Analysis of deep sequencing exosome‐microRNA expression profile derived from CP‐II reveals potential role of gga‐miRNA‐451 in inflammation

Abstract Mycoplasma gallisepticum (MG) can cause chronic respiratory disease (CRD) in chickens. While several studies have reported the inflammatory functions of microRNAs during MG infection, the mechanism by which exosomal miRNAs regulate MG‐induced inflammation remains to be elucidated. The expression of exosome‐microRNA derived from MG‐infected chicken type II pneumocytes (CP‐II) was screened, and the target genes and function of differentially expressed miRNAs (DEGs) were predicted. To verify the role of exosomal gga‐miR‐451, Western blot, ELISA and RT‐qPCR were used in this study. The results showed that a total of 722 miRNAs were identified from the two exosomal small RNA (sRNA) libraries, and 30 miRNAs (9 up‐regulated and 21 down‐regulated) were significantly differentially expressed. The target miRNAs were significantly enriched in the treatment group, such as cell cycle, Toll‐like receptor signalling pathway and MAPK signalling pathway. The results have also confirmed that gga‐miR‐451‐absent exosomes derived from MG‐infected CP‐II cells increased inflammatory cytokine production in chicken fibroblast cells (DF‐1), and wild‐type CP‐II cell–derived exosomes displayed protective effects. Collectively, our work suggests that exosomes from MG‐infected CP‐II cells alter the dynamics of the DF‐1 cells, and may contribute to pathology of the MG infection via exosomal gga‐miR‐451 targeting YWHAZ involving in inflammation.

microRNAs (miRNAs) are one of the short endogenous single non-coding RNAs of 18-26 nucleotides (nt) in length. 5,6 Recent studies have shown that miRNAs are involved in the occurrence and development of inflammatory diseases. 7 Our previous studies have also revealed that miRNAs are associated with MG infection and that they were differentially expressed in chicken lungs at 3 days after infection with 36 down-regulated and 9 up-regulated miRNAs belonging to 31 miRNA families. 8 gga-miR-146c represses the expression of MMP16, activating TLR6/MyD88/NF-κB pathway for promoting cell proliferation to defend against MG infection. 9 The mature sequences of miR-451 contain 22 nt in length and are highly conserved pending evolvement. 10 Recent data clarified the role of miR-451 in the cell apoptosis, migration and invasion. miR-451 has also been reported to play a critical role in regulating a variety of human and animal diseases, such as influenza, cancer, cerebral ischaemia/reperfusion injury and vascular endothelial dysfunction. [11][12][13] It is worth mentioning that gga-miR-451 modulates the MG-induced production of inflammatory cytokines by targeting YWHAZ. 14 Moreover, our previous study also confirmed that AhR:Arnt binds to the gga-miR-451 promoter and facilitates gga-miR-451 transcription in MGinfected DF-1 cells. 15 Exosomes, approximately 30-150 nm, are the small membranous vesicles, released by many kinds of cells (including B lymphocytes, endothelial cells and epithelial cells) and body fluids.
They are known to represent the characteristics of the cells which secrete them. 16,17 The mode of intercellular communication which had achieved positive acknowledgement is mediated by exosomes.
In juxtacrine or ectodomain cleavage-based signalling, exosomes can regulate the non-selective transfer of exosomal proteins and/ or RNA to the target cell via fusing with the target cell. 17 In addition to cellular proteins, lipids and functional RNA molecules and other non-coding RNAs (miRNAs and lncRNAs) were also found in exosomes. Growing evidence indicates that exosome-transferred miRNAs are also biologically active via regulating the mRNA level in target cells after entering the target cells. 18,19 Exosomes-miRNAs secreted from the donor cells to the extracellular environment can modulate gene expression and cell function, playing a crucial role in several processes such as inflammation and immune response. 20,21 However, much of our previous understanding regarding miRNA-mediated effects on mechanisms of MG infection is based on the use of 'unpackaged' miRNAs, which regulate the original cells and tissues. The function of exosomal miRNAs derived from MG-infected chicken lung epithelial cells on both neighbouring and distal cells is still unknown. To expand on our knowledge, it is now critical to assess the effect of miRNAs in a biologically relevant setting in which they are present as exosomal cargo. The purpose of this study is to screen and annotate the differentially expressed exosomal miRNAs in MG-infected CP-II cells and to evaluate the function and potential mechanism of exosomal gga-miR-451 in DF-1 cells, which provides a valuable miR-451 as diagnostic markers and therapeutic targets in MG-infected chickens.

