Exosomal miR-181a-5p Protects against MG Infection by Targeting PPM1B and Activating the TLR2-Mediated MyD88/NF-κB Pathway

Yingfei Sun Huazhong Agricultural University: Huazhong Agriculture University Yabo Zhao Huazhong Agricultural University: Huazhong Agriculture University Huiwen Pang Huazhong Agricultural University: Huazhong Agriculture University Mengyun Zou Huazhong Agricutural University Yingjie Wang Huazhong Agricutural University Xiuli Peng (  xiulipeng918@163.com ) Huazhong Agriculture University https://orcid.org/0000-0001-5210-6999


Introduction
Mycoplasma gallisepticum (MG) is the primary etiologic agent of chronic respiratory disease (CRD) in poultry and is known to invade, survive and multiply inside a variety of non-phagocytic cells such as chicken red blood cells, HeLa cells, and chicken broblasts [1][2][3]. MG has been proven to colonize its host mainly via the mucosal surfaces of the respiratory tract, causing air sacculates within a few days, and disseminates throughout the body [4]. Once avian is infected with MG, it is di cult to eradicate the effects on cells adhesion, metastasis, proliferation and differentiation in whole body as its long incubation period and strong infectivity [5]. Therefore, CRD induced by MG causes millions of economic losses to the poultry industry every year. MicroRNAs (miRNAs), as 19-22 nucleotide non-coding RNAs, involve in multiple important biological processes by binding partially complementary sequences of its target gene mRNA 3'-untanslated region (3'-UTR) and repressing target gene functions. Many studies have demonstrated that miRNAs play important roles in resisting kinds of disease in birds, like Marek's disease virus, avian in uenza virus, bursal disease virus and others virus infection [6][7][8]. Our previous studies have also highlighted the critical role of miRNAs during MG-HS (Mycoplasma gallisepticum-Huangshi) infection in lung tissue of chicken embryo, including miR-19a, miR-130b-3p, miR-451 ect [9][10][11][12][13]. miR-181a-5p belongs to the miR-181 family, and its nucleic acid sequence is highly conserved among species [14]. Several studies suggest that miR-181a differentially expressed in a variety of poultry diseases, such as avian in uenza [15,16], and Marek's disease [17,18], which indicates miRNA-181a may play an important role among various pathogenic microorganism infection. More interesting, our previous miRNAs deep sequencing showed miR-181a-5p was increased in the lungs of MG-infected chicken embryo [19], indicating its potential signi cant role in MG infection. Mg 2 + / Mn 2 + -dependent protein phosphatase 1B (PPM1B or PP2C) is a member of the Ser/Thr protein phosphatase (PP2C) family [20,21]. PPM1B regulates various cellular functions by dephosphorylating several substrates proteins, including TAK1 (also known as MAP3K7), PPARγ, p53, and IKKβ (also known as IKBKB), there by terminating IKKβ-mediated NF-κB activation [22,23]. Ectopic expression of PPM1B signi cantly suppressed proliferation and tumorigenicity in bladder cancer cells in vitro and in vivo [24]. These studies indicate that PPM1B plays a potential role in regulating various signaling pathways and cellular functions by affecting many downstream targets.
Exosomes are the small membranous vesicles (30-150 nm) released by various of cells, including B lymphocytes, endothelial cells, epithelial cells, and body uids. Increasing number of evidences indicate that exosomes play an important role in mediating cell-cell communication. Cell-derived exosomes cargo contains a variety of miRNAs, proteins, mRNAs and DNA, which can work locally or be stably transferred to recipient cells [25,26]. Among them, miRNA is the most investigated and has been shown to be involved in many physiological and pathological processes [27,28]. These ndings prompted us to investigate whether miR-181a-5p could be loaded and transferred by exosomes and its role in immune responses to MG both in vivo and in vitro.
In this study, we found that miR-181a-5p expression was signi cantly upregulated in MG-HS-infected chicken embryonic lungs and DF-1 cells. Exosomal miR-181a-5p derived from CP-II cells could be absorbed by DF-1 cells. And then the correlation between miR-181a-5p expression and MG infection progression was analyzed. The results demonstrated that miR-181a-5p played an important role in cell proliferation, cell cycle progression, and regulation of apoptosis in MG infection by targeting PPM1B. Furthermore, up-regulated miR-181a-5p could promote in ammatory response by down-regulating the expression of PPM1B via activation of the TLR2-mediated MyD88/NF-κB pathway to resist to MG infection.

