Characterizing TNF-alpha levels in the context of Dnmt3a-mutant bone marrow

As humans age, stem and progenitor cells in the bone marrow (BM) can acquire mutations. The most frequently observed mutation in is the gene DNA methyltransferase 3a (Dnmt3a). When comparing aged mice engineered to express a common Dnmt3a mutation to aged wild type (WT) mice, there is a significant increase in the amount of stem and progenitor cells in the bone marrow (BM). In addition, increased signaling of pro-inflammatory cytokines are observed, such as tumor necrosis factor alpha (TNF-α). The goal of this project was to test the hypothesis that Dnmt3a-mutant BM cells express or produce greater amounts of TNF-α, which contributes to their selective advantage. In this study, I first optimized Western blotting to detect TNF-α in the BM. Using this protocol, I examined TNF-α in BM samples isolated from young and old WT mice and found that the old BM samples had higher levels of TNF-α compared to young. This protocol can now be used to test Dnmt3a-mutant BM samples moving forward. In parallel, I examined Tnf transcript levels in Dnmt3a mutant compared to control mice and found that Tnf expression was decreased, rather than increased, in the Dnmt3a-mutant samples. In addition, I found that Tnf expression was further decreased by inflammatory stimulation of the Dnmt3a-mutant mice. Using knockout models of the two TNF-α receptors (TNFR1 and TNFR2), I found that this phenotype is mediated specifically by TNFR2. Together, my data support that Dnmt3a-mutant BM cells express less rather than more TNF-α and rely on TNFR2 signaling for this phenotype. Future work will focus on resolving and refining our model given the apparent discrepancies between past and current results, for which I provide ideas in the discussion.