An Arp2/3-cPLA2-NFκB axis acts as a Cell Shape Sensor to drive Homeostatic Migration of Dendritic Cells

Background

 The repertoire of cells that make up the body’s immune system are in a constant balancing act between steady state and activation. Within this repertoire, dendritic cells (DCs) are key players responsible for presenting foreign and self-antigens to T lymphocytes, thus fending off infectious diseases while maintaining self-tolerance^1,2. DCs travel throughout the body via vasculature or lymph vessels and return to lymph nodes to present their findings. DC migration to lymph nodes occurs even in the absence of pathogens in a process called homeostatic migration. To enter lymph nodes, DCs exit peripheral tissues and upregulate the cell surface receptor CCR7 which guides DCs along a trail of CCL21 or CCL19 chemokines^4,5. To date, most studies aimed at uncovering factors triggering DC migration to lymph nodes have focused on biochemical cues such as pathogen-associated molecular patterns (PAMPs) and cytokines. While these studies have yielded important insights, they have left the potential role of biophysical cues in homeostatic migration of DCs unexplored.

Using genetic mouse models, cell confinement assays, and transcriptional profiling, Alraies and colleagues illustrate a novel mechanosensitive signalling axis that guides DCs to lymph nodes at steady state. This work proposes that cell deformation regulates homeostatic migration of DCs and leads to an immunomodulatory profile distinct from one induced by foreign pathogens, ultimately affecting T cell activation.

Key Findings

CCR7 expression in DCs is sensitive to cell deformation

The authors observed that migrating DCs undergo dramatic cell deformations by performing intravital imaging in mouse ear skin, prompting the question of whether CCR7-dependent homeostatic migration of DCs could be triggered by mechanical cues. To investigate this question, they cultured bone marrow-derived DCs from mice expressing a CCR7/GFP reporter in a confinement device which could be adjusted to 3-4 μm in height. Immunofluorescence imaging as well as qPCR analyses revealed that cells confined to a height of 3 μm, but not 4 μm, strongly upregulated CCR7 relative to unconfined cells. Furthermore, these newly expressed CCR7 receptors were fully functional as revealed by migration assays in which a CCL19 gradient promoted chemotaxis in DCs under 3 μm confinement. Taken together, these results suggest that CCR7 expression in DCs is sensitive to changes in cell shape.

Above: DCs under confinement undergo cell shape changes and upregulate the cell surface receptor CCR7.

CCR7 upregulation upon DC deformation requires cPLA₂ and is mediated by Arp2/3

Previous studies have demonstrated that the lipid metabolism enzyme cPLA₂ is activated by increased nuclear envelope tension^6,7. Given their observation that DC nuclei were deformed by 3 μm confinement, the authors wondered whether cPLA₂ was involved in the upregulation of CCR7. Treating DCs with the cPLA₂ inhibitor AACOF3 or siRNA against cPLA₂ abolished CCR7 upregulation in response to cell confinement. In contrast, cPLA₂ inhibition or knockdown had no effect on the upregulation of CCR7 upon treatment with microbial lipopolysaccharide (LPS). Importantly, this result suggests that cPLA₂ mediates CCR7 expression in response to cell shape changes, but not microbial antigens.

The linker of nucleoskeleton and cytoskeleton (LINC) complex transmits force from the actin cytoskeleton to the nucleus, thus altering nuclear tension. Cytoskeletal remodelling by the actin nucleating complex Arp2/3 might therefore impact nuclear tension and ultimately CCR7 expression in DCs under confinement⁸. The authors treated confined cells with the Arp2/3 inhibitor CK666 and found that CCR7 upregulation and cPLA₂ nuclear translocation were both abrogated. Similar results were obtained in DCs from mice with a genetic deletion of Arpin, which encodes a negative regulator of Arp2/3. Strikingly, flow cytometry experiments revealed that the number of Arpin knockout (KO) migratory DCs in mouse ear skin-draining lymph nodes was increased compared to wildtype (WT), suggesting that Arpin normally suppresses DC migration from peripheral tissues to lymph nodes. From these results, the authors concluded that the Arp2/3 complex is an important mediator in the transduction of cell shape changes to CCR7 expression.