| Reagents
The primary antibody anti-YWHAZ was obtained from Sangon Biotech (Cat. D155211-0025), the primary antibodies anti-CD9 and anti-CD63 were purchased from Abcam (Cat. ab92726 and ab193349), and the primary antibody against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was acquired from Proteintech (Cat. 60004-1-1g). The secondary antibody horseradish peroxidase-conjugated antimouse/anti-rabbit was purchased from Sangon Biotech (Shanghai, China). The oligonucleotides of gga-miR-451 mimics and negative control (double-stranded chemically modified oligonucleotides) were synthesized by GenePharma (Shanghai, China). The sequences of all the primers and the sequences of RNA oligonucleotides used in the study are shown in Tables S1 and S2, respectively.

| CP-II isolation and DF-1 cell culture
CP-II cells were isolated as described previously, 22 with modifications.
Briefly, lungs were removed from 14-day-old White Leghorn specificpathogen-free (SPF) chicken embryos and were cut into tiny tissue blocks. Then, 0.25% trypsin and 0.1% IV collagenase (Invitrogen-Gibco) were used for digestion at 37°C for 15 and 20 minutes, respectively. Cell suspensions were filtrated by 200-mesh sieves, resuspended with 10% foetal bovine serum (FBS; Invitrogen) in a 150mm sterile culture plate and incubated for 1 hour. Supernatants with the unattached cells were then collected three times. The unattached cells were centrifuged at 1200 r/min for 5 minutes, resuspended in fresh Dulbecco's modified Eagle's medium (DMEM) for three times and filtrated by 400-mesh sieves. After the cell count was completed, cells were with 20% FBS and concentration was adjusted to 2 × 10 6 / mL and then inoculated into 150-mm sterile culture plate. Cells were incubated for 18 hours at 39°C. The attaching cells on culture dish were CP-II cells. Invert microscope and transmission electron microscope (TEM) were used to identify the cells.

| Mycoplasma strains
MG-HS, a virulent strain, was isolated from a chicken farm in Hubei, China, and conserved in the State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University (Wuhan, China). 23,24 The MG-HS strain was cultured, and the concentration was determined as previously described. The viable number of MG-HS in suspension was measured using a colourchanging unit (CCU) assay. 25

| Infection experiments
When CP-II cells in the experimental group reached 80-90% confluence, twelve 150-mm plates of the cells were incubated in medium without antibiotics. Six plates of the cells were infected with 2 mL/ plate of MG-HS at the mid-exponential phase (1 × 10 12 CCU/mL), while the other six plates of the cells were uninfected with MG-HS used as a control. At 12 hours after infection, the medium of the cells in both groups was changed to exosome-depleted media prepared by ultracentrifugation of FBS at 100 000 × g for 70 min. 26 The culture supernatant and the cells were collected at 12-60 hpi for further experiments.

| Exosome isolation and characterization
Sixty mL of CP-II cell culture supernatants in both groups was collected and immediately filtered through a 0.22-mm filter (Millipore, USA) and centrifuged immediately at 2000 × g for 10 minutes at 4°C. Cell culture exosomes were isolated and characterized as described previously. [27][28][29] Briefly, the supernatant was collected and then centrifuged at 10 000g for 40 minutes at 4°C. The resulting supernatant was transferred to a new ultracentrifuge tube and centrifuged at 100,000 × g for 2 hours at 4°C (Beckman Optima XE-90, SW32 Ti rotor). The supernatant was aspirated, and the pellet was suspended in pure PBS (HyClone, USA) and centrifuged at 100 000 × g for another 2 hours at 4°C. The purified exosomes were resuspended in 50 μL PBS and used for experimental procedures or stored at −80°C. For nanoparticle tracking analysis (NTA), exosomes were diluted with PBS over a range of concentrations to obtain between 10 and 100 particles per image before analysis. The ZetaView Nanoparticle Tracking Analyzer (Particle Metrix, Germany) was used to automatically measure the average diameter and concentration. For transmission electron microscopy, a 10 μL aliquot of the suspended exosomes was applied to a carbon-coated copper grid.
Then, the sample was negatively stained with 2% uranyl acetate after drying. Micrographs were obtained under a HITACHI H-7650 transmission electron microscope (HITACHI, Japan).

| Exosome labelling
Exosomes secreted by CP-II cells were labelled with a PKH67 green fluorescent labelling kit (Sigma-Aldrich, MINI67, MO, USA) to detect the uptake of exosomes by DF-1 cells in vitro. Labelled exosomes were incubated with DF-1 cells at 39°C for 6 hours and then fixed.
Fluorescent images were taken with a confocal laser scanning microscope (Zeiss LSM 800 META, Carl Zeiss Imaging, Germany).

