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Microbe-induced apoptosis in phagocytic cells and its role in innate immunity
Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
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 [en]
apoptosis, cell death, inflammation, innate immunity, microbiology, neutrophil, macrophage, phagocyte
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:liu:diva-7445ISBN: 91-85523-11-9 (print)OAI: oai:DiVA.org:liu-7445DiVA: diva2:22443
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
List of papers
1. Differential effects of invasion by and phagocytosis of Salmonella typhimurium on apoptosis in human macrophages: potential role of Rho–GTPases and Akt
Open this publication in new window or tab >>Differential effects of invasion by and phagocytosis of Salmonella typhimurium on apoptosis in human macrophages: potential role of Rho–GTPases and Akt
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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.

Keyword
macrophages, bacterial apoptosis, signal transduction
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14003 (URN)10.1189/jlb.1202586 (DOI)
Available from: 2006-09-27 Created: 2006-09-27 Last updated: 2017-12-13Bibliographically approved
2. Uropathogenic Escherichia coli triggers oxygen-dependent apoptosis in human neutrophils through the cooperative effect of type 1 fimbriae and lipopolysaccharide
Open this publication in new window or tab >>Uropathogenic Escherichia coli triggers oxygen-dependent apoptosis in human neutrophils through the cooperative effect of type 1 fimbriae and lipopolysaccharide
2004 (English)In: Infection and Immunity, ISSN 0019-9567, Vol. 72, no 8, 4570-4578 p.Article in journal (Refereed) Published
Abstract [en]

Type 1 fimbriae are the most commonly expressed virulence factor on uropathogenic Escherichia coli. In addition to promoting avid bacterial adherence to the uroepithelium and enabling colonization, type 1 fimbriae recruit neutrophils to the urinary tract as an early inflammatory response. Using clinical isolates of type 1 fimbriated E. coli and an isogenic type 1 fimbria-negative mutant (CN1016) lacking the FimH adhesin, we investigated if these strains could modulate apoptosis in human neutrophils. We found that E. coli expressing type 1 fimbriae interacted with neutrophils in a mannose- and lipopolysaccharide (LPS)-dependent manner, leading to apoptosis which was triggered by the intracellular generation of reactive oxygen species. This induced neutrophil apoptosis was abolished by blocking FimH-mediated attachment, by inhibiting NADPH oxidase activation, or by neutralizing LPS. In contrast, CN1016, which did not adhere to or activate the respiratory burst of neutrophils, delayed the spontaneous apoptosis in an LPS-dependent manner. This delayed apoptosis could be mimicked by adding purified LPS and was also observed by using fimbriated bacteria in the presence of D-mannose. These results suggest that LPS is required for E. coli to exert both pro- and antiapoptotic effects on neutrophils and that the difference in LPS presentation (i.e., with or without fimbriae) determines the outcome. The present study showed that there is a fine-tuned balance between type 1 fimbria-induced and LPS-mediated delay of apoptosis in human neutrophils, in which altered fimbrial expression on uropathogenic E. coli determines the neutrophil survival and the subsequent inflammation during urinary tract infections.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14004 (URN)10.1128/IAI.72.8.4570-4578.2004 (DOI)
Available from: 2006-09-27 Created: 2006-09-27 Last updated: 2009-04-28
3. Cathepsin-cleaved Bid promotes apoptosis in human neutrophils via oxidative stress-induced lysosomal membrane permeabilization
Open this publication in new window or tab >>Cathepsin-cleaved Bid promotes apoptosis in human neutrophils via oxidative stress-induced lysosomal membrane permeabilization
2007 (English)In: Journal of Leukocyte Biology, ISSN 0741-5400, E-ISSN 1938-3673, Vol. 81, 1213-1223 p.Article in journal (Refereed) Published
Abstract [en]

