Animal experiments were performed according to the protocols for the care of laboratory animals, Ministry of Science and Technology People’s Republic of China and approved animal care and use committee (IACUC) protocols at China Agricultural University of Beijing (20110611-01). All other experiments were carried out in accordance with the University Institutional Biosafety Committee approved protocol number 20110611-0.

MicroRNA miR-27a negative control, mimic, inhibitor control, and inhibitor were purchased from Shanghai GenePharma. Pam3Cys-Ser-(Lys)4 (ab142085), a TLR1/TLR2 agonist, was purchased from Abcam Biotechnology; lipofectamine 2000 from Invitrogen Thermo Fisher Scientific; and BBL Middlebrook OADC (211886) enrichment and mycobactin J from Becton, Dickinson and Sparks Company (USA), respectively. Macrophage colony-stimulating factor (M-CSF) was purchased from Peprotech Technology and total RNA extraction kit (ca. RN2802) from Aidlab Biotechnologies. The miR-27a (CD202-0033) and internal control S5 (CD202-0012) primers were from TIANGEN BIOTECH (Beijing). Primers for IL-1β, IL-6, IL-10, IL-12, interferon (IFN)-β, TNF-α, and GAPDH were purchased from Genewiz Technology. The calorimetric mouse IL-10, TNF-α, and IL-6 ELISA kit (KE10008, KE10007, and KE10002) were from Proteintech. Mouse IL-12 ELISA kit (ca. CSB-E07360m) was from Cusabio Co. Ltd. Rabbit polyclonal anti-p38 MAPK antibody (9211), rabbit polyclonal anti-phospho-p38 antibody (9211), rabbit monoclonal anti-phospho-JNK antibody (4668T), and rabbit monoclonal anti-phospho-ERK antibody (4370) were purchased from Cell Signaling Technology. Rabbit polyclonal anti-TAB 2 (MAP3K7IP2) polyclonal antibody (14410-1-Ap), rabbit polyclonal anti-GAPDH antibody (10494-1-AP), and goat anti-rabbit IgG (heavy and light chains) peroxidase-conjugated secondary antibody (SA000012) were purchased from Proteintech Biotechnology. Rabbit polyclonal anti-MAP3K7IP3 (TAB 3) antibody (bs5424R) was from Beijing Biosynthesis Biotechnology. Plasmid of mRNA for IL-10 and TAB 2 wild and mutant were constructed by Synbio Technology. Dual luciferase Reporter Assay kit (RFE: E1910, 205415) from Promega technology, USA. CellTiter 96Aqueous one solution cell proliferation assay kit (ca. G3580, 0000180214) from Promega technology, USA.

We used two strains of MAP (MAP-0908 and MAP k-10). MAP-0908 was isolated from a naturally infected cattle farm in the Shandong province of China. Molecular characterization of the MAP-0908 strain was done at the Transmissible Spongiform Encephalopathy Diagnostic Laboratory (China Agricultural University Beijing). Organisms were confirmed as MAP by cultural characteristics, while further species-specific DNA sequences by a standard PCR were performed for molecular characterization (41). K-10 strain of MAP 35716 was a kind gift from Prof. Paul Barrow, from the University of Nottingham at Leicestershire, UK. A stock culture of both strains of MAP was maintained in Middlebrook 7H9 medium supplemented with 1% glycerol, 10% oleic-acid-dextrose-catalase (OADC), and mycobactin J (2.0 mg/l) (Allied Monitor, Fayette, MO, USA) at 37°C. The organisms were grown to a concentration of 10⁸/ml for 2–3 weeks before being used for cell infection.

We used murine macrophages (RAW264.7 and BMDM) for our experiments. It has been demonstrated that MAP-infected RAW264.7 cells and sheep blood monocyte-derived macrophages show similar expression of TLR2 receptors (42). Furthermore, RAW264.7 cells are more differentiated than THP-1 cells, thus having higher phagocytic ability (43). Mouse bone marrow-derived macrophages were prepared and cultured as described previously (44). In brief, BMDMs were prepared from cells obtained from femurs, tibia, and humerus of 6- to 8-week-old C57BL/6J mice and cultured in RPMI supplemented with 10% heat-inactivated FBS (Hyclone) in the presence of 10 ng/ml of M-CSF (Peprotech) or GM-CSF (Peprotech) and 1% Penicillin-Streptomycin. At day four, non-adherent cells were collected and cultured for three more days in the presence of fresh RPMI containing M-CSF (10 ng/ml). These primary macrophages were cultured for 7–10 days in cell culture flasks. After that, the adherent BMDM cells (6–8 × 10⁶ cells per dish) were collected and plated in 12-well cell culture plates for further experiments (45). RAW264.7 macrophages were taken from cold storage (−80°C) and cultured in cell culture flask in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin for 3 days. The cells were transferred to 12-well cell culture plates 12–18 h before transfection.

