Periodic Reporting for period 4 - LNEXPANDS (The Mechanisms and Dynamics Controlling Cycles of Lymph Node Expansion )
Reporting period: 2022-09-01 to 2023-08-31
Lymph node swelling is a classical hallmark of immunity. The results of this project have given us new understanding about lymph node structure and function - we now know that lymph node swelling and tissue growth is regulated by mechanical signals such as cell stretching which means that the stromal architecture is instructed to expand to just the right degree to support the higher numbers of immune cells in the tissue. This new information now allows us to form hypotheses about how these mechanisms may fail in diseases states when the cell behaviour changes and fibrosis of the tissue can stiffen the environment senses by the cells in the tissue.
We focussed our research efforts on the structural cells (stromal cells) which form the tissue. The architecture of lymphoid organs is key to the effective operation of our immune system and is dictated by structures formed by non-haematopoetic stromal cells, including endothelial cells, and fibroblasts. In this proposal we focussed on the changing behaviour of an important cell type named fibroblastic reticular cells (FRCs) throughout cycles of lymph node remodelling.
Throughout this project we have used an extensive range of systems, ranging from proteomics and biochemistry to intravital imaging to: 1) To discover how spreading and stretching of the existing fibroblastic reticular network is directed in the acute phase of expansion: 2) To discover the cellular cues inducing the switch from stretching to proliferation and growth of the fibroblastic reticular network and 3) understand how homeostasis is regained as immune responses are resolved.
We focussed our research efforts on the structural cells (stromal cells) which form the tissue. The architecture of lymphoid organs is key to the effective operation of our immune system and is dictated by structures formed by non-haematopoetic stromal cells, including endothelial cells, and fibroblasts. In this proposal we focussed on the changing behaviour of an important cell type named fibroblastic reticular cells (FRCs) throughout cycles of lymph node remodelling.
Throughout this project we have used an extensive range of systems, ranging from proteomics and biochemistry to intravital imaging to: 1) To discover how spreading and stretching of the existing fibroblastic reticular network is directed in the acute phase of expansion: 2) To discover the cellular cues inducing the switch from stretching to proliferation and growth of the fibroblastic reticular network and 3) understand how homeostasis is regained as immune responses are resolved.
1) Aim1 - To discover how spreading and stretching of the existing fibroblastic reticular network is directed in the acute phase of expansion.
We firstly found and published mechanisms controlling the protrusion formation in fibroblastic reticular cells (de Winde et al. Journal of Cell Science 2021). Charlotte de Winde discovered that protrusion formation was regulated by the binding between Podoplanin and Tetraspanin CD9 and the receptor CD44. This initial study published in Journal of Cell Science was followed up with a collaborative study where were able to image PDPN clustering on FRCs at single molecule resolution to quantify and visualize this process at a molecular level (Lim et al., Open Biology 2023).
The major part of this aim is as yet still unpublished but we expect publication in 2024/25. PhD student Lindsey Millward pioneered the imaging of individual FRCs within the expanding lymph node to undertake quantification of protrusion formation, cell morphology and cell volume parameters. Lindsey identified that MARCKS phosphorylation was regulated downstream of RhoGTPases through PKN2.
Lindsey Millward PhD Thesis - https://discovery.ucl.ac.uk/id/eprint/10142040/
Published works
de Winde CM, Makris S, Millward LJ, Cantoral-Rebordinos JA, Benjamin AC, Martínez VG, Acton SE. Fibroblastic reticular cell response to dendritic cells requires coordinated activity of podoplanin, CD44 and CD9. J Cell Sci. 2021 Jul 15;134(14):jcs258610. doi: 10.1242/jcs.258610. Epub 2021 Jul 22. PMID: 34184727; PMCID: PMC8325952.
Lim SE, Joseph MD, de Winde CM, Acton SE, Simoncelli S. Quantitative single molecule analysis of podoplanin clustering in fibroblastic reticular cells uncovers CD44 function. Open Biol. 2023 May;13(5):220377. doi: 10.1098/rsob.220377. Epub 2023 May 10. PMID: 37161290; PMCID: PMC10170195.