miR-181a-5p Expression in Vivo and in Vitro Is Signi cantly Elevated after MG Infection
Our previous miRNAs deep sequencing showed miR-181a-5p expression was increased in the lungs of MGinfected chicken embryos [19]. To verify this result, we evaluated miR-181a-5p expression variation after MG infection in both chicken embryos and DF-1 cells. Chicken embryos were infected with MG on the 9th day of incubation (the eggs were hatched for a total of 21 days). On days 6 to 8 after infection (equivalent to days 15-17 of egg hatching), compared with non-infected groups, miR-181a-5p levels were increased by greater than 2-fold in MG-infected chicken embryos (p < 0.01; Figure 1A). Furthermore, the average expression level of miR-181a-5p in MG-infected DF-1 cells increased more than 5-fold compared to uninfected cells ( Figure 1B). These results indicates that miR-181a-5p was signi cantly increased in MG-infected chicken embryos and DF-1 cells and may have an important potential role in MG infection.

Exosomes
Derived from CP-II Cells Load and Transfer miR-181a-5p into DF-1 Cells To investigate whether miR-181a-5p could mediate cell-cell communication through exosomes in MG infection, we rst collected the conditioned medium of CP-II cells at 48 hours in vitro culture. Differential centrifugation was adopted to separate the exosomes from the CP-II cells medium. Exosomes were identi ed as rounded particles with approximately 80-100 nm size and a double-layer membrane by transmission electron Microscopy (Figure 2A). CD63 and CD9, the surface markers of exosome, were con rmed by western blot ( Figure 2B). These results indicate that exosomes secreted by CP-II cells isolated successfully.
We examined the expression levels of miR-181a-5p in MG-infected CP-II cells and its derived exosomes. Compared with normal cells, the expression level of miR-181a-5p in infected CP-II cells was signi cantly increased ( Figure 2C). Similarly, miR-181a-5p level was signi cant higher in exosomes isolated from MGinfected CP-II cells relative to control group ( Figure 2D).
To further determine whether exosomes with miR-181a-5p transfers from CP-II cells into the recipient DF-1 cells. Firstly, the primary CP-II cells were transfected with 500 ng miR-181a-5p mimics plate labeled with Cy3 red uorescence (Cy3 -miR-181a-5p) for 48h at 75 cm 2 ask, and then cell culture medium was used to extract exosomes. Secondly, the extracted exosomes were labeled by PKH67 (a green uorescence) and incubated at DF-1 cells for 48h at 37 °C. Both Cy3 uorescence and PKH67 lipid dye were observed in the incubated DF-1 cells ( Figure 2E). The above results indicate that exosomes derived from CP-II cells can package miR-181a-5p and transferred it into DF-1 cells.
2.3. miR-181a-5p Directly Targetes PPM1B in Recipient DF-1 Cells As above, we have identi ed that miR-181-5p could be absorbed by recipient DF-1 cells. To investigate the potential regulatory mechanism of miR-181a-5p in MG-HS infection, a combination of three algorithms, including Targetscan, miRDB, and miRTarBase were used. As a result, PPM1B was identi ed as a target gene of miR-181a-5p by all programs. In addition, Targetscan showed that the sequence of target sites in the 3'-UTR of PPM1B is highly conserved across species ( Figure 3A). RNAhybrid revealed that the minimum free energy (MFE) of the RNA duplex was -22.6 kcal/mol ( Figure 3B), suggesting a stable combination between miR-181a-5p and PPM1B. The expression of PPM1B in chicken embryos and DF-1 cells was then examined. On days 6 to 8 after infection (equivalent to days 15-17 of egg hatching), compared with normal solid tissues, PPM1B levels were signi cantly decreased in MG-infected chicken embryos ( Figure 3C). Furthermore, the expression level of PPM1B in MG-infected DF-1 cells was decreased more than 1.5-fold compared to uninfected cells ( Figure 3D). Western blot also showed that PPM1B was down-regulated after MG infection. This result shows contrary expression pattern compared with miR-181a-5p and indicates that PPM1B may be negatively related to miR-181a-5p expression after MG infection both in vivo and in vitro.
In order to verify whether PPM1B was the target gene of miR-181a-5p, the luciferase reporter gene plasmid containing the potential binding site for miR-181a-5p in 3'-UTR was constructed ( Figure 4A). The wild-type or mutant 3'-UTR luciferase constructs was transfected into DF-1 cells and treated with miR-181a-5p mimics or mimic NC. We found that transfection of the miR-181a-5p mimics decreased the luciferase activity of Luc-PPM1B 3'-UTR and transfection of miR-181a-5p inhibitor increased the luciferase activity. However, there was no effect on the mutant 3'-UTR type ( Figure 4B). Collectively, miR-181a-5p could directly bind to the nucleotide sequence in the 3 -UTR of PPM1B.
To further investigate whether PPM1B was regulated by miR-181a-5p, miR-181a-5p mimics and mimics-NC or inhibitor and inhibitor-NC were transiently transfected into DF-1 cells for 48 h. when miR-181a-5p expression was increased ( Figure 5A), PPM1B expression was signi cantly down-regulated at both mRNA and protein levels ( Figure 5B and 5C). On the contrary, when miR-181a-5p expression was decreased ( Figure 5D), the expression of PPM1B was signi cantly up-regulated at both mRNA and protein levels ( Figure 5E and 5F).
Taken together, all of the above results indicates that PPM1B is a direct target of miR-181a-5p and its expression is negatively regulated by miR-181a-5p.