Cells were allowed to attach overnight in 12-well plates (2 × 10⁵ cells in each well). The following day, miR-27a mimic, inhibitor, and control were individually transfected into RAW264.7 and BMDM cells using lipofectamine 2000 reagent, according to manufacturer’s instructions. After 6 h, the original medium was replaced with fresh medium supplemented with 10% FBS (46). After 48 h post-transfection, macrophages were infected at a multiplicity of infection (MOI) 20:1 (bacteria/cell) with live MAP in cell culture medium without antibiotic (47). Following incubation for 3 h at 37°C in 5% CO₂, the supernatant was discarded and each well was washed three times with sterile phosphate-buffered saline (PBS) to remove non-adherent MAP bacilli. After washing, fresh DMEM medium supplemented with 10% serum was added for the specified time period. The cells were harvested 6 and 18 h postinfection for analysis of total protein, total RNA, and cell supernatant. All samples were stored at −80°C until further use.

Total RNA (including miRNA) was extracted from RAW264.7 and BMDM cells with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s guidelines. To evaluate the expression levels of miRNAs and mRNAs, reverse transcription was done by using miRcute Plus miRNA First strand cDNA synthesis kits (obtained from Thermo Scientific and TIANGEN BIOTECH Beijing, China). To construct cDNA we used Thermo Scientific RevertAid First Strand cDNA synthesis kit according to the manufacturer’s instructions. Quantitative real-time PCR was performed by means of the miRcute miRNA qPCR detection kit (SYBR Green) for miRNA’s [miR-27a and internal references, small nucleolar S5 (mouse)]. Sybr Green Master Mix Kit (Vazyme Biotech Co., Ltd.) was used for the amplification of mRNA’s genes, including cytokines, by using the 700 Fast Real-Time PCR Systems (ViiA7 Real-time PCR, ABI). GAPDH was selected as housekeeping gene for the normalization of various cytokine genes because it is considered one of the most stable endogenous controls (48).

To normalize the expression of miRNAs, Ct values were obtained to calculate fold change for miRNA. A universal miR qPCR primer (possessing the binding site with universal adaptor primer, included in the kit), miRNA-27a primer, and 5S primer as internal reference (sequence complementary to the miRNA) were used to complete the real-time PCR reaction. miRNA qRT-PCR cycle parameters were as follows: 94°C for 2 min, followed by 45 cycles at 94°C for 20 s and 60°C for 34 s. The expression of mRNAs for various cytokines was determined by conducting qRT-PCR as described before (49). All the primers used in the present study for cytokines detection are mentioned in Table 1. To normalize the expression level of miRNAs and mRNAs, internal controls of S5 and GAPDH were used. The relative expression levels of miRNAs and mRNAs were determined as fold change, ΔCt values were obtained as follows: ΔCt = Ct of miRNAs or mRNAs − Ct of small nucleolar RNA S5or GAPDH. ΔΔCt values were obtained as follows: ΔΔCt = ΔCt of treated groups − ΔCt of untreated control groups. Fold change was calculated by the 2^−ΔΔCt method (50, 51).

The concentration of cytokines in the cells supernatant was measured by using an ELISA kit. Cells supernatant was collected at various time intervals (6 and 18 h) post-MAP (k-10 and 0908 strains) infection of different miR-27a transfected groups. All reagents, standard, and samples were prepared according to the manufacturer’s protocols. Standards and samples (100 µl each) were added into appropriate wells of 96-well ELISA plates and incubated for 2 h at 37°C in a humid environment. After incubation, the liquid was discarded and the plates washed 4–5 times with washing buffer. Then, 100 µl of detection antibody solution was added into each well, incubated in humid conditions at 37°C for 1 h, and washed as previously. Then we added 100 µl of HRP-conjugated antibodies to each well for 40 min and incubated at 37°C in a humid environment and washed again. After that, we added 100 µl of TMB substrate for 10–30 min at room temperature in the dark. To stop the reaction, we added 100 µl stop solution to each well. A standard curve was obtained using twofold dilutions of the standard for each independent experiment. To obtain the optical density (OD), plate was read at 450 nm with a wavelength correction of 630 nm by using an ELISA plate reader. The ELISA results were obtained from two independent experiments. Samples were added into triplicate wells in each independent experiment.

