Exosome-mediated delivery of gga-miR-20a-5p regulates immune response of chicken macrophages by targeting IFNGR2, MAPK1, MAP3K5, and MAP3K14

Objective This study aims to evaluate the target genes of gga-miR-20a-5p and the regulated immune responses in the chicken macrophage cell line, HD11, by the exosome-mediated delivery of miR-20a-5p. Methods Exosomes were purified from the chicken macrophage cell line HD11. Then, mimic gga-miR-20p or negative control miRNA were internalized into HD11 exosomes. HD11 cells were transfected with gga-miR-20a-5p or negative control miRNA containing exosomes. After 44 h of transfection, cells were incubated with or without 5 μg/mL poly(I:C) for 4 h. Then, expression of target genes and cytokines was evaluated by quantitative real-time polymerase chain reaction. Results Using a luciferase reporter assay, we identified that gga-miR-20a-5p directly targeted interferon gamma receptor 2 (IFNGR2), mitogen-activated protein kinase 1 (MAPK1), mitogen-activated protein kinase kinase kinase 5 (MAP3K5), and mitogen-activated protein kinase kinase kinase 14 (MAP3K14). Moreover, the exosome-mediated delivery of gga-miR-20a-5p successfully repressed the expression of IFNGR2, MAPK1, MAP3K5, and MAP3K14 in HD11 cells. The expressions of interferon-stimulated genes (MX dynamin like GTPase 1 [MX1], eukaryotic translation initiation factor 2A [EIF2A], and oligoadenylate synthase-like [OASL]) and proinflammatory cytokines (interferon-gamma [IFNG], interleukin-1 beta [IL1B], and tumor necrosis factor-alpha [TNFA]) were also downregulated by exosomal miR-20a-5p. In addition, the proliferation of HD11 cells was increased by exosomal miR-20a-5p. Conclusion The exosome-mediated delivery of gga-miR-20a-5p regulated immune responses by controlling the MAPK and apoptotic signaling pathways. Furthermore, we expected that exosomal miR-20a-5p could maintain immune homeostasis against highly pathogenic avian influenza virus H5N1 infection by regulating the expression of proinflammatory cytokines and cell death.


INTRODUCTION
Exosomes are cell membrane-derived small vesicles with diameters ranging from 30 to 150 nm that are generated by various cell types in biological fluids, such as plasma, lymph, saliva, semen, urine, and cerebrospinal fluid [1,2]. Exosomes contain various proteins and nucleic acids originating from "mother cells", which are transmitted to recipient cells [3,4]. Particularly, exosomal microRNAs (miRNAs) regulate the gene expression of recipient cells by repressing and degrading their target mRNAs [5,6]. MiRNAs are small non-coding RNAs that are 18 to 23 nucleotides long that bind to the 3′-untranslated regions (UTRs) of the target mRNA complementary sequences. The miRNAs then repress and degrade the target mRNAs by forming an RNA-induced silencing complex and inhibiting mRNA translation [7]. Exosomal miRNAs derived from various cell types, such as immune cells, cancer cells, and stem cells, have been demonstrated to regulate immune responses, cell migration, metastasis, proliferation, and invasion by repressing target gene translation [8][9][10].
Recently, studies have been conducted on exosomes as a gene delivery vehicle for molecules, such as RNA or DNA [11][12][13][14][15]. Exosomes as "nature's delivery system" have several advantages. Because exosomes are produced from host cells, they have low toxicity and immunogenicity and are easily internalized due to their exosomal host cell-derived membranes [16,17]. Moreover, exosomal membranes protect their nucleic acid contents from nuclease degradation [11].
In our previous study, the expression of gga-miR-20a-5p was down-regulated in exosomes derived from highly pathogenic avian influenza virus (HPAIV) H5N1-infected chickens compared with exosomes of non-infected chickens, and 32 immune-related genes were predicted as gga-miR-20a-5p targets [22]. However, there are limited studies about the regulatory function of miR-20a-5p in chickens. Therefore, in this study, we evaluated the target genes of gga-miR-20a-5p and demonstrated the immunomodulatory effects in a chicken macrophage cell line by the exosome-mediated delivery of miR-20a-5p.

