Arturo Zychlinsky

The apoptotic signaling pathway activated by Toll‐like receptor‐2

The innate immune system uses Toll family receptors to signal for the presence of microbes and initiate host defense. Bacterial lipoproteins (BLPs), which are expressed by all bacteria, are potent activators of Toll‐like receptor‐2 (TLR2). Here we show that the adaptor molecule, myeloid differentiation factor 88 (MyD88), mediates both apoptosis and nuclear factor‐κB (NF‐κB) activation by BLP‐stimulated TLR2. Inhibition of the NF‐κB pathway downstream of MyD88 potentiates apoptosis, indicating that these two pathways bifurcate at the level of MyD88. TLR2 signals for apoptosis through MyD88 via a pathway involving Fas‐associated death domain protein (FADD) and caspase 8. Moreover, MyD88 binds FADD and is sufficient to induce apoptosis. These data indicate that TLR2 is a novel ‘death receptor’ that engages the apoptotic machinery without a conventional cytoplasmic death domain. Through TLR2, BLP induces the synthesis of the precursor of the pro‐inflammatory cytokine interleukin‐1β (IL‐1β). Interestingly, BLP also activates caspase 1 through TLR2, resulting in proteolysis and secretion of mature IL‐1β. These results indicate that caspase activation is an innate immune response to microbial pathogens, culminating in apoptosis and cytokine production.

A bacterial invasin induces macrophage apoptosis by binding directly to ICE

Shigella, the etiological agent of dysentery, kills macrophages by inducing apoptosis. Deletion mutants in the invasion invasion plasmid antigen B (ipaB) of Shigella flexneri are not cytotoxic. Here, we localized IpaB to the cytoplasm of macrophages infected with S. flexneri. Purified IpaB induced apoptosis when microinjected into macrophages, indicating that IpaB is sufficient to induce apoptosis. Using a GST‐IpaB fusion protein as a ligand in affinity purification, we isolated four IpaB binding proteins from macrophages which were identified as the precursor and the mature polypeptides of interleukin‐1beta converting enzyme (ICE) or a highly homologous protease. We found that IpaB binds directly to ICE and this enzyme is activated during S. flexneri infection. Furthermore, specific inhibitors of ICE prevented Shigella‐induced apoptosis.

Caspase-1 Activation of IL-1β and IL-18 Are Essential for Shigella flexneri–Induced Inflammation

Caspases are intracellular proteases that mediate mammalian cell apoptosis. Caspase-1 (Casp-1) is a unique caspase because it activates the proinflammatory cytokines interleukin (IL)-1beta and IL-18. Shigella flexneri, the etiological agent of bacillary dysentery, induces macrophage apoptosis, which requires Casp-1 and results in the release of mature IL-1beta and IL-18. Here we show that casp-1(-/-) mice infected with S. flexneri do not develop the acute inflammation characteristic of shigellosis and are unable to resolve the bacterial infection. Using casp-1(-/-) mice supplemented with recombinant cytokines and experiments with IL-1beta(-/-) and IL-18(-/-) mice, we show that IL-1beta and IL-18 are both required to mediate inflammation in S. flexneri infections. Together, these data demonstrate the importance of Casp-1 in acute inflammation and show the different roles of its substrates, IL-1beta and IL-18, in this response.

Pathogen‐induced apoptosis of macrophages: a common end for different pathogenic strategies

Microbe–macrophage interactions play a central role in the pathogenesis of many infections. Several bacterial pathogens induce apoptosis specifically in macrophages, but the mechanisms by which it occurs differ, and the resulting pathology can take different courses. Macrophage death caused by Shigella flexneri and Salmonella spp. has been shown to result in the release of pro‐inflammatory cytokines. Conversely, Yersinia spp. induce apoptosis by suppressing the signalling pathways that lead to the production of tumour necrosis factor (TNF)‐α, a cytokine essential for the control of this infection. It is likely that there are a variety of reasons why macrophages are particularly susceptible to pathogen‐induced apoptosis. One reason may be the expression of surface receptors that recognize highly conserved bacterial components, such as lipopolysaccharide (LPS) and bacterial lipoproteins (BLPs). These receptors have recently been shown to activate pro‐apoptotic signalling pathways. The roles of macrophage apoptosis in different disease processes are discussed.

Apoptosis as a proinflammatory event: What can we learn from bacteria-induced cell death?

Infection of cells by some pathogenic bacteria triggers host cell apoptosis. Bacteria-induced apoptosis appears to promote an inflammatory response that causes tissue damage and further bacterial colonization. Shigella pathogenesis offers a paradigm for the role of apoptosis in bacterial infections.

The selC-associated SHI-2 pathogenicity island of Shigella flexneri.

