Ovotransferrin exerts bidirectional immunomodulatory activities via TLR4‐mediated signal transduction pathways in RAW264.7 cells

Abstract The immune regulation function of ovotransferrin (OVT) explored using the RAW264.7 was induced by lipopolysaccharide (LPS) as vitro model in this study. The results showed that RAW264.7 cultured with OVT (200 μg/ml) alone not only enhanced the phagocytic activity and the production and expression of inflammatory factors, but also expression of toll‐like receptor 4 (TLR4) gene was significantly promoted by OVT. OVT (50 μg/ml) significantly inhibited the secretion and expression of inflammatory factors in LPS‐stimulated RAW264.7, but CD14 and TLR4 genes expressions were no obvious effects. Inflammatory cytokines and NO secreted by OVT‐induced macrophages pretreated with inhibitors of TLR4 were down‐regulated. We further verified the effects of OVT on inflammatory signaling pathway‐related proteins through immunofluorescence and western blotting, MyD88, TLR4 and the phosphorylation of IκBα and p65 were significantly promoted by OVT, but there was no significant effects on the phosphorylation of IRF3. OVT promoted the phosphorylation of ERK and p38 in RAW264.7 and inhibited the phosphorylated expression of MAPK in LPS‐mediated inflammation. These results indicated that OVT had the bidirectional immunoregulatory function through TLR4‐mediated NF‐κB/MAPK signaling pathway, that is, anti‐inflammatory effect of low concentration and immune‐enhancing activity of high concentration were showed. That provides a theoretical utilization for the development and utilization of OVT.

| 6163 RU et al. proteins, accounting for approximately 12%-13% of total egg white content (Wu & Acero-Lopez, 2012). At the same time, it is the acidic soluble and good iron-binding glycoprotein consisting of 686 amino acid in the poultry transferrin family. In addition to its excellent immunoregulatory activity, OVT is also involved in antigen recognition and initiation of immune response. Furthermore, previous studies have shown that OVT currently plays an obvious role in antibacterial (Schade & Caroline, 1944), antiviral (Giansanti et al., 2002), antioxidant (Majumder et al., 2013) and immune-enhancing activity . There is still an increasing interest in the search for the immunity regulation of OVT.
Ovotransferrin is a component of natural immunity (Shang & Wu, 2018). Likewise, it participates in a series of immune regulation response. OVT, initially as an avian acute phase protein (Rath, 2005), is reported to modulate macrophage and heterophil function by stimulating the production of interleukin-6 (IL-6) and nitrite (Xie et al., 2002) and promote the proliferation of splenic lymphocytes (Xu, Lin, et al., 2012). In addition, oral administration of OVT reduces the secretion of inflammatory cytokines and slow the spread of the colitis induced by dextran sodium sulfate (Kobayashi et al., 2015) and enhances immune response in cyclophosphamide-induced immunosuppressed mice (Zhu et al., 2018). This suggests that OVT may have bidirectional immunomodulatory effects in vivo. Our previous research finds that OVT increases the secretion of TNFα though MAPK signaling pathway (Liu et al., 2017) in DCs. More recently, OVT exerts in vitro immune activity by MAPK signaling pathway.
These findings are based on inflammation models, but how OVT stimulates macrophages to exert immunomodulatory effects is currently less researched in noninflammatory models. In addition, there are few studies on whether OVT can play an anti-inflammatory role in LPS-induced inflammation by inhibiting the expression of signaling pathway-related proteins.
The immunomodulatory effect of protein may be mediated by the direct binding of food-derived proteins to immune cell surface receptors, thereby activating related signaling pathways mediated by cell surface receptors on the one hand . On the other hand, the binding of LPS and the plasma membrane CD14 protein, a GPI-anchored membrane protein in bone marrow cells, consults macrophage activation. LPS is next transferred to TLR4 triggering signal transduction (Prymas et al., 2020), which begins to recruit a single or specific receptor combination in the TIR domain containing the receptor protein. Then, NF-κB was activated by almost all known immune cell surface receptors (Oeckinghaus et al., 2011) and MAPK pathways including p38, JNK and ERK are stimulated. It is known that immunomodulatory activity of most food-derived protein is displayed by inhibiting the signal components of NF-κB or MAPK pathway (Han et al., 2009;Kim et al., 2016). Therefore, the purpose of the present study was to investigate the receptor to which OVT binds directly and whether OVT modulates MyD88-dependent and MyD88-independent, namely TRIF signaling pathways. Here, we determined that TLR4 may be the membrane receptor protein of OVT on the expression level of TLR1, TLR2 and TLR4. In addition, our results found that OVT regulated inflammation by TLR4/MyD88/NF-κB/MAPK inflammatory signaling using western blot. To our knowledge, it is reported for the first time that the TLR4 may be involved in the OVT binding on RAW 264.7 cells to trigger immune-enhancing response. That provides theoretical basis for the development of OVT as immunomodulator and enhancer.
Penicillin-streptomycin was purchased from Gibco BRL Co. Ltd.
Ovotransferrin from egg white, purity of which is over 98%, and LPS (from Escherichia coli, 0111:B4) were purchased from Sigma-Aldrich.
Mouse Enzyme-Linked Immune-Sorbent Assay (ELISA) kits were purchased from Thermo Fisher Scientific. Hoechst 33258 fluorescent dye (DAPI) was purchased from Bioworld Biotechnology CO., Ltd. CCK assay kit was obtained from TransGen Biotech Company.
TAK-242 inhibitor was purchased from MedChem Express. All associated antibodies except TLR4 were purchased from Cell Signaling Technology. TLR4 antibody, secondary antibodies and chemiluminescence (ECL) detection kit were purchased from ProteinTech Group, Inc. RAW264.7 cells were purchased from Procell Life Science Technology Co, Ltd, which were cultured in DEME high glucose medium containing 10% FBS and 1% penicillin and streptomycin and incubated in an incubator at 37°C in humidified 5% CO 2 atmosphere.

