This study was approved by the Animal Care Committee of South China Agricultural University (approval ID: SYXK-2014-0136). All study procedures and animal care activities were conducted per the recommendations in the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People’s Republic of China.

HD11 cells (20) were kindly provided by Dr. Liu Jue at the Beijing Academy of Agriculture and Forestry and cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS). DF-1 cells were cultured in Dulbecco modified Eagle medium (DMEM) containing 10% FBS. The specific‐pathogen‐free (SPF) chickens were purchased from the Guangdong DHN Poultry and Egg Products Co. Ltd., China.

A total of six -day-old SPF chickens were assigned to two groups (three chickens per group). Chickens in the experimental group were immunized with an inactivated NDV vaccine (Yebio, China), while chickens in the other group were immunized with PBS. All the chickens were euthanized to collect spleens at 5 days post-immunization. High-quality total RNAs were isolated from spleens using TRIzol reagent (Invitrogen), and miRNA sequencing was performed by LC Sciences (Hangzhou).

MiRNA mimics and inhibitors were synthesized by GenePharma Company (Shanghai). The sense sequences were as follows: for gga-miR-200b-3p mimics, 5′-UAAUACUGCCUGGUAAUGAUGAU-3′; for gga-miR-200b-3p inhibitors, 5′-AUUAUGACGGACCAUUACUACUA-3′; for the negative control mimics, 5′-UUUGUACUACACAAAAGUACUG -3′; and for negative-control inhibitors, 5′- UUUGUACUACACAAAAGUACUG-3′. MiRNA targets in the host were predicted by the use of Diana Tools (http://diana.imis.athena-innovation.gr/DianaTools/index.php?r=microT_CDS/index), PicTar (http://www.pictar.org/), Targetscan (http://www.targetscan.org/vert_72/), and miRDB (http://mirdb.org/).

Specific primer pairs for the gga-miR-200b-3p target sequence in MMD were designed as follows: for the gga-miR-200b-3p target sequence at position 37-43 in MMD, sense primer 5′-CCCTCGAGCTTGGCGTGGTGTTCTTC-3′ and antisense primer 5′-TGCTCTAGATACAGCACTTGCCGTCTG-3′; for the gga-miR-200b-3p target sequence at position 934-940 in MMD, sense primer 5′-CCCTCGAG CTTTACTTGTATTGCTGTC-3′ and antisense primer 5′-TGCTCTAGA GTTGGAAGATAGATAGGC-3′. The seed sites mutant in MMD gene were constructed by mutating nucleotides in the seed sequences (Position 37-43 in MMD: 5′-GCTGCACTTGGCCTCTGTAA CAGTATT ATTTGACTTAAGGAA-3′ mutated to 5′-GCTGCACTTGGCCTCTGTAA GCTCGCC ATTTGACTTAAGGAA-3′; Position 934-940 in MMD: 5′-ATTTACTCAGTTTTTCATGT CAGTATT TGAAAACAAAACCAA-3′ mutated to 5′-ATTTACTCAGTTTTTCATGT TGTCCGG TGAAAACAAAACCAA-3′) using a fast mutagenesis system (Transgene, China). All the primers were synthesized by Sangon Company (China).

All the fragments were amplified using a high-fidelity pfu DNA polymerase (Q5™ High-Fidelity DNA polymerase, NEB) under the following conditions: 98°C for 10 s, 40 cycles of 98°C for 10 s, 56°C for 30 s, and 72°C for 30 s, and 72°C for 10 min. The PCR products were cloned into a pEASY-Blunt plasmid (TransGen Biotech, Beijing) and sequenced commercially (Songon Biotech, Shanghai). The fragments of the MMD gene around the predicted gga-miR-200b-3p target sites were inserted into the site downstream of the firefly luciferase gene in the pGL3-Control vector to create the wild type or mutant 3′UTR vector.

HD11 cells were transfected with miRNA mimics, inhibitors, or controls using Lipofectamine 3000 reagent (Invitrogen). Twenty-four hours after transfection, cells were harvested for quantification of gene expression. Briefly, the specific primer pairs for chicken CD11b, F4/80, MHC-II, TNF-α, IL-1β, IL-6, IL-12α, CD14, CD68, RANTES, MIP-1, MMD, and GAPDH were designed according to the previous reports ( Table 1 ). Total RNA was isolated using TRIzol reagent and subjected to qRT-PCR detection. GAPDH was utilized as the reference gene. The transcriptional levels of genes were calculated relative to that of the GAPDH and are presented as fold increases or decreases relative to the control sample level.

