Mycobacterium tuberculosis poses a significant global health threat. MicroRNAs play an important role in regulating host anti-mycobacterial defense; however, their role in apoptosis-mediated mycobacterial elimination and inflammatory response remains unclear. In this study, we explored the role of microRNA-27b (miR-27b) in murine macrophage responses to M. tuberculosis infection. We uncovered that the TLR-2/MyD88/NF-κB signaling pathway induced the expression of miR-27b and miR-27b suppressed the production of proinflammatory factors and the activity of NF-κB, thereby avoiding an excessive inflammation during M. tuberculosis infection. Luciferase reporter assay and Western blotting showed that miR-27b directly targeted Bcl-2–associated athanogene 2 (Bag2) in macrophages. Overexpression of Bag2 reversed miR-27b–mediated inhibition of the production of proinflammatory factors. In addition, miR-27b increased p53-dependent cell apoptosis and the production of reactive oxygen species and decreased the bacterial burden. We also showed that Bag2 interacts with p53 and negatively regulates its activity, thereby controlling cell apoptosis and facilitating bacterial survival. In summary, we revealed a novel role of the miR-27b/Bag2 axis in the regulation of inflammatory response and apoptosis and provide a potential molecular host defense mechanism against mycobacteria.

Mycobacterium tuberculosis is the bacteria responsible for tuberculosis and is a highly successful intracellular pathogen that parasitizes the host’s macrophages (1). Macrophages can eliminate mycobacteria through different strategies, such as modulation of host cell-death programs (2), regulation of inflammatory responses (3), and acidification and maturation of phagosomes (4). However, M. tuberculosis can also overcome microbicidal mechanisms of macrophages to ensure survival and replication (5). Therefore, it is important to elucidate the immunological role of macrophages in the containment of M. tuberculosis infection to better understand the host defense mechanism of anti-mycobacterial response.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the immune response to various infectious microorganisms at the posttranscriptional level (6). To date, miRNAs have been reported to regulate the immune response of macrophages to mycobacterial infection. For example, let-7f increases the production of cytokines and diminishes M. tuberculosis survival (7), whereas miR-99b downregulates proinflammatory cytokines and increases the bacterial burden (8). miR-155 promotes autophagy to eliminate intracellular mycobacteria (9), whereas miR-125a inhibits the activation of autophagy (10). Although several miRNAs have been studied, the function and molecular basis of specific miRNAs in apoptosis and inflammation during M. tuberculosis infection requires further investigation.

We previously investigated the role of miRNAs in M. tuberculosis H37Ra infection using small RNA sequencing, which suggested that M. tuberculosis H37Ra induces the expression of miR-27b (11). In the current study, we investigated the potential role of miR-27b in regulating macrophage apoptosis and inflammatory response during M. tuberculosis infection. Our study demonstrated that upregulation of miR-27b is dependent on the TLR 2/MyD88/NF-κB signaling pathway, whereas overexpression of miR-27b restrains the activity of NF-κB and the production of proinflammatory factors, thereby avoiding excessive inflammation. Moreover, we found that miR-27b reinforced M. tuberculosis–induced macrophage apoptosis through the p53–reactive oxygen species (ROS) signaling pathway, thus facilitating bacterial elimination. Using various methods, we also showed a dual role of Bcl-2–associated athanogene 2 (Bag2), a target of miR-27b, as a positive regulator of NF-κB signaling and a negative regulator of p53 signaling. We believe our study provides new information regarding host defense molecular mechanisms against mycobacteria.

Female C57BL/6 and BALB/c mice (6–8 wk old) were purchased from the experimental animal center of the Fourth Military Medical University (Xi’an, Shaanxi, China). Animal experiments were conducted according to the Guidelines for the Care and Use of Animals of Northwest A&F University. BALB/c mice were injected with 1 × 106 CFUs of M. tuberculosis H37Rv, H37Ra, or PBS via the tail vein. After 3 wk, the lungs and spleens were harvested for RNA isolation using TRIzol reagent (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions. The infected mice were maintained in biosafety level 3 laboratory facilities.

HEK293T cells and murine macrophage-like RAW264.7 cells (American Type Culture Collection) were maintained in DMEM and RPMI 1640 media with 10% FBS, respectively. Mouse bone marrow–derived macrophages (BMDMs) were isolated from female 6- to 8-wk-old C57BL/6 mice and cultured in RPMI 1640 containing 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, and 50 ng/ml M-CSF (R&D Systems, Minneapolis, MN). All cells were incubated at 37°C and 5% CO2. M. tuberculosis H37Rv (25618; ATCC) and H37Ra (25177; ATCC) strains were grown in Middlebrook 7H9 broth medium or 7H10 agar plates supplemented with 10% oleic albumin dextrose catalase (Sigma-Aldrich, St. Louis, MO). Cells were infected for the indicated time at a multiplicity of infection (MOI) of 5.

Cells were infected with M. tuberculosis H37Ra for 6 h and then washed with PBS to remove extracellular bacteria. Infected cells were cultured for the indicated time and then lysed in sterile distilled water containing 0.05% SDS. The cell lysate was transferred to Middlebrook 7H10 agar plates supplemented with 10% oleic albumin dextrose catalase and incubated for 3–4 wk at 37°C for counting the colony number.

miRNA mimic, miRNA inhibitor (RiboBio, Guangzhou, China), and small interfering RNAs (siRNAs) (GenePharma, Shanghai, China) were transfected into cells using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. The siRNA sequences were as follows: si-NC: 5′-UUCUCCGAACGUGUCACGUTT-3′; si-Bag2: 5′-GCCACUUAAUGUCACUUUATT-3′; si-TLR2: 5′-CGAGCUGGGUAAAGUAGAATT-3′; si-MyD88: 5′-GGAGAUGAUCCGGCAACUATT-3′.

To test whether miR-27b directly targeted Bag2, HEK293T cells were cotransfected with a luciferase reporter and pCDH–miR-27b or miR-27b mimic using Lipofectamine 2000 reagent.

For promoter assays, promoters of miRNAs were cloned into the pGL4.10 Vector (Progema). RAW264.7 cells were cotransfected with the miR-27b promoter firefly luciferase reporter plasmids, p65 expression plasmids, and pRL-TK Renilla plasmid using Lipofectamine 2000.

After 24–48 h, the activity of luciferase was measured using the Dual-Luciferase Reporter Assay System (Promega).

Total RNA was isolated from mouse organs or cells using TRIzol reagent, according to the manufacturer’s instructions; RNA was reverse transcribed using a PrimeScript RT Reagent Kit (Takara, Dalian, China). For miRNA quantitative PCR (qPCR), cDNA was prepared using a miScript II RT Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. The qPCR analysis was conducted on an ABI StepOnePlus PCR system (Applied Biosystems, Foster City, CA) using TransStart Tip Green qPCR SuperMix (TransGen Biotech, Beijing, China). Small nuclear RNA (Rnu6) and GAPDH were used to normalize the relative expression of miRNA and mRNA, respectively. Primer sequences used for qPCR are provided in Supplemental Table I.

Cells were lysed in RIPA buffer (Pierce, Rockford, IL), according to the manufacturer’s instructions. Protein samples were separated by SDS-PAGE, then transferred to polyvinylidene difluoride membranes and incubated with appropriate Abs. Blots were visualized by autography using SuperSignal West Pico Substrate (Thermo Fisher Scientific). Primary Abs used in the study were TLR2, MyD88 (Santa Cruz Biotechnology, Dallas, TX), Bag2, hemagglutinin (HA)-tag (Abcam, Cambridge, U.K.), caspase-3, p53, NF-κB p65 (Cell Signaling Technology, Danvers, MA), Flag (Sigma-Aldrich), and actin (TransGen Biotech). Secondary Abs were purchased from Beyotime (Jiangsu, China).

Immunoprecipitation assays were conducted using a protocol provided by Pierce with some modifications. Briefly, cells were gently lysed in ice-cold immunoprecipitation buffer. Cell lysates were incubated with anti-Flag (Bag2) or mouse Ab at 4°C for 6–12 h on an orbital shaker. Immunocomplexes were captured with protein A agarose (Pierce) at 4°C for 2 h. The beads were collected, washed, and boiled; samples were analyzed by immunoblotting.

