The Spatial Distribution and Dynamics of CXCL13 in Lymphoid Tissues

Morphogens are soluble signalling molecules that regulate a broad spectrum of biological processes. However, the distances and scales over which this regulation occurs are unclear. To date, many studies have highlighted source-sink mechanisms for morphogen gradient formation but fail to take the role of the tissue microenvironment into account. Using a systems-based approach we show that the chemokine CXCL13 is regulated by the B-cell microenvironment on distinct but interconnected levels of biological organization. CXCL13 is a key determinant of humoral immune responses, regulating the localisation of lymphocytes within lymphoid tissues. Due to a complex and dynamic interaction network occurring over broad spatiotemporal scales, mapping the spatial distribution of CXCL13 in situ is challenging. To address this we have mapped the 3-dimensional organisation of CXCL13+ stromal cells in situ using a fluorescent reporter system, identifying three distinct but interconnected stromal subsets that are unique in their network properties. We quantify CXCL13 dynamics using high-speed narrowfield microscopy in collagen matrix and lymph node tissue sections with results suggesting that diffusion is highly constrained by local tissue microanatomy. However, this data alone is insufficient to describe CXCL13 gradient formation. To consolidate this data we employ a quantitative modelling approach hybridising different techniques into a high fidelity in silico representation of the B-follicle, where immune cells can interact with stroma capable of creating and shaping complex physiological gradients. Simulation analyses and immunohistochemistry suggest that chemokine fields within the follicle are dynamic and non-uniform, with multiobjective optimization analysis suggesting that this spatial configuration is designed to promote scanning rates. Taken in concert, our data suggests that CXCL13 acts over short distances creating a complex landscape of expression. Importantly, this study provides a basis for understanding the spatial distribution of morphogens with complex binding behaviours

Simulating CXCR5 Dynamics in Complex Tissue Microenvironments

To effectively navigate complex tissue microenvironments, immune cells sense molecular concentration gradients using G-protein coupled receptors. However, due to the complexity of receptor activity, and the multimodal nature of chemokine gradients in vivo, chemokine receptor activity in situ is poorly understood. To address this issue, we apply a modelling and simulation approach that permits analysis of the spatiotemporal dynamics of CXCR5 expression within an in silico B-follicle with single-cell resolution. Using this approach, we show that that in silico B-cell scanning is robust to changes in receptor numbers and changes in individual kinetic rates of receptor activity, but sensitive to global perturbations where multiple parameters are altered simultaneously. Through multi-objective optimization analysis we find that the rapid modulation of CXCR5 activity through receptor binding, desensitization and recycling is required for optimal antigen scanning rates. From these analyses we predict that chemokine receptor signaling dynamics regulate migration in complex tissue microenvironments to a greater extent than the total numbers of receptors on the cell surface

Using Emulation to Engineer and Understand Simulations of Biological Systems

Modeling and simulation techniques have demonstrated success in studying biological systems. As the drive to better capture biological complexity leads to more sophisticated simulators, it becomes challenging to perform statistical analyses that help translate predictions into increased understanding. These analyses may require repeated executions and extensive sampling of high-dimensional parameter spaces: analyses that may become intractable due to time and resource limitations. Significant reduction in these requirements can be obtained using surrogate models, or emulators, that can rapidly and accurately predict the output of an existing simulator. We apply emulation to evaluate and enrich understanding of a previously published agent-based simulator of lymphoid tissue organogenesis, showing an ensemble of machine learning techniques can reproduce results obtained using a suite of statistical analyses within seconds. This performance improvement permits incorporation of previously intractable analyses, including multi-objective optimization to obtain parameter sets that yield a desired response, and Approximate Bayesian Computation to assess parametric uncertainty. To facilitate exploitation of emulation in simulation-focused studies, we extend our open source statistical package, spartan, to provide a suite of tools for emulator development, validation, and application. Overcoming resource limitations permits enriched evaluation and refinement, easing translation of simulator insights into increased biological understanding

