Cyclic stretch enhances neutrophil extracellular trap formation

Abstract

Background: Neutrophils, the most abundant leukocytes circulating in blood, contribute to host defense and play a significant role in chronic inflammatory disorders. They can release their DNA in the form of extracellular traps (NETs), which serve as scaffolds for capturing bacteria and various blood cells. However, uncontrolled formation of NETs (NETosis) can lead to excessive activation of coagulation pathways and thrombosis. Once neutrophils are migrated to infected or injured tissues, they become exposed to mechanical forces from their surrounding environment. However, the impact of transient changes in tissue mechanics due to the natural process of aging, infection, tissue injury, and cancer on neutrophils remains unknown. To address this gap, we explored the interactive effects of changes in substrate stiffness and cyclic stretch on NETosis. Primary neutrophils were cultured on a silicon-based substrate with stiffness levels of 30 and 300 kPa for at least 3 h under static conditions or cyclic stretch levels of 5% and 10%, mirroring the biomechanics of aged and young arteries.

Results: Using this approach, we found that neutrophils are sensitive to cyclic stretch and that increases in stretch intensity and substrate stiffness enhance nuclei decondensation and histone H3 citrullination (CitH3). In addition, stretch intensity and substrate stiffness promote the response of neutrophils to the NET-inducing agents phorbol 12-myristate 13-acetate (PMA), adenosine triphosphate (ATP), and lipopolysaccharides (LPS). Stretch-induced activation of neutrophils was dependent on calpain activity, the phosphatidylinositol 3-kinase (PI3K)/focal adhesion kinase (FAK) signalling and actin polymerization.

Conclusions: In summary, these results demonstrate that the mechanical forces originating from the surrounding tissue influence NETosis, an important neutrophil function, and thus identify a potential novel therapeutic target.

Keywords: Cyclic stretch; Mechanotransduction; NETosis; Neutrophils; Substrate stiffness.

Fig. 1

Substrate stiffness and cyclic stretch modulate NETosis. A Schematic cartoon, showing different experimental conditions used in this study (figure created with BioRender.com). B Representative immunofluorescence images of human neutrophils stained with DAPI and CitH3 antibody under control or cyclic stretch. C Percentage of neutrophils with decondensed nuclei. D Percentage of cells stained positive for CitH3 after exposure to 5% and 10% cyclic stretch in the presence of two substrates with stiffness levels of 30 and 300 kPa. Boxes show the median and first and third quartiles. Whiskers represent minimum to maximum values. Bar graphs are representative of at least 3 independent experiments, and each dot represents a randomly selected field of view with at least 3 images analyzed per experiment. Statistical significance was determined using two-way ANOVA and multiple comparison tests. **P < 0.01 and ****P < 0.0001

Fig. 2

Cyclic stretch increased the sensitivity of neutrophils to NETs-inducing agents. A–D Representative immunofluorescence images and E–G Bar graphs showing NETs areas normalized to total cell number after exposure to 5% and 10% cyclic stretch or static condition in the presence or absence of PMA (75 nM), LPS (10 μg/mL), or ATP (100 nM). Boxes show the median and first and third quartiles. Whiskers represent minimum to maximum values. Bar graphs are representative of at least 4 independent experiments, and each dot represents a randomly selected field of view, with at least 3 images per experiment. Statistical significance was calculated using two-way ANOVA and multiple comparison tests. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001

Fig. 3

Contribution of PI3K signaling pathway in neutrophil responses to cyclic stretch and substrate stiffness. A Representative immunofluorescence images of human neutrophils pretreated with PF573228 (100 nM) or Ly294002 (10 μM) in the presence or absence of 10% cyclic stretch and substrate stiffness of 300 kPa and stained with DAPI and citrullinated histone H. Bar graphs show B normalized percentage of cells with decondensed chromatin, of neutrophils stained with DAPI and C normalized percentage of cells stained positive for citrullinated histone after neutrophils pretreated with PF573228 or Ly294002 in the presence of 10% cyclic stretch or static condition and a substrate stiffness of 300 kPa. D Representative confocal images of neutrophils pretreated with Ly294002 in the presence of 10% cyclic stretch or static condition and a substrate stiffness of 300 kPa and stained with pan-AKT and phospho-AKT. E Representative bar graph showing the percentage of phospho-AKT intensity. Boxes show the median and first and third quartiles. Whiskers represent minimum to maximum values. Bar graphs are representative of at least 3 independent experiments, and each dots represents a randomly selected field of view and at least three images have been analyzed per experiment. Statistical significance was calculated using two-way ANOVA and multiple comparison tests. ****P < 0.0001

Fig. 4

Contribution of cytoskeleton remodeling in neutrophil responses to cyclic stretch and substrate stiffness. A Representative immunofluorescence images of human neutrophils pretreated with ML-7 (10 μM) or latrunculin A (0.02 M), in the presence or absence of 10% cyclic stretch and substrate stiffness of 300 kPa and stained with DAPI and citrullinated histone H. Bar graphs show B normalized percentage of cells with decondensed chromatin, of neutrophils stained with DAPI and C normalized percentage of cells stained positive for citrullinated histone after neutrophils pretreated with ML-7 or latrunculin A in the presence of 10% cyclic stretch or static condition and a substrate stiffness of 300 kPa. D Representative confocal images of neutrophils in the presence of 10% cyclic stretch or static conditions and a substrate stiffness of 300 kPa, stained with Alexa Fluor 488 Deoxyribonuclease I to label G-actin and Atto 565 phalloidin to label F-actin. E Representative bar graph showing the ratio of F/G actin. Boxes show the median and first and third quartiles. Whiskers represent minimum to maximum values. Bar graphs are representative of at least 3 independent experiments, and each dots represents a randomly selected field of view and at least 3 images have been analyzed per experiment. Statistical significance was calculated using two-way ANOVA and multiple comparison tests. ***P < 0.001 and ****P < 0.0001

Fig. 5

Calpain activity controls stretch-induced NETosis. A Calpain activity assay in neutrophils pretreated with Calpain inhibitor; PD 151746 (20 μM) and vehicle control and exposed to 0–10% cyclic stretch. Bar graph in A represent 3 independent experiments. B Representative immunofluorescence images. C Percentage of decondensed chromatin in neutrophils pretreated with Calpain inhibitor; PD 150606 (3 μM), PD 151746 (20 μM) and inactive analogue; PD 145305 (20 μM) and vehicle control and exposed to 0–10% cyclic stretch. Boxes show the median and first and third quartiles. Whiskers represent minimum to maximum values. Bar graph in C represent at least 3 independent experiments, and each dot represents a randomly selected field of view, and at least 3 images have been analyzed per experiment. Statistical significance was calculated using two-way ANOVA and multiple comparison tests. ****P < 0.0001

Fig. 6

Schematic diagram showing the effects of cyclic stretch and substrate stiffness on NETosis (figure created with BioRender.com)