Annexin V Alexa Fluor 488 (Molecular Probes, Eugene, OR) was used for detecting cell apoptosis. RAW264.7 cells were stained with Annexin V Alexa Fluor 488 and propidium iodide, according to the manufacturer’s instructions. Cells were then selected and analyzed using a BD FACS Calibur flow cytometer (BD Biosciences, San Jose, CA). For each analysis, gate settings of flow cytometry were defined by suitable negative or positive controls.

Cell viability was tested using a cholecystokinin 8 (CCK8) kit (TransGen Biotech). Cells were plated at a density of 2 × 103 cells per well in a 96-well plate. After the cells were infected with H37Ra for the indicated time, CCK8 solution (10 μl) was added to each well, and the plates were incubated for 1–4 h at 37°C. Absorbance of each sample was detected at 450 nm using a microplate reader.

Postinfection, cell supernatants were collected and centrifuged for analysis by ELISA (Cloud-Clone, Houston, TX), according to the manufacturer’s instructions. The concentration of cytokines was determined using a standard curve.

Cell supernatants were collected for assessing the production of nitrite using Griess reagent (Promega), according to the manufacturer’s instructions. The microplate reader was used for detecting absorbance of samples at 540 nm; nitrite concentrations were calculated using a standard curve.

Intracellular ROS levels were measured using an ROS indicator, 2,7′-dichlorodihydrofluorescein diacetate (DCFDA; Invitrogen), at a final concentration of 10 μM. Cells were incubated for 30–60 min in the dark, then collected and analyzed using a BD FACSCalibur flow cytometer.

Data are presented as mean ± SD and were analyzed using the Student t test. Data were considered to be statistically significant at p < 0.05.

Our group recently performed small RNA sequencing and found that M. tuberculosis H37Ra (H37Ra) induced the expression of the cluster of miR-23b/27b (11). We hypothesized that the cluster of miR-23b/miR-27b/miR-24-1 could be regulated by M. tuberculosis. We performed qPCR to examine the expression of these miRNAs and found that precursors and mature forms of miR-27b showed a time-dependent increased expression in murine BMDMs and macrophage-like RAW264.7 cells treated with M. tuberculosis H37Ra and H37Rv (Fig. 1A, 1B, Supplemental Fig. 1A). We further examined the expression of miR-27a, miR-23a, miR-23b, and miR-24-1 using qPCR and found that each expression was changed in a time-independent manner during M. tuberculosis H37Rv infection (Supplemental Fig. 1B).

FIGURE 1.

Expression of miR-27b is induced post–mycobacterial infection both in vivo and in vitro. (A and B) Murine BMDMs (A) and RAW264.7 cells (B) were infected with M. tuberculosis H37Ra or H37Rv at an MOI of 5 for the indicated time; miR-27b expression levels were measured by qPCR. (C and D) Expression levels of miR-27b in the lungs (C) and spleens (D) isolated from H37Ra- or H37Rv-infected BALB/c mice were examined 3 wk postinfection. (E and F) RAW264.7 cells were transfected with miR-27b mimic (E) or miR-27b inhibitor (F) for the indicated time and then left uninfected or infected with H37Ra. Expression levels of miR-27b were determined by qPCR. (G) RAW264.7 cells were transfected with siTLR2 or siMyD88 for 24 h prior to H37Ra infection. Cells were collected and miR-27b expression measured using qPCR post–H37Ra infection. (H) RAW264.7 cells were cotransfected with miR-27b promoter-reporter constructs (empty vector and WT), p65-expressing plasmid, and Renilla plasmid. The activity of luciferase was measured 48 h posttransfection. (I) RAW264.7 cells were transfected with p65 or an empty vector without or with infection. Forty-eight hours later, relative levels of primary miR-27b were measured by qPCR (J) RAW264.7 cells were pretreated with BAY11-7082 prior to H37Ra infection, and the expression of miR-27b was measured by qPCR. Results are shown as the fold change compared with the control. All data are representative of three independent experiments. Data indicate mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 relative to the control.

FIGURE 1.

Expression of miR-27b is induced post–mycobacterial infection both in vivo and in vitro. (A and B) Murine BMDMs (A) and RAW264.7 cells (B) were infected with M. tuberculosis H37Ra or H37Rv at an MOI of 5 for the indicated time; miR-27b expression levels were measured by qPCR. (C and D) Expression levels of miR-27b in the lungs (C) and spleens (D) isolated from H37Ra- or H37Rv-infected BALB/c mice were examined 3 wk postinfection. (E and F) RAW264.7 cells were transfected with miR-27b mimic (E) or miR-27b inhibitor (F) for the indicated time and then left uninfected or infected with H37Ra. Expression levels of miR-27b were determined by qPCR. (G) RAW264.7 cells were transfected with siTLR2 or siMyD88 for 24 h prior to H37Ra infection. Cells were collected and miR-27b expression measured using qPCR post–H37Ra infection. (H) RAW264.7 cells were cotransfected with miR-27b promoter-reporter constructs (empty vector and WT), p65-expressing plasmid, and Renilla plasmid. The activity of luciferase was measured 48 h posttransfection. (I) RAW264.7 cells were transfected with p65 or an empty vector without or with infection. Forty-eight hours later, relative levels of primary miR-27b were measured by qPCR (J) RAW264.7 cells were pretreated with BAY11-7082 prior to H37Ra infection, and the expression of miR-27b was measured by qPCR. Results are shown as the fold change compared with the control. All data are representative of three independent experiments. Data indicate mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 relative to the control.

Close modal

To verify the expression of miR-27b in vivo in response to M. tuberculosis infection, BALB/c mice were intravenously infected with M. tuberculosis. qPCR data demonstrated that the expression of miR-27b was significantly induced in the lungs and spleens of infected BALB/c mice (Fig. 1C, 1D).

Compared with transfection of the control mimic, transfection of the miR-27b mimic further increased miR-27b transcription (Fig. 1E). Conversely, transfection of the miR-27b inhibitor repressed endogenous expression of miR-27b, which could also partially inhibit induction of the expression of miR-27b by H37Ra (Fig. 1F). These results validate our hypothesis that the expression of miR-27b is upregulated post–M. tuberculosis infection.

Sufficient evidence has demonstrated that TLR signaling can induce the expression of miRNA (1214). Therefore, we tested whether TLR signaling affected the expression of miR-27b during H37Ra infection. As exhibited in Fig. 1G, interference in the expression of TLR2 using RNA interference techniques (Supplemental Fig. 1C) significantly decreased H37Ra-induced expression levels of miR-27b. Most TLRs, except TLR3, use MyD88 as an adaptor protein in the TLR signaling cascade. Heat-killed mycobacteria have strong TLR2 agonist signaling through MyD88 (15). Next, we silenced the expression of MyD88 using siRNA (MyD88 siRNA) and assessed changes in the expression of MyD88 using qPCR and Western blot (Supplemental Fig. 1D). Inhibition of the expression of MyD88 resulted in a decreased expression of miR-27b in RAW264.7 cells during infection (Fig. 1G). NF-κB plays a pivotal role in cells of the immune system and functions as a potent transcriptional activator (16). To determine the regulation of miR-27b by NF-κB, we performed promoter assays with promoter constructs for miR-27b and found that NF-κB p65 increased miR-27b transcription through a segment located at −1664 to +336 relative to the putative transcription start site (Fig. 1H). Furthermore, ectopic expression of NF-κB p65 increased the expression of the endogenous miR-27b primary transcript (Fig. 1I); however, inhibition of NF-κB by Bay11-7082 reduced the expression of miR-27b in RAW264.7 cells during infection (Fig. 1J).

