IL-12 and IL-27 regulate the phagolysosomal pathway in mycobacteria-infected human macrophages
© Jung and Robinson; licensee BioMed Central Ltd. 2014
Received: 30 January 2014
Accepted: 7 March 2014
Published: 11 March 2014
The cytokine environment at the site of infection is important to the control of mycobacteria by host macrophages. During chronic infection immunosuppressive cytokines are likely to favor mycobacterial growth, persistence, and an avoidance of proper antigen processing and presentation. The activity of interleukin (IL)-27 toward macrophages is anti-inflammatory and this compromises control of mycobacteria. Modulation of the cytokine environment may enhance both protective and vaccine-induced responses.
In this study we showed that supplying IL-12 and neutralizing IL-27 enhanced acidification and fusion of mycobacterial-containing phagosomes with lysosomes. This was achieved by phagosomal acquisition of vacuolar ATPase (V-ATPase) and CD63. Both V-ATPase and CD63 protein levels were increased by the addition of IL-12 and neutralization of IL-27. In addition, cathepsin D associated with the bacteria and matured to the active form when IL-12 was supplied and IL-27 was neutralized. Lysosomal acidification and cathepsin D activity were associated with control of mycobacteria. The acidification of lysosomes, association with mycobacteria, and maturation of cathepsin D required macrophage production of IFN-γ and signaling through signal transducer and activator of transcription (STAT)-1. In contrast, STAT-3 signaling opposed these events.
Our results have identified novel influences of IL-12, IL-27, and STAT-3 on lysosomal activity and further demonstrate that modulating the cytokine environment promotes enhanced trafficking of mycobacteria to lysosomes in human macrophages. This has important implications in approaches to control infection and improve vaccination. Overcoming bacterial resistance to lysosomal fusion may expand the repertoire of antigens presented to the adaptive arm of the immune response.
Mycobacterium tuberculosis (MTB) is an intracellular human pathogen responsible for an enormous burden of human disease. In 2011, there were approximately 8.7 million incident cases of tuberculosis (TB) globally and nearly a third of the world population has been infected . The only currently available vaccine for TB is Mycobacterium bovis bacille Calmette-Guerin (BCG). BCG effectively protects against disseminated tuberculosis, such as miliary TB and tuberculous meningitis in children [2, 3]. However, the BCG vaccine has not been consistently effective at preventing pulmonary tuberculosis, and thus the effects of BCG on combating the global burden of tuberculosis have been limited.
Several hypotheses have been proposed to explain the limitations of BCG vaccination. These include interference by environmental mycobacteria, genetic differences in the human population, and differences between BCG substrains . Recently, it has been proposed that mycobacterial antioxidants, such as iron-cofactored superoxide mutase , and secA2 secretion suppress host immunity , resulting in reduction of vaccine efficacy. Suppression of host immunity could be mediated by anti-inflammatory cytokines. BCG-infected mice express high levels of the Th2 cytokines interleukin (IL)-5 and IL-13 . Similarly, IL-4 and TGF-β are known to be increased in tuberculosis patients . Thus, another possible explanation for limited effectiveness of BCG may be inhibition of host immunity through the action of immune suppressive cytokines.
IL-27 is produced by antigen presenting cells in response to a variety of activation stimuli, notably microbial-derived products . IL-27 activates Janus kinases (JAK) and signal transducer and activator of transcription (STAT)-1 and STAT-3 through its receptor composed of WSX-1 and gp130 . IL-27 was originally described as a soluble factor that promotes Th1 activity . STAT-1 and STAT-3 modulate the T-cell specific transcription factors such as T-bet (Th1) or GATA-3 (Th2) . However, IL-27 also negatively regulates Th1 cells, highlighting its paradoxical nature . Similarly, IL-27 inhibits differentiation of Th17 cells and production of IL-17 by inducing IL-10 producing Tr-1 cells through STAT-1 and STAT-3 . Immunosuppressive activity of IL-27 has been described toward a number of immune cell types involved in innate immune responses . IL-27 induces an immunosuppressive phenotype in murine DCs by increasing expression of B7-H1 in a STAT-3-dependent manner [15, 16]. Proinflammatory cytokine production is inhibited in both human and murine macrophages by IL-27 [17, 18].
