Basic Study
Copyright ©The Author(s) 2016. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Immunol. Mar 27, 2016; 6(1): 83-95
Published online Mar 27, 2016. doi: 10.5411/wji.v6.i1.83
Intracellular localisation of Mycobacterium marinum in mast cells
Sadiyo Siad, Simon Byrne, Galina Mukamolova, Cordula Stover
Sadiyo Siad, Simon Byrne, Galina Mukamolova, Cordula Stover, Department of Infection, Immunity and Inflammation, College of Medicine, Biological Sciences and Psychology, University of Leicester, Leicester LE1 9HN, United Kingdom
Author contributions: Siad S designed this study as part of her PhD thesis; Byrne S assisted in critical technical aspects; Mukamolova G and Stover C supervised the work and are joint last authors; Stover C assembled the data; all authors read and approved the contents.
Supported by Faculty for the Future Fellowship grant by the Schlumberger Foundation (recipient Siad S).
Institutional review board statement: The 3Rs (reduction, refinement, replacement) ethical subcommittee approved the animal experimentation described in this report as a way of refinement, avoiding severity.
Institutional animal care and use committee statement: All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Leicester and were in accordance with Home Office regulations (PPL60/4327; PIL60/13404).
Conflict-of-interest statement: None of the authors have a conflict of interest to declare.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Dr. med. Cordula Stover, PhD, Senior Lecturer in Immunology, Department of Infection, Immunity and Inflammation, College of Medicine, Biological Sciences and Psychology, University of Leicester, University Road, Leicester LE1 9HN, United Kingdom. cms13@le.ac.uk
Telephone: +44-116-2525032 Fax: +44-116-2525030
Received: May 18, 2015
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
Abstract

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.

Keywords: Mast cells; Mycobacterium marinum; Microscopy; Infection; Degranulation

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.