Biology subjects

Ellett, F.

Publications and source records attributed to Ellett, F..

7 recordsLinked to original sources

bioRxiv2024

Pulses of Class I PI3kinase activity identify the release and recapture of prey from neutrophil phagosomes

Class I PI3kinases coordinate the delivery of microbicidal effectors to the phagosome by forming the phosphoinositide lipid second messenger, phosphatidylinositol (3, 4, 5)-trisphosphate (PIP3). However, the dynamics of PIP3 in neutrophils during a bacterial infection are unknown. We have therefore developed an in vivo, live zebrafish infection model that enables visualisation of dynamic changes in Class 1 PI3kinases (PI3K) signalling on neutrophil phagosomes in real-time. We have identified that on approximately 12% of neutrophil phagosomes PHAkt-eGFP, a reporter for Class 1 PI3K signalling, re-recruits in pulsatile bursts. This phenomenon occurred on phagosomes containing structurally and morphologically distinct prey, including Staphylococcus aureus and Mycobacterium abscessus, and was dependent on the activity of the Class 1 PI3K isoform, PI3kinase {gamma}. Detailed imaging suggested that pulsing phagosomes represent neutrophils transiently reopening and reclosing phagosomes. This finding challenges the concept that phagosomes remain closed after prey engulfment and we propose that neutrophils occasionally use this alternative pathway of phagosome maturation to release phagosome contents and/or to restart phagosome maturation if digestion has stalled.

cell biology↗

bioRxiv2022

Mechanical competition promotes selective listening to receptor inputs to resolve directional dilemmas in neutrophil migration

As neutrophils navigate complex environments to reach sites of infection, they may encounter obstacles that force them to split their front into multiple leading edges, raising the question of how the cell selects which front to maintain and which front(s) to abandon. Here we challenge chemotaxing HL60 neutrophil-like cells with symmetric bifurcating microfluidic channels, enabling us to probe the cell-intrinsic properties of their decision-making process. Using supervised statistical learning, we demonstrate that cells commit to one leading edge late in the decision- making process, rather than amplifying early pre-existing asymmetries. Furthermore, we use optogenetic tools to show that receptor inputs only bias the decision similarly late, once mechanical stretching begins to weaken each front. Finally, optogenetic attempts to reverse cell decisions reveal that, once an edge begins retracting, it commits to this fate, with the kinase ROCK limiting its sensitivity to inputs until the retraction is complete. Collectively our results suggest a "selective listening" model in which both actively protruding cell fronts and actively retracting cell rears have strong commitment to their current migratory program.

cell biology↗

bioRxiv2021

Neutrophil Profiles of Pediatric COVID-19 and Multisystem Inflammatory Syndrome in Children

Multisystem Inflammatory Syndrome in Children (MIS-C) is a delayed-onset, COVID-19-related hyperinflammatory systemic illness characterized by SARS-CoV-2 antigenemia, cytokine storm and immune dysregulation; however, the role of the neutrophil has yet to be defined. In adults with severe COVID-19, neutrophil activation has been shown to be central to overactive inflammatory responses and complications. Thus, we sought to define neutrophil activation in children with MIS-C and acute COVID-19. We collected samples from 141 children: 31 cases of MIS-C, 43 cases of acute pediatric COVID-19, and 67 pediatric controls. We found that MIS-C neutrophils display a granulocytic myeloid-derived suppressor cell (G-MDSC) signature with highly altered metabolism, which is markedly different than the neutrophil interferon-stimulated gene (ISG) response observed in pediatric patients during acute SARS-CoV-2 infection. Moreover, we identified signatures of neutrophil activation and degranulation with high levels of spontaneous neutrophil extracellular trap (NET) formation in neutrophils isolated from fresh whole blood of MIS-C patients. Mechanistically, we determined that SARS-CoV-2 immune complexes are sufficient to trigger NETosis. Overall, our findings suggest that the hyperinflammatory presentation of MIS-C could be mechanistically linked to persistent SARS-CoV-2 antigenemia through uncontrolled neutrophil activation and NET release in the vasculature. One Sentence SummaryCirculating SARS-CoV-2 antigen:antibody immune complexes in Multisystem Inflammatory Syndrome in Children (MIS-C) drive hyperinflammatory and coagulopathic neutrophil extracellular trap (NET) formation and neutrophil activation pathways, providing insight into disease pathology and establishing a divergence from neutrophil signaling seen in acute pediatric COVID-19.

