A cardinal role for cathepsin D in co-ordinating the host-mediated apoptosis of macrophages and killing of pneumococci

The bactericidal function of macrophages against pneumococci is enhanced by their apoptotic demise, which is controlled by the anti-apoptotic protein Mcl-1. Here, we show that lysosomal membrane permeabilization (LMP) and cytosolic translocation of activated cathepsin D occur prior to activation of a mitochondrial pathway of macrophage apoptosis. Pharmacological inhibition or knockout of cathepsin D during pneumococcal infection blocked macrophage apoptosis. As a result of cathepsin D activation, Mcl-1 interacted with its ubiquitin ligase Mule and expression declined. Inhibition of cathepsin D had no effect on early bacterial killing but inhibited the late phase of apoptosis-associated killing of pneumococci in vitro. Mice bearing a cathepsin D-/- hematopoietic system demonstrated reduced macrophage apoptosis in vivo, with decreased clearance of pneumococci and enhanced recruitment of neutrophils to control pulmonary infection. These findings establish an unexpected role for a cathepsin D-mediated lysosomal pathway of apoptosis in pulmonary host defense and underscore the importance of apoptosis-associated microbial killing to macrophage function

Monocytes regulate the mechanism of T-cell death by inducing Fas-mediated apoptosis during bacterial infection.

Monocytes and T-cells are critical to the host response to acute bacterial infection but monocytes are primarily viewed as amplifying the inflammatory signal. The mechanisms of cell death regulating T-cell numbers at sites of infection are incompletely characterized. T-cell death in cultures of peripheral blood mononuclear cells (PBMC) showed 'classic' features of apoptosis following exposure to pneumococci. Conversely, purified CD3(+) T-cells cultured with pneumococci demonstrated necrosis with membrane permeabilization. The death of purified CD3(+) T-cells was not inhibited by necrostatin, but required the bacterial toxin pneumolysin. Apoptosis of CD3(+) T-cells in PBMC cultures required 'classical' CD14(+) monocytes, which enhanced T-cell activation. CD3(+) T-cell death was enhanced in HIV-seropositive individuals. Monocyte-mediated CD3(+) T-cell apoptotic death was Fas-dependent both in vitro and in vivo. In the early stages of the T-cell dependent host response to pneumococci reduced Fas ligand mediated T-cell apoptosis was associated with decreased bacterial clearance in the lung and increased bacteremia. In summary monocytes converted pathogen-associated necrosis into Fas-dependent apoptosis and regulated levels of activated T-cells at sites of acute bacterial infection. These changes were associated with enhanced bacterial clearance in the lung and reduced levels of invasive pneumococcal disease

A pneumococcal MerR-like regulator and S-nitrosoglutathione reductase are required for systemic virulence

Copyright © 2007 by the Infectious Diseases Society of America. All rights reserved.A transcriptional regulator, NmlR(sp), has been identified in Streptococcus pneumoniae that is required for defense against nitric oxide (NO) stress. The nmlR(sp) gene is cotranscribed with adhC, which encodes an alcohol dehydrogenase that is able to reduce S-nitrosoglutathione (GSNO) with NADH as reductant. nmlR(sp) and adhC mutants exhibited a reduced level of NADH-GSNO oxidoreductase activity and were more susceptible to killing by NO than were wild-type cells. Comparison of the virulence of wild-type and mutant strains by use of a mouse model system showed that NmlR(sp) and AdhC do not play a key role in the adherence of pneumococci to the nasopharynx in vivo. An intraperitoneal challenge experiment revealed that both NmlR(sp) and AdhC were required for survival in blood. These data identify novel components of a NO defense system in pneumococci that are required for systemic infection.Uwe H. Stroeher, Robert S. Kidd, Sian L. Stafford, Michael P. Jennings, James C. Paton and Alastair G. McEwa

Early apoptosis of porcine alveolar macrophages limits avian influenza virus replication and proinflammatory dysregulation

Pigs are evidently more resistant to avian than swine influenza A viruses, mediated in part through frontline epithelial cells and alveolar macrophages (AM). Although porcine AM (PAM) are crucial in influenza virus control, their mode of control is unclear. To gain insight into the possible role of PAM in the mediation of avian influenza virus resistance, we compared the host effects and replication of two avian (H2N3 and H6N1) and three mammalian (swine H1N1, human H1N1 and pandemic H1N1) influenza viruses in PAM. We found that PAM were readily susceptible to initial infection with all five avian and mammalian influenza viruses but only avian viruses caused early and extensive apoptosis (by 6 h of infection) resulting in reduced virus progeny and moderated pro- inflammation. Full length viral PB1-F2 present only in avian influenza viruses is a virulence factor that targets AM for mitochondrial associated apoptotic cell death. With the use of reverse genetics on an avian H5N1 virus, we found that full length PB1-F2 contributed to increased apoptosis and pro-inflammation but not to reduced virus replication. Taken together, we propose that early apoptosis of PAM limits the spread of avian influenza viruses and that PB1-F2 could play a contributory role in the process

