(The) effects of human breast milk-derived exosomes on the function of myeloid cells

다국어 초록 (Multilingual Abstract)

The effect of human breast milk-derived exosomes on the function of myeloid cells Dahyeon Kwon Major in Biopharmaceuticals Department of Pharmaceutical Regulatory Sciences Chung-Ang University Graduated School Human breast milk (HBM) is an ideal nutritional source for the growth and development of infants. In addition, HBM contains microorganisms, hormones, growth factors, miRNAs and exosomes that perform various physiological functions. This study investigates the effects of HBM-derived exosomes on macrophage polarization and implications for rheumatoid arthritis (RA). HBM- derived exosomes were isolated and characterized using nanoparticle track analyzer and western blotting for CD81, CD9, HSP70, Flotillin-1, Alix and Annexin V. The HBM-derived exosomes decreased the expression of inflammatory mediators, iNOS and COX-2 and the secretion of proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 in mouse peritoneal macrophages upon LPS stimulation. Flow cytometry analysis of bone marrow-derived macrophages (BMMs) indicated that exosomes promoted M2 polarization in a dose-dependent manner as evidenced by a decrease in cells expressing CD80 (M1 marker) and a concurrent increase in cells expressing CD206 (M2 marker). In addition, exosome treatment reduced the expression of matrix metalloproteinase-1(MMP-1), MMP-3, and MMP-13 in SW1353 chondrocytes following IL-1β stimulation. In vivo studies using a RA mouse model with DBA/1J mice revealed that exosome administration decreased tissue resident macrophages in the spleen and lymph node. Our findings suggest that HBM-derived exosomes modulate macrophage polarization towards an M2 phenotype and exhibit significant anti-inflammatory effects, which could offer new insights into the management of chronic inflammatory diseases. * Keywords: Human breast milk, exosome, rheumatoid arthritis, collage-induced arthritis, macrophage polarization

국문 초록 (Abstract)

사람 모유(Human breast milk, HBM)는 영유아의 성장과 발달을 위한 이상적인 영양 공급원이다. 또한, HBM에는 다양한 생리적 기능을 수행하는 미생물, 호르몬, 성장 인자, miRNA, 엑소좀이 포함되어 있다.
본 연구는 HBM 유래 엑소좀이 대식세포 극화 및 류마티스 관절염(RA)에 미치는 영향을 조사하였다. HBM 로부터 엑소좀을 분리한 후 나노 입자분석과 CD81, CD9, HSP70, Flotillin-1, Alix, Annexin V에 대한 웨스턴 블롯 분석을 통해 엑소좀을 확인하였다. HBM 유래 엑소좀은 LPS 자극을 받은 마우스 복막 대식세포에서 염증 매개체인 iNOS와 COX-2의 발현 및 TNF-α, IL-1β, IL-6과 같은 염증성 사이토카인의 분비를 감소시켰다. 골수 유래 대식세포(BMM)에 대한 유세포 분석 결과, 엑소좀 처리에 따라 M1 마커인 CD80 발현 세포는 감소하고, M2 마커인 CD206 발현 세포는 농도 의존적으로 증가하여 M2 분화를 촉진함을 확인하였다.
추가적으로, 엑소좀 처리는 IL-1β 자극을 받은 SW1353 연골 세포주에서 MMP-1, MMP-3, MMP-13의 발현을 감소시켰다. DBA/1J 마우스를 이용한 RA 마우스 모델에서 엑소좀 투여는 비장과 림프절 내에서 조직 상주 대식세포의 수를 감소시켰다. 본 연구 결과는 HBM 유래 엑소좀이 M2 표현형으로의 대식세포 분화를 조절하고 유의미한 항염증 효과를 나타냄을 시사하며, 이는 류마티스 관절염의 접근에 새로운 통찰을 제공할 수 있을 것이다.

목차 (Table of Contents)

• 1. Introduction 1
• 2. Materials and methods 6
• 2.1 Collection of human breast milk samples 6
• 2.2 Isolation of breast milk-derived exosomes 6
• 2.3 Nanoparticle tracking analysis (NTA) 7
• 2.4 Isolation of peritoneal macrophage 7
• 2.5 In vitro generation and differentiation of BMM 8
• 2.6 SW1353 cells culture 9
• 2.7 Enzyme-linked immunosorbent assay (ELISA) 9
• 2.8 Quantitative analysis of mRNA expression 11
• 2.9 Western blot analysis 11
• 2.10 Flow cytometry 12
• 2.11 Mice 14
• 2.12 Collagen-induced arthritis (CIA) mice model and treatment groups 14
• 2.13 Paw thickness and arthritis score 15
• 2.14 Statistical analysis 16
• 3. Results 17
• 3.1 Characterization of HBM-derived exosomes 17
• 3.2 Anti-inflammatory effects of HBM-derived exosomes in peritoneal macrophage 19
• 3.3 The effect of HBM-derived exosomes in BMM differentiation 22
• 3.4 Anti-inflammatory effects of HBM-derived exosomes in SW1353 cell 25
• 3.5 The effects of HBM-derived exosomes on paw-thickness and swelling in CIA mice model 27
• 3.6 Role of HBM-derived exosomes in immune cells 31
• 3.7 Modulation effect of HBM-derived exosomes on macrophage in CIA mice 34
• 4. Discussion 37
• 5. Conclusion 41
• References 42
• 국문초록 46