Rapamycin Re-Directs Lysosome Network, Stimulates ER-Remodeling, Involving Membrane CD317 and Affecting Exocytosis, in Campylobacter Jejuni-Lysate-Infected U937 Cells

Abstract

The Gram-negative Campylobacter jejuni is a major cause of foodborne gastroenteritis in humans worldwide. The cytotoxic effects of Campylobacter have been mainly ascribed to the actions of the cytolethal distending toxin (CDT): it is mandatory to put in evidence risk factors for sequela development, such as reactive arthritis (ReA) and Guillain–Barré syndrome (GBS). Several researches are directed to managing symptom severity and the possible onset of sequelae. We found for the first time that rapamycin (RM) is able to largely inhibit the action of C. jejuni lysate CDT in U937 cells, and to partially avoid the activation of specific sub-lethal effects. In fact, we observed that the ability of this drug to redirect lysosomal compartment, stimulate ER-remodeling (highlighted by ER–lysosome and ER–mitochondria contacts), protect mitochondria network, and downregulate CD317/tetherin, is an important component of membrane microdomains. In particular, lysosomes are involved in the process of the reduction of intoxication, until the final step of lysosome exocytosis. Our results indicate that rapamycin confers protection against C. jejuni bacterial lysate insults to myeloid cells.

Uptake and Intracellular Trafficking Studies of Multiple Dye-Doped Core-Shell Silica Nanoparticles in Lymphoid and Myeloid Cells

“… 27 Mitochondrial features were investigated by Tetramethylrhodamine ethyl ester perchlorate (TMRE) (Sigma-Aldrich, St. Louis, MO, USA) 40 nM, which is a ∆Ψm-specific stain able to selectively enter active mitochondria, was added to the samples 15 min before the acquisition. 28 50 μM monodansylcadaverine (MDC) (Sigma-Aldrich, St Louis, MO, USA), that is a specific autophagolysosomes marker typically used to investigate the autophagic machinery, was incubated for 10 min at 37°C. 29 MitoSOX Red (Thermo Fisher Scientific) is a florigenic dye specifically targeted to mitochondria in live cells and its oxidation by superoxide (O 2- ), that is contained in mitochondria, produces a red fluorescence.…”

Effect of calorie-restriction and rapamycin on autophagy and the severity of caerulein-induced experimental acute pancreatitis in mice

“…When we used rapamycin to induce autophagy, we observed enhanced induction of autophagy and also completion of autophagy, higher conversion to LC3-II, increased level of Beclin1 and number of autophagic vesicles, and decreased level of SQSTM1. The latter could be due to rapamycin-induced formation and recruitment of a larger number of lysosomes in the mammalian cells (45). As a consequence of this greater induction and completion of autophagic process, rapamycin treated mice had significantly lesser severity of pancreatitis with lower inflammation and necrosis scores (Figure 10).…”

Fluorescent Silica Nanoparticles Targeting Mitochondria: Trafficking in Myeloid Cells and Application as Doxorubicin Delivery System in Breast Cancer Cells

“…MTG 50 nM was added to the samples, incubated for 30 min at 37 °C and then acquired. TMRE (Sigma-Aldrich, St. Louis, MO, USA) 40 nM, which is a ∆Ψm-specific stain able to selectively enter active mitochondria, was added to the samples 15 min before the acquisition [ 52 ]. To establish the healthy condition of isolated mitochondria we also used carbonyl cyanide m-chlorophenylhydrazone (CCCP), which is a typical mitochondrial uncoupler, leading to the dissipation of mitochondrial membrane potential [ 53 ].…”

“…100 nM of the acidotropic dye LysoTrackerTM Green (LTG) (Thermo Fisher Scientific, Waltham, MA, USA) incubated for 45 min was used to investigate the involvement of the lysosomal compartment [ 55 ]. Mitochondrial features were investigated by tetramethylrhodamine ethyl ester perchlorate (TMRE) (Sigma-Aldrich, St. Louis, MO, USA) 40 nM, a ∆Ψm-specific stain able to selectively enter active mitochondria, was added to the samples 15 min before the acquisition [ 52 ]. Confocal microscopy analyses were applied by a Leica TCS SP5 II confocal microscope (Leica Microsystem, Wentzler, Germany) with 488, 543 and 633 nm illuminations and oil-immersed objectives.…”