NF-κB Signaling Pathway Activated by miR-181a-5p Overexpression
Bioinformatics prediction using Gene Ontology (GO),the Kyoto Gene and Genomic Encyclopedia (KEGG) database indicated that miR-181a-5p was involved in the NF-κB pathway. miR-181a-5p mimics or inhibitor was transiently transfected into DF-1 cells for 48 h, and qPCR evaluated the expression level of MAP3K7, IKBKB and NF-κB at the mRNA level. After transfection of miR-181a-5p mimics, the expression of MAP3K7, IKBKB, NF-κB were signi cantly up-regulated comparing to the NC group ( Figure 6A). In contrast, the expression of MAP3K7, IKBKB and NF-κB were signi cantly down-regulated after transfection with miR-181a-5p inhibitor ( Figure 6B). MAPK and IKBKB proteins further play a regulatory role through phosphorylation [29]. And we found that overexpression of miR-181a-5p induceed phosphorylation of MAP3K7 and IKBKB proteins comparing to the NC group ( Figure 6C). In contrast, p-MAP3K7 and p-IKBKB were high manifest upon knockdown of miR-181a-5p by western blot ( Figure 6D). To further investigate whether NF-κB was activated by miR-181a-5p overexpression, the NF-κB p65 expression was analyzed by an immuno uorescence. When DF-1 cells were transfected with the miR-181a-5p mimics, p65 (red) protein was observed to enter the nucleus from the cytosol, whereas the other groups had no clear change ( Figure 6E). Taken together, our results shows that up-regulation of miR-181a-5p signi cantly promotes phosphorylation of MAP3K7 and IKBKB to activate the NF-κB signaling pathways.

miR-181a-5p Activates NF-κB Signaling Pathway by Directly Inhibiting PPM1B
To further investigate whether miR-181a-5p actually activates the NF-κB signaling pathway via PPM1B, we constructed over-expression plasmid of PPM1B or siRNA directed against PPM1B, the e ciencies of which were examined by qPCR. Firstly, co-transfection of overexpressed PPM1B vector and miR-181a-5p-silenced into DF-1 cells for 48 hours, qPCR analysis demonstrated an 70% up-regulation of PPM1B mRNA level in the presence of overexpressing PPM1B vector compared with cells transfected with pcDNA3.1 vector. Interestingly, when co-transfection miR-181a-5p-Inh, PPM1B expression was further signi cantly increased ( Figure 7A). However, overexpression of PPM1B inhibited the expression of MAP3K7, IKBKB and NF-κB, and when cotransfection with miR-181a-5p-Inh, their expressions were signi cantly lower ( Figure 7A). The western blot assay showed the similar results at phosphorylation of MAP3K7 and IKBKB proteins ( Figure 7B).
In contrast, co-transfection of PPM1B siRNA and miR-181a-5p mimics enhanced down-regulation of PPM1B expression by PPM1B siRNA alone and increased MAP3K7, IKBKB and NF-κB expression as compared with cells transfected with PPM1B siRNA alone ( Figure 7C and D). These results indicates that miR-181a-5p mimics and PPM1B siRNA has synergistic effects on MAP3K7, IKBKB and NF-κB expression, and overexpression of miR-181a-5p could promote the activation of NF-κB pathway by directly inhibiting PPM1B.

