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Interaction Between Mycobacterium tuberculosis and Human Neutrophils
Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
2001 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Mycobacterium tuberculosis, the etiologic agent of tuberculosis, is responsible for more deaths each year than any other single pathogen. Mononuclear phagocytes and T cells are crucially involved in the control and local containment of this infection. Less is known about the contribution of neutrophils to control tuberculosis. As one of the most efficient phagocytic cells of the immune system, neutrophils restrict the initial, local replication of numerous pathogens and thereby delay their systemic spread. Neutrophils immigrate quickly to the site of mycobacterial entry and are found in granulomas after infection with M. tuberculosis. The aims of this study were to investigate how neutrophils control M. tuberculosis in the acute phase of mycobacterial infection and the signaling pathways regulating these processes.

When neutrophils are exposed to mycobacteria, they exhibit the typical early bactericidal responses: phagocytosis, generation of reactive oxygen intermediate (ROI), degranulation and the killing of mycobacteria. While production of ROI and M. tuberculosis killing in neutrophils are calcium dependent events, phagocytosis of M. tuberculosis is a calcium-independent process. Measuring intracellular calcium concentration [(Ca+2)]i, revealed that there is no increase in the level of [(Ca+2)]i in single neutrophils upon ingestion of M. tuberculosis. Investigation of the M. tuberculosis-induced phagocytic pathway showed that stimulation of neutrophils by M. tuberculosis triggers tyrosine phosphorylation of PLCγ2 and its association with sch, an adapter protein, and that such association are critical for the M. tuberculosis-stimulated ROI production through activating p38 MAPK. During phagolysosome biogenesis, phagosomes containing M. tuberculosis fused sequentially with secondary granule and late endosomal vacuoles, while delivery of azurophil granule was inhibited. A complex of Rab5a-GTP and syntaxin-4 controlled this fusion process. We suggested that the retention of this complex on the mycobacterial phagosome might allow mycobacteria to avoid the usual physiological destination of phagocytic maturation. Neutrophils infected by M. tuberculosis underwent rapid apoptosis that was mediated by activation of caspase-3 and the expression of Bax and Bcl-x1, two antagonizing members of Bcl-2 family. The level of ROI production controlled this M. tuberculosis induced apoptotic pathway. Apoptotic neutrophils are removed by macrophages, which leads to an augmented mycobactericidal effect in these cells. The results from this work show that neutrophils play an efficient and important role in the early innate immune response against mycobacterial infection, a process that may influence the subsequent specific immune response at the site of infection.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2001. , 71 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 679
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-28623Local ID: 13779ISBN: 91-7219-964-4 (print)OAI: oai:DiVA.org:liu-28623DiVA: diva2:249434
Public defence
2001-05-31, Berzeliussalen, Universitetssjukhuset, Linköping, 09:00 (Swedish)
Opponent
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2012-09-07Bibliographically approved
List of papers
1. Roles of calcium and annexins in phagocytosis and elimination of an attenuated strain of Mycobacterium tuberculosisin human neutrophils
Open this publication in new window or tab >>Roles of calcium and annexins in phagocytosis and elimination of an attenuated strain of Mycobacterium tuberculosisin human neutrophils
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1998 (English)In: Microbial Pathogenesis, ISSN 0882-4010, E-ISSN 1096-1208, Vol. 24, no 5, 309-320 p.Article in journal (Refereed) Published
Abstract [en]

