Immunoregulatory Effects of Yolkin on Cell Proliferation, Cytokine Production and Cell SignalingABSTRACT
Yolkin, an egg protein, is postulated to promote development of the immune system in embryos. The effects of yolkin on mitogen-induced mouse lymphocyte proliferation, cytokine production by mouse splenocytes, tumor cell line growth, as well as cell signaling in mouse splenocytes and mouse macrophage RAW 264.7 cells were investigated. Concanavalin A (Con A)-induced thymocyte proliferation was regulated depending on the mitogen concentration. The viability of splenocytes was enhanced in normal but inhibited in lipopolysaccharide (LPS)-treated cells. Yolkin inhibited the growth of mouse lymphocytic leukemia L-1210 cells, and exhibited an additive suppressive effect with cisplatin. Yolkin induced the production of tumor necrosis factor alpha (TNF α), interferon gamma (IFN γ), interleukin 6 (IL-6), and IL-10, although the latter only at a high concentration. LPS-induced TNF α, IFN γ and IL-6 were inhibited, but stimulated with regard to IL-10. The changes in expression of signaling molecules in splenocytes and cell lines indicated elicitation of activation and differentiation. Yolkin elicited significant levels of expression and production of cyclooxygenases COX-1 and COX-2 in splenocytes and cell lines. LPS-induced cyclooxygenase expression and production were regulated depending on the concentration of yolkin. The results contribute to explaining the mechanism of yolkin action and facilitate the interpretation of in vivo studies on yolkin.
Keywords:
Cell signaling; COX-1; COX-2; L-1210; splenocytes; RAW 264.7; yolkin
INTRODUCTION
Evolutionarily conserved proteins play a fundamental role in physiological processes between the classes and phyla of the animal kingdom. They have low-specificity, but are endowed with a bewildering spectrum of functions that act upon the immune, hormonal, and central nervous systems, as well as the host’s microbiome. A fine example of such a protein is lactoferrin, which is usually ascribed to mammals but can also be found in birds (Hou et al., 2015). Vitellogenin, in turn, occurs in all egg-laying animals, from insects (Amdam et al., 2004) to birds (Zambrowicz et al., 2017). However, its presence in the eggs of primitive mammals cannot be, in theory, excluded. Biologically active peptides and proteins such as colostrinin (proline rich polypeptide, PRP) (Zimecki, 2008; Janusz & Zabłocka, 2013) and lactoferrin (LF) (Zimecki & Kruzel, 2007), with a wide spectrum of activities, occur in colostrum and mature milk of mammals, and their actions are not species-specific.
The potential application of these peptides and proteins in the regulation of homeostasis and prevention or therapy of diseases renders them particularly attractive. In fact, LF and colostrums have undergone numerous clinical trials and are recognized as therapeutically effective preparations, used as functional foods and diet supplements (Artym et al., 2021; Artym & Zimecki 2023a; Artym & Zimecki 2023b; abd El-Hack et al., 2023). Egg proteins may also have such potential.
To date, there are gaps in the knowledge regarding the role of some eggs’ proteins in the embryo development of birds. The lack of a direct association between the maternal and embryo’s organisms, in contrast to mammals, indicates that an egg should contain all the bioactive constituents essential for a developing embryo. The roles of proteins such as ovotransferrin, phosvitin and immunoglobulin Y (IgY) in birds’ embryo development and protection have already been clarified (Giansanti et al., 2012; Li et al., 2014; Liu et al., 2018), but there is insufficient data relating to other proteins, such as yolkin, a product of vitellogenin degradation (Polanowski et al., 2013), in these processes.
Our recent results in experimental mouse models demonstrated that yolkin may affect the maturation and differentiation of immune system cells (Obminska-Mrukowicz et al., 2020). Based on these results, we assume that yolkin may fulfill, in egg-laying animals, an analogous role to that of immunotropic proteins and peptides (such as colostrinin and lactoferrin in colostrum/milk) in the immune system development and function of mammals. Yolkin also has a favorable impact on procognitive properties in a rat model, which suggests its significance in the development of the embryo nervous system (Lemieszewska et al., 2016). The protein was shown to induce the production and release of brain derived neurotrophic factor (BDNF) (Zambrowicz et al., 2017).
Yolkin was identified as a set of low molecular weight peptides and proteins (Polanowski et al., 2013). In recent years, knowledge on the activity of yolkin has expanded. Yolkin biological activity is attributed to the 35 kDa protein, containing glycan moieties and an amino acid sequence corresponding to the sequence of vitellogenin. Yolkin exhibits the characteristics of endogenous regulators of several immune and biochemical processes. In previous studies conducted by us and others, yolkin was shown to induce several cytokines (IL-6, IL-8, IL-10, TNF α and interferons) in human blood cell cultures (Zabłocka et al., 2014; Polanowski et al., 2016; Zambrowicz et al., 2017; Kazana et al., 2020). In addition, a modulating effect of yolkin on lipopolysaccharide (LPS)-induced nitric oxide (NO) secretion by a mouse macrophage J774 cell line, inhibition of cellular lipid peroxidation, and viral replication were described (Zabłocka et al., 2014; Kazana et al., 2020). Recently, a receptor for yolkin was identified on a mouse bone-marrow-derived macrophage cell line as Toll-like receptor 4 (TLR4) (Kazana et al., 2020). In these cells, yolkin induced production of nitric oxide, TNF α, and IFN α/β, thus indirectly indicating antiviral activity. A recombinant form of yolkin 40 kDa precursor with immunoregulatory activity was also obtained (Szmyt et al., 2021). A very recent report provided evidence that yolkin injected in ovo to developing embryos on the 18th day of egg incubation increased the content of T cells in the spleen and the circulating blood, as well as elevated the concentration of IL-1β and IL-2 in the blood (Szczypka et al., 2023).
