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The CLEC-2–podoplanin axis controls the contractility of fibroblastic reticular cells and lymph node microarchitecture

Nature Immunology volume 16, pages 75–84 (2015)Cite this article

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Abstract

In lymph nodes, fibroblastic reticular cells (FRCs) form a collagen-based reticular network that supports migratory dendritic cells (DCs) and T cells and transports lymph. A hallmark of FRCs is their propensity to contract collagen, yet this function is poorly understood. Here we demonstrate that podoplanin (PDPN) regulates actomyosin contractility in FRCs. Under resting conditions, when FRCs are unlikely to encounter mature DCs expressing the PDPN receptor CLEC-2, PDPN endowed FRCs with contractile function and exerted tension within the reticulum. Upon inflammation, CLEC-2 on mature DCs potently attenuated PDPN-mediated contractility, which resulted in FRC relaxation and reduced tissue stiffness. Disrupting PDPN function altered the homeostasis and spacing of FRCs and T cells, which resulted in an expanded reticular network and enhanced immunity.

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Figure 1: PDPN controls the spreading, elongation, and actomyosin contractility of FRCs.
Figure 2: The PDPN cytoplasmic tail controls FRC elongation but is dispensable for contraction.
Figure 3: PDPN signals through ERM and MLC to control contraction.
Figure 4: PDPN maintains normal proliferation and survival of FRCs.
Figure 5: In vivo blockade of PDPN results in enlarged LNs, FRC proliferation and reorganization of the FRC network.
Figure 6: PDPN is needed to control lymph node swelling and T cell proliferation following immunization.
Figure 7: Engagement of CLEC-2 by PDPN results in a phenocopy of the genetic deletion of PDPN.
Figure 8: CLEC-2 signals from migratory DCs are required for expansion of LNs and the FRC network in response to immunization.

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Acknowledgements

We thank L. Cameron for assistance at the Dana-Farber Cancer Institute confocal microscopy core, and R. Gelman for help with statistical analyses. Supported by the US National Institutes of Health (R0I DK074500, P01 AI045757 and R21 CA182598 to S.J.T.; 5R01 AI039246 to M.C.C.; and P01 HL085607 and GM103441 to L.X.), the American Cancer Society (S.J.T.), the Claudia Adams Barr Program in Innovative Cancer Research (S.J.T.), the Cancer Research Institute (to V.C.), the American Heart Association (SDG7410022 to J.F.) and the National Science Foundation (J.L.A.).

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Authors and Affiliations

  1. Division of Medical Sciences, Harvard Medical School, Boston, Massachusetts, USA

    Jillian L Astarita & Matthew C Woodruff

  2. Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, Massachusetts, USA

    Jillian L Astarita, Viviana Cremasco, James R Peck, Janice M Nieves-Bonilla & Shannon J Turley

  3. Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, USA

    Jianxin Fu, Kai Song, Yuji Kondo & Lijun Xia

  4. Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA

    Jianxin Fu, Kai Song, Yuji Kondo & Lijun Xia

  5. School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, USA

    Max C Darnell & David J Mooney

  6. Wyss Institute for Biologically Inspired Engineering at Harvard University, Cambridge, Massachusetts, USA

    Max C Darnell & David J Mooney

  7. Program in Cellular and Molecular Medicine, Children's Hospital Boston, Harvard Medical School, Boston, Massachusetts, USA

    Matthew C Woodruff & Michael C Carroll

  8. Department of Biomedical Imaging, Genentech, South San Francisco, California, USA

    Alvin Gogineni & Robby M Weimer

  9. Institute of Immunobiology, Kantonal Hospital St. Gallen, St. Gallen, Switzerland

    Lucas Onder & Burkhard Ludewig

  10. Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA

    Shannon J Turley

  11. Department of Cancer Immunology, Genentech, South San Francisco, California, USA

    Shannon J Turley

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Contributions

J.L.A. and S.J.T. designed experiments, analyzed results and wrote the manuscript; J.L.A., V.C., J.F., M.C.D., J.R.P., J.M.N.-B., A.G., M.C.W. and R.M.W. performed experiments; J.F., K.S., Y.K., and L.X. supplied reagents and mice; L.O. and B.L. provided mice; and V.C., B.L., M.C.C., D.J.M. and L.X. provided comments on the manuscript.

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Correspondence to Shannon J Turley.

