Methionine enkephalin upregulates the immune function of RAW264.7 cells to inhibit the infection of Nelson Bay orthoreoviruses

Abstract

Preliminary research has found that MENK can upregulate the antiviral state of macrophages to inhibit influenza virus infection. To further study the immunomodulatory mechanism of MENK in macrophages against reovirus, we used RNA-Seq technology to analyze the genomic changes between macrophages infected with Nelson Bay orthoreoviruses (Miyazaki) after MENK pretreatment and those infected with Miyazaki alone. A total of 3,624 genes were screened, with 1,817 genes upregulated and 1,447 genes downregulated. Differentially expressed genes were mainly enriched in the cell cycle (ko04110), FoxO signaling pathway (ko04068), cell adhesion molecules (ko04514), Fc receptor-mediated endocytosis (ko04666), and antigen processing and presentation pathways (ko04612). Key genes such as IL6ST, TNFR2, CCL24, MHC I, MHC II, CD28, FOXO-1, SYK, and CYCD1 were successfully docked with the MENK molecule. Genomic analysis showed that MENK enhanced the immune function of macrophages by upregulating cytokine-related molecules such as CCL24, IL6ST, and TNFR2 to recruit and induce inflammatory responses. Moreover, MENK upregulated the expression of MHC I, MHC II, and CD28 to promote antigen presentation and initiate adaptive immune responses and regulated the FOXO-CYCD1 pathway to inhibit the cell cycle, thereby exerting antiviral effects during reovirus infection.

Introduction

Nelson Bay orthoreoviruses (NBV) belong to the Orthoreovirus genus of the Reoviridae family and double-stranded nonenveloped RNA viruses. It has the ability to cause syncytial cytopathic effects in infected cells. NBV was first isolated from fruit bats in Australia more than 60 years ago. After mutation, it overcame species barriers and exhibited a broad host range, capable of infecting cell types from various mammalian groups, including humans, mice, monkeys, and black flies, becoming a zoonotic pathogen^1,2. In 2007, TAKAHIRO and others successfully isolated a strain of NBV from patients with acute respiratory infections and used reverse genetics to successfully prepare the Miyazaki–Bali/2007 (MB) virus strain^3,4. Pathogenic NBV can cause severe immune dysfunction in patients, who exhibit acute respiratory and intestinal inflammation, including symptoms such as fever, cough, pharyngitis, abdominal pain, watery diarrhea, and vomiting⁵. Therefore, NBV has an unknown potential for zoonotic transmission and may become an important pathogen causing respiratory and intestinal diseases in humans.

The innate immune system serves as the first line of defense against viral infections, where macrophages exhibit exceptional plasticity and participate in physiological functions such as anti-infection and immune regulation. They play a central role in the recognition, killing, and presentation of viral antigens. Innate immune responses played an important role in immune actions during viral infections in the respiratory and gastrointestinal tracts. Innate immune cells, including macrophages, have a key role in controlling influenza A virus infection by controlling early viral replication and promoting and regulating T cell and B cell responses. The cross-linking of Fcγ receptors in macrophages induces the phosphorylation of Spleen tyrosine kinase (Syk), which promotes the phagocytic action of macrophages against viruses and enhances the release of mediators to exert antiviral effects. The adaptive immune response mediated by B cells, initiated by the innate immune response, plays an irreplaceable role in mucosal infection immunity. Secretory immunoglobulin A (sIgA) is the most abundant and important isotype of antibody in the mucosal immune system, widely present in the gastrointestinal tract, respiratory tract, and other parts⁶. Reovirus infection can induce the production of intestinal IgA, which resists Miyazaki’s infection of the respiratory and gastrointestinal mucosa. Moreover, IgA plays a crucial role in timely clearing enteric infections and preventing re-infection by reovirus.

Methionine enkephalin (MENK) is an endogenous opioid peptide composed of five amino acids: Tyr-Gly-Gly-Phe-Met, produced by the adrenal glands, and is known for its ability to regulate neuroendocrine activity and cellular immune activity⁷. MENK positively modulates the functions of granulocytes, natural killer cells, dendritic cells, macrophages, and T cells^8,9. Our previous research has found that MENK regulated toll-like receptors (TLR)-nuclear factor-kappaB (NF-κB) signaling pathway and exerted anti-influenza virus effects in vitro and in vivo by upregulating the opioid receptor MOR^10,11. In proteomic analysis of the regulatory role of MENK on macrophage resistance to influenza infection, we discovered that MENK upregulated the regulatory receptor FcγR/CR3 and the chemokine signaling pathway, which enhanced the phagocytic killing and antigen presentation functions of macrophages¹². This has led us to consider whether MENK can exert anti-infection effects against reovirus by modulating immune cells. In this study, we used the Nelson Bay orthoreoviruses recombinant strain (NBV-MB) to infect macrophages RAW264.7 and applied a preventive intervention of MENK to macrophages infected with NBV-MB. By employing RNA-Seq technology to analyze the genomic changes in macrophages, we explored the immunological mechanisms by which MENK regulated macrophage resistance to NBV-MB. This research provides data support for the potential of MENK to become a new drug or vaccine adjuvant for NBV and offers new perspectives for the clinical prevention and treatment of reovirus.

