Abstract Rationale: Tumor Necrosis Factor-alpha (TNF) is elevated in myeloproliferative neoplasm (MPN) and plays a key role in expansion of the JAK2V617F neoplastic clone. A high TNF environment, as is the case in MPN patients, gives TNF resistant JAK2V617F mutant cells a selective advantage over their TNF sensitive non-mutant counterparts resulting in expansion of the neoplastic clone. To efficiently target TNF production therapeutically it is necessary to identify the mechanism driving this excessive TNF production. TNF is classically produced by monocytes after stimulation through Toll-like receptors (TLR), crucial pattern recognition receptors for microbial products. Because TLR signaling plays an integral role in inflammation and TNF production we hypothesized that exaggerated signaling of the TLR pathway is the mechanism by which TNF is overproduced in MPN. To test this hypothesis we quantified TLR responses in monocytes from MPN patients and normal controls. Results: We compared the response of peripheral blood monocytes from MPN versus normal controls to the TLR agonists R848 (TLR7/8), LPS (TLR4), or zymosan (TLR2). After stimulation with each of these TLR agonists for 24 hours, CD14+ monocytes from MPN patients (n=18) produced increased amounts of TNF (measured by ELISA) as compared to normal controls (n=10) at all concentrations tested (p<0.05). The increased TNF production in MPN monocytes could not be explained by a higher fraction of inflammatory (CD16+ CD14+) monocytes in MPN. We next used phosflow to detect whether MPN patients have abnormal activation of downstream signaling molecules following stimulation with TLR agonists. At early time points (15min) following stimulation, MPN patients (n=6) and normal controls (n=6) phosphorylated p38 and ERK1/2 equally. At later time points (2hrs) MPN patients maintained phosphorylation of p38 and ERK1/2, whereas in normal controls phosphorylation of p38 and ERK1/2 returned to baseline (p<0.05). These data suggest that the excessive production of TNF in MPN patients may be due either to persistent activation of signaling following TLR stimulation or failure to repress the activation state of these two proteins. TLR ligation induces a negative feedback loop culminating in the production of IL-10, an anti-inflammatory cytokine which serves to dampen TNF production. We next used intracellular flow cytometry to determine whether the excessive TNF production in MPN is due to a failure to dampen TNF production over time. Monocytes from normal controls (n=12) cease production of TNF by 9 hours post LPS stimulation, whereas MPN monocytes (n=8) continue producing TNF (p<0.05). This continued TNF production by MPN monocytes is coincident with a failure to produce the anti-inflammatory cytokine IL-10 in response to TLR ligation (p<0.05). Conclusions: We find that MPN monocytes produce excessive amounts of TNF in response to TLR ligation. This excessive production of TNF is due to a defect in the negative regulatory feedback loop which normally serves to dampen TNF production. Targeting the TLR pathway therapeutically in MPN may serve to reduce TNF production and neutralize the selective advantage of the JAK2V617F neoplastic clone, ultimately leading to a reduction in the JAK2V617F allele burden. Disclosures No relevant conflicts of interest to declare.