| Cell culture
The logarithmic growth phase cells are selected for subsequent seed plate experiments.

| Cell viability assay
The CCK assay (Li, Sun, et al., 2019) was used for determining effect of OVT with various concentrations on the viability of cell. Two hundred microliter of cell suspension with a density of 1 × 10 5 cells was added to the 96-well plate. The control group was added with complete culture medium; the LPS model group was added with 100 ng/ ml LPS solution; the OVT treatment group was added with mixed solutions containing LPS (100 ng/ml) and OVT of different concentrations; and the OVT control group was added with OVT of different concentrations, and the cells were furfure cultured for 24 h.
Subsequently, 10 µl CCK solution was added and incubated for 2 h.
Absorbance value was estimated at 450 nm by a microplate reader.
Cell viability was calculated by the following equation.

A (treated): OD of wells including cells and OVT solution.
A (blank): OD of wells including complete culture solution only.
A (control): OD of wells including cells and complete culture solution.

| Phagocytosis of neutral red assay
Macrophages phagocytosis was detected using neutral red uptake assay from Beijing solarbio science & technology co., Ltd . The cells were grouped and cultured according to the previous experiment. After incubation for 24 h, cells were cultured with 200 µl complete medium and 20 µl neutral red solution for a further 3 h incubation. PBS was used to wash cells for twice times to remove medium containing neutral red, and then, 200 µl neutral red detection lysis solution was added. The cells were lysed on the shaker at room temperature for 10 min to promote cell lysis in sync.
The absorbance values at 560 nm were acquired.

| Measurement of nitric oxide (NO)
The content of NO was assessed using Griess assay (Xiong et al., 2018) to indirectly reflect the degree of inflammation. The cells were grouped and cultured according to the previous experiment. The concentration of NO was measured by using NO assay kit (Beyotime Biotechnology Co., Ltd). Culture supernatant (50 µl) and 100 µl Griess solution (Griess A:Griess B = 1:1) were added to a 96well plate. Take care to avoid light and measure the absorbance at 540 nm using a microplate reader.