2) Aim 2- To discover the cellular cues inducing the switch from stretching to proliferation and growth of the fibroblastic reticular network.
Our most recent published study on lymph node tissue mechanics led by PhD student Harry Horsnell (Horsnell et al., Nature Immunology 2022). Here, we showed molecular signals controlling cellular physical properties, collectively determining tissue mechanics of lymph nodes. We find that increased tissue tension through the stromal meshwork gates the initiation of fibroblast proliferation. This was the first published study linking tissue mechanics to immune function. There is an associated news and views article published in the same issue https://doi.org/10.1038/s41590-022-01277-0
Harry Horsnell PhD Thesis - https://discovery.ucl.ac.uk/id/eprint/10141661/
Published works
Horsnell HL, Tetley RJ, De Belly H, Makris S, Millward LJ, Benjamin AC, Heeringa LA, de Winde CM, Paluch EK, Mao Y, Acton SE. Lymph node homeostasis and adaptation to immune challenge resolved by fibroblast network mechanics. Nat Immunol. 2022 Aug;23(8):1169-1182. doi: 10.1038/s41590-022-01272-5. Epub 2022 Jul 26. PMID: 35882934; PMCID: PMC9355877.
3) Aim 3 - To understand how homeostasis is regained as immune responses are resolved.
As planned in the proposal, we used PDGFRa-mGFP-CreERT2 mice crossed to PDPN floxed mice to examine whether PDPN-driven contractility was required for lymph nodes to return to homeostatic size and function following immune challenge. His results indicated that FRC contractility was not required and that in fact lymph node resolution was driven by the egress of lymphocytes, and FRC apoptosis which followed.
To continue the work on lymph node homeostasis, we have used 3D imaging methods to measure the mathematical ratios between stroma and lymphocytes, along with quantification of network topology through stages of expansion and resolution. This work will continue beyond the end of this funded project and will be published in 2024/25.
Published works (Preprint)
Podoplanin expression in fibroblasts determines lymph node architecture and adaptive immune function. Spyridon Makris, Yukti Hari-Gupta, Jesús A. Cantoral-Rebordinos, Victor G. Martinez, Harry L. Horsnell, Charlotte M. de Winde, Tanya Singh, Martina Jovancheva, Robin Kettler, Janos Kriston-Vizi, Sophie E. Acton
bioRxiv 2022.12.01.518753; doi: https://doi.org/10.1101/2022.12.01.518753
We firstly found and published mechanisms controlling the protrusion formation in fibroblastic reticular cells (de Winde et al. Journal of Cell Science 2021). Charlotte de Winde discovered that protrusion formation was regulated by the binding between Podoplanin and Tetraspanin CD9 and the receptor CD44. This initial study published in Journal of Cell Science was followed up with a collaborative study where were able to image PDPN clustering on FRCs at single molecule resolution to quantify and visualize this process at a molecular level (Lim et al., Open Biology 2023).
The major part of this aim is as yet still unpublished but we expect publication in 2024/25. PhD student Lindsey Millward pioneered the imaging of individual FRCs within the expanding lymph node to undertake quantification of protrusion formation, cell morphology and cell volume parameters. Lindsey identified that MARCKS phosphorylation was regulated downstream of RhoGTPases through PKN2.
Lindsey Millward PhD Thesis - https://discovery.ucl.ac.uk/id/eprint/10142040/
Published works
de Winde CM, Makris S, Millward LJ, Cantoral-Rebordinos JA, Benjamin AC, Martínez VG, Acton SE. Fibroblastic reticular cell response to dendritic cells requires coordinated activity of podoplanin, CD44 and CD9. J Cell Sci. 2021 Jul 15;134(14):jcs258610. doi: 10.1242/jcs.258610. Epub 2021 Jul 22. PMID: 34184727; PMCID: PMC8325952.
