EFFECT OF M2 MACROPHAGE-DERIVED SOLUBLE FACTORS ON DIFFERENTIATION OF SH-SY5Y CELLS

Macrophages play a key role in triggering and regulation of neuroregeneration. The characteristic feature of macrophages is pronounced plasticity, which manifests itself in the ability of macrophages to change their functional phenotype depending on the micromilieu. Apoptotic cell clearance (efferocytosis) is an important inducer of a macrophage polarization to M2 phenotype under pathological settings. Previously, we have developed an original protocol for the generation of M2-like macrophages, polarized by efferocytosis under serum-deprived conditions (M2 (LS), Low Serum). The present study was aimed to assess a neuroregenerative potential of M2 (LS) macrophages. We studied their effect on the differentiation of SH-SY5Y cells in comparison with retinoic acid (RA). As the morphological criteria of differentiation we have assessed the relative content of differentiated cells, i.e., cells with a neurite length exceeding the cell body length, and the average neurite length on days 3, 7, and 13. The ratio of neuron-like (N-type) and epithelial-like (S-type) cells in cultures was also assessed. SH-SY5Y cells were characterized by a low level of spontaneous differentiation, both under standard conditions (10% FBS) and serum deprivation (1% FBS). Upon RA treatment, SH-SY5Y cells stopped proliferating and underwent neuronal differentiation. Cultivation of SH-SY5Y cells in the presence of M2 (LS) conditioned medium also led to a significant increase in the relative content of differentiated cells, the average length of neurite-like processes, as well as a change in the balance of S- and N-type cells towards a pronounced predominance of the latter. The morphological features of differentiation were significantly less pronounced at early stage (day 3) of differentiation as compared with the RA-induced changes and reached the level of positive control only at later stages (day 13) (p < 0.05). In contrast to retinoic acid, M2 (LS) conditioned medium induced neuronal differentiation of SH-SY5Y cells without suppressing their proliferative activity. The data obtained may indicate a high neuroregenerative potential of M2 macrophages in vitro, which is realized through soluble factors and manifests itself in promoting SH-SY5Y differentiation

INTRANASAL INHALATIONS OF BIOACTIVE FACTORS PRODUCED BY M2 MACROPHAGES IN THE TREATMENT OF PATIENTS WITH ORGANIC BRAIN SYNDROME

The aim of present study was to evaluate safety and clinical efficacy of inhalatory immunotherapy based on intranasal delivery of bioactive factors produced by M2 macrophages applied for treatment of patients with organic brain syndrome (OBS).Materials and methods. The study under the NCT02957123 protocol (www.ClinicalTrails.gov) included thirty patients with OBS of various genesis (10 men and 20 women aged 18 to 81; Me, 62.5 years). Neurological assessment and the levels of 32 cytokines in the blood serum of patients were evaluated before and 2-3 days after completion of inhalation immunotherapy.Intranasal inhalations of cell-free culture medium of M2 macrophages (2 mL, once a day for 28-30 days) were safe and well tolerated. None of 30 treated patients had severe adverse events and serious treatmentrelated side reactions. One month after starting the inhalations, a positive dynamics in neurological status was noted in all the patients. A marked clinical response was documented in twenty out of thirty patients (67%), which manifested as improvement, according to all scales and questionnaires. The neurological improvement was not reversed over 6 months of follow-up period. In other ten patients (33%), a moderate clinical response was shown as improvement of individual scores. The positive changes were as follows: 1) a 43% decrease in anxiety and depression scores (according to HADS scale, pU = 0.0008); 2) an increase of total motor activity (stability and gait) by 25%, pU = 0.0001); 3) correction of cognitive functions (MoCa test, pU = 0.007); 4) reduced number and intensity of the disease symptoms by 52% (pU = 0.0001). This marked clinical response to immunotherapy is shown to be associated with correction/normalization of serum hepatocyte growth factor (HGF) level.Conclusion. Inhalation immunotherapy based on intranasal delivery of bioactive factors produced by M2 macrophages can improve neurological and functional recovery in patients with organic brain syndrome