| Exosome sRNA sequencing and data processing
Exosomes were isolated from samples and characterized as described above. Total RNA from exosomes in both groups (n = 3) was used for sRNA sequencing. Library preparation and sRNA sequencing were performed by RiboBio (Guangzhou, China). In brief, total RNA samples were fractionated and only small RNAs ranging from 18 to 40 nts were used for library preparation. After amplification by 15-cycle PCR, the libraries were sequenced using the Illumina HiSeq TM 2500 platform. Clean reads were collected from raw reads by removing the adapter dimers, glow quality and contaminated reads. Then, clean reads of sRNA were mapped to the chicken (G gallus) genome by BWA 0.7.12. 30 Mapped sRNA tags were used to look for known sRNA, and miRBase version 21.0 (www.mirba se.org), Rfam 12.1 (rfam.xfam.org) and piRNA-Bank (pirna bank.ibab.ac.in) were used for reference. The sequencing data were deposited at NCBI SRA under accession number PRJNA606684.  Table S1.

| Western blotting
The total proteins were extracted from exosomes or DF-1 cells at 48 hours after transfection or exosome-treated, and their concentrations were then determined using a BCA Protein Assay Kit (Transgen, China). Equal amounts of protein (5 μg of exosomal proteins and 50 μg of cell proteins) were transferred to nitrocellulose membranes after separation using 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Beyotime, China) and blocked in 5% (w/v) skimmed milk at 25 ± 1°C for 1 hours, followed by incubation overnight at 4°C with the previous antibodies (anti-CD9, CD63, 14-3-3ζ or GAPDH). After washing with TBST, the membranes were incubated with the secondary antibodies at room temperature for 1.5 hours. After another wash with TBST, the protein expressions on the membranes were detected using an ECL TM detection system (Bio-Rad).

| ELISA
After exosome treatment or transfection with siRNA in 6-well plates, the DF-1 cells were incubated for 36 hours; then, the culture supernatants were collected from the treated cells for determination of the inflammatory factor levels, including TNF-α and IL-1β. All experiments were performed according to the manufacturers' protocols of the enzyme-linked immunosorbent assay (ELISA) kits (Bio-Swamp).

| Statistical analysis
Data were presented as the mean ± SEM and analysed by SPSS 20.0 software for statistical analysis. ANOVA or Student's t test was used to determine significant differences between groups. A value of P < .05 was considered statistically significant and P < .01 considered extremely significant. (*P ＜ .05 and **P ＜ .01; different lowercase letters represent P < .01).

| CP-II cell-derived exosomes were isolated and identified in morphology and phenotype
The CP-II cells shaped monolayers when adherent cultured for 18 hours, were small and cycloid and grouped together to form islands ( Figure 1A). The morphology of CP-II cells was observed by  Figure 1D).
The exosomal surface markers [cluster of differentiation 9 (CD9) and CD63] could be detected using Western blotting ( Figure 1E). There was difference in the MG-infected and non-infected CP-II cells, and the group of MG infection is more consistent with previous reports on exosomes. 33 These results demonstrate that CP-II cell-derived particles collected in this experiment were identified as exosomes.

| Sequence analysis
To identify exosomal miRNAs and their expression levels from MGinfected and non-infected CP-II cells, two small RNA libraries were constructed for deep sequencing. The total reads reached more than thirteen million, and approximately 90.16% clean reads are listed in

| Differentially expressed miRNAs
A total of 722 mature chicken miRNAs were identified from the two exosomal sRNA libraries, and 279 novel miRNAs were discovered using miRDeep2 software (Table S4 and Figure S1). As shown in Figure (Table S5).
Among these differential expressed miRNAs, only 30 miRNAs (9 up-regulated and 21 down-regulated) were significantly differentially expressed (P ＜ .05) and 17 miRNAs (3 up-regulated and 14 down-regulated) were highly significantly different (P ＜ .01) in the MG-infected vs. non-infected groups ( Figure 2C). The details of the differentially expressed chicken miRNAs and their log2 (fold change) and P-value were shown in Table 1.

| Functional annotation and enrichment analysis of predicted target genes of the differentially expressed miRNAs
To further understand the biological function of miRNAs in CP-II cell-derived exosomes, a target prediction of miRNAs with log 2 (fold change) ≥ 1 and P ＜ 0.05 was performed with miRanda, Pita and RNAhybrid (Table S6)

| Exosomal miRNA interaction network for elements involved in cell cycle, apoptosis and Toll-like receptor signals
Our previous studies have shown that cell cycle, apoptosis and Tolllike receptor signalling pathways are activated and play an important role in the inflammatory response to MG infection. 14, 34 We found

| RT-qPCR verification
To validate the reliability of the sequencing results, a total of 8 dif-

| gga-miR-451-absent exosomes derived from MG-infected CP-II cells increase inflammatory cytokine production in DF-1 cells
Previous data showed that gga-miR-451 expression is significantly Exo-MG as compared with the control group ( Figure 6C). Recently, we reported that gga-miR-451 decreases the inflammatory cytokine production, including TNF-α and IL-1β. 14 We hypothesized that CP-II  Figure 7A,B). These data suggest that exosomes derived from CP-II cells may deliver less gga-miR-451 to DF-1 cells to induce inflammatory cytokines by targeting YWHAZ.