Despite growing knowledge on yolkin activity in vitro and in vivo, a deeper insight into its molecular mechanism of action is required. The aim of this study is to confirm and extend data on its mode of action, particularly regarding the potential effects of yolkin on cell growth and differentiation, cytokine production, cell signaling and the cyclooxygenase (COX) metabolism. In this work, comparisons of yolkin with LF will be frequently cited, since both proteins may fulfill similar roles in embryo and newborn developments in egg-laying animals and mammals. In addition, we will refer to some glycoproteins and saccharides of plant origin that induce similar activation pathways in the same cell models.
MATERIALS AND METHODS
Mice
8-12-week-old female BALB/c mice from the Mossakowski Institute of Experimental and Clinical Medicine, Polish Academy of Sciences, Warsaw, Poland, were used as organ donors. Such a procedure, in which animals are used only as organ donors, does not require approval by an ethics committee according to Directive 2010/63/EU of the European Union Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes.
Reagents
Fetal bovine serum (FCS) was from HyClone (Logan, Ut, US); L-glutamine, penicillin and streptomycin solution, 2-mercaptoethanol, lipopolysaccharide (LPS) O111:B4, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and concanavalin A (ConA) were purchased from Merck (St. Louis, Mo, US); Lymphocyte Separation Medium 1077, sodium chloride buffered with phosphates (PBS), ammonium chloride (NH4Cl), Roswell Park Memorial Institute-1640 (RPMI-1640), Dulbecco’s Modified Eagle’s Medium (DMEM), and Hanks’ Balanced Salt Solution (HBSS) media were obtained from Biowest (Nuaillè, France); cisplatin (P34394) came from Merck (St. Louis, Mo, US); TRIzol Reagent (Ambion) from Life Technologies (Warszawa, Poland); oligo (dT)12-18 primers Novazym VerteKit, AmpliQ 5× HOT EvaGreen® qPCR Mix Plus (noROX) from Novazym (Poznań, Poland); KH2PO4, and K2HPO4 were purchased from POCH (Gliwice, Poland); ammonium sulphate from Chempur (Piekary Śląskie, Poland); reagents for SDS-PAGE were from Bio-Rad (California, US); and sephacryl S-100 HR resin was obtained from GE Healthcare (Chicago, Illinois, US).
Cell Lines
All cell lines derived from the cell bank of the Institute of Immunology and Experimental Therapy, Wrocław, Poland. RAW 264.7 is a mouse macrophage cell line (ATCC, TIB 71), and L-1210 is a mouse lymphocytic leukemia cell line (ATCC, CCL-219).
Isolation of Yolkin from Hen’s Eggs
Yolkin was isolated from the egg yolks according to a procedure described by Polanowski et al. (2013). Yolk plasma was obtained after centrifugation of ten-half diluted yolks upon previous freezing and thawing. Then, Immunoglobulin Y (IgY) containing yolkin was salted out of the plasma using ammonium sulphate (40% saturation). The protein pellet was subsequently dissolved in water, dialyzed, and clarified by centrifugation. In a last step, yolkin was purified with the use of gel permeation chromatography on Sephacryl S-100 HR resin. The separation of IgY containing yolkin on Sephacryl s-100 HR resin resulted in a major peak, corresponding to IgY, and a small peak corresponding to low molecular weight proteins. These fractions were combined, dialyzed against water, freeze-dried, and analyzed by electrophoresis.
SDS-PAGE analysis
The electrophoresis was conducted according to a procedure described by Laemmli (1970). SDS/polyacrylamide slab gel (15%) was prepared using TXG Fast Cast Acrylamide solutions (Bio-Rad, California, US). The protein samples were diluted with the sample buffer containing a reducing reagent to a final protein concentration of 3 mg/mL, and loaded onto gel slabs (20 µg). At the end of the electrophoresis, the gel slab was stained with Coomassie G-250. For the in vitro experiments, yolkin was dissolved in RPMI-1640 medium supplemented with 10% of FCS to obtain 1 mg/mL stock solution, and filtered through 0.22 µm filters (Milipore). Figure 1 shows the electrophoretogram of yolkin preparation separated from the egg yolks.
SDS-PAGE analysis of the yolkin preparation. The yolkin preparation is visualized as three main proteins with MW lower than 45 kDa. The molecular weight markers were prepared in our laboratory and consisted of cytochrome C12 kDa, ovalbumin (OVA) 45 kDa, and bovine serum albumin (BSA) 66 kDa.
Proliferation of Cells from the Lymphoid Organs
Mice in isoflurane anesthesia were sacrificed by cervical dislocation. The cell suspension was prepared from pooled organs of three mice. Thymuses were isolated and placed in disposable Petri dishes containing sterile, ice-cold PBS. A single cell suspension was prepared by pressing them through a nylon mesh and separating by centrifugation (200 × g). The cells were then collected, washed with ice cold Hanks’ medium, and re-suspended in the culture medium (referred to below as the culture medium) consisting of RPMI-1640 supplemented with 10% FCS, L-glutamine, sodium pyruvate, 2-mercaptoethanol, and antibiotics. The cells were then distributed into 96-well flat-bottom tissue culture plates at a density of 5 × 105/well. 0.5 µg/mL or 2.5 µg/mL of ConA was added to induce cell proliferation. After 96 h of incubation, the cell proliferation was determined using the colorimetric MTT assay. The results of the proliferation assays are presented as the mean optical density (OD) at 550/630 nm ± standard error (SE) from quadruplicate determinations.