When invading mycobacteria are detected, proinflammatory factors, such as IL-1β, IL-6, TNF-α, and inducible NO synthase (iNOS), induce an innate immune response (17). The production of inflammatory cytokines is induced in human and mouse macrophages infected with M. tuberculosis (18, 19). We tested the role of miR-27b on the production of selected cytokines in infected macrophages. As shown in Fig. 2A–H, miR-27b inhibited the expression of IL-1β, IL-6, TNF-α, and iNOS protein and mRNA in H37Ra-infected BMDMs and RAW264.7 cells, whereas the miR-27b inhibitor significantly induced the release of cytokine mediators.

FIGURE 2.

miR-27b inhibits the expression of IL-1β, IL-6, TNF-α, and iNOS and increases the level of ROS in macrophages during H37Ra infection. (AH) BMDMs (A, C, E, and G) and RAW264.7 cells (B, D, F, and H) were transfected with control, miR-27b mimic, or miR-27b inhibitor followed by H37Ra infection. Protein levels of IL-6, IL-1β, TNF-α (A and B), and NO (E and F) were determined using a specific ELISA or Griess reagent. mRNA expression of IL-6, IL-1β, TNF-α (C and D), and NO (G and H) was determined by qPCR. (I and J) BMDMs (I) and RAW264.7 cells (J) were transfected with control or miR-27b mimic, then left uninfected or were infected with H37Ra. Production of ROS was determined by flow cytometry. Results represent mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 relative to the control mimic.

FIGURE 2.

miR-27b inhibits the expression of IL-1β, IL-6, TNF-α, and iNOS and increases the level of ROS in macrophages during H37Ra infection. (AH) BMDMs (A, C, E, and G) and RAW264.7 cells (B, D, F, and H) were transfected with control, miR-27b mimic, or miR-27b inhibitor followed by H37Ra infection. Protein levels of IL-6, IL-1β, TNF-α (A and B), and NO (E and F) were determined using a specific ELISA or Griess reagent. mRNA expression of IL-6, IL-1β, TNF-α (C and D), and NO (G and H) was determined by qPCR. (I and J) BMDMs (I) and RAW264.7 cells (J) were transfected with control or miR-27b mimic, then left uninfected or were infected with H37Ra. Production of ROS was determined by flow cytometry. Results represent mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 relative to the control mimic.

Close modal

ROS are important microbicidal mediators of macrophages and directly kill mycobacteria (20). Therefore, we tested the effects of miR-27b on the production of ROS. Macrophages were transfected with miR-27b mimic with or without H37Ra infection. DCFDA flow cytometry showed that ROS levels were enhanced by transfection with miR-27b mimic in BMDMs and RAW264.7 cells (Fig. 2I, 2J).

We investigated the effect of miR-27b on H37Ra-mediated macrophage apoptosis. After BMDMs and RAW264.7 cells were transiently transfected with the miR-27b mimic, an evident increase in apoptotic populations (Fig. 3A) and a marked induction of the activity of cleaved caspase-9 and caspase-3 (Fig. 3B) were observed in uninfected and infected macrophages compared with those in the control. Furthermore, inhibition of miR-27b attenuated cell apoptosis in H37Ra-treated and untreated macrophages (Fig. 3A). We further examined the effect of miR-27b on cell viability. BMDMs and RAW264.7 cells were transfected with control mimic, miR-27b mimic, or miR-27b inhibitor prior to H37Ra treatment; cell viability was measured by CCK8 assay. As shown in Fig. 3C and 3D, overexpression of miR-27b decreased cell viability in BMDMs and RAW264.7 cells, but inhibition of miR-27b increased cell viability.

FIGURE 3.

miR-27b promotes infected macrophage apoptosis and inhibits the survival of mycobacteria. (A and B) BMDMs and RAW264.7 cells were transfected with miR-27b mimic or miR-27b inhibitor, and 24 h posttransfection, cells were left uninfected or were infected with H37Ra. Cell apoptosis was tested by annexin V staining followed by flow cytometric analysis (A) and Western blotting (B). (C and D) BMDMs (C) and RAW264.7 cells (D) were transfected with miR-27b mimic or miR-27b inhibitor for 24 h and then left uninfected or infected with H37Ra. Cell viability was analyzed by CCK8 assay. (E and F) After transient transfection with miR-27b mimic or miR-27b inhibitor, BMDMs (E) and RAW264.7 cells (F) were infected with H37Ra for the indicated time; mycobacterial viability was detected by CFU assay. Data are shown as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 relative to the control mimic.

FIGURE 3.

miR-27b promotes infected macrophage apoptosis and inhibits the survival of mycobacteria. (A and B) BMDMs and RAW264.7 cells were transfected with miR-27b mimic or miR-27b inhibitor, and 24 h posttransfection, cells were left uninfected or were infected with H37Ra. Cell apoptosis was tested by annexin V staining followed by flow cytometric analysis (A) and Western blotting (B). (C and D) BMDMs (C) and RAW264.7 cells (D) were transfected with miR-27b mimic or miR-27b inhibitor for 24 h and then left uninfected or infected with H37Ra. Cell viability was analyzed by CCK8 assay. (E and F) After transient transfection with miR-27b mimic or miR-27b inhibitor, BMDMs (E) and RAW264.7 cells (F) were infected with H37Ra for the indicated time; mycobacterial viability was detected by CFU assay. Data are shown as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 relative to the control mimic.

Close modal

To determine the role of miR-27b in the survival of mycobacteria in macrophages, CFU assays were performed for evaluating the survival of H37Ra in miR-27b mimic– or miR-27b inhibitor–transfected BMDMs (Fig. 3E) and RAW264.7 cells (Fig. 3F). The results showed that the miR-27b mimic attenuated the survival of H37Ra in macrophages (Fig. 3E, 3F). Conversely, survival of H37Ra was facilitated in cells treated with the miR-27b inhibitor (Fig. 3E, 3F). Collectively, these results suggest that miR-27b accelerates macrophage apoptosis and decreases mycobacteria survival.

To determine how miR-27b modulates apoptosis and the expression of inflammatory cytokines, bioinformatics analysis was performed using three commonly used algorithms: TargetScan (http://www.targetscan.org/index.html), miRanda (http://www.microrna.org/), and PicTar ((http://pictar.bio.nyu.edu/). Bag2 displayed a potential seed match for miR-27b in its 3′-untranslated region (UTR) (Fig. 4A). To identify direct interaction between the Bag2 transcript and miR-27b, we performed a dual-luciferase reporter assay for measuring the expression level of a reporter gene fused to the 3′-UTR of Bag2 (wild-type [WT]-Bag2) or 3′-UTR containing a mutated (mut) miR-27b seed sequence (mut-Bag2). HEK293T cells were cotransfected with reporter plasmids together with miR-27b mimic or the overexpression plasmid. Enforced expression of miR-27b repressed the activity of luciferase fused to WT Bag2 3′-UTR but failed to suppress the expression of luciferase fused to the mut-Bag2 3′-UTR (Fig. 4B, 4C).

FIGURE 4.

miR-27b posttranscriptionally represses the expression of Bag2 by targeting its 3′-UTR. (A) The sequence of miR-27b and its predicted binding with the Bag2 3′-UTRs of different species and with mut-Bag2 3′-UTR are shown. (B) RAW264.7 cells were cotransfected with control or 27b mimic and a WT-Bag2 or mut-Bag2 3′-UTR (mut-Bag2) luciferase reporter plasmid and the activity of luciferase 24 h posttransfection was assessed. The activity of Renilla luciferase was normalized to that of firefly luciferase. (C) RAW264.7 cells were cotransfected with miRNA expression construct pCDH–miR-27b along with a reporter construct encoding WT-Bag2 or mut-Bag2, and a luciferase assay was performed. (D and E) Expression of the endogenous Bag2 protein in uninfected or infected BMDMs and RAW264.7 cells transfected with miR-27b mimic (D) or miR-27b inhibitor (E) was detected by Western blot. (F and G) BMDMs (F) and RAW264.7 cells (G) were infected with H37Ra (MOI 5:1) for the indicated time, and Bag2 protein levels were measured using qPCR and Western blotting. Data are shown as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 4.

