&lt;p&gt;Human Lung Macrophages Challenged to Oxidants ex vivo: Lysosomal Membrane Sensitization is Associated with Inflammation and Chronic Airflow Limitation&lt;/p&gt;

Persson, Hans Lennart; Sioutas, Apostolos; Jacobson, Petra; Vainikka, Linda

doi:10.2147/jir.s280419

Cited by 6 publications

(5 citation statements)

References 31 publications

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“…This method has been developed and tested in several different cell types, including human primary melanocytes obtained from fair-skinned donors through foreskin circumcision (0-7 years, parental written informed consent) and four different malignant melanoma Methods Protoc. 2023, 6, 72 3 of 11 cell lines (FM55P, Sigma Aldrich, St Louis, MO, USA; WM115 and WM278, Rockland Immunochemicals, Limmerick, PA, USA; and SkMel28, ATCC, Manassas, VA, USA) [16], murine macrophage-like lymphoma J774 cells (ATCC), patient samples from human alveolar macrophages isolated from bronchoalveolar lavage at Linköping University Hospital as described previously [36], and human foreskin fibroblasts (AG01518, ATCC). The suggested concentrations and conditions presented in this protocol are applicable for all the cell lines tested; however, specific cell culture conditions, medium and concentration of LMP-inducers need to be optimized for each individual cell type.…”

Section: Experimental Designmentioning

confidence: 99%

“…Also, fluorescence changes are measured over AO is a well-proven staining technique to monitor lysosomal membrane stability, mainly via fluorescence microscopy [32,33] and flow cytometry [34,35]. In two recent studies, we have adapted the AO staining to suit a high-scale approach using a fluorescence microplate reader [16,36]. In this protocol, both the material required and the analysis time is reduced, making it suitable for studying, for example, patient material, as well as making a high-throughput analysis.…”

Section: Introductionmentioning

confidence: 99%

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Real-Time Monitoring of Lysosomal Membrane Permeabilization Using Acridine Orange

Eriksson,

Vainikka,

Persson

et al. 2023

MPs

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Loss of lysosomal membrane integrity results in leakage of lysosomal hydrolases to the cytosol which might harm cell function and induce cell death. Destabilization of lysosomes often precede apoptotic or necrotic cell death and occur during both physiological and pathological conditions. The weak base acridine orange readily enters cells and accumulates in the acidic environment of lysosomes. Vital staining with acridine orange is a well-proven technique to observe lysosomal destabilization using fluorescence microscopy and flow cytometry. These analyses are, however, time consuming and only adapted for discrete time points, which make them unsuitable for large-scale approaches. Therefore, we have developed a time-saving, high-throughput microplate reader-based method to follow destabilization of the lysosomal membrane in real-time using acridine orange. This protocol can easily be adopted for patient samples since the number of cells per sample is low and the time for analysis is short.

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Section: Experimental Designmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Real-Time Monitoring of Lysosomal Membrane Permeabilization Using Acridine Orange

Eriksson,

Vainikka,

Persson

et al. 2023

MPs

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“…Lysosomal membrane permeability assay was prepared for experiments as previously described in more detail ( 37 , 59 ). In order to observe intracellular lysosome membrane permeability change, 2-µg/mL acridine orange (Sigma) was added to the cells infected with D39Δ ply for 2 h. Then, the slides were cultured for another 15 min with 5% CO 2 at 37°C, washed with PBS, and fixed by 4% paraformaldehyde.…”

Section: Methodsmentioning

confidence: 99%

hsdS _A regulated extracellular vesicle-associated PLY to protect Streptococcus pneumoniae from macrophage killing via LAPosomes

Wang,

Liu,

et al. 2024

Microbiol Spectr

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Streptococcus pneumoniae is a notorious human opportunistic pathogen which undergoes a spontaneous and reversible phenotypic change in response to the host environment. We demonstrated that the regulatory gene hsdS A of DNA methylation in the type I restriction modification system altered colony transparency and substantially contributed to S. pneumoniae virulence. Most importantly, hsdS A regulated the production of extracellular vesicles (EVs) which package cytosolic, surface, and secreted proteins, including pneumolysin (PLY). Interestingly, we confirmed that EV-associated PLY utilized internalization into macrophages to prolong the survival of intracellular bacteria as a major immune evasion strategy; that is, EV-associated PLY produced by the D39 strain (EVs-D39) could induce the formation of LC3-associated monolayer vacuoles [LC3-associated phagocytosis (LAP)] and co-localize with the NADPH oxidase 2 (NOX2) complex but not ULK1 when macrophages were infected with the D39Δ ply strain. In addition, EV-associated PLY derived from the EVs-D39 promoted macrophages to release more reactive oxygen species (ROS) and expression of p-p70s6k than EV-associated PLY derived from the D39Δ hsdS A strain (EVs-D39Δ hsdS A ), whereas the expression of p-ULK1 was reversed, indicating that EVs-D39Δ hsdS A was more likely to induce conventional xenophagy. Furthermore, we identified the β1 integrin receptor as a crucial inducer of ROS to mediate LAP activation. Bacterial evasion of host clearance is closely related to insufficient acidification after the fusion of autophagosomes or LAPosomes with lysosomes. Of note, we found EV-associated PLY damaged the integrity of the lysosome membrane and changed the pH gradient, resulting in lysosomes being unable to remove intracellular bacteria and ultimately prolonging the survival of S. pneumoniae in macrophages. Finally, the extracted mouse alveolar macrophages and mouse intranasal infection models were employed to further verify the above findings. IMPORTANCE S. pneumoniae is a major human pathogen that undergoes a spontaneous and reversible phase variation that allows it to survive in different host environments. Interestingly, we found hsdS A , a gene that manipulated the phase variation, promoted the survival and replication of S. pneumoniae in macrophages by regulating EV production and EV-associated PLY. More importantly, here we provided the first evidence that higher EV-associated PLY (produced by D39) could form LAPosomes that were single membrane compartments containing S. pneumoniae , which are induced by integrin β1/NOX2/ROS pathway. At the same time, EV-associated PLY increased the permeability of lysosome membrane and induced an insufficient acidification to escape the host killing, and ultimately prolonged the survival of S. pneumoniae in macrophages. In contrast, lower EV-associated PLY (produced by D39Δ hsdS A ) activated ULK1 recruitment to form double-layered autophagosomes to eliminate bacteria.

