key: cord-0273346-cfcm8hny authors: Huang, Frank Y.; Cunin, Pierre; Radtke, Felix A.; Grieshaber-Bouyer, Ricardo; Nigrovic, Peter A. title: Neutrophil transit time and localization within the megakaryocyte define morphologically distinct forms of emperipolesis date: 2021-04-27 journal: bioRxiv DOI: 10.1101/2021.04.26.441404 sha: 067e8114c0cd24755e98b8666e9a510975c0a138 doc_id: 273346 cord_uid: cfcm8hny In emperipolesis, neutrophils transit through megakaryocytes, but it is unknown whether this interaction represents a single type of cell-in-cell interaction or a set of distinct processes. Using an in vitro model of murine emperipolesis, we characterized neutrophils entering megakaryocytes using live-cell spinning disk microscopy and electron microscopy. Approximately half of neutrophils exited the megakaryocyte rapidly, typically in 10 minutes or less, displaying ameboid morphology as they passed through the host cell (fast emperipolesis). The remaining neutrophils assumed a sessile morphology, most remaining within the megakaryocyte for at least 60 minutes (slow emperipolesis). These neutrophils typically localized near the megakaryocyte nucleus. By ultrastructural assessment, all internalized neutrophils remained morphologically intact. Most neutrophils resided within emperisomes, but some could be visualized exiting the emperisome into the cell cytoplasm. Neutrophils in the cytoplasm assumed close contact with the platelet-forming demarcation membrane system or with the perinuclear endoplasmic reticulum, as confirmed by immunofluorescence microscopy. Together, these findings reveal that megakaryocyte emperipolesis reflects at least two processes, fast and slow emperipolesis, each with its own characteristic transit time, morphology, and intracellular localization, suggesting distinct functions. Key Points Neutrophil passage through megakaryocytes, termed emperipolesis, diverges into fast and slow forms that differ in transit time, morphology, and intracellular localization During emperipolesis, neutrophils can reside in vacuoles (emperisomes) or escape into the cell cytoplasm to assume positions near the megakaryocyte’s demarcation membrane system, endoplasmic reticulum, or nucleus. Megakaryocytes (MKs) are the largest cells in the bone marrow (50-100 µm) and constitute ~ 0.05% of marrow cells. 1 MKs produce platelets by extending long protrusions called proplatelets into sinusoids where shear stress causes platelet release into the circulation. 2, 3 This ability to generate platelets has been extensively studied. However, recent observations have begun to suggest important immune functions. 4, 5 MKs express Toll-like receptors [6] [7] [8] [9] and other immune receptors [10] [11] [12] and produce inflammatory cytokines and chemokines. 13-15 Early MK progenitors express major histocompatibility complex (MHC) class II. 16 Mature MKs crosspresent antigens to CD8 + T cells via MHC I 17 , to CD4 + T cells via MHC II 18 , and can exhibit antiviral potency. 19 In COVID-19 patients, the percentage of MKs in the PBMC fraction is increased and a hyperinflammatory MK subset, enriched in severe COVID-19 patients, constitutes a potential contributor to systemic inflammation. 20 Thus the functional portfolio of MKs has extended considerably beyond platelet production. An intriguing functional specialization of MKs is to interact directly with leukocytes, predominantly neutrophils, in a cell-in-cell interaction termed emperipolesis (EP). 21 Derived from the Greek for "inside round about wandering", EP was first described in 1956 by Humble et al. 22 Passage through MKs occurs without apparent harm to either cell. 23, 24 Efficient EP by neutrophils requires active cytoskeletal rearrangement in both the host MK and the transiting neutrophil. 24 These features clearly distinguish EP from cell-in-cell interactions such as phagocytosis or entosis in which the engulfed cell remains passive and is typically digested. 