The spleens were pressed against a plastic screen into 0.83% NH4Cl solution to lyse erythrocytes (5 min incubation at room temperature). The cells were then washed twice with Hanks’ medium, passed through a cell strainer to remove debris, and re-suspended in the culture medium. To induce cell activation, LPS was applied. Splenocytes were cultured for 20 or 48 h in 24-well plates at a density of 5 × 106/mL/well with 2, 10 and 50 µg/mL of yolkin or yolkin and LPS (1 µg/mL) given 2 h after the addition of yolkin. The viability of cells was measured by the MTT colorimetric method. The results are presented in percentage, where the OD at 550/630 nm in the control culture (medium only or LPS-treated cells) was accepted as 100% viability. The results are presented as the mean percentage ± SE from 3 independent experiments.
Induction of Cytokine Production in the Splenocyte Cultures
The spleens were pressed against a plastic screen into 0.83% NH4Cl solution to lyse erythrocytes (5 min incubation at room temperature). The cells were then passed through a plastic screen to remove debris, washed twice with Hanks’ medium and re-suspended in the culture medium (5 × 106/mL/well). The cells were then cultured for 20 h (determination of TNF alpha and IL-10) or 48 h (determination of IFN γ and IL-6) in 24-well plates, with or without LPS 1 µg/mL. LPS was added in the beginning of the culture, and yolkin (2, 10 and 50 µg/mL) was added 2 h later. The supernatants were collected and kept frozen at -80oC until cytokine determination was conducted using ELISA tests from BioLegend (San Diego, CA, US).
Effect of Yolkin on L-1210 Cell Growth
L-1210, a mouse lymphocytic leukemia, skin derived, of lymphoblast morphology, was used. The cells were cultured in a RMPI-1640 medium supplemented with 10% FCS and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin), at a density of 1.5 × 104 cells/well/100 µL. Cisplatin was dissolved in 0.9% NaCl to a concentration of 1 mg/mL, filtered, and a working solution was prepared in RPMI-1640 with 10% FCS, assuming that IC50 for cisplatin equals 0.795 µg/mL. Yolkin and cisplatin were used at concentrations indicated in the figure legends. The culture was conducted for 48 h in a cell culture incubator.
Colorimetric MTT Assay
Cell proliferation was measured using the colorimetric MTT assay (Hansen & Nielsen, 1989). In short, 25 µL from the stock solution (5 mg/mL) was added per well at the end of cell incubation time, and the plates were incubated for an additional 3 h in a cell culture incubator. Then, 100 µL of the extraction buffer (20% SDS with 50% DMF, pH 4.7) was added. After overnight incubation, the OD was measured at 550 nm with the reference wavelength of 630 nm (550/630 nm) in a Dynatech 5000 spectrophotometer.
Induction of Signaling Molecules in RAW 264.7 Cells and Splenocytes by Yolkin
RAW 264.7 cells, a mouse macrophage cell line, were cultured in DMEM containing 10% FCS and antibiotics (penicillin/streptomycin). The cultures were maintained for 18 h in 48-well plates at a density of 3 × 105 cells/mL/well. Yolkin was used at a 20 and 100 µg/mL concentration. For the determination of cell signaling molecule expression, mouse splenocytes at a density of 1 × 106/100 µL/well in the culture medium in 96-well plates were incubated for 18 h in a cell culture incubator using yolkin concentrations of 20 and 100 µg/mL.
Cell Cultures for Determinations of Cyclooxygenase Expression and Production
To determine cyclooxygenase expression and production, mouse splenocytes were incubated in the culture medium for 18 h using yolkin at a concentration of 10 and 100 µg/mL and 5 µg/mL of LPS in 24 well plates (5 × 105/mL). RAW 264.7 cells were incubated in the cell culture medium at a density of 3 × 105/well/100 µL in 96-well plates for 18 h. Yolkin was used at a concentration of 20 and 100 µg/mL. LPS was used at 2 µg/mL concentration.
Total RNA Isolation
Total RNA isolation was performed with TRIzol Reagent according to the manufacturer’s recommendations. The cell pellet (2 × 106 cells) was re-suspended in 1 mL of TRIzol reagent, shaken, incubated for 10 min at room temperature (RT), supplemented with 0.2 mL of chloroform, shaken vigorously for 15 s, incubated for 3 min at RT, and centrifuged at 12 000 × g for 15 min at 4oC. The water phase was collected, transferred to a new tube, supplemented with 0.5 mL of isopropanol, incubated at RT for 10 min, and centrifuged at 12 000 × g for 10 min at 4oC. The RNA pellet was washed with 1 mL of 75% ethanol, dried in air, and dissolved in 20-30 µL of sterile diethylpyrocarbonate-treated Mili-Q water. RNA samples were stored at -20oC.
Reverse Transcription Single Stranded Complementary DNA (cDNA)
The complementary DNA was synthesized with oligo (dT)12-18 primers from 5 µg of total RNA using Novazym VerteKit, according to the manufacturer’s instructions. The list of primers is presented in Table 1.