Materials and methods

Experimental cells and virus

The Nelson Bay orthoreoviruses (NBV-Miyazaki-Bali/2007, NBV-MB) was provided by the Laboratory of Pathogen Biology at Jinzhou Medical University. The mouse macrophage cell line (RAW264.7) was purchased from the China Center for Type Culture Collection (Wuhan, China).

Antibodies and reagents

MENK (≥ 99% purity) was provided by America peptide Inc. DAPI (4',6-diamidino-2-phenylindole, C1002, China) was purchased from Beyotime. A Goat Anti-Mouse IgG (H + L) FITC (S0007, China) was purchased from Affinity, and a mouse monoclonal antibody against β-actin was purchased from Zhongshan Golden Bridge (Beijing, China, TA-09). Polyclonal antibody against σ-nonstructural proteins (σNS) was prepared in rat immunized with σNS fusion protein. First, S3 gene encoding σNS protein was closed into a bacterial expression vector to make the recombinant plasmid which was transformed into Escherichia coli BL21 (DE3) cells. Then Fusion protein was purified on Ni–NTA agarose and used as an antigen.

Experimental group design

The experiment was divided into two groups: the NBV-MB control group (M group) and the MENK-NBV-MB group (MM group). RAW264.7 cells in the logarithmic growth phase were seeded into six-well plates, and when cell confluence reached 80%–90%, the following procedures were performed. For the NBV-MB control group, RAW264.7 cells were infected with the NBV-MB virus at an multiplicity of infection (MOI) of 30. The cells were cultured in DMEM maintenance medium containing 1% bovine serum albumin (Gibco), penicillin (100 U/mL, Gibco), streptomycin (100 μg/mL, Gibco) and HEPES (25 mM, Gibco) at 37 °C with 5% CO₂ for 1 h. After incubation, the virus solution was removed, cells were washed with PBS, and DMEM maintenance medium was added for further culture. In the MENK-NBV-MB group, RAW264.7 cells were seeded in six-well plates and treated with MENK (10 mg/ml, 2 ml per well) 48 h prior to infection with the NBV-MB virus strain. The cells were cultured in DMEM containing 10% fetal bovine serum (Gibco), penicillin (100 U/mL, Gibco) and streptomycin (100 μg/mL, Gibco) at 37 °C with 5% CO₂ for 48 h. After incubation, the cells were washed with PBS and Hank’s solution and then infected with the NBV-MB virus strain at an MOI 30. After 1 h of incubation at 37 °C with 5% CO₂, the virus solution was discarded, cells were washed with PBS, and DMEM maintenance medium was added for further culture. Samples were collected 48 h after virus infection.

RNA sequencing (RNA-seq) and analysis of sequencing data

Total RNA was extracted using the Total RNA Extractor (Trizol) kit (Sangon, China) following manufacturer’s instructions, with RNA integrity confirmed by a 1.0% agarose gel. RNA quality and quantity were assessed using a NanoPhotometer ® spectrophotometer (IMPLEN, CA, USA) and a Qubit® 2.0 Flurometer (Invitrogen). High-quality RNA samples were sent to Sangon Biotech (Shanghai) Co., Ltd. for library preparation using the VAHTSTM mRNA-seq V2 Library Prep Kit for Illumina®. mRNA was isolated, fragmented, and converted into cDNA, which was then ligated with adapters and amplified via PCR. The libraries were quantified and sequenced on an Illumina NovaSeq platform. Sequencing data quality was assessed with FastQC, and reads were filtered using Trimmomatic to remove low-quality sequences and adapters. Clean reads were aligned to the reference genome with HISAT2, and gene coverage was analyzed with BEDTools. Gene expression was quantified using StringTie, and DESeq2 (version 1.12.4) identified differentially expressed genes (DEGs) based on a q-value ≤ 0.001 and |FoldChange|≥ 2. After identifying the significantly differentially expressed genes, a volcano plot was used to visualize the number and expression levels of these genes. Functional enrichment analyses of DEGs were performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)^13,15,16, identifying significantly enriched pathways and biological functions with a false discovery rate (q-value) < 0.05. GO annotation analysis was visualized using bar charts, while KEGG significantly enriched pathways were presented with bubble charts. GO annotation analysis, GO enrichment analysis, and KEGG pathway enrichment were visualized using bar charts and bubble plots.

