Understanding the Effect of BVDV on Innate Immune Response of Neutrophils

Bovine viral diarrhea virus (BVDV) is one of the highly prevalent and economically important diseases of cattle industry worldwide. The two major consequences of this disease are persistent infection and immunosuppression. Several studies have been done to determine the underline mechanisms of BVDV-induced immunosuppression targeting antigen presenting cells, adaptive immune system cells and cytokine gene expression. However, very little research has been done to determine the effect of BVDV on neutrophils. Neutrophils are one of the most abundant while blood cells (WBC) in the peripheral blood, which play a critical role in the innate as well as adaptive immune response. The current study measured the effect of BVDV infection on viability of neutrophils, their surface marker expression and functional abilities including migration/chemoattraction, phagocytosis, reactive oxygen species production (oxidative burst) and neutrophil extracellular trap (NET) formation. These studies revealed that none of BVDV strains affected the viability of neutrophils in vitro. BVDV infection did affect surface marker expression. TGAC and TGAN reduced the expression of CD18 and Lselectin while increasing CD14 expression. All ncp BVDV strains used enhanced neutrophil migration while the cp BVDV strain reduced neutrophil migration as compared to mock-infected control treatment. Among the BVDV strains used in current study, highly virulent 1373 significantly enhanced neutrophil migration. The enhancement in neutrophil migration by 1373 was approximately 55% higher as compared to LPS-treated positive control macrophages. BVDV infection significantly enhanced neutrophil phagocytosis activity for 0.2 μm microsphere beads as compared to mock infection. Neutrophil phagocytic activity for rhodamine-labeled E. coli was reduced by BVDV infection as compared to LPS-control. TGAC, TGAN, 1373 or 28508 had 23%, 6%, 19% or 12% less phagocytic activity respectively as compared to mock-infected rhodamine-labeled E. coli-treated neutrophils. All BVDV strains used in the current study also reduced oxidative burst by approximately 50% as compared to positive control (

Cell death processes in immune cells of the shore crab, Carcinus maenas.

This thesis aimed to investigate programmed cell death processes and their role in invertebrate host defence, using the shore crab, Carcinus maenas as the main experimental model. Effort was focussed on two: namely apoptosis and ETosis; the latter being the controlled release of chromatin from the nucleus. Comparison of methods showed that for apoptosis, flow cytometry with bovine lactadherin-FITC and propidium iodide staining was the most effective for quantification in vitro for both mixed and separated haemocytes. By this method, different patterns of cell death were observed in different haemocyte populations and under different life conditions. Additionally, ETosis was demonstrated in two haemocyte types, specifically the hyaline cells and semi-granular cells. The process was found not only to entrap bacteria but also to aid defence by providing the scaffold upon which intact haemocytes assemble during encapsulation; a major cellular process that sequesters non-self particles from the circulation. Importantly, defence cells from the mussel, Mytilus edulis, and the sea anemone, Actinia equina, were also found to exhibit ETotic-like behaviour. As sea anemones lack a dedicated coelomic system, ETosis must predate the evolution of the coelom, showing that it has an evolutionary ancient origin

MerTK expressing hepatic macrophages promote the resolution of inflammation in acute liver failure

Objective: Acute liver failure (ALF) is characterised by overwhelming hepatocyte death and liver inflammation with massive infiltration of myeloid cells in necrotic areas. The mechanisms underlying resolution of acute hepatic inflammation are largely unknown. Here, we aimed to investigate the impact of Mer tyrosine kinase (MerTK) during ALF and also examine how the microenvironmental mediator, secretory leucocyte protease inhibitor (SLPI), governs this response. Design: Flow cytometry, immunohistochemistry, confocal imaging and gene expression analyses determined the phenotype, functional/transcriptomic profile and tissue topography of MerTK+ monocytes/macrophages in ALF, healthy and disease controls. The temporal evolution of macrophage MerTK expression and its impact on resolution was examined in APAP-induced acute liver injury using wild-type (WT) and Mer-deficient (Mer−/−) mice. SLPI effects on hepatic myeloid cells were determined in vitro and in vivo using APAP-treated WT mice. Results: We demonstrate a significant expansion of resolution-like MerTK+HLA-DRhigh cells in circulatory and tissue compartments of patients with ALF. Compared with WT mice which show an increase of MerTK+MHCIIhigh macrophages during the resolution phase in ALF, APAP-treated Mer−/− mice exhibit persistent liver injury and inflammation, characterised by a decreased proportion of resident Kupffer cells and increased number of neutrophils. Both in vitro and in APAP-treated mice, SLPI reprogrammes myeloid cells towards resolution responses through induction of a MerTK+HLA-DRhigh phenotype which promotes neutrophil apoptosis and their subsequent clearance. Conclusions: We identify a hepatoprotective, MerTK+, macrophage phenotype that evolves during the resolution phase following ALF and represents a novel immunotherapeutic target to promote resolution responses following acute liver injury