For total protein extraction, cells were washed twice with ice-cold PBS and homogenized with RIPA buffer containing a cocktail of protease and phosphatase inhibitor (Sigma Aldrich, St. Louis, MO, USA) for 20 min on ice. Afterward, samples were sonicated for 20 s and then centrifuged at 12,000 × g for 20 min at 4°C. The resulting supernatants were collected and boiled for 10 min after the addition of loading buffer (250 nM Tris HCl 6.8 pH, 10% sodium dodecyl sulfate, 0.5% bromophenol blue, 50% glycerol, and 0.5 M dithiothreitol) (52). Equal amounts of protein were separated by 12% or 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). After blocking with 5% skim milk prepared in Tris buffered saline Tween-20, membranes were incubated overnight at 4°C with primary antibodies. After incubation, membranes were rinsed three times in TBST and then incubated for 60 min at 37°C with HRP-labeled secondary antibodies. Signals were detected using an enhanced chemiluminescence detection kit (Bio-Rad, USA).

The binding elements for miR-27a at the 3′-UTR of IL-10 and TAB 2 mRNAs were obtained by PCR amplification using mouse genomic DNA as template and cloned into pMir-Reporter Luciferase vector (Synbio Tech). A mutant form of mouse IL-10 and TAB-2 3′-UTR was cloned into a pmirGLO vector by site-directed mutagenesis using a WT clone. HEK293 and BMDM cells were cultured in 24-well plates (2.5 × 10⁴ cells/well) overnight and transfected with 50 nM control mimics or 50 nM/ml miR-27a mimics using a Lipofectamine 2000. Six hours post-transfection, cells were again transfected with 100 ng pMir-Reporter constructs of IL-10 and TAB 2 wild- and mutant type of plasmid. Twenty-four hours after transfection, cells were lysed in lysis buffer, and a luciferase assay was performed using the Dual Luciferase reporter system (Promega), according to the manufacturer’s instructions (53). The results were obtained from three independent experiments, and all samples were collected in three replicates in each experiment.

To assess bacterial viability, BMDM and RAW264.7 macrophages (2 × 10⁵ cells in each well) were cultured in 12-well plates overnight and infected with MAP (0908 and k-10, MOI is 20:1) strains for 3 h and then washed thrice with warm PBS to remove extracellular bacteria. Thereafter, the infected cells were incubated for the indicated time periods and lysed at the specified time points with 0.1% Triton X 100. Appropriate dilutions were prepared for all transfected groups and plated on Middlebrook 7H10 agar plates supplemented with mycobactin-J and OADC in triplicate. Inoculated plates were incubated at 37°C, and colonies were counted 5 weeks after plating.

Cell viability was assessed by the MTS tetrazolium assay. BMDM and RAW264.7 cells were cultured in 96-well plates overnight before transfection. Cells were transfected with mimic control, mimic, inhibitor control, and inhibitor of miR-27a for 48 h. MTS reagent (20 µl) was added in each well, and then cells were incubated at 37°C for 3 h in a humidified, 5% CO₂ atmosphere. OD was quantified by measuring the absorbance at 490 nm wavelength with an ELISA plate reader.

All experiments were performed on three separate occasions. Data are expressed as mean ± SD. Student’s t-test was used for comparison between two groups. One-way ANOVA followed by Bonferroni’s multiple comparison tests was performed for multiple-group comparisons. Statistical analysis was carried out by using GraphPad Prism 5 software (GraphPad, La Jolla, CA, USA). Densitometric analysis of digitalized images was done by using ImageJ software (National Institute of Health, Bethesda, MD, USA). p Values < 0.05 were considered statistically significant.

To determine whether miR-27a plays a role in the immune responses against MAP infection, we investigated the expression of miR-27a in vivo and in vitro. We infected murine BMDMs and RAW264.7 cells with two strains of MAP (MAP k-10 and MAP 0908) at MOI 20:1 (bacteria/cell) and with TLR2 agonist. To ensure maximum infection of macrophages with MAP, we conducted phagocytosis assays at different levels of MOI (5:1, 10:1, and 20:1) before establishing the dose of infectivity (Figures S1A,B in Supplementary Material). The expression of miR-27a was measured by using qRT-PCR analysis. MAP infection decreased the expression of miR-27a in a time-dependent manner in BMDM (Figures 2A,B) and RAW264.7 cells (Figures 2D,E). Additionally, TLR2 agonist also showed a decrease in the expression of miR-27a in both types of macrophages (Figures 2C,F). The minimum level of miR-27a was detected at 12–24 h time intervals in all groups. Moreover, for in vivo determination of miR27a we challenged mice with MAP (0908) via intraperitoneal route. After 4 weeks postinfection, the expression level of miR-27a was significantly downregulated in different tissues of infected mice (Figures 2G–I). Various studies have reported that TLR2 receptors play an important role in the initiation of signaling cascades in macrophages to favor the persistent survival and growth of MAP (17). Our data suggest that downregulation of miR-27a in response to MAP strains and TLR2 treatment shares a common response in both types of macrophages and indicate that the expression of miR-27a in MAP- and TLR2-treated cells may share a common signaling mechanism.