Transfection of mimic miRNA into exosomes
We loaded miRNA to exosomes as described by Kim et al [25] with a slight modification. Mimic gga-miR-20a-5p and miR-NC (negative control miRNA) were synthesized by Bioneer (Daejeon, Korea). The sequence of the mimic gga-miR-20a-5p was 5′-UAAAGUGCUUAUAGUGCAG GUAG-3′ and that of the miR-NC was 5′-UUCUCCGAA CGUGUCACGU-3′. For loading of the miRNAs into the exosomes, 20 pmol of mimic gga-miR-20-5p or miR-NC were diluted in 100 μL of Opti-MEM I Reduced Serum Medium (Gibco, Waltham, MA, USA) and 6 μL of Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, San Diego, CA, USA) was diluted in 100 μL of Opti-MEM I Reduced Serum Medium. Diluted miRNA solutions were added to the diluted Lipofectamine RNAiMAX and incubated for 20 min at room temperature. Next, 100 μg of purified exosomes were incubated with miRNA-Lipofectamine RNAiMAX complexes for 6 h in a humidified incubator with 5% CO 2 at 41°C. Then, the complexes were filtered with a 100 kDa Amicon Ultra-0.5 Centrifugal Filter (Merck Millipore, Burlington, MA, USA) to remove un-loaded miRNA in the exosomes. Afterward, 1.2×10 6 cells/well of HD11 cells were plated in 12-well plates (SPL, Korea) containing 1.0 mL of exosome-depleted RPMI 1640 medium and incubated with the concentrated exosomes. After 44 h of incubation, cells were incubated with or without 5 μg/mL of polyinosine-polycytidylic acid (poly[I:C]) high molecular weight (HMW) (Invitrogen, USA) for 4 h. Then, the total RNA of the HD11 cells was extracted using TRIzol reagent (Thermo Fisher Scientific, USA) according to the manufacturer's protocol. The cDNA was then synthesized using 2 μg of RNA with the RevertAid first-strand cDNA synthesis kit (Thermo Fisher Scientific, USA) according to the manufacturer's protocol.

Analysis of miRNA expression by qRT-PCR
To quantify miRNA expression, cDNA was synthesized using 2 μg of RNA with the Mir-X miRNA First-Strand Synthesis Kit (TAKARA Bio Inc., Otsu, Shiga, Japan) according to the manufacturer's protocol. The forward primers of gga-miR-20a-5p, 5′-TAAAGTGCTTATAGTGCAGGTAG -3′, and small nuclear ribonucleoprotein polypeptide A (U1A), 5′-CTGCATAATTTGTGGTAGTGG -3′, were synthesized by Genotech (Korea). The miRNA expression level was evaluated using the Mir-X miRNA qRT-PCR TB Green Kit (TAKARA Bio Inc., Japan) with the CFX Connect Real-Time PCR Detection System (Bio-rad, USA). The miRNA expression level was calculated using the 2 -ΔΔCt method with U1A for inter- Table 1. Sequences of primers used for quantitative real-time polymerase chain reaction analysis nal reference.

Statistical analysis
Data are presented as the mean±standard error of the mean of three independent experiments. Statistical analyses were performed using the IBM SPSS software (SPSS 26.0; IBM, Chicago, IL, USA). Statistical comparison between two groups were evaluated using Student's t-test. Statistical significance is defined when p-values are less than 0.05.

Characterization of isolated exosomes
Exosomes were isolated from the cell culture supernatant of HD11 cells. The evaluated average size of the exosomes was 132.0 nm and the size range was from 82.33 to 279 nm ( Figure   1A). The exosomal markers CD9 and CD81 were detected by western blotting ( Figure 1B).

