微生物組中的細菌作為治療人類疾病的生物療法

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姓名

楊火龍(Albert Jackson Yang) 查詢紙本館藏

畢業系所

生醫科學與工程學系

論文名稱

微生物組中的細菌作為治療人類疾病的生物療法
(Bacteria in the microbiome as biotherapeutics for treatments of human diseases)

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摘要(中)

在青春痘痤瘡治療上，若使用抗生素可能會同時破壞對人體有益的微生物，這些微生物能有效對抗痤瘡丙酸桿菌（C. acnes），其是一種與炎症相關之尋常性痤瘡的細菌，但直接利用活表皮葡萄球菌（S. epidermidis）應用在開放性痤瘡病變部位的益生菌治療可能會導致血液感染。於是，我們製造了聚碸微管陣列膜（PSF MTAM）來封裝益生菌表皮葡萄球菌，使得表皮葡萄球菌在 PSF MTAM 中增強了對甘油的發酵活性。為了模擬肉芽腫類型的痤瘡炎症性尋常痤瘡，將小鼠的耳朵皮內注射痤瘡丙酸桿菌以誘導巨噬細胞炎症蛋白-2 (MIP-2) 的分泌，這是對應人類白細胞介素 (IL)-8 的小鼠分泌物質。在擁有甘油的環境下，注射痤瘡丙酸桿菌的小鼠耳朵被有或無表皮葡萄球菌的PSF MTAM 覆蓋著，結果顯示，同時擁有表皮葡萄球菌包裹的 PSF MTAM 並加入甘油應用於注射痤瘡丙酸桿菌的小鼠耳朵，大大減少了痤瘡丙酸桿菌的生長和 MIP-2 的產生。此外，沒有表皮葡萄球菌從 PSF MTAM 洩漏到小鼠皮膚中。藉此， PSF MTAM可作為益生菌痤瘡貼劑。
壓力是抑制毛髮生長的主要因素之一。
在這裡，我們研究了聲應力對炎症性皮膚中皮膚炎症和毛髮生長的誘導的影響。暴露於超聲波驅鼠器產生的聲波壓力下的小鼠，顯著上調磷酸二酯酶 4B (PDE4B) 的 mRNA 表達，增強了 4-羥基-2-壬烯醛 (4-HNE) 的產生，其是活性氧 (ROS)及促炎性白細胞介素 (IL)-6 細胞因子的蛋白質指示劑，並刺激巨噬細胞在皮膚中大量積累。聲壓引起的炎症性皮膚極大地阻礙了毛髮的生長。用重組 IL-6 的巨噬細胞治療增加了磷酸化的布魯頓酪氨酸激酶 (p-BTK)。在 PDE4B 基因剔除的小鼠中，由聲壓引發上升的4-HNE 和 IL-6亦受到抑制。頭狀葡萄球菌 (S. capitis) EHH 菌株透過甘油介導發酵，可有效抑制聲壓應力對抑制毛髮生長的炎症作用。使用 siRNA 下調的實驗驗證了游離脂肪酸受體 2 (FFAR2) 得以調控頭狀葡萄球菌 EHH 對聲壓的益生菌效果。該研究闡明了由聲壓引起的 PDE4B/4-HNE/IL-6 的信號通路，並展示了一種益生菌皮膚方法來減輕聲壓引起的皮膚炎症和延緩毛髮生長。
膜糖蛋白是嚴重急性呼吸系統綜合症冠狀病毒 2 (SARS-CoV-2) 中含量最豐富的蛋白質，但其在 2019 冠狀病毒病 (COVID-19) 中的作用尚未完全了解檢視。與接種綠色熒光蛋白 (GFP) 的小鼠相比，鼻內接種膜糖蛋白的小鼠在支氣管肺泡灌洗液 (BALF) 中顯著增加了細胞因子風暴標誌的白細胞介素 (IL)-6。在磷酸二酯酶 4 (PDE4B) 剔除的小鼠中，透過膜糖蛋白誘導的高度 IL-6 顯著降低，表明 PDE4B 在 IL-6 信號傳導中的重要作用。由鼠李糖乳桿菌 (L. rhamnosus) EH8 菌株的菌絲體發酵產生的丁酸，可下調巨噬細胞中 PDE4B 的表達和 IL-6 的分泌。用菌絲體餵養小鼠不只增加了共生鼠李糖乳桿菌的相對豐度，兩周後亦可顯著降低膜糖蛋白誘導的 PDE4B 表達和 IL-6 分泌。在用 GLPG-0974（游離脂肪酸受體 2 (Ffar2) 的拮抗劑）治療的小鼠中，鼠李糖乳桿菌加菌絲體對膜糖蛋白的益生菌活性被消除。在腸-肺軸中激活 Ffar2 以下調 PDE4B-IL-6 信號傳導可能作為開發模式的目標，包括用於治療 COVID-19 細胞因子風暴的益生菌。