It has been reported that TLR2-activated macrophages elevate the expression of IL-10 in response to MAP infection (20). IL-10 is one of the potent anti-inflammatory mediators that prevent tissue injury from excessive inflammatory reactions and also contribute to the pathogenesis of MAP (15, 54). More recently, it has been reported that miR-27a controls various targets to regulate the immune system (34, 39). We asked whether miR-27a participates in inflammatory responses associated with MAP (0908 and k-10) infection. We found that the level of miR-27a in BMDM cells increased by transfecting the cells with miR-27a mimic. We also found that upregulation of miR-27a reduced MAP-induced IL-10 expression at both 6 and 18 h postinfection (Figure 3A) and simultaneously reduced L-10 protein levels in BMDM cells after infection with MAP (Figure 3B). A similar IL-10 expression was recorded in RAW264.7 macrophages treated with miR-27a at both protein and mRNA levels (Figure 3D). In contrast, an upregulation of IL-10 was observed at protein and mRNA levels in both BMDM and RAW264.7 macrophages transfected with miR-27a inhibitor (Figures 3E,F,H). To check the transfection efficiency, BMDM cells were transfected with miR-27a control, mimic, and inhibitor for 48 h and then the expression of miR-27a observed with or without MAP infection (0908). The expression level of miR-27a was significantly higher in MAP-infected groups than in the MAP non-infected mimic-treated group (Figure 3C). In addition, miR-27a was downregulated in inhibitor treatment of both MAP-infected and MAP non-infected groups (Figure 3G). These data suggest that miR-27a negatively regulates the expression of IL-10 at both mRNA and protein levels in MAP-infected macrophages. We further analyzed the effects of miR-27a transfection on macrophage viability. BMDM and RAW264.7 cells were transfected with miR-27a (control, mimic, control-inhibitor, and inhibitor) for 48 h, and then cell viability was assessed by using the MTS assay (see Materials and Methods). Cell viability among all transfected groups showed no significant difference after miR-27a transfection (Figures S4A,B in Supplementary Material).

We have already shown that miR-27a is downregulated in MAP-infected macrophages and also observed the inhibitory effects of miR-27a on IL-10 expression. Previous studies reported that MAP inhibit the activation of macrophages by upregulating the expression of IL-10 while downregulating various pro-inflammatory mediators (15). The downregulation of miR-27a in MAP infection suggested that miR-27a could participate in the activation of macrophages. To test this hypothesis, we investigated whether miR27a participates in the expression of inflammatory mediators during MAP infection. First, we increased the level of miR-27a in BMDM cells by transfecting with miR-27a mimics for 48 h, and then cells were infected with MAP 0908 strain. After 6 h postinfection, we found a high level of pro-inflammatory cytokines, including IL-1β, IL-6, IL-12, IFN-β, and TNF-α in BMDM at both translational and transcriptional levels (Figures 4A,B). Similar results were observed for k-10 strain of MAP in infected macrophages (Figures 4C,D). Consistent with the findings in BMDM cells, upregulation of miR-27a also enhanced MAP-induced expression of IL-1β, IL-6, IL-12, IFN-β, and TNF-α in RAW264.7 cells (Figures 4E–H). In addition, we found that miR-27a also upregulates the expression of pro-inflammatory cytokines after 18 h of MAP infection, compared to miR-27a control (Figure S2 in Supplementary Material). These data suggest that miR-27a is a positive regulator for the induction of inflammatory cytokines in macrophages infected with MAP.

We observed that mimic treatment of miR-27a promoted inflammatory responses of macrophages infected with MAP and then asked whether downregulation of miR-27a will affect macrophage response against MAP. The level of miR-27a decreased in BMDM cells by transfecting the cells with miR-27a inhibitor for 48 h and then infected them with MAP 0908. After 6 h of infection, we found significantly decreased levels of pro-inflammatory cytokines, including IL-1β, IL-6, IL-12, IFN-β, and TNF-α induced by MAP 0908 in BMDM at both translational and transcriptional levels (Figures 5A,B). Similar findings were observed for MAP k-10-infected macrophages (Figures 5C,D). In addition, the downregulation of miR-27a also inhibited various cytokines expression in RAW264.7 macrophages after infection with MAP 0908 and k-10 strains (Figures 5E–H). Furthermore, we found that downregulation of miR-27a also inhibited the expression of pro-inflammatory cytokines after 18 h of MAP infection (Figure S3 in Supplementary Material). These findings suggest that miR-27a regulates macrophage activation by abrogating the anti-inflammatory environment created by MAP in infected macrophages for prolonged survival and growth.