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S.J.T., A.G. and R.M.W. are employees of Genentech.

Integrated supplementary information

Supplementary Figure 1 PDPN is required for cell adhesion and contraction.

(a) Representative histograms of FRC surface markers on wild-type and Pdpn−/− FRCs. (b) Graph depicting the area covered by FRCs as they spread on collagen at different time points after plating. (c) Graph representing the absorbance of crystal violet, which was used to stain FRCs adhering to collagen-coated wells at different time points after plating. (d-f). Wild-type FRCs were seeded into 3D collagen gels and then treated with the ROCK inhibitor Y27632. FRC morphology index (d) and the length (e) and number (f) of protrusions were measured. Data are representative of 3 independent experiments (mean±s.d., n>50 cells per condition per experiment). (g) Representative image of YAP staining in wild-type and Pdpn−/− FRCs. Data are representative of 3 independent experiments. NS, not significant *P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001 (Mann Whitney test).

Supplementary Figure 2 Generation of Δcyto mice.

(a) Schematic indicating the gene targeting strategy for the generation of the Δcyto mice. (b) Sequences of PCR fragments amplified from DNA from tail biopsies of wild-type and Δcyto pups indicating stop codons introduced after the transmembrane domain. (c) Representative histogram indicating surface levels of PDPN on wild-type, Pdpn−/−, and cyto FRCs. (d) Representative confocal images of PDPN staining in FRCs. Scale bar, 20 μm. (e) Immunoblot showing total levels of PDPN on wild-type, Pdpn−/−, and cyto FRCs. (f) Wild-type and Δcyto FRCs were treated with phosphatidylinositol-specific phospholipase C (PI-PLC) for 1 h at 37°C and the levels of Thy1 (a known GPI-anchored protein) or PDPN were measured by flow cytometry. (g) Representative image of YAP staining in wild-type and Δcyto FRCs. Data are representative of 3 independent experiments.

Supplementary Figure 3 Quantification of phosphorylated ERM, phosphorylated MLC and RhoA-GTP by immunoblot analysis.

The relative densities of the bands in immunoblots for p-ERM (a), p-MLC (b), and RhoA-GTP (c) were measured and set relative to those of wild-type FRCs. Graphs represent 3-5 independent experiments. *P<0.001; **P<0.0001 (one sample t-test).

Supplementary Figure 4 Treatment with anti-PDPN recapitulates genetic deletion of PDPN.

(a) Morphology index of either untreated or α-PDPN-treated wild-type and Pdpn−/− FRCs. Data represent 3 independent experiments (mean±s.d., n>60 cells per experiment). (b) Graph shows the amount that antibody-treated wild-type FRCs contracted collagen gels, relative untreated FRCs. Data represent 3 independent experiments (mean±s.d., three wells per experiment). (c) Representative image of HEVs and red blood cells (Ter119) in LNs of isotype- or α-PDPN-treated mice. (d-g) Graphs depicting the levels of the inflammatory markers IL-6 (d), neutrophil myeloperoxidase (MPO, e), IFNγ (f), and TNFα (g). (h) Representative flow cytometry plots indicating the forward scatter (FSC) and side scatter (SSC) profile of FRCs from LNs of mice treated with the isotype or α-PDPN antibodies. (i) Quantification of the percentage of FRCs with high FSC and SSC. Data are representative of 4 independent experiments, n=4 mice per experiment. *P<0.01; **P<0.001 (Mann Whitney test).

Supplementary Figure 5 Genetic deletion of PDPN results in enlarged LNs and an expanded FRC network.

(a-c) The LN masses (a), total cellularity (b), and FRC numbers (c) from Pdpnfl/fl and Pdpnfl/flPdgfrb-Cre+ mice. (d,e) Representative images of HEVs and red blood cells (Ter119) in LNs from Pdpnfl/fl and Pdpnfl/flPdgfr?-Cre+ mice (d) or wild-type and cyto mice (e). (f) Representative confocal images of conduits in isotype- and α-PDPN-treated mice that received a s.c. injection of FITC 4 h prior to imaging. FDCs are identified by CR1 staining (red) and collagen is visualized by second harmonic generation (blue). (g) Representative images of the FRC network. Confocal z-stack images of the FRC network in Pdpnfl/fl and Pdpnfl/flPdgfrb-Cre+ mice were analyzed in 3D and isosurfaces were generated. (h,i) Quantification of the surface area of desmin (h) and ER-TR7 (i) staining shown in g. (j) Representative histogram demonstrating that Ccl19-Cre+Rosa26-H2b-eGFPfl/fl mice only have a GFP signal in FRCs. (k) Representative images of FRC nuclei from Ccl19-Cre+Rosa26-H2b-eGFPfl/fl mice that were treated with the isotype or α-PDPN antibody. *P<0.05; **P<0.001; ***P<0.0001 (Mann Whitney test).

