Necroptosis of Glycolytic Dendritic Cells Enhances Activation of Gamma Delta T Cells

Gamma delta (gd) T lymphocytes are a poorly understood class of immune cells that accumulate at sites of inflammation due to infection or autoimmunity. Part of the mystery of this T cell subset derives from lack of knowledge of ligands that bind to the T cell receptor (TCR) of gd T cells. The conditions under which these ligands are induced remain ill-defined, but the Budd laboratory has recently reported that dendritic cell (DC) necroptosis exposes a previously unreported ligand for the gd TCR, leading to activation of gd T cells. Necroptosis is caspase-independent and can be induced via inhibition of caspase-8, leading to formation of a complex containing Receptor Interacting Protein Kinase 1 (RIPK1) known as the ripoptosome. In this study, we describe a new connection between glycolysis of DC and their susceptibility to necroptosis. DC grown with GM-CSF are highly glycolytic and susceptible to necroptosis induced by the pan-caspase blocker zVAD. However we demonstrate that GM-CSF-generated DCs treated with the glycolysis inhibitor 2-deoxyglucose are protected against necroptosis by zVAD. We propose that aerobic glycolysis is a prerequisite for DC necroptosis and accordingly their ability to activate gd T cells. Inhibition of glycolysis does not impact caspase activity levels, production of essential cytokines, or production of proteins essential for the ripoptosome. Instead, non-glycolytic DC have increased levels of cleaved RIPK, which serves as an inhibitor of Ripoptosome formation. It has been established that activation of DC leads to increased cellular rates of aerobic glycolysis. It may be that naïve dendritic cells are protected from necroptosis by increased cleavage of RIPK in order to prevent unnecessary cell death and the triggered activation of a downstream immune response

Neutrophil biomechanical properties and immune function in health and inflammatory disease

Low density granulocytes (LDGs) are a poorly understood class of immune cells found in patients with chronic inflammatory diseases including psoriasis and systemic lupus erythematosus (SLE). Research completed at the National Institutes of Health (NIH) revealed that in the context of SLE, LDGs release higher levels of type 1 interferons, undergo increased NETosis, and accordingly drive inflammation. Meanwhile, advances in mechanical phenotyping at the University of Cambridge have driven hypotheses of neutrophil trafficking and immune function being intimately linked to cellular biomechanical properties (e.g. density, stiffness, morphology). This thesis analyses the intersection of immune cellular biomechanical phenotypes and their function. Specifically, it focuses on the role of neutrophils and LDGs in inflammatory diseases. In this thesis, real-time deformability cytometry (RT-DC) was optimised as a high-throughput mechanical phenotyping technique for the analysis of neutrophils. This enabled development of a protocol to recover purified neutrophils to their whole blood mechanical phenotype. Neutrophil biomechanical properties were analysed by RT-DC, lattice light-sheet microscopy, confocal microscopy and scanning electron microscopy. Neutrophil immunologic functions (e.g. NETosis, macropinocytosis) were imaged using florescence microscopy. To analyse the contribution of biomechanical properties to neutrophil trafficking, a novel microfluidic microvasculature mimetic was developed. An endothelial flow assay was used to image neutrophils interacting with endothelial cells. Finally, the complete proteomes and phosphoproteomes of LDGs and normal dense neutrophils (NDNs) were obtained from five healthy donors and five SLE patients. Several key insights were gained. Firstly, hypotonic lysis and magnetic column-based isolation techniques are damaging to neutrophil biomechanical properties, but purification of neutrophils retaining their biomechanical properties can be achieved by using gradients and column-free magnetic systems followed by recovery at 37 degrees Celsius. Secondly, the biphasic biomechanical kinetics of neutrophil priming were described; cells contract briefly before immediately expanding. The expansion phase was determined to be macropinocytosis dependent. Thirdly, SLE LDGs are phenotypically rougher than autologous SLE NDNs or healthy LDGs. This appears to impact their microvasculature trafficking abilities, as SLE LDGs were increasingly trapped in the narrow channels of a three- dimensional microvasculature mimetic. These results suggest a role for biomechanical properties in modulation of neutrophil trafficking, indicating that SLE LDGs may be increasingly retained in microvasculature networks, similar to what has been described for primed neutrophils. Finally, unbiased proteomics quantified 4109 proteins and 875 phosphoproteins in four neutrophil subsets (healthy unstimulated NDNs, healthy primed NDNs, SLE NDNs, and SLE LDG). This shed new light into neutrophil heterogeneity at the protein level and to my knowledge, is the first proteomic profile of the SLE LDG. In addition to findings pertaining to SLE LDG biology and function, differential phosphorylation of proteins associated with cytoskeletal organisation were identified in SLE LDGs relative to SLE NDNs, suggesting a phosphoproteomic explanation for the SLE LDGs’ distinct biomechanical phenotype. When taken together, this work could have important pathogenic implications in the context of SLE manifestations in various organs and the development of small vessel vasculopathy.Kathleen Bashant was funded by the NIH-Cambridge Scholars Program This research was supported by the NIHR Cambridge Biomedical Research Centre (BRC-1215-20014*). The views expressed are those of the author and not necessarily those of the NIHR or the Department of Health and Social Care