Quantitative Analysis of Gene Expression by Real Time PCR
The expression of the studied genes was determined using AmpliQ 5× HOT EvaGreen® qPCR Mix Plus (noROX). Relative gene expression levels in samples treated with yolkin or LPS were assessed using the comparative Ct method (ΔΔCt). First, the Ct values of the target genes were normalized to the expression of the reference gene GAPDH (ΔCt). Subsequently, the ΔCt values of treated samples were compared to those of the untreated control culture to calculate the change in expression (ΔΔCt). All reactions were performed in triplicate, and negative controls (no-template controls) were included to exclude contamination. Melt curve analysis confirmed the specificity of each amplification product, consistently showing a single sharp peak for each primer set. Meanwhile, changes in gene expression in similar but separate cell samples after treatment with one or two stimulators were calculated as fold changes using the ΔΔCt method (2^-ΔΔCt).
Determination of Cyclooxygenase-2 Production by ELISA
The cells (mouse splenocytes and RAW 264.7) were collected from the culture flasks by vigorous pipetting, transferred to a centrifuge plastic tube, and centrifuged at 1400 rpm for 10 min at 4ºC. The supernatant was discarded, and the cell pellet was washed twice with PBS. The cell pellet was then dissolved in chilled Cell Extraction Buffer PTR from “Mouse COX2 SimpleStep ELISA® Kit” (Abcam, Cambridge, Great Britain). The total protein concentration in all cell lysates was estimated by spectrophotometrical measurements at l=280 nm, to ensure comparable sample amounts in ELISA measurements. COX-2 determination was done using the abovementioned ELISA kit, according to the manufacturer’s instructions. Each sample was tested in duplicate.
Statistical Analysis
The experiments were performed two or three times with similar results. The results are presented as mean values ± standard error (SE). Brown-Forsyth’s test was used to determine the homogeneity of variance between groups. When the variance was homogenous, analysis of variance (one-way ANOVA) was applied, followed by post hoc comparisons with the Tukey’s test to evaluate the significance of the difference between groups. Nonparametric data were evaluated with Kruskal-Wallis’s analysis of variance, as indicated in the text. Significance was determined at p<0.05. Statistical analysis was performed using STATISTICA 7.0 for Windows.
RESULTS
Effects of Yolkin on Mitogen-Induced Cell Proliferation
The effects of yolkin (2, 10 and 50 µg/mL) on concanavalin A (Con A)-induced mouse thymocyte proliferation was studied at optimal (2.5 µg/mL) and suboptimal (0.5 µg/mL) concentrations of Con A (Figure 2). In the optimal Con A concentration, yolkin showed concentration-dependent inhibition of the proliferative thymocyte response. On the other hand, the action in the suboptimal Con A concentration was co-stimulatory. In both cases, a 50 µg/mL concentration of yolkin gave the most distinct effects.
The effects of yolkin on mitogen-stimulated proliferation of mouse thymocytes. The cells were cultured in 96-well flat-bottom tissue culture plates at a density of 5 × 105/100 µL/well (control (-) culture) or with 2, 10 and 50 µg/mL of yolkin. 2.5 µg/mL or 0.5 µg/mL of Con A was added to induce cell proliferation. After a 3-day incubation, the cell proliferation was determined using the colorimetric MTT assay. The results are presented as the mean optical density (OD) at 550/630 nm ± standard error (SE) from quadruplicate determinations, which is representative for one of two separate experiments. Statistics: *, p<0.05 versus control cultures (Con A 2.5); #, p<0.05 versus control cultures (Con A 0.5).
The actions of yolkin were subsequently studied in mouse splenocyte cultures, or these cultures were treated with LPS (Figure 3ab). The supernatants from these cultures were subsequently used for determination of cytokine levels. In non-stimulated cultures, there was a tendency for yolkin to enhance splenocyte activation at a 10 µg/mL and 50 µg/mL concentration (Figure 3a). LPS alone elevated the splenocyte metabolism, which was lowered after adding yolkin (Figure 3b).
The effects of yolkin on the viability of normal (a) and LPS-treated (b) mouse splenocytes. Splenocytes were cultured for 48 h in 24-well plates at a density of 5×106/mL/well (control culture), and with 2, 10 and 50 µg/mL of yolkin or yolkin and LPS (1 µg/mL), given 2 h after adding yolkin. The viability of cells was measured by the MTT colorimetric method. The results are presented as percentages, where the OD at 550/630 nm in the control culture (medium only or LPS-treated cells) was accepted as 100% viability. The results are presented as the mean percentage ± SE from 3 independent experiments. * denote statistical significance at p<0.05.
Effect of Yolkin on Growth of Tumor Cell Lines
Yolkin was tested for a potential inhibitory effect on the growth of several tumor cells lines of various origins. Figure 4 presents its effect on the growth of L-1210 mouse leukemia cells when applied alone or in co-culture with cisplatin at a concentration close to that inducing 50 % growth inhibition in a preliminary determination. The results show that yolkin inhibits cell growth, in a dose-dependent fashion, and strengthens the antitumor action of cisplatin. The experiment was repeated (data not shown) with a very similar result.