Expression of arginase 1 and tyrosine kinase Mer by blood monocytes in the dynamics of physiological pregnancy

During pregnancy, the maternal immune system must maintain tolerance to paternal antigens, at the same time being able to eliminate pathogens, which is achieved by the weakening of adoptive immunity and the activation of innate immunity, in particular, monocytes. However, the question about the functional phenotype of monocytes, having not only pro-inflammatory, but also anti-inflammatory activity, remains open. In the given work, we have investigated the expression of M2-associated suppressive markers Arg1 and MerTK in monocyte subpopulations during uncomplicated pregnancy. Fifty-three pregnant women with uncomplicated gestation were recruited, including 14 pregnant in the 1st trimester, 20 – in the 2nd and 19 – in the third pregnancy trimester. The comparison group consisted of 15 fertile unpregnant women without aggravated somatic anamnesis, with a history of at least one childbirth. The findings showed that in the unpregnant group circulating Mo express Arg1 and MerTK, and the most relative number of Arg1+ and MerTK+ cells is concentrated in intermediate and nonclassic monocytes. During pregnancy the expression of researched molecules in monocytes reliably increases. An increase in MerTK expression is manifested by a simultaneous increase in the number of MerTK+ cells and the mean fluorescence intensity of this marker; it is observed in the 1st and 2nd trimesters and registered in all three monocyte subpopulations. At the same time, an increase in Arg1 expression is manifested either by an enhancement of Arg1+ cells, or an increase in receptor density; it is registered throughout pregnancy, including the 3rd trimester, and is maximally expressed in classic monocytes. There is a direct correlation between the number of Arg1+ and MerTK+ cells in intermediate Mo, which increases with the progression of pregnancy, and in the 3rd trimester is also detected in classical and non-classical Mo. In general, the revealed increase in the expression of Arg1 and MerTK by monocytes indicates an increase in the anti-inflammatory potential of monocytes during pregnancy, and the involvement of monocytes in the regulation of the inflammatory process at the system level. Moreover, the features of Arg1 and MerTK expression in various monocyte subpopulations during pregnancy suggest that monocytes expressing Arg1 and MerTK can mediate different mechanisms of immune adaptation during pregnancy

Expression of M2-associated molecules in circulating monocyte subsets in fertile non-pregnant women and pregnant women with uncomplicated pregnancy

In humans circulating monocytes include classical (CD14++CD16- ), intermediate (CD14++CD16+) and non-classical/alternative (CD14+CD16++) monocytes, which in turn can be activated via the classical or alternative pathway. Pregnancy is accompanied by significant changes in the monocyte compartment, which is manifested by an increase in the number of circulating monocytes, including the proportion of intermediate monocytes, and a change in their function. However, the functional properties of monocyte subsets during gestation remain largely unexplored. We hypothesized that circulating monocytes may be activated in an alternative pattern and acquire features of M2 polarization (anti-inflammatory / immunosuppressive properties). The aim of the investigation was to study M2-associated markers that characterize the anti-inflammatory and immunosuppressive potential of myeloid cells in subpopulations of circulating monocytes in fertile nonpregnant women and women with uncomplicated pregnancy in the 2nd trimester. It was shown that in fertile non-pregnant women intermediate and non-classical monocytes are characterized by a higher expression of M2-associated markers (CD206, Arginase 1, MerTK) compared to classical monocytes. In the 2nd trimester of pregnancy, the expression of these molecules on monocytes increases significantly, which is manifested by 1) an increase in the proportion of CD206+ cells in subpopulations of classical and intermediate monocytes, 2) an increase in the mean fluorescence intensity of Arginase 1 in all monocyte subsets, 3) an increase in the proportion of MerTK+ cells in subpopulations of classical and intermediate monocytes and mean fluorescence intensity across all monocyte subsets. The highest content of CD206+ and MerTK+ cells in pregnant women is detected in the subpopulation of intermediate monocytes, and the highest values of the mean fluorescence intensity of Arginase 1 and MerTK – in the subpopulations of intermediate and non-classical monocytes. The data obtained demonstrate that monocytes of pregnant women in the 2nd trimester of pregnancy are characterized by signs of M2 polarization. This is confirmed not only by an increase in the expression of the M2-associated mannose receptor CD206, but also by an increase in the expression of Arginase 1 and MerTK, which mediate the immunosuppressive activity of myeloid cells and, in particular, macrophages of the M2 phenotype. Further studies of M2-associated markers in monocyte subpopulations during gestation will allow a more detailed characterization of the regulatory role of circulating myeloid cells during pregnancy