miR-27b posttranscriptionally represses the expression of Bag2 by targeting its 3′-UTR. (A) The sequence of miR-27b and its predicted binding with the Bag2 3′-UTRs of different species and with mut-Bag2 3′-UTR are shown. (B) RAW264.7 cells were cotransfected with control or 27b mimic and a WT-Bag2 or mut-Bag2 3′-UTR (mut-Bag2) luciferase reporter plasmid and the activity of luciferase 24 h posttransfection was assessed. The activity of Renilla luciferase was normalized to that of firefly luciferase. (C) RAW264.7 cells were cotransfected with miRNA expression construct pCDH–miR-27b along with a reporter construct encoding WT-Bag2 or mut-Bag2, and a luciferase assay was performed. (D and E) Expression of the endogenous Bag2 protein in uninfected or infected BMDMs and RAW264.7 cells transfected with miR-27b mimic (D) or miR-27b inhibitor (E) was detected by Western blot. (F and G) BMDMs (F) and RAW264.7 cells (G) were infected with H37Ra (MOI 5:1) for the indicated time, and Bag2 protein levels were measured using qPCR and Western blotting. Data are shown as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

To explore whether miR-27b repressed endogenous Bag2, BMDMs and RAW264.7 cells were transfected with miR-27b mimic or control, and expression of Bag2 was measured by Western blot. miR-27b substantially reduced protein levels of Bag2 prior to infection and 24 h postinfection (Fig. 4D). Conversely, protein levels of Bag2 were augmented when cells were transfected with miR-27b inhibitor prior to H37Ra infection (Fig. 4E).

As shown in Fig. 1, the expression of miR-27b increased in a time-dependent manner in macrophages treated with M. tuberculosis. Expression of Bag2 post–M. tuberculosis infection was measured using qPCR and Western blot. Bag2 was downregulated in BMDMs (Fig. 4F, Supplemental Fig. 2) and RAW264.7 cells post–M. tuberculosis treatment (Fig. 4G), probably because of time-dependent induction of the expression of miR-27b. These data strongly support the fact that Bag2 is a target of miR-27b in macrophages.

We found that overexpression of miR-27b blocked the production of the proinflammatory factors IL-1β, IL-6, TNF-α, and iNOS (Fig. 2A–H). Considering that NF-κB is a key inflammatory gene in macrophages and that it mediates the production of proinflammatory cytokines and NO (21, 22), we next examined whether miR-27b modulates the activation of NF-κB in RAW264.7 cells. Nuclear translocation of the NF-κB p65 subunit was measured using Western blot and confocal microscopy to determine the activation of NF-κB. As demonstrated in Fig. 5A and 5B, activation of NF-κB was decreased in response to miR-27b with H37Ra infection. To further confirm these results, we tested whether overexpression of miR-27b affected the activation of NF-κB using dual-luciferase reporter assays. As shown in Fig. 5C, miR-27b inactivated the NF-κB reporter gene during infection. Collectively, these results demonstrated that miR-27b negatively regulated the activation of NF-κB in H37Ra-infected macrophages.

FIGURE 5.

miR-27b activates NF-κB signaling, and Bag2 reverses the effect of miR-27b on inflammatory factors in macrophages. (A and B) RAW264.7 cells were transfected with miR-27b mimic, followed by H37Ra infection for the indicated time. Protein levels of the NF-κB p65 subunit in the nucleus were detected by Western blot (A) and confocal microscopy (B). Scale bar, 10 μm. (C and D) RAW264.7 cells were cotransfected with control or miR-27b mimic (C), or Bag2 expression vector (D) and NF-κB luciferase reporter construct, and then they were infected with H37Ra. The activity of luciferase was measured for the indicated time. The activity of firefly luciferase was normalized to that of Renilla luciferase. (EH) RAW264.7 cells were cotransfected with control or miR-27b mimic together with empty vector or Bag2 plasmid, followed by H37Ra infection. Protein expression levels of IL-6, IL-1β, TNF-α (E), and NO (G) were measured by ELISA and Griess assay. mRNA levels were measured by qPCR (F and H). All data are shown as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 versus the control group.

FIGURE 5.

miR-27b activates NF-κB signaling, and Bag2 reverses the effect of miR-27b on inflammatory factors in macrophages. (A and B) RAW264.7 cells were transfected with miR-27b mimic, followed by H37Ra infection for the indicated time. Protein levels of the NF-κB p65 subunit in the nucleus were detected by Western blot (A) and confocal microscopy (B). Scale bar, 10 μm. (C and D) RAW264.7 cells were cotransfected with control or miR-27b mimic (C), or Bag2 expression vector (D) and NF-κB luciferase reporter construct, and then they were infected with H37Ra. The activity of luciferase was measured for the indicated time. The activity of firefly luciferase was normalized to that of Renilla luciferase. (EH) RAW264.7 cells were cotransfected with control or miR-27b mimic together with empty vector or Bag2 plasmid, followed by H37Ra infection. Protein expression levels of IL-6, IL-1β, TNF-α (E), and NO (G) were measured by ELISA and Griess assay. mRNA levels were measured by qPCR (F and H). All data are shown as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 versus the control group.

Close modal

The BAG family members BAG-1 (23) and BAG-3 (24) increase the activity of NF-κB. We speculated that Bag2 also enhances the activity of NF-κB. We performed dual-luciferase reporter assays to test this hypothesis. As shown in Fig. 5D, Bag2 upregulated the activity of the NF-κB luciferase reporter construct compared with the control at the indicated time postinfection. Therefore, we explored whether overexpression of Bag2 could recover proinflammatory mediators in miR-27b–upregulated macrophages. We found that when Bag2 was overexpressed, miR-27b failed to inhibit the production of IL-1β, IL-6, TNF-α, and iNOS (Fig. 5E–H). Taken together, miR-27b inhibits the activity of NF-κB and proinflammatory mediators by targeting Bag2 in macrophages during H37Ra infection.

Bag2 interacts with p53 in tumors (25); p53 regulates mycobacteria-induced apoptosis (26). Therefore, we first investigated the role of Bag2 in the activity of p53 using a dual-luciferase reporter assay. We confirmed that Bag2 suppressed p53 signaling (Fig. 6A). Furthermore, coimmunoprecipitation assays showed that with cotransfection or single transfection, Bag2 is associated with p53 in HEK293T and RAW264.7 cells (Fig. 6B, 6C).

FIGURE 6.

Bag2 negatively regulates p53 signaling to control apoptosis and production of ROS and enhances intracellular H37Ra survival in macrophages. (A) Control or Bag2 expression vector and p53-luciferase reporter vector were cotransfected into RAW264.7 cells prior to H37Ra infection. The activity of p53 luciferase was analyzed by dual-luciferase reporter assay. Results are shown as the fold change compared with the control with H37Ra infection for 6 h. (B) HEK293T cells were cotransfected with vectors expressing Bag2-Flag and p53-HA. The interaction of Bag2 with p53 in HEK293T cells was confirmed by coimmunoprecipitation assays followed by Western blot assays. (C) RAW264.7 cells were transfected with vectors expressing Bag2-Flag or p53-HA or cotransfected with Bag2-Flag and p53-HA. The interaction of Bag2 with p53 in RAW264.7 cells was confirmed by coimmunoprecipitation followed by Western blot assays. (DI) RAW264.7 cells were transfected with si-NC or siBag2 without or with PFTα (10 μM) treatment, followed by H37Ra infection. Protein levels of Bag2 were measured by Western blot (D). Cell apoptosis ratio was tested by annexin V staining followed by flow cytometric analysis (E and F). Cell viability was assayed by CCK8 (G). Intracellular survival of H37Ra was detected by CFU assay (H). ROS levels were measured by flow cytometry (I). Results are shown as the fold change compared with siNC. Data are mean ± SD of three separate experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 6.

