| Abstract |
Introduction – Background: MDSCs are a heterogeneous population of immature immunoregulatory myeloid cells, which are elevated in various human diseases that involve chronic inflammation and tumor progression. They are divided in two subpopulations, HLA-DRlow/-CD11b+CD33+CD15+ (polymorphonuclear-PMN-MDSCs) and HLA-DRlow/-CD11b+CD33+CD14+ (monocytic-M-MDSCs). Through activation of the enzymes arginase 1 and nitric oxide synthase 2, and production of reactive oxygen species, they lead to suppression of T-cell proliferation, inhibition of natural killer (NK) cell cytotoxicity, modulation of macrophage polarization and induction of development of regulatory T-cells (Bronte el al. 2016; Umansky et al. 2016; Millrud et al. 2017; Chesney et al. 2016).
Chronic Idiopathic Neutropenia (CIN) is the prolonged, otherwise unexplained reduction in the number of PMN below the lower limit of the normal range. The main pathogenetic mechanism for CIN implicates the increased, Fas-mediated apoptosis of the CD34+/CD33+ myeloid progenitor cells. Chronic inflammation driven by an inhibitory bone marrow (BM) microenvironment consisting of activated T-lymphocytes (oligoclonal profile) and pro-inflammatory mediators (TNF-α, TGF-1, Fas-ligand, IFN-γ, IL-1b, and IL-6) is also involved. Data from our lab have shown that CD14++/CD16+ monocyte subpopulation is increased in patients, which can be associated with the enhanced antigen presentation and T-cells over-activation in the disease (Papadaki et al. 2003; Papadaki et al. 2005; Papadaki et al. 2006; Velegraki et al. 2011; Pavlaki et al. 2014; Stavroulaki et al. 2011).
Aim of the study: The study aims to explore the possible involvement of the monocyte and MDSC subpopulations in the pathogenesis of CIN, through the investigation of the number and functional characteristics of the PMN-MDSCs and M-MDSCs and the correlation with those of CD16+ pro-inflammatory monocytes, in CIN patients compared to healthy subjects.
Methodology: We studied 35 CIN patients and 25 healthy subjects. The panels for cell staining for MDSCs from PBMCs were CD33-PC7/CD15-PC5/DR-ECD/CD14-PE/CD11b-
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FITC, for MDSCs from BMMCs were CD33-PC7/CD15-PC5/DR-ECD/CD14-PE/CD11b-FITC & CD45-FITC/CD33-PC7/CD15-PC5/DR-ECD/CD14-PE. FACS analysis was done with the Kaluza software and statistical analysis was done with the Graph Pad software and the Mann-Whitney test.
Results: CIN patients displayed decreased proportion of M-MDSCs in the PBMC fraction (0.657 ± 0.664) compared to the healthy controls (2.085 ± 2.496; p=0.0228). The proportion of M-MDSCs correlated with the number of ANC (r=0.2976, p=0.0273) suggesting that patients with more severe neutropenia display lower M-MDSCs numbers. A positive correlation was also found between the proportion of M-MDSCs and the number of PB monocytes (r = 0.2672, P = 0.0486).
Conclusions - Discussion: CIN patients display increased number of pro-inflammatory (intermediate CD14++/CD16+ and non-classical CD14dim/CD16++) monocytes in the PB that may contribute to the aberrant T-cell activation and chronic inflammation.
CIN patients display low proportions of PB PMN-MDSCs and M-MDSCs compared to the controls. These cells normally protect from uncontrolled immune responses, so the low number of these cells in CIN may contribute to the sustained chronic inflammation.
We will continue with further experiments focusing on the isolation of the MDSC populations and investigation of their T-cell suppression function and production of ARG1, NOS2, COX2, TGFβ, IL6, IL10.
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Association of myeloid derived suppressor cells (MDSCs) & monocyte subpopulations in patients with chronic idiopathic neutropenia (CIN)
