Characterisation of Fractalkine/CX3CL1 and Fractalkine Receptor (CX3CR1) Expression in Abdominal Aortic Aneurysm Disease

AbstractObjectivesFractalkine (CX3CL1) promotes adhesion and extravasation of leucocytes through interactions with fractalkine receptor (CX3CR1) expressed on CD56+/CD16+ NK cells and CD8+ T cells. The current study aims to test the hypothesis the CX3CL1–CX3CR1 interaction contributes to the inflammatory infiltrate in AAA tissue.Design and methodsImmunohistochemistry (IHC) was used to define expression of CX3CR1 in AAA tissue. Multi-parametric flow cytometry (FC) was used to determine CX3CR1 expression on T-cells (CD3+) and NK cells (CD56+) from AAA tissue and peripheral blood of AAA patients and healthy controls. Regulation of CX3CL1 expression by vascular endothelial (vEC) and smooth muscle cells (vSMC) was examined in vitro using primary cell cultures.ResultsCX3CR1+ cells were detected in 19/28 AAA tissue samples and predominately localised in the adventitia. PBMCs from patients with AAA demonstrated higher percentages of CX3CR1+ NK cells (60.0–88.6%) and T cells (7.5–39.4%) compared with healthy controls. Furthermore, the frequency of CX3CR1+ NK cells (91%) and T cells (94%) in inflammatory AAA tissue were higher than in atherosclerotic AAA tissue. The pro-inflammatory cytokine TNFα increased expression of fractalkine by vSMC and vEC.ConclusionCX3CL1+ and CX3CR1+ cells are present in AAA disease and their interaction may contribute to the recruitment of inflammatory cells seen in AAA tissue

Engineering of the gut commensal bacterium Bacteroides ovatus to produce and secrete biologically active murine interleukin-2 in response to xylan

Aims: The aim of this work was to engineer a gut commensal bacterium, Bacteroides ovatus, to produce and secrete a biologically active cytokine in a regulated manner as a basis for novel immunotherapies for chronic gut disorders. Methods and Results: Bacteroides ovatus was engineered to produce murine interleukin-2 (MuIL2) intracellularly in response to xylan in culture media by inserting the MuIL2 gene into the xylanase operon of the organism. A second strain was engineered to secrete MuIL2 by adding Bacteroides fragilis enterotoxin secretion signal sequence to the protein. The recombinant strains produced MuIL2 only in the presence of xylan as determined by ELISA of cell lysates and culture supernatants. The IL2-dependent cell line CTLL-2 was used to demonstrate that MuIL2 produced by both B. ovatus strains was biologically active. This activity could be blocked by an anti-IL2 neutralizing antibody. The xylan-inducible nature of this system was demonstrated by RT-PCR. Conclusions: Bacteroides ovatus was successfully engineered to produce and secrete biologically active MuIL2 in a xylan-inducible manner. Significance and Impact of the Study: The production and secretion of a biologically active mammalian protein by a member of the gut microflora could lead to the development of new long-term immunotherapies for inflammatory gut diseases. © 2005 The Society for Applied Microbiology

Modelling the influence of vitamin D and probiotic supplementation on the microbiome and immune response

The intestinal microbiota play a critical role in human health and disease, maintaining metabolic and immune/inflammatory health, synthesising essential vitamins and amino acids and maintaining intestinal barrier integrity. The aim of this paper is to develop a mathematical model to describe the complex interactions between the microbiota, vitamin D/vitamin D receptor (VDR) pathway, epithelial barrier and immune response in order to understand better the effects of supplementation with probiotics and vitamin D. This is motivated by emerging data indicating the beneficial effects of vitamin D and probiotics individually and when combined. We propose a system of ordinary differential equations determining the time evolution of intestinal bacterial populations, concentration of the VDR:1,25(OH)2D complexin epithelial and immune cells, the epithelial barrier and the immune response. The model shows that administration of probiotics and/or vitamin D upregulates the VDR complex, which enhances barrier function and protects against intestinal inflammation. The model also suggests co-supplementation to be superior to individual supplements. We explore the effects of inflammation on the populations of commensal and pathogenic bacteria and the vitamin D/VDR pathway and discuss the value of gathering additional experimental data motivated by the modelling insights

