The Role of TM4SF19 in TREM2 Positive Lipid Associated Macrophage of Adipose Tissue = 지방조직 내 TREM2 양성 지질관련 대식세포에서 TM4SF19의 기능 연구

국문 초록 (Abstract)

비만에 의한 지방조직의 재형성은 기능이 저하되고 스트레스를 받은 지방세포를 제거하고 대체하기 위해 면역세포의 동원이 조절하는 과정으로 이루어진다. 지질관련 대식세포는 비만에 의한 대사기능이 저 하된 지방조직 내에서 지질대사 및 대사 항상성 유지에 핵심적인 역할을 수행한다. 본 연구에서는 지질관련 대식세포에서 선택적으로 발현되는 리소좀 막 단백질인 Transmembrane 4 L six family member 19 (TM4SF19)를 규명하였으며, 해당 단백질이 Vacuolar-ATPase (V- ATPase) 소단위체와의 상호작용을 통해 리소좀 산성화를 조절함을 확 인하였다. Tm4sf19 유전자 결손 마우스는 리소좀 산성화가 항진되고, 사멸 지방세포의 제거가 가속화되며, 고지방식이(HFD) 섭취 수컷 마우 스의 증가된 대식세포는 지방조직내 대사 불균형을 회복시켰다. 또한, 고지방식이 섭취 Tm4sf19 결손 마우스는 비만 마우스 비교군에 비해 지방세포 과형성 증가, 인슐린 감수성 개선, 에너지 소비 증가를 보였다. 또한 HFD 섭취는 지방조직 내 지질관련 대식세포의 대표적 마 커인 TREM2 발현을 유의하게 증가시켰으며, 이는 HFD로 유도된 염증 반응 악화와 밀접히 연관되었다. 한편, ADAM10/17에 의해 매개되는 TREM2의 단백질 N-terminal 절단은 대식세포의 죽은 지방세포 제거기 능을 저해하고 지방조직 염증을 악화시키는 것으로 나타났다. ADAM10/17 억제제(GM6001) 투여는 TREM2 절단을 억제하고, 항염 증성 대식세포 분화를 촉진하며, 대사적 개선 효과를 보였다. 종합적으 로, 본 연구는 지질연관 대식세포가 대사 스트레스 하에서 지방조직 면 역세포 역학과 전신 건강을 조절하는 핵심 조절자임을 제시하며, 비만 관련 대사질환의 새로운 치료 표적 가능성을 제시한다.

다국어 초록 (Multilingual Abstract)

Adipose tissue remodeling in response to overnutrition is orchestrated through immune cells recruitments to remove and replace dysfunctional and stressed adipocytes. Among immune cells, lipid associated macrophages (LAMs) play a critical role in lipid metabolism and maintaining metabolic homeostasis in obese adipose tissue. Here, we identify transmembrane 4 L six family member 19 (TM4SF19) as a lysosome-membrane protein selectively expressed in LAMs, where it modulates lysosomal acidification via interaction with subunits of Vacuolar- ATPase (V-ATPase). Genetic deletion of Tm4sf19 enhances lysosomal acidification, accelerates the clearance of dying adipocytes, and shifts the macrophage landscape toward a restorative phenotype in adipose tissue of high-fat diet (HFD)-fed male mice. Tm4sf19 KO mice demonstrate increased adipocyte hyperplasia, improved insulin sensitivity, and elevated energy expenditure. Our findings indicate that HFD feeding upregulates the expression of triggering receptor expressed on myeloid cellsi 2 (TREM2), a representative marker of LAMs, in gonadal white adipose tissue (GWAT), and that this upregulation is closely associated with exacerbated pro- inflammatory responses induced by HFD. Proteolytic shedding of TREM2, mediated by a disintegrin and metalloprotease domain-containing protein 10/17 (ADAM10/ADAM17), impairs macrophage phagocytic function and exacerbates adipose tissue inflammation. Pharmacological inhibition of ADAM10/17 by GM6001 mitigates TREM2 cleavage, promotes anti-inflammatory macrophage polarization, and improves metabolic outcomes. Together, these findings highlight lipid associated macrophages govern adipose tissue immune cell dynamics and systemic health under metabolic stress, offering potential therapeutic targets for obesity-related metabolic disorders.

