Effect of Differently Polarized Human Macrophages on the SH-SY5Y Cells Damaged by Ischemia and Hypoxia <i>In Vitro</i>

Rashchupkin, Ivan Mikhailovich; Shevela, Ekaterina Yakovlevna; Maksimova, Aleksandra Aleksandrovna; Tikhonova, Marina Aleksandrovna; Ostanin, Aleksandr Anatolevich; Chernykh, Elena Removna

doi:https://doi.org/10.1155/2023/5595949

Journal of Immunology Research

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Abstract Introduction Results Discussion Conclusions Materials and Methods Data Availability Conflicts of Interest Authors’ Contributions Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2023 | Article ID 5595949 | https://doi.org/10.1155/2023/5595949

Effect of Differently Polarized Human Macrophages on the SH-SY5Y Cells Damaged by Ischemia and Hypoxia In Vitro

Ivan Mikhailovich Rashchupkin,¹Ekaterina Yakovlevna Shevela,¹Aleksandra Aleksandrovna Maksimova,¹Marina Aleksandrovna Tikhonova,¹Aleksandr Anatolevich Ostanin,¹and Elena Removna Chernykh¹

Academic Editor: Baohui Xu

Received25 May 2023

Revised16 Aug 2023

Accepted17 Aug 2023

Published31 Aug 2023

Abstract

Macrophages are the major cells of innate immunity with a wide range of biological effects due to their great plasticity and heterogeneity. Macrophages play a key role in neuroregeneration following nervous tissue injury. However, the neuroregenerative potential of various macrophage phenotypes, including those polarized by efferocytosis, remains unexplored. The aim of this study was to compare the neuroregenerative and neuroprotective activity of soluble factors secreted by variously activated human macrophages on the functions of neural progenitors in an in vitro model of ischemia or ischemia/hypoxia. Macrophages were polarized by interferon-γ (M1), IL-4 (M2a), or interaction with apoptotic cells (M2(LS)). The effect of macrophages conditioned media on the proliferation, differentiation, and survival of SH-SY5Y cells damaged by serum deprivation alone (ischemic conditions) or in combination with CoCl₂ (ischemic/hypoxic conditions) was assessed. All studied macrophages stimulated the proliferation and differentiation of SH-SY5Y cells. On day 3, the pro-proliferating effect of M1 and M2 was similar and did not depend on the severity of the damaging effect (ischemia or ischemia/hypoxia), while on day 7 and under ischemic/hypoxic conditions, the effects of M2(LS) exceeded those of M1 and M2a cells. The prodifferentiation effects of macrophages were manifested in both short- and long-term cultures, mainly under ischemic/hypoxic conditions, and were most characteristic of M2(LS) cells. Importantly, the ischemia/hypoxia model was accompanied by the pronounced death of SH-SY5Y cells. Only macrophages with the M2 phenotype demonstrated antiapoptotic activity, and the effect of M2(LS) was higher than that of M2a. The results obtained indicate that human macrophages have neuroprotective and neuroregenerative activity, which is mediated by soluble factors, is most characteristic for macrophages activated by efferocytosis (M2(LS)), and is most prominent under in vitro conditions simulating the combined effect of ischemia/hypoxia.

1. Introduction

At present, there is no doubt that the adult nervous system, due to its plasticity, is able to regenerate [1]. The concept of neuroregeneration comprises both the recovery of damaged nerve cells (intracellular regeneration) and the formation of new cells (cellular regeneration). The former case involves regeneration and remyelination of neurites, synaptogenesis, and restoration of damaged cell ultrastructures, and the latter case is related to the formation of new neurons from neural stem cells (NSCs) [2]. Replacement of damaged neurons with new cells requires enhancement of NSC proliferation, migration of NSCs to the damaged tissue, and their subsequent differentiation with integration into the existing neural network. These processes are regulated by immune system cells, in particular by macrophages that are represented in the central nervous system by microglia and recruited monocytic macrophages, with the latter playing an important role in pathological conditions.

Macrophages, which are characterized by pronounced plasticity, are able to alter their functional phenotype in response to various microenvironment signals and form many transitional phenotypes between opposite activation states: classical M1 and alternative M2 [3]. M1 phenotype is characterized by proinflammatory activity, whereas M2, on the contrary, exhibits anti-inflammatory properties, provides resolution of inflammation, and stimulates reparative processes [4]. In pathological conditions, the M1/M2 balance is drastically shifted toward the M1 phenotype that mediates neuroinflammation. Restoring M1/M2 balance and resolution of inflammation trigger neuroregeneration [5]. In this case, the intensity and coordination of these processes determine the degree and speed of impaired functional recovery.

According to the literature, the classical activation of macrophages by lipopolysaccharide and interferon-gamma (IFN-γ) induces the M1 phenotype, whereas the alternative activation by IL-4 and IL-13 induces the M2a phenotype. In addition, the M2 phenotype can be induced by other factors: immune complexes (M2b), IL-10, and dexamethasone (M2c) [3]. One of the most important inducers of the M2 phenotype is the uptake of apoptotic cells (efferocytosis) [6]. Previously, we developed an original protocol for the generation of M2-like macrophages (low serum, M2(LS)), which is based on culturing monocytes undergrowth/serum factor deprivation, which promotes deprivation apoptosis and subsequent uptake of apoptotic cells by macrophages [7]. Macrophages generated in this way are characterized by reduced allostimulatory activity, low production of proinflammatory cytokines, and high production of growth and neurotrophic factors (vascular endothelial growth factor (VEGF), erythropoietin (EPO), brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor, epidermal growth factor (EGF)) [7, 8].

