免疫疾患におけるRNA分解酵素Regnase-1調節機構解明とその操作法の開発

2019 Fiscal Year Annual Research Report

• Project/Area Number: 19J23450
• Research Institution: Kyoto University
• Principal Investigator: Tse Ka Man Carman 京都大学, 医学研究科, 特別研究員(DC1)
• Project Period (FY): 2019-04-25 – 2022-03-31
• Keywords: Regnase-1 / autoimmune diseases / mRNA stability

Outline of Annual Research Achievements

The landmark discovery that IL-17 producing CD4 T cells were the major drivers of autoimmune diseases positioned IL-17-Th17 pathway as a promising therapeutic target. Recent studies on how post-transcriptional mechanisms control immunity has attracted enormous attention. Being an essential ribonuclease controlling the activation and differentiation of CD4 T cells, and the expression of inflammatory cytokines upon TLR stimulation in macrophages, Regnase-1 was shown to have an important role in the suppression of autoimmunity in mice. However, the exact role of Regnase-1 plays in the development of autoimmune diseases was not known. In this study, we found that Regnase-1 is actively down-regulated in several autoimmune murine models and interfering with the structured RNA motifs of Regnase-1 can be a new strategy to influence autoimmune and inflammatory responses in a therapeutic setting.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

We have evaluated the role of Regnase-1 in the development of autoimmune diseases from several different view points: (1) using the spontaneous EAE model (2D2 TCR transgenic mouse), we found that the deficiency of Reg1 in CD4+ T cells renders mice to develop EAE more often than WT counterparts, suggesting down-regulation of Regnase-1 can exacerbate the progression of EAE; (2) it was also found that Reg1 expression (mRNA and protein) is significantly reduced in the PBMCs in patients with autoimmune diseases, compared to healthy controls; (3) we found that administrating antisense oligonucleotides targeting Regnase-1 stem-loop structures in the brain of EAE mice postponed and relived the EAE symptoms and allowed better weight recovery. This was associated with a significant reduction in spinal cord/brain-infiltrating immune cells.

Strategy for Future Research Activity

To investigate the potential mechanism by which Regnase-1 is degraded in the PBMCs (especially CD4 T cells) in patients with autoimmune diseases, different protein degradation inhibitors and antibodies will be screened in vitro, and RNA sequencing will be performed.
To better understand the function of Regnase-1 in certain cell(s) during EAE development, flow cytometry will be performed to analyze the Regnase-1 expression in various kinds of cell in the CNS after intracranial administration of PBS or antisense oligonucleotides, taking advantage of the flag-tag Regnase-1 knock-in mouse.
In addition, in order to reveal the mechanistic details of how antisense oligonucleotides ameliorates the EAE symptoms, we will perform single cell RNA sequencing to identify the set(s) of gene and pathway are altered when Regnase-1 is stabilized.