Quantifying spatial dynamics and regulators of Mycobacterium tuberculosis phagocytosis by primary human macrophages using microfabricated patterns

Abstract

Macrophages provide a first line of defense against invading pathogens, including the leading cause of bacterial mortality, Mycobacterium tuberculosis (Mtb). Phagocytosing extracellular organisms mediate pathogen clearance via a multitude of antimicrobial mechanisms, uniquely designed against an array of pathogens. Macrophages are able to execute different programs of activation in response to pathogenic challenge with host mediators, polarizing them to a variety of differentiation states, including the pro-inflammatory M1 and anti-inflammatory M2 states. The functional polarization of a macrophage prior to infection, thus impacts the outcome of host-pathogen interaction. One of the limitations when using in vitro differentiated human primary monocyte-derived macrophages (MDMs) is the heterogeneous nature of the mature population, which presents a challenge for quantitative characterization of various host-pathogen processes. Here, we describe an approach to minimize this heterogeneity, based on micropatterning of cells to reintroduce aspects of cellular homogeneity lost in a 2D tissue culture. Micropatterning consists of growing cells at the single cell level on microfabricated patterns, to constrain the size and shape of the cell, reducing cell-to-cell variation and mimicking the physiological spatial confinement that cells experience in tissues. We infected micropatterned GM-CSF- (M1) and M-CSF- (M2) derived human MDMs with Mtb, which allowed us to study host-pathogen interactions at a single cell level, at high resolution and in a quantitative manner, across tens to hundreds of cells in parallel. Using our approach, we were able to quantify phagocytosis of Mtb in MDMs, finding phagocytic contraction is increased by interferon-gamma stimulation, whilst contraction and bacterial uptake is decreased following silencing of phagocytosis regulator NHLRC2 or Tween80 removal of bacterial surface lipids. We also identify alterations in host organelle position within Mtb infected MDMs, as well as identifying differences in Mtb subcellular localization in relation to the microtubule organizing center (MTOC) and in line with the cellular polarity in M1 and M2 MDMs. Our approach described here can be adapted to study other host-pathogen interactions and co-infections in MDMs and can be coupled with downstream automated analytical approaches.