Although the role of M1 and M2 macrophages/microglia in inducing and resolving inflammation has been characterized quite fully, their involvement in the regulation of neurogenesis has not been well studied, and there are no data on efferocytosis-induced M2 cells. Therefore, the aim of this study was to comparatively assess the effect of human macrophages with M1, M2a, and M2(LS) phenotypes on the proliferative activity, viability, and differentiation of NSCs under simulated ischemic and combined ischemic and hypoxic conditions in vitro.

The SH-SY5Y neuroblastoma cell line was chosen as an in vitro model of human NSCs. The cells of this line exhibit properties of neural progenitors: they express NSC markers (nestin, doublecortin), actively proliferate, tend to form clusters and neurospheres [9], and are capable of differentiating into dopaminergic and cholinergic neuron-like cells under the action of various stimuli [10]. In vitro ischemic conditions were simulated by partial deprivation of growth/serum factors. Hypoxia was induced by the chemical hypoxia inducer CoCl₂, which is a hypoxia-induced factor stabilizer [11].

2. Results

2.1. Characterization of Macrophages

First of all, we evaluated the capacity of M1(IFN-γ), M2a(IL-4), and M2(LS) macrophages to stimulate T-cell proliferation in mixed leukocyte culture, or allostimulatory activity and the expression of the M2-associated marker (CD206) by macrophages. Allostimulatory activity is an integral parameter that reflects a number of macrophage characteristics (human leukocyte antigen-DR isotype expression, coinhibitory and costimulatory molecules expression, cytokine production, capacity to induce regulatory T cells, etc.) and allows identification of M1 and M2 macrophages [12]. The studied macrophages significantly differed from each other in allostimulatory activity. Thus, M1 macrophages polarized by IFN-γ were characterized by higher allostimulatory activity compared to M2a(IL-4) and M2(LS) (, Figure 1(a)). We also studied the level of CD206 expression, which is considered a characteristic marker of M2 macrophages. As expected, M2a(IL-4) and M2(LS) expressed CD206 at a higher level than M1 (, Figure 1(b)). Thus, M1(IFN-γ), M2a(IL-4), and M2(LS) macrophages corresponded to the characteristics typical for their phenotype.

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(b)

2.2. Effect of Soluble Factors of Human Macrophages on SH-SY5Y Cell Proliferation, Viability, and Yield in an Ischemic In Vitro Model

Under normal conditions (NC, 10% fetal bovine serum (FBS)), SH-SY5Y cells actively proliferated and reached a monolayer in 3–4 days. An analysis of the proliferative response kinetics showed a gradual increase in the proliferative activity of cells, which reached its maximum after 72 hr cultivation (data not shown). Under ischemia-like conditions (partial serum deprivation, SD, 1% FBS), SH-SY5Y cells also proliferated, but the intensity of their proliferation after 72 hr was significantly reduced compared with that in standard cultures () (Figure 2(a)). Conditioned media (30% v/v), which were derived from classically activated M1(IFN-γ), alternatively activated M2a (IL-4), and M2(LS) macrophages polarized through efferocytosis, exerted a comparable stimulating effect and increased cell proliferation 4.2–4.5-fold.

(a)

(b)

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Figure 2

Effect of conditioned media from differently activated macrophages on proliferative activity (a), survival (b), and the cell yield (c) of SH-SY5Y cells under serum deprivation. , , (the Mann–Whitney U test and t-test). cpm, counts per minute (proliferation was assessed using radioisotope method); 7-AAD, 7-aminoactinomycin D, NC, normal conditions; SD, serum deprivation conditions. Data are expressed as median and interquartile range (Me (IQR, 25%–75% quartiles)). (a) n = 9, (b) n = 4–8, (c) n = 4–12.

Since enhanced cell proliferation could be connected with increased survival due to a decrease in SH-SY5Y cell death, we next evaluated the number of nonviable cells (7-AAD⁺) in SH-SY5Y cell cultures supplemented with M1, M2a, and M2(LS) macrophage conditioned media (CM) (Figure 2(b)). SD in both 3-day and 7-day cultures did not lead to an increase in the percentage of nonviable cells. Neither M1 CM nor M2 CM significantly changed the number of dead cells. We observed only a slight decrease () in nonviable cells (compared with both normal conditions and deprivation control) on day 3 in the presence of M2(LS) CM.

In addition, we also evaluated the effect of macrophage CM on the number of SH-SY5Y cells since cell yield may reflect both proliferative activity and the level of cell apoptosis. Compared with NC, SD decreased the cell yield 1.2-fold on day 3 () and more than twofold on day 7 (). The addition of CM from all macrophage types caused a 1.4–1.6-fold increase in the number of cells compared with that in deprivation controls as early as on day 3, which was statistically significant only for M2(LS) (Figure 2(c)). At day 7, CM derived from all studied macrophages caused a more than twofold increase in the cell yield compared with that in deprivation controls (). Furthermore, M1, M2a, and M2(LS) CM did not differ significantly in the level of stimulating effect. Thus, M1, M2a, and M2(LS) soluble factors enhanced the growth of SH-SY5Y cells under simulated ischemic conditions in vitro, and this effect was observed regardless of the macrophage phenotype.

