Tet2-mediated clonal hematopoiesis improves neurological deficits and is associated with inflammation resolution in the subacute phase of stroke

Recent work has revealed that clonal hematopoiesis (CH) is associated with a higher risk of numerous age-related diseases, including ischemic stroke, however little is known about whether it influences stroke outcome. Studies suggest that leukocytes carrying CH driver mutations have an enhanced inflammatory profile, which could conceivably exacerbate brain injury after a stroke. Using a mouse model of Tet2-mediated CH, we tested the hypothesis that CH would lead to a poorer outcome after ischemic stroke by augmenting brain inflammation. In contrast to our hypothesis, Tet2-mediated CH had no effect on acute stroke outcome but led to reduced neurological deficits during the subacute phase. This improved neurological outcome was associated with lower levels of brain inflammation during this time, suggesting that Tet2-mediated CH may promote inflammation resolution in the brain post-stroke. While additional mechanistic studies are required, these findings may suggest that Tet2-mediated CH has beneficial actions on the post-stroke brain. RNA was extracted from brains using the methods described above and total RNA was sent to Azenta Life Sciences (formally Genewiz) for library construction, sequencing and analysis. For this, RNA samples were firstly quantified using Qubit 2.0 Fluorometer (Life Technologies, CA, USA) and RNA integrity was measured using the RNA Screen Tape on Agilent 2200 TapeStation (Agilent Technologies, CA, USA). rRNA depletion was performed using QIAseq® FastSelect™−rRNA HMR kit (Qiagen, MD, USA) according to the manufacturer’s protocol. RNA sequencing libraries were constructed with the NEBNext Ultra II RNA Library Preparation Kit for Illumina by following the manufacturer’s recommendations. Briefly, enriched RNAs are fragmented for 15 min at 94 °C. First strand and second strand cDNA are subsequently synthesized. cDNA fragments are end repaired and adenylated at 3’ends, and universal adapters are ligated to cDNA fragments, followed by index addition and library enrichment with limited cycle PCR. Sequencing libraries were validated using the Agilent Tapestation 4200 (Agilent Technologies, CA, USA), and quantified using Qubit 2.0 Fluorometer (ThermoFisher Scientific, USA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). The sequencing libraries were multiplexed and clustered on 1 lane of a flowcell. After clustering, the flowcell was loaded on the Illumina HiSeq 4000 instrument according to manufacturer’s instructions. The samples were sequenced using a 2x150 Pair-End (PE) configuration.