IL-27 produced by human macrophages during MTB infection opposes inflammatory responses [17, 19–21]. Treatment with IL-12 in conjunction with neutralization of IL-27 restricts the growth of MTB and requires the proinflammatory mediators IFN-γ, TNF-α, and IL-18 [17, 19]. This immunomodulation promotes more effective macrophage-mediated immunity. Even though immunological parameters involved with the treatment of IL-12 and sIL-27R that improve mycobacterial control have been revealed , the intracellular mechanisms involved have not been elucidated.
MTB arrest phagosomes at an early stage of endosomes by blocking phagosomal maturation . This prevents fusion with late endosomes and lysosomes. Beyond the implications in host-mediated control of mycobacteria, this limits antigen presenting cell processing of mycobacterial antigen. Mycobacterium bovis BCG also avoid phagosomal fusion with lysosomes as efficiently as MTB [23, 24]. In doing so, BCG may limit the range of antigens that are processed for presentation by major histocompatibility complex (MHC) class II. The phagosomal/lysosomal trafficking pathway involves a variety of host molecules including proteins and phospholipids. Phagosomal maturation occurs through the acquisition of several lysosomal markers, such as lysosome associated membrane protein LAMP-1, LAMP-2, and CD63. In the final stage of maturation, the phagosome acquires V-ATPase and cathepsins in a syntaxin6-dependent manner .
The proinflammatory cytokine IFN-γ promotes phagosomal maturation by inducing acidification of phagosomes . IFN-γ treatment in murine macrophages leads to acidification of mycobacterial-containing phagosomes . Additionally, IFN-γ treatment of monocyte-derived macrophages increased lysosomal fusion with endosomes . However, there is some evidence that anti-inflammatory cytokines can inhibit the phagosomal/lysosomal pathway [21, 23, 26]. MTB-infected macrophages from IL-10 knock-out mice exhibit an increased level of acidification . IL-10 decreased fusion of horse radish peroxidase (HRP) containing phagosomes with lysosomes . Recently we have demonstrated that IL-27 decreases phagosomal acidification through inhibition of V-ATPases .
Based on these reports, treatment of infected macrophages with IL-12 and sIL-27R could promote an environment that is unfavorable for mycobacterial growth by enhancing phagosomal maturation and fusion with lysosomes. The objective of this work was to evaluate the influence of IL-12 and IL-27 on the phagosomal/lysosomal pathway during infection by BCG. Promoting enhanced delivery of BCG to lysosomes may enhance the magnitude and diversity of antigen presentation. Treatment of IL-12 combined with neutralization of IL-27 increased the expression of CD63 and V-ATPase. The consequence was enhanced phagosomal acidification and localization of cathepsinD at the BCG-containing phagosome. This immunomodulatory approach that overcomes phagolysosomal resistance may not only be important for controlling bacterial growth but also increasing the repertoire of BCG antigens that are presented during vaccination and improve the efficacy of BCG.
BCG infection of human macrophages increases the production of IL-27
Supplying IL-12 and neutralizing IL-27 induced lysosomal acidification
Supplying IL-12 and neutralizing IL-27 induced the expression of CD63 and enhanced association with BCG
Supplying IL-12 and neutralizing IL-27 induced the expression of vacuolar ATPases
Supplying IL-12 and neutralizing IL-27 induced the formation of mature cathepsin D and enhanced association with BCG
Enhanced fusion of mycobacterial phagosomes with lysosomes restricts BCG growth
IFN-γ is important for the enhancement of phagosomal acidification when IL-12 is supplied and IL-27 is neutralized
STAT-1 is an important regulator of lysosomal changes mediated by supplying IL-12 and neutralizing IL-27
Effective innate immunity involves the activation of macrophages. Activated macrophages utilize a variety of molecules and intracellular pathways to combat microbial pathogens. Among these is the maturation of phagosomes to lysosomes that is accompanied by a decrease in pH. Mycobacteria evolved to avoid this pathway by excluding the effector proteins involved in phagosomal acidification and lysosomal fusion (CD63, V-ATPase, and cathepsin D) from their vacuoles . BCG has been safely used for nearly a century, but the protective efficacy against TB is highly variable . BCG is highly similar to MTB in antigenic composition [39, 40] and equally efficient at avoiding phagosomal maturation and lysosomal fusion [23, 24]. Consequently, only a limited repertoire of BCG antigens may be presented in the initiation of an adaptive response. This may contribute to the limited vaccine efficacy against tuberculosis. Although delayed clearance of BCG may promote immunity by providing continual antigenic stimulation, expanding the nature of the response through enhanced processing and presentation may be a more effective strategy for host protection. It is clear that a strong immune response is initiated during tuberculosis. However, the nature of the response may be equally as important as the magnitude of response. Our data provide new information to be considered on this front.
