Horsnell, Harry Lloyd; (2022) Investigating mechanical forces and communication in the fibroblastic reticular cell network of the lymph node. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis describes the investigation of how mechanical forces and intercellular signalling through fibroblastic reticular cells control lymph node expansion in response to infection. Chapter 1 reviews the current literature in lymph node biology and tissue mechanics, highlighting the lack of understanding of fibroblastic reticular cell mechanics in the tissue context. Chapter 2 describes the techniques, resources and reagents used in generating the following results. In chapter 3 we test the hypothesis that fibroblastic reticular cells are contractile units of the lymph node. We developed an ex vivo laser ablation protocol in combination with a novel mouse model to show that the fibroblastic reticular cell network generates mechanical tension in the steady state through actomyosin contractility. Furthermore, we show that in response to inflammation the mechanical tension in the network is first decreased and then increased beyond homeostatic levels. These observations led to the investigation as to why there is an initial decrease in mechanical tension and the functional consequence of increased tension as inflammation proceeds. In Chapter 4 we hypothesised that CLEC-2 (dendritic cells) and Podoplanin signalling (fibroblastic reticular cells), previously shown to regulate actomyosin contractility, contributes to the initial reduction in network tension. Using in vitro biophysical assays, we show that the cell intrinsic mechanical properties of fibroblastic reticular cells are regulated by the action of CLEC-2 through Podoplanin. In Chapter 5 we question whether the increased tension seen later in inflammation acts as a mechanical cue for fibroblastic reticular cell entry into division. In vivo we showed that induction of fibroblastic reticular cell division was correlated with the timing of tension increase. We then directly treated lymph nodes with ROCK inhibitor to abolish the increased tension in the network and found that specifically fibroblastic reticular cell numbers were reduced in this condition. We finish our mechanical investigations by highlighting potential mechanosensory pathways deployed by fibroblastic reticular cells. Finally in Chapter 6 we raise the question of why the fibroblastic reticular cell network remains connected during inflammation. We go on to find unexpected results that suggest fibroblastic reticular cells can communicate intercellularly using gap junction plaques and calcium signalling. We find that dendritic cell induced calcium waves may be a local signal that can be propagated across the fibroblastic reticular cell network to coordinate lymph node expansion. In the final chapter all the findings are discussed, and implications contextualised, and we suggest that the FRC network balances forces to control LN expansion.

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