Bag2 negatively regulates p53 signaling to control apoptosis and production of ROS and enhances intracellular H37Ra survival in macrophages. (A) Control or Bag2 expression vector and p53-luciferase reporter vector were cotransfected into RAW264.7 cells prior to H37Ra infection. The activity of p53 luciferase was analyzed by dual-luciferase reporter assay. Results are shown as the fold change compared with the control with H37Ra infection for 6 h. (B) HEK293T cells were cotransfected with vectors expressing Bag2-Flag and p53-HA. The interaction of Bag2 with p53 in HEK293T cells was confirmed by coimmunoprecipitation assays followed by Western blot assays. (C) RAW264.7 cells were transfected with vectors expressing Bag2-Flag or p53-HA or cotransfected with Bag2-Flag and p53-HA. The interaction of Bag2 with p53 in RAW264.7 cells was confirmed by coimmunoprecipitation followed by Western blot assays. (DI) RAW264.7 cells were transfected with si-NC or siBag2 without or with PFTα (10 μM) treatment, followed by H37Ra infection. Protein levels of Bag2 were measured by Western blot (D). Cell apoptosis ratio was tested by annexin V staining followed by flow cytometric analysis (E and F). Cell viability was assayed by CCK8 (G). Intracellular survival of H37Ra was detected by CFU assay (H). ROS levels were measured by flow cytometry (I). Results are shown as the fold change compared with siNC. Data are mean ± SD of three separate experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

Because Bag2 is known to limit apoptosis (27) and inhibit the activity of p53, we speculated that Bag2 modulates H37Ra-induced apoptosis via the inactivation of p53 signaling. To verify this hypothesis, we used siRNA to block the expression of Bag2 (siBag2) (Fig. 6D) and used pifithrin-α (PFTα) to chemically suppress nuclear translocation of p53 during infection of macrophages. Annexin V assays showed that transfection of cells with siBag2 enhanced cell apoptosis; this effect was significantly restricted in cells cotreated with PFTα and Bag2 siRNA during infection (Fig. 6E, 6F). Furthermore, in line with the above result, we observed that siBag2-transfected macrophages decreased cell viability (Fig. 6G) and survival of the M. tuberculosis H37Ra strain (Fig. 6H), whereas the addition of PFTα remarkably reduced these effects during infection. The above results indicate that Bag2 inhibits H37Ra-induced apoptosis via inactivating p53.

It is known that p53 acts as an upstream regulator of the production of ROS (28). To further test the relationship of Bag2 and p53 with the production of ROS, we used DCFDA flow cytometry to measure the activity of ROS. Our results showed that cells transfected with siBag2 markedly increased the formation of ROS compared with cells cotreated with siBag2 and PFTα during H37Ra stimulation (Fig. 6I). Taken together, these data show that Bag2 regulates the fate of H37Ra-infected macrophages and production of ROS by regulating the activity of p53.

Because Bag2 inhibits p53 signaling, we first investigated whether miR-27b could activate p53 signaling. We employed a dual-luciferase reporter assay and found that miR-27b enhanced the activity of the p53 luciferase reporter vector post–H37Ra infection compared with the control (Fig. 7A). Next, we studied the effect of miR-27b and p53 on H37Ra-induced apoptosis. PFTα greatly inhibited the ability of miR-27b to increase apoptosis (Fig. 7B, 7C) and decrease cell viability (Fig. 7D) and H37Ra survival (Fig. 7E) during infection. In addition, PFTα diminished the miR-27b–induced increase in the production of ROS (Figs. 7F, 8). Together, these data demonstrate that the function of miR-27b in increasing apoptosis and eliminating cellular bacterial depends on the p53 pathway in macrophages.

FIGURE 7.

miR-27b is required for activation of p53 and enhancement of apoptosis and ROS levels in macrophages during infection. (A) Control mimic or miR-27b mimic, Bag2 expression vector, and p53-luciferase reporter vector were cotransfected into RAW264.7 cells prior to H37Ra infection. The activity of p53 luciferase was analyzed by dual-luciferase reporter assay. (BF) RAW264.7 cells were transfected with control or miR-27b mimic and then left untreated or treated with PFTα (10 μM), followed by H37Ra infection. Cell apoptosis was analyzed by annexin V assay (B and C). Cells survival was assessed by CCK8 assay (D). H37Ra viability was detected by CFU assay (E). Production of ROS was determined by flow cytometry (F). Data are shown as mean ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed Student t-test.

FIGURE 7.

miR-27b is required for activation of p53 and enhancement of apoptosis and ROS levels in macrophages during infection. (A) Control mimic or miR-27b mimic, Bag2 expression vector, and p53-luciferase reporter vector were cotransfected into RAW264.7 cells prior to H37Ra infection. The activity of p53 luciferase was analyzed by dual-luciferase reporter assay. (BF) RAW264.7 cells were transfected with control or miR-27b mimic and then left untreated or treated with PFTα (10 μM), followed by H37Ra infection. Cell apoptosis was analyzed by annexin V assay (B and C). Cells survival was assessed by CCK8 assay (D). H37Ra viability was detected by CFU assay (E). Production of ROS was determined by flow cytometry (F). Data are shown as mean ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed Student t-test.

Close modal
FIGURE 8.

Model illustrating how miR-27b mediates the inflammatory response and cell apoptosis via targeting Bag2. M. tuberculosis–induced miR-27b is dependent on the TLR2/MyD88/NF-κB signaling pathway. Upregulation of miR-27b targets Bag2 and inhibits proinflammatory cytokines and the activity of NF-κB in macrophages. Furthermore, miR-27b enhances p53 signaling and production of ROS, thereby increasing cell apoptosis and controlling H37Ra survival.

FIGURE 8.

Model illustrating how miR-27b mediates the inflammatory response and cell apoptosis via targeting Bag2. M. tuberculosis–induced miR-27b is dependent on the TLR2/MyD88/NF-κB signaling pathway. Upregulation of miR-27b targets Bag2 and inhibits proinflammatory cytokines and the activity of NF-κB in macrophages. Furthermore, miR-27b enhances p53 signaling and production of ROS, thereby increasing cell apoptosis and controlling H37Ra survival.

Close modal

Several biological processes are associated with the expression of miRNA (29). In the current study, the role of miRNAs in regulating host immunity to M. tuberculosis was explored (30, 31). However, the biological role of miRNAs in host cell apoptosis during M. tuberculosis infection remains unclear. H37Ra-infected macrophage apoptosis is a defense response employed to control intracellular mycobacterial survival (32). Our study showed that miR-27b facilitates the ability of H37Ra to trigger apoptosis, suggesting an important function of miRNA in governing the microbicidal mechanism during M. tuberculosis infection. Mechanistically, we discovered that miR-27b positively regulates apoptosis by directly targeting Bag2 and increasing the activity of the p53–ROS signaling pathway.

In the current study, we demonstrated that the expression of miR-27b is upregulated by M. tuberculosis H37Ra (an avirulent strain of M. tuberculosis) and H37Rv (a virulent strain of M. tuberculosis) in vitro and in vivo to control the survival of M. tuberculosis, suggesting a potential correlation of miR-27b with mycobacterial infection. Our previous study also showed that H37Ra infection upregulates the expression of 41 miRNAs, including miR-21, miR-155, miR-23b, and miR-29a (data not shown). Recent studies found that the induced expression of miRNA-155 was essential for protective immune response and macrophage apoptosis (33, 34), thus decreasing the survival of intracellular mycobacteria in macrophages. Together, these studies indicate that the expression of multiple miRNAs is upregulated post–mycobacterial infection, which may benefit host defense mechanisms against mycobacteria. However, previous studies found that the expression of miR-27b in human monocyte-derived macrophages (MDMs) was different from that stated in our data (35). The contradiction in the expression of miR-27b between our findings using macrophages and those of others using human MDMs may be due to the difference in cell types, time of infection, and growth phases of the bacteria. This notion was tested in individual miRNAs. For instance, the expression of miR-26a at 72 h post–H37Rv infection was upregulated in human MDMs (36), whereas that of miR-26a was downregulated in RAW264.7 or BMDMs 24 h postinfection (37). These factors could cause differential expression patterns of miR-27b during mycobacterial infection and require further investigation.

