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Mycobacterium tuberculosis and the human macrophage: shifting the balance through inflammasome activation
Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
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: urn:nbn:se:liu:diva-100890DOI: 10.3384/diss.diva-100890ISBN: 978-91-7519-558-2 (print)OAI: oai:DiVA.org:liu-100890DiVA: diva2:664178
Public defence
2013-12-11, Berzeliussalen, Campus US, Linköpings universitet, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2013-11-14 Created: 2013-11-14 Last updated: 2013-11-14Bibliographically approved
List of papers
1. Validation of a Medium-Throughput Method for Evaluation of Intracellular Growth of Mycobacterium tuberculosis
Open this publication in new window or tab >>Validation of a Medium-Throughput Method for Evaluation of Intracellular Growth of Mycobacterium tuberculosis
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2010 (English)In: Clinical and Vaccine Immunology, ISSN 1556-6811, E-ISSN 1556-679X, Vol. 17, no 4, 513-517 p.Article in journal (Refereed) Published
Abstract [en]

Intracellular pathogens such as Mycobacterium tuberculosis have adapted to a life inside host cells, in which they utilize host nutrients to replicate and spread. Ineffective methods for the evaluation of growth of intracellular pathogens in their true environment pose an obstacle for basic research and drug screening. Here we present the validation of a luminometry-based method for the analysis of intramacrophage growth of M. tuberculosis. The method, which is performed in a medium-throughput format, can easily be adapted for studies of other intracellular pathogens and cell types. The use of host cells in drug-screening assays dedicated to find antimicrobials effective against intracellular pathogens permits the discovery of not only novel antibiotics but also compounds with immunomodulatory and virulence-impairing activities, which may be future alternatives or complements to antibiotics.

Place, publisher, year, edition, pages
American Society for Microbiology, 2010
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-54868 (URN)10.1128/CVI.00446-09 (DOI)000276170900004 ()
Available from: 2010-04-16 Created: 2010-04-16 Last updated: 2017-10-31
2. Human Macrophages Infected with a High Burden of ESAT-6-Expressing M. tuberculosis Undergo Caspase-1-and Cathepsin B-Independent Necrosis
Open this publication in new window or tab >>Human Macrophages Infected with a High Burden of ESAT-6-Expressing M. tuberculosis Undergo Caspase-1-and Cathepsin B-Independent Necrosis
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
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-68900 (URN)10.1371/journal.pone.0020302 (DOI)000291052200049 ()
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
3. Vitamin D enhances IL-1β secretion and restricts growth of Mycobacterium tuberculosis in macrophages from TB patients
Open this publication in new window or tab >>Vitamin D enhances IL-1β secretion and restricts growth of Mycobacterium tuberculosis in macrophages from TB patients
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2013 (English)In: International Journal of Mycobacteriology, ISSN 2212-5531, Vol. 2, no 1, 18-25 p.Article in journal (Refereed) Published
Abstract [en]

The emergence of multidrug-resistant strains of Mycobacterium tuberculosis (MTB), the bacterium responsible for tuberculosis (TB), has rekindled the interest in the role of nutritional supplementation of micronutrients, such as vitamin D, as adjuvant treatment. Here, the growth of virulent MTB in macrophages obtained from the peripheral blood of patients with and without TB was studied. The H37Rv strain genetically modified to express Vibrio harveyi luciferase was used to determine the growth of MTB by luminometry in the human monocyte-derived macrophages (hMDMs) from study subjects. Determination of cytokine levels in culture supernatants was performed using a flow cytometry-based bead array technique. No differences in intracellular growth of MTB were observed between the different study groups. However, stimulation with 100 nM 1,25-dihydroxyvitamin D significantly enhanced the capacity of hMDMs isolated from TB patients to control the infection. This effect was not observed in hMDMs from the other groups. The interleukin (IL)-1β and IL-10 release by hMDMs was clearly increased upon stimulation with 1,25-dihydroxyvitamin D. Furthermore, the 1,25-dihydroxyvitamin D stimulation also led to elevated levels of TNF-α (tumor necrosis factor-alpha) and IL-12p40. It was concluded that vitamin D triggers an inflammatory response in human macrophages with enhanced secretion of cytokines, as well as enhancing the capacity of hMDMs from patients with active TB to restrict mycobacterial growth.

