Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Direction of leukocyte polarization and migration by the phosphoinositide-transfer protein TIPE2

Abstract

The polarization of leukocytes toward chemoattractants is essential for the directed migration (chemotaxis) of leukocytes. How leukocytes acquire polarity after encountering chemical gradients is not well understood. We found here that leukocyte polarity was generated by TIPE2 (TNFAIP8L2), a transfer protein for phosphoinositide second messengers. TIPE2 functioned as a local enhancer of phosphoinositide-dependent signaling and cytoskeleton remodeling, which promoted leading-edge formation. Conversely, TIPE2 acted as an inhibitor of the GTPase Rac, which promoted trailing-edge polarization. Consequently, TIPE2-deficient leukocytes were defective in polarization and chemotaxis, and TIPE2-deficient mice were resistant to leukocyte-mediated neural inflammation. Thus, the leukocyte polarizer is a dual-role phosphoinositide-transfer protein and represents a potential therapeutic target for the treatment of inflammatory diseases.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: TIPE2 promotes leukocyte chemotaxis both in vivo and in vitro.
Figure 2: TIPE2 is required for chemoattractant-induced polarization of leukocytes.
Figure 3: Rac-dependent functions of TIPE2 in cells undergoing chemotaxis.
Figure 4: Rac-independent functions of TIPE2 in cells moving by chemotaxis.
Figure 5: TIPE2 functions as a PtdIns(4,5)P2-transfer protein in lipid bilayers enriched for PtdIns(3,4,5)P3.
Figure 6: TIPE2 controls phosphoinositide signaling through PtdIns(3,4,5)P3-dependent mechanisms.
Figure 7: Diminished encephalomyelitis and infiltration of leukocytes into the nervous tissue of Tipe2−/− mice.

References

  1. 1

    Swaney, K.F., Huang, C.-H. & Devreotes, P.N. Eukaryotic chemotaxis: a network of signaling pathways controls motility, directional sensing, and polarity. Annu. Rev. Biophys. 39, 265–289 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. 2

    Merlot, S. & Firtel, R.A. Leading the way: directional sensing through phosphatidylinositol 3-kinase and other signaling pathways. J. Cell Sci. 116, 3471–3478 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. 3

    Deng, Q. & Huttenlocher, A. Leukocyte migration from a fish eye's view. J. Cell Sci. 125, 3949–3956 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Iglesias, P.A. & Devreotes, P.N. Biased excitable networks: how cells direct motion in response to gradients. Curr. Opin. Cell Biol. 24, 245–253 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

    Huang, C.-H., Tang, M., Shi, C., Iglesias, P.A. & Devreotes, P.N. An excitable signal integrator couples to an idling cytoskeletal oscillator to drive cell migration. Nat. Cell Biol. 15, 1307–1316 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Tang, M. et al. Evolutionarily conserved coupling of adaptive and excitable networks mediates eukaryotic chemotaxis. Nat. Commun. 5, 5175 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

    Ahn, S.-H. et al. Two genes on A/J chromosome 18 are associated with susceptibility to Staphylococcus aureus infection by combined microarray and QTL analyses. PLoS Pathog. 6, e1001088 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

    Zhang, C. et al. Role of SCC-S2 in experimental metastasis and modulation of VEGFR-2, MMP-1, and MMP-9 expression. Mol. Ther. 13, 947–955 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

    Zhang, Y. et al. Tumor necrosis factor-α induced protein 8 polymorphism and risk of non-Hodgkin's lymphoma in a Chinese population: a case-control study. PLoS One 7, e37846 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Fayngerts, S.A. et al. TIPE3 is the transfer protein of lipid second messengers that promote cancer. Cancer Cell 26, 465–478 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. 11

    Ma, Y. et al. The expression and significance of TIPE2 in peripheral blood mononuclear cells from asthmatic children. Scand. J. Immunol. 78, 523–528 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. 12

    Wang, L., Song, Y. & Men, X. Variance of TNFAIP8 expression between tumor tissues and tumor-infiltrating CD4+ and CD8+ T cells in non-small cell lung cancer. Tumour Biol. 35, 2319–2325 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

    Xi, W. et al. Roles of TIPE2 in hepatitis B virus-induced hepatic inflammation in humans and mice. Mol. Immunol. 48, 1203–1208 (2011).

    Article  CAS  Google Scholar 

  14. 14

    Yang, M. et al. TNFAIP8 overexpression is associated with lymph node metastasis and poor prognosis in intestinal-type gastric adenocarcinoma. Histopathology 65, 517–526 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  15. 15

    Zhang, C. et al. The significance of TNFAIP8 in prostate cancer response to radiation and docetaxel and disease recurrence. Int. J. Cancer 133, 31–42 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Gus-Brautbar, Y. et al. The anti-inflammatory TIPE2 is an inhibitor of the oncogenic Ras. Mol. Cell 45, 610–618 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. 17

    Zhang, X. et al. Crystal structure of TIPE2 provides insights into immune homeostasis. Nat. Struct. Mol. Biol. 16, 89–90 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    Wang, Z. et al. TIPE2 protein serves as a negative regulator of phagocytosis and oxidative burst during infection. Proc. Natl. Acad. Sci. USA 109, 15413–15418 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  19. 19

