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Human Macrophages Infected with a High Burden of ESAT-6-Expressing M. tuberculosis Undergo Caspase-1-and Cathepsin B-Independent Necrosis
Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology.
Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
2011 (English)In: PLOS ONE, ISSN 1932-6203, Vol. 6, no 5Article in journal (Refereed) Published
Abstract [en]

Mycobacterium tuberculosis (Mtb) infects lung macrophages, which instead of killing the pathogen can be manipulated by the bacilli, creating an environment suitable for intracellular replication and spread to adjacent cells. The role of host cell death during Mtb infection is debated because the bacilli have been shown to be both anti-apoptotic, keeping the host cell alive to avoid the antimicrobial effects of apoptosis, and pro-necrotic, killing the host macrophage to allow infection of neighboring cells. Since mycobacteria activate the NLRP3 inflammasome in macrophages, we investigated whether Mtb could induce one of the recently described inflammasome-linked cell death modes pyroptosis and pyronecrosis. These are mediated through caspase-1 and cathepsin-B, respectively. Human monocyte-derived macrophages were infected with virulent (H37Rv) Mtb at a multiplicity of infection (MOI) of 1 or 10. The higher MOI resulted in strongly enhanced release of IL-1 beta, while a low MOI gave no IL-1 beta response. The infected macrophages were collected and cell viability in terms of the integrity of DNA, mitochondria and the plasma membrane was determined. We found that infection with H37Rv at MOI 10, but not MOI 1, over two days led to extensive DNA fragmentation, loss of mitochondrial membrane potential, loss of plasma membrane integrity, and HMGB1 release. Although we observed plasma membrane permeabilization and IL-1 beta release from infected cells, the cell death induced by Mtb was not dependent on caspase-1 or cathepsin B. It was, however, dependent on mycobacterial expression of ESAT-6. We conclude that as virulent Mtb reaches a threshold number of bacilli inside the human macrophage, ESAT-6-dependent necrosis occurs, activating caspase-1 in the process.

Place, publisher, year, edition, pages
Public Library of Science (PLoS) , 2011. Vol. 6, no 5
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-68900DOI: 10.1371/journal.pone.0020302ISI: 000291052200049OAI: oai:DiVA.org:liu-68900DiVA: diva2:422025
Note
Original Publication: Amanda Welin, Daniel Eklund, Olle Stendahl and Maria Lerm, Human Macrophages Infected with a High Burden of ESAT-6-Expressing M. tuberculosis Undergo Caspase-1-and Cathepsin B-Independent Necrosis, 2011, PLOS ONE, (6), 5. http://dx.doi.org/10.1371/journal.pone.0020302 Licensee: Public Library of Science (PLoS) http://www.plos.org/Available from: 2011-06-10 Created: 2011-06-10 Last updated: 2013-11-14
In thesis
1. Mycobacterium tuberculosis and the human macrophage: shifting the balance through inflammasome activation
Open this publication in new window or tab >>Mycobacterium tuberculosis and the human macrophage: shifting the balance through inflammasome activation
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Mycobacterium tuberculosis is a very successful pathogen and tuberculosis constitutes a major threat to global health worldwide. The World Health Organization (WHO) estimates that almost nine million new cases and 1.5 million deaths occur annually and the situation is worsened by increased antibiotic resistance and an extreme synergism with the HIV pandemic. M. tuberculosis primarily affects the lungs where the infection can lead to either eradication of the bacteria or the initiation of an immune response that culminates in the formation of a large cluster of immune cells termed granulomas. In these granulomas, the bacteria can either replicate and cause disease with the ultimate goal of spreading to new hosts or cause latent tuberculosis, which can persist for decades. The tools available to manage the disease are currently suboptimal and include lengthy antibiotic treatments and an inefficient vaccine resulting in poor protection. On a cellular level, M. tuberculosis primarily infects the cell designed to recognize, ingest and eradicate bacteria, namely the human macrophage. Following recognition, the macrophage phagocytoses the bacterium and tries to kill it using an array of different effector mechanisms including acidification of the bacterium-containing vacuole, different degradative enzymes and the generation of radicals. However, the bacterium is able to circumvent many of these harmful effects, leading to a tug-of-war between the bacterium  and host macrophage. This thesis aims at studying the interaction between the human macrophage and M. tuberculosis to identify host factors critical for controlling growth of the bacteria. More specifically, it focuses on the role of an intracellular receptor protein called NLRP3 and its downstream effects. NLRP3 is activated in human macrophages infected by M. tuberculosis and upon activation it forms a multi-protein complex known as the inflammasome. This protein complex is known to induce the production of the proinflammatory cytokine IL-1β and specialized forms of macrophage cell death. We hypothesized that stimulating this pathway would have a beneficial effect for the host macrophage during infection with M. tuberculosis.

To allow us to follow interaction between M. tuberculosis and the human macrophage, we first developed a luminometry-based method of measuring bacterial numbers and following bacterial growth over several days in infected cells. With this new assay we showed that low numbers of bacteria induced very low levels of IL-1β and failed to induce any type of cell death in the macrophage. However, when a critical number of bacteria were reached, the infected macrophages underwent necrosis, which was accompanied by high levels of IL-1β. We were also able to show that addition of vitamin D, which has been implicated as an important factor for increased killing capacity of infected macrophages, increased the production of IL-1β, which coincided with increased killing of M. tuberculosis. This effect was seen specifically in cells from patients with active tuberculosis, suggesting that these cells are primed to respond to vitamin D and increased levels of IL-1β. Furthermore, we also showed that increasing production of IL-1β by stimulating infected macrophages with apoptotic neutrophils in turn drives the production of other proinflammatory cytokines. Lastly, we showed that gain-of-function polymorphisms in inflammasome components linked to increased inflammasome activation and IL-1β production promotes bacterial killing in human macrophages. In conclusion, the work presented in this thesis shows  that by enhancing the functions of the inflammasome, it is possible to tip the balance between the human macrophage and M. tuberculosis in favor of the host cell.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2013. 97 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1372
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-100890 (URN)10.3384/diss.diva-100890 (DOI)978-91-7519-558-2 (ISBN)
Public defence
2013-12-11, Berzeliussalen, Campus US, Linköpings universitet, Linköping, 09:00 (English)
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Available from: 2013-11-14 Created: 2013-11-14 Last updated: 2013-11-14Bibliographically approved

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