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Autophagy. 2012 September 1; 8(9): 1357–1370.
PMCID: PMC3442882

ESX-1 dependent impairment of autophagic flux by Mycobacterium tuberculosis in human dendritic cells

Abstract

Emerging evidence points to an important role of autophagy in the immune response mediated by dendritic cells (DC) against Mycobacterium tuberculosis (Mtb). Since current vaccination based on Bacillus Calmette-Guerin (BCG) is unable to stop the tuberculosis epidemic, a deeper comprehension of the alterations induced by Mtb in DC is essential for setting new vaccine strategies. Here, we compared the capacity of virulent (H37Rv) and avirulent (H37Ra) Mtb strains as well as BCG to modulate autophagy in human primary DC. We found that Mtb H37Rv impairs autophagy at the step of autophagosome-lysosome fusion. In contrast, neither Mtb H37Ra nor BCG strains were able to hamper autophagosome maturation. Both these attenuated strains have a functional inhibition of the 6kD early secreted antigenic target ESAT-6, an effector protein of the ESAT-6 Secretion System-1(ESX-1)/type VII secretion system. Notably, the ability to inhibit autophagy was fully restored in recombinant BCG and Mtb H37Ra strains in which ESAT-6 secretion was re-established by genetic complementation using either the ESX-1 region from Mtb (BCG::ESX-1) or the PhoP gene (Mtb H37Ra::PhoP), a regulator of ESAT-6 secretion. Importantly, the autophagic block induced by Mtb was overcome by rapamycin treatment leading to an increased interleukin-12 expression and, in turn, to an enhanced capacity to expand a Th1-oriented response. Collectively, our study demonstrated that Mtb alters the autophagic machinery through the ESX-1 system, and thereby opens new exciting perspectives to better understand the relationship between Mtb virulence and its ability to escape the DC-mediated immune response.

Keywords: Mycobacterium tuberculosis, BCG, ESX1/type VII secretion system, RD1 region, autophagy, dendritic cells, vaccine

Introduction

Tuberculosis (TB) is still a leading cause of death worldwide despite the availability of the Bacillus Calmette-Guerin (BCG) vaccine and specific antibiotic treatments. The currently used BCG vaccine, based on a live attenuated strain of Mycobacterium bovis, offers variable protection efficacy and therefore needs to be either improved or replaced.1 Different TB vaccine candidates involving recombinant BCG, subunit vaccine and live attenuated Mycobacterium tuberculosis (Mtb) strains are currently under investigation.2 Comparative genomics of the Mtb complex revealed that the attenuation of BCG is related to the loss of the protein secretion system ESX-1 due to deletion of the region of difference 1 (RD1) locus.3 This segment is localized close to the origin of replication in all fully virulent members of the complex and harbors genes esxA (Rv3875), coding for the 6-kDa early secreted antigenic target (ESAT-6), and esxB (Rv3874), encoding the 10-kDa culture filtrate protein (CFP-10).4 ESAT-6 and CFP-10 are secreted via a member of type VII secretion system ESX-1, a dedicated secretion apparatus encoded by genes flanking esxA and esxB in the extended RD1 region.

A complex network of interactions between the host and pathogen controls the final outcome of Mtb infection.5 After phagocytosis, Mtb persists within macrophages through a variety of immune evasion strategies, such as preventing the recognition of infected macrophages by T cells and inhibiting phagosome maturation.6 Among several mechanisms used by the host to fight Mtb, the induction of the autophagic process represents a crucial step,7-11 which can lead to both the killing of mycobacteria and the production of Mtb peptides and lipids for antigen presentation pathways.12,13 An elegant indication of the role played by autophagy in reducing the viability of intracellular mycobacteria has been recently provided by Kumar and colleagues, who showed by means of a genome-wide RNA interference approach that the silencing of molecules involved in the process of autophagy enhanced the intracellular viability of Mtb.14

Autophagy functions in diverse aspects of innate and adaptive immunity mainly involved in the defense against intracellular pathogens.15-18 In addition to the well-characterized role of autophagy in regulating the major histocompatibility complex (MHC) class II presentation of cytoplasmic and nuclear antigens, and of those obtained from phagocytosed pathogens, this process has been shown to provide novel routes for MHC class I antigen presentation and enhanced CD8+ T-cell responses, for cross-presentation of dendritic cells (DC) to CD8+ T cells.19,20

DC display peculiar features in respect to autophagy since a high constitutive activity has been observed,21 which continuously provides peptidic antigens for MHC class II presentation in order to induce immunity or tolerance by immature and mature DC, respectively. In line with these findings, Jagannath and collaborators showed that in a mouse model the stimulation of autophagy by rapamycin enhanced the capacity of DC to present mycobacterial antigens into the MHC class II complex.13

Given the critical role played by DC in the regulation of the immune response against Mtb,22-24 we sought to investigate the modulation of the autophagic process in human primary DC infected by Mtb and the vaccine strain BCG. We show that Mtb, but not BCG, impairs the late steps of autophagy and a functional ESX-1 secretion system is required for this inhibition. In addition, rapamycin treatment restored autophagy in Mtb-infected DC, which displayed an enhanced capacity to promote the expansion of Th1-oriented CD4+ T cells. Collectively, these results highlight a unique crosstalk between Mtb and the autophagic process in DC, which could play a role in preventing an efficient antimycobacterial immune response.

