RESUMO
Tuberculosis remains a global pandemic and drives lung matrix destruction to transmit. Whilst pathways driving inflammatory responses in macrophages have been relatively well described, negative regulatory pathways are less well defined. We hypothesised that Mycobacterium tuberculosis (Mtb) specifically targets negative regulatory pathways to augment immunopathology. Inhibition of signalling through the PI3K/AKT/mTORC1 pathway increased matrix metalloproteinase-1 (MMP-1) gene expression and secretion, a collagenase central to TB pathogenesis, and multiple pro-inflammatory cytokines. In patients with confirmed pulmonary TB, PI3Kδ expression was absent within granulomas. Furthermore, Mtb infection suppressed PI3Kδ gene expression in macrophages. Interestingly, inhibition of the MNK pathway, downstream of pro-inflammatory p38 and ERK MAPKs, also increased MMP-1 secretion, whilst suppressing secretion of TH1 cytokines. Cross-talk between the PI3K and MNK pathways was demonstrated at the level of eIF4E phosphorylation. Mtb globally suppressed the MMP-inhibitory pathways in macrophages, reducing levels of mRNAs encoding PI3Kδ, mTORC-1 and MNK-1 via upregulation of miRNAs. Therefore, Mtb disrupts negative regulatory pathways at multiple levels in macrophages to drive a tissue-destructive phenotype that facilitates transmission.
Assuntos
Macrófagos/imunologia , Mycobacterium tuberculosis/imunologia , Tuberculose Pulmonar/imunologia , Animais , Humanos , Macrófagos/microbiologia , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/imunologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Complexos Multiproteicos/genética , Complexos Multiproteicos/imunologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/fisiologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/imunologia , Tuberculose Pulmonar/genética , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/patologiaRESUMO
Cell biology differs between traditional cell culture and 3-dimensional (3-D) systems, and is modulated by the extracellular matrix. Experimentation in 3-D presents challenges, especially with virulent pathogens. Mycobacterium tuberculosis (Mtb) kills more humans than any other infection and is characterised by a spatially organised immune response and extracellular matrix remodelling. We developed a 3-D system incorporating virulent mycobacteria, primary human blood mononuclear cells and collagen-alginate matrix to dissect the host-pathogen interaction. Infection in 3-D led to greater cellular survival and permitted longitudinal analysis over 21 days. Key features of human tuberculosis develop, and extracellular matrix integrity favours the host over the pathogen. We optimised multiparameter readouts to study emerging therapeutic interventions: cytokine supplementation, host-directed therapy and immunoaugmentation. Each intervention modulates the host-pathogen interaction, but has both beneficial and harmful effects. This methodology has wide applicability to investigate infectious, inflammatory and neoplastic diseases and develop novel drug regimes and vaccination approaches.