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“… 1 Breathlessness, either displayed at rest (BaR) or at physical activity (BaPA), is a consequence of chronic airflow limitation (CAL) and hyperinflation, 2 the latter being a result of alveolar wall destruction and development of emphysema. 3 CAL, on the other hand, is due to an airway inflammation, which is usually dominated by neutrophils, 4 and a sub-epithelial fibrosis, which is driven by epithelial–mesenchymal transition. 5 Metabolic disturbances of cells’ handling of iron, relevant for the resistance against oxidative damage, 6 , 7 and muscle tissue bioenergetics, important for normal muscle function, are some mechanisms implicated in COPD pathogenesis.…”

Section: Introductionmentioning

confidence: 99%

Applying the Rome Proposal on Exacerbations of Chronic Obstructive Pulmonary Disease: Does Comorbid Chronic Heart Failure Matter?

Jacobson,

Lind,

Persson

2023

COPD

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Background Chronic heart failure (CHF) is a common comorbidity among patients with chronic obstructive pulmonary disease (COPD). Both exacerbations of COPD (ECOPDs) and exacerbations of CHF (ECHFs) display worsening of breathlessness at rest (BaR) and breathlessness at physical activity (BaPA). Comorbid CHF may have an impact on the vital signs assessed, when the Rome proposal (adopted by GOLD 2023) is applied on ECOPDs. Thus, the aim of the present study was to investigate the impact of comorbid CHF on ECOPDs severity, particularly focusing on the influence of comorbid CHF on BaR and BaPA. Methods We analysed data on COPD symptoms collected from the telehealth study The eHealth Diary. Patients with COPD (n = 43) and patients with CHF (n = 41) were asked to daily monitor BaR and BaPA, employing a digital pen and scales for BaR and BaPA (from 0 to 10). Twenty-eight patients of the COPD patients presented with comorbid CHF. Totally, 125 exacerbations were analysed. Results Exacerbations in the group with COPD patients and comorbid CHF were compared to the group with COPD patients without comorbid CHF and the group with CHF patients. Compared with GOLD 2022, the GOLD 2023 (the Rome proposal) significantly downgraded the ECOPD severity. Comorbid CHF did not interfere significantly on the observed difference. Comorbid CHF did not worsen BaR scores, assessed at inclusion and at the symptom peak of the exacerbations. Conclusion In the present study, we find no evidence that comorbid CHF would interfere significantly with the parameters included in the Rome proposal (GOLD 2023). We conclude that the Rome proposal can be safely applied even on COPD patients with very advanced comorbid CHF.

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<p>Human Lung Macrophages Challenged to Oxidants ex vivo: Lysosomal Membrane Sensitization is Associated with Inflammation and Chronic Airflow Limitation</p>

Cited by 6 publications

References 31 publications

Real-Time Monitoring of Lysosomal Membrane Permeabilization Using Acridine Orange

Real-Time Monitoring of Lysosomal Membrane Permeabilization Using Acridine Orange

hsdS _A regulated extracellular vesicle-associated PLY to protect Streptococcus pneumoniae from macrophage killing via LAPosomes

Applying the Rome Proposal on Exacerbations of Chronic Obstructive Pulmonary Disease: Does Comorbid Chronic Heart Failure Matter?

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<p>Human Lung Macrophages Challenged to Oxidants ex vivo: Lysosomal Membrane Sensitization is Associated with Inflammation and Chronic Airflow Limitation</p>

Cited by 6 publications

References 31 publications

Real-Time Monitoring of Lysosomal Membrane Permeabilization Using Acridine Orange

Real-Time Monitoring of Lysosomal Membrane Permeabilization Using Acridine Orange

hsdS A regulated extracellular vesicle-associated PLY to protect Streptococcus pneumoniae from macrophage killing via LAPosomes

Applying the Rome Proposal on Exacerbations of Chronic Obstructive Pulmonary Disease: Does Comorbid Chronic Heart Failure Matter?

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hsdS _A regulated extracellular vesicle-associated PLY to protect Streptococcus pneumoniae from macrophage killing via LAPosomes