The donor of carbon monoxide (CORM-2) affects the level of serum immunoglobulins and the state of the bone marrow during the immune response in mice

Toxic carbon monoxide in small concentrations has pro-apoptotic, anti-allergic, vasodilator effects, and stimulates angiogenesis. The problem with the therapeutic use of low doses of carbon monoxide is that it is difficult to dose. To control the amount and gradual release of carbon monoxide, non-toxic preparation is used – CO donor based on carbonyl compound of ruthenium (CORM-2). The aim – is to identify the effect of CORM-2 on the level of immunoglobulins in the blood serum and bone marrow of mice under conditions of inducing an immune response. Materials and methods. 3 groups of 15 white laboratory mice each were formed. Induction of the immune response was due to the intraperitoneal administration of xenogenic red blood cells. The first experimental group on the first day of immunization received CORM-2 (20 mg/kg), group No. 2 – on the 5 th day after immunization (period of the productive phase). The control group consisted of immunized animals that did not receive CORM-2. The amount of IgA, IgM, and IgG in blood serum was determined by ELISA on the 2 nd and 6 th day after immunization. At the end of the experiment, bone marrow cell populations were counted. Results. After the injection of CORM-2 during the induction phase of the immune response, it inhibits the production of immunoglobulins. In comparison with the control, the level of IgA and IgG is reduced, but the amount of IgM remains unchanged. In the bone marrow, the number of monocytes, erythroblasts, and normoblasts, as well as lymphoblasts and plasma cells, increased. At the same time, the number of myeloblasts, myelocytes, basophilic normoblasts, and megakaryocytes decreased. The use of CORM-2 during the productive phase caused a decrease in the level of IgM and IgG with a simultaneous increase in the level of IgA. The number of neutrophils, eosinophils, monocytes, polychromophilic and oxyphilic normoblasts, lymphocytes, and plasma cells in the bone marrow increased. However, the number of myeloblasts, promyelocytes, myelocytes, metamyelocytes, basophilic normoblasts, and megakaryocytes decreased. Conclusions. The impact of the CORM-2 on the inductive phase of the immune response leads to inhibition of the production of immunoglobulins. The injection of CORM-2 during the productive phase of the immune response decreased the level of IgM and IgG, but at the same time, an increase in the level of IgA was observed. After the injection of CORM-2, in the bone marrow, the number of monocytes, lymphocytes, and plasma cells increased. The results indicate that CORM-2 is able to modulate the immune response.

Актуальные вопросы фармацевтической и медицинской науки и практики. 2020. Т. 13, № 3(34). С. 415-420 Among pharmacological drugs, there is a lot that has immunomodulatory properties [1]. These drugs can be divided into 2 groups: immunostimulants and immunosuppressants. The latter is widely used for the treatment of autoimmune diseases after an organ transplant procedure. Some antibiotics also belong to immunosuppressants, cytostatics, and hormonal drugs [1]. All of them cause adverse reactions, which increase with increasing dose. That is why search for drugs that can gently suppress the activity of the immune system and not cause catastrophic changes in other organs and systems of the body remains relevant. One of these applicants is a representative of the gas transmitter group -carbon monoxide (CO) [2,3].
CO is known as poison gas, however, in 1968, Tenhunen et al. reported that this gas is formed in the body during the breakdown of heme. After that, the period of studying the physiological effects of CO began [4,5]. Today, it is known that CO in a picomolar amount is able to suppress cell apoptosis [6], stimulates Ca 2+ dependent K-channels [7], blocks T-cell proliferation [8], and is able to affect mitochondrial activity [9]. ISSN 2306-8094 The donor of carbon monoxide (CORM-2) affects the level of serum immunoglobulins and the state of the bone marrow during the immune response... The only problem with using CO was that the dosage of this gas is difficult to carry out, poisoning is possible. In this regard, further studies of the therapeutic effects of CO are inhibited. This problem was solved by creating compounds -donors of CO [10]. After the introduction of such a compound into the body, a slow release of CO occurs in insignificant, controlled amounts. One of the representatives of these compounds is CORM-2 (tricarbonyldichlororuthenium (II) dimer), which is based on the carbonyl compound of ruthenium. Among several different CORMs synthesized, CORM-2 has been used extensively in vivo studies. CORM-2 rapidly liberates CO in physiological buffers (half-life of about 1 min at 37 °C, pH 7.4). Due to the fact that this compound is not toxic, it is therefore used in experimental studies. Its advantage is that this drug does not affect changes in carboxyhemoglobin levels. CORM-2 serves an important role in CO-mediated pharmacology.
It has been proven that CORM-2 has antioxidant activity in plasma [11], accelerates the healing of gastric ulcers [12], regulates the permeability of the mitochondrial membrane [13], and affects the duration of the cell cycle [14,15]. The above confirms the relevance of the study of the CO donor and the effect of this compound on bone marrow and the production of immunoglobulins under conditions of antigenic stimulation.