Modulation of Neutrophil Function by a Secreted Mucinase of Escherichia coli O157∶H7

Escherichia coli O157∶H7 is a human enteric pathogen that causes hemorrhagic colitis which can progress to hemolytic uremic syndrome, a severe kidney disease with immune involvement. During infection, E. coli O157∶H7 secretes StcE, a metalloprotease that promotes the formation of attaching and effacing lesions and inhibits the complement cascade via cleavage of mucin-type glycoproteins. We found that StcE cleaved the mucin-like, immune cell-restricted glycoproteins CD43 and CD45 on the neutrophil surface and altered neutrophil function. Treatment of human neutrophils with StcE led to increased respiratory burst production and increased cell adhesion. StcE-treated neutrophils exhibited an elongated morphology with defective rear detachment and impaired migration, suggesting that removal of the anti-adhesive capability of CD43 by StcE impairs rear release. Use of zebrafish embryos to model neutrophil migration revealed that StcE induced neutrophil retention in the fin after tissue wounding, suggesting that StcE modulates neutrophil-mediated inflammation in vivo. Neutrophils are crucial innate effectors of the antibacterial immune response and can contribute to severe complications caused by infection with E. coli O157∶H7. Our data suggest that the StcE mucinase can play an immunomodulatory role by directly altering neutrophil function during infection. StcE may contribute to inflammation and tissue destruction by mediating inappropriate neutrophil adhesion and activation

The role of neutrophil extracellular traps in hepatic immune homeostasis

The liver is a frontline immunological barrier tissue, an active detoxifying filter and the most important metabolic organ in human beings. The liver immune compartment is capable of detecting, capturing and clearing pathogens, pathogen-associated molecular patterns (PAMPs), gut-incoming antigens and diet-derived metabolites. Consequently, the hepatic immune compartment needs to ensure a delicate balance between tolerogenic and inflammatory responses, against non- and pathogenic insults, respectively. NETosis, a neutrophil-specific cell death program, is characterized by the release of web-like structures referred to as neutrophil extracellular traps (NETs). NETs are composed of extracellular DNA strands associated with modified histones and neutrophil granular proteins. NETs exert a key antimicrobial function that allows neutrophils to capture and kill pathogens. Here, we found that NET-like structures are present in murine immunological barrier tissues (e.g. spleen, lung, lymph nodes, liver) in the absence of pathology, likely supporting an alternative role of neutrophils and NETs during tissue homeostasis. In particular, NET-like structures emerged significantly during the night-time in the hepatic tissue. Absence of NET-like structures in different neutrophil-deficient knock-out mice, and their inhibition upon DNAse I or BB-Cl-Amidine treatment, confirmed the neutrophil-origin of the identified NETs and that their presence during tissue homeostasis helps maintaining a systemic anti-inflammatory condition. Homeostasis refers to the highly dynamic processes occurring within an organism internal environment in order to ensure a constant threshold of physiological parameters. Interestingly, mice are nocturnal creatures that have a night-associated active period, when they eat substantially more food compared to the diurnal phase and are exposed to an increased amount of potential insults. This brought up the idea of NETs being not just an inner circadian-regulated immunological function, but rather that food intake could work as an environmental entrainment cue for NET release. The NETosis dynamic profile in the liver was modified upon different dietary patterns, the diet´s nutritional composition and the chemical structure of the main fatty acid content of dietary fats. A diet high in fat and sugar induced the highest NETosis ratios, compared to diets with a low-fat content or fasted conditions. Fat-rich diets induce gut-permeability, intestinal dysbiosis and metabolic endotoxemia. NETs emerged as a key component of the hepatic immunological barrier system as sentinels against gut-derived PAMPs and DAMPs (reflected by a higher TLR2- and TLR4-night-associated activity), and as peacemakers within the low-profile pro-inflammatory scenario associated to postprandial conditions. Conditions gathering a higher and local hepatic presence of NETs at night were overall associated with a lower pro-inflammatory profile in the liver. Levels of IL-5 and IFN-γ (pro-fibrotic cytokines) were particularly augmented in the liver in absence of NETs. Altogether, our study spotlights NETs as key players in the maintenance of the hepatic tolerogenic and anti-inflammatory immune status and, ultimately, in the normal liver physiology