The cytokine environment at the site of infection is likely to influence the phagosomal/lysosomal pathway. IFN-γ promotes lysosomal fusion with endosomes and acidification . Via et al. showed that IFN-γ treatment increases the acidification of BCG-containing phagosomes in murine macrophages . In this report, bone marrow-derived BCG-infected macrophages from IL-10 deficient mice exhibited increased acidification of BCG phagosomes suggesting that IL-10 has negative influences on the phagosomal/lysosomal pathway . Recently, we have demonstrated that exogenous treatment of IL-27 decreases phagosomal acidification by inhibiting the protein expression of V-ATPases in latex bead-treated human macrophages . IL-27 is expressed by MTB [17, 20] and BCG-infected macrophages (Figure 1A). In addition, treatment with IL-12 and sIL-27R restricted growth of both species in human macrophages (Figure 1B, 17, 19, 20). This suggests that IL-27 may create a favorable intracellular environment for the bacteria. Alternatively, blocking IL-27 promotes host protection.
The major finding in this study is that altering the cytokine environment by supplying IL-12 and neutralizing IL-27 resolves the mycobacterial arrest of phagosome maturation. This involves several steps. V-ATPase reduces the pH to 5.0 in mycobacterial phagosomes (Figure 2 and 4) . Eventually BCG-containing phagosomes become phagolysosomes by acquiring CD63 (Figure 3), most likely through fusion with established acidic lysosomes or acquisition from other vesicles through the golgi or ER. Along with the phagosomal acidification, cathepsin D was processed to the mature form (Figure 5) indicating that this enzyme may be actively involved in mycobacterial degradation in an acidic milieu of phagolysosomes. Mature cathepsin D was associated with BCG phagosomes when macrophages were treated with IL-12 and sIL-27R. Cathepsin D may be recruited to phagosomes as a preform and subsequent acidification through acquisition of V-ATPase leads to generation of mature cathepsin D. Enhanced phagosomal acidification and cathepsin D activity during treatment with IL-12 and neutralization of IL-27 was important for limiting intracellular bacterial growth (Figure 6). Inhibition of V-ATPase by bafilomycin and cathepsin D by pepstatin reversed the inhibition of bacterial growth to the level of untreated macrophages (Figure 6C). This data indicates that supplying IL-12 and neutralizing IL-27 specifically influences the phagosomal/lysosomal pathway. IFN-γ was shown to be an important immunological mediator. Neutralizing IFN-γ reversed the enhancement of phagosomal acidification and cathepsin D activity that were responsible for control of mycobacterial growth (Figure 7). This was consistent with reduced V-ATPase and CD63 expression levels along with prevention of cathepsin D maturation (Figure 7). IFN-γ is known to enhance phagosomal acidification and lysosomal fusion . IFN-γ treatment enhanced the fusion of MTB-containing phagosomes with lysosomes in THP-1 cells, and these lysosomes originated from autophagy-stimulated autophagosomes . Since IFN-γ plays a major role in mediating phagosomal acidification during treatment with IL-12 and sIL-27R, an autophagic mechanism may be involved. This is currently under investigation. IFN-γ activates STAT-1 activity to induce anti-microbial mechanisms in macrophages . We found that STAT-1 inhibition reverses lysosomal acidification and association with BCG during treatment with IL-12 and sIL-27R (Figure 8). It is important to point out that although our experiments suggest that STAT-1 associated with IFN-γ signaling promotes lysosomal acidification and association with BCG, our approach using chemical inhibitors does not distinguish STAT-1 induced by signaling through the IFN-γ receptor from that through the IL-27 receptor. In contrast to the positive action of IFN-γ and STAT-1, IL-27 signals through STAT-3 to oppose lysosomal acidification and fusion with BCG. This was demonstrated by the observation that STAT-3 inhibition in the absence of additional influences on the cytokine environment allowed for lysosomal acidification and association with BCG.