Supplementary Figure 6 Anti-PDPN does not block the binding of CLEC-2 or impair DC migration.

(a) Histogram indicating CLEC-2-Fc staining of FRCs that were either untreated, pre-treated with a hamster isotype control antibody, or pre-treated with the 8.1.1 PDPN-specific antibody. Grey line indicates the secondary alone control for the CLEC-2-Fc staining. (b,c) DC numbers (gated on PI−CD45+CD11chi) in LNs of isotype- or -PDPN-treated mice in steady state (b) or after PBS or LPS/OVA administration (c). Data are representative of 4 independent experiments (n=3-4 mice per group per experiment). *P<0.05 (Mann Whitney test).

Supplementary Figure 7 CLEC-2–Fc induces elongation and impairs spreading in a way similar to a genetic deletion of PDPN.

(a) Graph depicting the morphology index for untreated or CLEC-2-Fc-treated wild-type, Pdpn−/−, and Δcyto FRCs. (b) The cell area of FRCs spreading on collagen in the presence or absence of CLEC-2-Fc. Data are representative of 3 independent experiments (mean±s.d., n>50 cells per experiment). (c) Graph representing the morphology index of wild-type FRCs that were pre-treated with cytochalasin D prior to CLEC-2-Fc treatment. (d,e) Representative images of YAP staining in wild-type (d) and Δcyto (e) FRCs that were untreated or treated with CLEC-2-Fc for 24 h. (f,g) Quantification of immunoblots for p-ERM (f) and p-MLC (g) levels in wild-type FRCs treated with CLEC-2-Fc for the indicated times. (h-k) Relative levels of inflammatory markers in LNs from mice treated with an isotype control or CLEC-2-Fc for 1 h. (l) Total cellularity from LNs of mice injected i.v. with an isotype control or CLEC-2-Fc for 1 h. (m) Morphology index for wild-type and Pdpn−/− LECs treated with an isotype control or CLEC-2-Fc for 24 h. (n) Representative images of HEVs in LNs from isotype- or CLEC-2-Fc-treated mice. Red blood cells are stained with Ter119 and can be seen leaving HEVs in CLEC-2-Fc-treated LNs. NS, not significant; *P<0.01; **P<0.001; ***P<0.0001 (Mann Whitney test).

Supplementary Figure 8 Proposed model of PDPN signaling in FRCs and LNs.

(a) LNs from both wild-type and Δcyto mice contain a dense FRC network and exhibit baseline levels of stiffness, due to maintained contraction and controlled proliferation of FRCs. (b) In wild-type FRCs, full-length PDPN associates with other transmembrane proteins to maintain high levels of ERM and MLC activation, which lead to strong adhesion to the ECM and contraction of the actomyosin skeleton within the cell. (c) Upon deletion of the cytoplasmic tail, PDPN is still able to maintain its lateral membrane interactions and localization in lipid rafts due to through its transmembrane and extracellular domains. This allows for the maintenance of ERM activation and normal contractility within the cells. In contrast, LNs from mice with treated with the α-PDPN antibody or CLEC-2-Fc (d) are enlarged and have an expanded FRC network with decreased LN stiffness. (e) In Pdpn−/− FRCs the lack of PDPN may disrupt the stability or signaling of other transmembrane binding proteins, which results in reduced ERM activation and reduced actomyosin contractility. (f) In wild-type FRCs treated with CLEC-2-Fc, ERM and MLC activation are also disrupted. While PDPN is still present in this scenario, our data suggest that CLEC-2 transiently blocks lateral membrane interactions and thus disrupts downstream signaling similarly to a complete Pdpn−/− FRC.

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Astarita, J., Cremasco, V., Fu, J. et al. The CLEC-2–podoplanin axis controls the contractility of fibroblastic reticular cells and lymph node microarchitecture. Nat Immunol 16, 75–84 (2015). https://doi.org/10.1038/ni.3035

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