Design and Build of an Aerosol Generator

Research was conducted to investigate different Laskin Nozzle aerosol generator designs. Based on previous designs an aerosol generator for use in the Union College wind tunnel lab was designed and built. A Laskin Nozzle is a device that uses compressed air to turn liquid into aerosol form. The aerosol is used as seeding particles for the particle image velocimetry (PIV) system utilized to measure flow in the Union College wind tunnel. The aerosol generator was designed with four nozzles to allow for a large amount of aerosol to be produced. Each nozzle contains four crosswise one-millimeter holes where the air exits into the liquid. This air flow causes a shearing process that creates aerosol particles whose diameter size distribution is centered on 1-2 microns. The concentration of the aerosol produced is dependent on the air pressure the nozzles are run at, with higher pressures corresponding to higher concentration of particles. Each nozzle can also be turned on and off, controlling the amount of aerosol generated. Ongoing preliminary testing investigates whether the generator provides sufficient amount of seeding at the desired particle size for PIV measurements

MP753: The Role of Interfering Plants in Regenerating Hardwood Stands of Northeastern North America

An annotated bibliography for American beech (Fagus grandifolia Ehrh.), striped maple (Acer pensylvanicum L.), hobblebush (Viburnum alnifolium Marsh.), hayscented fern (Dennstaedtia punctilobula L.), New York fern (Thelypteris noveborecensis L.), bracken fern (Pteridium aquilinum (L.) Kuhn), raspberries (Rubus spp.), and pin cherry (Prunus pensylvanica L.f.). While accessible literature includes many references to these species, the information remains scattered. No one has previously consolidated the separate reports for easy reference, nor summarized the findings relative to interference with tree regeneration. This annotated bibliography serves that purpose.https://digitalcommons.library.umaine.edu/aes_miscpubs/1023/thumbnail.jp

Real-time deformability cytometry reveals sequential contraction and expansion during neutrophil priming.

It has become increasingly apparent that the biomechanical properties of neutrophils impact on their trafficking through the circulation and in particularly through the pulmonary capillary bed. The retention of polarized or shape-changed neutrophils in the lungs was recently proposed to contribute to acute respiratory distress syndrome pathogenesis. Accordingly, this study tested the hypothesis that neutrophil priming is coupled to morpho-rheological (MORE) changes capable of altering cell function. We employ real-time deformability cytometry (RT-DC), a recently developed, rapid, and sensitive way to assess the distribution of size, shape, and deformability of thousands of cells within seconds. During RT-DC analysis, neutrophils can be easily identified within anticoagulated "whole blood" due to their unique granularity and size, thus avoiding the need for further isolation techniques, which affect biomechanical cell properties. Hence, RT-DC is uniquely suited to describe the kinetics of MORE cell changes. We reveal that, following activation or priming, neutrophils undergo a short period of cell shrinking and stiffening, followed by a phase of cell expansion and softening. In some contexts, neutrophils ultimately recover their un-primed mechanical phenotype. The mechanism(s) underlying changes in human neutrophil size are shown to be Na+ /H+ antiport-dependent and are predicted to have profound implications for neutrophil movement through the vascular system in health and disease

