Characterizing the Differential Response by Primary Macrophages to Infection by Pathogenic and Non-Pathogenic Mycobacteria

Mycobacteria are intracellular pathogens that are capable of surviving and persisting within host macrophages by using sophisticated evasion strategies. One of the most intriguing questions in mycobacterial pathogenesis concerns the initial interaction between mycobacteria and host macrophage and how mycobacteria modulate the macrophage signaling response. Interestingly infection with pathogenic strains of M. avium results in diminished activation of p38 and ERK1/2 in macrophages compared to infections with the fast-growing, non-pathogenic M. smegmatis and M. phlei. However, the upstream signals required for MAPK activation and the mechanisms behind the differential activation of the MAPKs remain undefined. In this study, using bone marrow-derived macrophages (BMMµÐ), we determined that ERK1/2 activation following mycobacterial infection is dependent on Ca2+ and sphingosine kinase (SPK) activation. Interestingly, in BMMµÐ infected with M. smegmatis compared to M. avium, there is sustained activation of Ca2+/CaM dependent CaMKII, which through induction of cAMP results in increased ERK1/2 activation and TNF-µÔ production. However, low TNF-µÔ production in M. avium infected BMMµÐ is independent of these pathways. Furthermore, there was increased activation of conventional PKC isoforms (cPKC) and PI-3 kinase in BMMµÐ infected with M. smegmatis compared to M. avium. This cPKC and PI-3 kinase activation was also dependent on SPK and PI-PLC. Finally, in BMMµÐ infected with M. smegmatis compared to M. avium, we observed enhanced secretion of TNF-µÔ, IL-6, RANTES and G-CSF, which is dependent on SPK, PI-PLC, cPKC and PI-3 kinase. We then initiated experiments using receptor knockout macrophages to determine the role of different macrophage Pattern Recognition Receptors (PRR) in the induction of macrophage pro-inflammatory response. We observed that mannose receptor is not required for mycobacterial phagocytosis or for macrophage activation. Additional analysis showed that MAPK activation and TNF-µÔ production in macrophage infected with M. avium or M. smegmatis is dependent on MyD88 and TLR2 (Toll-like receptor 2) but not TLR4. However, the TLR2 mediated production of TNF-µÔ by M. smegmatis infected macrophages was dependent on the µÕ-glucan receptor Dectin-1. A similar requirement for Dectin-1 in TNF-µÔ production was observed for macrophages infected with M. bovis BCG and M. phlei. Furthermore, IL-6, G-CSF and RANTES production by mycobacteria infected macrophages required Dectin-1. In conclusion, our data clearly demonstrate that multiple signaling pathways are targeted by mycobacteria to induce a differential pro-inflammatory response in macrophages and importantly this difference is due to the qualitative and quantitative differences in the stimulation of macrophage receptors by pathogenic and non-pathogenic mycobacteria.