miR-181a-5p Promotes Cell Proliferation and Inhibites Cell Apoptosis to Resist MG Infection
The NF-κB signaling pathway has been widely demonstrated to regulate various cellular processes, such as involvement in the regulation of cell proliferation and apoptosis. Therefore, we further investigated the effects of miR-181a-5p on cell proliferation, cell cycle and apoptosis.
DF-1 cells were infected with MG or not, after 6h of transfection with miR-181a-5p mimics, inhibitor or NC, for 24, 48 and 72h to detect cell proliferation. Detailed grouping as shown in Figure 9. Cell viability was measured by Cell Counting Kit-8 (CCK8) assay. The results showed that MG infection signi cantly decreased cell viability at infection 48 and 72h. Upregulated miR-181a-5p could signi cantly enhance cell viability compared with NC groups ( Figure 9A). On the contrary, depressed miR-181a-5p could signi cantly reduce cell viability at MG infection 48 and 72h ( Figure 9B).
In order to validate the protective effects of miR-181a-5p against MG infection in DF-1 cells, cell cycle and cell apoptosis were further checked through ow cytometry. Detailed grouping as shown in Figure 10, 11. We found that MG infection signi cantly inhibited cell mitosis by inducing G1 cell cycle arrested in DF-1 cells. Overexpression of miR-181a-5p could signi cantly reduce the percentage of cells in the G1 phase, while the percentage of cells in the S and G2 phases was signi cantly increased, which is opposite to depressed miR-181a-5p ( Figure 10).
Apoptosis experiments showed that MG infection signi cantly stimulated apoptosis of DF-1 cells (Figure 11). Apoptotic cell amount was signi cantly increased after MG infection. More importantly, at MG-infected DF-1 cell, overexpression of miR-181a-5p could reduce the apoptotic DF-1 cells amount (Q2 and Q3) compared to control groups, while the opposite was observed when miR-181a-5p was signi cantly decreased.
Taken together, above data suggestes that miR-181a-5p could promote cell proliferation and inhibit cell apoptosis to resist MG infection.
2.8. miR-181a-5p Depresses pMAG1.2 Expression by Directly Inhibiting PPM1B Finally, to verify whether miR-181a-5p resisted MG infection by directly targeting PPM1B. pMAG1.2 expression variation was tested by co-transformation trials and qPCR.pMAG1.2 is a major adhesion protein gene of MG, which is required for MG infection. PPM1B siRNA or/and miR-181a-5p mimics was/were (co-)transfected into DF-1 cells for 24 hours, which were then infected with the 8 μL of MG strain. We found that overexpression of miR-181a-5p could signi cantly down-regulate pMAG1.2 expression. Knockdown of PPM1B alone also extremely depressed the expression of pMAG1.2 and further signi cantly decreased pMAG1.2 expression, compared with overexpression of miR-181a-5p alone. While knockdown PPM1B along with overexpression of miR-181a-5p showed the same inhibitory effect on pMAG1.2 expression, as knockdown PPM1B alone ( Figure  12), which indicated that PPM1B played key role in depressing pMAG1.2 expression during MG infection and miR-181a-5p depressed pMAG1.2 expression by directly inhibiting PPM1B.