The phagocytic function of neutrophils is a crucial element in the host defence against invading microorganisms. We investigated phagocytosis and intracellular killing of an attenuated strain of Mycobacterium tuberculosis(H37Ra) by human neutrophils focusing on the role of the cytosolic free calcium concentration [Ca2+]iand certain cytosolic calcium-dependent membrane-binding proteins annexins. Phagocytic uptake did not trigger a calcium rise and occurred independently of different calcium conditions, and in a serum-dependent manner. Changes in the viability of H37Ra were determined by agar plate colony count and a radiometric assay. Neutrophils showed a capacity to kill ingested mycobacteria and this occurred without a rise in [Ca2+]i. The ability to kill H37Ra decreased in the absence of extracellular calcium and when intra-extracellular calcium was reduced. Immunofluorescence staining revealed that during phagocytosis of H37Ra, annexins III, IV and VI translocated from cytoplasm to the proximity of the H37Ra-containing phagosomes, whereas the localization of annexin I and V remained unchanged. The translocation of annexin IV occurred even when Ca2+-depleted neutrophils ingested H37Ra in the absence of extracellular calcium. We concluded that neutrophil-mediated killing of mycobacteria is a Ca2+-dependent process. The fact that the association of certain annexins to the membrane vesicle containing H37Ra differ from other phagosomes suggests a selective regulatory mechanism during phagocytosis of mycobacteria by neutrophils.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-81105 (URN)10.1006/mpat.1997.0200 (DOI)
Available from: 2012-09-07 Created: 2012-09-07 Last updated: 2017-12-07Bibliographically approved
2. Activation of Human Neutrophils by Mycobacterium tuberculosis H37Ra Involves Phospholipase Cγ2, Shc Adapter Protein, and p38 Mitogen-Activated Protein Kinase
Open this publication in new window or tab >>Activation of Human Neutrophils by Mycobacterium tuberculosis H37Ra Involves Phospholipase Cγ2, Shc Adapter Protein, and p38 Mitogen-Activated Protein Kinase
2000 (English)In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 164, no 2, 959-965 p.Article in journal (Refereed) Published
Abstract [en]

Recent studies have shown that human neutrophils play a significant protective role in mycobacteria infection. When encountered with mycobacteria, neutrophils exhibit the typical early bactericidal responses including phagocytosis and generation of reactive oxygen intermediates (ROI), but the underlying mechanisms are largely unknown. The present study shows that stimulation of neutrophils with an attenuated strain of Mycobacterium tuberculosis H37Ra (Mtb) led to a tyrosine kinase-dependent ROI production in these cells. Stimulation with Mtb induces a rapid and transient tyrosine phosphorylation of several proteins, one of which was identified as phospholipase Cγ2 (PLCγ2). Several tyrosine-phosphorylated proteins were associated with the PLCγ2 precipitates from Mtb-stimulated neutrophils, of which pp46 was characterized as the Shc adapter protein. A role for PLCγ2-Shc association in the generation of ROI is supported by the observations that stimulation with Mtb causes the activation of p38 mitogen-activated protein kinase (MAPK), a downstream target of the Shc/Ras signaling cascade, and that the effect of genistein on ROI production coincided with its ability to inhibit both PLCγ2-Shc association and p38 MAPK activation. Moreover, pretreatment of neutrophils with a PLC inhibitor markedly suppresses the Mtb-stimulated ROI production as well as p38 MAPK activation in these cells. Taken together, these results indicate that stimulation of neutrophils with Mtb triggers the tyrosine phosphorylation of PLCγ2 and its association with Shc, and that such association is critical for the Mtb-stimulated ROI production through activating p38 MAPK.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-49897 (URN)
Available from: 2009-10-11 Created: 2009-10-11 Last updated: 2012-09-07Bibliographically approved
3. Rab5a GTPase regulates fusion between pathogen-containing phagosomes and cytoplasmic organelles in human neutrophils
Open this publication in new window or tab >>Rab5a GTPase regulates fusion between pathogen-containing phagosomes and cytoplasmic organelles in human neutrophils
2002 (English)In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 115, no 6, 1321-1330 p.Article in journal (Refereed) Published
Abstract [en]

