Mechanical force modulates binding of Interleukin-7 to Fibronectin

Abstract

The extracellular matrix (ECM) plays an important role in cell regulation, development, and homeostasis. Not only does it provide a structural scaffold for cell migration and interaction, but also acts as a deposit for cytokines and chemokines that regulate cellular function. Cells can modify the structure, composition, and biochemical characteristics of the ECM, and are able to apply mechanical forces that can alter the ECM and it components. One essential component of the ECM is fibronectin (FN). A protein that forms fibers in the ECM, and has various binding sites that allow intermolecular interactions among other FN molecules, with other ECM constituent proteins, carbohydrates, cell-ECM binding proteins such as integrins, and various cytokines and signaling molecules. FN has several mechanosensitive cryptic binding sites that open upon protein unfolding due to mechanical tension. During inflammation and other physiological processes, swelling of tissue and ECM remodeling can induce mechanical stretching or relaxing of the ECM, which can in turn induce a change a in the conformation of Fn. This thesis contributes to the understanding of how force-induced conformational changes in FN can have an impact on cytokine binding, by focusing on how mechanically stretching FN fibers affect the binding of interleukin-7 (IL-7) to FN. IL-7 is a cytokine belonging to the g-c interleukin family that plays an essential role in the survival, proliferation and differentiation of naive and memory T-cells in lymphatic tissues, and the development of B and T-cells in the thymus. It is produced by stromal cells in the lymph nodes and thymus, and has been previously shown to bind to fibronectin (FN). In this thesis, a Forster Resonance Energy Transfer (FRET) based method to correlate ligand-binding to changes in FN conformation is presented. This method provides a quantitative tool to assess how changes in FN conformation result in modified cytokine binding. The developed method was used to assess whether FN’s conformational changes due to mechanical force application result in changes in binding of IL-7. We found that stretching of FN fibers increased IL-7 binding, and localized the FN binding site on the CD-loop region of IL-7 using a synthetic CD-loop peptide which also showed increased binding to stretched fibrillar FN. Upon structural analysis of IL-7 binding to its receptors IL7RA and IL2RG, we propose that the CD-loop is available for simultaneous interaction with FN while IL-7 is bound to its cognate cell receptors. We also propose a negative-feedback mechanism reciprocally regulating the tensional state of FN fibers and IL-7 signaling. Our data show then, for the first time, a mechano-regulated mechanism of IL-7 binding to the ECM, where stretching FN fibers might locally concentrate IL-7 in an extracellular matrix bound state. Finally, first steps towards determining what impact these findings may have on cellular function are presented, to motivate the refinement and/or development of adequate assays to test the physiological implications of our findings. Since IL-7 is produced by lymph node stromal cells (LNSCs), and stromal cells from the thymus, and these cells in turn respond to IL-7, we aimed to characterize mechano-biological behaviour of these cells, their ECM, and make the first steps towards elucidating whether these cells can bring about changes in the ECM that could result in differential binding of IL-7 to fibronectin in the ECM, and in turn, whether changes in IL-7 binding levels can have an effect on their function. The work presented here could be of relevance for the further understanding of how mechanically induced changes of the ECM affect chemokine and cytokine binding and the effects of this on cell function. These aspects should be considered in the future for the development of current and/or new IL-7 based immunotherapies that take into consideration possible changes in ECM due to disease, inflammation or ageing.