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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session E

Poster U19 - Extracellular Matrix regulates the Formation of Neutrophil Extracellular Traps (NETs)

Saturday, October 26, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Co-Author(s)

SS

Simrit Safarulla

Student
NC A&T State University, United States
Vikram Surendran

Graduate Student/ Graduate Research Assistant
Wake Forest Institute for Regenerative Medicine/Wake Forest School of Biomedical Engineering and Sciences
Winston-Salem, North Carolina, United States
SJ

Simone Josey

Student
NC A&T State University, United States

Presenting Author(s)

Arvind Chandrasekaran

Associate Professor (Bioengineering)
North Carolina A&T State University
Greensboro, North Carolina, United States

Introduction: Neutrophils are the most common leukocytes in circulation and are the first line of defense within the innate immune system. Under certain conditions of respiratory diseases, neutrophils release DNA into their surroundings, forming Neutrophil Extracellular Traps (NETs). Hyperconcentrated mucus associated with cystic fibrosis (CF) is compounded by the accumulation of DNA within the mucus layer that come from neutrophils, that form NETs. It is known that the NETs-DNA can entangle with and stiffen the mucin-laden mucus network. However, for NETs to adversely affect the mechanobiology of mucociliary clearance, they must spread through the mucus gel matrix such that the released DNA overlaps with adjacent NETs. There is not enough information to sufficiently explain the fundamental biomechanics of NETs generation within the construct of Extracellular Matrix (ECM), which we sought to understand through this work with a working hypothesis that NETs formation could be regulated by the extracellular matrix surrounding the neutrophils.

Materials and

Methods: Neutrophils were isolated from healthy human whole blood. For initial experiments, collagen was used as a surrogate ECM biomaterial to mucus. To prepare the collagen matrix, stock collagen (Type-I Bovine and Type-I Rat Tail (Stemcell Technologies), was diluted to the required concentration using a buffer of 10X DMEM, NaOH (1M), DI water, Sodium Bicarbonate as appropriate. Neutrophils were embedded into the collagen droplets and stimulated with 1.5 uM Phorbol 12-Myristate 13-Acetate (PMA) to form NETs. 1 µM of Sytox-green stain was added to visualize NETs. All statistical analysis was conducted using unpaired t-test or ordinary one-way ANOVA analysis (Prism; GraphPad Software, La Jolla, CA). p-values less than 0.05 were considered significant.

Results, Conclusions, and Discussions: Compared to NETs produced by the neutrophils cultured on regular well plates, NETs extrusion appeared constrained inside the collagen matrix (Fig. A) Increase in the concentration of collagen resulted in a reduction in the NETs extension (Fig. B). No significant difference was observed in the NETs spread area within Bovine collagen and Rat Tail collagen (Fig. C), suggesting that NETs formation could be independent of the pore size for the same collagen concentration (1.5 mg/ml). The introduction of collagen after NETs stimulation resulted in a decreased NETs area (Fig. D) indicating that the structure of the collagen fibers could mechanically suppress the extrusion of NETs. Similar behavior of NETs formation was also observed in human bronchial mucus of different concentrations which represented healthy and diseased states.
Our results show that the ECM could modulate the release of NETs by the neutrophils. NETs formation could be regulated by the properties of the ECM such as stiffness, pore size and composition. These novel, yet fundamental findings lay the foundation for further research to study the implications of these findings in different immunopathological conditions of respiratory diseases.

Acknowledgements (Optional): The authors would like to thank NC TraCS for funding this project, and NC A&T Student Health Center for providing blood samples used for neutrophil isolation.