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In human primary dendritic cells (DC) rapamycin-an autophagy inducer and protein synthesis inhibitor-overcomes the autophagy block induced by Mycobacterium tuberculosis (Mtb) and promotes a Th1 response via IL-12 secretion. Here, the immunostimulatory activity of rapamycin in Mtb-infected DC was further investigated by analyzing both transcriptome and translatome gene profiles. Hundreds of differentially expressed genes (DEGs) were identified by transcriptome and translatome analyses of Mtb-infected DC, and some of these genes were found further modulated by rapamycin. The majority of transcriptome-associated DEGs overlapped with those present in the translatome, suggesting that transcriptionally stimulated mRNAs are also actively translated. In silico analysis of DEGs revealed significant changes in intracellular cascades related to cytokine production, cytokine-induced signaling and immune response to pathogens. In particular, rapamycin treatment of Mtb-infected DC caused an enrichment of IFN-β, IFN-λ and IFN-stimulated gene transcripts in the polysome-associated RNA fraction. In addition, rapamycin led to an increase of IL-12, IL-23, IL-1β, IL-6, and TNF-α but to a reduction of IL-10. Interestingly, upon silencing or pharmacological inhibition of GSK-3β, the rapamycin-driven modulation of the pro- and anti-inflammatory cytokine balance was lost, indicating that, in Mtb-infected DC, GSK-3β acts as molecular switch for the regulation of the cytokine milieu. In conclusion, our study sheds light on the molecular mechanism by which autophagy induction contributes to DC activation during Mtb infection and points to rapamycin and GSK-3β modulators as promising compounds for host-directed therapy in the control of Mtb infection., Competing Interests: 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.​​​​​​​​​​​​​​​​​​​​, (Copyright © 2021 Etna, Severa, Licursi, Pardini, Cruciani, Rizzo, Giacomini, Macchia, Palumbo, Stallone, Carella, Livingstone, Negri, Pellegrini and Coccia.)

Autophagy is a primordial eukaryotic pathway, which provides the immune system with multiple mechanisms for the elimination of invading pathogens including Mycobacterium tuberculosis (Mtb). As a consequence, Mtb has evolved different strategies to hijack the autophagy process. Given the crucial role of human primary dendritic cells (DC) in host immunity control, we characterized Mtb-DC interplay by studying the contribution of cellular microRNAs (miRNAs) in the post-transcriptional regulation of autophagy related genes. From the expression profile of de-regulated miRNAs obtained in Mtb-infected human DC, we identified 7 miRNAs whose expression was previously found to be altered in specimens of TB patients. Among them, gene ontology analysis showed that miR-155, miR-155* and miR-146a target mRNAs with a significant enrichment in biological processes linked to autophagy. Interestingly, miR-155 was significantly stimulated by live and virulent Mtb and enriched in polysome-associated RNA fraction, where actively translated mRNAs reside. The putative pair interaction among the E2 conjugating enzyme involved in LC3-lipidation and autophagosome formation-ATG3-and miR-155 arose by target prediction analysis, was confirmed by both luciferase reporter assay and Atg3 immunoblotting analysis of miR-155-transfected DC, which showed also a consistent Atg3 protein and LC3 lipidated form reduction. Late in infection, when miR-155 expression peaked, both the level of Atg3 and the number of LC3 puncta per cell (autophagosomes) decreased dramatically. In accordance, miR-155 silencing rescued autophagosome number in Mtb infected DC and enhanced autolysosome fusion, thereby supporting a previously unidentified role of the miR-155 as inhibitor of ATG3 expression. Taken together, our findings suggest how Mtb can manipulate cellular miRNA expression to regulate Atg3 for its own survival, and highlight the importance to develop novel therapeutic strategies against tuberculosis that would boost autophagy.

Modern strategies to develop vaccines against Mycobacterium tuberculosis (Mtb) aim to improve the current Bacillus Calmette-Guerin (BCG) vaccine or to attenuate the virulence of Mtb vaccine candidates. In the present study, the impact of wild type or mutated region of difference 1 (RD1) variants on the immunogenicity of Mtb and BCG recombinants was investigated in human primary dendritic cells (DC). A comparative analysis of transcriptome, signalling pathway activation, maturation, apoptosis, cytokine production and capacity to promote Th1 responses demonstrated that DC sense quantitative and qualitative differences in the expression of RD1-encoded factors--ESAT6 and CFP10--within BCG or Mtb backgrounds. Expansion of IFN-γ producing T cells was promoted by BCG::RD1-challenged DC, as compared to their BCG-infected counterparts. Although Mtb recombinants acted as a strong Th-1 promoting stimulus, even with RD1 deletion, the attenuated Mtb strain carrying a C-terminus truncated ESAT-6 elicited a robust Th1 promoting phenotype in DC. Collectively, these studies indicate a necessary but not sufficient role for the RD1 locus in promoting DC immune-regulatory functions. Additional mycobacterial factors are likely required to endow DC with a high Th1 polarizing capacity, a desirable attribute for a successful control of Mtb infection.