FEATURES OF NK CELL PHENOTYPE VIRUS GENOTYPE-DRIVEN CHRONIC VIRAL HEPATITIS C

Elimination of the hepatitis C virus (HCV) due to direct antiviral drug (DAD) action affects alteration in virus phenotype and, accordingly, NK cell functional activity. However, the published data are very contradictory. Aim. Investigating alterations in NK cell subset phenotype after DAD treatment of HCV genotype-dependent chronic viral hepatitis C (CVHC) patients. Material. 111 CVHC patients and 21 healthy volunteers were examined. The diagnosis was established on epidemiological, clinical and laboratory data. All 111 subjects with CVHC received direct antiviral drugs Sofosbuvir and Velpatasvir for 12 weeks. The study of the NK cell phenotypes wwas analyzed by multicolor flow cytometry. Results. A decreased count of cytokine-producing along with increased frequency of cytotoxic NK cells were found in CVHC patients blood samples with various HCV genotypes prior to DAD treatment. The imbalance of cytotoxic cells with a high level of functional activity was also found in CVHC patients regardless of HCV genotype. The patients with HCV genotypes 1 and 3 showed significantly increased level of immunoregulatory NK cells. In addition, increased count of glycohydrolase (CD38) and ecto-5’-nucleotidase (CD73)-expressing NK cells were found in patients with HCV genotypes 1 and 3. Hence, such alterations in NK cell phenotype in CVHC patients were presented as sustained high viral load which peaking at carriers of HCV genotype 1 that was minimal in patients with HCV genotype 2. The most prominent change in NK cells after DAD treatment was found in CVHC patients with HCV genotype 2 (normalization of CD8-expressing NK cell subset composition and count). Only patients with HCV genotype 2 after treatment had increased frequencies of peripheral blood double-negative


Introduction.
1 Hepatitis C virus (Hepatitis C virus, HCV) is one of the main causes of chronic 2 liver infections in the world. The consequences of chronic viral hepatitis C (CVHC) 3 are highly variable, ranging from minimal histological changes to extensive fibrosis 4 and cirrhosis with or without hepatocellular carcinoma [5,13]. According to 5 available estimates, the number of patients with CVHC in the world is about 180 6 million people and most patients are unaware of the presence of this infection in 7 them [37]. Treatment of HCV-associated liver injury has improved significantly 8 over the past 20 years [4, 24]. It could be closely linked with our recent data on 9 disease pathophysiology as well as the improvement of methods for diagnostics, 10 treatment and disease prevention. 11 Currently, there are two main mechanisms of viral infection development. The 12 first one is determined by the pathogenic action of the virus itself against the 13 background of immune disorders associated with a lack of immunity components 14 and/or with a lack of activation for a specific pathogen (tolerance) [1,2]. The second 15 mechanism is realized by the activation of immune system on virus-infected cells. 16 Consequently, the prognosis of development, the nature of the course and the 17 outcome of an infectious disease vary significantly in depending on the state of the 18 immune system and the characteristics of its response to an infectious pathogen [6, 19 9]. 20 NK cells (Natural Killer) are defined as a separate population of lymphocytes 21 that perform the functions of innate immunity. Cytolytic activity without prior 22 stimulation of virus-infected and some tumor cells is the initially defined and main 23 function of NK cells [19,33]. The killer activity of NK cells is regulated by the 24 expression of MHC I molecules. Infected or malignant cells can downregulate MHC 25 I (also known as "missing-self hypothesis"), to become invisible for CD8+ T cells 26 but the loss of MHC I antigens for inhibitory receptors on NK cells sensitizes these 27 cells for NK-mediated killing [12,29]. However, at present, the regulation of some processes of innate and adaptive immunity is also determined by the function of NK 29 cells which is realized by these cells during functional activation due to the secretion 30 of a wide range of cytokines [7]. Thus, Pallmer K. and Oxenius A. (2016) showed 31 that NK cells stimulated the maturation and activation of dendritic cells, secrete 32 cytokines that promoted the differentiation of 'naïve' Th lymphocytes into Th1 cells 33 [29]. In a study by Anuforo O.U.U. et al. (2018) showed that NK cells maintained 34 apoptosis and regulated the functional activity of neutrophils against the background 35 of antigen-induced inflammation [8]. In this regard, there is a need to study the 36 phenotype of NK cells and the characteristics of their functional activity in patients 37 with CVHC. 38 A number of studies have shown that the treatment of the disease with direct 39 antiviral drugs (DADs) is effective and allows achieving complete elimination of 40 HCV from the body [4,30]. However, disturbances in the immune system may 41 persist while levels of inflammation and fibrosis in the liver have been reduced. It 42 was found that the treatment of CVHC patients with DADs led only to a temporary 43 restoration of the functional activity of cells and then the reactivity of these cells 44 decreased significantly below the norm [39]. without septa (5,2 kPa); F2 − portal and periportal fibrosis with single septa 67 (7,3 -9,5 kPa); F3 − portal and periportal fibrosis with multiple bridging porto-68 portal and porto-central septa (9,6 -12,5  RNA by polymerase chain reaction (PCR) was carried out before the start of 90 treatment, after 4 weeks of therapy, at the end of treatment and 24 weeks after the 91 end of therapy. Drug adherence was assessed using the Morisky-Green Test [10].