Quantitative real-time PCR (qPCR)

The qPCR method was used to validate the reproducibility of 10 key differentially expressed genes identified from transcriptome sequencing data. Total RNA was extracted from cells using Trizol, followed by phase separation with chloroform and RNA precipitation with isopropanol. The RNA precipitation was washed with 75% ethanol, dried, and dissolved in ddH₂O. Subsequently, reverse transcription was carried out using SYBR Prime Script RT-PCR Kit (Takara, Japan) with QuantStudio 6 Flex Real-time PCR system (ABI, USA). Primer sequences are provided in Supplementary Table 1. Gene expression was quantified and normalized to GAPDH RNA expression using the 2^−△△CT method. Among them, ΔCt = Ct value of the target gene—Ct value of the GAPDH gene, ΔΔCt = ΔCt value of the MM group—ΔCt value of the M group.

Immunofluorescence

The RAW264.7 cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100 and blocked with 2% BSA for 30 min at room temperature, incubated with NBV-σNS antibody at 4 °C overnight, followed by incubated for 1 h at room temperature with secondary antibody FITC IgG (H + L). Finally, added 25 μL DAPI-containing anti-fluorescence quencher to stain the cell nuclei. Images were acquired with a fluorescence microscope (Olympus, Japan) using DP Manager software.

Molecular docking

Key proteins involved in immune-related pathways, identified through GO and KEGG enrichment analyses, were selected for molecular docking using Autodock Vina software to validate interactions between MENK and key small-molecule compounds. The docking results between chemical components and key targets were visualized using PyMOL software.

Statistics analysis

All data were present as mean ± SEM and evaluated by One-way analysis of variance (ANOVA) with Dunnett’s multiple-comparison test using GraphPad Prism Ver.10 (GraphPad Software, San Diego, CA, USA). Every assay in this study was carried out in triplicate. Values of *P < 0.05,**P < 0.01 or ***P < 0.001 were considered statistically significant.

Result

Optimal concentration of MENK and inoculation dose of the virus strain

The MTS results showed that cell proliferation appeared to be increased by MENK. The OD value peaked at a concentration of 10 mg/ml of MENK, and gradually decreased as the concentration of MENK decreased, as shown in Fig. 1A. However, through cell counting, we found that the cell number was about 90% of that of normal macrophages when the MENK concentration was 20 mg/ml and 10 mg/ml, and the difference was statistically significant (Fig. 1B). The reason for the increased OD value in the MTS assay may reflect morphological changes (Fig. 2A) rather than true proliferation. Subsequent experiments used the optimal concentration of MENK at 10 mg/ml to intervene in RAW264.7 cells.

Fig. 1 [Images not available. See PDF.]

Influence of MENK concentration and MOI on RAW264.7 cells proliferation and immune function. (A) The effect of different concentrations of MENK on RAW264.7 cells proliferation. (B) The cell counting of RAW264.7 cells treated with different concentrations of MENK. (C) Copy number of NBV-σNS by qPCR. (D) The impact of different MOI on RAW264.7 cells immune function. NC represents uninfected control.

Fig. 2 [Images not available. See PDF.]

Morphological images of RAW264.7 cells under light microscopy and immunofluorescence image. (A) Morphological image of RAW264.7 cells in the NC, MENK, NBV-MB and MENK-NBV-MB group (× 200). (B) Immunofluorescence images of RAW264.7 cells in the NBV-MB group and the MENK-NBV-MB group (× 200). M Group was RAW264.7 cells infected with NBV-MB, and MM Group was RAW264.7 cells infected with NBV-MB after MENK intervention. The results were observed 48 h post-infection. (C) The ratio of infected RAW264.7 cells in NBV-MB and MENK-NBV-MB group by immunofluorescence images. Each sample was observed under 200 × magnification for 10 fields of view.

The Miyazaki virus strain was used to infect macrophages at an MOI levels of 10, 30, and 50 in Fig. 1C, and qPCR was used to detect the levels of pro-inflammatory cytokines interleukin-1 beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and monocyte chemoattractant protein-1 (MCP-1) produced by RAW264.7 cells in Fig. 1D. The results showed that the levels of all pro-inflammatory cytokines were significantly elevated compared to the control group when infected with the virus at an MOI levels of 10, 30, and 50 (P < 0.001). When the MOI was 30, the levels of IL-1β, IL-6, TNF-α, and MCP-1 reached their peak, indicating the most pronounced antiviral capability of the macrophages. Subsequent experiments used the Miyazaki strain at an MOI of 30 to infect RAW264.7 cells.

