Neutrophils Transiting Through Megakaryocytes During Emperipolesis Exhibit Distinct Fates and Acquire the Capacity to Guide Other Neutrophils via MK Trails

Frank Huang¹, Pierre Cunin², Felix Radtke³, Ricardo Grieshaber-Bouyer⁴, Roxane Darbousset⁵ and Peter Nigrovic⁶, ¹Division of Rheumatology, Inflammation and Immunity, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA; Department of Medicine V, Hematology, Oncology and Rheumatology, Heidelberg University Hospital, Heidelberg, Germany, Darmstadt, Baden-Wurttemberg, Germany, ²Division of Rheumatology, Inflammation and Immunity, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA, Boston, MA, ³Division of Rheumatology, Inflammation and Immunity, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA; Department of Medicine V, Hematology, Oncology and Rheumatology, Heidelberg University Hospital, Heidelberg, Germany, Boston, ⁴Department of Medicine V, Hematology, Oncology and Rheumatology, Heidelberg University Hospital, Heidelberg, Germany; Division of Rheumatology, Inflammation and Immunity, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA, Heidelberg, Germany, ⁵Division of Rheumatology, Inflammation and Immunity, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA, Boston, ⁶Division of Rheumatology, Inflammation and Immunity, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA; Division of Immunology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA, Boston

Meeting: ACR Convergence 2020

Keywords: Inflammation, neutrophils

Session Information

Date: Friday, November 6, 2020

Title: Innate Immunity Poster

Session Type: Poster Session A

Session Time: 9:00AM-11:00AM

Background/Purpose: Megakaryocytes (MKs) can contribute directly to experimental arthritis via pro-inflammatory microparticles containing IL-1 (Cunin et al. Journal of Clinical Investigation, 2017). MKs also interact with immune cells via emperipolesis (EP), a novel cell-in-cell interaction whereby neutrophils pass through MKs before egressing without apparent harm to either cell. EP is common in bone marrow, but its cell biology and implications for MKs and neutrophils are unknown.

Methods: We compared the frequency of EP between healthy mice and different inflammatory models (K/BxN serum transfer arthritis, LPS injection, cecal ligation and puncture). We developed an in vitro model of EP by culturing bone marrow MKs with neutrophils and quantitated the time course using live cell spinning disk microscopy. We used transmission electron microscopy and immunofluorescence microscopy to explore intermediate stages of EP.

Results: EP frequency increased 2-3-fold in inflammatory arthritis, after administration of LPS, and after cecal ligation and puncture (a model of polymicrobial sepsis). EP bifurcated into fast and slow events, with approximately 40% of neutrophils transiting through MKs in less than 10 minutes whereas 35% persisted for 50 minutes or more. Slow EP was often associated with direct physical contact between neutrophils and the MK nucleus. Electron microscopy revealed that most neutrophils within MKs were encased by a phagosome-like compartment, termed the “emperisome”. However, some neutrophils could be observed only exiting the emperisome to enter the MK cytoplasm, where we could observe physical association with the endoplasmic reticulum or the demarcation membrane system (the MK’s platelet membrane reserve). These observations suggest the existence of different forms of EP. Intriguingly, we observed that neutrophils pull long tubes consisting of CD41+ MK membrane (“MK trails”) during their egress from MKs. MK trails express markers for immune cell attachment and rolling. Post-EP neutrophils deposited fragments of these trails on endothelial cells during migration in vitro, marking paths then preferentially visited by other neutrophils.

Conclusion: EP increases markedly during inflammation, including inflammatory arthritis. We identified fast and slow modes of EP, as well as distinct intracellular areas of neutrophil localization within MKs, suggesting that EP can serve multiple functions. EP results in the generation of novel MK trails that are deposited by neutrophils on endothelial cells, suggesting a new mechanism by which EP enables coordination of neutrophil behavior and thereby an organized and efficient inflammatory response.

Disclosure: F. Huang, None; P. Cunin, None; F. Radtke, None; R. Grieshaber-Bouyer, None; R. Darbousset, None; P. Nigrovic, Novartis, 2, 5, BMS, 2, 5, Pfizer, 2, 5, Sobi, 5, Miach Orthopedics, 5, Simcere, 5, XBiotech, 5, Quench Bio, 5, Siglion, 5, Cerecor, 5, UpToDate, 7, American Academy of Pediatrics, 7, CARRA, 9.

To cite this abstract in AMA style:

Huang F, Cunin P, Radtke F, Grieshaber-Bouyer R, Darbousset R, Nigrovic P. Neutrophils Transiting Through Megakaryocytes During Emperipolesis Exhibit Distinct Fates and Acquire the Capacity to Guide Other Neutrophils via MK Trails [abstract]. Arthritis Rheumatol. 2020; 72 (suppl 10). https://acrabstracts.org/abstract/neutrophils-transiting-through-megakaryocytes-during-emperipolesis-exhibit-distinct-fates-and-acquire-the-capacity-to-guide-other-neutrophils-via-mk-trails/. Accessed .

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