Results

Mtb and BCG have different impacts on the autophagic process in human primary DC

To set up our experimental model, we faced an issue related to the use of IL4 and granulocyte-macrophage colony stimulating factor (CSF2) for the in vitro differentiation of monocyte-derived DC. Given the inhibitory effect exerted by IL4 on autophagy25 and the activation of MTOR pathway by CSF2 and IL4 along monocyte-derived DC,26 we compared in preliminary experiments the effect of Mtb infection on the autophagic process in the presence of CSF2 and IL4 or following the replacement of the differentiating medium 20 h before infection. Having found that both the basal and Mtb-driven autophagic response was altered by the presence of IL4 and CSF2 (data not shown), we decided to perform our study in DC starved from these differentiating cytokines.

Autophagy levels were analyzed in primary human DC infected with either Mtb or BCG at a multiplicity of infection (MOI) of 1. Cells were harvested 24 h after infection and the occurrence of autophagy was monitored by both immunoblotting and immunostaining using an antimicrotubule-associated protein 1 light chain 3 (MAP1LC3) antibody. As shown in Figure 1A, Mtb induced a significant increase of MAP1LC3-II, the processed isoform of MAP1LC3 associated with autophagosomes. This increase was accompanied by an accumulation of MAP1LC3-positive vesicles observed by confocal microscopy (Fig. 1B). Notably, MAP1LC3-II increase was not observed when cells were infected with either BCG or stimulated with heat-inactivated Mtb, suggesting that only live, virulent mycobacteria are able to modulate MAP1LC3-II levels (Fig. 1A and B). Autophagosome accumulation observed during Mtb infection was prevented by incubating DC with the autophagy inhibitor wortmannin, confirming the specificity of MAP1LC3-positive vescicles observed in Mtb-infected DC (Fig. S1).

figure auto-8-1357-g1
Figure 1. Modulation of autophagy by Mtb. (A and B) DC were infected with Mtb, BCG or heat-inactivated Mtb (Mtb HI). After 24 h, autophagy levels were analyzed for MAP1LC3 expression by immunoblotting (A) or immunofluorescence using an MAP1LC3 ...

We then asked whether the accumulation of MAP1LC3-II was an early or late event during Mtb infection. MAP1LC3 levels were analyzed in DC harvested by immunoblotting (Fig. 1C) and confocal microscopy (Fig. 1D) at different time points after Mtb infection. MAP1LC3 II increase occurs at late times, being slightly induced at 8 h and increasing at 16 h, although Mtb was already detected intracellularly at earlier times (Fig. 1E), as revealed by confocal microscopy performed with a Cherry-expressing Mtb following the visualization of DC plasma membrane using a FITC-conjugated DR antibody.

Mtb causes a block of autophagosome maturation

The accumulation of MAP1LC3-II in Mtb-infected cells could be due to an enhanced autophagosome formation or a reduced degradation of autophagic material.17,25 To discriminate between these events, we assessed the occurrence of a functional autophagic flux in Mtb-infected cells. To this aim, we analyzed the lysosome-mediated autophagosome degradation by monitoring either the effect of lysosomal inhibitors on MAP1LC3 levels or the colocalization of autophagosomes with lysosomal markers. Both analyses indicated that autophagosome accumulation in Mtb-infected cells was due to a defect in the late steps of the autophagic process. As shown in Figure 2A, treatment of Mtb-infected DC with the lysosomal protease inhibitors E64d and Pepstatin A (PepA) resulted in a reduced increase of MAP1LC3-II protein levels [1.3 fold, calculated as the ratio between MAP1LC3 levels in Mtb-infected DC treated with E64D+PepA (6.4) and those in untreated Mtb-infected DC (5.1)] as compared with the effect promoted by the same treatment in not-infected cells (3.8 fold), suggesting that MAP1LC3-II degradation by lysosomal enzymes is impaired during Mtb infection. Similar results were obtained when the levels of SQSTM1 protein, a well-characterized target of the autophagic process, were analyzed in the same experimental setting. Indeed, Mtb infection leads to the accumulation of SQSTM1 protein (5.7 fold) which is not further increased by treatment with lysosomal inhibitors (1.1 fold), different from what was observed in control cells (2.4 fold, Fig. 2A). It should be pointed out that SQSTM1 increase in Mtb-infected DC is not only due to an impairment of protein degradation, since an induction at the transcriptional level was also observed during infection (2.5 fold induction, Fig. S2A). Similar results were obtained when lysosome activity in Mtb-infected DC was interfered using bafilomycin A1, an inhibitor of the vacuolar-type H+-ATPase (1.2 fold in Mtb-infected DC vs. 3.9 fold in not-infected cells, Fig. S2B). The impairment of autophagosome maturation was further investigated by confocal microscopy. We observed a reduced colocalization between MAP1LC3 and the lysosomal markers lysosomal-associated membrane protein 1 (LAMP1) and cathepsin B (CTSB) in Mtb-infected cells (Fig. 2B–D), confirming that Mtb hampers the autophagic flux.

figure auto-8-1357-g2
Figure 2. Effects of rapamycin on autophagic flux in Mtb-infectd DC. (A) DC were infected with Mtb and treated with rapamycin (Rapa, 0.2 μM; 4 h after infection) or E64d plus PepA A (E64d+PepA, 10 μg/ml each, 4 h before cell harvesting) ...