High-speed single-molecule tracking of CXCL13 in the B-Follicle

Soluble factors are an essential means of communication between cells and their environment. However, many molecules readily interact with extracellular matrix components, giving rise to multiple modes of diffusion. The molecular quantification of diffusion in situ is thus a challenging imaging frontier, requiring very high spatial and temporal resolution. Overcoming this methodological barrier is key to understanding the precise spatial patterning of the extracellular factors that regulate immune function. To address this, we have developed a high-speed light microscopy system capable of millisecond sampling in ex vivo tissue samples and sub-millisecond sampling in controlled in vitro samples to characterize molecular diffusion in a range of complex microenvironments. We demonstrate that this method outperforms competing tools for determining molecular mobility of fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) for evaluation of diffusion. We then apply this approach to study the chemokine CXCL13, a key determinant of lymphoid tissue architecture, and B-cell mediated immunity. Super-resolution single-molecule tracking of fluorescently labeled CCL19 and CXCL13 in collagen matrix was used to assess the heterogeneity of chemokine mobility behaviors, with results indicating an immobile fraction and a mobile fraction for both molecules, with distinct diffusion rates of 8.4 ± 0.2 µm2s-1 and 6.2 ± 0.3 µm2s-1 respectively. To better understand mobility behaviors in situ we analyzed CXCL13-AF647 diffusion in murine lymph node tissue sections and observed both an immobile fraction and a mobile fraction with a diffusion coefficient of 6.6 ± 0.4 µm2s 1, suggesting that mobility within the follicle is also multimodal. In quantitatively studying mobility behaviors at the molecular level, we have obtained an increased understanding of CXCL13 bioavailability within the follicle. Our high-speed single-molecule tracking approach affords a novel perspective from which to understand the mobility of soluble factors relevant to the immune system

Augmentation of Staphylococcal α-Toxin Signaling by the Epidermal Platelet-Activating Factor Receptor

Staphylococcal α-toxin is a cytolytic toxin secreted by many strains of Staphylococcus aureus that has proinflammatory and cytotoxic effects on human keratinocytes. α-toxin exerts its effects by forming a transmembrane pore that behaves like an ionophore for ions such as calcium. Because cellular membrane disruption with resultant intracellular calcium mobilization is a potent stimulus for the synthesis for the lipid mediator platelet-activating factor, the ability of α-toxin to induce platelet-activating factor production was assessed, and whether the epidermal platelet-activating factor receptor could augment toxin-induced signaling in epithelial cells examined. Treatment of the human keratinocyte-derived cell line HaCaT with α-toxin resulted in significant levels of platelet-activating factor, which were approximately 50% of the levels induced by calcium ionophore A23187. α-toxin also stimulated arachidonic acid release in HaCaT keratinocytes. Pretreatment of HaCaT cells with platelet-activating factor receptor antagonists, or overexpression of the platelet-activating factor metabolizing enzyme acetylhydrolase II blunted α-toxin-induced arachidonic acid release by approximately one-third, suggesting a role for toxin-produced platelet-activating factor in this process. Finally, retroviral-mediated expression of the platelet-activating factor receptor into the platelet-activating factor receptor-negative epithelial cell line KB resulted in an augmentation of α-toxin-mediated intracellular calcium mobilization and arachidonic acid release. These studies suggest that α-toxin-mediated signaling can be augmented via the epidermal platelet-activating factor receptor

The Grizzly, October 4, 1994

1994 Homecoming Court Spotlighted • Research Opportunities • Family Day \u2794 a Success • Arms Embargo Still Intact • Mining Industry Saved From Taxation • Homecoming Queen Nominees • New Lab Expands Horizons of Freshman Biologists • Recycling Lets Everyone Breathe a Little Easier • Kilmartin Lightens up Lower Lounge • Bright Moments and Sweet Sounds • Executing Justice with Pro-Theatre • Suggestions, Please! • Exploring Secrets & Truths Otherwise Unknown • Jam at the Trench • A Lesson in Life • The Real World: Collegeville • Prejudice Can Eclipse Unique Differences • Swarthmore Hands Bears Second Straight Defeat • UC Volleyball Loses Pair • UC Captain\u27s Council Set to Make Changeshttps://digitalcommons.ursinus.edu/grizzlynews/1342/thumbnail.jp