Fibroblastic Reticular Cell Response to Dendritic Cells Requires Coordinated Activity of Podoplanin, CD44 and CD9

Abstract

Lymph node expansion is pivotal during immune activation. It is controlled by interactions between CLEC-2 hi migratory dendritic cells and podoplanin + fibroblastic reticular cells (FRCs) resulting in rapid loss of actomyosin contractility permitting FRC spreading. Podoplanin is an inflammatory marker in many pathologies, reported to facilitate both contractility and cell protrusions. However, how podoplanin expression is regulated, and how this one membrane protein can elicit these opposing phenotypes is unknown. We report that CLEC-2 binding to FRCs induces transcriptional upregulation of podoplanin, increasing surface protein expression. Further, we find that tetraspanin CD82 is required for trafficking of podoplanin to the plasma membrane. At the cell surface, podoplanin elicits multiple functions, balanced by its membrane binding partners hyaluronan receptor CD44 and tetraspanin CD9. FRCs lacking expression of both CD44 and CD9 are hypercontractile, and upon binding to CLEC-2 hi dendritic cells, both CD44 KO and CD9 KO FRCs exhibit defective protrusion formation and fail to spread. Podoplanin co-localises with CD44 or CD9 in distinct membrane domains, and both CD44 and CD9 are required for FRCs to spread in response to dendritic cells, however they control formation of cell protrusions via different and complimentary mechanisms. In vivo, surface expression levels of podoplanin, CD44 and CD9 are upregulated on T-cell zone FRCs in the early phase of lymph node expansion. Our data support a model whereby podoplanin resides in distinct plasma membrane domains, and that CLEC-2 binding serves as a molecular switch to change podoplanin function. Podoplanin | CD44 | CD9 | CD82 | Tetraspanins | Fibroblastic reticular cell | Lymph node Correspondence: s.acton@ucl.ac.uk de Winde et al. | bioRχiv | October 3, 2019 | 1-2

Tissue homeostasis and adaptation to immune challenge resolved by fibroblast network mechanics

“…F-actin intensity through the T cell FRC network was quantified by using PDPN staining as a mask (Figure 1B). Whilst PDPN intensity increased (Figure 1C) as inflammation proceeded, as has been noted in other studies (10,27), F-actin cables were maintained in the network through to day 5 post immunisation (Figure 1D).…”

“…Cell lines were maintained at 10% CO 2 , 37°C in Dulbecco's modified eagle medium (DMEM) (Thermo Fischer scientific) supplemented with 10% FBS, 1% P/S and 1% ITS unless otherwise stated. Cells were treated with recombinant CLEC2-Fc or Control-Fc supernatant (16,27) for approximately 2 hours, where indicated, to assay the effect of CLEC2 signalling through PDPN. Figure 3C is a paired analysis of the same cell before and 15 minutes after CLEC-2 treatment.…”

Molecular mechanisms of dendritic cell migration in immunity and cancer

“…Interestingly, podoplanin and CCL21 interact with each other on LECs [128], but a role of this interaction in DC migration has not been directly addressed. Loss of tetraspanin CD9, a partner protein of podoplanin [129], or tetraspanin CD82 causes a decrease in podoplanin surface expression [130,131]. Although DCs could still bind to and interact with CD9-deficient FRCs [130], the role of tetraspanins in CLEC-2/podoplanindependent DC migration has not been studied.…”

Interplay between Podoplanin, CD44s and CD44v in Squamous Carcinoma Cells

“…In FRCs, podoplanin promotes actomyosin contractility through its binding to ERM proteins and activation of RhoA GTPase, and binding of CLEC-2 located on migratory dendritic cells inhibits podoplanin-induced contractility, resulting in FRC spreading and elongation to allow a rapid lymph node expansion [ 21 , 74 ]. In a recent report, de Winde and colleagues suggest that upon binding of CLEC-2 to FRCs, podoplanin is recruited to cholesterol-rich domains (lipid rafts) where interacts with membrane partners CD44 and CD9 to form cell protrusions and to spread [ 75 ]. In fibrosarcoma cells, podoplanin–CD9 interaction neutralizes podoplanin-mediated platelet aggregation via binding to CLEC-2 and suppresses metastasis [ 29 ].…”