Lysosomal membrane permeabilization (LMP) is emerging as an important regulator of cell apoptosis. Human neutrophils are highly granulated phagocytes, which respond to pathogens by exhibiting increased production of reactive oxygen species (ROS) and lysosomal degranulation. In a previous study, we observed that intracellular, nonphagosomal generation of ROS triggered by adherent bacteria induced ROS-dependent neutrophil apoptosis, whereas intraphagosomal production of ROS during phagocytosis had no effect. In the present study, we measured lysosomal membrane stability and leakage in human neutrophils and found that adherent, noningested, Type 1-fimbriated Escherichia coli bacteria induced LMP rapidly in neutrophils. Pretreatment with the NADPH oxidase inhibitor diphenylene iodonium markedly blocked the early LMP and apoptosis in neutrophils stimulated with Type 1-fimbriated bacteria but had no effect on the late LMP seen in spontaneously apoptotic neutrophils. The induced lysosomal destabilization triggered cleavage of the proapoptotic Bcl-2 protein Bid, followed by a decrease in the antiapoptotic protein Mcl-1. Involvement of LMP in initiation of apoptosis is supported by the following observations: Bid cleavage and the concomitant drop in mitochondrial membrane potential required activation of cysteine-cathepsins but not caspases, and the differential effects of inhibitors of cysteine-cathepsins and cathepsin D on apoptosis coincided with their ability to inhibit Bid cleavage in activated neutrophils. Together, these results indicate that in microbe-induced apoptosis in neutrophils, ROS-dependent LMP represents an early event in initiation of the intrinsic apoptotic pathway, which is followed by Bid cleavage, mitochondrial damage, and caspase activation.

Keyword
bacteria, Bcl-2 family proteins, Escherichia coli, lysosomes, Mcl-1, reactive oxygen species
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14005 (URN)doi:10.1189/jlb.0506359 (DOI)
Available from: 2006-09-27 Created: 2006-09-27 Last updated: 2017-12-13
4. Pathogen-Induced Apoptotic Neutrophils Express Heat Shock Proteins and Elicit Activation of Human Macrophages
Open this publication in new window or tab >>Pathogen-Induced Apoptotic Neutrophils Express Heat Shock Proteins and Elicit Activation of Human Macrophages
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2004 (English)In: Journal of immunology, ISSN 0022-1767, Vol. 173, no 10, 6319-6326 p.Article in journal (Refereed) Published
Abstract [en]

Ingestion of aged or irradiated apoptotic neutrophils actively suppresses stimulation of macrophages (MΦ). Many bacterial pathogens can also provoke apoptosis in neutrophils, but little is known about how such apoptotic cells influence MΦ activation. We found that neutrophils undergoing apoptosis induced by UV irradiation, Escherichia coli, or Staphylococcus aureus could either stimulate or inhibit MΦ activation. In contrast to MΦ that had ingested irradiated apoptotic neutrophils, MΦ that had phagocytosed bacteria-induced apoptotic neutrophils exhibited markedly increased production of the proinflammatory cytokine TNF-α, but not the anti-inflammatory cytokine TGF-β. Moreover, ingestion of bacteria, but not UV-induced apoptotic neutrophils, caused increased expression of FcγRI on MΦ, and this effect was not provoked directly by bacteria associated with the apoptotic neutrophils. Instead, we found that a link between pathogen-induced apoptotic neutrophils and up-regulation of the heat shock proteins HSP60 and HSP70, and we also observed that recombinant HSP60 and HSP70 potentiated LPS-stimulated production of TNF-α in MΦ. The opposing macrophage responses to neutrophils undergoing apoptosis induced in different ways may represent a novel mechanism that regulates the extent of the immune response to invading microbes in two steps: first by aiding the functions of MΦ at an early stage of infection, and subsequently by deactivating those cells through removal of uninfected apoptotic neutrophils. HSP induction in neutrophils may provide the danger signals required to generate a more effective macrophage response.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14006 (URN)
Available from: 2006-09-27 Created: 2006-09-27 Last updated: 2009-06-09

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