Peer-review started: May 20, 2015
First decision: October 17, 2015
Revised: January 19, 2015
Accepted: February 14, 2016
Article in press: February 16, 2016
Published online: March 27, 2016
Processing time: 314 Days and 17.6 Hours
AIM: To study the bacteriocidal or bacteriostatic role of mast cells during infection with Mycobacterium.
METHODS: Mycobacterium marinum (M. marinum) (BAA-535/M strain) was investigated for its ability to grow at a temperature relevant to the mammalian host. Primary mast cells were differentiated from bone marrows of mice, a human mast cell line (HMC-1) and a human monocytic cell line (MonoMac6) were maintained in culture. Mice were stimulated by intraperitoneal injection of heat-killed M. marinum to study cytochemically the degranulation of peritoneal mast cells. HMC-1 cells were stimulated with M. marinum to analyse mRNA expression for inflammatory reactant genes, while HMC-1 and primary mouse mast cells were infected with M. marinum to establish in parallel cell viability (lactate dehydrogenase release and cell counts) and viable mycobacterial counts. Flow cytometry was used to assess intracellular presence of fluorescein isothiocyanate labelled M. marinum after trypan blue quenching and to measure the extent of infection-induced apoptosis or necrosis in HMC-1. A GFP expressing recombinant M. marinum strain was used to assess intracellular location by fluorescence microscopy. Light microscopy of osmium tetroxide and Gram Twort stained sections of 0.5 μm and transmission electron microscopy were undertaken as sensitive methods.
RESULTS: Since its isolation, M. marinum has adapted to grow at 37 °C. This study found that M. marinum infects HMC-1 cells and primary murine mast cells, where they survive, replicate, and cause dose dependent cell damage over the analysis period of up to 120 h. Amikacin was an effective aminoglycoside antibiotic to eliminate extracellular or membrane attached M. marinum in order to adequately quantify the intracellular bacterial loads. In vivo, intraperitoneal injection of heat-killed M. marinum led to the release of mast cell granules in mice. HMC-1 cells stimulated with M. marinum showed a biphasic pattern of increased mRNA expression for LL-37 and COX-2/TNF-α during 24 h of stimulation. In HMC-1, M. marinum localised to the cytoplasm whereas in primary mast cells, M. marinum were found in vacuoles.
CONCLUSION: The effector role of mast cells in infection with M. marinum can be studied in vitro and in vivo.
Core tip: Mycobacterium marinum (M. marinum) is easily culturable and shows promise as a model to understand in mammalian cells the pathogenicity of M. tuberculosis. We used M. marinum to study uptake and elimination of M. marinum by mast cells, being abundant immune effector cells. A range of imaging techniques was used to unequivocally show the intracellular presence of M. marinum. Mast cells did not control the replication of M. marinum but reacted in a pro-inflammatory way. This is consistent with mast cells being orchestrators of inflammation. In summary, we clearly show that M. marinum can infect mast cells, survive and replicate within.