25 Under physiological conditions, conventional paraffin sections identify EP in approximately 1-4% of MKs in mice. 24 This frequency can more than double with systemic inflammation 24 , chronic blood loss 26 , myelofibrosis [27] [28] [29] , myeloproliferative diseases 30 , and gray platelet syndrome. [31] [32] [33] [34] Distinct forms of emperipolesis 4 While regularly observed, basic questions regarding the cell biology of EP remain unanswered. We showed previously that neutrophils undergoing EP can fuse transiently with the MK demarcation membrane system (DMS), thereby transferring neutrophil membrane to daughter platelets to enhance platelet production. 24 Earlier authors had hypothesized that EP may serve as a transmegakaryocytic route for neutrophils in the bone marrow to enter the circulation 26 or that MKs might provide a "sanctuary" for neutrophils. 35 Since EP is observed in multiple states of health and disease, the possibility remains that EP could serve several functions. We hypothesized that, if EP represented a heterogeneous set of processes, then the transit of neutrophils through MKs could exhibit corresponding morphological heterogeneity. We therefore employed immunofluorescence and electron microscopy (EM) to investigate the time course and fate of neutrophils engaged in EP. We demonstrate that EP diverges into fast and slow forms, with multiple distinct intermediate stages, suggesting distinct processes with potentially divergent physiological roles. Mice 8-12 weeks old WT C57BL/6J mice were purchased from the Jackson Laboratory (#000664) and housed at specific pathogen-free conditions. All animal studies were approved by the Institutional Animal Care and Use Committee of the Brigham and Women's Hospital. Anti-CD41 APC (MWReg30), anti-CD41 AF488 (MWReg30), anti-Ly6G AF594 (1A8) were from BioLegend. Polyclonal anti-calnexin and FluorSave Reagent were from Sigma-Aldrich. Distinct forms of emperipolesis 5 Polyclonal anti-golgin-97, donkey anti-rabbit AF488, DRAQ5, Hoechst 33342, RPMI 1640 with and without phenol red, ACK lysing buffer, paraformaldehyde and glutaraldehyde were from Thermo Fisher. Femurs and tibias were flushed with PBS using 22-gauge needles. Cell suspensions were then filtered through 40 µm cell strainers to remove pieces of bone or tissue and centrifuged, followed by lysis of red blood cells using ACK lysing buffer. Bone marrow cells were then washed with PBS and resuspended in complete RPMI medium containing 1% thrombopoietin (TPO medium). Hematopoietic progenitor cells were isolated from bone marrow using EasySep TM Mouse Hematopoietic Progenitor Cell Isolation Kit (negative selection) and cultured 1, 2 or 4 days in TPO medium (5´10 6 cells/ml). Alternatively, bone marrow cells were cultured in TPO medium (10 7 cells/ml) for 4 days at 37°C, 5% CO2. MKs were then enriched using an albumin step gradient. 36 Emperipolesis assay 2´10 6 bone marrow cells and 2´10 4 MKs were co-cultured in P96 round bottom wells for 12 hours at 37°C, 5% CO2. After 12 hours of co-culture, bone marrow cells and MKs were fixed in PFA 2% for 30 minutes at room temperature. After washing with PBS, cells were resuspended in PBS containing 0.2% Distinct forms of emperipolesis 6 saponin and 10% fetal bovine serum (permeabilization buffer) and stained with Hoechst 33342 (5 µg/mL), anti-CD41 AF488 and anti-Ly6G AF594 for 4 hours at RT or overnight at 4°C. In some experiments, cells were stained with Hoechst 33342 (5 µg/mL), anti-CD41 APC, anti-Ly6G AF594 and anti-calnexin or anti-golgin-97 (2.5 µg/mL respectively). Cells were then washed with PBS and resuspended in permeabilization buffer containing donkey anti-rabbit AF488 secondary antibody (10 µg/mL) for 4 hours at RT or overnight at 4°C. After staining, cells were washed and cytospun onto coverslips and mounted on glass microscope slides (Fisher Scientific) using FluorSave Reagent. Images were obtained using a Zeiss LSM 800 with Airyscan attached to a Zeiss Axio Observer Z1 Inverted Microscope using a Plan-Apochromat 63´ objective. Zen 2.3 blue edition software was used for image acquisition. Image analysis was performed using ImageJ 1.52p. Neutrophils and MKs were stained with anti-CD41 AF488 and anti-Ly6G AF594 (1.5 µg/mL respectively) for 1 hour prior to the experiment. DNA was stained with Hoechst 33342 (5 µg/mL) or DRAQ5 (5 µM). Cells were resuspended in