Anti-inflammatory cytokines may strongly influence the intracellular fate of mycobacteria in human macrophages. Previous reports show that anti-inflammatory cytokines, IL-4 and TGF-β are increased in MTB-infected individuals . Another anti-inflammatory cytokine, IL-10 is also involved in preventing acidification of mycobacteria-containing phagosomes . Consistent with this observation, blocking IL-10 signaling following BCG vaccination enhanced protective Th1 and Th17 responses . Since IL-10 also signals through STAT-3, IL-27 and IL-10 may operate similarly to oppose lysosomal acidification and protective immunity. Thus, regulating the cytokine environment to establish an antibacterial state in macrophages will not only be beneficial during infection but may also enhance vaccine-induced responses.
Bacterial strains and growth conditions
Mycobacterium bovis Calmette Guérin (BCG) was purchased from ATCC (Manassas, VA). Bacteria were maintained in Middlebrook 7H9 broth supplemented with ADC enrichment media (Albumin, Dextrose, Catalase) at 37°C with 5% CO2.
Staining of Mycobacterium bovis BCG
BCG were pre-stained with SYTO-9® or SYTO-61® (100 μM, Molecular Probes, Life Technologies) according to the manufacturer’s instructions. Briefly, 5 × 106 CFUs were pelleted at 2,000 × g for 10 min at room temperature. The supernatant was discarded and the pellet was suspended in SYTO probes at a final volume of 1 mL and passed through a 27-guage needle to disperse the bacteria completely. Following 30 min incubation in the dark, the stained bacteria were centrifuged at 2000 × g for 10 min. The bacterial pellet was suspended in 1 ml of infection medium (DMEM supplemented with 1% human serum, 2 mM glutamine, 25 mM HEPES) and passed through a 27-gauge needle several times. Stained BCG were diluted to a MOI of 10 with infection medium.
Human buffy coats were purchased from the New York Blood Center (New York, NY). Eligible donors were 16 years of age or older, at least 110 pounds, and in good physical health. The donor samples were anonymous and deidentifed. Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats by Ficoll gradient centrifugation. Monocytes were then isolated from PBMCs by Optiprep (Sigma-Aldrich) gradient centrifugation as described previously . Monocytes were adhered to plastic 60 mm culture dishes in serum-free DMEM. The media was then replaced with DMEM supplemented with 2 mM glutamine, 25 mM HEPES, 20% fetal bovine serum (FBS), and 10% human serum and incubated at 37°C with 5% CO2 for 7 days. Macrophages were removed from the culture dish with PBS that contained 5 mM EDTA and 4 mg/ml lidocaine. The cells were washed with PBS and plated onto new culture dishes in DMEM supplemented with infection medium. These cells are routinely >95% CD14 positive.
Macrophage infection and enumeration of BCG
Human macrophages were cultivated in 24-well plates (2 × 105/well) and treated with medium alone, IL-12 (5 ng/ml), sIL-27R (10 μg/ml), or their combination for 6 h prior to infection with SYTO-stained BCG (~MOI 10). Infected cultures were incubated 48 h at 37°C with 5% CO2. Culture supernatants were removed and macrophages were permeabilized with 1% saponin to release bacteria. Tenfold serial dilutions were plated on Middlebrook 7H10 agar and incubated 10 days at 37°C with 5% CO2.