Rapamycin has been reported to promote autophagy-mediated clearance of Mtb in macrophages.12 Thus, we decided to test whether rapamycin is able to overcome the impaired autophagic flux in Mtb-infected DC. Rapamycin was added 4 h after infection when the majority of cells showed the internalization of Mtb (see Fig. 1E). Western blot and confocal analyses showed that rapamycin treatment restores a functional autophagic flux as indicated by the reduction of SQSTM1 levels (Fig. 2A) as well as by the increased colocalization of MAP1LC3 with LAMP1 and CTSB, two markers of late endocytic/lysosomal compartments (Fig. 2B–D). Notably, rapamycin treatment led to a significant increase in Mtb-containing autophagosomes and autolysosomes, as evaluated by both electron and confocal microscopy (Fig. 3). Interestingly, confocal microscopy analysis showed not only an increased Mtb-MAP1LC3 colocalization following rapamycin treatment (Fig. 3B and E), but also a robust formation of Mtb-containing autolysosomes positive to LAMP1 (Fig. 3C and E). Mtb-MAP1LC3 colocalization induced by rapamycin is prevented by pretreating cells with wortmannin (Fig. S2C). To further characterize the intracellular Mtb localization after rapamycin treatment, the acidification of Mtb-containing phagosome was evaluated using LysoTracker, a fluorescent dye for labeling acidic organelles (Fig. 3D and E). Mtb was found within LysoTracker positive lysosome only after rapamycin treatment. Interestingly, the ability to rescue the inhibition of autophagic flux imposed by Mtb is not restricted to rapamycin, as it was also observed by treating cells with IFNG (Fig. S3), a known inducer of autophagosome formation.12 Indeed, in IFNG stimulated DC Mtb localized with MAP1LC3 in LAMP1 positive compartment, likely autolysosome. Altogether these results indicate that, although a high number of autophagosomes accumulate in Mtb-infected DC, their maturation into the autolysosome is reached only after the treatment with autophagy inducers, such as rapamycin and IFNG.

figure auto-8-1357-g3
Figure 3. Inhibition of autophagosome maturation by Mtb (A) Ultrastructural analysis of Mtb-infected DC 24 h post-infection either untreated (upper panel) or incubated with rapamycin (bottom panel) as described in Figure 2A. Autophagic ...

ESX-1 is responsible for the inhibition of autophagosome maturation by Mtb

The different modulation of autophagy by Mtb and BCG prompted us to better characterize the mycobacterial factors responsible for this phenomenon. DC were infected with recombinant Mtb strains, which were deleted for either the entire RD1 region (MtbΔRD1::pYUB412),27 or the promoter region of the CFP-10/ESAT-6 operon, preventing ESAT-6 and CFP-10 expression. Furthermore we tested a recombinant Mtb strain secreting an ESAT-6 variant lacking 12 aa of C-terminus (ESAT-6Δ84–95). These recombinant strains resulted attenuated in the TB mouse infection model.28,29 Notably, all tested deletion mutants have lost the ability to accumulate MAP1LC3-II as well as to prevent MAP1LC3/LAMP1 colocalization (Fig. 4A–C), thus indicating that secretion of biologically functional ESAT-6/CFP-10 is a requirement for the inhibition of autophagosome maturation by Mtb.

figure auto-8-1357-g4
Figure 4. Autophagy inhibition by ESX1/type VII secretion system. (A) DC were infected with wild-type Mtb (H37Rv) or with the following Mtb strains: H37RvΔRD1::empty vector pYUB412, H37RvΔRD1::RD1–2F9-ESAT-6-Δ84–95, ...

In order to better elucidate the functional relationship between the RD1 encoded ESX-1 secretion system and the autophagic process, we decided to test the modulation of autophagy in DC infected with BCG complemented with a 32 kb fragment from Mtb spanning the extended RD1 region (BCG::RD1),30 which actively secretes ESAT-6 and CFP-10. Cells infected with the BCG::RD1 strain depicted an accumulation of MAP1LC3-II protein similar to what was observed for Mtb (Fig. 4D). Finally, we tested the attenuated Mtb H37Ra strain, which shows an impaired secretion of ESAT-6 due to a point mutation in the DNA-binding region of the regulator PhoP. We analyzed the effect on autophagy of Mtb H37Ra and recombinant H37Ra strain complemented with a wild-type copy of phoP (Rv0757), which has been described to restore the ability of the H37Ra (H37Ra::PhoP) to secrete ESAT-6 and to elicit specific T-cell responses,31 in comparison with a strain complemented with an unrelated gene (H37Ra::RpsL). Strikingly, infection of DC with H37Ra::PhoP promoted MAP1LC3-II accumulation (Fig. 4D), whereas this effect was not observed with the Mtb H37Ra and H37Ra::RpsL strains. Taken together, these findings indicate that mycobacterial inhibition of autophagy in DC is dependent on the ability of mycobacteria strains to secrete intact ESAT-6/CFP-10.