Aim
To reveal the effect of CORM-2 on the level of immuno globulins in the blood serum and bone marrow of mice under conditions of inducing an immune response.

Materials and research methods
The study was conducted on white outbred laboratory mice weighing 22 ± 3 g. 2 experimental groups of 15 males each were formed. Sheeps red blood cells were intraperitoneally injected into all animals to induce an immune response ("Pharmstandard Biolek", Ukraine) at a dose of 100 cells per 1 kg of body weight. In the first experimental group, during the inductive phase (1 st day of immunization), CORM-2 (20 mg/kg) dissolved in dimethyl sulfoxide (DMSO) and physiological saline was administered. The animals of the experimental group No. 2 were injected with the same solution but during the period of the productive phase (5 th day after immunization). The control group consisted of 15 male white mice that were also immunized but did not receive CORM-2. The study was conducted on white outbred laboratory mice weighing 22 ± 3 g. Two experimental groups of 15 males each were formed. Sheeps red blood cells were intraperitoneally administered to all animals to induce an immune response.
To determine the amount of IgA, IgM, and IgG immunoglobulins, an enzyme-linked immunosorbent assay kit ("Granum", Ukraine) was used. Detection of the results of the study was performed using an ELISA analyzer Immunohem-21100, HTI (USA) on the 2 nd and 6 th day after immunization.
At the end of the experiment (6 th day), euthanasia was performed by an overdose of isoflurane. Femur bones were isolated and bone marrow imprints were obtained. The obtained imprints were stained with Romanovsky-Giemsa paint and bone marrow cell populations were counted using a microscope.
All procedures with laboratory animals were carried out in compliance with the Council of Europe directives of the EU in 2010/63/EU and were approved by the bioethical commission of Kherson State University. Statistical processing was performed using the Mann-Whitney and Wilcoxon criteria. In this case, the result was considered reliable at P ≤ 0.05. The degree of correlation of indicators was calculated using the Pearson correlation coefficient.

Results
Comparison of serum immunoglobulin levels under conditions of induction an immune response showed differences. In the control group, the level of IgA increased by 81.0 ± 4.0 %, IgM -by 35.3 ± 1.8 %, and IgG -by 47.0 ± 1.9 % (Fig. 1).
The result of the level of immunoglobulins in the serum of animals that were injected with CORM-2 on the first day of the immune response turned out to be interesting. In this group, the content of IgA increased by 100 ± 5 %, IgM remained at the same level, IgG increased only by 6.8 ± 0.3 % (Fig. 2).
But, after comparing this group with the control group, it was found that the level of IgA on the next day (after CORM-2 injection) was reduced by 22.0 ± 1.1 % (1.09 g/L versus 1.41 g/L of the control group). On the 6 th day, it was reduced by 15.0 ± 0.75 % (2.18 g/L versus 2.56 g/L of the control group).
The IgM level (on the 2 nd day) after comparison with the control group was reduced by 12.0 ± 0.6 % (1.49 g/L versus 1.7 g/L of the control). On the 6 th day, it was reduced by 35.0 ± 1.75 % (1.49 g/L versus 2.3 g/L of the control group). Similar results were obtained after comparing the IgG values. Its level was reduced on the 2 nd day by 10.5 ± 0.5 % (9.32 g/L versus 10.42 g/L of the control). On the 6 th day, the level was also reduced, but already by 35.0 ± 1.75 % (9.95 g/L versus 15.32 g/L of the control group).
The level of immunoglobulins in the serum of the group of mice that were injected with CORM-2 in the productive period of the immune response was radically different from the other two (Fig. 3). The amount of IgA in serum increased by 67.0 ± 3.3 %, while IgM decreased by 48.7 ± 2.4 %, IgG decreased by 17.0 ± 0.8 %.
A study of bone marrow changes showed that the injection of CORM-2 influenced the proliferation of individual cell types after stimulation of the immune response (Fig. 4).
A correlation analysis revealed a direct, close relationship between immunoglobulin counts and a decrease in the population level of certain bone marrow cells. In group No. 1, a close correlation was observed between the level of serum immunoglobulins and the content of polychromic and oxyphilic normoblasts, lymphocytes/lymphoblasts, plasma cells, megakaryocytes.
In group No. 2, the level of immunoglobulins correlates with the levels of myeloblasts, basophilic normoblasts, polychrome, and oxyphilic normoblasts, lymphocytes, and plasma cells.