| D ISCUSS I ON
Mycoplasma provokes rock-ribbed inflammation in organisms, resulting in the production of various pro-inflammatory cytokines in many different cell types. 35 MG is well known as a common pathogen that can cause chronic respiratory inflammation in poultry worldwide.
After MG-HS enters the chicken's body, it adheres to the respira-  process', 'metabolic process regulation of response to stimulus' and 'biological regulation'). The KEGG pathways are widely used to predict the gene function and genome information, which is helpful for us to study the metabolism function, genetic information processing, cellular processes and diseases. 45 The target genes of DEGs in this study were plotted to the reference pathways in KEGG, the most Our results showed that the concentration of exosomes derived from MG-infected CP-II cells was increased as non-infected cells ( Figure 1E; Table S3), which was similar to the results of previous reports in other diseases. 33,47 Moreover, respiratory secretions of patients with cystic fibrosis are often found with protease-rich exosomes that are produced by gram-negative bacteria that colonize F I G U R E 4 Network of miRNA target pathways involved in cell cycle, apoptosis and Toll-like receptor signalling pathway for differential expressed miRNAs. The pink represents the pathways, green represents the genes, the red represents the up-regulated miRNAs, and the blue represents the down-regulated miRNAs

F I G U R E 5
The validation of the selected exosome-microRNA expression profile approach by using RT-qPCR. 5s RNA was used as the internal control.
Data represent means ± SDs of three independent experiments (two-tailed Student's t test, **P < .01, *P < .05) the airways. LPS stimulation of respiratory epithelial cells also could lead to release of exosomes in human. 48   Exo-NC ( Figure 6D,E). It is reported that exosomes, like vesicles released by adipose tissue of obese mice, are taken up by mononuclear cells inducing their differentiation into active macrophages with an increased release of TNF-α and IL-6. 49 The blood microparticle treatment led to remarkable increases in myeloperoxidase (MPO), TNF-α, IL-1β and IL-10 productions in bronchoalveolar lavage fluid and plasma. 50 Interestingly, the Exo-NC decreased TNF-α and IL-1β expressions compared to Blank ( Figure 6D,E); it may be that gga-miR-451 in exosomes had negative effect on TNF-α and IL-1β protein levels. We confirmed previously that gga-miR-451 expression significantly decreased TNF-α and IL-1β secretions in DF-1 cells. 14 These were consistent with results in this study. It is reasonable to believe that gga-miR-451-absent exosomes derived from MGinfected CP-II cells increase inflammatory cytokine production, including TNF-α and IL-1β.
Studies on YWHAZ protein (14-3-3ζ) in multiple systems have found that it is a target gene and is negatively regulated by miR-451 in mammals and chickens. 14, 51 We also found that gga-miR-451-absent exosomes can regulate the expression of YWHAZ protein in DF-1 cells (Figure 7). Our previous data showed that YWHAZ protein in the miR-451-NC group is no significantly different from the mock transfection group (Blank). 14 These results suggest that the exosomes derived from CP-II cells may deliver less gga-miR-451 to DF-1 cells to induce inflammatory cytokines by targeting YWHAZ and increasing its expression. In addition to gga-miR-451, other exosomal gga-miRNAs may also regulate the immunity processes in MG infection. For example, exosomal miR-223 targets the transcription factor STAT5 and is implicated in inflammatory reactions in multiple sclerosis. 52 The serum exosome-derived miR-193 was reported to be a potential Alzheimer's disease biomarker. 53 However, the molecular mechanism of these exosomal miRNAs that shuttle and regulate chronic respiratory inflammation needs to be further studied.
The present study provides evidence to prove that the mode of intercellular communication between chicken cells is mediated

| E THI C S APPROVAL AND CON S ENT TO PARTI CIPATE
Our experimental protocols for chicken embryo treatment were approved by the Institutional Animal Care and Use Committee of Huazhong Agricultural University. The procedures were carried out in accordance with the approved guidelines.

ACK N OWLED G EM ENTS
This study was supported by the National Natural Science

CO N FLI C T O F I NTE R E S T
All authors listed declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this published article [and its supplementary information files].