Low Dose BCG Infection as a Model for Macrophage Activation Maintaining Cell Viability

Abstract

Mycobacterium bovis BCG, the current vaccine against tuberculosis, is ingested by macrophages promoting the development of effector functions including cell death and microbicidal mechanisms. Despite accumulating reports on M. tuberculosis, mechanisms of BCG/macrophage interaction remain relatively undefined. In vivo, few bacilli are sufficient to establish a mycobacterial infection; however, in vitro studies systematically use high mycobacterium doses. In this study, we analyze macrophage/BCG interactions and microenvironment upon infection with low BCG doses and propose an in vitro model to study cell activation without affecting viability. We show that RAW macrophages infected with BCG at MOI 1 activated higher and sustained levels of proinflammatory cytokines and transcription factors while MOI 0.1 was more efficient for early stimulation of IL-1β, MCP-1, and KC. Both BCG infection doses induced iNOS and NO in a dose-dependent manner and maintained nuclear and mitochondrial structures. Microenvironment generated by MOI 1 induced macrophage proliferation but not MOI 0.1 infection. In conclusion, BCG infection at low dose is an efficient in vitro model to study macrophage/BCG interactions that maintains macrophage viability and mitochondrial structures. This represents a novel model that can be applied to BCG research fields including mycobacterial infections, cancer immunotherapy, and prevention of autoimmunity and allergies.

Interferon-gamma promotes iron export in human macrophages to limit intracellular bacterial replication

“…bovis BCG was used as model for M. tuberculosis in several assays that required analysis at biosafety level 2 (Fig 3 and 4). However, we feel M. bovis BCG is an adequate substitute for these studies and the data generated here are comparable to M. tuberculosis for a number of reasons: It is well known that M. bovis BCG maintains a high genetic homology and similar cell wall composition to M. tuberculosis (50, 51), and it has been observed here (Fig 1) and in other studies (52)(53)(54) that within the 24-48 hour infection times used in this study, no differences in the bacterial replication rates, trafficking patterns, or host cell viability rates are observed between M. tuberculosis and M. bovis BCG infected cell lines. These common traits at least in the earliest stages of infection allow M. bovis BCG to extrinsically activate TLR signaling and induce hepcidin secretion and intracellular iron retention much like M. tuberculosis (Fig 2 and 3).…”

Transmembrane Tumor Necrosis Factor Controls Myeloid-Derived Suppressor Cell Activity via TNF Receptor 2 and Protects from Excessive Inflammation during BCG-Induced Pleurisy

“…The density of the bands was analyzed using the online ImageJ 1.39c software. Actin was used as loading control as reported ( 34 ).…”

Gain of function in Mycobacterium bovis BCG Moreau due to loss of a transcriptional repressor

“…Numerous studies have focused on macrophage activation and death in response to M. tuberculosis , but much less is known about the BCG-mediated cellular activities in the phagocytic cells. 25 Under the experimental conditions used in our study, significant loss in macrophage viability was observed at all times due to the infection, but no significant loss in macrophage viability was observed due to the recombinant BCG strain used ( Fig. 3 ).…”