摘要(英)

Antibiotics without selectivity for acne treatment may destroy the beneficial microbes in the human microbiome that helps to fight Cutibacterium acnes (C. acnes), a bacterium associated with inflammatory acne vulgaris. Probiotic treatment by direct application of live Staphylococcus epidermidis (S. epidermidis) onto the open acne lesions may run the risk of bloodstream infections. Here, we fabricated the polysulfone microtube array membranes (PSF MTAM) to encapsulate probiotic S. epidermidis. We demonstrate that the application of the encapsulation of S. epidermidis in PSF MTAM enhanced the glycerol fermentation activities of S. epidermidis. To mimic the granulomatous type of acne inflammatory acne vulgaris, the ears of mice were injected intradermally with C. acnes to induce the secretion of macrophage inflammatory protein-2 (MIP-2), a murine counterpart of human interleukin (IL)-8. The C. acnes-injected mouse ears were covered with a PSF MTAM encapsulated with or without S. epidermidis in the presence of glycerol. The application of S. epidermidis-encapsulated PSF MTAM plus glycerol onto the C. acnes-injected mouse ears considerably reduced the growth of C. acnes and the production of MIP-2. Furthermore, no S. epidermidis leaked from PSF MTAM into mouse skin. The S. epidermidis-encapsulated PSF MTAM functions as a probiotic acne patch.
Stress is one of the major causal factors in the inhibition of hair growth. Here, we investigate the effect of sonic stress on the induction of skin inflammation and hair growth in inflammatory skin. Mice exposed to sonic stress created by ultrasonic rodent repellants significantly up-regulated mRNA expression of phosphodiesterase 4B (PDE4B), enhanced the production of 4-hydroxy-2-nonenals (4-HNE), a marker of reactive oxygen species (ROS) and a pro-inflammatory interleukin (IL)-6 cytokine, and stimulated substantial accumulation of macrophages in skin. The inflammatory skin elicited by sonic stress considerably impeded hair growth. Treatment of macrophage cells with recombinant IL-6 increased phosphorylated Bruton′s tyrosine kinase (p-BTK). The elevated 4-HNE and IL-6 as well as the restricted hair growth caused by sonic stress were subdued in PDE4B knockout mice. A probiotic Staphylococcus capitis (S. capitis) EHH strain-mediated glycerol fermentation to effectively suppress the inflammatory effect of sonic stress on the inhibition of hair growth. Experiments using siRNA knockdown validated that free fatty acid receptor 2 (FFAR2) mediated probiotic action of S. capitis EHH against sonic stress. The study elucidated a signaling pathway of PDE4B/4-HNE/IL-6 provoked by sonic stress and demonstrated a probiotic skin approach to mitigate the sonic stress-induced skin inflammation and retarded hair growth.
Membrane glycoprotein is the most abundant protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but its role in coronavirus disease 2019 (COVID-19) has not been fully characterized. The mice were intranasally inoculated with membrane glycoprotein substantially increased the interleukin (IL)-6, a hallmark of the cytokine storm, in bronchoalveolar lavage fluid (BALF), compared to mice inoculated with green fluorescent protein (GFP). The high level of IL-6 induced by membrane glycoprotein was significantly diminished in phosphodiesterase 4 (PDE4B) knockout mice, demonstrating the essential role of PDE4B in IL-6 signaling. Mycelium fermentation of Lactobacillus rhamnosus (L. rhamnosus) EH8 strain yielded butyric acid, which can down-regulate the PDE4B expression and IL-6 secretion in macrophages. Feeding mice with mycelia increased the relative abundance of commensal L. rhamnosus. Two-week supplementation of mice with L. rhamnosus plus mycelia considerably decreased membrane glycoprotein-induced PDE4B expression and IL-6 secretion. The probiotic activity of L. rhamnosus plus mycelia against membrane glycoprotein was abolished in mice treated with GLPG-0974, an antagonist of free fatty acid receptor 2 (Ffar2). Activation of Ffar2 in the gut-lung axis for down-regulation of the PDE4B-IL-6 signaling may provide targets for developing modalities, including probiotics for treating the cytokine storm in COVID-19.

