Abstract 3293: The TLR-2 signal pathway is enhanced in myeloproliferative neoplasm - related to the increased inflammatory cytokine and pathogenesis
Authors: Shi Guanfang , Yarotska Maryna , chen Hui , Abdelmalek Cherif , Wong Ching , Kalaovar Madhumati , Gotlieb VLadimir , Wang Jen Chin 
Published: July 2016

Introduction

Although MPNs have been regarded as stem cell disease. Recent evidence suggest that chronic inflammation induces a state of chronic oxidative stress with elevated reactive oxygen species (ROS) and then induce alternation in epigenetic changes or DNA mutations.. Also, MPNs are associated with constitutional symptoms and high levels of circulating inflammatory cytokines. The etiology of this increase in inflammatory cytokines remains elusive, we investigated the role of TLRs (Toll-like receptors) and related cytokines in MPN as they are known to play a role in innate and adaptive immunity in inflammation.

Materials and Methods:

TLR assay: TLR-2, 4, 7, 9 quantification was performed by TLR staining of 5×105 mononuclear cells (MNC) which were incubated with 5 μg/ml of conjugated TLR-2, 4, 7, 9 antibody and assayed by flow cytometry.

Monocyte-derived dendritic cells (mdDC) and Inflammatory Cytokines.

mdDC were generated from isolated monocytes (anti-CD14-coated magnetic beads) then by incubation with IL-4 and GM-CSF for 4 days. Immature mdDC (1×104/well) were either left untreated or stimulated with Pam3CSK4, in 96 well plates. After 48h incubation, cells were stained with fluorescence-labeled mAb specific to CD80, CD83 (BD Biosciences). Cytokine levels were determined in supernatants harvested after 48h using the MSD (Meso Scale Discovery) System.

Results. 1) Levels of TLR-2 were significantly elevated in patients with MPNs especially in those with polycythemia vera and essential thrombocythemia, but only marginally elevated in myelofibrosis patients. In all patient subsets, TLR-4, 7, 9 levels did not differ significantly from controls. 2) Maturation of mdDC were done by stimulation of TLR-2 ligand, Pam3CSK4. Higher numbers of CD83+ cells were generated more from patients with elevated TLR levels, confirming these MPN patients have elevated TLR-2 levels which portend the ability to generate more DC by TLR-2 ligand. 3) In patients with elevated TLR-2 level, Pam3CSK4 stimulated more cytokines including IFNγ, IL12p70, TNFα, IL6, IL8, IL10, and IL13 than those patients with low levels of TLR-2. 4). Moreover, patients with elevated TLR-2 levels have more of IL8, TNFα, IL13, IL6, in the plasma, consistent with inflammatory cytokines which is a likely consequence of elevated TLR-2.

Conclusion. TLR-2 and not TLR-4, 7, 9 were found to be significantly elevated in MPNs. Stimulation of the TLR-2 ligand promotes the generation of more mature DCs, greater inflammatory cytokines production and Higher plasma inflammatory cytokines are associated with patients with higher TLR-2 levels. We conclude that TLR-2 is important in the production of inflammatory cytokines in MPNs and this evidence suggests that MPNs are in a TLR induced chronic inflammatory state and may play a significant role in the pathogenesis of MPNs.

Citation Format: Guanfang Shi, Maryna Yarotska, Hui chen, Cherif Abdelmalek, Ching Wong, Madhumati Kalaovar, VLadimir Gotlieb, Jen Chin Wang. The TLR-2 signal pathway is enhanced in myeloproliferative neoplasm - related to the increased inflammatory cytokine and pathogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3293.