The effects of yolkin and cisplatin on the growth of the L-1210 mouse leukemia cell line. L-1210 cells were cultured for 48 h at a density of 1.5 × 104 cells/100 µL/well. Cisplatin was dissolved in 0.9% NaCl to a concentration of 1 mg/mL, filtered, and a working solution was prepared in the culture medium. Yolkin and cisplatin were used at concentrations indicated in the figure legends (µg/mL). The results are presented as a percentage in relation to a control value (growth of the L-1210 cells in culture medium only). The data are presented as the mean ± standard error (SE) from sixfold determinations, which is representative for one of two separate experiments. Statistical significance versus appropriate concentration of yolkin (p<0.05) was denoted by *
Effects of Yolkin on LPS-Inducible Cytokine Production in Mouse Splenocyte Cultures
The effects of yolkin on LPS-inducible production of cytokines in mouse splenocyte cultures are presented in Figure 5abcd. Yolkin was used at 2, 10 and 50 µg/mL concentrations in 3 independent experiments. Yolkin alone induced TNF α, attaining the highest level of the cytokine at 10 µg/mL (around half of the LPS-induced production) (Figure 5a). Its effects on LPS-inducible TNF α production were down regulatory in a dose-dependent fashion. The production of IFN γ by yolkin was also the highest at 10 µg/mL. In LPS-treated splenocyte cultures, yolkin was inhibitory at 50 µg/mL but stimulatory to various degrees at 2 µg/mL and 10 µg/ml (Figure 5b). Interestingly, yolkin alone was almost as potent as LPS in inducing IL-6 production at 10 µg/mL and 50 µg/mL. Its inhibitory actions on LPS-induced IL-6 production were minor, even at 50 µg/mL (Figure 5c). Yolkin only showed a detectable ability to induce IL-10, and only at 50 µg/mL. However, at 50 µg/mL, it demonstrated distinct upregulating action on LPS-induced IL-10 production (Figure 5d).
The effects of yolkin on LPS-inducible cytokine production in mouse splenocyte cultures: TNF α (a), IFN γ (b), IL-6 (c), IL-10 (d).The splenocytes in the culture medium (5×106/mL/well) in 24-well plates were cultured for 24 h to determine TNF α and IL-10, and for 48 h to determine IFN γ and IL-6, with or without LPS 1 µg/mL. Yolkin (2, 10 and 50 µg/mL) was added to the cultures 2 h before LPS. The levels of the cytokines were measured by ELISA kits. The results are presented as mean percentage ± SE from 3 independent experiments, showing effects of yolkin alone (-) or yolkin in combination with LPS in relation to LPS-induced values, assumed as a 100 % control level.
Effects of Yolkin on Expression of Signaling Molecules
Changes in expression of selected signaling molecules, associated with cell activation and apoptosis, were subsequently investigated in mouse resident cells and cell lines. Yolkin was used at a concentration of 20 and 100 µg/mL in overnight cell cultures. The changes in the expression of the molecules induced by yolkin in the splenocyte cultures are shown in Table 2. At 20 µg/mL of yolkin concentration, the most significant change was observed for NFκB (3.54 × increase). The expression of MAP kinase molecules was reduced (ERK1 and ERK2), and in the case of JNK, almost blocked. A strong inhibition of expression was also noted with caspase 9. The concentration of 100 µg/mL of yolkin did not increase expression of NFκB, but a marked elevation of caspase 3 was observed. The expression of other molecules did not differ much to that described for 20 µg/mL of yolkin.
Relative gene expression in mouse spleen cells treated with yolkin at 20 and 100 µg/mL. Mouse splenocytes at a density of 1 × 106/200 µL/well in the culture medium in 96-well plates were incubated for 18 h in a cell culture incubator using a yolkin concentration of 20 and 100 µg/mL. Values were normalized to a housekeeping gene and calculated using the 2^-ΔΔCt method. Each result represents the mean of three independent biological replicates ± SE.
Potential changes in expression of signaling molecules are of special interest in the case of RAW 264.7 macrophages, given the significant effects of yolkin on cytokine and NO production by macrophages (Zabłocka et al., 2014; Kazana et al., 2020; Kazana et al., 2022). In this cell line, yolkin induced a high expression of NFκB at 20 µg/mL (Table 3). The expression of caspase 9 was also elevated, and there were moderate increases in the expression of ERK and JNK. The effects of yolkin at a 100 µg/mL concentration revealed a stronger induction of caspase 9, but a lower expression of NFκB and JNK and the inhibition of Bcl-2, caspase 3, p53, ERK-1, and ERK-2 expression.
Relative gene expression in mouse RAW 264.7 macrophages incubated at a density of 3 × 105/200 µL/well in 96-well plates for 18 h and treated with yolkin at 20 and 100 µg/mL. Values were normalized to a housekeeping gene and calculated using the 2^-ΔΔCt method. Each result represents the mean of three independent biological replicates ± SE.
The Effects of Yolkin on Expression and Production of Cyclooxygenases
The effects of yolkin on the expression of COX-1 and COX-2 in macrophage RAW 264.7 cultures are presented in Table 4. Yolkin at concentrations of 20 µg/mL stimulated 2× expression of COX-1, but lowered it (2×) at 100 µg/mL. LPS did not change COX-1 expression in the control culture, but yolkin at concentrations of 20 and 100 µg/mL acted synergistically with LPS by inducing the 5× and 63× expression of COX-1, respectively. On the other hand, the induction of COX-2 by both yolkin concentrations was strong at both concentrations (250× and 155×), as well as for LPS (370×). The expression of COX-2 was further enhanced by the culture of yolkin with LPS (360× and 1090× for the respective yolkin concentrations).
Fold change in expression of cyclooxygenase genes (COX-1 and COX-2) in RAW 264.7 macrophages treated with yolkin and/or LPS. RAW 264.7 cells were incubated in the cell culture medium at a density of 3 × 105/100 µL/well in 96-well plates for 18 h (control culture). Yolkin was used at concentrations of 20 and 100 µg/mL. LPS (2 µg/mL) was added to the cultures 1 h after yolkin. Gene expression was calculated using the 2^-ΔΔCt method, with GAPDH as the reference gene. Results are presented as fold change (mean) relative to the control (unstimulated, untreated cells).