Morphological changes of RAW264.7 cells

The RAW264.7 cells exhibited an initial state, mostly round and translucent. With the intervention of MENK, the cells changed from a round shape to a long fusiform shape. The cells in the NBV-MB group exhibited an irregular shape, with many vacuoles and granular materials within the cytoplasm. In the MENK-NBV-MB group, the RAW264.7 cells were mostly spindle-shaped with pseudopodia, and occasional vacuoles and granular materials were observed inside the cells in Fig. 2A. Immunofluorescence results showed that the fluorescence intensity in the MENK-NBV-MB group was reduced compared with the NBV-MB group, as shown in Fig. 2B. The number of infected macrophages in the MENK-NBV-MB group was significantly lower than that in the NBV-MB group in Fig. 2C, indicating that MENK intervention in macrophages can enhance the cells’ antiviral capacity.

RNA-Seq data quality control assessment

Following the quality control analysis of the data obtained from RNA-Seq, the Trimmomatic software was utilized for data processing, resulting in a range of 52,399,922 to 53,456,976 clean reads. The Q20 value was ≥ 99.27%, and the Q30 value was ≥ 97.05%. This demonstrated that the sequencing data was highly reliable and suitable for application in subsequent experiments.

Differential gene expression analysis

The differential analysis revealed that, compared to the NBV-MB group, the MENK-NBV-MB group had a total of 3,264 differentially expressed genes that met the criteria of qValue < 0.05 and |log2FoldChange|> 1, with 1,817 genes upregulated and 1,447 genes downregulated in the MENK-NBV-MB group in Supplementary Table 2 and Significantly DEGs in Table 1. For the differentially expressed genes in each sample group, we conducted screening and statistical analysis and constructed a volcano plot to visually display the distribution of the differentially expressed genes between the two groups in Fig. 3.

Table 1. Significant DEGs.

Gene id	GeneName	GeneDescription	log2FoldChange	pValue	Result
ENSMUSG00000070348	Ccnd1	cyclin D1 [Source:MGI Symbol;Acc:MGI:88313]	− 1.67	2.15501791465e-27	Down
ENSMUSG00000004814	Ccl24	chemokine (C–C motif) ligand 24 [Source:MGI Symbol;Acc:MGI:1928953]	11.51	0.012459939755599999	Up
ENSMUSG00000021457	Syk	spleen tyrosine kinase [Source:MGI Symbol;Acc:MGI:99515]	2.45	3.3917489532399994e-41	Up
ENSMUSG00000024610	Cd74	CD74 antigen (invariant polypeptide of major histocompatibility complex, class II antigen-associated) [Source:MGI Symbol;Acc:MGI:96534]	2.60	0.0029108025668499998	Up
ENSMUSG00000044167	Foxo1	forkhead box O1 [Source:MGI Symbol;Acc:MGI:1890077]	1.17	6.34402784341e-05	Up
ENSMUSG00000026471	Mr1	major histocompatibility complex, class I-related [Source:MGI Symbol;Acc:MGI:1195463]	1.55	1.96136012945e-06	Up
ENSMUSG00000021756	Il6st	interleukin 6 signal transducer [Source:MGI Symbol;Acc:MGI:96560]	1.11	0.00138448634111	Up
ENSMUSG00000026012	Cd28	CD28 antigen [Source:MGI Symbol;Acc:MGI:88327]	5.16	1.39512036691e-17	Up
ENSMUSG00000045362	Tnfrsf26	tumor necrosis factor receptor superfamily, member 26 [Source:MGI Symbol;Acc:MGI:2651928]	1.86	1.57668799446e-23	Up

Fig. 3 [Images not available. See PDF.]

Volcano plot of DEGs between MENK-NBV-MB group and NBV-MB group.

GO annotation analysis

To elaborate on the functional differences in gene expression between samples, we conducted a statistical analysis of the distribution of differential genes in terms of annotated functions, as shown in Fig. 4A. In the GO analysis comparing the MENK-NBV-MB group with the the NBV-MB group, the biological process (BP) mainly pertained to immune system processes, biological adhesion, and cellular defense responses to cytokines. The cellular component (CC) included organelles, intercellular junctions, cell membranes, and inclusion bodies. The molecular function (MF) was related to enzyme binding and activity.

Fig. 4 [Images not available. See PDF.]

