Crosstalk between Oxidative Stress, Chronic Inflammation and Disease Progression in Essential Thrombocythemia

AMELIA MARIA GAMAN1,2, CORNEL MOISA1,3*, CAMELIA CRISTINA DIACONU4,5, MIHNEA ALEXANDRU GAMAN4* 1University of Medicine and Pharmacy of Craiova, Department of Pathophysiology, 2 Petru Rares Str., 200349, Craiova, Romania 2Clinic of Haematology, Filantropia City Hospital, 1 Filantropiei Str.,200143, Craiova, Romania 3County Emergency Hospital Slatina, Department of Haematology, 5 Crisan Str., Slatina 230008, Romania 4Carol Davila University of Medicine and Pharmacy, 8 Eroii Sanitari Blvd., 050474, Bucharest, Romania 5Internal Medicine Clinic, Clinical Emergency Hospital of Bucharest, 8 Calea Floreasca Str., 014461, Bucharest, Romania

The aim of the study was to evaluate oxidative stress levels in patients with ET and to investigate the relationship between ROS, TAC, chronic inflammation parameters, leukocytosis, JAK2V617F mutation and progression of disease to myelofibrosis or leukemic transformation. * email: mccor45@yahoo.com; mihneagaman@yahoo.com

Experimental part
Sixty-two patients diagnosed with ET based on the 2016 revised World Health Organization diagnostic criteria and 20 healthy volunteers with similar demographic characteristics were included in the study [1]. Informed consent was obtained from all subjects involved, the study protocol had the approval of the Ethics Committee of the University of Medicine and Pharmacy of Craiova (approval no. 79/23.02.2017) and was carried out in accordance with the standards imposed by the Declaration of Helsinki and the European Union 679/2016 regulation.
Patients were classified based on age, sex, presence of JAK2V617F mutation and inflammation status. Haematological parameters (haemoglobin value, haematocrit levels, MCV, MCH, MCHC, RDWR, RDWA, leukocyte count, leukocyte formula, platelet count, mean platelet volume, PCT, PDW, LPCR, bone marrow aspiration or biopsy), lactate dehydrogenase, fibrinogen, C-reactive protein (CRP), serum ferritin level and neutrophil/ lymphocyte ratio were measured. JAK2V617F detection was performed in a specialised molecular biology laboratory. Haematological parameters were analysed using a SYSMEX XN-450 analyser and biochemical parameters were determined using a KONELAB601 analyser by spectrophotometry. Serum ferritin level was estimated using the Enzyme-Linked Immunosorbent Assay (ELISA) method as per the manufacturer's guidelines. Oxidative stress was evaluated by measuring the level of reactive oxygen species (ROS) using a CyFlow Space Sysmex flow-cytometer (reagents from Abcam). Negative control was analysed immediately, patient samples were analysed after incubation at 37 0 Celsius for 30 minutes and the positive control was analysed after incubation at 37 0 Celsius for four hours. The total antioxidant capacity (TAC) was analysed using a FLUOstar Omega multi-detection microplate reader (reagents from Sigma-Aldrich) according to manufacturer 's instructions. We compared inflammation markers (fibrinogen, CRP, serum ferritin, NLR, leukocytosis) and oxidative status parameters (ROS and TAC) in: 1) ET patients vs. the control group; 2) ET JAK2V617F-positive cases vs. JAK2V617F-negative cases; 3) ET patients vs. patients with disease progression (myelofibrosis or leukemic transformation). Statistical analysis was performed and a p-value < 0.05 was considered significant.