| Measurement of cytokine production
RAW 264.7 cells with a density of 1 × 10 6 cells/ml were seeded onto 24-well plates and incubated for 6 h. The supernatant was discarded.
The control group was added with complete culture medium; the LPS model group was added with LPS solution containing 100 ng/ ml LPS; the OVT treatment group was added with mixed solutions containing LPS (100 ng/ml) and OVT (50, 100 and 200 μg/ml); and the OVT control group was added with OVT at 50, 100 and 200 μg/ ml and the cells were cultured for 24 h. And the supernatant was collected and centrifugal. Levels of TNFα, IL-6 and IL-10 in the supernatant were measured using a microplate reader at 450 nm according to the manufacturer's instructions.

| Real-time quantitative reverse transcription PCR (qRT-PCR)
Cells were seeded in a 6-well plate at 1 × 10 6 cells/ml, cultured for 6 h and then adhered to the wall for group treatment. Total RNA was collected from the RAW264.7 cells using the RNA extraction kit from TransGen Biotech Company. To ensure that subsequent experiments can be carried out, the BioDrop µLife+ (BioDrop) is needed to detect purity and integrity. Total RNA (2 µg) was revamped into cDNA by cDNA Synthesis SuperMix kit. The PCR amplification of cDNA was performed with specific primers for TNFα, IL-6, IL-10, CD14, TLR1, TLR2, TLR4 and β-actin as the control. The forward and reverse primers sequences which are designed and synthesized are listed in Table 1. Amplification conditions were as follows: 94°C initial denaturation for 30 s followed by 42 cycle of 94°C for 5 s and 62°C for 30 s. Relative expression levels of the target genes were calculated using 2 −ΔΔCt .

| Immunofluorescence assay
Lipopolysaccharide is known to activate NF-κB, which regulates the inflammatory response by modulating multiple proinflammatory cytokines in macrophages (Ren et al., 2020). To further investigate whether the anti-inflammatory effects of OVT are mediated by the NF-κB signaling pathway, p65 NF-κB expression in macrophage nucleus was observed by the immunofluorescence in this study. Cells (2 × 10 5 /ml) were set in 24-well plates and treated with 200 µg/ml OVT, LPS and LPS+OVT for 24 h. After being fixed with

| Western blot analysis of protein expression
Cells were seeded in a 6-well plate at 1 × 10 6 cells/ml, cultured for The antibody-specific protein was viewed by enhanced chemiluminescent detection system with ECL kit.

RAW264.7
RAW 264.7 cells with a density of 1 × 10 6 cells/ml were seeded onto 24-well plates and incubated for 6 h. The supernatant was discarded.
The control group was added with complete culture medium, the

| Statistical analysis
Data are showed as means ± standard error of the mean (SEM) and statistical significant analysis is determined by Duncan's test with a p < .05 taken as value of significance. All the figures were drawn using the GraphPad Prism 7.0 software.

| No inhibitory effect of OVT on the cell viability of RAW264.7
CCK assay is used to detect whether OVT with various concentrations has damage to cell viability (Li, Sun, et al., 2019). CCK is a reagent for cell proliferation and cytotoxicity detection based on water-soluble tetrazolium salt. In the presence of electron coupling reagent 1-methoxy PMS, it can be reduced to soluble orange-yellow formazan by dehydrogenase in mitochondria, and the shade of color can directly reflect whether the protein is toxic to cells. As shown in

RAW264.7 cells and inhibited phagocytosis activity of LPS-stimulated RAW264.7
When pathological changes occur in tissues and organs, macrophages can eliminate antigens by phagocytosis (Xiong et al., 2018).
Neutral red phagocytosis experiment is commonly applied to evaluate the phagocytic activities of RAW264.7 cells treated with various concentrations of OVT (Wang et al., 2010). Cell phagocytosis was promoted slightly by OVT compared with the normal control group (p > .05) and increasing effect of 200 µg/ml OVT treatment group was significant (p < .05; Figure 2a). In the LPS-stimulated RAW264.7, the phagocytic rate of cells was significantly inhibited of OVT in Figure 2b (p < .05). As the concentration increases, the stimulating effect of OVT on the phagocytic ability of RAW264.7 cells was also continuously decreasing.