Visualization of GO annotations and enrichment analysis for MENK-NBV-MB and NBV-MB Groups. (A) Bar plot of GO annotation categories for MENK-NBV-MB group and NBV-MB group. The left vertical axis (percentage of genes) represent the ratio of DEGs to all genes. The light-colored bars represent DEGs, while the dark-colored bars represent all genes. On the vertical axis, the gray numbers indicate the DEGs, and the black numbers indicate all genes. (B) Bubble plot of BP. (C) Bubble plot of CC. (D) Bubble plot of MF.

GO enrichment analysis

The GO enrichment analysis between the MENK-NBV-MB group and the NBV-MB group, the BP enrichment category showed that the differential genes were predominantly enriched in immune system processes, signal transduction, and cellular responses to cytokine stimuli, as seen in Fig. 4B. In the CC enrichment category, the differential genes were mainly enriched in cellular components such as the cell, cytoplasm, and vesicles, as depicted in Fig. 4C. In the MF enrichment category, the differential proteins were primarily enriched in functions related to enzyme binding, cytokine binding, and regulatory activities, as illustrated in Fig. 4D

KEGG enrichment analysis

KEGG enrichment analysis aims to annotate pathways for differential genes, thereby visually presenting the impact of gene expression differences on metabolic pathways. By conducting KEGG enrichment analysis, we can better understand the functions and roles of differential genes in various metabolic pathways.

In the comparison between MENK-NBV-MB group and NBV-MB group, the main pathways involved include osteoclast differentiation (ko04380), cell cycle (ko04110), FoxO signaling pathway (ko04068), cell adhesion molecules (ko04514), cytokine-cytokine receptor interaction (ko04060), Fc gamma receptor-mediated phagocytosis (ko04666), and antigen processing and presentation (ko04612), as seen in Fig. 5. In ko04514 (Fig. 6A) and ko04060 (Fig. 6B), cytokine-related interleukin 6 signal transducer (IL6ST), tumor necrosis factor receptor 2 (TNFR2), and C–C motif chemokine ligand 24 (CCL24) were highly expressed, indicating that MENK played a role in recruiting immune cells to the site of infection and promoting inflammatory responses to clear the virus. In ko04666 (Fig. 6C), the high expression of syk suggested that MENK enhanced the phagocytic ability of macrophages, which was crucial for virus clearance and antigen presentation. In ko04514 and ko04612 (Fig. 6D), the high expression of antigen presentation related molecules such as major histocompatibility complex class I (MHC Ⅰ), major histocompatibility complex class II (MHC Ⅱ), and cluster of differentiation 28 (CD28) indicated that MENK promoted the antigen presentation capability of macrophages, inducing the initiation of adaptive immune responses. Additionally, ko04672 (Fig. 6E) showed antigen presentation related factors of MHC and CD28 that promoted the production of IgA by B cells in the intestinal immune network. ko04068 (Fig. 6F) and ko04110 (Fig. 6G) respectively showed differential expression of forkhead box O1 (FOXO-1) and cell cycle-related molecules such as cyclinD1 (CYCD1). The enrichment analysis indicated that MENK regulated the antiviral functions of macrophages by promoting inflammatory responses, enhancing antigen presentation, and affecting the cell cycle.

Fig. 5 [Images not available. See PDF.]

Bubble plots of KEGG enrichment analysis. (A) Enrichment of upregulated differentially expressed genes. (B) Enrichment of downregulated differentially expressed genes.

Fig. 6 [Images not available. See PDF.]

KEGG pathway enrichment map. (A) Cell adhesion molecules pathway (ko04514). (B) Cytokines-cytokine receptor interaction(ko04060). (C) Fc gamma R-mediated phagocytosis pathway (ko04666) (D) Antigen processing and presentation pathway (ko04612) (E) Intestinal immune network for IgA production pathway (ko04672). (F) FOXO signaling pathway (ko04068). (G) Cell cycle pathway (ko04110). Red indicates upregulated genes, green indicates downregulated genes, and yellow indicates no difference.

qPCR validation

To verify the reliability of the RNA-Seq results, we conducted qPCR validation for key indicators. In the differential analysis between NBV-MB control group and MENK-NBV-MB group, the levels of the genes IL6ST, TNFR2, CCL24, MHC Ⅰ, MHC Ⅱ, CD28, FOXO-1, and SYK in the macrophages of the MENK-NBV-MB group showed an increasing trend (P < 0.05), while the level of CYCD1 genes were downregulated (P < 0.05), as shown in Fig. 7. These results were consistent with the trends observed in the RNA-Seq Data.

Fig. 7 [Images not available. See PDF.]

qPCR validation. M represents the NBV-MB group, and MM represents the MENK-NBV-MB group.