immunology↗

bioRxiv2021

Directional reorientation of migrating neutrophils is limited by suppression of receptor input signaling at the cell rear through myosin II activity

To migrate efficiently to target locations, cells must integrate receptor inputs while maintaining polarity: a distinct front that leads and a rear that follows. Here we investigate what is necessary to overwrite pre-existing front/rear polarity in neutrophil-like HL60 cells migrating inside straight microfluidic channels. Using subcellular optogenetic receptor activation, we show that receptor inputs can reorient weakly polarized cells, but the rear of strongly polarized cells is refractory to new inputs. Transient stimulation reveals a multi-step repolarization process, confirming that cell rear sensitivity to receptor input is the primary determinant of large-scale directional reversal. We demonstrate that the RhoA/ROCK/myosin II pathway limits the ability of receptor inputs to signal to Cdc42 and reorient migrating neutrophils. We discover that by tuning the phosphorylation of myosin regulatory light chain we can modulate the activity and localization of myosin II and thus the amenability of the cell rear to listen to receptor inputs and respond to directional reprogramming.

cell biology↗

bioRxiv2019

Megakaryocytes Display Innate Immune Cell Functions and Respond during Sepsis

Megakaryocytes (MKs) are precursors to platelets, the second most abundant cells in the peripheral circulation. However, while platelets are known participate in immune responses and play significant roles during infections, the role of MKs within the immune system has not been explored. Here we utilize in vitro techniques to show that both cord blood-derived MKs (CB MKs) and MKs from a human megakaryoblastic leukemia cell line (Meg-01) chemotax towards pathogenic stimuli, phagocytose bacteria, and release chromatin webs in response to bacteria. Moreover, in patients with sepsis, we found that MK counts were significantly higher in the peripheral blood, and CD61+ staining was increased in the kidneys and lungs, correlated with the development of organ dysfunction. Overall, our study suggests that MK cells display basic innate immune cell functions and respond during infections and sepsis.

immunology↗

bioRxiv2019

β-glucan dependent shuttling of conidia from neutrophils to macrophages occurs during fungal infection establishment

The initial host response to fungal pathogen invasion is critical to infection establishment and outcome. However, the diversity of leukocyte-pathogen interactions is only recently being appreciated. We describe a new form of interleukocyte conidial exchange called \"shuttling\". In Talaromyces marneffei and Aspergillus fumigatus zebrafish in vivo infections, live imaging demonstrated conidia initially phagocytosed by neutrophils were transferred to macrophages. Shuttling is unidirectional, not a chance event, involves alterations of phagocyte mobility, inter-cellular tethering, and phagosome transfer. Shuttling kinetics were fungal species-specific, implicating a fungal determinant. {beta}-glucan serves as a fungal-derived signal sufficient for shuttling. Murine phagocytes also shuttled in vitro. The impact of shuttling for microbiological outcomes of in vivo infections is difficult to specifically assess experimentally, but for these two pathogens, shuttling augments initial conidial redistribution away from fungicidal neutrophils into the favourable macrophage intracellular niche. Shuttling is a frequent host/pathogen interaction contributing to fungal infection establishment patterns.

cell biology↗

bioRxiv2017

Lipid mediator class-switching downstream of PGE2 determines the outcome of inflammation resolution in vivo.

Neutrophils are the first immune cells recruited to a site of injury or infection, where they perform many functions. Having completed their role, neutrophils must be removed from the inflammatory site - either by apoptosis and efferocytosis or by reverse migration away from the wound - for restoration of normal tissue homeostasis. Disruption of these tightly controlled physiological processes of neutrophil removal can lead to a range of inflammatory diseases. We used an in vivo zebrafish model to understand the role of lipid mediator production in neutrophil removal. Following tailfin amputation in the absence of macrophages, neutrophillic inflammation does not resolve. This is due to loss of macrophage-dependent production of eicosanoid prostaglandin E2, which drives neutrophil removal via promotion of reverse migration. Knockdown of endogenous prostaglandin E synthase gene reveals PGE2 as essential for neutrophil inflammation resolution. Furthermore, PGE2 is able to signal through EP4 receptors to enhance Alox15 production, causing a switch towards anti-inflammatory eicosanoid signalling, specifically Lipoxin A4. Our data confirm regulation of neutrophil migration by PGE2 and LXA4 in an in vivo model of inflammation resolution. This pathway may contain therapeutic targets for driving inflammation resolution in chronic inflammatory disease.

immunology↗