2.3. Effect of Soluble Factors of Human Macrophages on SH-SY5Y Cell Differentiation in an Ischemic In Vitro Model

Because successful neuroregeneration requires not only proliferation but also neural progenitor differentiation, we evaluated the effect of macrophage CM on the differentiation of SH-SY5Y cells. Retinoic acid (RA) is a classic inducer of SH-SY5Y cell differentiation. During differentiation, cell bodies are elongated, and neurites are significantly elongated; at the later stages of differentiation, cells form synaptic-like connections with each other (Figure 3).

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(b)

(c)

One of the main morphological criteria for differentiation is the predominance of the neurite length over the cell body length. Another criterion for cell differentiation is the average neurite length [13]. We evaluated the number of differentiated cells and the average neurite length at the early (day 3) and late (day 7) stages. As expected, the number of differentiated cells in the presence of RA (positive differentiation control) increased significantly on day 3 and continued increasing till day 7. The average length of cell neurites in RA-induced cultures was also significantly higher than that in deprivation controls at both time points () (Table 1).

Similar to RA, although to a lesser extent, all studied macrophage CM increased the number of differentiated cells in comparison with deprivation controls on day 3 with a statistically significant effect only for M2(LS) (). Of note, the stimulatory effect of M2(LS) was more than twofold higher compared with that of M1 and M2a. In addition, M2(LS) enhanced the length of cell neurites more effectively than M1 and M2a () (Table 1).

Cell culture prolongation up to 7 days did not result in a further increase in the relative number of differentiated cells and the average neurite length, which may be explained by active cell proliferation under these conditions. Similar to 3-day cultures, only M2(LS) had a significant stimulatory effect on SH-SY5Y cell differentiation. Thus, the prodifferentiation effects of M2(LS) exceeded the effects of M1 and M2a under SD.

2.4. Effect of Soluble Factors of Human Macrophages on SH-SY5Y Cell Proliferation in an Ischemic/Hypoxic In Vitro Model

As has already been shown, SD (1% FBS) decreased SH-SY5Y cell proliferation (1.3-fold) compared with that under NC (10% FBS) on day 3. The addition of CoCl₂ to simulate the combined action of serum deprivation and hypoxia (SD/HC: serum deprivation/hypoxic conditions) at a concentration of 50 μM did not enhance the inhibitory effect on proliferation. However, an increase in CoCl₂ concentration (100 and 200 μM) was associated with a dose-dependent decrease in proliferation. On day 7, cell proliferation under SD/HC was significantly reduced compared with that under NC in a dose-dependent manner (Figure 4(a)). Therefore, the SD/HC conditions demonstrated a more pronounced inhibitory effect on SH-SY5Y cell proliferation. Based on dose-dependence data, a concentration of 100 µM CoCl₂ was chosen for further experiments.

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(b)

(c)

Figure 4

Effect of conditioned media from differently activated macrophages on proliferative activity and viability of SH-SY5Y cells under serum deprivation and hypoxia conditions: (a) proliferative response of SH-SY5Y cells in the presence of various doses of CoCl₂ under normal (10% FBS) and serum deprivation (1% FBS) conditions; (b) effect of macrophage CM on SH-SY5Y proliferation; (c) effect of macrophage CM on SH-SY5Y viability. Percentage of 7-AAD⁺ cells among all SH-SY5Y cells is shown. , (the Mann–Whitney U test and t-test). NC, normal conditions, SD/HC, serum deprivation, and hypoxia conditions; FBS, fetal bovine serum. Data are expressed as the median and interquartile range (Me (IQR, 25%–75% quartiles)). (a) n = 5, (Bb) n = 8, (c) n = 4–6.

At the next stage, we evaluated the effect of CM derived from M1(IFN-γ), M2a (IL-4), and M2(LS) on SH-SY5Y cell proliferation under SD/HC (Figure 4(b)). Compared with NC, the proliferation of SH-SY5Y cells under SD/HC decreased 1.4-fold on day 3 () and 4.8-fold on day 7 (), i.e., the cells almost ceased to proliferate. Macrophage CM significantly increased the level of proliferation both in 3-day and 7-day cultures. In 3-day cultures, the effects of CM of all studied macrophages were comparable and ranged from 1.6 to 1.65 (). In 7-day cultures, the stimulating effect of macrophage CM was also retained (). However, the effect of M2(LS) CM was higher compared with M1 CM and M2a CM (). Thus, soluble factors of all studied macrophages enhanced the proliferation of SH-SY5Y cells under SD/HC, with M2(LS) exerting a significantly more pronounced effect compared with that of M1 and M2a with longer exposure to damaging factors.

2.5. Effect of Soluble Factors of Human Macrophages on SH-SY5Y Cell Viability in an Ischemic/Hypoxic In Vitro Model

Further, we evaluated the neuroprotective effect of M1(IFN-γ), M2a (IL-4), and M2(LS) soluble factors, namely, the effect of CM on the viability of SH-SY5Y cells under SD/HC. Under SD/HC, the median number of nonviable SH-SY5Y cells increased by day 3 and amounted to 14.2% (9.6; 22.8) (median and (Q1; Q3 values)) versus 6.3% (3.0; 13.1) under NC (; Figure 4(c)). At this stage, the protective effect was observed only for M2(LS) CM, which decreased the number of nonviable cells to 6.9% (5.7; 8.0) (). However, the viability of SH-SY5Y cells added with M1 CM (12.9% (12.2; 13.8) 7-AAD⁺ cells) and M2a CM (13.7% (9.8; 16.5)) did not differ from that in controls ().

