A Bayesian view of murine seminal cytokine networks

It has long been established that active agents in seminal fluid are key to initiating and coordinating mating-induced immunomodulation. This is in part governed by the actions of a network of cytokine interactions which, to date, remain largely undefined, and whose interspecific evolutionary conservation is unknown. This study applied Bayesian methods to illustrate the interrelationships between seminal profiles of interleukin (IL)-1alpha, IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 (p70), IL-13, IL-17, eotaxin, granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), interferon (IFN)-gamma, keratinocyte-derived chemokine (KC), monocyte chemoattractant protein (MCP-1), macrophage inflammatory protein (MIP-1) alpha, MIP-1beta, regulated on activation normal T cell expressed and secreted (RANTES), tumour necrosis factor (TNF)-alpha, leptin, inducible protein (IP)-10 and vascular endothelial growth factor (VEGF) in a rat model. IL-2, IL-9, IL-12 (p70), IL-13, IL-18, eotaxin, IFN-gamma, IP-10, KC, leptin, MCP-1, MIP-1alpha and TNF-alpha were significantly higher in serum, whilst IL-1beta, IL-5, IL-6, IL-10, IL-17, G-CSF and GM-CSF were significantly higher in seminal fluid. When compared to mouse profiles, only G-CSF was present at significantly higher levels in the seminal fluid in both species. Bayesian modelling highlighted key shared features across mouse and rat networks, namely TNF-alpha as the terminal node in both serum and seminal plasma, and MCP-1 as a central coordinator of seminal cytokine networks through the intermediary of KC and RANTES. These findings reveal a marked interspecific conservation of seminal cytokine networks

Modelling physiological reproductive inflammatory networks in vivo.

The immune and reproductive systems have long been known to be inextricably linked, with components of immune pathways, particularly cytokines, mediating processes such as ovarian/menstrual cyclicity, endometrial remodelling, mating-induced immunomodulation, implantation, pregnancy, parturition and lactation. The nature of this involvement has often been investigated at the level of single mediators, with little consideration of the fact that cytokines are increasingly understood to function as complex networks. This study aimed to characterise inflammatory networks using both traditional and novel machine-learning Bayesian network-based methods in the context of keystone aspects of reproduction, viz., in the endometrial response to seminal plasma, cytokine:hormone interactions during lactation, and oocyte maturation following controlled ovarian hyperstimulation. ‘Traditional’ pathway analyses used to examine the murine endometrial response to seminal plasma revealed previously unidentified mediators and showed compartmentalised epithelium/stroma-specific responses. However, they proved ineffective in describing novel cytokine interactions. This led to the development a highly effective novel Bayesian network-based approach to explore cytokine:hormone networks during murine lactation. This revealed that prolactin, a putative potent immunomodulator, was far less influential than expected in vivo. The method also identified previously unknown cytokine interactions and described features such as synergy and antagonism. Further refinement of these network analyses as modified variational Bayesian state space models enabled the display of core, conserved subnetworks (communities) of human follicular fluid cytokines whose interactions varied with oocyte maturity. Moreover, these cytokine signatures also allowed the prediction of an oocytes’ fertilisability potential, with potential attendant benefits to assisted conception. This thesis represents the first endeavour to model inflammatory networks in vivo in any setting to date. It has revealed their central role, functional conservation and key features of cytokine interactions across a spectrum of reproductive processes. Further development of this methodology appears set to offer invaluable new insights into the complex immune signalling that underpins reproductive biology

Discovery and application of immune biomarkers for hematological malignancies

Introduction: Haematological malignancies originate and progress in primary and secondary lymphoid organs, where they establish a uniquely immune-suppressive tumour microenvironment. Although high-throughput transcriptomic and proteomic approaches are being employed to interrogate immune surveillance and escape mechanisms in patients with solid tumours, and to identify actionable targets for immunotherapy, our knowledge of the immunological landscape of haematological malignancies, as well as our understanding of the molecular circuits that underpin the establishment of immune tolerance, is not comprehensive. Areas covered: This article will discuss how multiplexed immunohistochemistry, flow cytometry/mass cytometry, proteomic and genomic techniques can be used to dynamically capture the complexity of tumour-immune interactions. Moreover, the analysis of multi-dimensional, clinically annotated data sets obtained from public repositories such as Array Express, TCGA and GEO is crucial to identify immune biomarkers, to inform the rational design of immune therapies and to predict clinical benefit in individual patients. We will also highlight how artificial neural network models and alternative methodologies integrating other algorithms can support the identification of key molecular drivers of immune dysfunction. Expert comment: High-dimensional technologies have the potential to enhance our understanding of immune-cancer interactions and will support clinical decision making and the prediction of therapeutic benefit from immune-based interventions

In vitro expanded human CD4+CD25+ regulatory T cells suppress effector T cell proliferation.