TPO medium without red phenol to minimize autofluorescence and plated onto Nunc TM Glass Bottom Dishes (Thermo Fisher) for imaging. Images were obtained using a W1 Yokogawa Spinning Disk Confocal attached to a Nikon Ti inverted microscope with a Plan Fluor 40x/1.3 Oil DIC H/N2 objective and Nikon Elements Acquisition Software AR 5.02 or using a Perkin Elmer Ultraview Vox Spinning Disk Confocal attached to a Nikon Ti inverted microscope with a 60x (1.4NA) objective and Volocity Acquisition Software 6.3. Microscopy chambers were kept at 37°C and 5% CO2 throughout the experiment. In each experiment, three regions of interest and 10-12 z-stacks were imaged, with approximately 90 seconds between two timepoints. Image analysis was performed using ImageJ 1.52p. Distinct forms of emperipolesis 7 The migration of neutrophils through the cytoplasm of MKs was tracked using the ImageJ plugin TrackMate v4.0.1. After 12 hours of co-culture, bone marrow cells and MKs were washed twice with PBS and fixed in PFA 2% and glutaraldehyde 0.1% for 4 hours at room temperature. Specimens were postfixed in 1% osmium tetroxide and 1.5% potassium ferrocyanide, stained with 1% uranyl acetate, followed by gradual dehydration in 70%, 90%, 100% ethanol and propylene oxide. Specimens were then embedded in Epon. 80 nm sections were imaged using a JEOL 1200EX transmission electron microscope. EM imaging was performed in the Harvard Medical School Electron Microscopy Facility. Statistical analyses were performed using Graphpad Prism 8 or R. One-way ANOVA and posthoc Tukey test were performed to compare the frequency of EP across developmental stages. Hartigan's dip test was performed to determine bimodality in transit time. Unpaired t test was performed to compare neutrophil migration speed between fast and slow EP. All values were displayed as mean ± standard error of the mean. A p-value ≤ 0.05 was considered statistically significant. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. To study EP, we employed a model wherein murine MKs differentiated from hematopoietic progenitor cells in TPO medium are incubated together with unfractionated murine bone Distinct forms of emperipolesis 8 marrow. We performed EP with MKs at different stages in culture, corresponding to different maturational states, grading MK maturity using the extent of DMS. 37 In each of three experiments, we evaluated EP as performed by 100 MKs at each maturational stage. Mature MKs proved most efficient at EP, displaying neutrophil uptake by approximately 20% of cells in comparison with 0.33% of immature MKs (Figures 1A-G) . Uptake of > 1 neutrophil was largely restricted to the most mature MKs (Figures 1H-I) . We therefore employed mature day 4 MK cultures for our studies going forward. To understand whether neutrophil transit through MKs represents a uniform or heterogeneous process, we employed spinning disk confocal microscopy. MKs were cultured together with whole bone marrow cells and visualized over 90 minutes, obtaining images every 90 seconds and acquiring 10-12 z-stacks per MK to distinguish internalized from superimposed neutrophils. Prior studies have shown that neutrophils engaged in EP may reside either in MK vacuoles, termed emperisomes, or directly within the MK cytoplasm. 24 We sought to better understand the relationship between these compartments in EP using transmission EM. We co-cultured murine MKs and bone marrow cells as above and processed them for EM after 12 hours of culture, analyzing 45 EP events across five independent experiments. All MKs and neutrophils remained morphologically intact, without membrane blebbing, nuclear fragmentation, or other evidence of apoptosis. Residence within an emperisome was the most common location for internalized neutrophils (18 of 45 events, 40%), yet the interaction between neutrophil and emperisome was heterogeneous. In some cases, the emperisome membrane was smooth and separated from the neutrophil by a large pericellular space ( Figure 4AI) . Alternately, the emperisome membrane could exhibit small protrusions extending towards the Distinct forms of emperipolesis 10 engulfed neutrophil ( Figure 