Impaired Mitochondrial Microbicidal Responses in Chronic Obstructive Pulmonary Disease Macrophages

RATIONALE: Chronic obstructive pulmonary disease (COPD) is characterized by impaired clearance of pulmonary bacteria. OBJECTIVES: The effect of COPD on alveolar macrophage (AM) microbicidal responses was investigated. METHODS: Alveolar macrophages (AMs) were obtained from bronchoalveolar lavage from healthy donors or COPD patients and challenged with opsonized serotype 14 Streptococcus pneumoniae. Cells were assessed for apoptosis, bactericidal activity and mitochondrial reactive oxygen species (mROS) production. A transgenic mouse line, in which the CD68 promoter ensures macrophage specific expression of human Mcl-1 (CD68.hMcl-1), was used to model the molecular aspects of COPD. MEASUREMENTS AND MAIN RESULTS: COPD AM had elevated levels of Mcl-1, an anti-apoptotic Bcl-2 family member, with selective reduction of delayed intracellular bacterial killing. CD68.hMcl-1 AM phenocopied the microbicidal defect since transgenic mice demonstrated impaired clearance of pulmonary bacteria and increased neutrophilic inflammation. Murine bone marrow-derived macrophages (BMDM) and human monocyte-derived macrophages (MDM) generated mitochondrial reactive oxygen species (mROS) in response to pneumococci, which co-localized with bacteria and phagolysosomes to enhance bacterial killing. The Mcl-1 transgene increased oxygen consumption rates and mROS expression in mock-infected BMDM but reduced caspase-dependent mROS production after pneumococcal challenge. COPD AM also increased basal mROS expression, but failed to increase production after pneumococcal challenge, in keeping with reduced intracellular bacterial killing. The defect in COPD AM intracellular killing was associated with a reduced ratio of mROS /superoxide dismutase 2. CONCLUSIONS: Upregulation of Mcl-1 and chronic adaption to oxidative stress alters mitochondrial metabolism and microbicidal function, reducing the delayed phase of intracellular bacterial clearance in COPD

Downregulation of Mcl-1 has anti-inflammatory pro-resolution effects and enhances bacterial clearance from the lung

Phagocytes not only coordinate acute inflammation and host defense at mucosal sites, but also contribute to tissue damage. Respiratory infection causes a globally significant disease burden and frequently progresses to acute respiratory distress syndrome, a devastating inflammatory condition characterized by neutrophil recruitment and accumulation of protein-rich edema fluid causing impaired lung function. We hypothesized that targeting the intracellular protein myeloid cell leukemia 1 (Mcl-1) by a cyclin-dependent kinase inhibitor (AT7519) or a flavone (wogonin) would accelerate neutrophil apoptosis and resolution of established inflammation, but without detriment to bacterial clearance. Mcl-1 loss induced human neutrophil apoptosis, but did not induce macrophage apoptosis nor impair phagocytosis of apoptotic neutrophils. Neutrophil-dominant inflammation was modelled in mice by either endotoxin or bacteria (Escherichia coli). Downregulating inflammatory cell Mcl-1 had anti-inflammatory, pro-resolution effects, shortening the resolution interval (R(i)) from 19 to 7 h and improved organ dysfunction with enhanced alveolar–capillary barrier integrity. Conversely, attenuating drug-induced Mcl-1 downregulation inhibited neutrophil apoptosis and delayed resolution of endotoxin-mediated lung inflammation. Importantly, manipulating lung inflammatory cell Mcl-1 also accelerated resolution of bacterial infection (R(i); 50 to 16 h) concurrent with enhanced bacterial clearance. Therefore, manipulating inflammatory cell Mcl-1 accelerates inflammation resolution without detriment to host defense against bacteria, and represents a target for treating infection-associated inflammation

Knowledge generation about care-giving in the UK: a critical review of research paradigms