Discussion
The results indicate that intraperitoneal injection of CORM-2 directly affects the course of the immune response. CORM-2 inhibits the production of immunoglobulins during the induction phase of the immune response. In comparison with the control, the level of IgA and IgG was reduced, but the amount of IgM did not change. It is known that an increase in serum IgM is characteristic of the primary immune response stage. This indicates that CORM-2 is a promising anti-inflammatory agent. This property of CORM-2 indicates the possibility of its use in autoimmune diseases or to prevent the development of chronic inflammation.
The injection of CORM-2 during the productive phase of the immune response decreased the level of IgM and IgG, however, an increase in the level of IgA was observed at the same time (it is known that IgA is "responsible" for the so-called local immunity). This property of CORM-2 is promising to use in cases of immunodeficiency, reactions after transfusion measures, or medications that cause selective IgA deficiency.
Injection of CORM-2 during the inductive period of the immune response stimulated (or led to a delay in the bone marrow) separation of monocytes, erythroblasts and normoblasts, as well as lymphoblasts, and plasma cells. This was observed against a background of a decrease in the number of myeloblasts, myelocytes, basophilic normoblasts, and megakaryocytes. Injection of CORM-2 during the productive period also showed a decrease in the number of myeloblasts, promyelocytes, myelocytes, metamyelocytes, basophilic normoblasts, and megakaryocytes. At the same time, the level of neutrophils, eosinophils, monocytes, polychromophilic and oxyphilic normoblasts, lymphocytes and plasma cells increased. Thus, it was found that CORM-2 stimulates an increase in the number of monocytes, lymphocytes, and plasma cells, regardless of the stage of the immune response.
After the action of CO, which is released from CORM-2, inducible heme oxygenase (HO-1) is activated. It should be noted that inhalation of CO does not increase the activity of HO-1 [14]. Its activity is enhanced only by CORM-2, which once again confirms that CORMs are suitable for these purposes.
Thus, the release of CO leads to the activation of the HMOX1 gene. Activation of this gene increases HO-1 activity. HO-1 is known to be a metabolite to fight inflammation. The anti-inflammatory ability of HO-1 is realized by increasing the expression of interleukin 10 (IL-10) and an antagonist of the pro-inflammatory interleukin 1 receptor (IL-1RA) [9].
After we stimulated the immune response and introduced anti-inflammatory CORM-2, HO-1, likely, affects the cells of the immune system, bone marrow stem cells. This mechanism may explain the findings of the study.

Conclusions
1. The injection of CORM-2 during the induction phase of the immune response enhances the production of IgA and IgG. The amount of IgM remained at the same level. An increase in the number of monocytes, erythroblasts, and normoblasts, as well as lymphoblasts and plasma cells, was observed in the bone marrow. At the same time, there was a decrease in the number of myeloblasts, myelocytes, basophilic normoblasts, and megakaryocytes.
2. During the productive phase of the immune response, CORM-2 lowered the level of IgM and IgG with a simultaneous increase in IgA level. The number of neutrophils, eosinophils, monocytes, polychromophilic and oxyphilic normoblasts, lymphocytes, and plasma cells in the bone marrow increased. However, there was a decrease in the number of myeloblasts, promyelocytes, myelocytes, metamyelocytes, basophilic normoblasts, and megakaryocytes.
Prospects for further research. In the future, a study of the properties of CORM-2 regarding the processes of tissue regeneration and healing will be conducted in vivo.