DF-1 cells were transfected with luciferase reporter plasmids (pGL3-MMD-wt or pGL3-MMD-mut) and miRNA mimics, inhibitors or controls. pRL-TK Renilla luciferase reporter plasmid was used as the reference control to normalize the transfection efficiency. Forty-eight hours after transfection, relative luciferase activities were detected using a dual-luciferase assay kit (Promega, USA).

siRNAs were designed by GenePharma Co. (Shanghai, China) to knock down MMD in this study. The siRNAs for targeting MMD in HD11 cells included the followings: RNAi#1 (sense, 5′-ACACGCATTCCTCATTGtt-3′; antisense, 5′- CTTUGAGGAAUGCGUGUtt -3′), RNAi#2 (sense, 5′-AGACATCATTCGTCACTTAtt-3′; antisense, 5′-UAAGUGACGAAUGAUGUCUtt-3′), RNAi#3 (sense, 5′-CCACAGTGTTTCATATCGTtt-3′; antisense, 5′-ACGAUAUGAAACACUGUGGtt-3′), and negative control (sense, 5′-UUCUCCGAACGUGUCACGUtt-3′; antisense, 5′-ACGUGACACGUUCGGAGAAtt-3′). HD11 cells were transfected with siRNA or controls twice at a 20 h interval. Twenty-four hours after the second transfection, cells were harvested for further analysis.

Polyclonal antibodies against chMMD were prepared in mice and used to investigate the expression of MMD in HD11 cells. Briefly, 8-weeks-old BALB/c mice were immunized with chMMD protein for three times at 3-weeks intervals. The blood samples were collected from the orbital sinus and processed to collect sera for the detection of anti-chMMD antibodies by indirect ELISA and Western Blot.

Western blot was used to examine the effect of gga-miR-200b-3p and knockdown of MMD on the expression of MHC-II in HD11 cells. The HD11 cells were firstly transfected with miRNA mimics, control miRNA, MMD-specific siRNA or control siRNA, respectively. LPS-treated HD11 cells were used as a positive control. Twenty-four hours after transfection, cells were lysed and centrifuged for sample collection. Samples were fractionated by SDS–PAGE and subsequently transferred to a polyvinylidene difluoride membrane, the followingblock with 5% skimmed milk and probe with indicated antibodies. Blots were developed using an ECL kit (ThermoFisher, USA).

HD11 cells were transfected with miRNA mimics or control miRNA, or MMD-specific siRNA or control siRNA. LPS-treated HD11 cells were used as a positive control. Cells were harvested and stained with FITC-conjugated anti-Chicken MHC-II (8350-09, SouthernBiotech). The cell fluorescence was examined with flow cytometry and analyzed with CellQuest software (BD Biosciences).

HD11 cells were transfected with miRNA mimics or control miRNA, or MMD-specific siRNA or control siRNA. LPS-treated HD11 cells were used as a positive control. The cell culture supernatants were collected at 24 h after transfection or treatment. The concentrations of TNF-α, IL-1β, IL-6 and IL-12α in cell culture supernatants were quantified using sandwich ELISA kits (Cloud-Clone).

Intracellular ROS was detected by measuring the oxidative conversion of permeable 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) to fluorescent dichlorofluorescein (DCF) using a commercial kit (Beyotime, China). Briefly, HD11 cells were transfected with miRNA mimics or control miRNA, or MMD-specific siRNA or control siRNA. LPS-treated HD11 cells were used as a positive control. After incubation with DCFH-DA for 20 min, cells were harvested to detect DCF fluorescence distribution an excitation wavelength of 488 nm and at an emission wavelength of 525 nm.

The significance of differences between miRNA mimics-treated cells and controls in gene expression, cell activation, or intracellular ROS levels, between miRNA mimics-treated cells and mutant controls in gene expression, between MMD RNAi cells and controls in gene expression, cell activation, or intracellular ROS levels was determined by Mann-Whitney test or analysis of variance, respectively.