While discourse about care and caring is well developed in the UK, the nature of knowledge generation about care and the research paradigms that underpin it have been subjected to limited critical reflection and analysis. An overarching synthesis of evidence – intended to promote debate and facilitate new understandings – identifies two largely separate bodies of carer-related research. The first body of work – referred to as Gathering and Evaluating – provides evidence of the extent of caregiving, who provides care to whom and with what impact; it also focuses on evaluating policy and service efficacy. This type of research tends to dominate public perception about caring, influences the type and extent of policy and support for carers and attracts funding from policy and health-related sources. However, it also tends to be conceptually and theoretically narrow, has limited engagement with carers’ perspectives and adopts an atomistic purview on the care-giving landscape. The second body of work – Conceptualising and Theorising – explores the conceptual and experiential nature of care and aims to extend thinking and theory about caring. It is concerned with promoting understanding of care as an integral part of human relationships, embedded in the life course, and a product of interdependence and reciprocity. This work conceptualises care as both an activity and a disposition and foregrounds the development of an ‘ethic of care’, thereby providing a perspective within which to recognise both the challenges care-giving may present and the significance of care as a normative activity. It tends to be funded from social science sources and, while strong in capturing carers’ experiences, has limited policy and service-related purchase. Much could be gained for citizens, carers and families, and the generation of knowledge advanced, if the two bodies of research were integrated to a greater degree

The role of pneumolysin in mediating lung damage in a lethal pneumococcal pneumonia murine model

BACKGROUND: Intranasal inoculation of Streptococcus pneumoniae D39 serotype 2 causes fatal pneumonia in mice. The cytotoxic and inflammatory properties of pneumolysin (PLY) have been implicated in the pathogenesis of pneumococcal pneumonia. METHODS: To examine the role of PLY in this experimental model we performed ELISA assays for PLY quantification. The distribution patterns of PLY and apoptosis were established by immunohistochemical detection of PLY, caspase-9 activity and TUNEL assay on tissue sections from mice lungs at various times, and the results were quantified with image analysis. Inflammatory and apoptotic cells were also quantified on lung tissue sections from antibody treated mice. RESULTS: In bronchoalveolar lavages (BAL), total PLY was found at sublytic concentrations which were located in alveolar macrophages and leukocytes. The bronchoalveolar epithelium was PLY-positive, while the vascular endothelium was not PLY reactive. The pattern and extension of cellular apoptosis was similar. Anti-PLY antibody treatment decreased the lung damage and the number of apoptotic and inflammatory cells in lung tissues. CONCLUSION: The data strongly suggest that in vivo lung injury could be due to the pro-apoptotic and pro-inflammatory activity of PLY, rather than its cytotoxic activity. PLY at sublytic concentrations induces lethal inflammation in lung tissues and is involved in host cell apoptosis, whose effects are important to pathogen survival

Effects of deletion of the Streptococcus pneumoniae lipoprotein diacylglyceryl transferase gene lgt on ABC transporter function and on growth in vivo

Lipoproteins are an important class of surface associated proteins that have diverse roles and frequently are involved in the virulence of bacterial pathogens. As prolipoproteins are attached to the cell membrane by a single enzyme, prolipoprotein diacylglyceryl transferase (Lgt), deletion of the corresponding gene potentially allows the characterisation of the overall importance of lipoproteins for specific bacterial functions. We have used a Δlgt mutant strain of Streptococcus pneumoniae to investigate the effects of loss of lipoprotein attachment on cation acquisition, growth in media containing specific carbon sources, and virulence in different infection models. Immunoblots of triton X-114 extracts, flow cytometry and immuno-fluorescence microscopy confirmed the Δlgt mutant had markedly reduced lipoprotein expression on the cell surface. The Δlgt mutant had reduced growth in cation depleted medium, increased sensitivity to oxidative stress, reduced zinc uptake, and reduced intracellular levels of several cations. Doubling time of the Δlgt mutant was also increased slightly when grown in medium with glucose, raffinose and maltotriose as sole carbon sources. These multiple defects in cation and sugar ABC transporter function for the Δlgt mutant were associated with only slightly delayed growth in complete medium. However the Δlgt mutant had significantly reduced growth in blood or bronchoalveolar lavage fluid and a marked impairment in virulence in mouse models of nasopharyngeal colonisation, sepsis and pneumonia. These data suggest that for S. pneumoniae loss of surface localisation of lipoproteins has widespread effects on ABC transporter functions that collectively prevent the Δlgt mutant from establishing invasive infection

Selected isotope ratio measurements of light metallic elements (Li, Mg, Ca, and Cu) by multiple collector ICP-MS

The unique capabilities of multiple collector inductively coupled mass spectrometry (MC-ICP-MS) for high precision isotope ratio measurements in light elements as Li, Mg, Ca, and Cu are reviewed in this paper. These elements have been intensively studied at the Geological Survey of Israel (GSI) and other laboratories over the past few years, and the methods used to obtain high precision isotope analyses are discussed in detail. The scientific study of isotopic fractionation of these elements is significant for achieving a better understanding of geochemical and biochemical processes in nature and the environment