DISCUSSION
In the present study, we delivered gga-miR-20a-5p via exosomes derived from a chicken macrophage cell line and identified the gene regulatory functions of gga-miR-20a-5p, which repressed the expression of IFNGR2, MAPK1, MAP3K5, and MAP3K14, in chicken macrophage cells.
MiR-20a-5p is a known regulator of immune responses. For example, miR-20a-5p was downregulated during liver fibrosis and transfection of miR-20a-5p reduced the production of the proinflammatory cytokines IL-6, TNF-α, IL-18, and IFN-γ in a murine hepatoma cell line by targeting transforming growth factor beta 2 (TGFB2) [27]. MiR-20a-5p is also upregulated in certain cancers, such as thyroid cancer [28], gastric cancer [29], breast cancer [30], and cervical cancer [31], by inducing cancer cell proliferation and inhibiting cell apoptosis. On the other hand, miR-20a-5p is normally downregulated in infectious and proinflammatory environments, such as tuberculosis [20], liver fibrosis [27], and rheumatoid arthritis [19], by stimulating proinflammatory cytokine production and apoptosis. In our previous study, the expression of exosomal miR-20a-5p was downregulated in H5N1-infected chickens [22]. Therefore, we predicted the target genes and validated the regulatory functions in chicken macrophages.
In this study, we stimulated chicken macrophages with poly(I:C) after transfection with exosomal miR-20a-5p. Macrophages as antigen presenting cells have crucial immune functions, such as phagocytosis of pathogens and production of cytokines and chemokines [32]. Moreover, poly(I:C) is a viral like double-stranded RNA (dsRNA) that binds to TLR3, RNA helicases, retinoic acid-inducible gene-I, and melanoma differentiation-associated protein 5. Upon recognition, the transcription of pro-inflammatory cytokines and interferons are activated by the transcription factors such as interferon regulatory factor-3 (IRF-3), active protein-1 (AP-1), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) [33,34].
HPAIV H5N1 infection induces a massive production of proinflammatory cytokines, called a cytokine storm, which causes multiple organ dysfunction syndrome, acute respiratory distress syndrome, and sudden death [43][44][45][46]. Moreover, the MAPK signaling pathway is an important mediator of cytokine production against influenza A virus infection [47]. Therefore, we expected that the exosome-mediated delivery of gga-miR-20a-5p to H5N1-infected chicken cells could regulate proinflammatory cytokine expression by targeting the MAPK signaling pathway molecules, MAPK1, MAP3K5, and MAP3K14, and inhibit cell death by targeting the apoptosis signaling pathway molecules, MAP3K5 and MAP3K14.

CONCLUSION
In the present study, we evaluated gga-miR-20a-5p func-tions by delivery of exosomes into chicken macrophage cell lines. The expression of the candidate target genes, IFNGR2, MAPK1, MAP3K5, and MAP3K14, was repressed by exosomal miR-20a-5p. Moreover, the expression of ISGs and proinflammatory cytokines was downregulated by exosomal miR-20a-5p targeting IFNGR2 and MAPK signaling pathway molecules (MAPK1, MAP3K5, and MAP3K14). Cell proliferation was increased by the exosomal miR-20a-5p targeting the apoptosis pathway molecules (MAP3K5 and MAP3K14). However, although we identified repressed expression of miR-20a-5p target genes via a luciferase reporter assay and qRT-PCR, protein expression levels of the target genes were not measured because there are no commercially available antibodies against these chicken proteins. Moreover, additional studies such as evaluation of macrophage phenotypic changes and antiviral activity would be helpful in clarifying these results and conclusions, so continuous research on exosomal miR-20a-5p is needed. Taken together, the exosome-mediated delivery of gga-miR-20a-5p will help to maintain immune homeostasis against virus infections like HPAIV H5N1 by repressing the expression of proinflammatory cytokines and cell death.

AUTHOR CONTRIBUTIONS
YH and YHH conceived and designed the experiments. YH, THV, JH, and SK performed the experiments. YH analyzed the data. YH and YHH wrote the manuscript. HSL and YHH reviewed and revised the paper. All authors read and approved the final manuscript.

CONFLICT OF INTEREST
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.

FUNDING
This research was funded by a grant (NRF-2021R1A2C200 5236) from the National Research Foundation, Republic of Korea.