Muralidharan, Sujatha;
Sasi, Sharath;
Yan, Xinhua;
Enderling, Heiko;
Goukassian, David
Abstract 184: TNF-TNFR1/p55 or TNFR2/p75 Receptor-Ligand Interactions Inhibit Early and Increase Delayed Radio-Biological Bystander Responses in BM-Derived EPCs
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Abstract 184: TNF-TNFR1/p55 or TNFR2/p75 Receptor-Ligand Interactions Inhibit Early and Increase Delayed Radio-Biological Bystander Responses in BM-Derived EPCs
Beschreibung:
<jats:p>TNF-α (TNF) binds two receptors TNFR1-p55 and TNFR2-p75. Ionizing radiation (IR) increases tissue TNF levels. TNF signaling regulates numerous cyto- and chemokines, known to mediate radiation-induced non-targeted effects (NTE), a phenomenon where cells that are not directly “hit” by IR exhibit IR effects as a result of signals received from nearby or distant IR cells. Little is known about the role of p55 or p75 in regulating NTEs in bone marrow (BM)-derived EPCs. We hypothesized that inhibition of TNF signaling either via p55, or p75 may alter TNF-mediated inflammatory responses, inducing NTEs. Medium transfer experiments (MTE) were performed in WT, p55 knockout(KO) and p75KO BM-derived EPC ex-vivo, where one set of EPCs was irradiated with 1Gy of γ-IR, then IR-conditioned medium (CM) was collected from these dishes at 1, 5, 24h and 3, 5d post-IR. Filtered (0.22µm) IR-CM was transferred to naïve non-IR same genotype EPCs and 24h post-incubation naïve EPCs were processed for p-y-H2AX staining for presence and decay of double strand breaks (DSB). CM from IR EPCs were processed for ELISA profiling (16 genes) and IR EPCs were processed for transcriptional profiling. In WT EPC the peak of detectable mean p-y-H2AX foci/cell were at 24h, whereas in p55 and p75KOs p-y-H2AX were the lowest at 24h (9±0.8 vs 4.8±0.6 and 3.7±0.5, p<0.01 and 0.001, WT vs p55 and p75KOs). This finding indicates that altered TNF signaling inhibits early NTEs (hours) in EPCs. Compared to WT, delayed (5 days) NTEs were amplified in naïve p55 and p75KO EPCs (3.8±0.4 vs 8.5±1 and 5.9±0.8, p<0.02 and 0.01, WT vs p55 and p75KOs), suggesting significant role for TNF signaling in mediating delayed NTEs. ELISA profiling of 16 proteins in CM over 5 days post-IR showed 200-1600% increases (p<0.002, p55KO vs WT) in cumulative levels of TNF, IFNr, IL6, EGF, MIP-1α, GM-SCF, Rantes, and 200-1000%, increases (p<0.05, p75KO vs WT) in IL1α, IL1β, G -CSF, MCP-1, SCF. Transcriptional profiling of γ-IR EPC revealed 139 genes (>2 fold) up or down regulated, clustered into 9 groups. Other aspects of gene array data are being considered. We conclude that TNF ligand-receptor axis regulates NTEs in naïve EPCs and suggest that restoring TNF signaling balance could be used to prevent delayed NTEs in distant from primary IR target tissues.</jats:p>