the work


Effectors versus Protectors:
How Anti-inflammatory Tissue Macrophages Maintain Tissue Homeostasis

On January 24th, 2018, I had the honor of giving a lecture for the Immunonology IG seminar series at NIAID/NIH on “Effectors versus Protectors: How Anti-inflammatory Tissue Macrophages Maintain Tissue Homeostasis“. The talk was recorded and is permanently archived here.

King et al., Science 2020

Water loss regulates cell and vesicle volume

Jason S. King and Elizabeth Smythe wrote a preview to our recent paper in Science.

Check out the paper here [$] or contact to request a copy.

SUMMARY When cells take up extracellular fluid by endocytosis, they internalize a considerable proportion of the cell volume quickly and yet maintain their volume and ionic composition. This is particularly striking in the case of macropinocytosis, which is the bulk uptake of extracellular fluid. Through this pathway, macrophages can be stimulated to internalize ∼25% of their cellular volume per hour into large vacuoles known as macropinosomes. An intriguing question is how cells and organelles are able to maintain their size while internalizing such large volumes. On page 301 of this issue, Freeman et al. reveal a molecular mechanism underpinning homeostatic regulation of cell size. They demonstrate that newly formed macropinosomes rapidly lose volume by osmosis driven by two-pore channel (TPC)–mediated outflow of sodium ions. This reduces hydrostatic pressure within the macropinosome, facilitating the extension of tubules from the macropinosome surface and recycling of membrane lipids and proteins back to the cell surface.

Freeman et al., Science 2020

Lipid-gated monovalent ion fluxes regulate endocytic traffic and support immune surveillance

>>Ion fluxes resolve organellar volume<<

EDITOR’s SUMMARY Animal cells continuously sample the surrounding medium, a feature accentuated in immune cells. Sampling is accomplished by trapping external medium into membrane-bound vesicles or vacuoles. These structures are promptly resolved, thus avoiding accumulation of endomembranes and volume expansion. In a variety of cultured cells, Freeman et al. found that this resolution entails conversion of spherical vacuoles into thin tubules, a process that involves marked changes in surface-to-volume ratio (see the Perspective by King and Smythe). Shrinkage of membrane-bound structures is driven by ion fluxes and subsequent osmotic transfer of water. Shriveled vacuoles attract curvature-sensing proteins that promote the extension of fine tubules. Ion channels thereby control membrane remodeling, enabling receptor recycling and proper routing of cellular cargo.

Check out the paper here [$] or contact to request a copy.

ABSTRACT Despite ongoing (macro)pinocytosis of extracellular fluid, the volume of the endocytic pathway remains unchanged. To investigate the underlying mechanism, we used high-resolution video imaging to analyze the fate of macropinosomes formed by macrophages in vitro and in situ. Na+, the primary cationic osmolyte internalized, exited endocytic vacuoles via two-pore channels, accompanied by parallel efflux of Cl and osmotically coupled water. The resulting shrinkage caused crenation of the membrane, which fostered recruitment of curvature-sensing proteins. These proteins stabilized tubules and promoted their elongation, driving vacuolar remodeling, receptor recycling, and resolution of the organelles. Failure to resolve internalized fluid impairs the tissue surveillance activity of resident macrophages. Thus, osmotically driven increases in the surface-to-volume ratio of endomembranes promote traffic between compartments and help to ensure tissue homeostasis.

Failure of endosome resolution in PIKfyve-deficicent tissue macrophages (right) compared to wildtype cells (left).

The techniques & methods we used in this study:
+ Intravital 2P-microscopy
+ Multiplex confocal microscopy
+ In vivo cell biology (tissue biology)
+ Ex vivo cell biology

+ Dynamic spinning disk microscopy
+ Electron microscopy

Failure of endosome resolution (right) abrogates tissue surveillance and promotes neutrophil-driven inflammation (magenta) at stromal micro-lesions which are usually cloaked (left) by macrophages (green).
Blériot et al., Cell 2019

“Cloaking” on Time: A Cover-Up Act by Resident Tissue Macrophages.

Camille Blériot, Lai Guan Ng and Florent Ginhoux wrote a preview to our recent paper in Cell.

SUMMARY In this issue of Cell, Uderhardt et al. employed intravital two-photon microscopy to examine tissue-resident macrophage responses to sterile cellular injuries of variable size. They observed that while multi-cell “macrolesions” are characteristically pro-inflammatory, resident macrophages can “cloak” single-cell microlesions to prevent excessive neutrophil recruitment and limit subsequent tissue damage.

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Uderhardt et al., Cell 2019

Resident Macrophages Cloak Tissue Microlesions to Prevent Neutrophil-Driven Inflammatory Damage

HIGHLIGHTS Resident tissue macrophages (RTMs) respond to tissue damage and cloak the debris. Cloaking by RTM prevents neutrophil activation and neutrophil-driven inflammation. Cloaking by RTM prevents excess tissue damage and preserves tissue homeostasis.

Check out the paper here [$] or the PMC version or contact to request a copy.
I’ve also uploaded all the supplementary files and videos here (Pssst!).
Enjoy and get in touch!

SUMMARY Neutrophils are attracted to and generate dense swarms at sites of cell damage in diverse tissues, often extending the local disruption of organ architecture produced by the initial insult. Whether the inflammatory damage resulting from such neutrophil accumulation is an inescapable consequence of parenchymal cell death has not been explored. Using a combination of dynamic intravital imaging and confocal multiplex microscopy, we report here that tissue-resident macrophages rapidly sense the death of individual cells and extend membrane processes that sequester the damage, a process that prevents initiation of the feedforward chemoattractant signaling cascade that results in neutrophil swarms. Through this “cloaking” mechanism, the resident macrophages prevent neutrophil-mediated inflammatory damage, maintaining tissue homeostasis in the face of local cell injury that occurs on a regular basis in many organs because of mechanical and other stresses.

Check out our official video abstract (credits: SU & AR)

The techniques & methods we used in this study:
+ Intravital 2P-microscopy
+ Multiplex confocal microscopy
+ Optical clearing (Ce3D)
and volume imaging
+ Spatiotemporal tracking
+ low-input bulk RNA sequencing
+ In vivo cell biology

Resident tissue macrophages (green) cloaking a stromal microlesion (brown).