Longer cultivation under SD/HC (7 days) led to a further increase in the percentage of nonviable cells: up to 64.0% (52.7; 73.8) versus 4.3% (3.1; 5.5) under NC (; Figure 4(c)). Only the M2 phenotype of macrophages exhibited a significant protective effect in these cultures, while M1 CM reduced the death of SH-SY5Y cells only in the form of a trend (35.6% (30.6; 51.8), compared with SD/HC). It is noteworthy that the protective effect of M2(LS) CM was significantly higher compared with that of M2a CM: the percentage of nonviable cells decreased to 37.2% (26.8; 48.9) for M2a CM and to 16.0% (4.7; 27.8) for M2(LS) CM. Thus, under combined SD/HC, soluble factors of M2 macrophages enhance the survival of SH-SY5Y cells. In this case, the protective effect of M2(LS) is observed at both early and late stages of cultivation and exceeds the similar effect of M2a cells.

2.6. Effect of Soluble Factors of Human Macrophages on SH-SY5Y Cell Differentiation in an Ischemic/Hypoxic In Vitro Model

Previously, we have shown that under SD, macrophage-derived soluble factors are capable of stimulating not only proliferation but also differentiation of SH-SY5Y cells, with M2(LS) exhibiting a significantly more pronounced prodifferentiation effect. Next, we evaluated whether a similar effect of macrophage CM persisted under SD/HC (i.e., in more pronounced injury). On day 3, the relative number of differentiated cells increased in the presence of CM from all studied macrophage phenotypes (Table 2). However, the percentage of differentiated cells in cultures with M2(LS) was 2.1–2.6-fold greater than that in cultures with M1 and M2a CM. In addition, an increase in the average neurite length was observed in the presence of macrophage CM, with all three studied macrophage phenotypes exerting a comparable effect (). Similar effects were seen in 7-day cultures, with the highest number and average neurite length being observed in M2(LS) cultures (Table 2).

Therefore, prodifferentiation effects of macrophage CM on SH-SY5Y cells observed under serum deprivation persisted in an SD/HC when damaging effects were enhanced. Importantly, M2(LS) CM had the highest stimulatory effect.

3. Discussion

The activation of neurogenesis has been considered an important mechanism of regenerative macrophage activity [14]. However, the efficiency of neurogenesis and its regulation by macrophages in neuropathology may be significantly affected by ischemia and hypoxia. Therefore, studying the effect of macrophages on NSCs under simulated ischemic or combined ischemic and hypoxic conditions seems to be an important aspect for a deeper understanding of the immune control of neuroregeneration and the development of new therapies for the treatment of neuropathology.

In the current study, we have studied the effects of soluble factors derived from differently activated human macrophages (M1(IFN-γ), M2a(IL-4), and M2(LS)) on proliferation, differentiation, and survival of SH-SY5Y cells under serum deprivation (an in vitro ischemia model) and a combination of deprivation and CoCl₂-induced hypoxia (an in vitro ischemia/hypoxia model). According to the data obtained, both M1 and M2 macrophages are able to stimulate the proliferation and differentiation of SH-SY5Y cells. In 3-day cultures, the pro-proliferative effects of differently activated macrophages were similar and did not depend on the severity of the damaging effect (SD or SD/HC), while in 7-day cultures and under combined SD/HC, M2(LS) effects exceeded those of M1 and M2a cells. The prodifferentiation effects of macrophages were manifested both in short- and long-term cultures, mainly under SD/HC, and were most characteristic of M2(LS) cells. The neuroprotective effect of macrophage CM was revealed only for macrophages with the M2 phenotype, with the effect of M2(LS) significantly exceeding that of M2a macrophages. Altogether, neuroprotective and neuroregenerative activity is most characteristic of macrophages activated by efferocytosis and is associated with more pronounced damage to SH-SY5Y cells.

Despite M1 and M2 macrophages are capable of secreting pro- and anti-inflammatory cytokines and trophic and growth factors [7], each functional macrophage phenotype has a unique secretory profile [15]. For example, M2 cells are characterized by lower production of pro-inflammatory cytokines and higher production of trophic and growth factors [16]. The similar stimulating effects of soluble factors from all three macrophage types on the proliferation of SH-SY5Y cells may be explained by relatively low concentrations of the factors that are required to activate SH-SY5Y cells.

Data concerning the neuroregenerative capacity of microglia, i.e., tissue-specific macrophages, are restricted mainly to experimental animals and are often contradictory. For example, Vay et al. [17] showed that regardless of their polarization (with LPS or IL-4), rat microglia accelerated the differentiation of NSC into neurons while inhibiting NSC proliferation in vitro. On the contrary, according to the results by Osman et al. [18], neither NSC proliferation nor neuronal differentiation changed upon exposure to CM from LPS- or IL-4-stimulated mouse BV2 microglial cells.

Despite the fact that macrophages can engraft into the brain and replenish the functions of damaged microglia, there are only a few data on the neuroregenerative activity of monocyte-derived macrophages. As an example, Zhang et al. [19] demonstrated that CM from M2 but not M1 macrophages induced differentiation of NSC into neurons in vitro. In this study, the authors utilized macrophages obtained from mouse bone marrow and activated with M-CSF. In contrast, we first reported the stimulatory effect of human macrophages generated from blood monocytes in the presence of granulocyte macrophage colony–stimulating factor (GM-CSF), i.e., a factor simulating inflammatory conditions in vitro. Our results clearly demonstrated that not only M2 but also M1 macrophages are able to exhibit neuroprotective activity and enhance the proliferation/differentiation of neural progenitors. But as the damaging effect increases (e.g., combined ischemia and hypoxia), the neuroprotective effects of macrophages with the M2 phenotype exceed those of M1 cells. In addition, we obtained novel data showing that macrophages polarized by efferocytosis (M2(LS)) possess the most potent pro-regenerative activity.