M-CSF and GM-CSF determinate fibromodulatory activity of polarized human macrophages

GM-CSF and M-CSF, the hematopoietic colony-stimulating factors, induce various phenotypic changes in macrophage lineage populations and promote cell differentiation, respectively, into M1- and M2-like macrophages. The pro- and anti-inflammatory properties of macrophages generated by these colony-stimulating factors are well described, but the contribution of differentiation and polarization signals to the fibromodulatory activity of macrophages remains unexplored. To clarify the differences in the fibrogenesis regulation mechanisms inherent in differently activated macrophages, we studied the effects of macrophage-conditioned media on proliferation and differentiation of dermal fibroblasts. In this study, the human macrophages generated from peripheral blood monocytes were investigated. They were induced for differentiation by M-CSF or GM-CSF, being further polarized in the M1 direction with lipopolysaccharide and, in the M2 direction, with IL-4 or dexamethasone. Proliferative response of the fibroblasts was determined radiometrically by [3H]-thymidine incorporation. Differentiation into myofibroblasts was determined with flow cytometry technique, as expression of a specific marker α-smooth muscle actin (α-SMA). The level of macrophage TGF-β1 production was assessed using an appropriate ELISA kit. The data obtained indicate that the macrophages differentiated under the influence of “homeostatic” M-CSF are characterized by a moderate stimulating effect upon fibroblast proliferation, and the effects of M2 (IL-4) and M2 (Dex) macrophages exceed that of M1 (LPS), but do not differ significantly from each other. The M-CSF-induced M1 (LPS) and M2 (IL-4) macrophages, but not M2 (Dex), enhance the fibroblast differentiation and show similar level of stimulation. In contrast to M-CSF, the macrophages induced by “pro-inflammatory” GM-CSF exhibit a pronounced stimulatory effect on fibroblast proliferation, and the effects of M2 macrophages exceed those of M1 cells, being most pronounced for M2 (Dex). At the same time, only GM-CSF-induced M2 (IL-4) macrophages enhance fibroblast differentiation. Dexamethasone-polarized macrophages do not significantly affect fibroblast differentiation regardless of the CSF used (M-CSF or GM-CSF). The content of TGF-β1 in the supernatants of differently activated macrophages does not correlate with the level of stimulating effect of macrophage-conditioned media upon fibroblast differentiation. In general, the data obtained suggest the involvement of differentiation and polarization signals into modulation of pro- and anti-fibrogenic properties of macrophages

SPONTANEOUS AND LPS-INDUCED PRODUCTION OF 26 CYTOKINES SECRETED BY BLOOD CELLS OF PATIENTS WITH LIVER CIRRHOSIS DURING OF CELL THERAPY