Complex signaling cascades are triggered by MAP after interaction with TLR2/4 for the activation of MAPK-p38 pathway (15). TAB 1, TAB 2, and TAB 3 are involved in the activation of TAK1 (55). Various kinases are activated by downstream signaling of TAK1 including MAPKs. We investigated whether miR-27a participates in the regulation of signaling cascades of MAPK pathway in MAP-infected macrophages. BMDM cells were transfected with miR-27a control, mimic, and inhibitor. Forty-eight hours after transfection, cells were infected with MAP 0908 strain, and the expression of various proteins of the MAPK pathway was determined by immunoblot assays. As shown in Figure 6A, upregulation of miR-27a diminished MAP (0908) induced TAB 2 (Figure 6B) and TAB 3 (Figure 6C) expression, while downregulation of miR-27a counteracted these effects. Furthermore, we examined whether miR-27a affects the components of MAPK pathway that regulate the expression of IL-10 during MAP infection. We found that upregulation of miR-27a reduced the expression of phosphorylated p38 (Figure 6D) and p-JNK (Figure 6E), while downregulation of miR-27a significantly promoted the expression of p-p38 and p-JNK at all time intervals. However, no significant difference was observed in p-ERK (Figure 6F) in BMDM cells at various time intervals among all treated groups after MAP (0908) infection. These findings suggest that miR-27a modulates MAPK signaling pathways activated by MAP in infected macrophages.

To clarify whether miR-27a targets IL-10 alone or IL-10 and TAB 2 together at their 3′-UTR region, we used TargetScan and miRanda algorithm to predict miRNA targets as previously described (56, 57). We identified a putative binding site of miR-27a/b at the 3′-UTR of IL-10 and TAB 2 transcript in mice and also in other species (Figure 7A). Luciferase reporter vectors were constructed for wild and mutant 3′-UTR for IL-10 and TAB 2 target site of miR-27a (Figure 7B). We found that upregulation of miR-27a reduced the activity of the luciferase reporter in BMDM and HEK293 cells containing IL-10 wild-type reporter, while miR-27a failed to inhibit luciferase activity in cells having IL-10 mutant-type reporter (Figures 7D,E). We also found that miR-27a decreased the activity of the luciferase reporter that contained the wild-type 3′-UTR of TAB 2 mRNA in both BMDM and HEK293 cells. However, miR-27a had no effect on the activity of a luciferase reporter that contained the mutant 3′-UTR of TAB 2 mRNA (Figures 7F,G). Additionally, qRT-PCR data demonstrated that BMDM transfected with miR-27a mimic significantly increased the expression levels of miR-27a. On the other hand, BMDMs transfected with miR-27a inhibitor significantly reduced the expression level of miR-27a (Figure 7C). These findings suggest that both IL-10 and TAB 2 are direct targets of miR-27a.

Mycobacterium avium subspecies paratuberculosis adopts various strategies for intracellular survival in mononuclear phagocytic cells. The activation of MAPK-p38 signaling pathway and upregulation of IL-10 is one of the important strategies of MAP survival and pathogenesis (58, 59). We already found that miR-27a positively regulates the expression of pro-inflammatory cytokines in MAP-infected macrophages and that overexpression of miR-27a promotes macrophage activation by inhibition of IL-10. We next investigated whether miR-27a affects the intracellular survival of MAP. To test this hypothesis, BMDM cells were transfected with miR-27a control, mimic, and inhibitor. Upregulation of miR-27a reduced the survival of MAP (0908) in BMDM cells, while miR-27a inhibitor counteracted that effect (Figure 8A). Additionally, transfection of RAW264.7 cells with miR-27a mimic showed a clearer difference in the intracellular survival of MAP than cells transfected with miR-27a inhibitor (0908) (Figure 8C). We observed similar results between miR-27a mimic and inhibitor for MAP (k-10)-infected macrophages (Figures 8B,D). Although the difference for total viable bacterial count between the control and miR-27a inhibitor was less evident, there was a clear significant difference between miR-27a mimic and inhibitor group (Figures 8B,C). These results indicate that miR-27a inhibits the intracellular survival of MAP by promoting bactericidal activities of macrophages.