Place, publisher, year, edition, pages
Netherlands: Elsevier, 2013
Keyword
Vitamin D, Human macrophages, Intracellular growth, TB patients, IL-1β
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-90356 (URN)10.1016/j.ijmyco.2012.11.001 (DOI)
Available from: 2013-03-25 Created: 2013-03-25 Last updated: 2015-03-12
4. Apoptotic neutrophils augment the inflammatory response to Mycobacterium tuberculosis infection in human macrophages
Open this publication in new window or tab >>Apoptotic neutrophils augment the inflammatory response to Mycobacterium tuberculosis infection in human macrophages
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2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 7, e101514- p.Article in journal (Refereed) Published
Abstract [en]

Macrophages in the lung are the primary cells being infected by Mycobacterium tuberculosis (Mtb) during tuberculosis. Innate immune cells such as macrophages and neutrophils are first recruited to the site of infection, and mount the early immune protection against this intracellular pathogen. Neutrophils are short-lived cells and removal of apoptotic cells by resident macrophages is a key event in the resolution of inflammation and tissue repair. Such anti-inflammatory activity is not compatible with effective immunity to intracellular pathogens. We therefore investigated how uptake of apoptotic neutrophils by Mtb-activated human monocyte-derived macrophages modulates their function. We show that Mtb infection exerts a potent pro-inflammatory activation of human macrophages with enhanced gene activation and release of several cytokines (TNF, IL-1ß, IL-6, IL-18 and IL-10). This response was augmented by apoptotic neutrophils. Macrophages containing both Mtb and apoptotic cells showed a stronger cytokine expression than non-infected cells. The enhanced macrophage response is linked to apoptotic neutrophil-driven activation of the NLRP3 inflammasome and subsequent IL-1β signalling. We also demonstrate that apoptotic neutrophils not only modulate the inflammatory response, but also enhance the capacity of infected macrophages to control intracellular growth of virulent Mtb. Taken together, these results suggest a novel role for apoptotic neutrophils in the modulation of the macrophage-dependent inflammatory response, which can contribute to the early control of Mtb infection.

Place, publisher, year, edition, pages
PLoS, 2014
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-100888 (URN)10.1371/journal.pone.0101514 (DOI)000338637300054 ()
Available from: 2013-11-14 Created: 2013-11-14 Last updated: 2017-12-06Bibliographically approved
5. Human gene variants linked to enhanced NLRP3 activity limit intramacrophage growth of Mycobacterium tuberculosis
Open this publication in new window or tab >>Human gene variants linked to enhanced NLRP3 activity limit intramacrophage growth of Mycobacterium tuberculosis
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2014 (English)In: The Journal of infectious diseases, ISSN 1537-6613, Vol. 209, no 5, 749-753 p.Article in journal (Refereed) Published
Abstract [en]

Activation of the NLRP3 inflammasome and subsequent generation of IL-1β is initiated in macrophages upon recognition of several stimuli. In the present work, we show that gain-of-function gene variants of inflammasome components known to predispose individuals to inflammatory disorders have a host-protective role during infection with Mycobacterium tuberculosis. By isolation of macrophages from patients and healthy blood donors with genetic variants in NLRP3 and CARD8 and subsequently infecting the cells by virulent M. tuberculosis, we show that these gene variants, combined, are associated with increased control of bacterial growth in human macrophages.

Place, publisher, year, edition, pages
University of Chicago Press / Oxford University Press (OUP), 2014
National Category
Clinical Medicine
Identifiers
urn:nbn:se:liu:diva-100889 (URN)10.1093/infdis/jit572 (DOI)000331873700016 ()24158955 (PubMedID)
Available from: 2013-11-14 Created: 2013-11-14 Last updated: 2015-04-10Bibliographically approved

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