Lim SE, Joseph MD, de Winde CM, Acton SE, Simoncelli S. Quantitative single molecule analysis of podoplanin clustering in fibroblastic reticular cells uncovers CD44 function. Open Biol. 2023 May;13(5):220377. doi: 10.1098/rsob.220377. Epub 2023 May 10. PMID: 37161290; PMCID: PMC10170195.
2) Aim 2- To discover the cellular cues inducing the switch from stretching to proliferation and growth of the fibroblastic reticular network.
Our most recent published study on lymph node tissue mechanics led by PhD student Harry Horsnell (Horsnell et al., Nature Immunology 2022). Here, we showed molecular signals controlling cellular physical properties, collectively determining tissue mechanics of lymph nodes. We find that increased tissue tension through the stromal meshwork gates the initiation of fibroblast proliferation. This was the first published study linking tissue mechanics to immune function. There is an associated news and views article published in the same issue https://doi.org/10.1038/s41590-022-01277-0
Harry Horsnell PhD Thesis - https://discovery.ucl.ac.uk/id/eprint/10141661/
Published works
Horsnell HL, Tetley RJ, De Belly H, Makris S, Millward LJ, Benjamin AC, Heeringa LA, de Winde CM, Paluch EK, Mao Y, Acton SE. Lymph node homeostasis and adaptation to immune challenge resolved by fibroblast network mechanics. Nat Immunol. 2022 Aug;23(8):1169-1182. doi: 10.1038/s41590-022-01272-5. Epub 2022 Jul 26. PMID: 35882934; PMCID: PMC9355877.
3) Aim 3 - To understand how homeostasis is regained as immune responses are resolved.
As planned in the proposal, we used PDGFRa-mGFP-CreERT2 mice crossed to PDPN floxed mice to examine whether PDPN-driven contractility was required for lymph nodes to return to homeostatic size and function following immune challenge. His results indicated that FRC contractility was not required and that in fact lymph node resolution was driven by the egress of lymphocytes, and FRC apoptosis which followed.
To continue the work on lymph node homeostasis, we have used 3D imaging methods to measure the mathematical ratios between stroma and lymphocytes, along with quantification of network topology through stages of expansion and resolution. This work will continue beyond the end of this funded project and will be published in 2024/25.
Published works (Preprint)
Podoplanin expression in fibroblasts determines lymph node architecture and adaptive immune function. Spyridon Makris, Yukti Hari-Gupta, Jesús A. Cantoral-Rebordinos, Victor G. Martinez, Harry L. Horsnell, Charlotte M. de Winde, Tanya Singh, Martina Jovancheva, Robin Kettler, Janos Kriston-Vizi, Sophie E. Acton
bioRxiv 2022.12.01.518753; doi: https://doi.org/10.1101/2022.12.01.518753
This project is now complete and we plan to have all of the new data and outputs published in peer reviewed journals within the next year.
During this project we have:
- Generated MARCKS KO cell lines and shown that MARCKS is required for FRCs to respond to dendritic cells in vitro.
- Performed laser ablation experiments show that network tension increases during early phases of lymph node expansion and that this increased network tension is a mechanical cue to stimulate proliferation of the fibroblast network.
- We have generated a new mouse model (PDGFRa-mGFP- CreERT2) as proposed to enable the conditional and specific deletion or expression of genes in fibroblasts, during resolution phase. Using this tool we have altered expression of PDPN which we find to be a mechanical sensor in fibroblasts in lymph nodes. Podoplanin deletion causes changes to lymph node structure and immune function.
During this project we have:
- Generated MARCKS KO cell lines and shown that MARCKS is required for FRCs to respond to dendritic cells in vitro.
- Performed laser ablation experiments show that network tension increases during early phases of lymph node expansion and that this increased network tension is a mechanical cue to stimulate proliferation of the fibroblast network.
- We have generated a new mouse model (PDGFRa-mGFP- CreERT2) as proposed to enable the conditional and specific deletion or expression of genes in fibroblasts, during resolution phase. Using this tool we have altered expression of PDPN which we find to be a mechanical sensor in fibroblasts in lymph nodes. Podoplanin deletion causes changes to lymph node structure and immune function.