Tuberculosis, a human infectious disease caused by Mycobacterium tuberculosis (M.tb), is still a major cause of morbidity and mortality worldwide. The success of M.tb as a pathogen relies mainly on its ability to divert the host innate immune responses. One way by which M.tb maintains a persistent infection in a “silent” granuloma is to inhibit inflammation and induce an immunoregulatory phenotype in host macrophages (MΦs). However, M.tb effectors governing the switch of MΦs from the pro-inflammatory M1 to the anti-inflammatory M2 phenotype remain to be determined. The Early Secreted Antigenic Target 6 kDa or ESAT-6, has been implicated in the virulence and pathogenesis of tuberculosis. Here, we investigated roles of ESAT-6 in MΦ differentiation and polarization. We found that treatment of human monocytes with ESAT-6 did not interfere with differentiation of M1 MΦs. However, ESAT-6 promoted differentiation of M0 and M2 MΦs toward the M1 phenotype, as indicated by secretion of pro-inflammatory cytokines IL-6, IL-12, and TNF-α, and induction of a typical M1 transcriptional signature. Interestingly, we found that ESAT-6 switched terminal full activation of M1 polarized MΦs to the M2 phenotype. Indeed, in the pro-inflammatory M1 MΦs, ESAT-6 was able to inhibit IL-12 and TNF-α secretion and stimulate that of IL-10. Moreover, gene expression profiling of these cells showed that ESAT-6 induced downregulation of M1 MΦ cell surface molecules CD80 and CD86, transcription factors IRF5 and c-MAF, cytokines IL-12, IL-10, and IL-6, as well as chemokines CXCL10 and CXCL1. Overall, our findings suggest ESAT-6 as being one of the effectors used by M.tb to induce the pro-inflammatory M1 phenotype at the primo-infection; a prerequisite step to promote granuloma formation and subsequently drive the phenotype switch of MΦ polarization from M1 to M2 at a later stage of the infection. Our study improves current knowledge regarding mechanisms of virulence of M.tb and may be helpful to develop novel tools targeting ESAT-6 for a better and more efficient treatment of tuberculosis.

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Early secreted antigenic target 6 kDa (ESAT‐6) and culture filtrate protein 10 kDa (CFP‐10) are complex proteins secreted by Mycobacterium tuberculosis that play a major role in the pathogenesis of tuberculosis. However, studies focusing on the biological functions of ESAT‐6 led to discordant results and the role of ESAT‐6 remains controversial. In the present study, we aim to address a potential explanation for this discrepancy and to highlight the physiological impact of two conformational states of ESAT‐6. Analysis of a recombinant form of ESAT‐6 by native gel electrophoresis, size exclusion chromatography and CD spectroscopy revealed that ESAT‐6 forms dimers/multimers with higher molecular weight, which disappeared under the action of the detergent amidosulfobetaine‐14 (ASB), giving rise to another conformational state of the protein. NMR has further indicated that ASB‐treated versus nontreated ESAT‐6 adopted distinct structural forms but with no well defined tertiary structure. However, protein–protein docking analysis favored a dimeric state of ESAT‐6. Interestingly, the two preparations presented opposing effects on mycobacterial infectivity, as well as macrophage survival, interferon‐γ secretion and membrane pore formation. Thereafter, we generated a recombinant form of the physiological heterodimer ESAT‐6/CFP‐10 that ASB was also able to dissociate and which showed functions similar to those of ESAT‐6 dimers/multimers. Our data suggest that, in the absence of CFP‐10, the hydrophobic regions of the ESAT‐6 can form dimers/multimers, mimicking the ESAT‐6/CFP‐10 heterodimer, whereas their dissociation generates a protein presenting entirely different activities. Overall, the present study clarifies the intriguing divergences between reports that could be attributed to the ESAT‐6 oligomeric state and sheds light on its importance for a better comprehension of the physiopathology of tuberculosis.

Enhanced apoptosis of BCG-infected macrophages has been shown to induce stronger dendritic cell-mediated cross-priming of T cells, leading to higher protection against tuberculosis (TB). Uncovering host effectors underlying BCG-induced apoptosis may then prove useful to improve BCG efficacy through priming macrophage apoptosis. Her we report that BCG-mediated apoptosis of human macrophages relies on FOXO3 transcription factor activation. BCG induced a significant apoptosis of THP1 (TDMs) and human monocytes (MDMs)-derived macrophages when a high moi was used, as shown by annexin V/7-AAD staining. BCG-induced apoptosis was associated with dephosphorylation of the prosurvival activated threonine kinase (Akt) and its target FOXO3. Cell fractionation and immunofluorescence microscopy showed translocation of FOXO3 to the nucleus in BCG-infected cells, concomitantly with an increase of FOXO3 transcriptional activity. Moreover, FOXO3 expression knock-down by small interfering RNA (siRNA) partially inhibited the BCG-induced apoptosis. Finally, real-time quantitative PCR (qRT-PCR) analysis of the expression profile of BCG-infected macrophages showed an upregulation of two pro-apoptotic targets of FOXO3, NOXA and p53 upregulated modulator of apoptosis (PUMA). Our results thus indicate that FOXO3 plays an important role in BCG-induced apoptosis of human macrophages and may represent a potential target to improve vaccine efficacy through enhanced apoptosis-mediated cross-priming of T cells.

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