Molecular docking

After determining the changes in gene expression by MENK treatment through transcriptomic analysis, we then used molecular docking techniques to verify the interaction between MENK and the proteins encoded by these genes, thereby revealing the mechanism of MENK action. Incorporating KEGG pathways, we assessed the interactions between MENK and 9 small molecular core proteins: IL6ST, TNFR2, CCL24, MHC Ⅰ, MHC Ⅱ, CD28, FOXO-1, SYK and CYCD1. Computational calculations determined the binding affinities of MENK with these core proteins to be as follows: IL6ST (− 6.84), TNFR2 (− 5.27), CCL24 (− 4.58), MHC Ⅰ (− 5.29), MHC Ⅱ (− 4.9), CD28 (− 4.97), FOXO−1 (− 6.91), SYK (− 6.9), and CYCD1 (− 7.21). Molecular docking was successful for all core proteins with MENK, including IL6ST, TNFR2, CCL24, MHC Ⅰ, MHC Ⅱ, CD28, FOXO-1, SYK and CYCD1, as illustrated in Fig. 8.

Fig. 8 [Images not available. See PDF.]

Molecular docking simulation of MENK with core proteins. The MENK is orange stick models and the protein molecules at the docking site are represented as blue stick models. The connected hydrogen bonds are indicated by yellow dotted lines.

Discussion

The RNA sequencing analysis results showed that the DEGs between MENK-NBV-MB group and NBV-MB group were mainly enriched in pathways such as cell adhesion molecules (ko04514), Fc gamma receptor-mediated phagocytosis (ko04666), antigen processing and presentation (ko04612), FoxO signaling pathway (ko04068), and cell cycle (ko04110). In the cytokine-cytokine receptor interaction (ko04060) and cell adhesion molecule pathways (ko04514), high expression of CCL24, IL6ST, TNFR, and ITGA9 were observed. The chemokine CCL24 is considered a key molecule that plays a major role in the recruitment process of macrophages and is involved in the chemotaxis of M1 macrophages¹⁴.CCL24 recruited mononuclear macrophages to sites of infection and tissue damage. MENK enhanced the chemotactic and recruitment ability of macrophages through CCL24, thereby promoting the body’s effective resistance to the NBV-MB strain. IL6ST (gp130) is an important factor in immune homeostasis during infection. The expression of OnIL6R and Ongp130 in mononuclear macrophages and lymphocytes was significantly upregulated after the tilapia was attacked by extracellular pathogens, participating in the process of resisting bacterial or viral infections and playing an important role in immune regulation¹⁷. Ongp130 is highly homologous to human gp130 in sequence and structure, and it is speculated that gp130 is involved in the defense process against pathogenic infections. In this experiment, gp130 was significantly upregulated in the MENK intervention group, indicating that MENK promoted the high expression of gp130 in macrophages and upregulated the ability of macrophages to resist viral infections. TNF-α is a cytokine produced by activated monocytes, macrophages, and lymphocytes in response to pathogen immune reactions^18,19. The TNF-TNFR2 signal transduction helps to regulate the function of macrophages, such as anti-apoptosis and the production of inflammatory factors, to maintain immune balance in the body²⁰. The upregulation of TNFR2 in macrophages can increase the production of anti-apoptotic and inflammatory factors within the cell, playing a role in anti-infection. Therefore, the promotion of TNFR expression by MENK helped to inhibit the apoptosis of macrophages and enhance the ability of macrophages to resist reovirus.

Additionally, the TNFR2-Etk-VEGFR2 pathway is involved in cell adhesion, migration, survival, and proliferation. Integrin subunit alpha 9 (ITGA9) is a member of a unique integrin subclass that preferentially promotes cell migration, inducing rapid egress of leukocytes from the vascular system to migrate to extravascular sites of inflammation and immune response²¹. ITGA9 played a critical role in the recruitment of inflammatory cells, ITGA9 antibodies inhibited the production of cytokines and chemokines in the joints of arthritis, thereby suppressing the recruitment of macrophages and neutrophils into the joints, suggesting that ITGA9 was a key molecule in promoting the migration of immune cells²². In this study, MENK promoted the high expression of ITGA9 and TNFR2 in macrophages, further enhancing the ability of macrophages to migrate to the site of infection and improving the efficiency of resistance to NBV-MB infection. Interestingly, the two negative regulatory factors, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and programmed death receptor ligand-1 (PD-L1), were both highly expressed in macrophages intervened by MENK. Staples KJ reported that influenza infection induced the upregulation of PD-L1 in macrophages in the lungs of mice, which rapidly increased the immune response of CD8⁺ T cells through PD-L1. The PD-1/PD-L1 pathway may play a role in suppressing acute inflammatory responses²³. In porcine alveolar macrophages infected with porcine reproductive and respiratory syndrome virus, the upregulation of PD-L1 and CTLA-4 has been found to help prevent excessive inflammation and protect the lungs from immune-mediated damage²⁴. In this study, MENK upregulated the negative regulatory gene in macrophages, which to some extent helped to prevent cell damage mediated by excessive inflammation.