A hierarchical Bayesian framework for understanding the spatiotemporal dynamics of the intestinal epithelium

AbstractOur work addresses two key challenges, one biological and one methodological. First, we aim to understand how proliferation and cellular migration rates in the intestinal epithelium are related under healthy, damaged (Ara-C treated) and recovering conditions, and how these relations can be used to identify mechanisms of repair and regeneration. We analyse new data, presented in more detail in a companion paper, in which BrdU/IdU cell-labelling experiments were performed under these respective conditions. Second, in considering how to more rigorously process these data and interpret them using mathematical models, we develop a probabilistic, hierarchical framework. This framework provides a best-practice approach for systematically modelling and understanding the uncertainties that can otherwise undermine drawing reliable conclusions - uncertainties in experimental measurement and treatment, difficult-to-compare mathematical models of underlying mechanisms, and unknown or unobserved parameters. Both discrete and continuous mechanistic models are considered and related via hierarchical conditional probability assumptions. This allows the incorporation of features of both continuum tissue models and discrete cellular models. We perform model checks on both in-sample and out-of-sample datasets and use these checks to illustrate how to test possible model improvements and assess the robustness of our conclusions. This allows us to consider - and ultimately decide against - the need to retain finite-cell-size effects to explain a small misfit appearing in one set of long-time, out-of-sample predictions. Our approach leads us to conclude, for the present set of experiments, that a primarily proliferation-driven model is adequate for predictions over most time-scales. We describe each stage of our framework in detail, and hope that the present work may also serve as a guide for other applications of the hierarchical approach to problems in computational and systems biology more generally.Author SummaryThe intestinal epithelium serves as an important model system for studying the dynamics and regulation of multicellular populations. It is characterised by rapid rates of self-renewal and repair; failure of the regulation of these processes is thought to explain, in part, why many tumours occur in the intestinal and similar epithelial tissues. These features have led to a large amount of work on estimating rate parameters in the intestine. There still remain, however, large gaps between the raw data collected, the experimental interpretation of these data, and speculative mechanistic models for underlying processes. In our view hierarchical statistical modelling provides an ideal, but currently underutilised, method to begin to bridge these gaps. This approach makes essential use of the distinction between ‘measurement’, ‘process’ and ‘parameter’ models, giving an explicit framework for combining experimental data and mechanistic modelling in the presence of multiple sources of uncertainty. As we illustrate, the hierarchical approach also provides a suitable framework for addressing other methodological questions of broader interest in systems biology: how to systematically relate discrete and continuous mechanistic models; how to formally interpret and visualise statistical evidence; and how to express causal assumptions in terms of conditional independence.</jats:sec

Toll-like receptor-mediated responses of primary intestinal epithelial cells during the development of colitis

noThe interleukin-2-deficient (IL-2¿/¿) mouse model of ulcerative colitis was used to test the hypothesis that colonic epithelial cells (CEC) directly respond to bacterial antigens and that alterations in Toll-like receptor (TLR)-mediated signaling may occur during the development of colitis. TLR expression and activation of TLR-mediated signaling pathways in primary CEC of healthy animals was compared with CEC in IL-2¿/¿ mice during the development of colitis. In healthy animals, CEC expressed functional TLR, and in response to the TLR4 ligand LPS, proliferated and secreted the cytokines IL-6 and monocyte chemoattractant protein-1 (MCP-1). However, the TLR-responsiveness of CEC in IL-2¿/¿ mice was different with decreased TLR4 responsiveness and augmented TLR2 responses that result in IL-6 and MCP-1 secretion. TLR signaling in CEC did not involve NF-B (p65) activation with the inhibitory p50 form of NF-B predominating in CEC in both the healthy and inflamed colon. Development of colitis was, however, associated with the activation of MAPK family members and upregulation of MyD88-independent signaling pathways characterized by increased caspase-1 activity and IL-18 production. These findings identify changes in TLR expression and signaling during the development of colitis that may contribute to changes in the host response to bacterial antigens seen in colitis