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The control group included 21 practically healthy individuals who were

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The level of viral load in CVHC patients is presented in

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NK cells in patients with HCV genotype 2 was found relative to the level detected 157 in patients with HCV genotype 1.

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It is known that about 30% of peripheral blood NK cells express the CD8 159 receptor, the functional activity of such cells is considered high and they demonstrate 160 increased survival in the process of target cell lysis [18,24]. We found that the 161 maximum (among CVHC patients) percentage of NK cells expressing CD8 162 (CD3 − CD56 + CD8 + CD45 + ) before DAD treatment was detected in patients with 163 HCV genotype 2 (table 3). However, there were practically no differences in the   are capable of producing a wide range of cytokines [12,19,33]. We carried out a 241 study of the content of the main fractions of blood NK cells in CVHC patients 242 according to CD16 and CD56 markers. CD16 is a low-affinity G III type 243 immunoglobulin receptor (FcRIII), which mediates the mechanism of cellular 244 antibody-dependent cytotoxicity [11,19]. The CD56 marker (NCAM,245 NKH-1) is a glycoprotein belonging to the immunoglobulin superfamily and is ChemR23, migrate to the zones of immune-inflammatory processes [22,23,35]. 263 We found that the restructuring of the subset composition of NK cells in patients with HCV genotype 2 (more than 10 times higher than control values). family of Ca 2+ -dependent lectins (type C) [27]. were characterized as IFN producing cells with cytolytic activity [20]. Therefore, 320 we can conclude that the treatment of CVHC patients with HCV genotype 2 led to 321 an increase in the functional activity of NK cells.

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In recent years, attention has been drawn to the fraction of NK cells expressing 323 the CD8 receptor [16,25]. It has been proven that CD8 + NK cells are highly sensitive 324 to activation stimuli, have an increased level of cytotoxicity, and co-express 325 molecules such as IFN-, CD107a, TNF-α, and MIP-1β [16,25]  Also, the number of NK cells expressing and non-expressing CD38 and CD73 361 in CVHC patients before and after DAD treatment was studied. CD38 is a about 45 362 kDa glycoprotein that is expressed on the surface of many cells of the immune 363 system and is defined as a glycohydrolase (EC 3.2.2.6) that catalyzes the degradation 364 of NAD + or NADP + to form cyclic ADP-ribose and nicotinamide [32]. The products 365 of this reaction are necessary for the regulation of the intracellular pool of Ca 2+ .

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CD38 is involved in the regulation of cellular metabolism through the regulation of 367 the NAD pool and in the pathogenesis of many conditions, including aging, obesity, 368 diabetes, heart disease, asthma, and inflammation. It was shown that the expression 369 of the CD38 receptor on the membrane of NK cells led to a decrease in their 370 functional activity [34]. CD73 (NT5E) is an ecto-5'-nucleotidase (EC 3.1.3.5) − an 371 enzyme that cleaves adenosine monophosphate (AMP) to adenosine [36]. It has been 372 proven that NK cells expressing CD73 have reduced cytotoxic activity and can 373 implement the functions of regulatory cells [28]. 374 We found a decrease in the number of double-negative NK cells as well as    Примечание: p 1 − статистически значимые различия с показателями контрольной группы.
Note: p 1 − significant differences versus control.