The Fc receptor-mediated phagocytosis pathway (ko04666) showed increased expression of Syk and linker for activation of T cells (LAT) in the MENK intervention group. The phagocytic action mediated by Fcγ receptors in macrophages was dependent on the presence of Syk. After the engagement of Fc receptors on macrophages, the immunoreceptor tyrosine-based activation motifs within the receptor were phosphorylated by Src family kinases, recruiting and activating the protein tyrosine kinase Syk. The recruitment and activation of Syk led to the formation of a signaling complex on the plasma membrane, where multiple adaptor proteins were phosphorylated by Syk, activating downstream pathways, and ultimately promoting the engulfment of pathogens by phagocytic cells²⁵. Cross linking of Fcγ receptors induced the phosphorylation and activation of Syk PTK, leading to enhanced phagocytosis and the release of inflammatory mediators²⁶. LAT was expressed in mononuclear phagocytes and was a functional component of myeloid cell FcR signaling. LAT recruited signaling proteins containing SH2 domains to the plasma membrane, thereby supporting phagocytosis. The adaptor protein LAT contributed to the innate immune response by facilitating FcR induced signal transduction for the engulfment of pathogens and the release of inflammatory factors. Additionally, studies have found that the phagocytic efficiency of bone marrow derived macrophages from LAT deficient mice was significantly reduced²⁷. Therefore, in this study, the preventive intervention of MENK in macrophages could promote Fc receptor-mediated endocytosis and enhance the phagocytic and virucidal functions of macrophages through the high expression of Syk and LAT.

Macrophages, as important professional antigen presenting cells in the body, played a crucial role in inducing adaptive immune responses. Macrophages presented antigenic peptides to T cell receptors through MHC molecules and activate T cells under the influence of co-stimulatory molecules such as CD28²⁸. The impaired activation of CD28 significantly reduced the capacity for humoral immune responses²⁹. The binding of the T cell receptor to the antigen-MHC and the interaction between CD28 and B7 molecules provided signals for T cells activation³⁰. The activation of T cells not only promoted the release of cytokines but also assisted the activation and differentiation of B cells. In this study, the KEGG enrichment pathway (ko04514) showed that MENK upregulated the expression of MHC Ⅰ, MHC Ⅱ, and CD28, driving the antigen presenting capacity of macrophages to activate T cells and enhanced the ability to mount adaptive immune response. In addition, IgA played an anti-infective role in mucosal tissues such as the gastrointestinal tract, respiratory tract, and vagina, and protective immune responses against reovirus were strictly dependent on the presence of IgA ³¹. SIgA can protect mucosa from the adhesion and invasion of enteric pathogens and prevent the virus from entering mouse Peyer’s patches ³². SIgA can directly block or interfere with the attachment of microorganisms to epithelial cells through spatial blocking mechanisms by promoting the capture of pathogens in mucus. The intestinal immune network producing IgA pathway (ko04672) showed high expression of MHC molecules in macrophages, promoting T cell activation, and further providing signals for B cells activation, inducing B cells to produce sIgA antibodies. Su F et al.³³, in their study of the infection process of porcine epidemic diarrhea virus, found that the activation of T cells induced the activation of B cells in vivo, increasing the level of intestinal IgA to resist diarrhea virus infection. Preventive intervention with MENK played an active role in promoting macrophage MHC molecule antigen presentation and inducing B cells to produce sIgA antibodies.

The FOXO signaling pathway (ko04068) showed significant changes in FOXO1 and CYCD1. FOXO1 regulated the expression of various important components of cyclin-dependent kinase inhibitors, playing a role in cell cycle regulation³⁴. FOXO1 is positively regulated by different kinases, such as JNK and AMPK, and negatively regulated by ERK-MAPK and CDK2³⁵. FOXO1 modulates the development and function of immune cells³⁶, and autophagy mediated by FOXO1 is important for the development of NK cells and their induce innate immune responses. Mutations or deletions of FOXO1 can eliminate the initiation of autophagy in immature NK cells, thereby impairing the development of NK cells and their effect on virus clearance³⁷. In this study, MENK may regulate the cell cycle of macrophages before viral infection, inhibit their proliferation, promote the polarization of macrophages to present an antiviral state, and respond rapidly after NBV-MB infection to exert phagocytic and virucidal effects.