Several studies have demonstrated that miR-27b plays an important role in regulating various physical and pathological process by repressing distinct targets, such as KH-type splicing regulatory protein (38) and apoptotic protease activating factor-1 (Apaf-1) (39). Most of these miR-27b targets modulate inflammatory response and apoptosis. Studies have demonstrated that the function of individual miRNAs may be cell-type specific (40). For example, miR-27b inhibits neuron apoptosis by targeting Apaf-1 (41), whereas miR-27b enhances tamoxifen-induced breast cancer cell apoptosis by targeting nuclear receptor subfamily 5 group A member 2 and cAMP-response element binding protein 1 (42).

Bag2 is a member of the BAG family that is characterized by the BAG domain. As a group of multipurpose proteins, Bag2 is linked with various transcription factors and administrates a series of intracellular courses (43, 44); however, no precise function of Bag2 has been reported in the host defense mechanism against M. tuberculosis. Our study validates Bag2 as a critical target of miR-27b and shows that the activity of the Bag2 3′-UTR reporter plasmid is suppressed in the presence of a miR-27b mimic during infection. Similarly, the expression of miR-27b was upregulated, and the endogenous expression of Bag2 was inhibited post–H37Ra infection, suggesting that M. tuberculosis–induced overexpression of miR-27b is associated with the inhibition of Bag2. Furthermore, recent studies have shown that Bag2 binds to Sp110 (a positive anti-mycobacterial regulator) (45) and unphosphorylated STAT1 (46) to alter the cell death pathway. These results indicate a critical role of Bag2 in modulating macrophage resistance to M. tuberculosis.

Known as the guardian of the genome, p53 exhibits numerous roles in biological functions, particularly proapoptotic effects. To date, underlying mechanisms of p53-mediated apoptosis have been widely investigated (47, 48). In the current context, we uncovered a new relationship between p53 and Bag2 in macrophage apoptosis. The silencing of Bag2 enhanced miR-27b–induced p53-dependent apoptosis during M. tuberculosis infection, suggesting that Bag2 can act downstream of miR-27b and upstream of p53. In addition, mutp53 promotes BAG2 nuclear translocation and BAG2 inhibits the binding of MDM2 to mutp53, thereby restricting ubiquitination and degradation of the mutp53 protein mediated by MDM2 in tumors (25). Of note, our results revealed that Bag2 can interact with wtp53 and inhibit the p53–ROS pathway in macrophages. ROS is important in the innate immune response to various intracellular bacterial infections, including mycobacterial infections (49), and is required for the induction of p53-dependent apoptosis (50). We demonstrated that Bag2 controls infected cell apoptosis by inactivating the p53–ROS pathway.

In infected macrophages, elimination of M. tuberculosis requires a proper immune response; however, an abnormal inflammatory response may result in the transmission and diffusion of pathogens (51, 52). Therefore, effective modulation of inflammation is vital for controlling the progress of M. tuberculosis infection. We demonstrated that the H37Ra-trigged TLR2/MyD88/NF-κB pathway induces expression of miR-27b, and, in turn, miR-27b represses the activity of NF-κB and proinflammatory genes. Negative feedback could represent an important link between the host and M. tuberculosis, suggesting that miR-27b may play a vital role in avoiding an excessive inflammatory reaction and sustaining proinflammatory mediators at optimal levels to balance mycobacterial survival and tissue lesions, thereby facilitating host resistance to pathogens.

As a major transcription factor, NF-κB is not only an antiapoptotic regulator in macrophages but also regulates a series of inflammatory responses (53). Several miRNAs are involved in regulating the NF-κB pathway. For example, miR-26b enhances the LPS-induced NF-κB signaling pathway and the production of proinflammatory factors by targeting PTEN (54). Furthermore, miR-3570 inhibits the bacteria-induced inflammatory response of miiuy croaker via the MyD88-mediated NF-κB signaling pathway by targeting MyD88 (55). The function of miR-27b in the regulation of the NF-κB signaling pathway remains unclear. In this study, we reported a new regulatory network in which miR-27b targets a positive regulator of NF-κB signaling in macrophages.

In summary, our study highlights signaling networks by which M. tuberculosis–induced miR-27b controls infected cell survival and eliminates intracellular pathogens via activation of the p53 pathway by directly targeting Bag2. Meanwhile, miR-27b and the NF-κB pathway form a negative feedback loop to avoid an excessive inflammatory response (Fig. 8). Our findings offer the basis to further investigate pathogenesis and therapies for tuberculosis using miRNAs and also explain the molecular basis for the enhancement of apoptosis by avirulent (H37Ra) strains of M. tuberculosis.

We thank Jingcheng Zhang and Kezhen Yao for reading the manuscript and providing valuable insights and Life Science Research Core Services for technological assistance using the confocal microscope.

This work was supported by the National Natural Science Foundation of China (31530075) and the National Science and Technology Major Project in New Varieties Cultivation of Transgenic Organisms (2016ZX08007-003).

The online version of this article contains supplemental material.

Abbreviations used in this article:

     
  • Bag2

    Bcl-2–associated athanogene 2

  •  
  • BMDM

    bone marrow–derived macrophage

  •  
  • CCK8

    cholecystokinin 8

  •  
  • HA

    hemagglutinin

  •  
  • iNOS

    inducible NO synthase

  •  
  • MDM

    monocyte-derived macrophage

  •  
  • miRNA

    microRNA

  •  
  • MOI

    multiplicity of infection

  •  
  • mut

    mutated

  •  
  • PFTα

    pifithrin-α

  •  
  • qPCR

    quantitative PCR

  •  
  • ROS

    reactive oxygen species

  •  
  • siRNA

    small interfering RNA

  •  
  • UTR

    untranslated region

  •  
  • WT

    wild-type.