In splenocyte cultures a very strong induction of COX-1 expression with a 20 µg/mL concentration of yolkin was registered (239×), with a negligible one at 100 µg/mL (14.6×) (Table 5). LPS did not induce COX-1 expression. However, the combination of yolkin (20 µg/mL) and LPS led the expression of COX-1 to fall to the background level. It was nevertheless strongly elevated (37×) at 100 µg/mL of yolkin. The background level of COX-2 expression was completely blocked by 20 µg/mL of yolkin, and enhanced at 100 µg/mL. However, LPS-induced COX-2 expression (6×) was downregulated by 20 µg/mL of yolkin, but strongly elevated (46 ×) at a 100 µg/mL concentration of yolkin.
Fold change in expression of cyclooxygenase genes (COX-1 and COX-2) in splenocytes treated with yolkin and/or LPS. Mouse splenocytes at a density of 1 × 106/200µL/well were incubated for 18h (control culture). Yolkin was used at concentrations of 20 and 100 µg/mL. LPS (5µg /mL) was added to the cell cultures 1h after yolkin. Gene expression was calculated using the 2^-ΔΔCt method, with GAPDH as the reference gene. The results are presented as fold change (mean) relative to the control (unstimulated, untreated cells).
Yolkin was also studied in terms of its ability to induce COX-2 production, as well as its influence on altering LPS-inducible COX-2 production in RAW 264.7 cells (mouse macrophages), and mouse splenocytes by immunoassay (Table 6). Yolkin alone, at 20 µg/mL, was an efficient inducer of COX-2 production in RAW 264.7 cells, with a lesser effect at 100 µg/mL. LPS at a concentration of 2 µg/mL was a stronger but comparable inducer of COX-2 production as yolkin. The production of COX-2 by LPS was slightly inhibited by 20 µg/mL of yolkin, but upregulated by the higher dose of yolkin. In splenocytes, yolkin was a weak inducer of COX-2, and only at a concentration of 100 µg/mL. LPS at 5 µg/mL induced production of 0.31 ng/mL of COX-2. This production was essentially reduced to the background level upon addition of 20 µg/mL of yolkin, but significantly elevated at 100 µg/mL of yolkin.
Effect of yolkin on COX-2 production in RAW 264.7 macrophages and splenocytes. RAW 264.7 cells were seeded at 3 × 105 cells per 200 µL in 96-well plates and incubated for 18 hours. One hour after adding yolkin, LPS (2 µg/mL) was introduced. Splenocytes were seeded at 5 × 106 cells per well in 24-well plates and also incubated for 18 hours. LPS (5 µg/mL) was added 1 hour after yolkin. COX-2 levels (ng/mL) were measured in the cell pellets using an immunoassay, as described in the Materials and Methods section. Each result represents the mean of three independent biological replicates ± SE.
DISCUSSION
The results of these studies provided more information on the mechanism of action of yolkin. Additionally, they confirmed the original data of other authors and enabled interpretation of our previous in vivo investigations on the effects of yolkin on the humoral immune response in mice. In general, the data confirmed the fine immunoregulatory nature of yolkin. Although the in vivo study revealed a wide range of immunotropic doses of yolkin (Obmińska-Mrukowicz et al., 2020), the effective range of yolkin concentration in vitro is rather narrow, where 20 µg/mL appeared to be regulatory, but 100 µg/mL enhanced the actions of LPS, as evidenced by the increase in COX-2 gene expression and protein production. The immunoregulatory nature of yolkin was also evident in determining Con A-induced thymocyte proliferation at suboptimal and optimal mitogen concentrations, as well for the LPS-activated splenocyte metabolism. We had already reported a stimulatory effect of yolkin on ConA-induced thymocyte proliferation at a concentration range of 10-50 µg/mL (Obmińska-Mrukowicz et al., 2020), but at a very low proliferation index. In this study, yolkin also stimulated low thymocyte proliferation at a suboptimal mitogen dose, but inhibited the very high control proliferative response. In the case of splenocytes, we showed that yolkin enhanced the metabolic activity of the cells, while inhibiting this activity upon LPS stimulation.
The molecular studies in this model showed an increase of Bcl-2, inhibition of caspases 3 and 9, as well as inhibition of MAP kinases, with a concomitant increase in NFκB. Such changes characterize cell activation and are probably associated with residing macrophages. These alterations in the expression of signaling molecules also correlated with a strong induction of pro-inflammatory cytokines in the splenocyte cultures. A standby, activating effect of IL-6 production on residing B cells in this culture also cannot be excluded. On the other hand, an LPS-induced increase in the splenocyte metabolism, which primarily affects macrophages and B cells, was partly inhibited by yolkin. This effect correlated well, at 20 µg/mL of yolkin, with a 3.6 × decrease in COX-2 expression (Table 5) and a similar drop (about 75%) in COX-2 production (Table 6). The inhibition of the LPS-stimulated mouse splenocytes metabolism, as demonstrated by decreased levels of inflammatory mediators, including pro-inflammatory cytokines and COX-2 by flavokawain A (plant-derived glycoprotein), was reported by other authors (Yang et al., 2020).
