Multiscale Invasion Assay for Probing Macrophage Response to Gram-Negative Bacteria.

Kimberly A Wodzanowski,April M Kloxin,Catherine L Grimes,Jeffrey L Caplan

doi:10.3389/fchem.2022.842602

Abstract

The immune system is a complex network of various cellular components that must differentiate between pathogenic bacteria and the commensal bacteria of the human microbiome, where misrecognition is linked to inflammatory disorders. Fragments of bacterial cell wall peptidoglycan bind to pattern recognition receptors within macrophages, leading to immune activation. To study this complex process, a methodology to remodel and label the bacterial cell wall of two different species of bacteria was established using copper (I) catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition (SPAAC). Additionally, an approach for three-dimensional (3D) culture of human macrophages and their invasion with relevant bacteria in a well-defined hydrogel-based synthetic matrix inspired by the microenvironment of the gut was established. Workflows were developed for human monocyte encapsulation and differentiation into macrophages in 3D culture with high viability. Bacteria invaded into macrophages permitted in situ peptidoglycan labeling. Macrophages exhibited biologically-relevant cytokine release in response to bacteria. This molecularly engineered, multi-dimensional bacteria-macrophage co-culture system will prove useful in future studies to observe immunostimulatory, bacterial fragment production and localization in the cell at the carbohydrate level for insights into how the immune system properly senses bacteria.

Highlights

The innate immune system is the body’s first line of defense against invading pathogens
The CuAAC click reaction allows for the visualization of the cell wall, reaction requires the cells to be fixed due to the toxic nature of copper to cells, eliminating the ability of tracking the bacteria in real time to get engulfed by macrophages
Through a detailed study of DBCO concentrations and click reaction times, we determined that 40 min at 37°C with 30 μM DBCO-488 was sufficient to label the cell wall in E. coli for microscopy (Figure 2, Supplementary Figure S1), demonstrating that the DBCO dye can cross the outer membrane of the bacterial cell

Summary

Introduction

The innate immune system is the body’s first line of defense against invading pathogens. Macrophages have many roles in the innate immune system, including ingesting pathogens by phagocytosis, scavenging dead cells and cell debris, and remodeling tissues after injury (Galli et al, 2011). Mature macrophages express receptors that identify pathogens, allowing their proper uptake into the cell for degradation and response (Galli et al, 2011) These receptors include membraneassociated Toll-like receptors (TLRs) and cytosolic NOD-like receptors (NLRs), which are known to bind to fragments of the bacterial peptidoglycan (PG), a component of the bacterial cell wall. There is a need for development of physiologically-relevant co-culture systems to model invasion of bacteria within human macrophages and begin probing these complex interactions toward addressing these important questions These systems should be amenable to downstream chemical biology labeling techniques of both the host and invading bacterial species

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Conclusion