Since we used macrophage-conditioned media, the described effects may be mediated by soluble factors. Notably, we characterized 47 cytokines in the supernatants of GM-CSF-differentiated macrophages polarized by efferocytosis, [8, 20] many of which (e.g., insulin-like growth factor 1 (IGF-1), VEGF, migration inhibitory factor (MIF), BDNF, EPO, EGF, TGFα, etc.) may contribute to the neuroprotective and neuroregenerative effect of macrophages [21–23]. Consistent with the above observations, we propose that M2 macrophages can significantly enhance neuronal differentiation of NSC-like SH-SY5Y cells due to the secretion of IGF-1, BDNF, VEGF, EPO, MIF, and many others.

Indeed, IGF-1 plays a key role in mediating the neuroprotective effect of macrophages. According to our previous data, the level of IGF-1 production by M2(LS) is tenfold higher than that by M- and GM-CSF-induced nonpolarized macrophages [7]. IGF-1 is able to induce neural differentiation of dental pulp stem cells and stimulate differentiation of SH-SY5Y cells in vitro [24, 25]. We suggest that IGF-1 could significantly contribute to the more pronounced induction of neural differentiation demonstrated by M2(LS). In addition, IGF-1 is able to stimulate the proliferation of SH-SY5Y cells [26]. Moreover, IGF-1 is known to be a potent protective factor; in particular, it exerts a glycogen synthase kinase-3-beta (GSK3b)-mediated antiapoptotic effect on neurons [27]. Therefore, a high level of IGF-1 secretion may largely explain the protective effect of M2(LS).

Another important inducer of neurodifferentiation, which is involved in a number of protocols for SH-SY5Y cell differentiation, is BDNF [28, 29]. According to our previous data, M2(LS) actively produced BDNF [7].

The VEGF and EPO may also be factors that contribute to mediating the neuroregenerative effects of macrophages. VEGF is not only an angiogenic factor but also has neuroprotective properties, which have been demonstrated both in vitro and in vivo [30, 31]. In addition, VEGF is involved in the regulation of neurogenesis by stimulating the proliferation of neuronal progenitors [32]. EPO is currently considered to exert neuroprotective, neurotrophic, and antiapoptotic effects [23]. In addition, EPO is known to stimulate the proliferation of neural progenitors [33]. According to our data, the level of VEGF production by M2(LS) significantly exceeds that by M1 and M2a [7]; also, efferocytosis significantly increases EPO production [34]. Altogether, VEGF may significantly contribute to mediating the anti-apoptotic effect of M2(LS).

Along with neurotrophic and angiogenic factors, the macrophage MIF has been considered another factor possessing neuroregenerative activity. Actually, MIF was shown to increase the survival and proliferation of mouse NSCs [35]. In a pilot clinical trial, the increased serum MIF in patients with cerebrovascular diseases following intranasal therapy with M2(LS) CM was associated with a potent clinical response [20].

Given the wide range of cytokines and growth factors that exhibit neuroregenerative activity, the effects of macrophages observed in our study are apparently mediated not by one specific factor but rather by their combination.

4. Conclusions

The results of the present study show that human macrophages possess neuroprotective and neuroregenerative activity, which is mediated by soluble factors, is most characteristic for macrophages activated by efferocytosis (M2(LS)), and is most pronounced under in vitro conditions simulating the combined effects of ischemia and hypoxia. The data obtained allow us to consider M2 macrophages as a promising cellular platform for the development of new methods for the treatment of inflammatory and degenerative diseases of the nervous system.

5. Materials and Methods

5.1. Isolation and Generation of Macrophages

Human blood samples were obtained from healthy donors with informed consent. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood through density gradient centrifugation (Ficoll-Paque, Sigma-Aldrich, St. Louis, MO, USA). Then PBMCs were plated at 3–5 × 10⁶/ml in tissue culture dishes (TPP, Trasadingen, Switzerland) and cultivated in RPMI-1640 (Biolot, Saint-Petersburg, Russia) supplemented with 2 mM sodium pyruvate, 0.3 mg/ml L-glutamine, 0.05 mM 2-mercaptoethanol, 1% nonessential amino acids, 100 µm/ml gentamicin, 50 ng/ml recombinant human GM-CSF, and 10% FBS (FBS was added only for M1 and M2a macrophages) (all reagents of Sigma-Aldrich, St. Louis, MO, USA). After 1 hr, the nonadherent cells were discarded, and adhesive cells were cultured at 37°C with 5% CO₂ for 7 days. At day 5, the polarizing stimuli were added: 200 ME/ml IFN-γ (Sigma-Aldrich) for M1 macrophages and 20 ng/ml IL-4 (Sigma-Aldrich) for M2a. M2(LS) generated under serum deficiency conditions were cultivated in a similar culture medium supplemented with 2% of autoplasma and without FBS. The adhesion time for M2(LS) was increased to 18 hr. After 7 days of cultivation, the conditioned medium of macrophages was collected, centrifuged (1,500 rpm, 7 min), and then stored at −80°C. For use in experiments, the conditioned media were thawed, filtered using a 0.22 μm pore size filter, and added to SH-SY5Y cell cultures at a concentration of 30% (v/v).

