Dataset Information

Effect of 1-MHz ultrasound on the proinflammatory interleukin-6 secretion in human keratinocytes.

ABSTRACT: Keratinocytes, the main cell type of the skin, are one of the most exposed cells to environmental factors, providing a first defence barrier for the host and actively participating in immune response. In fact, keratinocytes express pattern recognition receptors that interact with pathogen associated molecular patterns and damage associated molecular patterns, leading to the production of cytokines and chemokines, including interleukin (IL)-6. Herein, we investigated whether mechanical energy transported by low intensity ultrasound (US) could generate a mechanical stress able to induce the release of inflammatory cytokine such IL-6 in the human keratinocyte cell line, HaCaT. The extensive clinical application of US in both diagnosis and therapy suggests the need to better understand the related biological effects. Our results point out that US promotes the overexpression and secretion of IL-6, associated with the activation of nuclear factor-κB (NF-κB). Furthermore, we observed a reduced cell viability dependent on exposure parameters together with alterations in membrane permeability, paving the way for further investigating the molecular mechanisms related to US exposure.

SUBMITTER: Giantulli S

PROVIDER: S-EPMC8463532 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Effect of 1-MHz ultrasound on the proinflammatory interleukin-6 secretion in human keratinocytes.

Giantulli Sabrina S Tortorella Elisabetta E Brasili Francesco F Scarpa Susanna S Cerroni Barbara B Paradossi Gaio G Bedini Angelico A Morrone Stefania S Silvestri Ida I Domenici Fabio F

Scientific reports 20210924 1

Keratinocytes, the main cell type of the skin, are one of the most exposed cells to environmental factors, providing a first defence barrier for the host and actively participating in immune response. In fact, keratinocytes express pattern recognition receptors that interact with pathogen associated molecular patterns and damage associated molecular patterns, leading to the production of cytokines and chemokines, including interleukin (IL)-6. Herein, we investigated whether mechanical energy tra ...[more]

PMID: 34561481

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Project description:Aims/hypothesisThe aim of this study was to examine the effects of proinflammatory cytokines on cells of different developmental stages during the generation of stem cell-derived beta cells (SC-beta cells) from human pluripotent stem cells (hPSCs). We wanted to find out to what extent human SC-beta cells are suitable as an experimental cellular model and, with regard to a possible therapeutic use, whether SC-beta cells have a comparable vulnerability to cytokines as bona fide beta cells.MethodshPSCs were differentiated towards pancreatic organoids (SC-organoids) using a 3D production protocol. SC-beta cells and non-insulin-producing cells were separated by FACS and differential gene expression profiles of purified human SC-beta cells, progenitor stages and the human beta cell line EndoC-βH1, as a reference, were determined after 24 h incubation with the proinflammatory cytokines IL-1β, TNF-α and IFN-γ via a transcriptome microarray. Furthermore, we investigated apoptosis based on caspase cleavage, the generation of reactive oxygen species and activation of mitogen-activated protein-kinase (MAPK) stress-signalling pathways.ResultsA 24 h exposure of SC-beta cells to proinflammatory cytokines resulted in significant activation of caspase 3/7 and apoptosis via the extrinsic and intrinsic apoptosis signalling pathways. At this time point, SC-beta cells showed a markedly higher sensitivity towards proinflammatory cytokines than non-insulin-producing cells and EndoC-βH1 cells. Furthermore, we were able to demonstrate the generation of reactive oxygen species and rule out the involvement of NO-mediated stress. A transient activation of stress-signalling pathways p38 mitogen-activated protein kinases (p38) and c-Jun N-terminal kinase (JNK) was already observed after 10 min of cytokine exposure. The transcriptome analysis revealed that the cellular response to proinflammatory cytokines increased with the degree of differentiation of the cells. Cytokines induced the expression of multiple inflammatory mediators including IL-32, CXCL9 and CXCL10 in SC-beta cells and in non-insulin-producing cells.Conclusions/interpretationOur results indicate that human SC-beta cells respond to proinflammatory cytokines very similarly to human islets. Due to the fast and fulminant cellular response of SC-beta cells, we conclude that SC-beta cells represent a suitable model for diabetes research. In light of the immaturity of SC-beta cells, they may be an attractive model for developmentally young beta cells as they are, for example, present in patients with early-onset type 1 diabetes. The secretion of chemotactic signals may promote communication between SC-beta cells and immune cells, and non-insulin-producing cells possibly participate in the overall immune response and are thus capable of amplifying the immune response and further stimulating inflammation. We demonstrated that cytokine-treated SC-organoids secrete IL-32, which is considered a promising candidate for type 1 diabetes onset. This underlines the need to ensure the survival of SC-beta cells in an autoimmune environment such as that found in type 1 diabetes.

Project description:Interleukin-1β (IL-1β), a major pro-inflammatory cytokine, is a leaderless cytosolic protein whose secretion does not follow the classical endoplasmic reticulum-to-Golgi pathway, and for which a canonical mechanism of secretion remains to be established. Neutrophils are essential players against bacterial and fungi infections. These cells are rapidly and massively recruited from the circulation into infected tissues and, beyond of displaying an impressive arsenal of toxic weapons effective to kill pathogens, are also an important source of IL-1β in infectious conditions. Here, we analyzed if an unconventional secretory autophagy mechanism is involved in the exportation of IL-1β by these cells. Our findings indicated that inhibition of autophagy with 3-methyladenine and Wortmannin markedly reduced IL-1β secretion induced by LPS + ATP, as did the disruption of the autophagic flux with Bafilomycin A1 and E64d. These compounds did not noticeable affect neutrophil viability ruling out that the effects on IL-1β secretion were due to cell death. Furthermore, VPS34IN-1, a specific autophagy inhibitor, was still able to reduce IL-1β secretion when added after it was synthesized. Moreover, siRNA-mediated knockdown of ATG5 markedly reduced IL-1β secretion in neutrophil-differentiated PLB985 cells. Upon LPS + ATP stimulation, IL-1β was incorporated to an autophagic compartment, as was revealed by its colocalization with LC3B by confocal microscopy. Overlapping of IL-1β-LC3B in a vesicular compartment peaked before IL-1β increased in culture supernatants. On the other hand, stimulation of autophagy by cell starvation augmented the colocalization of IL-1β and LC3B and then promoted neutrophil IL-1β secretion. In addition, specific ELISAs indicated that although both IL-1β and pro-IL-1β are released to culture supernatants upon neutrophil stimulation, autophagy only promotes IL-1β secretion. Furthermore, the serine proteases inhibitor AEBSF reduced IL-1β secretion. Moreover, IL-1β could be also found colocalizing with elastase, suggesting both some vesicles containing IL-1β intersect azurophil granules content and that serine proteases also regulate IL-1β secretion. Altogether, our findings indicate that an unconventional autophagy-mediated secretory pathway mediates IL-1β secretion in human neutrophils.

Dataset Information

Effect of 1-MHz ultrasound on the proinflammatory interleukin-6 secretion in human keratinocytes.

Publications

Effect of 1-MHz ultrasound on the proinflammatory interleukin-6 secretion in human keratinocytes.

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