Derangements of Toll-like Receptors, Inflammatory Cytokines, and Reactive Oxygen Species in Philadelphia Chromosome-Negative Myeloproliferative Neoplasm: Implicate Roles of Inflammation in the Pathogenesis
Authors: Shi Guanfang , Chen Hui , Abdelmalek Cherif , Bandarchuk Andrei , Mahmood Abdullah Khawer , Ching Wong , Kalavar Madhumati , Gotlieb Vladimir , Cheung Tony W. , Wang Jen-Chin 
Published: October 2019

Abstract
Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) including essential thrombocythemia(ET), polycythemia vera(PV), and myelofibrosis (MF) have been regarded as a stem cell diseases with malignant clonal proliferation; but recently, inflammatory processes have been proposed as playing found a major role in the pathogenesis of MPN. Toll-like receptors (TLRs) are a family of pattern-recognition receptors that function as key initiators of innate immunity signaling, then induce inflammatorycytokines, and ROS formation. Therefore we measured TLRs, inflammatory cytokines, and ROS in patients with MPN to assess the role of inflammation in MPN. Methods: TLR assay.TLR-2, 4, 7, 9 quantification was performed by immuno-staining of 1×106 mononuclear cells which were incubated with fluorescence-conjugated anti-TLR-2, 4, 7, 9 antibodies and assayed by flow cytometry. Monocytes culture and dendritic cells differentiation.Human monocytes were isolated from human peripheral blood mononuclear cells (PBMNC) using Pan Monocyte Isolation Kit. Isolated monocytes were incubated with IL-4 and GM-CSF in cultures to differentiate to dendric cells . Immature dendritic cells were either left untreated or stimulated with Pam3CSK4. The maturation of dendritic cells was determined by flow cytometry using CD80, CD83, CD11c, and HLA-DR. Multiplex ELISA. Human plasma and cell culture supernatants were analyzed in duplicates by Meso Scale Discovery Multi-Spot Assay system. In total, ten cytokines were assayed: IFN-g, IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and TNF-a. Cell ROS measurement. Cellular ROS was determined by a dichlorofluorescein (DCF) assay. PBMNCs were incubated with 10 µM 2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) (Life Technology) for 30 min at 37°C. The oxidation of H2DCFDA was measured and analyzed by flowcytometery.
Results. 1)TLR2 was the only TLR found to be elevated in PV or ET patients (Fig 1).
2) PlasmaIL-1b levels were elevated in TLR2 high patients than TLR-2 low patients. 3) No difference between TLR-2 high and low patients in the maturation of monocytes to dendritic cells. 4) Monocyte derived dendritic cells with high TLR-2 patientsreleased more IL-8 and TNF-a after Pam3CSK4 stimulation (Fig 2). 5) ROS were more elevated in MF patients than PV, ET patients, and controls (Fig 3).
Conclusion. 1) TLR 2 is significantly elevated in PV and some ET patients and TLR-2 high patients were found to have elevated plasma IL-1b,and IL-8, and TNF-α in monocytes-derived dendric cell cultures afterPam3CSK4 stimulation than TLR-2 low patients. This confirms that TLR-2 is deranged in PV and ET. 2) ROS is elevated in MF patients compared to ET and PV patients, or controls. Thus, this study suggests that inflammatory processes likely play a role in the pathogenesis of Ph- MPN through first TLR-2 derangement in PV and ET , then through years of chronic inflammatory process , with the accumulation of more ROS seen in MF, which caused more damage to the DNA resulting more malignant clonal proliferation. A similar phenomenon was observed that in JAK2V617F mutation , allele-burden was observed gradually increased from ET , PV then to MF .

Disclosures
No relevant conflicts of interest to declare.