Increase Expression of the Genes PADI4, MPO and ELANE in the Chronic Phase of DVT Patients
Authors: Toni Isabella Macedo , Zapponi Kiara , Hounkpe Bidossessi Wilfried , Martinelli Beatriz Moraes , Montalvão Silmara Aparecida De Lima , De Paula Erich V , Annichino- Bizzacchi Joyce Maria 
Published: November 2019

Venous thromboembolism (VTE) is a common multifactorial disease, and the knowledge in the pathophysiology can improve prophylaxis and treatment. The myeloid lineage, especially neutrophils, have been implied in the pathophysiology of VTE, but it is still unclear whether its activation is persistent after the acute phase of the disease.
In addition to phagocytosis, neutrophils participate of the innate immunity through the release of NETs (Neutrophil Extracellular Traps): Reactive Oxygen Species (ROS)-triggered antimicrobial structures composed of DNA lined with granular components (Brinkmann et al., 2004). NETs also contribute to the coagulation, as they facilitate the interaction between erythrocytes, leukocytes, platelets and endothelium, besides potentially inhibit anticoagulant pathways (Fuchs et al., 2012). The molecular mechanisms underlying the "NETosis" are still being characterized, but it is known that following inflammatory stimuli, the generation of ROS leads to the dissolution of intracellular membranes and translocation of Myeloperoxidase (MPO) and Neutrophil Elastase (ELANE) to nucleus. Histones are then citrullinated by Peptidyl Arginine Deiminase 4 (PADI4) and degraded by ELANE, promoting chromatin decondensation, plasma membrane rupture and NET's release.
Previously we showed increased neutrophil activation, adhesive properties and the presence of NETs serum markers in patients with VTE in the chronic phase (Zapponi et al., 2014; Zapponi, manuscript in preparation). However, assessment of NETs activity is challenging and, until now, there is no consensus in markers or gold standards measurements. We decided to investigate if the expression of the genes PADI4, ELANE, MPO, could provide additional information regarding NETosis activity, through an indirect analysis of the process by Real-Time PCR. We analyzed samples from 20 controls and 19 patients previously included in the NET study, respecting the time window in which the NETs plasma markers were assessed (between 3 and 43 months after the TVP event). RNA extraction was performed from peripheral blood leukocytes using Trizol reagent, according to the manufacturer's protocol. Real time-PCR reactions were performed with qPCRBIO SyGreen mix (PCRBio), on RotorGene Q (Qiagen) with a reaction efficiency above 0.99. The relative fold changes (Fc) of the genes PADI4, ELANE, MPO were calculated using the Pfaffl method (Pfaffl, 2001 - Nucleic Acids Res.) and the significance level of Mann-Whitney test was considered if p <0.05.
The 39 cDNA samples were amplified in duplicates and a standard sample was included for normalization between different runs. The threshold values (Ct) from PADI4, ELANE, MPO were adjusted for the individual's neutrophil proportion and the geometric means of the housekeeping genes (Actin Beta and Glyceraldehyde-3-Phosphate Dehydrogenase) were used for normalization between samples. The standard deviation was 0.30 between runs and ranged between duplicates from 0 to 1.16 (0.096 on average).
The patients showed significant increased expression of PADI4 (p <0.01), MPO and ELANE (p <0.05) when compared to controls (Figure 1).
Neutrophils emerges fully differentiated from the bone marrow and ranges from 40 to 70% of circulating leukocytes (Cowland and Borregaard, 2016). Isolating these cells can be challenging and the process itself may influence expression profile. The use of total leukocytes would not be the most appropriate process but considering that the analyzed genes are predominantly expressed in neutrophils, the Ct values were weighted according to the neutrophil to leucocytes proportions of each individual.
Our results indicate that the increased neutrophils activation in patients can be represented in PADI4, ELANE and MPO gene expression of peripheral blood leukocytes, which could better represent NETotic activity in these patients. We cannot rule out that the expression of these genes in myeloid precursors could better represent NETotic activity, but our results indicate that this analysis may also be viable in peripheral blood.

Disclosures
No relevant conflicts of interest to declare.