The ability of yolkin to induce expression or production of cytokines has already been demonstrated by other researchers. Yolkin was shown to induce production of TNF α, IL-1β, IL-6, and IL-10 in human whole blood cultures (Polanowski et al., 2013; Zabłocka et al., 2014); as well as of TNF α and IL-10 in the case of yolkin precursor (Szmyt et al., 2021); and in the case of bone marrow-derived macrophages, TNF α and IFN α/β (Kazana et al., 2020); as well as IL-6, IL-10 and TGF β in these same cells (Kazana et al., 2022). Yolkin’s ability to induce cytokine production was confirmed in this work in the model of mouse splenocyte cultures. Yolkin preferentially induced TNF α, IFN γ and IL-6 production, having a weak stimulatory effect on IL-10. However, we additionally showed that LPS-elicited cytokine production was differentially regulated by yolkin, i.e., pro-inflammatory cytokine production was inhibited and anti-inflammatory cytokine IL-10 production was enhanced. The actions of yolkin resemble the properties of LF, which had the ability to induce production of TNF α and IL-6 in human PBMC cultures (Zaczyńska et al., 2014), while enhancing LPS-mediated IL-10 production (Zimecki & Kruzel, 2000). Yolkin demonstrated distinct anti-inflammatory properties, as shown with regard to inhibition of LPS-induced lipid peroxidation and NO production (Zabłocka et al., 2014). Analogous properties were reported for LF (Choe & Lee, 1999; Xia et al., 2022). Strong indirect evidence showed that the ability of yolkin to induce pro-inflammatory cytokines is associated with the interaction of the protein with TLR-4 (Kazana et al., 2020). The production of pro-inflammatory cytokines by LF also involves TLR-4 (Ando et al., 2010; Wright et al., 2020). It is proposed that TLR4 is responsible for the activation of cells representing innate immunity through LF carbohydrate chains. On the other hand, the polypeptide moiety of LF interferes with the activation of cells by LPS (Ando et al., 2010).
In this study we showed that yolkin inhibited the cell growth of L-1210 mouse leukemia and deepened the antitumor action of cisplatin. Such an action resembles the synergistic action of LF with doxorubicin on prostate cancer in vitro and in mice (Shankaranarayanan et al., 2016), which indicates the possibility of applying yolkin in a combined therapy with other antimetabolic drugs. On the other hand, we could not demonstrate any suppressive effects of yolkin, even in a co-culture with cisplatin, on the growth of other cell lines (data not shown), such as WEHI 231 (immature mouse B cells), Jurkat (immature human T cells), THP-1 (human monocytes/macrophages), A-549 (human lung epithelial cells), HT-29 (human colon epithelial cells) and T98-G (human glioblastoma). Only limited and inconclusive suppressive effects of yolkin on the growth of NK-92 (natural killer human cells) were achieved. A possible explanation for the inhibitory action of yolkin on L-1210 cell growth is a finding that cartilage polysaccharides induce apoptosis in both L-1210 mouse and human K562 leukemic cell lines (Liu et al., 2007). Therefore, it cannot be excluded that the glycan portion of yolkin could be involved in this process. Studies are in progress to establish potential yolkin mechanisms of tumor cell lines growth inhibition.
Our previous study (Obmińska-Mrukowicz et al., 2020) showed that yolkin induced MAP kinase signaling in immature Jurkat T and WEHI 231 B cell lines, indicating promotion of cell differentiation. Moreover, for WEHI 231 cells, evidence revealed the acquisition of a functional differentiation phenotype by these cells. In this study, yolkin at an optimal 20 µg/mL concentration in the splenocyte cultures induced an increase (3.5 ×) of NFκB expression, a small increase in Bcl-2, and almost a block of JNK. These changes could reflect cell activation, probably of residing macrophages, and may be correlated with the induction of cytokine production shown in this work. Yolkin at 20 µg/mL in mouse macrophage RAW 264.7 cells induced the expression of caspase 9, NFκB, and MAP kinases such as ERK-1, ERK-2 and JNK. Similar effects of yolkin on bone marrow derived macrophages with regard to MAP kinases were found by other researchers (Kazana et al., 2020). These changes suggest that a cell differentiation process was elicited by yolkin in these cells, since other studies stress the importance of ERK-1/2 and JNK in differentiating tumor cells as one of the therapeutic strategies of different compounds (Uchida et al., 2001; Jerjees et al., 2014; Wang et al., 2014). In addition, the exceptionally strong increase of NFκB and caspase 9 may contribute to the proapoptotic process (Chen et al., 2011). Caspase 9 expression was higher at 100 µg/mL of yolkin, which may indicate a stronger proapoptotic pathway. However, it should be kept in mind that NFκB activation ensures the viability of RAW 264.7 cells (Pagliari et al., 2000), and activation of caspase 9 can inhibit necrosis of RAW 264.7 cells infected with Mycobacterium tuberculosis (Uchiyama et al., 2007). Furthermore, proapoptotic caspase 3 expression was virtually unchanged at 20 µg/ml of yolkin, and deeply inhibited at 100 µg/ml, indicating a prosurvival effect of yolkin in this model. The differences between the viability of bone marrow-derived macrophages depending on the yolkin concentration has already been shown (Kazana et al., 2020), whereby the concentration of 10 µg/mL enhanced cell viability, while 100 µg/mL did not.
