M1 macrophage dependent-p53 regulates the intracellular survival of mycobacteria
Tumor suppressor p53 is not only affects immune responses but also contributes to antibacterial activity. However, its bactericidal function during mycobacterial infection remains unclear. In this study, we found that the p53-deficient macrophages failed to control Mycobacterium tuberculosis (Mtb), manifested as a lower apoptotic cell death rate and enhanced intracellular survival. The expression levels of p53 during Mtb infection were stronger in M1 macrophages than in M2 macrophages. The TLR2/JNK signaling pathway plays an essential role in the modulation of M1 macrophage polarization upon Mtb infection. It facilitates p53-mediated apoptosis through the production of reactive oxygen species, nitric oxide and inflammatory cytokines in Mtb-infected M1 macrophages. In addition, nutlin-3 effectively abrogated the intracellular survival of mycobacteria in both TB patients and healthy controls after H37Ra infection for 24 h, indicating that the enhancement of p53 production effectively suppressed the intracellular survival of Mtb in hosts. These results suggest that p53 can be a new therapeutic target for TB therapy.
KeywordsApoptosis Tuberculosis Mycobacteria Macrophages p53 Polarization
Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is estimated to affect more than one-fourth of the world’s population, and it is the most important bacterial infection worldwide. During Mtb infection, macrophages provide a critical first line of host defense . Macrophages recognize Mtb antigens on the bacterial cell surface and secrete proteins in response to various receptors, including Toll-like receptors (TLRs), to modulate inflammatory responses and bactericidal functions . Macrophages are classified as M1 and M2 according to their functions. Classically activated (M1) macrophages are polarized by lipopolysaccharide (LPS) and interferon γ (IFNγ), while alternatively activated (M2) macrophages are polarized by interleukin-4 (IL-4) and IL-13 [3, 4]. Generally, M1 macrophages produce pro-inflammatory cytokines, reactive oxygen species (ROS), and nitric oxide (NO), leading to bacterial death . In a previous study, we showed that virulent mycobacterial infection skewed macrophages from the M1 to M2 type .
Apoptosis is an important mechanism in immune cells during bacterial and viral infection [7, 8]. Because many intracellular bacterial or viral pathogens evade the defenses of the immune system and hide within the host cell for their replication, the induction of apoptosis in infected cells can be used to limit their survival . Recent studies have suggested that inducing the apoptosis of Mtb-infected host cells helps maintain host defense [10, 11]. Our previous studies have consistently indicated that apoptosis mediated by endoplasmic reticulum (ER) stress in macrophages benefits the host against Mtb infection [12, 13, 14]. Importantly, ER stress-mediated apoptosis effectively removes Mtb in M1-polarized macrophages more so than in M2 macrophages . In this study, we hypothesized that M1-polarized macrophages might be useful for eliminating intracellular Mtb via the induction of pro-apoptotic-associated mechanisms such as p53-dependent apoptosis.
The tumor suppressor gene p53 is a transcription factor that promotes target genes associated with DNA repair, cell cycle arrest, senescence and programmed cell death, thereby limiting tumorigenesis [15, 16]. Upon phosphorylation and acetylation, activated p53 can directly bind to specific DNA sequences in the promoter regions of target genes including those regulating apoptosis, DNA repair, and the cell cycle [16, 17, 18]. p53 plays a key modulating role in the pro-apoptotic effect between the extrinsic and intrinsic pathways, through transcriptional regulation of its target genes such as p53 upregulated modulator of apoptosis (PUMA), NOXA, Bcl2-associated X (Bax) and BH3 interacting-domain death agonist (Bid), which release apoptotic proteins from the mitochondria, activating caspases and apoptosis .