Regulatory T cells (Tregs) have been shown to be critical in the balance between autoimmunity and tolerance and have been implicated in several human autoimmune diseases. However, the small number of Tregs in peripheral blood limits their therapeutic potential. Therefore, we developed a protocol that would allow for the expansion of Tregs while retaining their suppressive activity. We isolated CD4+CD25 hi cells from human peripheral blood and expanded them in vitro in the presence of anti-CD3 and anti-CD28 magnetic Xcyte Dynabeads and high concentrations of exogenous Interleukin (IL)-2. Tregs were effectively expanded up to 200-fold while maintaining surface expression of CD25 and other markers of Tregs: CD62L, HLA-DR, CCR6, and FOXP3. The expanded Tregs suppressed proliferation and cytokine secretion of responder PBMCs in co-cultures stimulated with anti-CD3 or alloantigen. Treg expansion is a critical first step before consideration of Tregs as a therapeutic intervention in patients with autoimmune or graft-versus-host disease

Alternative activation of dendritic cells

The alternative activation of macrophage populations by Interleukin-4 (IL-4) is well characterised. Alternatively activated macrophages (AAM) express high levels of the arginine converting enzyme arginase-1, and express a plethora of IL-4 driven molecules including the resistin like molecule alpha (RELMα) and the chitinase like molecule Ym1/2. Dendritic cells (DCs) are the professional antigen presenting cells (APC) of the immune system, responsible for the detection of invading pathogens, secretion of cytokines and the subsequent activation of T-cells. This thesis addresses whether IL-4 is able to ‘alternatively activate’ DCs both in vitro and in vivo, in a manner similar to that of AAM. The impact of IL-4 on DC and macrophage activation was compared and contrasted, and it was confirmed for the first time that IL-4 can alternatively activate DCs, inducing high level expression of a range of alternative activation associated markers including RELMα, Ym1/2, CCL24 and dectin-1, with the exception of arginase. DCs were significantly more capable at the in vivo priming of T-cell responses in the context of both Th1 and Th2 polarising antigens than similarly exposed macrophages, confirming their superior capacity as APC. The requirements for DC IL-4Rα expression were assessed, and IL-4 responsiveness was found to be required for the optimal induction of Th1 responses. Conversely, selective loss of only one facet of the IL-4 response, namely RELMα expression, limited the ability of IL-4 exposed DCs to induce the regulatory cytokine IL-10 both in vitro and in vivo. Furthermore, alternatively activated DCs (AADCs) were found in the spleen following 8 weeks of infection with the parasitic trematode Schistosoma mansoni, highlighting a role for DC alternative activation in a disease setting. IL-4 was shown to induce expression of the vitamin A converting enzyme aldehyde dehydrogenase, and the product of such activity, retinoic acid (RA), was found to promote the expression of RELMα in IL-4 exposed DCs. Aldehyde dehydrogenase activity was found to inversely correlate with DC expression of Ym1/2 and inhibition of RA signalling limited IL-4 driven RELMα and promoted Ym1/2