The objective of the present study was to assess the level of 26 cytokines secreted by peripheral blood cells of the patients with liver cirrhosis (LC; n = 20) during the cell therapy (CT). All the patients were administered with intravenously injected autologous bone marrow-derived mononuclear cells (MNCs) in a dose of 1.3±0.3 × 109 (Ме 1.0 × 109 ) followed by 14 days later intravenous injection of ex vivo generated mesenchymal stromal cells (MSCs) in a dose of 22.3±5.0 × 106 (Ме 16.0 × 106 ). The patients were examined before the CT, 2-3 days after the administration of MNCs and, then, 2-3 days after the introduction of MSCs. Cytokine-secretory function of peripheral blood cells was evaluated in a 24-hour whole blood cultures both in the absence of any stimulation and in response to lipopolysaccharide (LPS). The control group consisted of 10 healthy donors. The administration of patients’ bone marrow cells (both MNCs and MSC) was safe and well tolerated and caused no any adverse (toxic or allergic) events. Compared with donors, LC patients (especially, with class B+C by Child-Pugh) differed by an initially increased production of several cytokines and chemokines. Actually, there was a statistically significant increase of the spontaneous production of IL-9, MIP-1β, and IP-10, as well as a distinct trend to an increase in TNFα, IL-1ra, IL-4, IL-5, IL-6, IL-13, of MCP-1, MIP-1α, RANTES and Eotaxin. Moreover, the blood cells of LC patients were susceptible to the stimulatory effect of LPS, and the LPS-induced production of 11 out of 26 cytokines (IL-1ra, IL-6, IL-9, IL-15, IL-17, IL-7, IL-8, IP-10, MIP-1α, MIP-1β, Eotaxin) significantly exceeded the normative values. Transplantation of bone marrow MNCs had minimal impact on cytokine production. Meanwhile, the MSCs introduction resulted in a significant decrease in spontaneous and LPS-induced production of, respectively, 20 and 18 analytes including pro-/anti-inflammatory and immunoregulatory cytokines, chemokines and growth factors. The normalization of cytokine-secretory function following transplantation of MSCs revealed in the patients with LC indicates the weakening of an inflammatory activity of circulating blood cells and the decrease in their reactivity to endotoxin. MSC suppressive effect on cytokine production was dose-dependent, and most pronounced in patients with decompensated LC (class B+C by Child-Pugh) of viral etiology

Therapeutic effect of soluble factors of M2 phenotype macrophages in children with language impairments

The aim of the present study was to assess safety and clinical efficacy of inhalation immunotherapy based on intranasal administration of bioactive factors produced by the M2 phenotype macrophages in children with language impairments, as well as to study the effect of inhalation immunotherapy on the cytokine profile in the patients' blood serum. The study was carried out according to the NCT04689282 protocol (www.ClinicalTrials.gov) and included 14 children (9 boys / 5 girls), aged 3 to 8 years, with language impairments associated with perinatal or postnatal CNS lesions of various origin. The children recruited into the study were assessed by a neurologist and speech therapist before the therapy, at the end of the course (1 month), and 6 months later. Serum samples for cytokine analysis were obtained before and 1 month after therapy. The course of intranasal inhalations by the conditioned M2 media (2 ml one time per day for 28-30 days) was safe and well tolerated. None of the 14 treated children had significant adverse reactions and severe undesirable events. Intranasal immunotherapy led to a decrease in the severity of language problems, which manifested by improved speech understanding by 45%; the sensorimotor level of speech, by 51%; word formation skills, by 72%, as well as a twofold increase in general and fine motor skills. In children with signs of autism spectrum disorders, along with a language improvement, a decrease in the severity of autistic symptoms was registered, as evidenced by statistically significant decrease in the CARS score from 42.5 to 38.5 after 1 month, and to 33 points after 6 months (p < 0.05). The clinical effect was revealed rather soon, i.e., within a month after the first procedure, being maintained or intensified during a follow-up for 6 months. At the same time, two-thirds of the children showed a clear clinical improvement, with insignificant effect in the rest of patients. Comparative analysis of the serum cytokine levels in these subgroups showed that children with a pronounced positive response to inhaled immunotherapy differed in the following parameters: (1) initially higher level of VEGF and IGF-1, and (2) decrease the level of TNFα in response to intranasal immunotherapy. In summary, we first tested a fundamentally new approach based on the use of soluble factors from M2-type macrophages and intranasal route of their administration in order to treat the children with severe language impairments, demonstrating safety and obtained preliminary data on effectiveness of such approach