The activation of p53 is initiated by oxidative stresses, including ROS and NO, which may in turn upregulate inflammation and programmed cell death. In addition, p53 promotes cytochrome c release and caspase activation, resulting in apoptotic cell death though mitochondrial ROS and NO generation. p53 interacts with the nuclear factor κB (NF-κB)  and mitogen-activated protein kinase (MAPK) pathways  in inflammatory and immune responses. Owing to these regulatory functions, we hypothesized that p53 is involved in the modulation of macrophage polarization. Recent evidences have revealed that the presence of p53 is important for infected cells to have a bactericidal effect in various infectious diseases, including influenza, pneumonia, chlamydia, listeriosis and Helicobacter pylori infections [22, 23, 24, 25, 26, 27]. Mtb infection also increases p53 gene expression in a human monocytic cell line  and peripheral blood human monocytes . A previous study showed that Mtb-induced tumor necrosis factor (TNF)-α modulates p53 expression in macrophages cell line . However, the detailed functions of p53 during mycobacterial infection remain poorly understood.
In this study, we investigated the role of p53 in abrogating the intracellular survival of Mtb in macrophages. Our results revealed an antibacterial role of p53 through apoptotic cell death of M1-polarized macrophages during mycobacterial infection.
The p53 expression in Mtb-infected macrophages controls intracellular survival
TLR2-MAPK signaling activation is essential for p53 activation in Mtb-infected macrophages
Mtb-increased p53 activation is related to the anti-mycobacterial effects of M1 macrophages
Next, we checked TLR2-dependent p53 activation in M1-polarized macrophages during H37Ra infection. Notwithstanding skewing toward M1 macrophages, the absence of TLR2 resulted in significantly lower levels of p53 protein in M1 macrophages (Fig. 3e). Furthermore, apoptosis was significantly lower in TLR2-deficient M1 macrophages than in WT controls (Fig. 3f). Subsequently, increased intracellular survival of Mtb was observed at 48 h in TLR2 KO BMDMs (Fig. 3g). Regardless of the M1 or M2 macrophage subtype, the survival of Mtb was reduced in TLR2 KO BMDMs (Fig. S3g). Collectively, these data suggest that TLR2-dependent p53 activation is essential for the clearance of intracellular Mtb in M1 macrophages.
p53 activation in M1 macrophages is closely associated with JNK activation
p53 activation in M1 occurs though the production of NO, ROS and inflammatory cytokines
p53 activation controls intracellular Mtb survival in the lungs of mice and in the monocyte-derived macrophages (MDMs) of TB patients
The activity of p53 is modulated by interactions with the E3 ligase MDM2, which binds stably to p53, leading to its degradation . We found that MDM2 protein expression was upregulated in H37Rv-infected macrophages compared to H37Ra-infected cells, and was upregulated to a greater extent in Mtb-infected M2 macrophages than in M1 types. Indeed, p53 signaling can regulate M2 macrophage polarization. Earlier studies have shown that p53 activated by nutlin-3 treatment powerfully decreases the expression of M2-associated genes , resulting in the suppression of M2-like tumor-associated macrophages (TAMs) [36, 37]. Meanwhile, the functions of p53 are disrupted in TAMs, which promotes invasion, metastasis, proliferation, and survival . Similarly, virulent H37Rv infection can skew toward M2 macrophages and in turn suppress p53 by inducing MDM2 activation. Of note, nutlin-3 treatment also effectively decreased intracellular Mtb in M2-polarized macrophages, although the M2 phenotypes benefit bacterial survival. These results suggest that M2-skewed polarization suppresses p53-mediated cell death via MDM2 induction, which might be considered a unique survival strategy of Mtb. Therefore, p53 activation could be a good strategy for TB treatment.
The balance between M1 and M2 macrophage polarization plays an important role in TB . The macrophage polarization ratio determines immune functions, including inflammation and antimicrobial activity within the granuloma model, and NF-κB signaling is particularly important to promote M1 polarization during early TB infection . Most discussions concerning the relationship between macrophage polarization and p53 have focused on their roles in cancer [35, 36, 41, 42], but not in infectious diseases such as TB. Previously, we suggested that ER stress-mediated apoptosis in M1 macrophages is important and required for the elimination of intracellular Mtb . M1 macrophages highly promote the generation of ROS/NO and secretion of pro-inflammatory cytokines, which are well known activators of the ER stress response. In addition to the ER stress response, p53-dependent signaling is regulated by ROS/NO production or inflammation. Here, we note that p53 activation in M1-polarized macrophages plays critical roles in the apoptosis-mediated bactericidal effect against Mtb, an effect that is similar to the ER stress response. It is important to determine the effective defense mechanism against Mtb in M1 phenotypes.