Neuroinflammmation in alcohol dependence

Alcohol dependence damages the brain through a multiplicity of factors including thiamine deficiency, liver disease, head injuries, and repeated episodes of alcohol withdrawal. Alcohol withdrawal is a potential opportunity for reducing damage as it is an intensive time of contact between doctors and patients. Pre-clinical models of alcohol dependence have demonstrated activation of microglia, resident tissue macrophages, and expression of cytokines and other inflammatory mediators both in the brain and peripheral blood during alcohol withdrawal. These changes were associated with neuronal death, and learning deficits. Similar processes may occur in man as increased microglial numbers, increased chemokine expression and raised circulating pro-inflammatory cytokines have been reported in alcohol dependence. The aim of the thesis was to characterise the peripheral cytokine profile during alcohol detoxification, to investigate whether there are relationships between peripheral cytokines and clinical features of alcohol withdrawal, to investigate neuroinflammation in alcohol dependence by using [11C]PBR28 Positron Emission Tomography to assess microglial activation in recently abstinent alcohol dependent patients in vivo and to investigate how these processes relate to elevated cortisol and elevated cerebral glutamate reported in alcohol withdrawal respectively. The longitudinal study undertaken in 51 alcohol dependent patients during detoxification demonstrated that both pro- and anti-inflammatory cytokines, and chemokines, decreased significantly during detoxification while T cell cytokines increase. IL-6 was positively associated with withdrawal severity and depressive symptoms during withdrawal. The chemokine CCL-2 was positively associated with performance on cognitive tasks. Serum cortisol was in the high normal range and decreased during detoxification. The salivary Cortisol Awakening Response (CAR) was also in the normal range at all time points. Both serum cortisol and the CAR were positively correlated with IL-6 concentrations suggesting hyperfunction of the HPA axis during alcohol detoxification may relate in part to inflammatory stimulation. The PET study comparing alcohol dependent men in early abstinence and healthy controls was undertaken using the ligand, [11C]PBR28 that binds to the Translocator Protein 18 kDa (TSPO) richly expressed in microglia. Alcohol dependent patients had lower TSPO binding in the hippocampi than healthy controls. TSPO binding in the hippocampus was also positively correlated with performance on tests of verbal memory. This suggests that hippocampal microglial loss or dysfunction may be related to memory problems in alcohol dependence. Given that benzodiazepines are used to treat alcohol withdrawal, an in vitro binding study was conducted to investigate whether benzodiazepines would significantly block [11C]PBR28 binding and found that benzodiazepines do not block a significant proportion of TSPO at all but the highest clinical doses. The relationship between brain glutamate, as measured with Magnetic Resonance Spectroscopy, and microglial activation was investigated. Alcohol dependent patients had significantly lower glutamate + glutamine (Glx) concentrations in the occipital cortex with no difference in glutamate concentrations in the anterior cingulate cortex (ACC). In summary, there are changes in both peripheral and brain inflammatory processes in early abstinence from alcohol dependence that are related to clinical symptoms. Peripheral pro-inflammatory cytokines are raised early in detoxification relative to late detoxification and are related to withdrawal and affective symptoms. Surprisingly, evidence of decreased microglial function in the hippocampus was found and this related to poorer cognitive function, suggesting a positive role for immune cells in the brain in alcohol dependence. Inflammatory processes were related to HPA axis function during detoxification but not to changes in brain glutamate concentrations. In conclusion, characterisation of inflammation through multiple approaches in this series of studies demonstrates the likely importance of such processes and provides novel approaches for treatment to reduce brain damage due to alcoholism.Open Acces

Exploring Genetic Susceptibility: Using a combined systems biology, in vitro and ex vivo approach to understand the pathology of ulcerative colitis

The overall aim of this PhD is to use a multidisciplinary approach to determine the function of Ulcerative Colitis (UC) associated SNPs, to help understand the role of SNPs in the pathogenesis of UC in general and in a patient specific context. UC is a chronic, relapsing inflammatory disease of the large bowel for which the aetiology is thought to be a trifecta of 1) dysregulation of the immune system in response to 2) an environmental trigger in a 3) genetically susceptible host. Genetic susceptibility or susceptibility loci for UC have been identified by Genome Wide Associations Scanning and subsequent fine mapping and deep sequencing. This work intended to further characterise these susceptibility loci at a global level and a patient specific level using both a systems biology approach and experimental validation of the in-silico work. Using publicly available datasets non exonic UC associated SNPs were functionally annotated to regulatory regions within the genome. Exonic SNPs were also analysed looking at impacts in protein linear motifs and splice enhancement motifs. Bioinformatics was used to identify interacting proteins and create a UC-interactome network. This suggested that UC was a disease of fine regulators as opposed to a disease of specific target proteins. Analysis of the UC-interactome identified the focal adhesion complex (FAC) that is involved in regulating wound healing as major component of the network. One member of the FAC, Leupaxin (LPXN), was identified as a potential target for validation. Using CRISPR-Cas9 technology, LPXN overexpressing cell lines and knock out cell lines were created. Wound healing assays and cytokine analysis identified that overexpression of LPXN impaired wound healing and reduced the secretion of MCP-1. In addition, using genotyped colonic biopsies from UC patients and control patients in a polarised in vitro organ culture (pIVOC) system we show that the LPXN risk allele may impact on cytokine production. Finally, UKIBD genetics consortium data was used to access a pilot dataset of 58 patients’ SNP profiles from Immunochip data who were patients at the Norfolk and Norwich University Hospital to create patient-specific UC-interactomes. Analysis of these footprints identified convergent interacting proteins affected by multiple SNPs and novel pathogenic pathways