2024 AIChE Annual Meeting

Effects of Extracellular Matrix Composition on the Neutrophil Response

Neutrophils are the body’s first line of defense in combating infection. The neutrophil response is incredibly effective and powerful when adopted correctly; however, it is prone to dysfunction in diseases. The chemical factors that govern the neutrophil response are well studied, but our understanding of how the physical environment plays a role remains incomplete. Interestingly, diseases worsened by neutrophil dysfunction are often also accompanied by physical changes in the protein composition of the surrounding extracellular matrix (ECM). Specifically, collagen, the predominant protein in the ECM, is responsible for altering its mechanical and structural properties. The impact of the ECM composition is difficult to study in traditional experimental systems. Typical in-vitro models are composed of hard plastics and glass with little flexibility that fail to mimic physiological conditions (geometry of arteries and veins, 3-D interactions, etc). On the other hand, most in-vivo models do not allow for customization of the ECM composition. As a solution, we are using a biologically inspired microfluidic device that enables us to mimic human physiology while maintaining the tailoring we seek from in-vitro models. This project aims to better understand the effects of ECM composition on the neutrophil response through microfluidic devices.

Previous work on the project reveals a biphasic connection between collagen concentration and neutrophil extravasation1. Lysyl oxidase (LOX) was introduced into the system to improve the physiological relevance of this study since both LOX and collagen define the ECM in disease states such as fibrosis, cancer, and general aging. As an added benefit, LOX enables us to decouple the role of ECM mechanical and structural properties on the neutrophil response since the enzyme stiffens matrices without changing its pore size. First, we confirmed LOX treatment does not affect the pore structure within the gels. Using confocal reflectance microscopy (CRM), we found no changes in the major or minor axis pore length of collagen gels treated with LOX. Next, we observed a 1.2-1.4X increase in the stiffness of LOX-treated gels compared to the untreated condition at five days (Figure 1a-b). While this difference is not statistically significant, literature suggests it is biologically relevant on the neutrophil scale. We discovered LOX treatment does not change the extravasation behavior of neutrophils (Figure 1c). Analyzing confocal microscopy images of the neutrophil response to Pseudomonas aeruginosa (PAK), we saw no significant differences in extravasation trends between LOX treated and pure collagen gels. Together, the CRM, mechanical testing, and extravasation results suggest mechanical properties such as matrix stiffness have negligible effects on the neutrophil response. Further work on this study involves microscale mechanical testing of LOX gels and quantification of neutrophil antimicrobial activities to better predict the mechanism driving the neutrophil response in varying tissue environments.

  1. Calo, C. J., Patil, T., Palizzi, M., Wheeler, N., & Hind, L. E. (2024). Collagen concentration regulates neutrophil extravasation and migration in response to infection in an endothelium dependent manner. Frontiers in immunology, 15, 1405364.