p53 modulates inflammation and immune responses resulting from the production of inflammatory cytokines via activation of the NF-κB and MAPK pathways [43, 44]. In addition, it is activated by the increased generation of ROS and reactive nitrogen species (RNS) during inflammation [45, 46]. We propose that p53 activation in Mtb-infected macrophages is involved in the TLR2 signaling pathway, which triggers both NF-κB and MAPK activation. We suggest that activation of the NF-κB and MAPK pathways during Mtb infection contributes to the production of ROS/NO and inflammatory cytokines including IL-12, IL-6, and TNFα, and that such stressful stimuli might lead to p53-mediated apoptosis via MAPK activation. Furthermore, recent studies have shown that NO- or ROS-related mechanisms in macrophages are used for host defense and inflammatory responses in various diseases. In particular, M1 macrophages produce NO, ROS, and pro-inflammatory cytokines at high levels compared to M2 types . Thus, increased production of NO, ROS, and pro-inflammatory cytokines in M1 macrophages likely has a positive influence on p53 activation during Mtb infection.
We observed that p53 activation in M1 polarization is tightly linked to the JNK-dependent pathway. The expression levels of iNOS and M1-related p53 activation were decreased by the JNK specific inhibitor or JNK siRNA transfection even when macrophages were treated with M1-polarizing stimuli. Our findings are consistent with the literature; most recent studies have reported that JNK activation is an essential factor driving M1 macrophage polarization via the secretion of pro-inflammatory cytokines [48, 49, 50, 51]. Moreover, signal transducer and activator of transcription 1 (STAT1) has emerged as a major transcriptional factor in M1 polarization. By contrast, ERK1/2, STAT3, and STAT6 signaling are associated with TAMs . Furthermore, Mtb-infected macrophages promote anti-inflammatory responses via TLR2-dependent ERK signaling activation . It is well known that the MEKK1/JNK signaling is important for stabilization and activation of p53 to mediate apoptosis . Similarly, our results showed that p53-mediated apoptosis depends on JNK signaling activation in M1 types. Importance of dynamic epigenetic regulation during monocyte to macrophage differentiation has been suggested after infection or vaccination . Macrophage activation can be trained by environmental signals such as cytokines and microbial components . Based on our observations, p53 signaling pathway is important for the protective effects of trained immunity during mycobacterial infection. Although p53 was not directly associated with macrophage polarization during Mtb infection, but we found that p53 expression was increased in Mtb-infected M1 macrophages though TLR2-JNK pathway. Thus, it is estimated that the apoptotic machinery of JNK-dependent p53 activation in M1 might be a promising strategy for the inhibition of Mtb survival.
Materials and Methods
Mice and cells
Pathogen-free female WT, TLR4-deficient, TLR2-deficient, MyD88-deficient, p53flox/flox, and LysM-Cre;p53flox/flox conditional knockout mice (C57BL/6 background) were maintained in specific-pathogen-free conditions and used at 6–8 weeks of age in all experiments. BMDMs were isolated and polarized to M1 or M2 types as described previously . Briefly, BMDMs were isolated and differentiated for 4–5 days in Dulbecco’s minimal essential medium (DMEM) containing 10% fetal bovine serum (FBS), penicillin (100 IU/mL), streptomycin (100 μg/mL), and 25 ng/mL macrophage colony-stimulating factor (M-CSF; R&D Systems, Minneapolis, MN, USA). For M1 polarization, macrophages were incubated with 10 ng/mL lipopolysaccharide (LPS; InvivoGen, San Diego, CA, USA) plus 10 ng/mL mouse IFN-γ (R&D Systems) for 24 h. For M2, cells were incubated with 10 ng/mL mouse IL-4 (R&D Systems) plus 10 ng/mL mouse IL-13 (R&D Systems) for 24 h. WT and JNK-/- mouse embryonic fibroblasts (MEFs) were cultured in DMEM containing 10% FBS, penicillin and streptomycin at 37 °C and 5% CO2. Human PBMCs were isolated from heparinized venous blood using Lymphoprep (Axis-Shield, Dundee, UK) as described previously . For macrophage differentiation, adherent monocytes were incubated in RPMI 1640 with 10% pooled human serum, 1% l-glutamine, 50 IU/ml penicillin, and 50 μg/ml streptomycin for 1 h at 37 °C, and nonadherent cells were removed. Human MDMs were prepared by culturing peripheral blood monocytes for 4 days in the presence of 4 ng/ml human CSF/macrophage colony-stimulating factor (Sigma-Aldrich, St. Louis, MO, USA) as described previously .
