2020 Fiscal Year Research-status Report
The role of B cell-derived secretory autophagosome-like vesicles in immune regulation
Project/Area Number |
19K07606
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Research Institution | Osaka University |
Principal Investigator |
TSAI CHAO・YUAN 大阪大学, 免疫学フロンティア研究センター, 寄付研究部門助教(常勤) (10727001)
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Project Period (FY) |
2019-04-01 – 2022-03-31
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Keywords | B cells / Autophagy / Secretory / IL-4/CD40 signaling / Immune regulation / Autophagosome / Extracellualr vesicles / ALVs |
Outline of Annual Research Achievements |
Cell-derived extracellular vesicles (EVs) are small membrane-enclosed vesicles with the diameter ranging from 30 to 1000 nm including exosomes and microvesicles et al. and act as cargos to deliver the biomolecular messages between cells. Recent reports showed that autophagy is involved in the unconventional secretion of cytosolic proteins. However, the secretion of autophagy-associated vesicles is still controversial. Whether external stimulation can induce such secretion remains largely unknown. We previously found that T cell-dependent signals through IL-4 and CD40 (IL-4/CD40) specifically induced the secretion of LC3-II+ autophagosome-like vesicles (ALVs) in B cells. Comparing to IL4/LPS signals, IL4/CD40 signals increased the autophagosome-endosome fusion but not the autophagosome-lysosome fusion, suggesting that IL-4/CD40 axis promoted the secretion of ALVs through the recycling endosome pathway. To further investigate the functions of ALVs, the protein and RNA compositions of EVs isolated from wild-type and autophagy-deficient B cells were analyzed by mass spectrometry and small RNA-sequencing (RNA-seq), respectively. To achieve this, B cell-specific Atg7 KO (Atg7 B-KO) mice were generated by crossing Atg7F/F mice with CD19-Cre or Mb1-Cre mice. Then, EVs including microvesicles and exosomes from activated wild-type (WT) or Atg7 KO B cells can be isolated. The characterization of bio-information carried by EVs can help us understand the physiological role of ALVs.
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Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
In the second fiscal year, we first continued to explore the molecule mechanism of how IL-4/CD40 signaling induced the ALVs secretion in B cells. IL4/CD40 signals not only increased the EEA1 expression, a endosome marker, but also increased the gene expressions related to the endocytosis and exocytosis, such as rab21 and rab27A, comparing to IL-4/LPS signals. Additionally, the inhibition of autophagosome-lysosome fusion by bafilomycin A1 only slightly increased the secretion of LC3-II+ vesicles in both IL4/CD40 and IL4/LPS stimulations. In contrast to IL4/LPS signals, IL-4/CD40 signals increased the fusion of autophagosomes to endosomes in B cells observed by high-resolution confocal microscopy and transmission electronic microscopy. Together our results suggest that IL4/CD40 axis induces ALVs secretion through the recycling endosome pathway. Second, we investigated whether autophagy plays a role in the transportation of cytosolic biomolecules including proteins and nuclear acids. We compared the RNA composition between exosomes isolated from IL4/LPS or IL4/CD40-activated B cells by small RNA-seq. However, the miRNA expression profile of IL4/CD40-induced exosomes was similar to that of IL4/LPS-induced exosomes, suggesting that ALVs may not regulate the extracellular transportation of miRNA. We next analyzed the protein compositions of EVs isolated from activated WT or autophagy-deficient B cells by mass spectrometry. Such bio-informations will be used to investigate the physiological role of ALVs.
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Strategy for Future Research Activity |
In the third fiscal year, we will focus on the investigation of physiological significance of ALVs. In order to examine the biological functions of ALVs during B-T cell interaction, the CD4+ and CD8+ T cells will be isolated and cultured with ALVs. The proliferation, activation marker and cytokine production will be evaluated by flow cytometry. Additionally, the characterization of bio-information carried by ALVs can help us understand the molecular mechanism if those ALVs modulate T cell functions. To further confirm in vitro results, the WT and Atg7 B-KO mice will be used for some disease models, such as cancer. Additionally, previous reports showed that B cells expressing high autophagy corelated to human SLE autoimmune diseases, the LC3-II+ EVs may be detected in the sera of mice with autoimmune diseases. Thus, we will investigate the potential of ALVs as a diagnostic maker for autoimmune diseases. Finally, we will prepare the manuscript to publish our finding.
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