목차 (Table of Contents)

• Abstract i
• Table of Contents iii
• List of Figures vii
• List of Tables xiii
• Abbreviation xiv
• Chapter 1. 1
• 1. Introduction 2
• 1.1. Adipose tissue macrophage in Obesity 3
• 1.2. Role of lipid associated macrophages (LAMs) in adipose tissues 7
• 1.3. Lysosomal exocytosis in LAMs 9
• 1.4. Purpose of Research 12
• 2. Material and Methods 13
• 2.1. Mice 14
• 2.2. Generation of Macrophage-Specific TM4SF19 Knockout Mice 14
• 2.3. Diet-Induced Obesity and Metabolic Assessments 14
• 2.4. Bone Marrow Transplantation 16
• 2.5. Human Sample Collection 16
• 2.6. Histological Analysis 16
• 2.7. BrdU Labeling and Adipocyte Cellularity Analysis 17
• 2.8. Western Blotting and Quantitative PCR 17
• 2.9. Flow Cytometric Analysis 18
• 2.10. Adipose Tissue Fractionation and Magnetic Cell Sorting 18
• 2.11. Two-Photon Intravital Imaging of Adipose Tissue 18
• 2.12. Cell Culture and Colocalization Studies 19
• 2.13. Subcellular Fractionation and Lysosome Isolation 19
• 2.14. Gene Overexpression 20
• 2.15. Immunoprecipitation 20
• 2.16. Phagocytosis Assays 20
• 2.17. Promoter and Transcription Factor Binding Analysis 21
• 3. Results 22
• 3.1. Upregulation of TM4SF19 in Adipose Tissue Macrophages Triggered by Obesity 23
• 3.2 Transcriptional Regulation of Tm4sf19 by Pro-Inflammatory and Cholesterol Biosynthesis Pathways 29
• 3.3 TM4SF19 is Highly Expressed in Human Adipose Tissue Macrophages and Correlates with Increased BMI and NF-κB Expression. 34
• 3.4 Lysosomal membrane protein TM4SF19 disrupts lysosomal acidification by interfering with Vacuolar ATPase V1V0 assembly. 37
• 3.5 TM4SF19 deletion in macrophages enhances the clearance of dead adipocytes. 45
• 3.6 NCD Fed TM4SF19 Whole-Body Knockout (WKO) Mice Show Limited Phenotypic Differences. 49
• 3.7 SnRNA-seq analysis of GWAT reveals distinct cellular responses associated with TM4SF19 knockout 54
• 3.8. TM4SF19 KO attenuates HFD-induced adipose tissue inflammation in GWAT. 63
• 3.9 TM4SF19 deficiency promotes the restoration of insulin-responsive adipocytes in the GWAT of HFD fed mice. 70
• 3.10. TM4SF19 deficiency protects against HFD-induced insulin resistance and energy metabolism. 78
• 3.11. Macrophage-specific TM4SF19 KO alleviates obesity-associated metabolic dysfunction. 85
• 4. Discussion 95
• 4.1. Lysosomal membrane protein TM4SF19 impairs metabolic balance of HFD-fed mice. 96
• 4.2. TM4SF19 inhibits V-ATPase assembly and dead adipocyte clearance in macrophage. 97
• 4.3. Macrophage-specific Tm4sf19 KO increase HFD-impaired energy expenditure and metabolic dysfunction 98
• 4.4. Limitation of Study 99
• Chapter 2. 100
• 1. Introduction 101
• 1.1. TREM2 Positive Macrophage in Obesity 102
• 1.2. Unresolved Function of TREM2 in Adipose Tissue 105
• 1.3. Roles of TREM2 in Neurodegeneration and Metabolic Disease 107
• 1.4. Purpose of Research 110
• 2. Material and Methods 111
• 2.1. Ethics Statement 112
• 2.2. Animal Study 112
• 2.3. RNA extraction and QPCR analysis 112
• 2.4. Isolation of Mature Adipocytes and Stromal Vascular Fraction (SVF) 113
• 2.5. Western Blot Analysis 113
• 2.6. Cell Culture 113
• 2.7. Histology 114
• 2.8. Human Samples 114
• 2.9. Statistics 114
• 2.10. Data Availability 115
• 3. Results 116
• 3.1. HFD feeding produces the cleavage of TREM2 and soluble TREM2 in GWAT. 117
• 3.2. Obesity leads to upregulated expression of ADAM10 and ADAM17 in adipose tissue macrophages in both humans and mice. 120
• 3.3. Co-culture with pyroptotic adipocytes, but not apoptotic adipocytes, leads to the activation of ADAM10/17 and subsequent cleavage of TREM2 in RAW264.7 cells. 127
• 3.4. GM6001 enhances the clearance of pyroptotic adipocytes by RAW264.7 cells. 129
• 3.5. GM6001 mitigates HFD-induced TREM2 shedding and metabolic imbalance. 133
• 4. Discussion 141
• 4.1. The Paradox of TREM2 in Adipose Inflammation 142
• 4.2. Pyroptotic Adipocyte Death Drives TREM2 Cleavage via STING/ADAM signaling in macrophage. 143
• 4.3. GM6001 Restores Macrophage Function and Metabolic Homeostasis by Inhibiting TREM2 Shedding in GWAT of HFD-fed Mice. 144
• 4.4. Limitations of Study 146
• 5. Reference 147