Blood samples were collected from 9 healthy controls and 13 patients with active TB. The mean age of TB patients (male: n = 7; female: n = 6) was 56.153 ± 22.345 years, and that of the healthy group (male: n = 8, female: n = 1) was 28.111 ± 7.975 years. All patients were newly diagnosed with TB disease during 2017–2018, and blood samples were collected from patients before treatment began.
Mtb infection and intracellular survival analyses in vitro and in vivo
Mycobacterial culture and in vitro macrophage infection were performed as described previously . Briefly, the Mtb strain H37Ra (ATCC 25177) was grown in Middlebrook 7H9 liquid medium supplemented with 10% OADC (oleic acid, albumin, dextrose, catalase) and 5% glycerol and then was suspended in phosphate-buffered saline (PBS) at a concentration of 1 × 108 CFU/mL. Cells were infected with live at an MOI of 1, and were incubated for 3 h at 37 °C, 5% CO2. After allowing time for phagocytosis, cells were washed with PBS to remove extracellular bacteria and then were incubated with fresh medium without antibiotics for an additional 24 or 48 h. In vivo mice were challenged by intranasal infection with Mtb (1 × 106 in 50 µL PBS) into the lungs. Then, 3, 7, and 20 days after infection, five mice per group were sacrificed in duplicate. To test the intracellular survival of Mtb in vitro and in vivo, infected cells or lung tissues from infected mice were lysed in sterile distilled water to allow intracellular bacteria to be collected. The lysates were serially diluted in 7H9 broth, plated separately onto 7H10 agar plates, and incubated for 2–3 weeks. Colonies were counted in triplicate.
Antibodies, reagents and transfections
Cells were pretreated with inhibitors or inducers for 2 h prior to Mtb infection. Specific inhibitors of JNK (SP600125), ERK (PD098059), p38 (SB203580), and NF-κB (BAY11-7082) were purchased from Calbiochem (San Diego, CA, USA). The ROS scavenger (N-acetyl-l-cysteine; NAC) and NOS inhibitor (nitric oxide synthase inhibitor; L-NMMA) were purchased from Sigma-Aldrich. Nutlin-3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as a p53 activator by MDM2 inhibition. Western blotting was performed using antibodies against phospho-MDM2, caspase-9, caspase-3, phospho-JNK, phosphor-ERK, phosphor-p38, p65, p50 (Cell Signaling, Danvers, MA, USA) p53, iNOS, arginase 1, β-actin, and laminB1 (Santa Cruz). Goat anti-rabbit IgG (Santa Cruz) and goat anti-mouse IgG (1:2000; Calbiochem) were used as secondary antibodies. Transfection of siControl (200 nM; Santa Cruz) and siJNK (200 nM; Bioneer Corporation, Daejeon, South Korea) into macrophage cells was performed using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions.