4AII) . Finally, some emperisomes were tightly wrapped around the neutrophil, with zipper-like approximation of neutrophil and emperisome membranes ( Figure 4AIII ). Intriguingly, some neutrophils appeared within a vacuolar space but in contact with the cytoplasmic DMS without an interposed MK membrane, suggesting an egress event mediated by reshaping or dissolution of the emperisome (Figure 4BI) . Others were entirely surrounded by DMS ( Figure 4BII) . Importantly, some neutrophils could be visualized transiting directly from an emperisome into the MK cytoplasm, far from the DMS (Figure 4CI) , or fully resident within the MK cytoplasm without any interposed MK membrane (Figure 4CII ). The frequencies of these different EP stages are shown in Figure 4D . Of note, despite the proximity of the neutrophil to the MK nucleus in some instances, our EM studies identified no examples of direct membranemembrane contact. While the fixed nature of EM images precludes assignment of these stages to fast or slow EP, these images confirm the highly varied interaction between an internalized neutrophil and its MK, suggesting further that penetration through the emperisome membrane represents the most common mechanism of neutrophil egress into the MK cytoplasm. The DMS is easily recognized by its dilated appearance ( Figure 5A , bottom left), but not all interactions between cytoplasmic neutrophils and MK organelles were with the DMS. Identifying membranes as belonging to the endoplasmic reticulum (ER) or the Golgi apparatus can be difficult by EM. 7 of 45 neutrophils (16%) were surrounded by membranes that we could not unambiguously assign to one of these structures (Figures 4D and 5A) . Given our observation that neutrophils undergoing slow EP frequently reside near the MK nucleus, we hypothesized that they might interact with the perinuclear ER. Immunofluorescence staining confirmed that the perinuclear ER sometimes surrounded intracytoplasmic neutrophils (Figures 5B-C) , enclosing neutrophils between ER and nucleus. This location is distinct from that of the DMS, as reflected in the inverse distribution of the calnexin + ER and the CD41 + DMS (Figures 5B-C) . The Golgi apparatus, another intracellular membrane network marked by golgin-97, did not colocalize with internalized neutrophils (Figure 5D) . These data further confirm the diversity of intramegakaryocytic localization by neutrophils in EP, supporting the heterogeneity of this process. Emperipolesis is a cell-in-cell interaction at the interface of hemostasis and immunity. Neutrophils pass through MKs without disrupting the integrity of either cell, penetrating into the MK cytoplasm in at least some cases. Other granulocytes, lymphocytes, erythrocytes or monocytes are occasionally observed inside MKs, but neutrophils predominate and are observed more frequently than any other lineage even after adjusting for their abundance in the bone marrow. 24, 39, 40 Despite the frequency of EP, it remains unknown why neutrophils enter MKs, whether different forms of EP exist, and what roles this interaction plays in health and disease. Understanding the cell biology of EP will be key to answering these questions. In the present study, we employed immunofluorescence and electron microscopy to study EP in a system that had previously been shown to model key elements of EP in vivo. 24 Based on the duration of transit, we found that EP diverged into fast (generally < 10 minutes) and slow (generally > 60 minutes) forms. Neutrophils sinusoids. 45 Thus, preferential conduct of EP by mature MKs restricts the process to cells in direct contact with the lumen of blood vessels. Alternately, enhanced EP by mature MKs could reflect their larger cell size and more extensive DMS, providing a larger cell surface for cell-cell contact and more space to accommodate neutrophils. Intriguingly, approximately half of neutrophils remain within MKs for an extended period, a process we term slow EP. These cells exhibit a sessile morphology. Failing to observe any degraded neutrophils within MKs, we assume that most eventually exit, though since almost all slow EP events extended beyond our video observations we cannot confirm this assumption. De Pasquale et al 35 proposed that EP might serve as a "sanctuary" for neutrophils in an unfavorable bone marrow environment, though why sanctuary should be necessary is unclear. Localization between the nucleus and the perinuclear ER raises additional novel possibilities, such as to "intercept" mRNA emerging from the nucleus or modulating ER function. 