Rapamycin enhances the capacity of Mtb-infected DC to promote a Th1-oriented response

Having found that rapamycin treatment overcomes the block in autophagosome maturation by Mtb, we sought to investigate the impact of this reversion on DC function. Initially, we evaluated whether rapamycin could modulate Mtb-induced DC maturation. Previous findings showed that rapamycin suppressed immunostimulatory molecules and the allostimulatory potential of LPS-stimulated DC.26 However, when we investigated by flow cytometry analysis the immunophenotype of DC, no major differences were observed on the Mtb-induced expression of the molecules involved in T cell activation, such as CD86, CD83, HLA-DR and CD38 (Table 1). Also, no modulation of Mtb-induced tumor necrosis factor (TNF) production was observed upon rapamycin treatment (Fig. 5A). Conversely, the expression of cytokines involved in the polarization of Th1 lymphocytes was enhanced in Mtb-infected DC cultures upon rapamycin addition (Fig. 5B and C), namely interleukin (IL)12 p70 and interferon (IFN)B1. In particular, a significant induction of Mtb-stimulated production of interleukin (IL)12 p70 was observed following rapamycin treatment, (Fig. 5B). A similar result was obtained by studying interferon (IFN)B1 expression performed by real-time RT-PCR (Fig. 5C). Indeed, the induction of IFNB1 mRNA observed in Mtb-infected DC was further enhanced after exposure to rapamycin, although this increase was not statistically significant. Interestingly, also the treatment with another autophagy inducer, IFNG, enhances the IL12 production in Mtb-infected cultures (Fig. S4A), suggesting that the expression of this Th1 promoting cytokine could be linked to the autophagic process. In line with this result, E64D and PepA treatment abolishes Mtb-induced IL12 expression, while exerts a limited effect on TNF release (Fig. S4B).

Table thumbnail
Table 1. Rapamycin effect on the expression of maturation markers in DC infected with Mtb
figure auto-8-1357-g5
Figure 5. Effect of rapamycin on DC maturation. (A and B) Cell culture supernatants were collected from DC cultures infected with Mtb and treated with rapamycin as in Figure 2A, or left untreated. The production of TNF (A) IL12 (B) was ...

To evaluate whether the effect induced by rapamycin on the secretion of Th1 cytokines from Mtb-stimulated DC resulted in an enhanced capacity to expand a Th1-oriented CD4+ T cell population, we studied T-cell polarization by mixed lymphocyte reaction (MLR) experiments. As shown in Figure 5D, the rapamycin treatment enhanced the production of IFNG. IL12 neutralization with specific monoclonal antibodies drastically reduced IFNG levels in co-cultures with DC infected with Mtb both in presence or absence of rapamycin, suggesting that the enhanced expression of IL12 induced by rapamycin may represent a key event for the expansion of an anti-Mtb Th1 response mediated by DC. No induction of IL4 and IL5 was observed when T cells were co-cultured for 5 d with Mtb-stimulated DC in the presence or absence of rapamycin (data not shown).

Finally, we evaluated the autophagy-mediated killing of the tubercle bacilli by CFU assay (Fig. S5). A bacteriostatic effect on mycobacterial growth rather than a reduction in Mtb viability was observed upon rapamycin treatment at 3 d, pointing at a major role of autophagy in DC in the induction of an efficient T cell response rather than Mtb killing, a function mainly exerted by macrophages.13

Collectively, these results provide evidence of the capacity of rapamycin to promote Mtb-infected DC to stimulate a Th1 response likely by overcoming the mycobacterial phagosome maturation block and enhancing IL12 production.

Discussion

In light of the importance of the autophagic process in different facets of antigen presentation,32 there is an increasing interest in exploiting this pathway for optimizing Mtb vaccine strategies. For this reason, particular attention is currently devoted to the role of autophagy in DC, which represent key effectors of the immune response against Mtb.22,23,33 At variance with macrophages, which are primarily involved in the containment of Mtb growth, DC are engaged in inducing a protective T cell response. A recent study in mouse DC indicated that both secreted Ag85B antigen and intact mycobacteria can be targeted into lysosomes for peptide production through autophagy.13 These authors provided also the first in vivo evidence on the effects promoted by rapamycin in mouse DC infected with attenuated vaccine candidates. The rapamycin-mediated increase of Mtb antigen presentation occurring in DC, resulted in a strong expansion of a Th1 population in the spleen, in the improvement of Mtb clearance and in in vivo protection against virulent Mtb, generating a novel proof of principle that the efficacy of anti-TB vaccines can be enhanced through the modulation of autophagy. In line with these findings, autophagy is currently investigated as novel therapeutic target for preventing acquisition or reactivation of latent TB or to treat multidrug resistence and extensively-drug resistant TB.34

To extend this study to human primary DC, we investigated the autophagic process in DC infected with virulent or avirulent Mtb as well as the vaccine BCG strain. We found that Mtb promotes a significant increase of MAP1LC3-II level and an accumulation of autophagosomes, that was specific for the virulent strain since it was not observed during BCG or avirulent Mtb H37Ra infection, or stimulation with heat-inactivated Mtb. The increased levels of autophagosomes in Mtb-infected DC was determined by a defective autophagic flux likely due to an impaired ability of autophagosomes to fuse with lysosomes, as shown by the reduced localization of MAP1LC3 with the lysosomal markers LAMP1 and cathepsin B, as well as by the impaired lysosomal degradation activity, assessed by comparing MAP1LC3-II and SQSTM1 levels in the presence or absence of lysosome inhibitors, such as E64D plus PepA or bafilomycin A1.

No differences have been outlined so far on the capacity of these mycobacterial strains to modulate the autophagic process. This is possibly due to the fact that previous studies were mainly performed on cell lines rather than primary cultures, and they were focused on the effect of autophagy induced by cytokines or pharmacological agents rather than on the modulation of basal autophagy.12 We believe that our results provide an important indication for future studies, underlining that the use of attenuated strains such as BCG or Mtb H37Ra in the study of the interaction of Mtb with the host immune system might not reflect what is occurring with fully virulent strains.

