Effects of Helicobacter suis γ-glutamyl transpeptidase on lymphocytes: modulation by glutamine and glutathione supplementation and outer membrane vesicles as a putative delivery route of the enzyme

Helicobacter (H.) suis colonizes the stomach of the majority of pigs as well as a minority of humans worldwide. Infection causes chronic inflammation in the stomach of the host, however without an effective clearance of the bacteria. Currently, no information is available about possible mechanisms H. suis utilizes to interfere with the host immune response. This study describes the effect on various lymphocytes of the γ-glutamyl transpeptidase (GGT) from H. suis. Compared to whole cell lysate from wild-type H. suis, lysate from a H. suis ggt mutant strain showed a decrease of the capacity to inhibit Jurkat T cell proliferation. Incubation of Jurkat T cells with recombinantly expressed H. suis GGT resulted in an impaired proliferation, and cell death was shown to be involved. A similar but more pronounced inhibitory effect was also seen on primary murine CD4+ T cells, CD8+ T cells, and CD19+ B cells. Supplementation with known GGT substrates was able to modulate the observed effects. Glutamine restored normal proliferation of the cells, whereas supplementation with reduced glutathione strengthened the H. suis GGT-mediated inhibition of proliferation. H. suis GGT treatment abolished secretion of IL-4 and IL-17 by CD4+ T cells, without affecting secretion of IFN-γ. Finally, H. suis outer membrane vesicles (OMV) were identified as a possible delivery route of H. suis GGT to lymphocytes residing in the deeper mucosal layers. Thus far, this study is the first to report that the effects on lymphocytes of this enzyme, not only important for H. suis metabolism but also for that of other Helicobacter species, depend on the degradation of two specific substrates: glutamine and reduced glutatione. This will provide new insights into the pathogenic mechanisms of H. suis infection in particular and infection with gastric helicobacters in general

Clostridium perfringens Beta-Toxin Induces Necrostatin-Inhibitable, Calpain-Dependent Necrosis in Primary Porcine Endothelial Cells

International audienceClostridium perfringens β-toxin (CPB) is a β-barrel pore-forming toxin and an essential virulence factor of C. perfringens type C strains, which cause fatal hemorrhagic enteritis in animals and humans. We have previously shown that CPB is bound to endothelial cells within the intestine of affected pigs and humans, and that CPB is highly toxic to primary porcine endothelial cells (pEC) in vitro. The objective of the present study was to investigate the type of cell death induced by CPB in these cells, and to study potential host cell mechanisms involved in this process. CPB rapidly induced lactate dehydrogenase (LDH) release, propidium iodide uptake, ATP depletion, potassium efflux, a marked rise in intracellular calcium [Ca(2+)]i, release of high-mobility group protein B1 (HMGB1), and caused ultrastructural changes characteristic of necrotic cell death. Despite a certain level of caspase-3 activation, no appreciable DNA fragmentation was detected. CPB-induced LDH release and propidium iodide uptake were inhibited by necrostatin-1 and the two dissimilar calpain inhibitors PD150606 and calpeptin. Likewise, inhibition of potassium efflux, chelation of intracellular calcium and treatment of pEC with cyclosporin A also significantly inhibited CPB-induced LDH release. Our results demonstrate that rCPB primarily induces necrotic cell death in pEC, and that necrotic cell death is not merely a passive event caused by toxin-induced membrane disruption, but is propagated by host cell-dependent biochemical pathways activated by the rise in intracellular calcium and inhibitable by necrostatin-1, consistent with the emerging concept of programmed necrosis ("necroptosis")

pEC exposed to rCPB do not exhibit apoptotic but typical features of necrotic cell death.

<p>(A) Presence of cleaved caspase 3 was assessed by Western blotting at different time points after exposure to 30 ng/ml rCPB or staurosporine. Tubulin was used as loading control. Representative results of 3 independent experiments. (B) Internucleosomal DNA fragmentation was assessed after 16 h of exposure to rCPB. Staurosporine was used as a control for apoptosis, while incubation with 5 mM of H₂O₂ or freeze thawing served as controls for necrosis. Representative results of 3 independent experiments. (C) Intracellular ATP levels were determined at the indicated time points. Results represent mean ± SEM of 3 independent experiments. Statistical difference to untreated control cells was assessed by 2-way ANOVA and Bonferroni multiple comparisons test. *P<0.05, **P<0.01, †P<0.001, ††P<0.0001. (D) Cytoplasmic translocation of HMGB-1 (red) was detectable by immunofluorescence in pEC already 30 min after exposure to 30 ng/ml rCPB. Control cells incubated with toxin-free medium exhibit typical nuclear localization. Nuclear counterstain with Hoechst 33258 (blue). (E) Electron microscopic image of pEC 6 h after incubation with 30 ng/ml rCPB. Cells exhibit small irregular clumps of chromatin abutting to the nuclear membrane (asterisk), swelling of cell organelles with disappearance of the elongated mitochondria with cristae (arrows), and plasma membrane discontinuities (arrowhead).</p

