 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Dec 23, 2022 |
| Title |
Thioredoxin-interacting protein is essential for memory T cell formation via the regulation of the redox metabolism |
| Organism |
Mus musculus |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
Expression profiling by high throughput sequencing
|
| Summary |
CD4+ memory T cells are central to long-lasting protective immunity and are involved in shaping the pathophysiology of chronic inflammation. While metabolic reprogramming is critical for the generation of memory T cells, the mechanisms controlling the redox metabolism in memory T cell formation remain unclear. We found that reactive oxygen species (ROS) metabolism changed dramatically in T helper-2 (Th2) cells during the contraction phase in the process of memory T cell formation. Thioredoxin-interacting protein (Txnip), a regulator of oxidoreductase, regulated apoptosis by scavenging ROS via the nuclear factor erythroid 2-related factor 2 (Nrf2)-biliverdin reductase B (Blvrb) pathway. Txnip regulated the pathology of chronic airway inflammation in the lung by controlling the generation of allergen-specific pathogenic memory Th2 cells in vivo. Thus, the Txnip-Nrf2-Blvrb axis directs ROS metabolic reprogramming in Th2 cells and is a potential therapeutic target for intractable chronic inflammatory diseases.
|
| |
|
| Overall design |
RNA-seq and ATAC-seq samples of Wild-type or Txnip KO mice that were immunized with 100 μg of OVA (Sigma-Aldrich) and 20% hydroxypropyl-β-cyclodextrin (HP-β-CD) (FUJIFILM, Tokyo, Japan) or 1×PBS administered intranasally (i.n.) and were collected on 0h and 48h in spleen. Th2 cells recovered from the spleen and sorted using a BD FACS Aria III (10,000 cells each, cell viability >98%) were encapsulated into droplets. Libraries were then prepared using Chromium Single Cell 3’ Reagent Kits v3.1 following the manufacturer’s protocol (10X Genomics; California, USA). The generated scRNA-seq libraries were sequenced using a total 127 cycle (paired-end reads) with a NovaSeq 6000 (Illumina). RNA sequencing (RNA-seq) was carried out as previously described. Total cellular RNA was extracted with TRIzol reagent (Invitrogen). For cDNA library construction, we used a SMARTer Stranded Total RNA-Seq Kit v2-Pico Input Mammalian (TaKaRa, Shiga, Japan) following the manufacturer’s protocol. Sequencing of library fragments was performed on an Illumina HiSeq 1500 System (Illumina, California, USA). ATAC-Seq was that 10,000 cells were used to extract the nuclei. The nuclei were treated with Tn5 transposase (15027865 and 15027866; Illumina) to tag and fragmentize the accessible chromatin. The fragmentized DNA was purified using a QIAGEN MinElute kit (QIAGEN, Venlo, Netherlands) and amplified with 14 PCR cycles, based on the amplification curve. The libraries were sequenced for 50 cycles.
The scRNA-seq samples of Th2 at day7 and day11 in vivo were that Naïve CD4+ cells were administered intravenously (i.v.) through the tail vein to BALB/c mice (1×10^6 cells per mouse, day 0). After cell transfer, OVA/Alum was administered intranasally (i.p.) on days 1 and 6, and were collected on day 7 and day11 in spleen. The scRNA-seq samples of Effector Th2 and transferred Th2 were that Th2 in vitro cells were administered intravenously (i.v.) through the tail vein to BALB/c mice (5×10^6 cells per mouse, day 0), and were collected Effector Th2 and transferred Th2 on 48h in spleen. The scRNA-seq , RNA-seq and ATAC-seq samples of Wild-type or Txnip KO mice were that these mice immunized with 100 ?g of OVA (Sigma-Aldrich) and 20% hydroxypropyl-?-cyclodextrin (HP-?-CD) (FUJIFILM, Tokyo, Japan) or 1×PBS administered intranasally (i.n.) , and were collected on 0h and 48h in spleen.Th2 cells recovered from the spleen and sorted using a BD FACS Aria III (10,000 cells each, cell viability >98%) were encapsulated into droplets. Libraries were then prepared using Chromium Single Cell 3? Reagent Kits v3.1 following the manufacturer?s protocol (10X Genomics; California, USA). The generated scRNA-seq libraries were sequenced using a total 127 cycle (paired-end reads) with a NovaSeq 6000 (Illumina). RNA sequencing (RNA-seq) was carried out as previously described (30). Total cellular RNA was extracted with TRIzol reagent (Invitrogen). For cDNA library construction, we used a SMARTer Stranded Total RNA-Seq Kit v2-Pico Input Mammalian (TaKaRa, Shiga, Japan) following the manufacturer?s protocol. Sequencing of library fragments was performed on an Illumina HiSeq 1500 System (Illumina, California, USA). ATAC-Seq was that 10,000 cells were used to extract the nuclei. The nuclei were treated with Tn5 transposase (15027865 and 15027866; Illumina) to tag and fragmentize the accessible chromatin. The fragmentized DNA was purified using a QIAGEN MinElute kit (QIAGEN, Venlo, Netherlands) and amplified with 14 PCR cycles, based on the amplification curve. The libraries were sequenced for 50 cycles.
|
| Web link |
https://www.pnas.org/doi/10.1073/pnas.2218345120?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%20%200pubmed
|
| |
|
| Contributor(s) |
Kokubo K, Hirahara K, Kiuchi M, Tsuji K, Shimada Y, Sonobe Y, Shinmi R, Hishiya T, Iwamura C, Onodera A, Nakayama T |
| Citation(s) |
36595680 |
| |
| Submission date |
Dec 19, 2022 |
| Last update date |
Mar 28, 2023 |
| Contact name |
Masahiro Kiuchi |
| E-mail(s) |
aema2758@chiba-u.jp
|
| Organization name |
Chiba univercity
|
| Street address |
cyuoku inohana1-8-1
|
| City |
chiba city |
| State/province |
chiba |
| ZIP/Postal code |
2608670 |
| Country |
Japan |
| |
|
| Platforms (2) |
| GPL18480 |
Illumina HiSeq 1500 (Mus musculus) |
| GPL24247 |
Illumina NovaSeq 6000 (Mus musculus) |
|
| Samples (29)
|
|
| Relations |
| BioProject |
PRJNA913845 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE221291_RAW.tar |
1.4 Gb |
(http)(custom) |
TAR (of BIGWIG, FPKM_TRACKING, MTX, TSV) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
Less...
More...