In the attempt to identify the molecular basis underlying autophagy inhibition in Mtb-infected DC, we investigated a variety of attenuated Mtb strains that were either lacking the major part of the ESX-1 secretion system, the promoter region of the CFP-10/ESAT-6 operon, or the C-terminus of the secreted effector protein ESAT-6. We also studied a variant of the attenuated Mtb H37Ra strain, which regained the ability of secreting ESAT-6 and CFP-10 by complementation with the phoP regulator from Mtb H37Rv. Both approaches showed that impairment of the autophagic flux only occurred if the engulfed live mycobacteria had the capacity to secrete intact ESAT-6/CFP-10 via the ESX-1/type VII secretion system. In line with these results, our data obtained with heat killed Mtb (Fig. 1A) showed that the interaction and the internalization of the bacteria inside DC are not yet sufficient for the inhibition of autophagy since the accumulation of the membrane-bound form MAP1LC3-II was observed when production and secretion of different Mtb effector proteins is likely to occur.

How the ESX-1 system could negatively regulate the autophagic process remains to be elucidated. The lack of ESAT-6 or CFP-10 antibodies functioning in intracellular detection assays prevented us to verify whether these proteins localize with autophagosomes. However, it is important to underline that also the truncated form of ESAT-6 lacking 12 residues at C-terminus seems to be unable to affect the autophagic flux since no MAP1LC3-II accumulation was observed. As the extreme C terminus of ESAT-6 represents a floppy, structurally not well-defined region of the protein,35 it is likely that this region is free to interact with host proteins or might help the interaction of ESAT-6 with the lipid bilayers of the subcellular organelles or the cell membrane.27,36 As an alternative to a direct interaction of ESAT-6 with the autophagic machinery, ESX-1 proteins could allow other Mtb-encoded factors to translocate into the cytosol to inhibit the autophagy flux. In this regard, a potential candidate for the antiautophagic activity of Mtb could be the phosphatidylinositol 3-phosphate phosphatase SapM, which has been described to block BCG-containing phagosome maturation.37 Interestingly, a recent observation by Shin and collaborators38 showed that the Mtb enhanced intracellular survival (Eis) factor, which is also released in the cytoplasm of infected macrophages,39 negatively regulates autophagy induction in murine macrophages. Although the specific contribution of Eis in infected DC remains to be characterized, these results suggest that Mtb could have adopted different strategies to interfere with distinct steps of autophagy.

It has also been reported that ESX-1 is required for the translocation of M. marinum and/or Mtb to the host cell cytosol.40-44 However, since Mtb is still detected in vesicle structures 16 h after infection (ref. 44 and data not shown) when autophagy block is already fully visible, it is likely that the effect on autophagy was not exerted by cytosolic Mtb. In this context, it is conceivable that the membrane-lysing or pore-forming activity exerted by ESAT-6 and/or other effector proteins of the ESX-1 system might be responsible for autophagy blockage during the early phases of infection while at later stages it allows the phagosome-to-cytosol translocation of Mtb.40-44 Although in our experimental model it is not yet clear whether ESAT-6 modulates directly or indirectly the autophagic process, the results obtained so far strongly suggest that the functional interaction between ESAT-6 and the DC autophagosome/autolysosome machinery is crucial to influence the immune response against TB.30

We also showed that rapamycin treatment restores a functional autophagic flux in Mtb-infected cells, as evaluated by the analysis of SQSTM1 levels and the colocalization of MAP1LC3 with the lysosomal markers LAMP1 and cathepsin B. These results also showed that, although a high number of autophagosomes are present in infected DC, autophagy does not target Mtb for lysosome degradation unless cells are treated with rapamycin, an effect that was prevented by the simultaneous addition of the conventional inhibitor of autophagy, wortmannin.

Since rapamycin is known to induce autophagy by inhibiting the activity of MTOR, an upstream negative regulator of this process, it is plausible that the effect of rapamycin is due to a rise of the levels of autophagy above a threshold, which cannot be further blocked by Mtb. In addition, we observed that IFNG has positive effects on autophagy similar to rapamycin, as shown previously by Gutierrez and colleagues.12 Indeed, activation of Mtb-infected DC with IFNG resulted in an enhanced degradation of SQSTM1 level and increased colocalization of Mtb with MAP1LC3 and LAMP1.

To provide functional relevance to these observations, we investigated the effect of rapamycin on autophagy-mediated killing of the tubercle bacilli and on Mtb-induced DC maturation. Rapamycin induced a bacteriostatic effect on mycobacterial growth rather than a reduction in Mtb viability, which conversely occurs in Mtb-infected macrophages.14 Moreover, while no significant differences were detected in the level of maturation markers in Mtb-infected DC upon rapamycin treatment, we observed a statistically significant enhancement of IL12 production. In addition, the secretion of IL12 in Mtb-infected cells was also promoted by IFNG, confirming the existence of a link between the intracellular pathways regulating autophagy process and the release of this Th1 promoting cytokine in DC. Accordingly, treatment of infected DC with E64D plus PepA abolishes Mtb-mediated induction of IL12, irrespective to the presence of rapamycin or IFNG, indicating that lysosomal activity is required for essential events of DC maturation process, such as the release of IL12, a crucial Th1 promoting cytokine. Moreover, an increased expression of IFNB1 was also evidentiated in Mtb-infected DC upon rapamycin treatment, which likely synergizes with IL12 in promoting a Th1 response as previously described.34 In this scenario it is likely that the expansion of IFNG producing cells may potentiate the autophagic processes in DC as well as in macrophages,15,16,45 which represent crucial regulators of both innate and adaptive immune response against pathogens, including intracellular mycobacteria.22,23 Although the involvement of components of autophagic machinery in the production of inflammatory cytokines in response to endotoxin or viral infections has been reported,46-48 no data are available so far on the regulation of cytokine expression by autophagy in Mtb-infected cells. Therefore, it would be important to identify the molecular mechanisms underlying the enhancement of IL12 and IFNB1 expression by rapamycin and IFNG in Mtb-infected DC in order to improve their immunoregulatory properties.