Macrophage metabolic reprogramming presents a therapeutic target in lupus nephritis.

IgG antibodies cause inflammation and organ damage in autoimmune diseases such as systemic lupus erythematosus (SLE). We investigated the metabolic profile of macrophages isolated from inflamed tissues in immune complex (IC)-associated diseases, including SLE and rheumatoid arthritis, and following IgG Fcγ receptor cross-linking. We found that human and mouse macrophages undergo a switch to glycolysis in response to IgG IC stimulation, mirroring macrophage metabolic changes in inflamed tissue in vivo. This metabolic reprogramming was required to generate a number of proinflammatory mediators, including IL-1β, and was dependent on mTOR and hypoxia-inducible factor (HIF)1α. Inhibition of glycolysis, or genetic depletion of HIF1α, attenuated IgG IC-induced activation of macrophages in vitro, including primary human kidney macrophages. In vivo, glycolysis inhibition led to a reduction in kidney macrophage IL-1β and reduced neutrophil recruitment in a murine model of antibody-mediated nephritis. Together, our data reveal the molecular mechanisms underpinning FcγR-mediated metabolic reprogramming in macrophages and suggest a therapeutic strategy for autoantibody-induced inflammation, including lupus nephritis

Proteomic, biomechanical and functional analyses define neutrophil heterogeneity in systemic lupus erythematosus

Funder: NHLI FoundationFunder: NIHR Imperial Biomedical Research Centre; FundRef: http://dx.doi.org/10.13039/501100013342Funder: National Heart Lung and Blood InstituteFunder: Medical Research Council; FundRef: http://dx.doi.org/10.13039/501100000265Funder: National Institute of Biomedical Imaging and Bioengineering; FundRef: http://dx.doi.org/10.13039/100000070Funder: Gates Cambridge ScholarshipFunder: NIH/OXCAM FellowshipObjectives: Low-density granulocytes (LDGs) are a distinct subset of proinflammatory and vasculopathic neutrophils expanded in systemic lupus erythematosus (SLE). Neutrophil trafficking and immune function are intimately linked to cellular biophysical properties. This study used proteomic, biomechanical and functional analyses to further define neutrophil heterogeneity in the context of SLE. Methods: Proteomic/phosphoproteomic analyses were performed in healthy control (HC) normal density neutrophils (NDNs), SLE NDNs and autologous SLE LDGs. The biophysical properties of these neutrophil subsets were analysed by real-time deformability cytometry and lattice light-sheet microscopy. A two-dimensional endothelial flow system and a three-dimensional microfluidic microvasculature mimetic (MMM) were used to decouple the contributions of cell surface mediators and biophysical properties to neutrophil trafficking, respectively. Results: Proteomic and phosphoproteomic differences were detected between HC and SLE neutrophils and between SLE NDNs and LDGs. Increased abundance of type 1 interferon-regulated proteins and differential phosphorylation of proteins associated with cytoskeletal organisation were identified in SLE LDGs relative to SLE NDNs. The cell surface of SLE LDGs was rougher than in SLE and HC NDNs, suggesting membrane perturbances. While SLE LDGs did not display increased binding to endothelial cells in the two-dimensional assay, they were increasingly retained/trapped in the narrow channels of the lung MMM. Conclusions: Modulation of the neutrophil proteome and distinct changes in biophysical properties are observed alongside differences in neutrophil trafficking. SLE LDGs may be increasingly retained in microvasculature networks, which has important pathogenic implications in the context of lupus organ damage and small vessel vasculopathy