論文目次

Table of Contents
CHINESE ABSTRACT i
ABSTRACT iii
ACKNOWLEDGEMENTS v
LIST OF FIGURES ix
LIST OF TABLES xiv
CHAPTER I INTRODUCTION 1
1.1. Human Skin and its Microorganism 1
1.2. Probiotic and Prebiotic Human Skin 2
1.3. Human gut prebiotic from mushroom 3
1.4. Short-chain fatty acids (SCFAs) 4
1.5. Coronavirus disease 2019 (Covid-19) 5
1.7. Gut microbiota treatment in COVID-19 pneumonia 6
CHAPTER II A MICROTUBE ARRAY MEMBRANE (MTAM) ENCAPSULATED LIVE FERMENTING STAPHYLOCOCCUS EPIDERMIDIS AS A SKIN PROBIOTIC PATCH AGAINST CUTIBACTERIUM ACNES 8
2.1. Introduction 8
2.2. Materials and methods 10
2.2.1. Culture of Microorganisms 10
2.2.2. Fermentation of Bacteria 11
2.2.3. Fermentation of S. epidermidis in PSF MTAM Against C. acnes In Vivo 11
2.2.4. SEM 12
2.2.5. Bacterial Loads in Mouse Ears 12
2.2.6. ELISA 13
2.2.7. Detection of S. epidermidis Leaking from PSF MTAMs 13
2.2.8. Statistics 14
2.3. Result 14
2.3.1. The S. epidermidis-encapsulated PSF MTAM 14
2.3.2. Glycerol Fermentation of S. epidermidis in PSF MTAM 15
2.3.3. In Vivo Inhibition of C. acnes Growth and Inflammation by S. epidermidis Fermentation 17
2.3.4. Detection of Bacterial Leaking from PSF MTAM 21
2.4. Discussion 22
CHAPTER III THE SIGNALING OF PED4B/4-HNE/IL-6 IN SONIC STRESS-INDUCED INHIBITION OF HAIR GROWTH WAS SUPPRESSED BY PROBIOTIC STAPHYLOCOCCUS CAPITIS 27
3.1. Introduction 27
3.2. Materials and methods 29
3.2.1. Ethics statement 29
3.2.2. Anagen induction 29
3.2.3. Sonic stress 30
3.2.4. Macrophage cell culture 30
3.2.5. Bacterial culture and fermentation 31
3.2.6. Western blotting 31
3.2.7. Skin tissue immunostaining 32
3.2.8. Reverse transcription-polymerase chain reaction (RT-PCR) 33
3.2.9. Small interfering RNA (siRNA)-mediated gene knockdown of FFAR2 34
3.2.10. Statistical analysis 34
3.3. Results 34
3.3.1. Sonic stress up-regulated PED4B, 4-HNE and IL-6 and inhibited hair growth 34
3.3.2. Sonic stress-induced macrophage accumulation in skin 37
3.3.3. PDE4B was essential in blocking sonic stress-induced inhibition of hair growth 39
3.3.4. S. capitis EHH fermentation abrogated sonic stress-induced inhibition of hair growth 41
3.3.5. The probiotic effect of S. capitis EHH was subdued by FFAR2 knockdown 44
3.4. Discussion 46
CHAPTER IV- GUT PROBIOTIC LACTOBACILLUS RHAMNOSUS ATTENUATES PDE4B-MEDIATED INTERLEUKIN-6 INDUCED BY SARS-COV-2 MEMBRANE GLYCOPROTEIN 50
4.1. Introduction 50
4.2. Material and method 52
4.2.1. Ethics statement 52
4.2.2. Cloning, expression, and purification of SARS-CoV-2 membrane glycoprotein 53
4.2.3. Detection of IL-6 in BALF 53
4.2.4. Real-time polymerase chain reaction (RT PCR) 53
4.2.5. Mycelium preparation 54
4.2.6. Mycelium fermentation of L. rhamnosus EH8 55
4.2.7. Quantification of L. rhamnosus 55
4.2.8. Cell culture 56
4.2.9. Detection of butyric acid by high performance liquid chromatography (HPLC) 56
4.2.10. Inhibition of Ffar2 57
4.2.11. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay 57
4.2.12. Statistical analysis 57
4.3. Results 58
4.3.1. PDE4B is essential for induction of IL-6 by SARS-CoV-2 membrane glycoprotein 58
4.3.2. Mycelium fermentation of L. rhamnosus EH8 mitigates SARS-CoV-2 membrane glycoprotein-induced IL-6 59
4.3.3. Butyric acid was produced by mycelium fermentation of L. rhamnosus EH8 and reduced IL-6 secretion and PDE4B expression in macrophages 62
4.3.4. Ffar2 mediates the effect of mycelium fermentation of L. rhamnosus EH8 on the reduction of PDE4B and IL-6 63
4.4. Discussion 65
CHAPTER V CONCLUSIONS AND OUTLOOKS 71
BIBLIOGRAPHY 73
APPENDIX 1 LIST OF PUBLICATION 84

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指導教授

黃俊銘(Eric Chun-Ming Huang)

審核日期

2021-7-26

推文