RAW264.7 cells and inhibited production of NO in LPS-stimulated RAW264.7
NO, an important transmembrane molecular signal, positively reflects the severity of inflammation in the inflammatory process, and it is also a basic condition for macrophages to play phagocytic function Ye et al., 2020). To confirm the extent of the inflammatory response to OVT, the amount of NO produced was analyzed. As shown in Figure 3a, as expected, a minimum amount of NO was produced in control group; while LPS-stimulated macrophages released NO at a level of 6.68 ± 0.15 μM. NO production is promoted in a dose-dependent manner in OVT-induced RAW264.7.
Among them, the production of NO treated with OVT at 200 µg/ ml was higher than those in other groups, but it was lower than LPS group (p < .01). As shown in Figure 3b, compared with the LPS group, the treatment of OVT (50 µg/ml) significantly reduced the levels of NO in LPS-induced macrophages (p < .01). In addition, the production of NO in the RAW264.7 cocultured with LPS and OVT was higher than that of OVT-induced RAW264.7 macrophages only, indicating that OVT and LPS had a synergistic effect on NO production.

| Effect of OVT on transfer of p65 NF-κB proteins into nucleus
Activation of NF-κB was confirmed by immunofluorescence staining to determine whether p65, the major subunit of NF-κB, was trans-

F I G U R E 3 (a)
Nitric oxide (NO) production promotion in OVTinduced RAW264.7 and (b) NO production inhibition of OVT in LPS-stimulated RAW264.7. Macrophages were incubated with various concentrations of OVT in the presence or absence of LPS (100 ng/ml) for 24 h. NO level in culture media was displayed using Griess assay. Data are shown as means ± SEM (n = 6). Compared with the control group: *p < .05, **p < .01; compared with the LPS group: # p < .05, ## p < .01

MyD88, NF-κB and MAPK in RAW264.7 cells and inhibited expression of these proteins in LPSstimulated RAW264.7
In order to confirm whether MyD88, IRF3, NF-κB and MAPK are involved in the immunity activity of OVT in RAW264.7, western blotting must be employed. It was clear (shown in Figure 8a,b) that expression of MyD88 protein, TLR4, phosphorylation of IκBα and p65 were increased by OVT (200 µg/ml) in RAW264.7 cells (p < .05).
In addition, OVT significantly reduced expression of MyD88, TLR4, phosphorylation of IκBα and p65 in LPS-stimulated RAW 264.7(p < .01). As shown in Figure 8a, compared with the control group, the level of IRF3 protein in the LPS-stimulated group was decreased. However, following treatment with OVT, the expression levels of the phosphorylated proteins found no effect compared with the LPS-only group. No apparently differences in the expression of IRF3 in RAW264.7 cells were found between control group and OVT group (p > .05). As expected in Figure 8c, the phosphorylation of JNK, p38 and ERK1/2 was significantly increased in LPSstimulated RAW264.7 cells (p < .01). Phosphorylation of p38 and ERK of RAW264.7 cells treated with OVT was increased (p < .05), but OVT did not affect phosphorylation of JNK. Phosphorylation of ERK, JNK and p38 was significantly decreased by OVT in LPSstimulated RAW264.7 (p < .05).