Germination of <it>Aspergillus fumigatus</it> inside avian respiratory macrophages is associated with cytotoxicity

Abstract Although aspergillosis is one of the most common diseases in captive birds, the pathogenesis of avian aspergillosis is poorly known. We studied the role of avian respiratory macrophages as a first line of defense against avian aspergillosis. The phagocytic and killing capacities of avian respiratory macrophages were evaluated using pigeon respiratory macrophages that were inoculated with Aspergillus fumigatus conidia. On average, 25% of macrophage-associated conidia were phagocytosed after one hour. Sixteen percents of these cell-associated conidia were killed after 4 h and conidial germination was inhibited in more than 95% of the conidia. A. fumigatus conidia were shown to be cytotoxic to the macrophages. Intracellularly germinating conidia were located free in the cytoplasm of necrotic cells, as shown using transmission electron microscopy. These results suggest that avian respiratory macrophages may prevent early establishment of infection, unless the number of A. fumigatus conidia exceeds the macrophage killing capacity, leading to intracellular germination and colonization of the respiratory tract.</p

Characterization of rCBP-induced cell death by FACS analysis.

<p>pEC were either left untreated or exposed to 30 or 250 ng/ml of rCPB, or staurosporine, detached from the culture dishes by gentle trypsinization, and analyzed by FACS for PI uptake and GFP-annexin V staining at 2, 4 and 16 h of incubation. (A) Representative cytograms and bar graphs (B) in which cells in the lower left quadrant (PI/annexin V double-negative) were considered to be viable (white), cells in the upper left quadrant (PI single-positive) to be necrotic (gray), cells in the lower right quadrant (annexin V single-positive) to be apoptotic (light gray), and cells in the upper right quadrant (PI/annexin double-positive) to be either necrotic or secondary necrotic after apoptosis (dark gray). Bar graphs show quantitative summary of FACS analysis from 3 to 6 independent experiments. Results are expressed as mean ± SD. Statistical difference to control cells was assessed by non-parametric Kruskal-Wallis and Dunn’s post-hoc test. *P<0.05, **P<0.01, †P<0.001.</p

rCPB-induced efflux of K⁺ and accumulation of intracellular Ca²⁺.

<p>(A) Incubation of confluent pEC with 30 ng/ml rCPB resulted in rapid and significant reduction of intracellular K⁺. rCPB preincubated with mAb-CPB did not cause K⁺ efflux. Values represent means ± SEM from 3 independent experiments. Statistical difference to cells treated with neutralized toxin ( = control) was assessed by 2-way ANOVA and Bonferroni multiple comparisons test. *P<0.05, **P<0.01, ****P<0.0001. (B) Intracellular calcium ([Ca²⁺]_i) levels were measured by Fluo-4 fluorescence in the presence of 3 mM extracellular CaCl₂. Results are expressed as mean ± SEM from 3 independent experiments.</p

Contrast-enhanced reperfusion of Thiel embalmed and dehydrated pig kidneys with PP and diluted PEG.

<p>(A) Planar CT image shows two distinct areas of filling: the arterial and venous system (bright) demonstrating that PP recruits the major renal vessels and the renal tissue (dark grey). ra, renal artery; sa, segmental artery; la, lobar artery; ila, interlobar artery; aa, arcuate artery; illa, interlobular artery; iv, interlobar vein; sv, segmental vein. (B) Three-dimensional representation of the same kidney; red, arterial and venous system; transparent blue-grey, renal tissue showing less contrast. (C) Planar CT image illustrates one uniform area of filling: the arterial and venous system together with the renal tissue (light grey) and centrally a darker area (dark grey) representing the renal calices. (D) Three-dimensional representation of the same kidney. The renal tissue is transparent purple, and the segmental vessels are highlighted in red for illustration purposes. The central white polylined structure depicts the renal calices. (E) The white horizontal line represents the cross-sectional slice through the mid-central part of the kidney containing renal tissue, segmental vessels and the renal calices. (F) Top view of the caudal part following the virtual slicing illustrated in E. Transparent purple, renal tissue; the segmental vessels are highlighted in red and have the same contrast as the renal tissue; white polylines border the calices, which are not filled or are less filled by the contrast fluid. (G) Cross-section through frozen Thiel embalmed pig kidney reperfused with PP. Red PP is present in the major renal vessels. (H) Cross-section through frozen Thiel embalmed pig kidney reperfused with diluted PEG. Blue diluted PEG diffusely stains the sectioned renal surface.</p