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Differential effects of invasion by and phagocytosis of Salmonella typhimurium on apoptosis in human macrophages: potential role of Rho–GTPases and Akt
Linköping University, Department of Molecular and Clinical 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.
Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
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2003 (English)In: Journal of Leukocyte Biology, ISSN 0741-5400, E-ISSN 1938-3673, Vol. 74, no 4, 620-629 p.Article in journal (Refereed) Published
Abstract [en]

In addition to direct activation of caspase-1 and induction of apoptosis by SipB, invasive Salmonella stimulates multiple signaling pathways that are key regulators of host cell survival. Nevertheless, little is known about the relative contributions of these pathways to Salmonella-mediated death of macrophages. We studied human monocytic U937 cells and found that apoptosis was induced by invading wild-type Salmonella typhimurium but not by phagocytosed, serum-opsonized, noninvasive Salmonella mutants. Pretreating U937 cells with inhibitors of tyrosine kinases or phosphatidylinositol-3 kinase (PI-3K) completely blocked phagocytosis of opsonized Salmonella mutants but did not affect invasion by wild-type Salmonella or the apoptosis caused by invasion. However, pretreatment with GGTI-298, a geranylgeranyltransferase-1 inhibitor that prevents prenylation of Cdc42 and Rac1, suppressed Salmonella-induced apoptosis by ∼70%. Transduction of Tat fusion constructs containing dominant-negative Cdc42 or Rac1 significantly inhibited Salmonella-induced cell death, indicating that the cytotoxicity of Salmonella requires activation of Cdc42 and Rac. In contrast to phagocytosis of opsonized bacteria, invasion by S. typhimurium stimulated Cdc42 and Rac1, regardless of the activities of tyrosine- or PI-3K. Moreover, Salmonella infection activated Akt protein in a tyrosine-kinase or PI-3K-dependent manner, and a reduced expression of Akt by antisense transfection rendered the cells more sensitive to apoptosis induced by opsonized Salmonella. These results indicate that direct activation of Cdc42 and Rac1 by invasive Salmonella is a prerequisite of Salmonella-mediated death of U937 cells, whereas the simultaneous activation of Akt by tyrosine kinase and PI-3K during receptor-mediated phagocytosis protects cells from apoptosis.

Place, publisher, year, edition, pages
2003. Vol. 74, no 4, 620-629 p.
Keyword [en]
macrophages, bacterial apoptosis, signal transduction
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-14003DOI: 10.1189/jlb.1202586OAI: oai:DiVA.org:liu-14003DiVA: diva2:22439
Available from: 2006-09-27 Created: 2006-09-27 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Microbe-induced apoptosis in phagocytic cells and its role in innate immunity
Open this publication in new window or tab >>Microbe-induced apoptosis in phagocytic cells and its role in innate immunity
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Apoptosis, or programmed cell death, is a controlled process by which aged or damages cells are eliminated in multicellular organisms. Neutrophils, short-lived phagocytes of the innate immune system, are highly equipped effectors that can sense, locate, ingest and kill bacterial pathogens. Inflammatory mediators and the presence of bacterial products at the foci of infection regulate the function and life span of these cells. Modulation of neutrophil apoptosis and the subsequent clearance by scavenger cells, such as macrophages, is part of a balanced inflammatory process leading to resolution of inflammation. Many pathogens are capable of modulating host cell apoptosis, and thereby influence the progression of disease. Hence, this thesis was aiming at elucidating mechanisms involved in pathogen- and host-modulated apoptosis and its contribution to the inflammatory process.

We found that different routes of bacterial entry, i.e. through invasion or by receptor-mediated phagocytosis, triggered different signaling pathways within phagocytes. Invasion of virulent Salmonella caused apoptosis, a process requiring activation of the Rho GTPases Rac1 and Cdc42. On the other hand, phagocytosis of the non-invasive Salmonella inhibited apoptosis despite similar intracellular survival as the invasive bacteria. Protection against phagocytosis-induced apoptosis was regulated by tyrosine- and PI3-kinase-dependent activation of AKT (also called PKB for protein kinase B). Furthermore, inhibiting the intraphagosomal production of reactive oxygen species (ROS) in neutrophils during phagocytosis of E. coli decreased apoptosis below spontaneous apoptosis, further indicating that both pro- and anti-apoptotic pathways are triggered by receptor-mediated phagocytosis.

Type 1 fimbria-expressing E. coli adhering to neutrophils resisted ingestion, and induced a ROS-dependent apoptosis by a cooperative effect of the FimH adhesin and LPS. To explore how compartmentalization of ROS during neutrophil activation was involved in modulating apoptosis, we evaluated the stability of lysosomes. In contrast to phagocytosis of E. coli, the adhesive strain induced intracellular non-phagosomal ROS production which triggered early permeabilization and release of lysosomal enzymes to the cytosol. Cathepsin B and/or L were responsible for targeting of the pro-apoptotic Bcl-2 protein Bid, thereby inducing mitochondrial damage, and apoptosis. These data propose a novel pathway for ROS-induced apoptosis in human neutrophils, where the location of the ROS rather than production per se is important.