Since prostanoids play a fundamental role in the regulation of innate and adaptive immunity (Hirata et al., 2012), the effects of yolkin on cell signaling in RAW 264.7 cells were further investigated by determining its effects on the expression and production of cyclooxygenases 1 (COX-1) and 2 (COX-2), alone or in combination with LPS. Both isoforms of cyclooxygenases may coexist in cells and tissues; COX-1 being constitutive, while COX-2 is an inducible form of the enzyme (Faki et al., 2021). Prostaglandin E2 (PGE2) is a product of both enzymes, and may be both pro- or anti-inflammatory, depending on the receptor used (Zimecki, 2012; Yu et al., 2014). In this work we showed that yolkin, both in RAW 264.7 - representing mouse macrophages - and splenocytes, elicited significant expression and production of COX-1 and COX-2, while also regulating the LPS-inducible levels of these enzymes (Tables 4, 5 and 6). In RAW 264.7 cells, yolkin was a strong inducer of COX-2, comparable to that of LPS. Although at 20 µg/mL yolkin weakly (2×) enhanced LPS-induced COX-2 expression, its stimulatory effect at 100 µg/mL was much stronger. The induction of COX-1 expression was small, with no observable effect of LPS, but a synergistic stimulatory action with LPS was registered at 100 µg/mL of yolkin. The effects of yolkin and LPS on COX-2 production in RAW 264.7 cells were closely correlated with the respective results on COX-2 expression in this cell model. The results on the effects of yolkin on the COX-2 production in RAW 264.7 cells (Table 6) are in accord with the data presented in Table 3 regarding changes in cell signaling, thus indicating elicitation of activation and differentiation processes in these cells by yolkin. These alterations include a role for COX-2 (Wu et al., 2016) and COX-1 (Rocca et al., 2004) in cell differentiation, and as the markers of cell survival (Perrone et al., 2010). Interestingly, both COX-1 and COX-2 expression and production were further elevated upon co-culture with LPS. This phenomenon could be similar to that described by Hinz et al. (2000), who demonstrated that adding PGE2 to LPS-stimulated RAW 264.7 cells (in our model yolkin-induced PGE2) further increased expression of COX-2, a process that was mediated by EP2/4 receptors. As the action of PGE2 via EP2/4 receptors mediates anti-inflammatory processes, the increased COX-2 levels in combination with higher doses of yolkin and LPS may explain the inhibition of production pro-inflammatory mediators and cytokines by these cells. It also appears that the immunotropic characteristics of yolkin resembles that of plant origin polysaccharides - TLR4 agonists, which induce cytokine production but inhibit all markers associated with LPS-activation, including COX-2 and TLR4 expression (Zhou et al., 2015; Lu et al., 2022). These results indirectly indicate that the glycan moiety in the immunoregulatory property of yolkin cannot be underestimated.
The effect of yolkin on cyclooxygenase expression and production in splenocytes (Table 5 and 6) differed from that found in RAW 264.7 cells (Tables 4 and 6). It was characterized by a potent induction of COX-1 expression (but not COX-2) in splenocytes and induction of COX-2 expression and production in RAW 264.7 cells. The most plausible explanation for this discrepancy are the differences in the target cell for yolkin, whereby RAW 264.7 is an immortalized immature macrophage, and splenocytes are a heterologous cell population with residing B, T and macrophages as potential yolkin targets. In terms of effects on production of COX-2, yolkin behaved similarly to LPS (Table 6), probably because both compounds are agonists of TLR-4.
Nevertheless, the results on the effects of yolkin in splenocyte cultures on COX-1 expression (Table 5) were of special interest and crucial to explaining our in vivo data on the recruitment of immunologically competent T and B cells from their precursors, as well as its stimulatory effects on the development of the humoral immune response to sheep red blood cells (SRBC) in mice (Obmińska-Mrukowicz et al., 2020). In this work we found that yolkin stimulated IgM and IgG-mediated immune responses in vivo to SRBC, promoted differentiation of immature Jurkat and WEHI 231 cells, and recruited mouse mature B cells from the bone marrow. These results are supported by findings on the essential role of COX-1 in B-cell development (Baldari, 2014; Yand et al., 2014), antibody class switch (Blaho et al., 2009), and T-cell development (Liu et al., 2019) in mouse models. It should be highlighted that the ability of lactoferrin to induce COX-1 expression (Kruzel et al., 2013) may also explain its ability to promote T cell (Zimecki et al., 1991) and B cell differentiation (Zimecki et al., 1995).
CONCLUSION
In summary, this work provided additional information on the in vitro mechanism of action of yolkin and contributed to the understanding of the mode of action of yolkin in our previous and present in vivo studies. The results revealed the ability of yolkin to differentially regulate LPS-induced production of pro- and anti-inflammatory cytokines, and elicit differentiation and activation pathways and cyclooxygenase production in cell lines representing major cell types of the immune system. Further investigations require more extensive research and engagement of other research institutions to precisely determine the mechanism of action of yolkin and evaluate its potential utility to treat clinically relevant immune disorders in experimental models. The hitherto collected data on the effects of yolkin on the immune and nervous system not only support benefits of egg consumption for human health (Rehault-Godbert, 2019), but also offer a perspective for preventive and therapeutic application for some egg-derived proteins and fractions as nutraceutics and ingredients of functional foods (Zambrowicz et al., 2023).
ACKNOWLEDGEMENTS
None.
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FUNDING
This research was funded in whole by National Science Centre, Poland (no. 2021/41/B/NZ6/01167). For the purpose of Open Access, the author has applied a CC-BY public copyright license to any Author Accepted Manuscript (AAM) version arising from this submission.
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DATA AVAILABILITY STATEMENT
Data are available from authors on request.
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Data are available from authors on request.