Analysis of lysosomal acidification and immunolabeling
Human macrophages cultured in 24-well plates were treated as indicated above. In the last hour of infection, culture supernatants were replaced with medium that contained Lysotracker DND-99 Red (Life technologies) (100 nM). The slides were examined using a Zeiss Meta 510 laser confocal microscope with a plan-Apochromat 63× objective lens. A total of 10 fields containing 5–10 macrophages per field were examined in each experiment. The mean fluorescent intensity (MFI) for each macrophage was calculated using Image J software. Each cell from the image was selected and histogram analysis was performed. For immunostaining, mouse monoclonal antibodies for CD63 (sc-5275, Santa Cruz Biotechnology) and V1-ATPase H (sc-166227, santa Cruz Biotechnology) were visualized with anti-mouse-Alexafluor 588-conjugated secondary antibody. Goat polycolonal antibodies for active cathepsin D (sc-6486, Santa cruz) were visualized with anti-goat-Alexafluor 488-conjugated secondary antibody (Life technologies).
Quantitative bacterial association analysis
To quantify bacterial association with lysosomes, CD63, V-ATPase, or cathepsin D, we employed Pearson’s correlation coefficient analyses. The analyses were performed as follows. During imaging, the microscope pinhole size was set to acquire the same amount of signal for each channel. To minimize the bleed-through effect, the image was scanned sequentially. To avoid saturation, a range indicator for identification of excessive bright and excessive high contrast images was established. This allowed for adjustment using an offset function. Each image was analyzed by image J software. This software produces a number of coefficients for estimating the degree of association. Pearson’s correlation was employed to analyze association between green (BCG) and red (Lysotracker, CD63, V-ATPase) or red (BCG) and green (cathepsin D)) since this analysis considers similarity between shapes .
Quantitative real time PCR
Human macrophages (2×105/well) cultivated in 24-well dishes were treated as indicated. At appropriate time points, media was removed from cultures, the cells were lysed with PureZol® (Bio-Rad), and RNA was isolated according to commercial product protocol. First strand cDNA synthesis was performed using iScript™ cDNA synthesis reagents (Bio-Rad) according to protocol. For IL-27 p28 and EBI3 gene expression analysis, real time cycling of reactions that included cDNA diluted 20-fold from above, gene-specific primer probe sets (Applied Biosystems), iQ™ Supermix (Bio-Rad) was performed in triplicate using iQ5™ cycler (Bio-Rad). GAPDH was used as an internal reference gene.
Human macrophages (2 × 105 cells/well) cultivated in 24-well plates were left untreated or treated with the combination of IL-12 (5 ng/ml) and sIL-27R (10 μg/ml) for 6 h prior to infection with BCG (~MOI 10). Infected cultures were incubated for 48 h at 37°C with 5% CO2. Briefly, after infection with BCG, 40 μl of PBS supplemented with 1% Tx-100 was applied to each sample and lysates collected by scraping. They were subsequently sonicated and then stored at 4°C. Equal amounts of cell lysates were separated on SDS-PAGE gels and transferred to nitrocellulose by standard technique. Primary antibodies used in this study were mouse monoclonal anti-CD63, V-ATPase H, Cathepsin D antibodies that recognize all forms (sc-374381, Santa Cruz Biotechnology), and rabbit polyclonal anti-actin (Sigma) antibodies. Primary antibodies were revealed with horse radish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies. ECL substrate (Amersham Biosciences) was applied to visualize proteins.
Bafilomycin and pepstatin titration
Bafilomycin and pepstatin were purchased from Sigma. Bafilomycin titration was performed as follows. Macrophages were treated with varying concentrations of bafilomycin (0–1000 nM) for 6 h and then treated with fluorescent-conjugated latex beads for an additional 48 h. The macrophages were examined by confocal microscopy as described earlier in this section. Pepstatin titration was performed as follows. Macrophages were either untreated or treated with varying concentrations (0–100 μM) of pepstatin for 6 h, and then subsequently infected with BCG (MOI 10) for 48 h. Auramine O staining was performed as described previously  and fluorescence (λex = 420 and λem = 508 nm) was measured with a Synergy HT Multi-Mode Microplate reader (Biotek, VT).
This work was supported by NIH grant HL093300.
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