Discussion
MG is the main pathogen of chronic respiratory diseases in chicken, it can adhere to respiratory tract mucous membrane, and cause in ammation and the damage of respiratory tract tissue. MG-HS strain is a virulent strain with markedly pathogenicity. The adhesin protein pMGA1.2, a crucial adhesin on the surface of MG-HS strain, is responsible for MG-HS to propagate in animal tissues through binding to apolipoprotein A-I (ApoA-I) on host [30]. Many researches have shown that miRNAs play an important role in the pathogenesis of various diseases. And in recent years, a raising number of studies indicates exosomal miRNAs exert important regulatory effects on recipient cells, suggesting that the exosomal transfer of miRNAs could be a novel mechanism for intercellular communication [27,31].
Our previous study had indicated that miR-181a-5p expression was signi cantly up-regulated in the lungs of MG-infected chicken embryos [19]. Therefore, we rstly veri ed its expression veri cation in vivo and in vitro. The upregulated miR-181a-5p result implied its potential role in MG infection. Recently, the function of miR-181a-5p in immune system was also unveiled in many researches. The down-regulated expression of miR-181a-5p in non-small cell lung cancer is directly related to the low survival rate of patients, suggesting that miR-181a may be a diagnostic biomarker for non-small cell lung cancer [32]. miR-181a-5p expression was upregulated in the lung tissues of LPS-challenged mice to regulate LPS-induced apoptosis in A549 cells and in vivo by targeting Bcl-2 [33]. Meanwhile, up-regulated serum miR-181a is found to be associated with the early pathogenic process of chronic obstructive pulmonary disease in asymptomatic heavy smokers [34]. miR-181a-5p is also reported to inhibit non-small cell lung cancer A549 cells proliferation and migration by targeting KRAS [35]. All these studies suggest and consolidate that miR-181a-5p plays important regulatory role in diseases and host infection.
MG infection is systemic and throughout whole body. Once MG enter the body, almost all tissues and organs, especially lung tissues will be infected. Exosomes are well documented to be enriched with miRNAs and able to deliver miRNA from host cells to target cells, and can further regulate the function of recipient cells throughout the body. Previous study shows that miR-181-5p of exosome suppresses hepatic stellate cells activation and induces autophagy activation through direct targeting Bcl-2 and STAT3 [36]. Exosomal miR-124 derived from M2 microglia can be delivered to neurons and attenuates ischemic brain injury, consequently promotes neuronal survival [37]. In this study, we found that Cy3 uorescence could be detected in DF-1 cells incubated with exosomes derived from CP-II cells which before were transfected with Cy3-labeled miR-181a-5p. This result uncovered that exosomes derived from MG-infected CP-II cells could load and delivery miR-181a-5p into recipient DF-1 Cells. miRANs play their regulatory role in kinds of biological process is through the regulation by repressing the translation of downstream target mRNAs into protein. PPM1B is a member of the metal-dependent serine/threonine protein phosphatase (PPM) family with important regulatory functions in cellular signaling pathways [38]. Further, we identi ed PPM1B was the target gene of miR-181a-5p. One common functional feature of PPM family members is their involvement in the cellular stress response. According to the report, PP2Cβ negatively regulates the TAK1 pathways by dephosphorylating and inactivating TAK1 [22]. TAK1 is a key component required for cytokine-induced IKK activation [39]. And IKKβ knock-out mice con rms that IKKβ is the dominant kinase in regulating NF-κB activity [40]. PP2Cβ negatively regulates the NF-κB pathway post-TNFα treatment by dephosphorylating IKKβ and thus reducing its kinase activity [23]. In our study, we found the phosphorylated MAP3K7 and IKBKB were signi cantly induced through PPM1B directly depressed by miR-181a-5p, which indicated that PPM1B played an important role in MG infection. NF-κB is a general nuclear transcription factor consisting of two glutenin subunits (p65 and p50). More interesting, we found p65 subunit protein was expressed from the cytoplasm to the nucleus when overexpressed miR-181a-5p in MG infection, indicating the NF-κB pathway being activated. Therefore, we further investigated the underlying mechanism of NF-κB activation. And we found that MG highly increased the expression of TLR-2, MYD88, TNF-α and IL-1β in DF-1 cells. Overexpression or knockdown of miR-181a-5p resulted in an up-regulation or down-regulation in the expression of TLR-2, MYD88 and pro-in ammatory cytokines (IL-1β and TNF-α). NF-κB acts at the center of the in ammatory response and controls the gene expression of numerous in ammation-associated substances, including IL-1β, IL-6, IL-8, and TNF-α, which induce a cascade of in ammatory responses and related lung damage [41][42][43][44]. Several researches reported that MG activates IL-1β production through the NF-κB pathway via TLR2 and MyD88. And up-regulate in ammatory genes in chicken tracheal epithelial cells via TLR-2 ligation through an NF-κB Dependent Pathway [45,46]. Our previous research also indicated that MG infection stimulated the IL2/IL6-mediated in ammatory responses through TLR6-MyD88-NF-κB pathway [47].
The NF-κB pathway involves various cellular processes, such as involvement in the regulation of cell proliferation and apoptosis. Last, we found that miR-181a-5p promoted MG-infected DF-1 cell proliferation, accelerated cell cycle transition, and inhibited apoptosis through down-regulation of PPM1B. Other studies also suggested that upregualated miR-181a promoted cell proliferation and G1/S transition, and suppressed apoptosis in gastric cancer cell lines [48]. Overexpression of miR-181a promoted proliferation and G1/S transition apoptosis in ccRCC 786-O and 769-P cells, and inhibited their apoptosis [49]. In addition, miR-181a is also reported to enhanced G1/S transition and cell proliferation in pediatric acute myeloid leukemia by regulating EGR1 expression [50]. And miR-181a is identi ed to increase cell proliferation and inhibits apoptosis through activating the AKT pathway in keloid broblasts. Moreover, PPM1B often negatively regulates cell reaction pathways, such as regulating stress response, cell cycle, cell proliferation and apoptosis [24,51]. Correctly, we highly believe miR-181a-5p directly targets PPM1B and further activate NF-κB pathway to against MG infection in DF-1 cells.
In summary, we found that miR-181a-5p expression was up-regulated after MG infection in the lungs of chicken embryos and DF-1 cells. miR-181a-5p could be packaged in exosome derived from MG-infected CP-II cells and transferred into recipient DF-1 cells to further resist MG infection through its target gene PPM1B, activating TLR2-mediated MyD88/NF-κB pathway and depressing pMAG1.2 expression (Figure 13). Therefore our study suggests the TLR2-mediated MyD88/NF-κB pathway may be the therapeutic target for protecting against MG-induced CRD diseases.