5.2. Cultivation of SH-SY5Y Cells

SH-SY5Y cells (American Type Culture Collection, #CRL-2266, Manassas, VA) were cultured in DMEM/F12 (Biolot) supplemented with 10% FBS (normal conditions, NC) in T-25 cell culture flasks (TPP). Cells were passaged when 70%–80% of the monolayer was reached and then plated at 0.5–1 × 10⁶/ml. The number of passages did not exceed 15.

5.3. Ischemia and Hypoxia Modeling

In all experiments, ischemia was modeled by the partial deprivation of growth/serum factors: the concentration of FBS was reduced from 10% to 1% (serum deprivation, SD). Hypoxia was simulated by adding 100 µM of the chemical hypoxia inducer cobalt chloride(II) (Sigma-Aldrich) (hypoxic conditions, HC).

5.4. Allostimulatory Activity and Phenotypic Characterization of Macrophages

The allostimulatory activity of macrophages was determined by measuring allogeneic T-cell proliferation in the mixed leukocyte culture. PBMCs were plated at 1 × 10⁵ cells in 96-well tissue culture plates in RPMI-1640 (BioloT, St. Petersburg, Russia) with 10% of autoplasma in the absence or presence of different macrophage subtypes (PBMCs : macrophages 10 : 1). Cells were incubated for 4 days, followed by pulse-labeling with 1 μCi/well of [3H] thymidine for an additional 18 hr. The allostimulatory activity was expressed by a stimulation index calculated as PBMC proliferation in the presence of macrophages/spontaneous PBMC proliferative response. For evaluation of the phenotype, macrophages were stained with CD206 (BD Biosciences, USA).

5.5. Evaluation of SH-SY5Y Cells Proliferation, Cell Yield, and Viability

Cells were cultured for 3 and 7 days in 96-well plates (TPP) at 1 × 10⁴ cells per well for the radioisotope method and at 2.5 × 10⁴ per well for the WST test. For assessing cell viability and yield, cells were cultured in 6-well plates at 4 × 10⁶ cells per well. SH-SY5Y cell proliferation was assessed after 5 days by adding [3H] thymidine (1 µCi/well) for 18 hr. Cells were then harvested, and the thymidine incorporation was measured in a liquid scintillation counter SL-30 (Intertechnic, France). Also, proliferation was evaluated by the WST test (Merck, Darmstadt, Germany) according to the manufacturer’s instructions. The cell yield was assessed using a LUNA-II automated cell counter (Logos Biosystems, Annandale, VA, USA). Cell viability was assessed by flow cytometry using FACSCalibur and CellQuest software (BD Biosciences, San Jose, CA, USA), 7-aminoactinomycin-D (7-AAD) (Merck, Darmstadt, Germany) positive cells were determined to be nonviable.

5.6. Evaluation of SH-SY5Y Cell Differentiation

To assess SH-SY5Y cell differentiation, morphometric analysis was performed using the ImageJ program (National Institutes of Health (NIH), Bethesda, MD, USA) and the NeuronGrowth plugin [36, 37]. At least 100 cells in four different fields of view were analyzed, and three independent experiments were performed. The number of cells with neurites, the length of which exceeded the length of the cell body, and the average neurite length were used as morphological criteria of differentiation. Morphometric analysis was performed on days 3 and 7. For each cell, the length of the primary neurite, defined as a single neurite, was measured. For cells with more than one neurite, the length of the longest neurite was measured. RA (Sigma-Aldrich) at a concentration of 10 µM was used as an inductor of differentiation (positive control).

5.7. Statistical Analysis

Statistical analysis was performed using STATISTICA 8.0 software (StatSoft Inc., Tulsa, OK, USA). The data is presented as a median and an interquartile range (Me (IQR, 25%–75% quartiles)). To reveal a significant difference between the compared values, the Mann–Whitney nonparametric U-test, the Wilcoxon signed-rank test, and the t-test were used. Differences were considered statistically significant at .

Data Availability

The authors confirm that the data supporting the findings of this study are available within the article.

Conflicts of Interest

The authors declare that they have no competing interests.

Authors’ Contributions

Rashchupkin IM contributed to the methodology, investigation, writing—original draft, writing—review and editing, and formal analysis; Shevela EY contributed to the methodology, writing—original draft, writing—review and editing, data curation, validation, and project administration; Maksimova AA contributed to the investigation, writing—original draft and visualization; Tikhonova MA contributed to the investigation, visualization, and validation; Ostanin AA contributed to the data curation, supervision, project administration, and writing—review and editing; Chernykh ER contributed to the data curation, supervision, project administration, writing—review and editing, conceptualization, and funding acquisition.

Acknowledgments

The work was funded by the Federal Budget for Fundamental Scientific Research (project no. 122011800324-4).