In molecular docking, MENK has successfully docked with nine core molecules, including IL6ST, TNFR2, CCL24, MHC Ⅰ,MHC Ⅱ, CD28, FOXO-1, SYK, and CYCD1. Among them, the binding of MENK to CYCD1 may influence the expression of CYCD1 through multiple mechanisms. On the one hand, MENK may directly bind to the CYCD1 protein, interfering with its normal function in the cell cycle, which could lead to its degradation or inactivation. On the other hand, MENK may also indirectly regulate the transcriptional level of CYCD1 by affecting upstream MAPK signaling pathway (ko04110). CYCD1 plays a key role in the regulation of the cell cycle, and its reduced expression is usually associated with the inhibition of cell proliferation. The binding of MENK to CYCD1 may be a negative regulatory mechanism that inhibited the proliferation of macrophages by reducing the expression of CYCD1. This also indicated that MENK’s preventive intervention in macrophages induced polarization of macrophages to some extent, rather than stimulating their proliferation. The polarization of macrophages induced by MENK played a significant antiviral role in subsequent resistance to NBV infection. In Silico Study, Benzimidazole derivatives, acting as protein kinase inhibitors, inhibited cell proliferation through two key targets CDK4/CYCD1 and Aurora B, possessed the ability to treat cancer, reduce viral load, and enhance immunity³⁸. This was consistent with our observations and further supported the possibility that MENK exerted its antiviral functions through binding to CYCD1.

Conclusion

The preventive intervention of MENK induced an antiviral state in macrophages to some extent before NBV-MB infection, increased the release of inflammatory and chemotactic factors, promote phagocytosis and antigen presentation functions within a certain range. It may also enhance the immune functions of macrophages against reovirus infection in multiple aspects by upregulating the mucosal immune effect of IgA and modulating the FOXO-CYCD1 pathway to suppress cell cycle. This study provided data support for MENK to become a novel drug or vaccine adjuvant for NBV and offers new perspectives for the clinical prevention and treatment of reovirus infections.

Acknowledgements

The authors would like to thank all the participants involved in this study. We also thank Sangon Biotech Co., Ltd. for performing the RNA sequencing.

Author contributions

X.T.: Conceptualization, Formal analysis, Funding acquisition, Resources, Supervision, Validation, Writing—review and editing. X.W.: Conceptualization, Validation, Visualization, Writing—original draft. Z.M.: Formal analysis, Validation, Writing—original draft. M.C.: Conceptualization, Validation, Writing—original draft. J.T.: Conceptualization, Formal analysis, Resources, Supervision, Writing—review and editing.

Funding

This research was supported by the National College Students Innovation and Entrepreneurship Training Program (202310160004), 2021 Youth Science and Technology Talents Support Plan from Boze Project of Jinzhou Medical University (JYBZQT2112) and Liaoning Provincial Department of Education (LJ212410160068).

Data availability

The RNA sequencing dataset generated during the current study is available on the NCBI GEO platform (GSE277277). Other data and figures from this study are available upon reasonable request.

Competing interests

The authors declare no competing interests.

Abbreviations

NBV

Nelson Bay orthoreoviruses

Syk

Spleen tyrosine kinase

sIgA

Secretory immunoglobulin A

MENK

Methionine enkephalin

TLR

Toll-like receptors

NF-κB

Nuclear factor-kappaB

σNS

σ-Nonstructural proteins

DAPI

4',6-Diamidino-2-phenylindole

MOI

Multiplicity of infection

RNA-seq

RNA sequencing

DEGs

Differentially expressed genes

GO

Gene Ontology

KEGG

Kyoto Encyclopedia of Genes and Genomes

qPCR

Quantitative real-time PCR

IL-1β

Interleukin-1 beta

IL-6

Interleukin-6

TNF-α

Tumor necrosis factor alpha

MCP-1

Monocyte chemoattractant protein-1

BP

Biological process

CC

Cellular component

MF

Molecular function

IL6ST

Interleukin 6 signaltransducer

TNFR2

Tumor necrosis factor receptor 2

CCL24

C–C motif chemokine ligand 24

MHC Ⅰ

Major histocompatibility complex class I

MHC Ⅱ

Major histocompatibility complex class II

CD28

Cluster of differentiation 28

FOXO-1

Forkhead box O1

CYCD1

CyclinD1

ITGA9

Integrin subunit alpha 9

CTLA-4

Cytotoxic T lymphocyte-associated antigen-4

PD-L1

Programmed death receptor ligand-1

LAT

Linker for activation of T cells

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1038/s41598-025-16284-y.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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