Collectively, our results shed light on novel aspect of DC-Mtb interaction, which could offer a new target for vaccine design and adjunctive immunotherapy with autophagy-promoting agents for the improvement of TB control.

Material and Methods

Antibodies and other reagents

Monoclonal antibodies (BD Bioscience) specific for CD1a (555806), CD14 (555398), CD38 (555460), CD86 (555657), HLA-DR (555811), CD83 (556855), IgG1 (555748), IgG2a (553457) were used as direct conjugates to FITC (Fluorescein Isothiocyanate) or PE (Phycoerythrin). Rabbit anti-MAP1LC3 for immunoblotting analysis (Cell Signaling, 2775), rabbit anti-MAP1LC3 for confocal microscopy (Sigma-Aldrich, L7543), mouse anti-LAMP1 (Abcam, ab25630), mouse anti-actin (Sigma-Aldrich, A2066), mouse anti-SQSTM1 (SQSTM1 D3, Santa Cruz Biotechnology, sc28359), mouse anti-cathepsin B (Calbiochem, IM27L). For autophagy induction, rapamycin (0.2 μM; Sigma-Aldrich, R0395) or with 10 ng/ml of IFNG (Pepro Tech EC LTD, 300-02) was added to DC cultures. To inhibit lysosomal activity, cells were incubated with 10 μg/ml E64d (Sigma-Aldrich, E8460) together with 10 μg/ml PepA (Sigma-Aldrich, P5318), bafilomycin A1 (Sigma-Aldrich, B1793), while wortmannin 1 μM (Sigma-Aldrich, W1628) was used as a pharmacological inhibitor of autophagy. The optimal concentration of rapamycin and E64d plus PepA was evaluated in preliminary dose-response experiments, while the treatment time is specified in each figure legend.

DC preparation

DC were prepared as previously described.49 Briefly, peripheral blood mononuclear cells were isolated from freshly collected buffy coats obtained from healthy voluntary blood donors (Blood Bank of University “La Sapienza,” Rome, Italy) by density gradient centrifugation using Lympholyte-H (Cedarlane, CL 5020). Monocytes were purified by positive sorting using anti-CD14 conjugated magnetic microbeads (Miltenyi, 130-050-201). The recovered cells were > 99% CD14+ as determined by flow cytometry with anti-CD14 antibody. DC were generated by culturing monocytes in 6-well tissue culture plates (Costar Corporation, 3516) with 50 ng/ml CSF2 (R&D Systems, 215-GM) and 1000 U/ml of IL4 (R&D Systems, 204-IL) for 5 d at 0.5 × 106 cells/ml in RPMI 1640 (BioWhittaker, BE12-167F) supplemented with 2 mM L-glutamine (BioWhittaker, BE17-605E) and 15% FCS (BioWhittaker, DE14-801F). No antibiotics were added to the cultures. At day 5 the cells were 90% CD1a+ and 95% CD14-. The differentiation medium containing IL4 and CSF2 was removed and replaced with RPMI supplemented with 2 mM L-glutamine and 15% FCS before Mtb infection to avoid cytokine-driven modulation of the autophagic and MTOR pathway.

Bacteria preparation and infection of DC

Mtb H37Rv (ATCC 27294; American Type Culture Collection) and M. bovis BCG (ATCC 27291) were used to infect DC cultures. As regards the recombinant strains used in this study, they were derived from Mtb H37RvΔRD127 or BCG Pasteur (1173P2) by integrating either the empty vector pYUB412, or pRD1-2F9 that carries a 32 kb fragment from Mtb H37Rv overlapping the extended RD1 region, or modified versions of pRD1-2F9 deleted for the promoter region of the CFP-10/ESAT-6 operon (ΔCFP-10prom) or the 11 C-terminal codons of ESAT-6 (ESAT-6-Δ84-95).28,29 Mtb H37Ra, the recombinant strain H37Ra::PhoP containing the wild-type PhoP gene and the strain complemented with an unrelated gene (H37Ra::RpsL) were previously described in Frigui et al.31 The plasmid pSMT3-DsRed (kindly by Wilbert Bitter, VU University, Amsterdam), and containing a cassette with the gene encoding the red fluorescent protein cherry under the control of the hsp60 promoter, was electroporated in Mtb H37Rv following standard procedures.50 The cloning of the recombinant Mtb H37Rv GFP was previously described by Delogu and collaborators.51 Recombinant Mtb H37Rv Ds-Red cherry as well as Mtb H37Rv GFP were cultured on 7H11 agar (BD Bioscience, 283810) supplemented with OADC (BD Bioscience, 211886) and with 50 μg/ml of hygromycin (Sigma-Aldrich, H9773) or in 7H9 liquid medium (BD Bioscience, 271310) supplemented with ADC (BD Bioscience, 211887), glycerol (Sigma-Aldrich, G5516) (0.5% vol/vol), 0.05% Tween 80 (Sigma-Aldrich, P1754), and containing 50 μg/ml of hygromycin until late log phase. One-ml of culture aliquots containing 10% of glycerol were stored at -80°C until use.