1
Russell
,
D. G.
2001
.
Mycobacterium tuberculosis: here today, and here tomorrow.
Nat. Rev. Mol. Cell Biol.
2
:
569
577
.
2
Behar
,
S. M.
,
C. J.
Martin
,
M. G.
Booty
,
T.
Nishimura
,
X.
Zhao
,
H. X.
Gan
,
M.
Divangahi
,
H. G.
Remold
.
2011
.
Apoptosis is an innate defense function of macrophages against Mycobacterium tuberculosis.
Mucosal Immunol.
4
:
279
287
.
3
Cooper
,
A. M.
2009
.
Cell-mediated immune responses in tuberculosis.
Annu. Rev. Immunol.
27
:
393
422
.
4
van Crevel
,
R.
,
T. H. M.
Ottenhoff
,
J. W. M.
van der Meer
.
2002
.
Innate immunity to Mycobacterium tuberculosis.
Clin. Microbiol. Rev.
15
:
294
309
.
5
de Chastellier
,
C.
2009
.
The many niches and strategies used by pathogenic mycobacteria for survival within host macrophages.
Immunobiology
214
:
526
542
.
6
Lu
,
L. F.
,
A.
Liston
.
2009
.
MicroRNA in the immune system, microRNA as an immune system.
Immunology
127
:
291
298
.
7
Kumar
,
M.
,
S. K.
Sahu
,
R.
Kumar
,
A.
Subuddhi
,
R. K.
Maji
,
K.
Jana
,
P.
Gupta
,
J.
Raffetseder
,
M.
Lerm
,
Z.
Ghosh
, et al
.
2015
.
MicroRNA let-7 modulates the immune response to Mycobacterium tuberculosis infection via control of A20, an inhibitor of the NF-κB pathway.
Cell Host Microbe
17
:
345
356
.
8
Singh
,
Y.
,
V.
Kaul
,
A.
Mehra
,
S.
Chatterjee
,
S.
Tousif
,
V. P.
Dwivedi
,
M.
Suar
,
L.
Van Kaer
,
W. R.
Bishai
,
G.
Das
.
2013
.
Mycobacterium tuberculosis controls microRNA-99b (miR-99b) expression in infected murine dendritic cells to modulate host immunity.
J. Biol. Chem.
288
:
5056
5061
.
9
Wang
,
J.
,
K.
Yang
,
L.
Zhou
,
Y.
Minhaowu
,
Y.
Wu
,
M.
Zhu
,
X.
Lai
,
T.
Chen
,
L.
Feng
,
M.
Li
, et al
.
2013
.
MicroRNA-155 promotes autophagy to eliminate intracellular mycobacteria by targeting Rheb.
PLoS Pathog.
9
:
e1003697
.
10
Kim
,
J. K.
,
J. M.
Yuk
,
S. Y.
Kim
,
T. S.
Kim
,
H. S.
Jin
,
C. S.
Yang
,
E. K.
Jo
.
2015
.
MicroRNA-125a inhibits autophagy activation and antimicrobial responses during mycobacterial infection.
J. Immunol.
194
:
5355
5365
.
11
Wu
,
Y.
,
Z.
Guo
,
K.
Yao
,
Y.
Miao
,
S.
Liang
,
F.
Liu
,
Y.
Wang
,
Y.
Zhang
.
2016
.
The transcriptional foundations of Sp110-mediated macrophage (RAW264.7) resistance to Mycobacterium tuberculosis H37Ra.
Sci. Rep.
6
:
22041
.
12
O’Neill
,
L. A.
,
F. J.
Sheedy
,
C. E.
McCoy
.
2011
.
MicroRNAs: the fine-tuners of Toll-like receptor signalling.
Nat. Rev. Immunol.
11
:
163
175
.
13
Li
,
M.
,
J.
Wang
,
Y.
Fang
,
S.
Gong
,
M.
Li
,
M.
Wu
,
X.
Lai
,
G.
Zeng
,
Y.
Wang
,
K.
Yang
,
X.
Huang
.
2016
.
microRNA-146a promotes mycobacterial survival in macrophages through suppressing nitric oxide production.
[Published erratum appears in 2016 Sci. Rep. 6: 24555.]
Sci. Rep.
6
:
23351
.
14
Xie
,
N.
,
H.
Cui
,
S.
Banerjee
,
Z.
Tan
,
R.
Salomao
,
M.
Fu
,
E.
Abraham
,
V. J.
Thannickal
,
G.
Liu
.
2014
.
miR-27a regulates inflammatory response of macrophages by targeting IL-10.
J. Immunol.
193
:
327
334
.
15
Fremond
,
C. M.
,
V.
Yeremeev
,
D. M.
Nicolle
,
M.
Jacobs
,
V. F.
Quesniaux
,
B.
Ryffel
.
2004
.
Fatal Mycobacterium tuberculosis infection despite adaptive immune response in the absence of MyD88.
J. Clin. Invest.
114
:
1790
1799
.
16
Li
,
Q.
,
I. M.
Verma
.
2002
.
NF-κB regulation in the immune system.
[Published erratum appears in 2002 Nat. Rev. Immunol. 2: 975.]
Nat. Rev. Immunol.
2
:
725
734
.
17
Koul
,
A.
,
T.
Herget
,
B.
Klebl
,
A.
Ullrich
.
2004
.
Interplay between mycobacteria and host signalling pathways.
Nat. Rev. Microbiol.
2
:
189
202
.
18
Giacomini
,
E.
,
E.
Iona
,
L.
Ferroni
,
M.
Miettinen
,
L.
Fattorini
,
G.
Orefici
,
I.
Julkunen
,
E. M.
Coccia
.
2001
.
Infection of human macrophages and dendritic cells with Mycobacterium tuberculosis induces a differential cytokine gene expression that modulates T cell response.
J. Immunol.
166
:
7033
7041
.
19
Drennan
,
M. B.
,
D.
Nicolle
,
V. J. F.
Quesniaux
,
M.
Jacobs
,
N.
Allie
,
J.
Mpagi
,
C.
Frémond
,
H.
Wagner
,
C.
Kirschning
,
B.
Ryffel
.
2004
.
Toll-like receptor 2-deficient mice succumb to Mycobacterium tuberculosis infection.
Am. J. Pathol.
164
:
49
57
.
20
Dharmaraja
,
A. T.
,
M.
Alvala
,
D.
Sriram
,
P.
Yogeeswari
,
H.
Chakrapani
.
2012
.
Design, synthesis and evaluation of small molecule reactive oxygen species generators as selective Mycobacterium tuberculosis inhibitors.
Chem. Commun. (Camb.)
48
:
10325
10327
.
21
Pautz
,
A.
,
J.
Art
,
S.
Hahn
,
S.
Nowag
,
C.
Voss
,
H.
Kleinert
.
2010
.
Regulation of the expression of inducible nitric oxide synthase.
Nitric Oxide
23
:
75
93
.
22
Lawrence
,
T.
2009
.
The nuclear factor NF-kappaB pathway in inflammation.
Cold Spring Harb. Perspect. Biol.
1
:
a001651
.
23
Clemo
,
N. K.
,
T. J.
Collard
,
S. L.
Southern
,
K. D.
Edwards
,
M.
Moorghen
,
G.
Packham
,
A.
Hague
,
C.
Paraskeva
,
A. C.
Williams
.
2008
.
BAG-1 is up-regulated in colorectal tumour progression and promotes colorectal tumour cell survival through increased NF-kappaB activity.
Carcinogenesis
29
:
849
857
.
24
Ammirante
,
M.
,
A.
Rosati
,
C.
Arra
,
A.
Basile
,
A.
Falco
,
M.
Festa
,
M.
Pascale
,
M.
d’Avenia
,
L.
Marzullo
,
M. A.
Belisario
, et al
.
2010
.
IKKγ protein is a target of BAG3 regulatory activity in human tumor growth.
Proc. Natl. Acad. Sci. USA
107
:
7497
7502
.
25
Yue
,
X.
,
Y.
Zhao
,
J.
Liu
,
C.
Zhang
,
H.
Yu
,
J.
Wang
,
T.
Zheng
,
L.
Liu
,
J.
Li
,
Z.
Feng
,
W.
Hu
.
2015
.
BAG2 promotes tumorigenesis through enhancing mutant p53 protein levels and function
.
Elife.
DOI: 10.7554/eLife.08401.001
.
26
Cruz
,
A.
,
P.
Ludovico
,
E.
Torrado
,
J. B.
Gama
,
J.
Sousa
,
J.
Gaifem
,
R.
Appelberg
,
F.
Rodrigues
,
A. M.
Cooper
,
J.
Pedrosa
, et al
.
2015
.
IL-17A promotes intracellular growth of Mycobacterium by inhibiting apoptosis of infected macrophages
.
Front. Immunol.
6
:
498
.
27
Kabbage
,
M.
,
M. B.
Dickman
.
2008
.
The BAG proteins: a ubiquitous family of chaperone regulators.
Cell. Mol. Life Sci.
65
:
1390
1402
.
28
Liu
,
B.
,
Y.
Chen
,
D. K.
St Clair
.
2008
.
ROS and p53: a versatile partnership.
Free Radic. Biol. Med.
44
:
1529
1535
.
29
Ebert
,
M. S.
,
P. A.
Sharp
.
2012
.
Roles for microRNAs in conferring robustness to biological processes.
