Immune-related mechanisms, molecular and genetic characteristics of patients with the systemic connective tissue diseases with cryoglobulinemic syndrome

demonstrated statistically lower miR-146 а expression which resulted in the abnormal production of pro-in ﬂ ammatory cytokines, the highest TLR9 expression on monocytes, slightly lower on lymphocytes, and the lowest on granulocytes; the increase in the relative amount of cytolytic T-lymphocytes, IL2 receptor lymphocytes, activated CD HLA DR+-lymphocytes against the reduction of NK-cells and regulatory suppressor CD4 + /25 + -cells was observed. The idiopathic and initiated oxidative monocyte capacity in CGS patients distinctly tended to increase, as compared to patients without CGS and normal individuals. Conclusions. Cryoglobulins may act as the so-called bridge between viral infections and the autoimmune processes. CGS was diagnosed in 31.1% of patients. Despite a substantial number of studies dedicated to the cryoglobulinemic syndrome, the peculiarities of the immune reaction of such patients need further research, since they create the risks of secondary vasculitis against SAD.

Introduction. Cryoglobulinemic syndrome (CGS) is an immune-related process caused by abnormal protein (cryoglobulins) composition in the blood that undergoes reversible precipitation at temperatures lower than 37 о С in small or medium vessels which frequently results in systemic vasculitis [1]. Lymphotropic viruses, including herpesviruses (herpes simplex virus, cytomegaloviruses, Epstein-Barr virus), immune-related and oncological diseases are the most frequent CGS. CGS development is determined by the interaction between the genetic virus factors and the host [2]. The immune reactions causing the response to the virus replication in the immunocompetent cells resulting in the chronic immune system stimulation are of paramount concern.
Research testifi es to the high EBV viral load in patients with the systemic diseases of connective tissue correlating with the disease activity regardless of the immunosuppressor therapy [2].

Objectives:
We planned the studying of the immune-related mechanisms, molecular and genetic characteristics of patients with systemic autoimmune diseases (SAD) against cryoglobulinemic syndrome.
Materials and methods. The study involved 380 SAD patients (70 patients with systemic lupus erythematosus, 90 with systemic vasculitis, 120 with rheumatoid arthritis, 100 with psoriasis) hospitalized or undergoing outpatient treatment at the rheumatology department of Lviv Region Clinical Hospital and Lviv Region Clinical Diagnostic Center related to the clinical sites of the Department of Clinical Immunology and Allergology of Danylo Halytsky Lviv National Medical University. The control group involved 20 healthy individuals of the specifi c group and sex. DNA HSV 1/2, EBV was determined using the polymerase chain reaction (PCR) method using «Ampli Sens» (Russia) and «Rotor Geen 6000» (Corbett Research, Australia) diagnostic simultaneously in three biological media (blood serum, saliva, and scraping of the posterior pharynx mucus membrane). To defi ne the level of specifi c immunoglobulins, enzyme immunoassay was conducted with the application of immunoassay analyzer Stat Fax® 303 Plus. The phenotypic characteristics of lymphocytes were defi ned using the fl ow cytometry with the involvement of monoclonal antibodies on the Becton Dickinson (USA) cytofl uorometer. MiR-146а and miR-155 expressions; miR-BART 13, 15 in blood samples were determined in several stages. At fi rst, the general RNA was released using mirVana TM PARIS TM (Ambion, USA); afterwards, micro-RNA was determined using the real-time reverse transcription and PCR with the application of High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA), specifi c primers for each miRNA and general RNA. Quantitative real-time PCR was performed using TaqMan MicroRNA (Applied Biosystems, USA) analyses. The miRNA level was shown in conventional units (CU) using the specifi c formula. The amplifi cation was carried out using 7500 Fast Real_time PCR (Applied Biosystems, USA). The received data were analyzed using the 7500 Fast Real_time PCR database [3,4]. The research was carried out at the Department of General and Molecular Pathophysiology of Bogomoletz Institute of Physiology of NAS of Ukraine as part of the cooperation between universities. TLR9 study was carried out based on CD123 + detection on the peripheral mononuclear blood cells through the ductal cytofl uorometry using ductal cytofl ow meter and «Beckton Dickenson» (USA) test kit [5].