Biogenesis of phagolysosomes proceeds through a sequential series of interactions with endocytic organelles, a process known to be regulated by Rab and SNARE proteins. The molecular mechanisms underlying phagosome maturation in neutrophils are, however, not clearly understood. We investigated fusion between phagosomes containing the intracellular pathogen Mycobacterium tuberculosis versus the extracellular pathogen Staphylococcus aureus (designated MCP for mycobacteria-containing phagosome and SCP for S. aureus-containing phagosome) and cytoplasmic compartments in human neutrophils. Western blot analysis of phagosomes isolated after internalisation revealed that lactoferrin (a constituent of secondary granules) and LAMP-1 were incorporated into both SCP and MCP, whereas hck (marker of azurophil granules) interacted solely with SCP. The subcellular distribution of the proteins Rab5a and syntaxin-4 suggested a role in docking of granules and/or endosomes to the target membrane in the neutrophil. We observed that during phagocytosis, Rab5a in GTP-bound form interacted with syntaxin-4 on the membrane of MCP and were retained for up to 90 minutes, whereas the complex was recruited to the SCP within 5 minutes but was selectively depleted from these vacuoles after 30 minutes of phagocytosis. Downregulation of Rab5a by antisense oligonucleotides efficiently reduced the synthesis of Rab5a, the binding of syntaxin-4 to MCP and SCP and the capacity for fusion exhibited by the pathogen-containing phagosomes, but it had no effect on bacteria internalisation. These data indicate that the difference in granule fusion is correlated with a difference in the association of Rab5a and syntaxin-4 with the phagosomes. Intracellular pathogen-containing phagosomes retain Rab5a and syntaxin-4, whereas extracellular pathogen-containing phagosomes bind briefly to this complex. These results also identified Rab5a as a key regulator of phagolysosome maturation in human neutrophils.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26512 (URN)11069 (Local ID)11069 (Archive number)11069 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-09-07Bibliographically approved
4. Mycobacterium tuberculosis Promotes Apoptosis in Human Neutrophils by Activating Caspase-3 and Altering Expression of Bax/Bcl-xL Via an Oxygen-Dependent Pathway
Open this publication in new window or tab >>Mycobacterium tuberculosis Promotes Apoptosis in Human Neutrophils by Activating Caspase-3 and Altering Expression of Bax/Bcl-xL Via an Oxygen-Dependent Pathway
2002 (English)In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 168, no 12, 6358-6365 p.Article in journal (Refereed) Published
Abstract [en]

In addition to direct bactericidal activities, such as phagocytosis and generation of reactive oxygen species (ROS), neutrophils can regulate the inflammatory response by undergoing apoptosis. We found that infection of human neutrophils with Mycobacterium tuberculosis (Mtb) induced rapid cell death displaying the characteristic features of apoptosis such as morphologic changes, phosphatidylserine exposure, and DNA fragmentation. Both a virulent (H37Rv) and an attenuated (H37Ra) strain of Mtb were equally effective in inducing apoptosis. Pretreatment of neutrophils with antioxidants or an inhibitor of NADPH oxidase markedly blocked Mtb-induced apoptosis but did not affect spontaneous apoptosis. Activation of caspase-3 was evident in neutrophils undergoing spontaneous apoptosis, but it was markedly augmented and accelerated during Mtb-induced apoptosis. The Mtb-induced apoptosis was associated with a speedy and transient increase in expression of Bax protein, a proapoptotic member of the Bcl-2 family, and a more prominent reduction in expression of the antiapoptotic protein Bcl-xL. Pretreatment with an inhibitor of NADPH oxidase distinctly suppressed the Mtb-stimulated activation of caspase-3 and alteration of Bax/Bcl-xL expression in neutrophils. These results indicate that infection with Mtb causes ROS-dependent alteration of Bax/Bcl-xL expression and activation of caspase-3, and thereby induces apoptosis in human neutrophils. Moreover, we found that phagocytosis of Mtb-induced apoptotic neutrophils markedly increased the production of proinflammatory cytokine TNF-α by human macrophages. Therefore, the ROS-dependent apoptosis in Mtb-stimulated neutrophils may represent an important host defense mechanism aimed at selective removal of infected cells at the inflamed site, which in turn aids the functional activities of local macrophages.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26523 (URN)11082 (Local ID)11082 (Archive number)11082 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-09-07Bibliographically approved

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