Cell
149
:
515
524
.
30
Rajaram
,
M. V.
,
B.
Ni
,
J. D.
Morris
,
M. N.
Brooks
,
T. K.
Carlson
,
B.
Bakthavachalu
,
D. R.
Schoenberg
,
J. B.
Torrelles
,
L. S.
Schlesinger
.
2011
.
Mycobacterium tuberculosis lipomannan blocks TNF biosynthesis by regulating macrophage MAPK-activated protein kinase 2 (MK2) and microRNA miR-125b.
Proc. Natl. Acad. Sci. USA
108
:
17408
17413
.
31
Rothchild
,
A. C.
,
J. R.
Sissons
,
S.
Shafiani
,
C.
Plaisier
,
D.
Min
,
D.
Mai
,
M.
Gilchrist
,
J.
Peschon
,
R. P.
Larson
,
A.
Bergthaler
, et al
.
2016
.
MiR-155-regulated molecular network orchestrates cell fate in the innate and adaptive immune response to Mycobacterium tuberculosis.
Proc. Natl. Acad. Sci. USA
113
:
E6172
E6181
.
32
Lee
,
J.
,
M.
Hartman
,
H.
Kornfeld
.
2009
.
Macrophage apoptosis in tuberculosis.
Yonsei Med. J.
50
:
1
11
.
33
Yang
,
S.
,
F.
Li
,
S.
Jia
,
K.
Zhang
,
W.
Jiang
,
Y.
Shang
,
K.
Chang
,
S.
Deng
,
M.
Chen
.
2015
.
Early secreted antigen ESAT-6 of Mycobacterium Tuberculosis promotes apoptosis of macrophages via targeting the microRNA155-SOCS1 interaction.
Cell. Physiol. Biochem.
35
:
1276
1288
.
34
Ghorpade
,
D. S.
,
R.
Leyland
,
M.
Kurowska-Stolarska
,
S. A.
Patil
,
K. N.
Balaji
.
2012
.
MicroRNA-155 is required for Mycobacterium bovis BCG-mediated apoptosis of macrophages.
Mol. Cell. Biol.
32
:
2239
2253
.
35
Furci
,
L.
,
E.
Schena
,
P.
Miotto
,
D. M.
Cirillo
.
2013
.
Alteration of human macrophages microRNA expression profile upon infection with Mycobacterium tuberculosis.
Int. J. Mycobacteriol.
2
:
128
134
.
36
Ni
,
B.
,
M. V.
Rajaram
,
W. P.
Lafuse
,
M. B.
Landes
,
L. S.
Schlesinger
.
2014
.
Mycobacterium tuberculosis decreases human macrophage IFN-γ responsiveness through miR-132 and miR-26a.
J. Immunol.
193
:
4537
4547
.
37
Sahu
,
S. K.
,
M.
Kumar
,
S.
Chakraborty
,
S. K.
Banerjee
,
R.
Kumar
,
P.
Gupta
,
K.
Jana
,
U. D.
Gupta
,
Z.
Ghosh
,
M.
Kundu
,
J.
Basu
.
2017
.
MicroRNA 26a (miR-26a)/KLF4 and CREB-C/EBPβ regulate innate immune signaling, the polarization of macrophages and the trafficking of Mycobacterium tuberculosis to lysosomes during infection.
PLoS Pathog.
13
:
e1006410
.
38
Zhou
,
R.
,
A. Y.
Gong
,
A. N.
Eischeid
,
X. M.
Chen
.
2012
.
miR-27b targets KSRP to coordinate TLR4-mediated epithelial defense against Cryptosporidium parvum infection.
PLoS Pathog.
8
:
e1002702
.
39
Chen
,
Q.
,
F.
Zhang
,
Y.
Wang
,
Z.
Liu
,
A.
Sun
,
K.
Zen
,
C. Y.
Zhang
,
Q.
Zhang
.
2015
.
The transcription factor c-Myc suppresses MiR-23b and MiR-27b transcription during fetal distress and increases the sensitivity of neurons to hypoxia-induced apoptosis.
PLoS One
10
:
e0120217
.
40
Faraoni
,
I.
,
F. R.
Antonetti
,
J.
Cardone
,
E.
Bonmassar
.
2009
.
miR-155 gene: a typical multifunctional microRNA.
Biochim. Biophys. Acta
1792
:
497
505
.
41
Chen
,
Q.
,
J.
Xu
,
L.
Li
,
H.
Li
,
S.
Mao
,
F.
Zhang
,
K.
Zen
,
C. Y.
Zhang
,
Q.
Zhang
.
2014
.
MicroRNA-23a/b and microRNA-27a/b suppress Apaf-1 protein and alleviate hypoxia-induced neuronal apoptosis.
Cell Death Dis.
5
:
e1132
.
42
Zhu
,
J.
,
Z.
Zou
,
P.
Nie
,
X.
Kou
,
B.
Wu
,
S.
Wang
,
Z.
Song
,
J.
He
.
2016
.
Downregulation of microRNA-27b-3p enhances tamoxifen resistance in breast cancer by increasing NR5A2 and CREB1 expression.
Cell Death Dis.
7
:
e2454
.
43
Wang
,
H. Q.
,
H. Y.
Zhang
,
F. J.
Hao
,
X.
Meng
,
Y.
Guan
,
Z. X.
Du
.
2008
.
Induction of BAG2 protein during proteasome inhibitor-induced apoptosis in thyroid carcinoma cells.
Br. J. Pharmacol.
155
:
655
660
.
44
Hauser
,
B.
,
Y.
Zhao
,
X.
Pang
,
Z.
Ling
,
E.
Myers
,
P.
Wang
,
J.
Califano
,
X.
Gu
.
2015
.
Functions of MiRNA-128 on the regulation of head and neck squamous cell carcinoma growth and apoptosis.
PLoS One
10
:
e0116321
.
45
Wu
,
Y.
,
Z.
Guo
,
F.
Liu
,
K.
Yao
,
M.
Gao
,
Y.
Luo
,
Y.
Zhang
.
2017
.
Sp110 enhances macrophage resistance toMycobacterium tuberculosisvia inducing endoplasmic reticulum stress and inhibiting anti-apoptotic factors.
Oncotarget
8
:
64050
64065
.
46
Yao
,
K.
,
Q.
Chen
,
Y.
Wu
,
F.
Liu
,
X.
Chen
,
Y.
Zhang
.
2017
.
Unphosphorylated STAT1 represses apoptosis in macrophages duringMycobacteriumtuberculosisinfection.
J. Cell Sci.
130
:
1740
1751
.
47
Bieging
,
K. T.
,
S. S.
Mello
,
L. D.
Attardi
.
2014
.
Unravelling mechanisms of p53-mediated tumour suppression.
Nat. Rev. Cancer
14
:
359
370
.
48
Giorgi
,
C.
,
M.
Bonora
,
G.
Sorrentino
,
S.
Missiroli
,
F.
Poletti
,
J. M.
Suski
,
F.
Galindo Ramirez
,
R.
Rizzuto
,
F.
Di Virgilio
,
E.
Zito
, et al
.
2015
.
p53 at the endoplasmic reticulum regulates apoptosis in a Ca2+-dependent manner.
Proc. Natl. Acad. Sci. USA
112
:
1779
1784
.
49
Roca
,
F. J.
,
L.
Ramakrishnan
.
2013
.
TNF dually mediates resistance and susceptibility to mycobacteria via mitochondrial reactive oxygen species.
Cell
153
:
521
534
.
50
Johnson
,
T. M.
,
Z. X.
Yu
,
V. J.
Ferrans
,
R. A.
Lowenstein
,
T.
Finkel
.
1996
.
Reactive oxygen species are downstream mediators of p53-dependent apoptosis.
Proc. Natl. Acad. Sci. USA
93
:
11848
11852
.
51
O’Garra
,
A.
,
P. S.
Redford
,
F. W.
McNab
,
C. I.
Bloom
,
R. J.
Wilkinson
,
M. P.
Berry
.
2013
.
The immune response in tuberculosis.
Annu. Rev. Immunol.
31
:
475
527
.
52
Cilfone
,
N. A.
,
C. B.
Ford
,
S.
Marino
,
J. T.
Mattila
,
H. P.
Gideon
,
J. L.
Flynn
,
D. E.
Kirschner
,
J. J.
Linderman
.
2015
.
Computational modeling predicts IL-10 control of lesion sterilization by balancing early host immunity-mediated antimicrobial responses with caseation during mycobacterium tuberculosis infection.
J. Immunol.
194
:
664
677
.
53
Lawrence
,
T.
,
C.
Fong
.
2010
.
The resolution of inflammation: anti-inflammatory roles for NF-kappaB.
Int. J. Biochem. Cell Biol.
42
:
519
523
.
54
Zhang
,
L.
,
C.
Huang
,
Y.
Guo
,
X.
Gou
,
M.
Hinsdale
,
P.
Lloyd
,
L.
Liu
.
2015
.
MicroRNA-26b modulates the NF-κB pathway in alveolar macrophages by regulating PTEN.
J. Immunol.
195
:
5404
5414
.
55
Chu
,
Q.
,
Y.
Sun
,
J.
Cui
,
T.
Xu
.
2017
.
MicroRNA-3570 modulates the NF-κB pathway in teleost fish by targeting MyD88.
J. Immunol.
198
:
3274
3282
.

The authors have no financial conflicts of interest.

Supplementary data