The design of the work was agreed upon with the Commission on Bioethics at Danylo Halytsky National Medical University of Lviv (Minutes No. 5 of February 17, 2016) with a conclusion on the compliance with moral and ethical standards of bioethics according to ICH/GCP, Helsinki Declaration of Human Rights (1964 ), the Council of Europe Convention on Human Rights and Biomedicine (1997), as well as current legislation of Ukraine.
The descriptive statistics methods were applied to describe the initial condition of treatment groups. For quantitative indexes, the normality of data distribution within the groups was verifi ed using the Shapiro-Wilk test. In most cases, the Gauss distribution is identifi ed. The groups were compared using the Independent Samples t-Test or Paired T-Test. In the case of non-Gaussian distributions, the groups were compared using the Mann-Whitney test. The null hypothesis was rejected at р<0.05 [6]. Праці  MicroRNA plays an important role in the control of the immune response, miR-146а, miR-155 are the major immune system regulators [3]. Therefore, our next task was to study the level of miR-146а, -155 expression in the blood serum of patients with the systemic connective tissue disease with the cryoglobulinemic syndrome ( Table 2).
According to Table 2, the level of miR-146а expression in SAD patients tended to reduce, and in patients with cryoglobulinemic syndrome, it was statistically twice as low as compared to healthy individuals and 1.7 lower as com- Thus, patients with systemic connective tissue diseases and the cryoglobulinemic syndrome demonstrated statistically lower miR-146а expression, which resulted in the abnormal production of pro-infl ammatory cytokines with the following aggravation of the chronic infl ammatory process and activation of autoimmune reactions [3]. Statistical increase of miR-155 expression is frequently the reason for anti-infectious protection disturbances due to the defect of Т-, В-, and dendritic cell functions, and thus, the activation of herpesvirus infection which is present in the body [7].
The next studies were dedicated to determining TLR9 expression on immunocompetent cells of SAD patients against hypercomplecte-my (Table 3). TLR is known to trigger the inborn immunity cells.
According to Table 3, TLR 9 expression on monocytes of SAD patients with the cryoglobulinemic syndrome was statistically 4 times higher (0.12 ± 0,045, р<0.05) as compared to the index in healthy individuals (0.03 ± 0.01%) and 1.5 times higher as compared to patients with no CGS (р>0.05). The expression of this receptor on lymphocytes with CGS also turned out to be 1.6 times statistically higher (1.30 ± 0.22, р<0,05) as compared to healthy individuals and 1.2 times higher as compared to patients with normal CG. Statistically, TLR 9 expression on granulocytes of CGS patients did not diff er from the same index of normal individuals and patients without CGS.
Phagocytosis is the process of detoxifi cation and elimination of foreign substances. The most informative indexes of phagocytosis include the following: the phagocytic index (PhI) and the oxygen-dependent digestion mechanism through the oxidative burst (OB) of monocytes and neutrophils [4]. Therefore, our next study was dedicated to the phagocytic activity of SAD patients with the cryoglobulinemic syndrome (table 4).
According to Table 4, the idiopathic and initiated absorbing capacity of neutrophils of SAD patients with cryoglobulinemic syndrome tended to reduce as compared to healthy individuals and SAD patients with the normal CG level. The absorbing capacity of monocytes (4.68 ± 1.16%) was mostly aff ected in CGS patients as compared to healthy individuals (8.80 ± 1.86%, р<0.05) against their reduced reserve capacity. The idiopathic oxygen-dependent processing of neutrophils was statistically higher in CGS patients (13.7±2.45%) as compared to patients without CGS (13.7 ±2.45%, р<0.05) and healthy individuals (7.14 ± 2.21%, р<0.05). The initiated oxidative capacity of neutrophils was also statistically higher (99.8 ±3.56%, р<0.05) in CGS patients as compared to patients without CGS (87.6 ±5.14%). The idiopathic and initiated oxidative monocyte capacity in CGS patients showed a distinct tendency towards increas-ing as compared to patients without CGS and healthy individuals.