| Effect of TLR4 receptor inhibitor on NO and cytokine secretion in RAW264.7 cells
To determine whether OVT can induce the release of related cytokines through activation of TLR4 receptors, RAW264.7 macrophages were pretreated with a TLR4 inhibitor (Wen et al., 2019). Figure 9a shows the effect of different concentrations of TLR4 on cell viability at the concentration of blocker. We found that the 50 nM TAK-242 blocker had no effect on cell viability, which could be used for subsequent experiments. The results showed in Figure 9b,c that 200 μg/ml OVT significantly increased the secretion of NO and TNFα, which were 3.8 µM and 1800 pg/ml, respectively. The levels of NO and TNFα in RAW264.7 macrophages treated with TLR4 inhibitor were significantly reduced than that of OVT group alone (p < .05).

| D ISCUSS I ON
Macrophages, as a group of mononuclear phagocytes, are widely found in the body and are critical in host defense due to coordination innate immunity and inflammatory responses. Inflammation is the immune defense response of the body for the purpose of self-protection (Mosher et al., 2001). It is a common pathological process of the human body. It is closely related to atherosclerosis, asthma, rheumatoid arthritis, diabetes and other diseases. If proinflammatory cytokines are released in large quantities and inflammatory response is too strong, it will lead to increased tissue damage and induce serious immune damage of the body. Therefore, moderate inflammatory immune response is conducive to the body to remove pathogens and achieve self-repair; once the inflammatory immune response exceeds a certain range, it will cause immune damage and harm to the body health (Laskin & Pendino, 1995).
Cytokines and inflammatory mediators were also secreted by activated macrophage in the host defense of the immune system. It has been reported in the literature that yupingfeng fermentation polysaccharide promotes the expression of inflammatory cytokines in noninflammatory models on the one hand, and inhibits the expression of inflammatory cytokines in inflammatory models on the other hand (Sun et al., 2017). A growing body of support suggests that activated RAW264.7 macrophages can secrete some inflammation cytokines. Among them, TNFα and IL-6 are representative major factors which often used to measure the severity of inflammation . When the inflammatory response reaches a certain level, IL-10 is secreted to suppress inflammation. In our present study, OVT showed immunoregulatory activity, with NO production of LPS-induced RAW264.7 treated with low concentration of OVT being inhibited, and increased secretion of the NO in a dosedependent manner in OVT-induced RAW264.7 cells. These observations are in accordance with the previous finding that food-derived protein anti-inflammatory extracts markedly inhibit the release of NO in LPS-stimulated RAW264.7 macrophages (Kim et al., 2020;Tagashira et al., 2018). Kim et al. (2020) found that the production of NO is inhibited by ovalbumin hydrolysate through reducing the phosphorylation of JNK and ERK in LPS-stimulated RAW264.7. It is speculated that the possible reason is that OVT can control the NO release process by MAPK signaling pathway. Furthermore, the result of the study also showed that secretion and mRNA expression of IL-6, TNFα and IL-10 in RAW264.7 were promoted, which were part consistent with the previous reporting that OVT stimulated the release of IL-6 from HD11 macrophages (Xie et al., 2002). And secretion and expression of the IL-6, TNFα and IL-10 were inhibited by OVT in LPS-induced RAW264.7 cells. TNFα, as main upstream cytokines, induces the activation of the NF-κB pathway and stimulates production and expression of pro-inflammatory factors like IL-6 (Li, Chang, et al., 2019). Fifty microgram per milliliter OVT suppressed level of IL-10 in LPS-stimulated RAW264.7 macrophages. This effect might be due to the significant reduction of TNFα and IL-6 in LPS-stimulated RAW264.7 cultured with 50 µg/ml OVT (Meram & Wu, 2017). Anti-inflammatory activity of phosphopeptides (PPPs) from hen egg yolk phosvitin is reflected in inhibitory release of cytokine expression (Xu, Yang, et al., 2012). In summary, these results indicate that OVT has the ability to activate macrophages to secrete inflammatory cytokines and inflammatory mediators, and it is particularly important to study its mechanism of action.