References

X. Xu, A. E. Warrington, A. J. Bieber, and M. Rodriguez, “Enhancing CNS repair in neurological disease: challenges arising from neurodegeneration and rewiring of the network,” CNS Drugs, vol. 25, pp. 555–573, 2011.
View at: Publisher Site | Google Scholar
M. M. Steward, A. Sridhar, and J. S. Meyer, “Neural regeneration,” in New Perspectives in Regeneration, E. Heber-Katz and D. Stocum, Eds., vol. 367 of Current Topics in Microbiology and Immunology, pp. 163–191, Springer, Berlin, Heidelberg, 2013.
View at: Publisher Site | Google Scholar
X. Huang, Y. Li, M. Fu, and H.-B. Xin, “Polarizing macrophages in vitro,” in Macrophages, G. Rousselet, Ed., vol. 1784 of Methods in Molecular Biology, pp. 119–126, Humana Press, New York, NY., 2018.
View at: Publisher Site | Google Scholar
N. A. Devanney, A. N. Stewart, and J. C. Gensel, “Microglia and macrophage metabolism in CNS injury and disease: the role of immunometabolism in neurodegeneration and neurotrauma,” Experimental Neurology, vol. 329, Article ID 113310, 2020.
View at: Publisher Site | Google Scholar
H. S. Kwon and S.-H. Koh, “Neuroinflammation in neurodegenerative disorders: the roles of microglia and astrocytes,” Translational Neurodegeneration, vol. 9, Article ID 42, 2020.
View at: Publisher Site | Google Scholar
J. Lin, A. Xu, J. Jin et al., “MerTK-mediated efferocytosis promotes immune tolerance and tumor progression in osteosarcoma through enhancing M2 polarization and PD-L1 expression,” Oncoimmunology, vol. 11, no. 1, Article ID 2024941, 2022.
View at: Publisher Site | Google Scholar
E. R. Chernykh, E. Y. Shevela, L. V. Sakhno, M. A. Tikhonova, Y. L. Petrovsky, and A. A. Ostanin, “The generation and properties of human M2-like macrophages: potential candidates for CNS repair?” Cellular Therapy and Transplantation, vol. 2, no. 6, pp. 1–8, 2010.
View at: Google Scholar
L. V. Sakhno, E. Y. Shevela, M. A. Tikhonova, A. A. Ostanin, and E. R. Chernykh, “The phenotypic and functional features of human M2 macrophages generated under low serum conditions,” Scandinavian Journal of Immunology, vol. 83, no. 2, pp. 151–159, 2016.
View at: Publisher Site | Google Scholar
F. C. Ferlemann, V. Menon, A. L. Condurat, J. Rößler, and J. Pruszak, “Surface marker profiling of SH-SY5Y cells enables small molecule screens identifying BMP4 as a modulator of neuroblastoma differentiation,” Scientific Reports, vol. 7, Article ID 13612, 2017.
View at: Publisher Site | Google Scholar
J. Kovalevich, M. Santerre, and D. Langford, “Considerations for the use of SH-SY5Y neuroblastoma cells in neurobiology,” in Neuronal Cell Culture, S. Amini and M. K. White, Eds., vol. 2311 of Methods in Molecular Biology, pp. 9–23, Humana, New York, NY, 2021.
View at: Publisher Site | Google Scholar
J. Muñoz-Sánchez and M. E. Chánez-Cárdenas, “The use of cobalt chloride as a chemical hypoxia model,” Journal of Applied Toxicology, vol. 39, no. 4, pp. 556–570, 2019.
View at: Publisher Site | Google Scholar
A. A. Yankovskaya, E. Y. Shevela, L. V. Sakhno et al., “Аllostimulatory activity as a criterion of the functional phenotype of human macrophages,” Human Immunology, vol. 80, no. 10, pp. 890–896, 2019.
View at: Publisher Site | Google Scholar
S. Dwane, E. Durack, and P. A. Kiely, “Optimising parameters for the differentiation of SH-SY5Y cells to study cell adhesion and cell migration,” BMC Research Notes, vol. 6, Article ID 366, 2013.
View at: Publisher Site | Google Scholar
E. Manole, C. Niculite, I. M. Lambrescu et al., “Macrophages and stem cells—two to tango for tissue repair?” Biomolecules, vol. 11, no. 5, Article ID 697, 2021.
View at: Publisher Site | Google Scholar
A. Shapouri-Moghaddam, S. Mohammadian, H. Vazini et al., “Macrophage plasticity, polarization, and function in health and disease,” Journal of Cellular Physiology, vol. 233, no. 9, pp. 6425–6440, 2018.
View at: Publisher Site | Google Scholar
A. Mantovani, S. K. Biswas, M. R. Galdiero, A. Sica, and M. Locati, “Macrophage plasticity and polarization in tissue repair and remodeling,” The Journal of Pathology, vol. 229, no. 2, pp. 176–185, 2013.
View at: Publisher Site | Google Scholar
S. U. Vay, L. J. Flitsch, M. Rabenstein et al., “The plasticity of primary microglia and their multifaceted effects on endogenous neural stem cells in vitro and in vivo,” Journal of Neuroinflammation, vol. 15, Article ID 226, 2018.
View at: Publisher Site | Google Scholar
A. M. Osman, J. Rodhe, X. Shen, C. A. Dominguez, B. Joseph, and K. Blomgren, “The secretome of microglia regulate neural stem cell function,” Neuroscience, vol. 405, pp. 92–102, 2019.
View at: Publisher Site | Google Scholar
K. Zhang, J. Zheng, G. Bian et al., “Polarized macrophages have distinct roles in the differentiation and migration of embryonic spinal-cord-derived neural stem cells after grafting to injured sites of spinal cord,” Molecular Therapy, vol. 23, no. 6, pp. 1077–1091, 2015.
View at: Publisher Site | Google Scholar
