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SRX18337354: GSM6749036: CD8_Prf1_Ruxo_2; Mus musculus; RNA-Seq
1 ILLUMINA (Illumina NovaSeq 6000) run: 87.1M spots, 17.3G bases, 1.9Gb downloads

External Id: GSM6749036_r1
Submitted by: Nichols Lab, Oncology/ Division of Cancer Predisposition, St. Jude Children's Research Hospital
Study: Cellular and transcriptional impacts of ruxolitinib versus IFN-gamma neutralization in mouse models of HLH
show Abstracthide Abstract
Hemophagocytic lymphohistiocytosis is a cytokine storm syndrome characterized by the excessive activation of myeloid and T cells. In primary HLH (pHLH), disease pathophysiology is driven by CD8 T cells proliferation and IFNg production. Therefore, targeting IFNg has been a viable option for the treatment of HLH. Since many cytokines that signaling throught the JAK/STAT pathway, including IFNg, largely drive cytokine storm in HLH, we and others have demonstrated that targeting JAK1/2 with ruxolitinib is effective in ameliorating disease manifestations in pre-clinical models of HLH. Both IFNg and ruxolitinib treatments have also shown great efficacy in dampening inflammation in patients with HLH, albeit not completely. Since ruxolitinib treatment does not fully inhibit IFNg production, we sought to determine the effect of combining ruxolitinib treatment with IFNg neutralization as a treatment option in a LCMV-infected Prf1??? mice. We aimed to elucidate the effects of these treatments on the main hematologic and cellular parameters of disease. Moreover, we determined the main changes in the transcriptional landscape on CD8 T cells isolated from mice infected with LCMV either untreated or treated with aIFNg, ruxolitinib, or combination treatments. We demonstrate that combination treatment was very effective in reversing anemia and thrombocytopenia, but failed to reduce splenomegaly, hypercytokinemia, and myeloid and T cell numbers compared to ruxolitinib treatment. Transcriptional analysis revealed distinct pathways targeted by aIFNg and ruxolitinib; while aIFNg was more effective in downregulating the expression of interferon gamma response genes, allograft rejection, and complement genes, ruxolitinib treated CD8 T cells displayed a reduction in expression of genes involved in reactive oxygen species, glycolysis, E2F targets, and protein secretion pathways. Therefore, this study provide novel insights on the effects of combining ruxolitinib treatment with IFNg neutralization in the treatment of primary HLH. Overall design: RNA sequencing analysis of CD8 T cells sorted on day 9 p.i. from Prf1-/- naïve mice, LCMV-infected untreated, or treated in vivo with aIFNg, ruxolitinib, or combination treatment (day 4-8 p.i.)
Sample: CD8_Prf1_Ruxo_2
SAMN31823131 • SRS15826339 • All experiments • All runs
Organism: Mus musculus
Library:
Name: GSM6749036
Instrument: Illumina NovaSeq 6000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: RNA was extracted from sorted cell populations using the RNeasy Micro Kit (Qiagen, Germantown, Maryland). The quality and quantity of the initial RNA was determined by using the Agilent 2100 Bioanalyzer with the Eukaryote Total RNA Pico kit. For each good quality sample, 2.5-25 nanograms of total RNA was used in the Tecan Ovation RNA sequencing system v2 protocol as written to create SPIA (single primer isothermal amplification)-cDNA. Once the cDNA was purified, five hundred nanograms of each sample was sheared using the Covaris LE220 focused ultra sonicator. The shearing plate used was the 96 microtube-50 AFA fiber plate and the target base pair size was 300. The settings for the shearing were the following: Peak incident Power (W) of 450, duty factor of 15%, cycles per burst 1000 with a 100 second treatment time. Once the cDNA was sheared, libraries were created using the KAPA Hyper-Prep kit from Roche. Based on the starting cDNA input amount, the Kapa Hyper protocol recommended four pcr cycles for the amplification step while the rest of the protocol was followed as described. The indexes used were the UDI DNA indexes from Illumina. Eukaryote Total RNA Pico kit (catalog: 5067-1513) Tecan (formally NuGen) Ovation RNA sequencing system v2 (catalog: 7102-32) Covaris 96 tube plate (catalog:520168) KAPA Hyper-Prep (catalog: KK8504) UDI DNA Indexes from Illumina (catalog: 20022370)
Runs: 1 run, 87.1M spots, 17.3G bases, 1.9Gb
Run# of Spots# of BasesSizePublished
SRR2236672187,059,67817.3G1.9Gb2023-04-24

ID:
25350908

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