The immune-related receptors (PRR) on the cell surface are activated by foreign pathogen stimulation and initiate a series of tight signal cascades in the cells. The toll-like receptor family (TLRs) is the most widely studied PRR. Over the past few years, it has been bound to a variety of ligands including sugars , proteins (Ren et al., 2010), and lipids (Diao et al., 2019) in LPS-stimulated RAW264.7 and have anti-inflammatory effect . Similar with study of Ganoderma protein receptor (Li, Chang, et al., 2019), the data showed that OVT may combine with TLR4 to exert immune enhancement functions, but it did not exert anti-inflammatory function by inhibiting the expression of these two genes according the current observations (Lee et al., 2019).
This also explains why OVT can significantly enhance the phagocytosis of macrophages to effectively eliminate potential pathogens.
Lactoferrin suppresses LPS-induced expression of NF-кB pathway in RAW264.7 cells through TLR4 (Nemati et al., 2020), similar with which, inflammatory cytokines secreted by macrophages induced by OVT were down-regulated, when cells were pretreated with inhibitors of TLR4. It indicated that OVT activated macrophages through TLR4 receptors to initiate downstream signaling pathways. Whey proteolytes treated with high pressure inhibited the secretion of TNFα and IL-8 in LPS-induced respiratory epithelial cells, but had no effect on the secretion of IL-8 in TNFα and IL-1-induced cells, suggesting that whey proteolytes may reduce the expression of proinflammatory factors through toll-like receptor pathways. On the other hand, the hydrolysates did not down-regulate TLR4 receptor expression, suggesting that whey protein may play a role in regulating inflammation by inhibiting LPS binding to toll-like receptors and inhibiting NF-κB signaling pathway activation, thereby regulating cytokine secretion (Iskandar et al., 2013).
As we all know, in the presence of external stimuli such as LPS, NF-κB as a nuclear factor is responsible for regulating immune responses through regulating gene transcription in RAW264.7 cells (Li, Ye, et al., 2019). IκBα is degraded by phosphorylation and dissociated from the trimer, exposing the p65/p50 dimer. The dimer enters the nucleus from the cytoplasm through the translocation signal on the p65 subunit, thereby exerting a transcriptional regulatory role (Ren et al., 2020). In this study, the OVT group promoted the phosphorylation of IκB and p65 in macrophages and LPS-induced cells were inhibited the phosphorylation of IκB and p65. MAPKs including the p38, ERK1/2 and JNK subgroups can regulate cell functions such as cell growth and gene expression as well as also plays a key role in the transcriptional activation of NF-κB . Recently, OVT activates RAW264.7 macrophages through the MAPK pathway, promoting NO secretion, cytokine expression and phagocytic activity, and has immune enhancing activity . However, OVT is shown to significantly increase the phosphorylation of p38 and ERK, except that of JNK in our research. The one possibility is that the concentration of OVT may be relatively low, leading to an insignificant change in JNK. When OVT was added to LPS-induced macrophages, the phosphorylation levels of JNK, ERK and p38 were significantly inhibited. These findings suggest that NF-κB and MAPK signaling cascades both contribute to OVT related the process of immune regulation.
In conclusion, our study demonstrated immunomodulatory activities of OVT in RAW264.7 in vitro. The study indicates that high concentration of OVT promotes the accumulation of inflammatory mediator in RAW264.7. TLR4 was demonstrated to be a membrane receptor by which OVT may act on RAW264.7 cells, and the MAPK and NF-κB signaling pathways were involved in OVT-induced macrophage immunoregulation. Besides, low concentration of OVT suppressed LPS-induced NO, TNFα and IL-6 production by inhibiting NF-κB and MAPK associated protein expression. Following our experimental results, OVT could be researched and developed as one of the potentially functional foods for immunological reagents.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

AUTH O R CO NTR I B UTI O N S
Huaying Du: Funding acquisition (supporting); Supervision (equal).

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.

ACK N OWLED G M ENTS
We gratefully acknowledge the financial support for this study was from the National Natural Science Foundation of China (no:

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.