Pathogenicity islands are chromosomal gene clusters, often located adjacent to tRNA genes, that encode virulence factors present in pathogenic organisms but absent or sporadically found in related non‐pathogenic species. The selC tRNA locus is the site of integration of different pathogenicity islands in uropathogenic Escherichia coli, enterohaemorrhagic E. coli and Salmonella enterica. We show here that the selC locus of Shigella flexneri, the aetiological agent of bacterial dysentery, also contains a pathogenicity island. This pathogenicity island, designated SHI‐2 (Shigellaisland 2), occupies 23.8 kb downstream of selC and contains genes encoding the aerobactin iron acquisition siderophore system, colicin V immunity and several novel proteins. Remnants of multiple mobile genetic elements are present in SHI‐2. SHI‐2‐hybridizing sequences were detected in all S. flexneri strains tested and parts of the island were also found in other Shigella species. SHI‐2 may allow Shigella survival in stressful environments, such as those encountered during infection.

The regulation of apoptosis by microbial pathogens.

In the past few years, there has been remarkable progress unraveling the mechanism and significance of eukaryotic programmed cell death (PCD), or apoptosis. Not surprisingly, it has been discovered that numerous, unrelated microbial pathogens engage or circumvent the hosts apoptotic program. In this chapter, we briefly summarize apoptosis, emphasizing those studies which assist the reader in understanding the subsequent discussion on PCD and pathogens. We then examine the relationship between virulent bacteria and apoptosis. This section is organized to reflect both common and diverse mechanisms employed by bacteria to induce PCD. A short discussion of parasites and fungi is followed by a detailed description of the interaction of viral pathogens with the apoptotic machinery. Throughout the review, apoptosis is considered within the broader contexts of pathogenesis, virulence, and host defense. Our goals are to update the reader on this rapidly expanding field and identify topics in the current literature which demand further investigation.

Effects of low-intensity AC and/or DC electromagnetic fields on cell attachment and induction of apoptosis

Rat tendon fibroblast (RTF) and rat bone marrow (RBM) osteoprogenitor cells were cultured and exposed to AC and/or DC magnetic fields in a triaxial Helmholtz coil in an incubator for up to 13 days. The AC fields were at 60 and 1000 Hz and up to 0.25 mT peak to peak, and the DC fields were up to 0.25 mT. At various combinations of field strengths and frequencies, AC and/or DC fields resulted in extensive detachment of preattached cells and prevented the normal attachment of cells not previously attached to substrates. In addition, the fields resulted in altered cell morphologies. When RTF and RBM cells were removed from the fields after several days of exposure, they partially reattached and assumed more normal morphologies. An additional set of experiments described in the Appendix corroborates these findings and also shows that low-frequency EMF also initiates apoptosis, i.e., programmed cell death, at the onset of cell detachment. Taken together, these results suggest that the electromagnetic fields result in significant alterations in cell metabolism and cytoskeleton structure. Further work is required to determine the relative effect of the electric and magnetic fields on these phenomena. The research has implications for understanding the role of fields in affecting bone healing in fracture nonunions, in cell detachment in cancer metastasis, and in the effect of EMF on organisms generally.

The regulatory protein PhoP controls susceptibility to the host inflammatory response in Shigella flexneri

The PhoP/PhoQ two‐component regulatory system controls transcription of several key virulence genes essential for Salmonella survival in the host cell phagosome. Here, we determine that the PhoP/PhoQ system also regulates virulence in the aetiological agent of bacillary dysentery, Shigella flexneri, even though this pathogen escapes from the phagosome into the cytoplasm of the host cell. A phoP mutant of Shigella established infections and induced an acute inflammatory response in two different animal models. However, infections with phoP mutant bacteria were resolved more rapidly than infections with wild‐type Shigella. Moreover, the Shigella phoP mutant was more sensitive than the wild‐type strain to killing by polymorphonuclear leucocytes (PMNs), cationic polypeptides extracted from PMNs and other animal‐derived antimicrobial peptides. The phoP mutant, however, invaded epithelial cells, spread intercellularly, induced apoptosis in macrophages and tolerated extreme acid pH as efficiently as the wild‐type strain. PhoP appears to regulate Shigella susceptibility to PMNs and antimicrobial molecules that are important for the late stages of infection with this enteric bacterium.

Do macrophages kill through apoptosis

Macrophages can kill target cells independent of conventional immune specificity. Based on a re-examination of literature three decades old and recent experiments, Antonios Aliprantis and colleagues propose that macrophages kill target cells by inducing apoptosis. For this purpose, macrophages employ a selection of pro-apoptotic mediators including reactive oxygen and nitrogen species and tumour necrosis factor alpha.