Mycobacteria were grown with gentle agitation (80 rpm) in 7H9 liquid medium supplemented with 0.05% Tween 80 and 10% OADC. Logarithmically growing cultures were centrifuged at 800 rpm for 10 min to eliminate clumped mycobacteria and then washed three times in RPMI 1640. Mycobacteria were resuspended in RPMI 1640 containing 10% FCS and then stored at -80°C. Vials were thawed and bacterial viability was determined by counting the number of colony forming units (CFU) on Middlebrook 7H10 agar plates (BD Bioscience, 262710). All bacteria preparations were analyzed for LPS contamination by the Limulus lysate assay (BioWhittaker, 50-647U) and contained less than 1 EU/ml. DC cultures were infected with a MOI of 1 bacteria/cell. Where indicated, Mtb was heat killed at 80°C for 1 h.

CFU assay

Four h after infection, the cell cultures were gently washed (three times) with RPMI 1640. DC were centrifuged at 150 × g for 10 min to selectively spin down cells while extracellular bacteria remain in the supernatants. Cells (~300.000) were resuspended in complete medium and cultured for the times indicated. Triplicate samples were assayed for CFU. At each time point cells were lysed with water containing 0.05% saponin (Sigma-Aldrich, 84510). Serial dilutions of the bacterial suspensions were plated (in duplicate) on 7H10 agar plates and viable bacteria were evaluated after 3 weeks of incubation at 37°C. Values are expressed as CFU/well.

Flow cytometry analysis

Approximately 1–2 × 105 cells were aliquoted into tubes and washed once in PBS containing 2% FCS. The cells were incubated with indicated antibodies (BD Bioscience) at 4°C for 30 min. The DC were then washed and fixed overnight with 4% paraformaldehyde (Pro-Labo, 20909290) before analysis on a FACSCan using CellQuest software (BD Bioscience).

Cytokine determination

Supernatants from DC cultures infected with Mtb and treated with rapamycin (0.2 μM) or E64d/PepA (10 μg/ml each) were harvested at 24 h after infection, filtered (0.2 μm) and stored at -80°C. The production of TNF (A) and IL12 (B) was measured by CBA Flex Set (BD Bioscience, 558472 for TNF, 558459 for IL12p70). The production of cytokines in MLR experiments was measured with human Th1/Th2 CBA Flex Set (BD Bioscience).

RNA isolation and real-time PCR quantifications

RNA was extracted from DC with RNeasy kit (Qiagen Inc., 74104) according to the manufacturer’s instructions. A phenol/chloroform extraction was performed to inactivate residual mycobacterial particles. Reverse transcriptions and quantitative PCR assays were performed as previously described.49 Primer pairs have been previously described.49 The primers used for IFNB1 and GAPDH were previously described33 while the oligonucleotides for SQSTM1 mRNA quantification were: 5′-ACAGATGCCAGAATCCGAAG-3′ (forward) and 5′-TGGGAGAGGGACTCAATCAG -3′ (reverse). Levels of IFNB1 and SQSTM1 mRNA are normalized to the GAPDH level using the Equation 2-ΔCt; the values are means ± SD of triplicate determinations

Immunoblotting analysis

Proteins were separated on 12% NuPAGE Bis-Tris gel (Invitrogen, NP0341) and electroblotted onto polyvinylidene fluoride membranes (Millipore, IPVH20200). Blots were incubated with primary antibodies in 5% nonfat dry milk in TBS plus 0,1% Tween20 (Sigma-Aldrich, P1379) overnight at 4°C. Detection was achieved using horseradish peroxidase-conjugate secondary antibody anti-mouse (Jackson Immunoresearch, 711-035-152), anti-rabbit (Jackson Immunoresearch, 715-035-150) and anti-goat (Jackson Immunoresearch, 705-035-147) and visualized with Enhanced Chemiluminescence plus (GE Healthcare, RPN2132). MAP1LC3-II/actin and SQSTM1/actin ratios were quantified by densitometric analysis using the ImageQuant software according to Rubinsztein et al.52

Confocal microscopy

Cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, P6148) in PBS followed by permeabilization with 0.1% triton X-100 (Sigma-Aldrich, 93443) in PBS. Primary antibodies were incubated for 1 h at room temperature and visualized by means of Cy2-conjugated secondary antibodies (Jackson Immunoresearch, 711-226-152) and Cy3-conjugated secondary antibodies (Jackson Immunoresearch, 715-166-150). Coverslips were mounted in SlowFade-Anti-Fade (Invitrogen, S36939) and examined under a confocal microscope (Leica TCS SP2). For lysosome staining 500 nM Lysotracker Red (Invitrogen, L7528) was added to cells 30 min before fixation. Digital images were acquired with Leica software, and processed and quantified with ImageJ (National Institutes of Health) using a set of defined intensity thresholds that were applied to all images. A minimum of 50 cells per sample was counted for triplicate samples per condition in each experiment.