Results and discussions
The study group had a median age of 59.50 years and consisted in 25 males (40.30%) and 37 females (59.70%), revealing a female predominance. The JAK2V617F mutation was positive in 36 ET patients (58.06%) (heterozygous genotype in 30 patients and homozygous genotype in six patients) and negative in 26 ET patients (41.94%), according with literature data. ROS levels were increased (with high levels in JAK2V617F-positive ET patients vs. JAK2V617F-negative cases; mean ROS value = 2.73 mM/L vs. 2.60 mM/L; p<0.05) and TAC levels were decreased in patients with ET vs. the control group (with low levels in JAK2V617F-positive patients vs. JAK2V617Fnegative patients, mean TAC value = 0.46 mM/L vs. 0.51 mM/L; p<0.05) [12,15].
It has been shown that the acquisition of the JAK2V617F mutation induces constitutive activation of JAK-STAT, NF-kB and PI3K/AKT signalling pathways and hypersensitivity of myeloid progenitors to hematopoietic cytokines [16]. STAT3 is responsible for neutrophil activation and STAT5 for cell proliferation [16]. STAT3 activates the release of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, IL-11, TNFα, growth factors: vascular endothelial growth factor VEGF, fibroblast growth factor FGF) and metalloproteinases. IL-1β sustains induction of cytokines and inhibits the activation of transcription factor Nrf2 (nuclear erythroidrelated factor 2) with subsequent downregulation of antioxidants genes. AKT is responsible for the negative regulation of the Forkhead O transcription factor family (FoxO) with downregulation of several antioxidant enzymes: glutathione peroxidase, catalase and superoxide dismutase [17][18]. Uncontrolled cytokine secretion induces a pro-inflammatory status in the bone marrow microenvironment and in the blood. Chronic inflammation in the bone marrow activates immune cells, increases NF-kB activity in hematopoietic and stromal cells. NF-kB is a redox-sensitive transcription factor with a major role in inflammation, innate immunity and carcinogenesis. The association between chronic inflammation, sustained proliferation of the myeloid lineage and the activation of cell signaling pathways ultimately damages the DNA of hematopoietic cells via ROS accumulation, tissue destruction, remodeling and fibrosis [16]. Leukocytosis and thrombocytosis reflect clonal myeloproliferation and the effect of chronic inflammation per se on malignant cells which are inherently hypersensitive to the stimuli of growth factors and cytokines [16].
It is known that inflammation is associated with increased ROS levels. H 2 O 2 , the most potent ROS, activates pro-inflammator y pathways (NF-kB) which in turn generate more ROS. The excessive ROS production in MPNs induces a proliferative advantage to JAK2-positive clones [17][18]27]. On the other hand, the JAK2V617F mutation per se is responsible for an accumulation of ROS in the hematopoietic stem cell compartment, the excessive ROS production acting as a mediator of genomic instability, JAK2V617F-induced oxidative stress and DNA strand breaks and mutations [19]. Sevin et al. (2015) revealed that heat shock proteins (HSP) have a major role in inflammation in MPNs through their chaperone activity. HSP90 stabilizes many oncogenes, including JAK2, and, in association with HSP70, regulates the NF-5ØßB signaling pathway [31]. Hasselbalch et al. (2014) reported that, in MPNs, there is a deregulation of oxidative stress and anti-oxidative stress genes. Their study revealed that ATOX1, DEFB122, GPX8, PRDX2, PRDX6, PTGS1, and SEPP1 genes were progressively upregulated, while AKR1B1, CYBA, SIRT2, TTN, UCP2, SOD2, CAT and Nrf2 genes were progressively downregulated [8]. Bjorn and Hasselbalch (2015) revealed that the hematopoietic stem cell niche in MPNs downregulates catalase and superoxide dismutase activity [7]. UCP2 and SIRT2 induce constitutively increased NF-kB activity and, when downregulated, production of proinflammatory cytokines [17]. An important role in the promotion of DNA repair, protection of telomere stability and differentiation of stem cells is played by sirtuins [24]. In MPNs, a downregulation of the Nrf2 gene, which encodes the transcription factor nuclear factor erythroid 2-related factor 2, occurs. Since Nrf2 modulates the migration and retention in the stem cell niche of hematopoietic stem cells, Nrf2 depletion results in the expansion of the compartment of hematopoietic stem and progenitor cells [20]. Inactivation of the tumor suppressor gene p53 and of FoxO3 has been shown to increase ROS levels and was linked to a loss of function of the hematopoietic stem cells, excessive oxidation of DNA, acquired mutations, genomic instability and disease progression to myelofibrosis or leukemic transformation [17,[21][22].
In MPNs, chronic inflammation has been suggested as a major player in disease progression, clonal evolution and myelofibrotic and leukemic transformation via TNF-α as a tumor promoter and inducer of clonal expansion of JAK2V617-positive cells, since the JAK2V617F mutation per se has been demonstrated to induce ROS overproduction, oxidative stress, DNA damage and genomic instability [6-7, 16, 25-27]. Oxidative stress activates the redox-sensitive transcription factor NF-kB, leading to an excessive release of pro-inflammatory cytokines which, in turn, can cause oxidative stress in hematopoietic cells. Thus, a self-perpetuating vicious circle develops: chronic inflammation -NF-kB activation -proinflammatory cytokines release -ROS generation -oxidative stress -acquired mutations -genomic instability -clonal evolution to myelofibrosis or leukemic transformation [6][7][8]. The sustained release of inflammatory cytokines and chronic oxidative stress cause a high-risk bone marrow microenvironment for induction of oxidative DNA damage in hematopoietic cells, mutations and epigenetic changes, such as DNA methylation and histone deacetylation, which contribute to tumor initiation, tumor progression and leukemogenesis [28][29]. Tefferi et al. (2016) and Hermouet & Vilaine (2011) reported that the JAK2 46/1 haplotype predisposes to additional mutations in the JAK2 gene and genomic instability, and associated with cytokine-mediated expansion of malignant clones, determines an inferior survival in primary myelofibrosis and increases the risk of myelofibrotic transformation in ET and polycythemia vera patients [1,30]. According to recent studies, sustained JAK2 overexpression, as well as the presence of the JAK2V617F mutation, may induce genomic instability and disease progression from an early disease stage to the advanced myelofibrosis stage [6,30].

Conclusions
In our study, we found high levels of inflammation markers in ET patients vs. controls, but without statistical significance between JAK2V617F-positive and JAK2V617Fnegative cases. We found positive weak correlations between oxidative stress markers and fibrinogen, CRP, serum ferritin and a moderate positive correlation between oxidative stress -leukocyte count and NLR, respectively. Disease progression to AML associated increased levels of oxidative stress, elevated values of inflammation markers and increased leukocyte counts, while in patients who progressed to myelofibrosis, ROS and serum ferritin increased after vs. before transformation. Thus, we may hypothesize that chronic inflammation and oxidative stress are involved both in ET initiation and also in disease progression to myelofibrosis or leukemic transformation. In this respect, anti-inflammatory and oxidative stressmodulating therapy, in addition to JAK2 inhibitors and IFN-α2, might be of aid in ET to slow down disease progression to myelofibrosis or leukemic transformation.