin and lipopolysaccharide (LPS) stimulation. I also found that LPS-induced IκB degradation was inhibited in PBMCs from obese subjects. By using THP-1 cells with GSK-3β knockdown or cells treated with hyperinsulinemic and high fatty acid conditions, I found that LPS-induced NF-κB activation was inhibited and cAMP response element-binding protein (CREB) activation was enhanced. I also found that bariatric surgery corrected the abnormal TLR-induced cytokine secretions in obese subjects. Moreover, type 2 DM, which may be developed from obesity, also showed abnormal TLR-induced cytokine secretions. These findings indicated that GSK-3β is important in the regulation of NF-κB and CREB activation in leukocytes under the metabolic condition of obesity. Our study hence reveals a key mechanism through which metabolic abnormalities, even before the onset of type 2 DM, compromise leukocyte functions in people with obesity.
Table of contents VI
Figure list IX
Table list XI
Chapter 1: Introduction 1
1-1 The paradox: chronic immune activation and susceptibility to infections in obesity 3
1-1-1 The characteristic metabolic abnormality in obesity 3
1-1-2 Chronic inflammation in obesity 4
1-1-3 Aberrant leukocyte responses and increased incidence of infection in obesity 7
1-1-4 Secondary immunodeficiency in type 2 DM. 9
1-1-5 Weight loss interventions reduce abnormal immune conditions in obesity 11
1-2 Innate immune defense against infections 13
1-2-1 TLR-induced cytokine secretion 13
1-2-2 NADPH oxidase-induced ROS production 14
1-2-3 Regulation of innate immunity by GSK-3β and IL-10 16
1-3 The complex interaction between obesity and immunity 19
Chapter 2: Objective of this study 20
Chapter 3: Materials and methods 21
3-1 Subjects of lean and obese groups 21
3-2 Reagents and antibodies 22
3-3 TLR-induced cytokine secretion by stimulated blood cells 22
3-4 PBMCs preparation and treatment 23
3-5 BSA-Palmitate preparation 24
3-6 Cell culture and treatment 24
3-7 ROS production measured by Chemiluminescence 25
3-8 ROS production measured by flow cytometry 25
3-9 SDS-PAGE and Western blot analysis 26
3-10 Lentiviral targeting GSK-3 for gene knockdown 26
3-11 Immunofluorescence analysis 27
3-12 Chromatin immunoprecipitation (ChIP) 27
3-13 Statistics 29
Chapter 4: Phosphorylation of GSK-3β in metabolically abnormal obesity affects immune stimulation-induced cytokine production 30
4-1 Demographic and clinical characteristics of the lean and obese study groups 30
4-2 Decreased IFN-gamma, IL-6, TNF-alfa and increased IL-10 secretion by TLR-stimulated whole blood samples from obese subjects 34
4-3 Correlation between TLR-induced IL-6 and IL-10 secretion with BMI and serum insulin concentration 36
4-4 Mononuclear leukocytes of obese subjects had elevated basal Akt and GSK-3β phosphorylation which did not further increase after insulin and LPS stimulation 39
4-5 Decrease in LPS-induced IκB degradation in PBMCs of obese subjects and in monocytic cells treated with high-insulin and high-free fatty acid conditions 43
4-6 Inhibition of GSK-3β decreased LPS-induced NF-kB activation but increased CREB activation 46
4-7 Basal phosphorylated GSK-3β level was enhanced in splenocytes of ob/ob mice. 55
4-8 Discussion 56
Chapter 5: Innate immune functions in obese subjects with bariatric surgery and in subjects with type 2 DM 61
5-1 Bariatric surgery corrected TLR-induced cytokine secretions of obese subjects 61
5-2 Whole blood leukocytes isolated from type 2 DM patients had lower cytokine production ability compared with control subjects 65
5-3 High glucose conditions inhibited PMA-induced ROS production in differentiated HL-60 cells. 69
5-4 There were no differences in granulocyte ROS production between lean and obese subjects 70
5-5 Discussion 71
Chapter 6: Conclusion and perspectives 74
Reference List 77
Curriculum Vitae 94
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