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Abstract Number: 2804

Neutrophil Passage Through the Megakaryocyte Cytoplasm via Emperipolesis Modulates Neutrophil Migration

Pierre Cunin1, Alexandra Wactor 2, Li Guo 3, Andrew Weyrich 3, Éric Boilard 4 and Peter Nigrovic 5, 1Department of Medicine, Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, 2Department of Medicine, Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Harvard Medical School, Boston, 3Program in Molecular Medicine and Department of Internal Medicine, University of Utah, Salt Lake City, 4Axe maladies infectieuses et inflammatoires, Centre de recherche du CHU de Québec – Université Laval, Québec, Qc, Canada, Québec, QC, Canada, 5Department of Medicine, Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA ; Department of Medicine, Division of Immunology, Boston Children’s Hospital, Harvard Medical School, Boston, MA, USA., Boston

Meeting: 2019 ACR/ARP Annual Meeting

Keywords: neutrophils

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Session Information

Date: Tuesday, November 12, 2019

Session Title: 5T110: Innate Immunity (2804–2809)

Session Type: ACR Abstract Session

Session Time: 4:30PM-6:00PM

Background/Purpose: Emperipolesis (EP) is a little-understood phenomenon referring to the presence neutrophils within megakaryocytes (MKs). We developed methods to study EP in vitro and in vivo, finding that EP is a highly regulated cell-in-cell interaction, requires cytoskeleton rearrangement by neutrophils and MKs, and results in the reciprocal exchange of cell membrane and proteins between both lineages (Cunin P et al. eLife 2019;8:e44031). At the end of EP, neutrophils exit MK cytoplasm alive and intact, but the consequences for neutrophil function are unknown

Methods: We assessed the frequency of EP in healthy mice, in the K/BxN mouse model of inflammatory arthritis and in the cecal ligation and puncture model. A model of EP was developed through incubation of MKs together with neutrophils and characterized using confocal imaging and electron microscopy. The biology of EP was interrogated in vitro by time-lapse microscopy on µ-slide chemotaxis chambers, and in vivo using whole-mount immunofluorescence of bone marrow. The role of EP on neutrophil migration was investigated in vitro using transwell assay and in vivo through engraftment of labelled neutrophils co-cultured or not with MKs back into mice subjected to inflammation

Results: Histological sections of bone marrow revealed that EP is increased 2-3 fold in inflammatory arthritis and after cecal ligation and puncture, a model of polymicrobial sepsis (Figure 1). Whole-mount immunostaining in bone marrow showed that EP is restricted to MKs in contact with vascular sinusoids, and neutrophils can be observed in the space between MKs and blood vessels, suggesting egress directly into blood (Figure 2). In vitro, we observed that neutrophils enter MKs through a membrane-lined vacuole, termed “emperisome”. We routinely noted the appearance of MK-derived exosomes in the emperisome, and the uptake of MK exosomes by internalized neutrophils. Migration assay on µ-slide chemotaxis chambers revealed that neutrophils enter and exit MKs following a chemotactic gradient, using a “trans-MK” route (Figure 3A-B). After their passage into MK, neutrophils deposit MK material behind them during their migration, forming a “trail”-like structure made of MK proteins involved in immune cell attachment, and these neutrophils exhibit a clear migratory advantage (Figure 3B-E and not shown). In transwell assay, we observed that neutrophils previously co-cultured with MKs migrate more efficiently toward an LTB4 gradient. Neutrophil migration is not modulated after culture with MK supernatant or PFA-fixed MKs, excluding an effect from MK-derived microparticles, cytokines, or surface proteins and suggesting a role for EP. In vivo, neutrophils previously incubated with MKs migrated more efficiently into the peritoneal cavity during LPS- or IL-1β-induced peritonitis

Conclusion: EP is dramatically increased during inflammation and mediates transfer of membranes and MK-exosome contents to internalized neutrophils. 3-D bone marrow microscopy and migration assays suggest that neutrophils can exit from MKs directly into the circulation, resulting in enhanced migratory capacity. Additional consequences of EP for neutrophil biology remain to be defined

Percent of MK containing neutrophil -A- 8 days after arthritis induction and -B- 4 days after cecal ligation and puncture -CLP- surgery.

Whole bone marrow stained for CD41 -MK, green- Ly6G -neutrophil, red-, CD31/CD144 -bone marrow sinusoids, white- and Hoescht -DNA, blue-. -A- MK attached on a sinusoid and containing PMN. -B- Neutrophil in a vascular gap, at the interface between circulation and MK in the bone marrow compartment.

-A- µslide chemotaxis chamber -B- Neutrophils -Ly6G, green- migrating through a MK -CD41, white-. Each colored arrow follows a neutrophil over time. The passage inside MK is confirmed by analyzing each Z-stacks for each time point -not shown-. The image at 48 min shows trails left by neutrophils -colored arrows- -C- 3D reconstitution in high resolution of the MK in -B-. Asterisks show neutrophils attached on trails. -D- Trails left by neutrophils are Ly6G negative and CD41 positive, demonstrating a MK origin. -E- Average speed of neutrophils positive for the MK marker CD41 -green circles- or CD41 negative -black squares-. Representative of 3 experiments.

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