Transmission electron microscopy

DC cultures were analyzed 24 h after infection. Cells cultured in different conditions were fixed with 2.5% glutaraldehyde (Assin Spa, R1012) in 0.1 M cacodylate buffer for 1 h at 4°C (sodium cacodylate trihydrate, Sigma-Aldrich, C4945), and postfixed in 1% osmium tetroxide (Sigma-Aldrich, 75632) in 0.1 M cacodylate buffer for 1 h. The cells were then dehydrated in graded ethanol and embedded in Epon resin (AGAR 100, Agar Scientific R1045). Ultrathin sections were stained with 2% uranyl acetate (Sigma-Aldrich, 73943) and observed under a Zeiss EM900 transmission electron microscope. Images were captured digitally with a Mega View II digital camera (SIS; Zeiss).

MLR experiment

MLR was conducted as previously described.49 CD4+ T cells were purified from cord blood cells obtained from healthy voluntary donors (Cord Blood Bank of UOC Transfusion Medicine, S. Eugenio Hospital-ASL Roma C, Rome, Italy) by indirect magnetic sorting with a CD4+ T-cell isolation kit (Miltenyi, 130-091-155). DC were infected with Mtb and 4 h after the infection rapamycin was added for additional 20 h. Immature DC, Mtb-infected DC with or without a rapamycin treatment were resuspended in 5% human AB serum (Euroclone, ECS0219D) complete medium prior to co-culture with naïve allogeneic cord blood CD4+ T cells. Where indicated, neutralizing mouse monoclonal anti-IL12 antibody (R&D Systems, AB-219-NA) and its isotype control antibody (R&D Systems, MAB002) were added to DC/CD4+ T cell co-culture at 20 μg/ml to neutralize the IL12. Supernatants from T cell/DC co-culture (ratio 10:1) were harvested at day 5 and analyzed for IFNG (560111), IL4 (558462) and IL5 (558463) release by CBA Flex set (BD Bioscience).

Ethics statement

Human blood was collected at the Blood Bank of University “La Sapienza” (Rome, Italy) and the Cord Blood Bank of UOC Transfusion Medicine, S. Eugenio Hospital (Rome, Italy) from healthy volunteers after written informed consent. Both blood centers operate under license from the Italian Ministry of Health. Therefore, the use of blood from these sources is exempt from our institutional review board.

Statistical analysis

Statistical analysis was calculated using a two-tailed for paired-data Student’s t-test. A p value < 0.05 was considered statistically significant.

Supplementary Material

Additional material

Acknowledgments

The study was supported by grants from Ministry for Health of Italy: “Ricerca Corrente” to M.P. G.M.F. and D.G.; “Ricerca Finalizzata” RF06.76.1, RF-IMI-2009-1302952 to D.G.; RF07.103 to E.M.C., G.M.F. and D.G.; by grants from the FP7 program of the European Community n°241745 to R.B. and E.M.C., n°40H58 to D.G. and APO-SYS Health F4-2007-200767 to M.P. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The authors wish to thank Marco Tilotta and Tiziana Vescovo for the help provided in some of the experimental settings. We are deeply grateful to Blood Bank of University “La Sapienza” (Rome, Italy) and UOC Transfusion Medicine, S. Eugenio Hospital-ASL Roma C (Rome, Italy) for providing us with blood donations.

Glossary

Abbreviations:

ADC
albumin, dextrose, catalase
Ag85B
antigen 85 B
BCG
Bacillus Calmette-Guerin
CTSB
cathepsin B
CBA
cytometric bead assay
CD
cluster of differentiation
CFP-10
10 kD culture filtrate protein
CFU
colony forming units
Cy
cyanine
DC
dendritic cells
DMSO
dimethyl sulfoxide
ECL
enhanced chemiluminescence
ESAT-6
6 kD early secreted antigenic target
ESX-1
ESAT-6 secretion system-1
FITC
fluorescein isothiocyanate
FCS
fetal calf serum
GAPDH
glyceraldehyde-3-phosphate dehydrogenase
CSF2/GM-CSF
granulocyte-macrophage colony stimulating factor
HLA-DR
human leucocytes antigen-DR
IF
immunofluorescence
IFN
interferon
IL
interleukin
LAMP1
lysosomal-associated membrane protein 1
MAP1LC3/LC3
microtubule-associated protein 1A/1B-light chain 3
LPS
lipopolysaccharide
MDR-TB
multidrug-resistant tuberculosis
MHC
major histocompatibility complex
MLR
mixed lymphocyte reaction
MOI
multiplicity of infection
mRNA
messenger RNA
Mtb
Mycobacterium tuberculosis
MTOR
mechanistic target of rapamycin
OADC
oleic acid albumin dextrose complex
PAF
paraformaldehyde
PBMC
peripheral blood mononuclear cell
PBS
phosphate buffered saline
PE
phycoerythrin
pepA
pepstatin A
PVDF
polyvinylidene fluoride
Rapa
rapamycin
RD1
region of differentiation 1
RT-PCR
real time-polymerase chain reaction
SapM
secreted acid phosphatase of Mycobacterium tuberculosis
SE
standard error
SEM
standard error of the mean
SQSTM1/p62
sequestosome 1
TB
tuberculosis
TBS
tris buffered saline
Th
T helper
TNF
tumor necrosis factor-α
WB
western blot
XDR-TB
extensively-drug-resistant tuberculosis

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Supplemental Materials

Supplemental materials may be found here: www.landesbioscience.com/journals/autophagy/article/20881

Footnotes

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