2015 Fiscal Year Annual Research Report
| Project/Area Number |
15F15908
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| Research Institution | Osaka University |
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Principal Investigator |
黒崎 知博 大阪大学, 免疫学フロンティア研究センター, 特任教授(常勤) (50178125)
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| Co-Investigator(Kenkyū-buntansha) |
TSAI CHAO-YUAN 大阪大学, 免疫学フロンティア研究センター, 外国人特別研究員
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| Project Period (FY) |
2015-04-24 – 2017-03-31
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| Keywords | Autophagy / Rubicon / B cell / Germinal center |
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| Outline of Annual Research Achievements |
Autophagy deficiency resulted in the defect of memory B cell survival and plasma cell differentiation. However, no reports have showed whether enhanced autophagy process alters the B cell development and differentiation in the bone marrow (BM) or during germinal center (GC) reaction. Rubicon (Run domain protein as Beclin-1 interacting and cystein-rich containing) was up-regulated in activated B cells, suggesting that Rubicon may play a role by controlling autophagy process in the B cells. To address these questions, we generated a CD19-cre driven, pan-B cell-specific Rubicon knockout mouse (B/Rubicon-/- mice). Normal B cell development and differentiation in the blood, BM, spleen and GC B cell differentiation in the Peyer’s Patches were found in the non-immunized B/Rubicon-/- mice. However, the B-1a and B2 cell number in the peritoneal cavity were increased in the non-immunized B/Rubicon-/- mice comparing to control mice. B/Rubicon-/- mice also expressed normal serum immunoglobulin (Ig) production and did not produce anti-double-stranded DNA (dsDNA) antibody. In addition, Rubicon-deficient B cells expressed higher IgG1 antibody production but did not alter cell proliferation and surface Ig expression in vitro. In conclusion, Rubicon deficiency did not alter B cell development and differentiation but may promote the B-1a and B2 cell survival. Loss of Rubicon may promote plasma B cell survival or increase the IgG1 production by enhancement of autophagy.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
In the first year, we have stably generated B/Rubicon-/- mice and examined the B cell development and differentiation in the blood, bone marrow, spleen, Peyer’s Patches and peritoneal cavity by flow cytometry. We found that normal B cell development and differentiation in the blood, bone marrow, spleen, and Peyer’s Patches, but higher B-1a and B2 cell number in the peritoneal cavity in the B/Rubicon-/- mice with age over 15 weeks. Since B-1a cells were decreased in the autophagy-deficient mice, enhanced autophagy by Rubicon deletion may promote the cell survival of B-1a cells. In addition, no significant elevation of anti-dsDNA antibody was found in these mice, suggesting that enhanced autophagy by Rubicon deletion did not cause the generation of autoreactive antibody. Furthermore, only serum IgG1 was slightly increased in the B/Rubicon-/- mice, but not other Ig subtypes, indicating that Rubicon deficiency did not alter the endogenous Ig production. Our in vitro results also showed that Rubicon-deficient B cells expressed higher IgG1 antibody production but did not alter cell proliferation and surface Ig expression. However, we could not detect any significant accumulation of LC3 puncta by fluorescent microscopy or by flow cytometry and LC3-II conversion by western blot. Since our results supported that Rubicon-deficiency may result in the enhancement of autophagic process to promote plasma B cell survival or increase the IgG1 production, other methods may be required to determine the level of autophagy formation in Rubicon-deficient B cells.
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| Strategy for Future Research Activity |
In the second year, we will continue to clarify whether Rubicon deficiency enhanced autophagy in B cells. Previous studies showed that Rubicon not only suppressed the fusion between autophagosome and lysosome but also regulated the endocytic trafficking. Since we could not detect any enhancement of autophagy inside the Rubicon-deleted B cells, loss of Rubicon might promote the secretion of autophagosomes, a process called secretory autophagy. We will isolate the secretory vesicles from anti-CD40 antibody- or LPS-stimulated B cells by ultracentrifugation and determine the expression level of autophagosome marker, LC3-II. For investigation of humoral responses in the Rubicon-deficient mice, these mice immunized with NP-KLH emulsified in aluminum. The GC B cell and antigen-specific B cell differentiation will be determined by flow cytometry and the production of high-affinity and low-affinity anti-NP antibodies will be determined by ELISA. In addition, we will also infect mice with a genetic modified, non-lethal subtype of influenza virus and then test the cross-protective immunity by challenging them with a lethal influenza virus subtype. Finally, whether enhanced autophagy by Rubicon-deletion changes the antibody repertoire will also be investigated.
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Research Products
(1 results)