MG Culture
The MG virulent strain used in this study was MG-HS strain isolated from henneries in Hubei, China [52] and was donated by the State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University (Wuhan, China). MG-HS was cultured at 37 ℃ in modi ed FM-4 medium supplemented with 12% (v/v) porcine serum and 10% yeast extract until the mid-log phase. The concentration of MG-HS was determined by the acid-mediated shift of phenol red dye from red to orange as previously described. The number of viable Mycoplasmas in a suspension was then determined by a color-changing unit (CCU) assay.

Infection Experiments
One hundred embryos of White Leghorn speci c-pathogen-free (SPF) chickens were incubated on the ninth day and the all were injected with 300 µL of MG-HS at 10 CCU/mL. Other 100 chicken embryos were injected with the same dosage of the diluent to serve as controls. The viability of the chicken embryos was examined by eye under a candling machine. The dead embryos were eliminated. The mortality rates of the chicken embryos of the infection and control groups were 12.3% and 7%, respectively. Whole-lung tissue samples from six infected live chicken embryos and six controls were collected on days 6, 10, and 11 post-infection and stored in RNA xer (BioTeke Co., Ltd., Beijing, China).

Cell Culture and Treatment
The chicken embryonic broblast cell line (DF-1) was obtained and authenticated from American Type Culture Collection (Rockville, MD, USA). Cells were maintained in Dulbecco's modi ed eagle medium (DMEM, Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS, Invitrogen, USA) and 1% penicillin-streptavidinglutamine (PSG, Invitrogen, USA). Cells were incubated at 39 °C in a 5% CO 2 humidi ed atmosphere. For transient transfection, DF-1 cells were plated evenly in 6-, 24-or 96-well plates and grown to 60% con uency without antibiotics and subsequently transfected with RNAs and/or plasmids using Lipofectamine 3000 (Invitrogen, USA). After 48 h, the cells in different groups were collected for further use. For MG-HS infection experiments, at 24 h post-transfection, cells were infected with MG-HS at the log phase (1 × 1010 CCU/ml) for the times mentioned in the gure legends.