To identify the potential host miRNAs regarding the activation and differentiation of macrophage in birds, we screened the spleens of vaccine-immunized chickens and mock-immunized chickens by deep sequencing. As a result, 514 and 494 miRNAs were identified from the vaccine-immunized and mock-immunized chickens, respectively. Compared to that of mock-immunized chickens, 43 differentially expressed miRNAs were screened in vaccine-immunized chickens (threshold: P < 0.05 and fold change ≥ 2), including 25 up-regulated miRNAs and 18 down-regulated miRNAs. Among these differentially expressed miRNAs, a total of 9 miRNAs were selected for further study according to the functional analysis and the threshold value ( Figure 1A ), including three down-regulated miRNAs (gga-miR-1a-3p, gga-miR-122-5p and gga-miR-1663-3p) and six up-regulated miRNAs (gga-miR-1416-5p, gga-miR-122-5p, gga-miR-9-3p, gga-miR-200b-3p, gga-miR-31-5p and gga-miR-194).

Although miRNA-Seq possesses the ability to measure transcriptional levels, the accuracy of miRNA-Seq analysis needed to be verified using different methods. Therefore, we determined the differential expression of miRNAs mentioned above using qRT-PCR assay. As a result, compared to that in the mock-immunized chickens, the transcriptional levels of gga-miR-1a-3p, gga-miR-122-5p and gga-miR-1663-3p were significantly decreased in the vaccine-immunized chickens. In contrast, the transcriptional levels of gga-miR-1416-5p, gga-miR-122-5p, gga-miR-9-3p, gga-miR-200b-3p, gga-miR-31-5p, and gga-miR-194 were upregulated considerably ( Figure 1B ), yielding change trends similar to that determined by miRNA-Seq. These data provided solid evidence for the validity of miRNA-Seq analysis in the whole variations of the expression of miRNAs.

Since miR-200b-3p plays a critical role in the regulation of macrophage differentiation in mammals (19), it was essential to determine whether miR-200b-3p involves macrophage activation and differentiation in birds. Considering CD11b and F4/80 are the macrophage surface markers, we examined the effect of gga-miR-200b-3p on the expression of CD11b and F4/80 in HD11 cells. As a result, compared to that of the control group, the transcription of CD11b and F4/80 were significantly increased in miRNA mimic-treated cells. In contrast, miRNA inhibitor effectively lowered the transcriptional levels of CD11b and F4/80 ( Figure 2A ), indicating that gga-miR-200b-3p promotes macrophage activation in birds.

Because the expression of MHC-II in the cytoplasm is a typical characteristic during the macrophage activation, we proposed that gga-miR-200b-3p might enhance the expression of MHC-II. Therefore, we examined the effect of gga-miR-200b-3p on MHC-II expression using diverse assays. As a result, compared to that of the control group, over-expression of gga-miR-200b-3p mimics in HD11 cells effectively enhanced the transcriptional levels and expression level of MHC-II ( Figures 2A, B ). To consolidate this finding, we examined the effect of gga-miR-200b-3p on MHC-II expression using Flow cytometry assay. Interestingly, we found that over-expression of gga-miR-200b-3p mimics markedly enhanced the expression level of MHC-II in HD11 cells ( Figures 2C, D ). Considering the production of ROS during macrophage activation, we wondered whether gga-miR-200b-3p affects the intracellular ROS level in HD11 cells. As a result, gga-miR-200b-3p effectively enhanced the production of ROS in HD11 cells ( Figure 2E ). All these data provided solid evidence for the positive effect of gga-miR-200b-3p on macrophage activation in birds.

The gga-miR-200b-3p enhances macrophage activation in birds, and the activated macrophage can secret pro-inflammatory cytokines and chemokines. This led us to examine the effect of gga-miR-200b-3p on the expression of pro-inflammatory cytokines and chemokines in HD11 cells. Thus, we detected the production of cytokines in HD11 cells with or without gga-miR-200b-3p treatment using qRT-PCR and ELISA. As a result, the transcriptional levels of TNF-α, IL-1β, IL-6, IL-12α, CD14, CD68, RANTES, and MIP-1 were significantly increased in miRNA mimic-treated HD11 cells, but decreased in miRNA inhibitor-treated HD11 cells ( Figures 3A–H ). To further substantiate the positive role of gga-miR-200b-3p in promoting the expression of pro-inflammatory cytokines, ELISA was employed in this study. As a result, the expression of TNF-α, IL-1β, IL-6, and IL-12α were significantly increased in HD11 cells transfected with gga-miR-200b-3p ( Figures 4A–D ), yielding the trend similar to that determined by qRT-PCR. All these data strongly suggested that gga-miR-200b-3p enhances macrophage activation and differentiation in birds.

