Quantifying spatial dynamics of Mycobacterium tuberculosis infection of human macrophages using microfabricated patterns

Highlights

• Microfabricated patterns enable quantitative measurements of MtbMDM interactions
• Interferon-γstimulation increases phagocytic contraction of Mtb-infected MDMs
• Removal of bacterial surfacelipids attenuates phagocytic contraction and entry
• Mtbinfection leads to alterations in host organelle positioning

Motivation

Mycobacterium tuberculosis (Mtb) manipulates a plethora of macrophage responses to survive within its intracellular niche. Limited knowledge exists regarding the events that occur during phagocytosis and if/how Mtb manipulates the spatial organization of its new home. The morphological heterogeneity of differentially polarized primary human monocyte-derived macrophages (hMDMs) presents a hurdle for quantitative analysis of these earliest host-pathogen interactions. This study introduces a micropatterning, image-based approach to reduce this heterogeneity, instilling uniform cell morphology and organelle positioning. This toolbox facilitates high-resolution, image-based, quantitative investigations of previously under-studied early interactions between Mtb and differentially derived hMDMs at a single-cell level.

Summary

Macrophages provide a first line of defense against invading pathogens, including the leading cause of bacterial mortality, Mycobacterium tuberculosis (Mtb). A challenge for quantitative characterization of host-pathogen processes in differentially polarized primary human monocyte-derived macrophages (MDMs) is their heterogeneous morphology. Here, we describe the use of microfabricated patterns that constrain the size and shape of cells, mimicking the physiological spatial confinement cells experience in tissues, to quantitatively characterize interactions during and after phagocytosis at the single-cell level at high resolution. Comparing pro-inflammatory (M1) and anti-inflammatory (M2) MDMs, we find interferon-γ stimulation increases the phagocytic contraction, while contraction and bacterial uptake decrease following silencing of phagocytosis regulator NHLRC2 or bacterial surface lipid removal. We identify host organelle position alterations within infected MDMs and differences in Mtb subcellular localization in line with M1 and M2 cellular polarity. Our approach can be adapted to study other host-pathogen interactions and coupled with downstream automated analytical approaches.

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Authors: Anca F. Savulescu , Nashied Peton, Delia Oosthuizen, Rudranil Hazra, Robert P. Rousseau, Musa M. Mhlanga, Anna K. Coussens

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