Diversity and function of anti-modified protein autoantibodies in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease particularly affecting synovial joints. Anti-citrullinated protein autoantibodies (ACPA) are detected in the serum of about 2/3 of RA patients and are being used to classify the disease. These autoantibodies may occur years before any signs of arthritis which implies that they are a cause rather than a consequence of disease. By using modified autoantigens to detect ACPAs in large patient cohorts and by purifying polyclonal autoantibodies from patients, the hunt for disease promoting autoantigens has been going on for decades. However, the relationship between ACPA specificity and any functional effects remains unclear. In order to understand the evolution, specificity, and function of autoreactive B cells in RA, the focus of this thesis is the generation of monoclonal antibodies (mAbs) from paired variable heavy- and light-chain immunoglobulin (Ig) sequences from identified single B cells from various RA tissue compartments. We have identified citrulline-reactive autoantibody producing plasma cells in the synovium of RA patients with established disease. By generating single plasma cell derived mAbs, we learned that such autoantibodies may be directly involved in the pathogenesis of RA by promoting bone degrading osteoclasts. The mAbs were all multireactive to citrulline-peptides and citrullinated proteins, but with unique distinct binding patterns. We recognized glycine in +1 position to the citrulline and a fraction of the citrulline-reactive mAbs cross-reacted with carbamyl-peptides. In addition, the identified plasma cells displayed features of high somatic mutations and fragment antigen binding (Fab) variable N-glycosylation sites introduced by affinity maturation. By analyzing a selection of RA patient B cell derived mAbs for reactivity against apoptotic cells and activated neutrophils, we learned that a subset of the citrulline reactive mAbs bound nuclear antigens. Interestingly, a fraction of these mAbs could target nuclear histones independently of the citrullinating enzyme PAD by binding to acetylated histones. We explored the extent of the ACPA mAb multireactivity and cross-reactivity by acknowledging the importance of neighboring amino acids in addition to glycine in +1 to the citrulline. By analyzing the bone marrow plasma cell repertoire of RA patients, we observed differences in Ig-frequencies and variable Fab N-linked glycosylation sites between ACPA+ and ACPA- patients. We also found RA patient bone marrow plasma cell clonotypes. In addition, we identified citrulline-reactive bone marrow plasma cells that could bind activated neutrophils which strengthen previous reports of citrullinated histones as ACPA targets. Lastly, we identified autoantibodies against the oxidation-induced post translational protein modification adducts malondialdehyde (MDA) and malondialdehyde-acetaldehyde (MAA) in the bone marrow and lung of RA patients and individuals that also harbor ACPAs. A majority of these autoantibodies, that can promote osteoclastogenesis, need cross-linked MAA-protein for recognition independent of protein backbone. Taken together, the generation of RA patient single B cell derived mAbs, have revealed remarkable features of the autoreactivity that increases the understanding of B cell involvement in the pathogenesis of RA

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

Investigations into the contributions of mitochondrial dynamics and function to platelet ageing and reactivity

PhD ThesisPlatelets are essential for the physiological process of haemostasis, but also drive pathological thrombosis. Platelet lifespan is a tightly controlled process through which platelets exist for approximately 10 days within the circulation of healthy individuals. However, in a number of disease states this process is dysregulated leading to an accelerated platelet turnover. Indeed, there are a number of reports suggesting that newly formed platelets are hyper-reactive and their presence has been associated with a higher risk of thrombosis. Whilst there are these indications of hyper-reactivity in young platelets, there are few systematic studies. Here I have used proteomics coupled with functional studies and immunofluorescence to show that there is a progressive decline in mitochondrial and cytoskeletal proteins as platelets age and an increase in apoptotic pathways. Given the apparent importance of mitochondria in supporting the predetermined platelet lifespan, it raised the question as to whether mitochondria are important for other platelet functional processes. Therefore, I sought to elucidate the impact of platelet activation on mitochondrial function and dynamics. Physiological stimulation causes an increase in mitochondrial respiration, consistent with an increase in energy demand. Interestingly, P2Y12 receptor inhibition causes a reduction in basal oxygen consumption, suggesting a dysregulation in mitochondrial function. Furthermore, this work highlights a role for mitochondria beyond energy production, with indications that stimulation causes platelets to package and release their mitochondria into microvesicles. Interestingly, these mitochondria-containing microvesicles have high P-selectin expression suggesting they may be more likely to interact with neutrophils than the rest of the microvesicle population. Indeed, incubation of neutrophils with mitochondria-positive microvesicles but not mitochondria-negative microvesicles causes alterations in the expression of surface markers; CD11b, CD66b and CXCR2, indicative of neutrophil activation potentially as a result of phagocytosis. This work highlights an important role of mitochondria in both platelet ageing and activation