Using the prediction software mentioned above, we found a putative gga-miR-200b-3p targeted gene MMD that likely modulate macrophage activation and differentiation. Since miRNA usually serves as a guide RNA to target mRNAs to degrade mRNAs or inhibit translation, it was logical to examine the relation of gga-miR-200b-3p with MMD. Therefore, we constructed firefly luciferase reporter pGL3-MMD-wt (21-43 and 918-940), and two mutant plasmids pGL3-MMD-mut1 (21-43) and pGL3-MMD-mut2 (918-940) with mutations in the seed regions ( Figure 5A ), and then transfected DF-1 cells with these reporter plasmids and miRNA mimics. As a result, gga-miR-200b-3p markedly inhibited the luciferase activities of pGL3-MMD-wt ( Figure 5B ), but do not affect the luciferase activities of pGL3-MMD-mut ( Figure 5C ), indicating that MMD is targeted by gga-miR-200b-3p. Moreover, overexpression of epigenetic gga-miR-200b-3p reduced the expressions of MMD at both mRNA and protein levels ( Figures 5D, E ). All these data indicated that MMD is targeted and regulated by gga-miR-200b-3p.

The fact that vaccination-induced upregulation of gga-miR-200b-3p in birds, gga-miR-200b-3p directly targets MMD, and miR-200b-3p plays a critical role in the regulation of macrophage activation in mammals (19) prompted us to investigate the possibility that the expression of gga-miR-200b-3p and MMD are related to macrophage activation in birds. Considering the function of LPS on triggering the macrophage activation and differentiation, we examined the expression of gga-miR-200b-3p and MMD in HD11 cells treated with LPS. As a result, gga-miR-200b-3p was markedly upregulated in HD11 cells upon LPS stimulation ( Figure 6A ). Interestingly, consistent with a previous report (27), the expression of MMD was down-regulated at mRNA and protein levels after LPS stimulation ( Figures 6B–D ). All these results suggest that macrophage activation is highly correlated to the expression of gga-miR-200b-3p and MMD in birds.

To examine the role of MMD in the macrophage activation and differentiation in birds, we made three MMD RNAi constructs. We identified one construct that could effectivelyreduce the cellular level of MMD without causing any cytopathic effects ( Figure 7A ). We subsequently examined the expression of marker molecules in HD11 cells receiving siRNA or control siRNA. As a result, knockdown of MMD effectively enhanced the transcriptional levels of CD11b and F4/80 in HD11 cells ( Figure 7B ); the low level expression of MMD markedly enhanced MHC-II expression at both mRNA and protein levels in HD11 cells ( Figures 7B, C ). We also examined the effect of MMD on MHC-II expression in HD11 cells using Flow cytometry assay. As a result, knockdown of MMD by RNAi effectively enhances the expression of MHC-II and activates the macrophage ( Figures 7D, E ). The intracellular ROS levels were detected in HD11 cells receiving this siRNA or control siRNA. As expected, cells with lower expression of MMD produced more ROS ( Figure 7F ). These data provide clues that MMD plays a critical role during macrophage activation in birds. To further substantiate the regulation of MMD on macrophage activation and differentiation, we examined the effect of MMD on the expression of pro-inflammatory cytokines, surface markers, and chemokines in HD11 cells receiving siRNA or control siRNA. As expected, knockdown of MMD by RNAi effectively increased the expression of TNF-α, IL-1β, IL-6, IL-12α, CD14, CD68, RANTES, and MIP-1 at mRNA level in HD11 cells ( Figure 8 ). Furthermore, knockdown of MMD by RNAi significantly enhanced the expression of TNF-α, IL-1β, and IL-6, IL-12α at protein level ( Figure 9 ). As gga-miR-200b-3p inhibited MMD expression, these data strongly suggested that gga-miR-200b-3p enhances macrophage activation and differentiation via degradation of MMD.