Graphene Oxide Membranes: Controlled Laser Reduction for Sensing Applications

Reduced graphene oxide (rGO) has attracted attention as an active electrode material for flexible electrochemical devices due to its high electric conductivity and large surface area. Compared to other reduction processes, laser reduction is a precise, low-cost, and chemical-free process that is directly applied to graphene oxide (GO) membranes. This study aims to develop rGO through laser irradiation for application as electrodes in thin flexible electrochemical sensors. Laser irradiation parameters will be optimized to achieve reduction of a low oxygen to carbon (O/C) ratio and surface impedance. The influence of humidity on the impedance of rGO electrodes will be studied. The observed instability of the rGO electrode is related to incomplete reduction and oxygenated defects involved in reduction. Partially removed oxygenated functional groups not only influence the impedance of the electrode but make it sensitive to the humidity of the working environment. The result provides references for GO’s laser reduction optimization, demonstrates the potential of applying rGO as an electrode in sensing applications, but also reveals the limitation of applying the laser reduced rGO electrode in a non-constant humidity environment

Idiopathic Acroosteolysis: A Novel Cutaneous Sign Can Help Identify the Condition Early

Acroosteolysis (AO) is a rare condition characterized by resorption of the distal phalanges of the fingers and/or toes. It can be familial, idiopathic (IAO), occupational, or secondary. Other authors suggest a classification into primary (genetic disorders, lysosomal storage disorders) or secondary AO. Various skin and nail changes have been reported in this condition. However, the cutaneous change on the affected digit(s)/toe(s) during the natural course of AO has been poorly documented. A 5-year-old girl presented with a 3-month history of a distinct transverse boundary between normal skin proximally and affected crusted skin overlying osteolysis distally (“split” sign) on the plantar surface of the third toe. This boundary gradually elongated circumferentially to involve the dorsal surface. The mother gave a similar history of a delimitation line on the 2nd, 4th, and 5th toes of the right foot with durations of 3 months, 1 year, and 2 years, respectively, that disappeared before she noticed a shortening of those toes. X-rays revealed partial resorption of the terminal phalanx of the third toe and several lytic changes in the middle and terminal phalanx of the second, fourth, and fifth toes. The clinical features, radiology findings, and a workup that helped rule out conditions associated with AO (secondary AO) helped establish the diagnosis of IAO in our patient. This case study highlights that the natural course of IAO includes distinct skin findings, such as the “split” sign that we describe. This sign can help identify the condition early

Facilitation or competition? Effects of lions on brown hyaenas and leopards

Intra-guild interactions related to facilitation and competition can be strong forces structuring ecological communities and have been suggested as particularly prominent for large carnivores. The African lion (Panthera leo) is generally thought to be a dominant predator where it occurs and can be expected to have broad effects on sympatric carnivore communities. We used data from two small game reserves in northern South Africa to relate the presence of African lions to abundance, habitat use, diet, and prey selection of two sympatric large carnivores, brown hyaenas (Parahyaena brunnea) and leopards (Panthera pardus). Our results offered some support for the facilitative effects of lions on brown hyaenas, and competitive effects on leopards. However, differences between populations living without and with lions were restricted to broad diet composition and appear not to have permeated into differences in either prey selection, abundance or habitat use. Therefore, we suggest that the potential effects of lions on the predator–prey interactions of sympatric predators may have been context dependent or absent, and subsequently argue that lions may not necessarily influence the predator–prey dynamics in the landscapes they live in beyond those caused by their own predatory behaviour.This research was funded by the National Geographic/Wait’s Foundation (grant number W32-08 to FD), the National Research Foundation in South Africa (grant numbers SFP2008072900003 to FD, IFR2011032400087 and UID 115040 to LS), and the Ministry of Economy and Competitiveness in Spain (grant number RYC2013-14662 to FD).Peer reviewe