Forkhead box O3 transcription factor fine-tunes IL-6 expression in mycobacteria-infected macrophages.

INTRODUCTION: Tuberculosis (TB) remains the deadliest infectious disease in humans; however, a robust immune response can either protect against or control the infection. Household contacts of TB patients can remain uninfected for decades, while a quarter of the world's population that is latently infected can remain healthy for years. Characterization of the effectors of such an immune response would help in developing novel host-directed tools to better treat and/or prevent TB. IL-6 is a crucial cytokine that plays a central role in orchestrating a protective TB immune response; however, a delicate balance between its beneficial and detrimental effects must be maintained to effectively control the infection. Here, we aimed to characterize the cellular effectors that regulate IL-6 expression in mycobacteria-infected cells. METHODS: We used PI3K and AKT inhibitors, as well as FOXO3-specific siRNA, to modulate FOXO3 activity in BCG-infected macrophages and assessed the effect on IL-6 expression. Reporter gene assays were performed using four IL-6 promoter constructs (-600 bp, -900 bp, -1.5 kb, and -2 kb). In silico analysis was carried out using Genomatix software and the Eukaryotic Promoter Database to identify Forkhead binding motifs, followed by mutagenesis experiments to validate their function. RESULTS: The PI3K/AKT/FOXO3 axis regulates IL-6 expression in human macrophages: FOXO3 activation (via PI3K or AKT inhibition) induced IL-6 expression, while FOXO3 silencing reduced IL-6 production in BCG-infected macrophages. FOXO3 positively regulated IL-6 promoter activity across all cloned fragments, with the strongest effect at -1.5 kb; extension to -2 kb modulated this FOXO3-mediated induction. In silico analysis identified three FOXO3-specific binding sites (-786, -1331, and -1631). Mutagenesis identified the motif at -1331 as the major positive regulator of IL-6 expression, while the motif at -1631 mediated the negative regulation exerted by the distal promoter fragment. DISCUSSION: We report for the first time that FOXO3 exerts both positive and negative regulation of IL-6 transcription, fine-tuning its expression in mycobacteria-infected macrophages. Together with our previous report on FOXO3-mediated suppression of IL-10, these results highlight the crucial role of FOXO3 in the TB immune response and support targeting the PI3K/Akt/FOXO3 axis as a host-directed approach to improve TB treatment and prevention.