E. Shevela, M. Davydova, N. Starostina, A. Yankovskaya, A. Ostanin, and E. Chernykh, “Intranasal delivery of M2 macrophage-derived soluble products reduces neuropsychological deficit in patients with cerebrovascular disease: a pilot study,” Journal of Neurorestoratology, vol. 7, no. 2, pp. 89–100, 2019.
View at: Publisher Site | Google Scholar
S. Bhalla, S. Mehan, A. Khan, and M. U. Rehman, “Protective role of IGF-1 and GLP-1 signaling activation in neurological dysfunctions,” Neuroscience & Biobehavioral Reviews, vol. 142, Article ID 104896, 2022.
View at: Publisher Site | Google Scholar
M. Sayyah, M. Seydyousefi, A. E. Moghanlou et al., “Activation of BDNF- and VEGF-mediated neuroprotection by treadmill exercise training in experimental stroke,” Metabolic Brain Disease, vol. 37, pp. 1843–1853, 2022.
View at: Publisher Site | Google Scholar
S. Hemani, O. Lane, S. Agarwal, S. P. Yu, and A. Woodbury, “Systematic review of erythropoietin (EPO) for neuroprotection in human studies,” Neurochemical Research, vol. 46, pp. 732–739, 2021.
View at: Publisher Site | Google Scholar
W. Lu, W. Xu, J. Li, Y. Chen, Y. Pan, and B. Wu, “Effects of vascular endothelial growth factor and insulin growth factor-1 on proliferation, migration, osteogenesis and vascularization of human carious dental pulp stem cells,” Molecular Medicine Reports, vol. 20, no. 4, pp. 3924–3932, 2019.
View at: Publisher Site | Google Scholar
E. Lavenius, V. Parrow, E. Nånberg, and S. Påhlman, “Basic FGF and IGF-I promote differentiation of human SH-SY5Y neuroblastoma cells in culture,” Growth Factors, vol. 10, no. 1, pp. 29–39, 1994.
View at: Publisher Site | Google Scholar
M. E. Mattsson, G. Enberg, A. I. Ruusala, K. Hall, and S. Påhlman, “Mitogenic response of human SH-SY5Y neuroblastoma cells to insulin-like growth factor I and II is dependent on the stage of differentiation,” The Journal of Cell Biology, vol. 102, no. 5, pp. 1949–1954, 1986.
View at: Publisher Site | Google Scholar
C. A. Bondy and C. M. Cheng, “Signaling by insulin-like growth factor 1 in brain,” European Journal of Pharmacology, vol. 490, no. 1–3, pp. 25–31, 2004.
View at: Publisher Site | Google Scholar
L. Agholme, T. Lindström, K. Kågedal, J. Marcusson, and M. Hallbeck, “An in vitro model for neuroscience: differentiation of SH-SY5Y cells into cells with morphological and biochemical characteristics of mature neurons,” Journal of Alzheimer’s Disease, vol. 20, no. 4, pp. 1069–1082, 2010.
View at: Publisher Site | Google Scholar
L. Hromadkova, D. Bezdekova, J. Pala et al., “Brain-derived neurotrophic factor (BDNF) promotes molecular polarization and differentiation of immature neuroblastoma cells into definitive neurons,” Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, vol. 1867, no. 9, Article ID 118737, 2020.
View at: Publisher Site | Google Scholar
C. Lange, E. Storkebaum, C. R. de Almodóvar, M. Dewerchin, and P. Carmeliet, “Vascular endothelial growth factor: a neurovascular target in neurological diseases,” Nature Reviews Neurology, vol. 12, pp. 439–454, 2016.
View at: Publisher Site | Google Scholar
J. N. Nicoletti, S. K. Shah, D. P. McCloskey et al., “Vascular endothelial growth factor is up-regulated after status epilepticus and protects against seizure-induced neuronal loss in hippocampus,” Neuroscience, vol. 151, no. 1, pp. 232–241, 2008.
View at: Publisher Site | Google Scholar
K. Jin, Y. Zhu, Y. Sun, X. O. Mao, L. Xie, and D. A. Greenberg, “Vascular endothelial growth factor (VEGF) stimulates neurogenesis in vitro and in vivo,” Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 18, pp. 11946–11950, 2002.
View at: Publisher Site | Google Scholar
S. Pavlica, J. Milosevic, M. Keller et al., “Erythropoietin enhances cell proliferation and survival of human fetal neuronal progenitors in normoxia,” Brain Research, vol. 1452, pp. 18–28, 2012.
View at: Publisher Site | Google Scholar
L. V. Sakhno, E. Y. Shevela, A. P. Lykov, O. V. Poveshchenko, A. A. Ostanin, and E. R. Chernykh, “Effect of apoptotic neutrophils on the production of erythropoietin, MMP-9, and TIMP-1 in cultures of human macrophages,” Bulletin of Experimental Biology and Medicine, vol. 167, pp. 755–758, 2019.
View at: Publisher Site | Google Scholar
S. Ohta, A. Misawa, R. Fukaya et al., “Macrophage migration inhibitory factor (MIF) promotes cell survival and proliferation of neural stem/progenitor cells,” Journal of Cell Science, vol. 125, no. 13, pp. 3210–3220, 2012.
View at: Publisher Site | Google Scholar
Z. Fanti, M. E. Martinez-Perez, and F. F. De-Miguel, “NeuronGrowth, a software for automatic quantification of neurite and filopodial dynamics from time-lapse sequences of digital images,” Developmental Neurobiology, vol. 71, no. 10, pp. 870–881, 2011.
View at: Publisher Site | Google Scholar
I. M. Rashchupkin, E. Y. Shevela, and E. R. Chernykh, “Effect of soluble factors of M2-phenotype macrophages on differentiation of SH-SY5Y cells,” Medical Immunology, vol. 23, no. 4, pp. 677–684, 2021.
View at: Publisher Site | Google Scholar

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Copyright © 2023 Ivan Mikhailovich Rashchupkin et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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