microRNA Target Prediction and Sequences
Putative miR-181a-5p target genes were identi ed using a miRNA database (http://www.mirbase.org/) and target prediction tools: miRDB (http://www.mirdb.org/miRDB/), PicTar (http://pictar.mdc-berlin.de/), and TargetScan (http://www.targetscan.org/). The conservation of the target gene was analyzed by TargetScan (http://www.targetscan.org/). The duplex and mfe between miR-181a-5p and the 3'-UTR of the potential targets were analyzed by RNAhybrid (https://bibiserv.cebitec.unibielefeld.de/rnahybrid/). AmiGO (http://amigo.geneontology.org) was used to analyze the functions of the target genes of gga-miR-181a-5p in Gallus gallus.The sequences of all of the primers used in this study are shown in Table 1. All RNA oligonucleotides were designed and synthesized by GenePharm (Shanghai, China) and are shown in Table 2.  [53]. Brie y, it was prepared from lung tissue of 15 day SPF chicken embryos. The lung tissue was cut, washed three times with Hank's solution; then, 0.25% trypsin was added, each embryo was added in an amount of 1 ml, digested in a water bath at 37 °C for 10 minutes, centrifuged at 800 rpm/min for 10 minutes, and the supernatant was discarded; then, 0.1% IV collagenase was added, each embryo was added in an amount of 1 ml, digested in a water bath at 37 °C for 15 min, centrifuged at 800 rpm/min for 10 minutes, the supernatant was discarded, and a moderate volume of 10% FBS in DMEM complete medium was gently pipetted. The cells were mixed and ltered through a 75 μm mesh to a sterile plate for adherence. The unattached suspension was centrifuged at 1200 rpm/min for 5 min, resuspended and centrifuged, and repeated 3 times. After resuspending the cells in DMEM complete medium supplemented with 20% FBS, the cells were ltered through a 38 μm sieve into a culture ask, and the cells were cultured in a humidi ed incubator at 37 °C, 5% CO 2 for 18 hours, and then the culture solution was changed. At this time, the adherent cells are chicken embryo type II epithelial cells.

Exosome isolation, identi cation and labeling
Exosomes were puri ed from the cell culture supernatant of CP-II cells. Prior to culture medium collection, CP-II cells were washed twice with PBS, and the medium was switched to exosome-free medium (ultracentrifugation at 100,000X g for 16 h at 4 ℃) upon MG stimulation. The cells were then cultured for 48 h. The supernatant was collected and went through sequential ultracentrifugation at 2000 X g for 30 min, 10,000X g for 30 min, and 100,000 X g for 70 min at 4 ℃. The exosomes were washed once with PBS at 100,000X g for 70 min and suspended for further characterization.
A transmission electron microscope (TEM, Thermo Scienti c, Waltham, MA) was used to identify the form of the exosomes. Nanoparticle tracking analysis (NTA, Brookhaven, New York) was used to measure exosome diameter and particle number. The protein content was measured using BCA protein assay (Thermo Scienti c, Waltham, MA), and exosome markers CD9 and CD63 were detected by western blot analysis.
Fluorescence labeling of exosomes was performed according to the protocol previously described [54]. The PKH26 kit was used according to the instruction manual (Sigma-Aldrich, San Louis, MO). The labeled exosomes were washed at 100,000 g for 1 h, and the exosome pellet was diluted in PBS and used for the uptake experiment.

Dual-Luciferase Reporter Assay
The psi−CHECK™-2 dual-luciferase reporter vector (Promega, Madison, WI, USA) harboring the wild-type and mutant PPM1B 3′-UTR, which were inserted into the Xho I and Not I restriction sites 3′ to the end of the Renilla gene, were used to check the effect of miR-181a-5p on Renilla activity. The psi−CHECK™-2 mutant PPM1B′-UTR construct was generated by inducing a point mutation using the overlap extension PCR method. DF-1 cells were seeded on 24-well plates at a density of 3 × 10 5 cells per well and cultured until the cells reached approximately 60% con uence. Cells were then transfected with 200 ng of the luciferase reporter plasmid and 10 pmol of miR-181a-5p, miR-181a-5p-NC, miR-181a-5p-Inh or miR-181a-5p-Inh-NC using Lipofectamine 3000 (Invitrogen, USA

Conclusions
We conclude that the newly identi ed primary CP-II cells exosomal miR-181a-5p activates the TLR2-mediated MyD88/NF-κB pathway by directly targeting PPM1B to promote pro-in ammatory cytokines expression for defending against MG infection in recipient DF-1 cells. These ndings provide potential novel targets for prevention or treatment of MG infection in chicken.