NFkB and IKKb induce CCR7 expression upon changes to DC shape

Prior studies have suggested that the NFkB pathway is involved in homeostatic migration of DCs to lymph nodes⁹. Thus, the authors investigated whether the NFkB pathway was activated in confined DCs. Inhibiting IKKb, the main activating kinase in the NFkB signalling axis, with B1605906, abolished CCR7 upregulation in confined DCs. Furthermore, while cPLA₂ KO abrogates the nuclear localization of NFkB, IKKb inhibition did not affect nuclear accumulation of cPLA₂. Together with their previous results, these experiments suggest that cPLA₂ functions upstream of NFkB to upregulate CCR7 expression.

The Arp2/3-cPLA2-NFkB signalling axis alters the DC transcriptome and endows DCs with distinct immunomodulatory properties

The authors next investigated how the transcriptional landscape of DCs is altered by confinement. This was of particular interest since alterations to the transcriptome of DCs may affect the type of response they evoke from T cells. Thus, the authors performed bulk RNA-sequencing on confined or unconfined cPLA₂ WT or KO cells. Pathway analysis revealed that genes related to innate immunity and antigen processing were upregulated in confined cells, and this upregulation was lost in cPLA₂ KO cells. They also observed that genes related to Arp2/3 actin nucleation were upregulated in confined cells, as well as interferon response-related genes.

Additional transcriptional analyses in cPLA₂ WT and KO DCs treated with LPS revealed that cPLA₂ expression had no effect on the types of transcriptional changes induced by LPS treatment. Genes that were upregulated in LPS-treated DCs, but not confined DCs, were mainly related to the immune response. To test this further, the authors analyzed the cell surface expression of CD80, CD86 and MHCII via flow cytometry, and found that confined DCs expressed these immune stimulatory molecules to a lesser extent than LPS-treated DCs. Co-culturing confined versus LPS-treated DCs with T-cells revealed that confined DCs were less effective at activating T-cells. Importantly, these findings indicate that microbial activation and cell confinement endow DCs with distinct immunomodulatory profiles, which ultimately leads to differential activation of regulatory T cells.

Above: a schematic illustrating the mechanosensitive signalling pathway that guides migrating DCs to lymph nodes.

Why I chose this preprint

I found this preprint interesting due to its proposal that a mechanical cue (i.e., cell deformation) could be a key factor in guiding DCs to lymph nodes. Previous work has focused almost exclusively on biochemical signalling, while less attention has been paid to the diverse physical forces experienced by DCs while patrolling tissues throughout the body. Although this study focuses specifically on homeostatic DC migration, I believe the findings from this preprint will encourage other researchers studying cell migration to explore not just biochemical signalling pathways, but mechanical ones too. In my own research, I am interested in uncovering the roles of tissue mechanics in glioblastoma (GBM) cell migration. Like DCs, GBM tumour cells experience diverse physical cues while invading healthy brain tissue. It would be fascinating to determine whether similar mechanosensitive signalling axes are at play during metastasis as well.

Question for the Authors

Dendritic cells would likely experience cell deformations while patrolling dense peripheral tissues in contexts beyond homeostatic migration to lymph nodes. Do you anticipate that CCR7 will still be upregulated in these contexts, or could there be additional mechanisms (ex. Arpin activity) that limit CCR7 upregulation to tissues that are in proximity to lymph nodes?

References

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2. Steinman, R. M., & Hemmi, H. (2006). Dendritic cells: translating innate to adaptive immunity. Current topics in microbiology and immunology, 311, 17–58. https://doi.org/10.1007/3-540-32636-7_2
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7. Ward, K. E., Ropa, J. P., Adu-Gyamfi, E., & Stahelin, R. V. (2012). C2 domain membrane penetration by group IVA cytosolic phospholipase A₂ induces membrane curvature changes. Journal of lipid research, 53(12), 2656–2666. https://doi.org/10.1194/jlr.M030718
8. Thiam, H. R., Vargas, P., Carpi, N., Crespo, C. L., Raab, M., Terriac, E., King, M. C., Jacobelli, J., Alberts, A. S., Stradal, T., Lennon-Dumenil, A. M., & Piel, M. (2016). Perinuclear Arp2/3-driven actin polymerization enables nuclear deformation to facilitate cell migration through complex environments. Nature communications, 7, 10997. https://doi.org/10.1038/ncomms10997
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doi: https://doi.org/10.1242/prelights.32793

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