PCR, Western blotting and enzyme-linked immunosorbent assay
Mtb-infected macrophages were processed by PCR, Western blotting, and sandwich enzyme-linked immunosorbent assay (ELISA) as described previously . Briefly, total RNA was isolated from Mtb-infected BMDMs, and mRNA was reverse transcribed into cDNA. Reverse transcription–PCR was performed using Prime Taq Premix (Genet Bio, Daejeon, Korea) to detect the mRNA levels of target genes. For quantitative real-time PCR, total RNA from the MDMs of healthy controls and TB patients was extracted, cDNA was synthesized, and then p53 gene expression was quantified by SYBR green (Qiagen, Hilden, Germany). The mean value of triplicate reactions was normalized against the mean value of β-actin.
For Western blotting, Mtb-infected cells were lysed, and the lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by transfer to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% nonfat milk for 1 h at room temperature. Primary antibodies were diluted 1:1000, and horseradish peroxidase (HRP)-conjugated secondary antibodies were diluted 1:2000. For the detection of target proteins, the membranes were developed using a chemiluminescent reagent (ECL; Millipore) and were subsequently quantified using the Alliance Mini 4 M (UVITEC, Cambridge, UK).
The secretion levels of cytokines in the cell culture supernatants were measured using sandwich ELISA with detection kits for mouse IL-12p40, TNF, IL-6, and IL-10 (BD Biosciences, Franklin Lakes, NJ, USA). The sample absorbances were measured using a microplate reader at 450 nm and were compared to a standard curve.
To confirm the ratio of apoptotic cells, BMDMs were stained using an Annexin-V/PI staining kit (BD Biosciences) as described in the manufacturer’s instructions and then were analyzed using a FACS Canto II flow cytometer (BD Immunocytometry Systems, Franklin Lakes, NJ, USA).
ROS and NO measurement assays
To detect intracellular ROS production, Mtb-infected BMDMs were measured using the dihydroethidium (DHE) assay. Macrophages were infected with Mtb for 30 min, stained with 2 μM DHE for 30 min, and then washed with Krebs-Hepes buffer. Positive cells were identified using a laser-scanning confocal microscope (TCS SP8; Leica Microsystems, Wetzlar, Germany).
To evaluate NO levels during Mtb infection, macrophage cell culture supernatant fractions were analyzed using the Griess assay. Briefly, culture medium (100 µL) was incubated with the Griess reagent (100 µL) at room temperature for 10 min, and then the absorbance was measured at 541 nm. Sodium nitrite was used to create a standard concentration curve.
Data analysis and statistics
All experimental results were statistically evaluated using Student’s t test or one-way analysis of variance followed by Bonferroni’s multiple comparison tests. Statistical significance between groups was determined using the appropriate nonparametric Mann–Whitney or Kruskal–Wallis test. Differences were deemed significant when the p-value was < 0.05, and a difference of p < 0.001 was deemed highly significant. All experiments were performed three to five times, and the data are presented as means ± SDs. In vivo assays were performed in triplicate, and a minimum of three mice was used per group. Statistical analyses were performed using GraphPad Prism software (version 5.01).
This work was supported by the research fund of Chungnam National University. The funders had no role in study design, data collection and analysis decision to publish, or preparation of the manuscript.
YJL designed the study, performed the majority of the experiments, analyzed the data and wrote the manuscript; JHL and JAC performed experiments and analyzed data; SNC and SSH provided expert technical assistance; SJK and JWS provided clinical advice and critical discussion of work; CHS designed the study, supervised the project, and wrote the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
All animal experiments were performed in accordance with the Korean Food and Drug Administration (KFDA) guidelines. The experimental protocols used in this study were reviewed and approved by the Ethics Committee and Institutional Animal Care and Use Committee of Chungnam National University, Daejeon, South Korea (permit no. CNU-00425). This study was approved by the Institutional Review Board of Konyang University Hospital (Daejeon, South Korea) after receiving informed consent from the subjects (IRB approval no., KYUH-2015-06-007-002).
- 53.Richardson ET, Shukla S, Sweet DR et al (2015) Toll-like receptor 2-dependent extracellular signal-regulated kinase signaling in Mycobacterium tuberculosis-infected macrophages drives anti-inflammatory responses and inhibits Th1 polarization of responding T cells. Infect Immun 83:2242–2254PubMedPubMedCentralCrossRefGoogle Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.