Thus, patients with systemic connective tissue diseases with cryoglobulinemic syndrome showed the tendency to reducing idiopathic and initiated absorbing capacity of neutrophils and its statistical reduction among monocytes against the statistically high idiopathic and initiated oxidative activity of neutrophils as compared to patients with the normal cryoglobulinemic level [9].
At the next stage of our study, we defi ned major populations and sub-populations of lymphocytes in SAD patients with the cryoglobulinemic syndrome (Table 5) According to Table 5, SAD patients with CGS show the tendency to the reduction of Т-cells and Т-helper cells against the statistical increase of cytolytic T-lymphocytes (28.9 ±2.33%) and statistical reduction of killer CD3+/8+-lymphocytes (6.01±1.22%) as compared to healthy individuals ( 20.6 ± 2.91% and 9.77 ± 1.49% respectively, р<0.05). The relative amount of CD3 + /8 + -lymphocytes in CGS patients was 1.2 times higher as compared to patients without CGS, and the number of CD16 + /56 + -lymphocytes just tended to increase. In CGS patients, activated CD HLA DR + -lymphocytes and IL2 receptor lymphocytes (CD25 + ) tended to increase and the number of CD4 + /25 + -suppressor lymphocytes was 1.5 times lower as compared to patients without CGS.

Indices of the phagocytic and oxidative activity of neutrophils and monocytes of the peripheral blood of healthy individuals and patients with systemic connective tissue diseases and the cryoglobulinemic syndrome (М ± m)
Thus, SAD patients with the cryoglobulinemic syndrome as compared to SAD patients with the normal cryoglobulins composition demonstrated the increase of the relative amount of cytolytic T-lymphocytes (1.2 times), IL2 receptor lymphocytes, activated CD HLA DR+-lymphocytes (1.4 times) against the reduction of NK-cells (1.5 times) and regulatory suppressor CD4 + /25 + -cells (1.5 times), however, without the statistical diff erence (р>0.05).
Discussion. Hyperproduction of a certain type of cryoglobulins is caused by viruses resulting in damage to the macrophage system by the hyperproduction which brings about the reduction of antigens and immunoglobulin clearance which includes B-cells activation and hyperproduction of specifi c antibodies [10]. The defects of the macrophage system cause the accumulation of cryoglobulins in circulation [11]. Today, the materials concerning the cryoglobulins involvement in the regulation of the immune response are abundant. The long-lasting antigenic stimulation of the immune system on the condition of increased Th2-dependent cytokines (interleukine-3, -4, -5, etc.) facilitates excess immunoglobulin production. The antigenic shift of the trigger initiates the new types of antibodies. Several, even the smallest molecular modifi cations of the structure of these proteins, may have a dramatic impact on their physical and chemical properties and the development of cryoglobulinemic vasculitis [12].
In conclusion, cryoglobulins may act as the so-called bridge between viral infections and autoimmune processes. The capacity of such globulins to form cryoprecipitates separately or in interaction with other antigens results in the sedimentation of the latter on the vascular endothelium and the activation of the complement system. Cryoglobulinemic vasculitis is the consequence of vascular depositions of circulating immune complexes in the vessels. Despite the substantial number of studies dedicated to the cryoglobulinemic syndrome, many important aspects of the problem remain understudied. Table 5 Indices of population and sub-population structure of lymphocytes in healthy individuals and patients with the systemic connective tissue disease and the cryoglobulinemic syndrome (М ± m)