46, 47 Without direct evidence, all such possibilities remain purely speculative. A central topological problem presented by EP is how neutrophils leave the emperisome to enter the MK cytoplasm. 21, 24 We approached this problem via EM of 45 EP events. The most common localization of neutrophils was within a clearly demarcated vesicle, termed the emperisome. 24 In other instances, only a single membrane separated the cytoplasm of the neutrophil from that of the MK, consistent with intracytoplasmic residence (Figure 4CII) . We observed intermediate steps in which part of the neutrophil remained in the emperisome while part exhibited contact with the cell cytoplasm ( Figure 4CI) . These images suggest penetration of the neutrophil through part of the vesicle wall, rather than for example wholesale resorption or disintegration of the emperisome membrane. Other images are more difficult to categorize definitively with respect to emperisome vs. cytoplasm, in particular where the neutrophil is surrounded by the DMS (Figures 4BI-BII) . Earlier EM studies had described these cells as Distinct forms of emperipolesis 14 residing "loosely in the canalicular system" 48 , though further study will be require to understand the topological relationship of these cells with respect to the intra-vesicular, cytoplasmic, and extracellular compartments. The technical limitations of EM do not allow us to determine whether these morphological phases represent distinct neutrophil fates, restricted for example to either fast or slow EP, or instead sequential stages undertaken by many or most neutrophils during EP. Our study has several important limitations. We studied murine EP, employing a useful but nevertheless in vitro system. How our findings translate to human and in vivo contexts remains unknown. While fast and slow EP appear distinct from each other, suggesting distinct functions, we could not here define these functions and cannot exclude the possibility that they fulfil similar roles. Further research is required to elucidate how neutrophils transition between different intermediate stages; whether EP modulates the behavior of neutrophils, MKs, or platelets; and whether MKs outside the bone marrow compartment, for example in the lung, also engage neutrophils and other cells via EP. Despite these limitations, our studies provide the first evidence that MK EP is a heterogeneous process through which neutrophils may engage with MKs either for a short or long duration, interacting with intracellular structures including the emperisome, the DMS, the cytoplasm, the perinuclear ER, and potentially the nucleus itself. Preserved in all mammalian species studied, across millions of years of otherwise divergent evolution 21 , these observations suggest that EP will likely serve a range of roles to be defined through further investigations. (AII) The emperisome extends membrane protrusions toward the engulfed neutrophil. (AIII) The emperisome tightly wraps around the engulfed neutrophil. Magnification shows close membrane approximation between neutrophil and emperisome membranes (arrowheads). (BI) Internalized neutrophil partly covered by the emperisome and partly exposed to the DMS of the MK. (BII) Neutrophil residing within the cavities of the DMS. (CI) Internalized neutrophil partly covered by the emperisome (CIa) and partly exposed to organelles of the MK cytoplasm (CIb). (CII) Two neutrophils fully reside inside the MK cytoplasm. Only the neutrophil membranes remain visible (arrowheads). (D) Frequency of the previously described EP stages (n = 45). Distinct forms of emperipolesis 24 MKs were allowed to engage in EP for 12 hours followed by processing for EM or laser scanning confocal microscopy. 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