Moreover, we found that pathogen-induced apoptotic neutrophils, in contrast to uninfected apoptotic neutrophils, activated blood-monocyte derived macrophages to increase their FcγRI surface expression and to produce large quantities of the pro-inflammatory cytokine TNF-α. This demonstrates that during the early phase of infection, pathogen-induced neutrophil apoptosis will help local macrophages to gain control over the microbes. Furthermore, we suggest that heat shock protein 60 and 70 represent a stress signal that enables macrophages to distinguish between, and react differently to, uninfected and inflammatory apoptotic neutrophils.

Place, publisher, year, edition, pages
Institutionen för molekylär och klinisk medicin, 2006
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 956
Keyword
apoptosis, cell death, inflammation, innate immunity, microbiology, neutrophil, macrophage, phagocyte
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:liu:diva-7445 (URN)91-85523-11-9 (ISBN)
Public defence
2006-09-28, Linden, Campus US, Linköpings Universitet, Linköping, 13:00 (English)
Opponent
Supervisors
Available from: 2006-09-27 Created: 2006-09-27
2. Signal transduction in human phagocytic cells during phagocytosis, oxidative activation and apoptosis
Open this publication in new window or tab >>Signal transduction in human phagocytic cells during phagocytosis, oxidative activation and apoptosis
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Neutrophils and macrophages are professional phagocytic cells that play a crucial role in host defense against invading microorganisms. They bind to, internalize, and subsequently kill microbes with an arsenal of reactive oxygen metabolites and microbicidal agents. The microbes are recognized by cell surface receptors, mainly by the phagocytic receptors FcγR and complement receptor 3 (CR3) that recognize IgG and complement fragments C3b/C3bi, respectively. Microbial pathogens such as Salmonella typhimurium have developed sophisticated mechanisms to avoid the host defense system and enter the cells by invasion, mediated by a type III secretion system.

The objective of this thesis was to investigate the signaling pathways during receptor-mediated phagocytosis by FcγRIIa, FcγRIIIb and complement receptor 3 (CR3), or during invasion by Salmonella typhimurium in human phagocytic cells. We have focused on the intracellular signaling pathways controlling phagocytosis, production of reactive oxygen metabolites, and apoptosis. Paper I-III focus on signal transduction events triggered after ligation of CR3, FcγRIIa, and FcγRIIIb in human neutrophils. Both activation of CR3 and FcγR induced production of reactive oxygen metabolites (ROM), where CR3 induced the most prominent response. The ROM production was dependent on intracellular Ca2+, tyrosine kinase activation, and phospholipase D (PLD) activity. FcγRIIa induced a strong phosphorylation Syk, which was less pronounced following FcγRIIIb ligation, and absent after CR3 activation. Our data indicate that CR3 and FcγR activate different signaling pathways. By exposing neutrophils to TNF-α prior to ligation of CR3, the oxidative response was strongly enhanced, whereas the response to FcγR-ligation was unaffected. This increase was in part due to a p38 MAPK-dependent upregulation of CR3 on the cell surface, but also due to modulation of intracellular signaling pathways since Syk was activated by CR3 as well as FcγR in TNF-α treated cells. In contrast to macrophages where only FcγR activates Rac, Cdc42, and the subsequent ROM production, we show that CR3 as well as FcγR activate the GTPases Rac2 and Cdc42 in human neutrophils. Their downstream target p21 activated kinase was also activated, and Rac2 translocated to the membrane fraction. Correct function of these small GTP-binding proteins was necessary for generating a proper signal for ROM production in these cells.

One survival strategy exploited by microbial pathogens might be to induce apoptosis of tbe host. Invasive Salmonella typhimurium efficiently entered U937 cells and induced a pronounced degree of apoptosis in contrast to its opsonized mutants, which were internalized by receptor-mediated phagocytosis but failed to induce apoptosis. Invasion by Salmonella typhimurium activated Rac1 and Cdc42 independently of PI3 K and tyrosine kinase activation. Inhibition of Racl and Cdc42 inhibited both invasion and the induction of apoptosis. Receptor-mediated phagocytosis activated the survival signals Akt/PKB which protected the cells from apoptosis. Thus, control of apoptosis is a fine tuned balance between pro- and anti-apoptotic signaling proteins.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2003. 58 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 789
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26651 (URN)11216 (Local ID)91-7373-548-5 (ISBN)11216 (Archive number)11216 (OAI)
Public defence
2003-05-09, Berzeliussalen, Hälsouniversitetet, Linköping, 13:00 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-10-09Bibliographically approved

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Forsberg, MariaBlomgran, RobertLem, MariaStendahl, OlleZheng, Limin

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