    Sun, H. et al. TIPE2, a negative regulator of innate and adaptive immunity that maintains immune homeostasis. Cell 133, 415–426 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

    Wrighton, K.H. Sensing and controlling protein dynamics. Nat. Rev. Mol. Cell Biol. 11, 680–681 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Schaaf, G. et al. Functional anatomy of phospholipid binding and regulation of phosphoinositide homeostasis by proteins of the sec14 superfamily. Mol. Cell 29, 191–206 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

    Ghosh, M. et al. Cofilin promotes actin polymerization and defines the direction of cell motility. Science 304, 743–746 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Bravo-Cordero, J.J., Magalhaes, M.A.O., Eddy, R.J., Hodgson, L. & Condeelis, J. Functions of cofilin in cell locomotion and invasion. Nat. Rev. Mol. Cell Biol. 14, 405–415 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. 24

    Steinbach, K., Piedavent, M., Bauer, S., Neumann, J.T. & Friese, M.A. Neutrophils amplify autoimmune central nervous system infiltrates by maturing local APCs. J. Immunol. 191, 4531–4539 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. 25

    Steinman, L. Multiple sclerosis: a two-stage disease. Nat. Immunol. 2, 762–764 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. 26

    Luster, A.D., Alon, R. & von Andrian, U.H. Immune cell migration in inflammation: present and future therapeutic targets. Nat. Immunol. 6, 1182–1190 (2005).

    Article  CAS  Google Scholar 

  27. 27

    Friedl, P. & Weigelin, B. Interstitial leukocyte migration and immune function. Nat. Immunol. 9, 960–969 (2008).

    Article  CAS  Google Scholar 

  28. 28

    de Oliveira, S., Rosowski, E.E. & Huttenlocher, A. Neutrophil migration in infection and wound repair: going forward in reverse. Nat. Rev. Immunol. 16, 378–391 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Fritsch, R. et al. RAS and RHO families of GTPases directly regulate distinct phosphoinositide 3-kinase isoforms. Cell 153, 1050–1063 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

    Sadhu, C., Masinovsky, B., Dick, K., Sowell, C.G. & Staunton, D.E. Essential role of phosphoinositide 3-kinase delta in neutrophil directional movement. J. Immunol. 170, 2647–2654 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    Boulven, I. et al. Class IA phosphatidylinositide 3-kinases, rather than p110 gamma, regulate formyl-methionyl-leucyl-phenylalanine-stimulated chemotaxis and superoxide production in differentiated neutrophil-like PLB-985 cells. J. Immunol. 176, 7621–7627 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. 32

    Ferguson, G.J. et al. PI(3)Kγ has an important context-dependent role in neutrophil chemokinesis. Nat. Cell Biol. 9, 86–91 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. 33

    Tang, W. et al. A PLCβ/PI3Kγ-GSK3 signaling pathway regulates cofilin phosphatase slingshot2 and neutrophil polarization and chemotaxis. Dev. Cell 21, 1038–1050 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Miao, Y. et al. Altering the threshold of an excitable signal transduction network changes cell migratory modes. Nat. Cell Biol. 19, 329–340 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    Wilson, K. et al. Mechanisms of leading edge protrusion in interstitial migration. Nat. Commun. 4, 2896 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. 36

    Vargas, P. et al. Innate control of actin nucleation determines two distinct migration behaviours in dendritic cells. Nat. Cell Biol. 18, 43–53 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. 37

    Leithner, A. et al. Diversified actin protrusions promote environmental exploration but are dispensable for locomotion of leukocytes. Nat. Cell Biol. 18, 1253–1259 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Yoo, S.K. et al. Differential regulation of protrusion and polarity by PI3K during neutrophil motility in live zebrafish. Dev. Cell 18, 226–236 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. 39

    Sun, H. et al. TIPE2 controls innate immunity to RNA by targeting the phosphatidylinositol 3-kinase-Rac pathway. J. Immunol. 189, 2768–2773 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    Sun, J., Hilliard, B., Xu, L. & Chen, Y.H. Essential roles of the Fas-associated death domain in autoimmune encephalomyelitis. J. Immunol. 175, 4783–4788 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. 41

    Xu, L. et al. Arginase and autoimmune inflammation in the central nervous system. Immunology 110, 141–148 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. 42

    Ruan, Q. et al. Development of Foxp3(+) regulatory t cells is driven by the c-Rel enhanceosome. Immunity 31, 932–940 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. 43

    Ruan, Q. et al. The Th17 immune response is controlled by the Rel-RORγ-RORγ T transcriptional axis. J. Exp. Med. 208, 2321–2333 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. 44

    Wang, T., Wei, J.J., Sabatini, D.M. & Lander, E.S. Genetic screens in human cells using the CRISPR-Cas9 system. Science 343, 80–84 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. 45

    Hirayama, A., Adachi, R., Otani, S., Kasahara, T. & Suzuki, K. Cofilin plays a critical role in IL-8-dependent chemotaxis of neutrophilic HL-60 cells through changes in phosphorylation. J. Leukoc. Biol. 81, 720–728 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. 46

    JoVE Science Education Database. The Transwell Migration Assay. Cell Biology. JoVE, Cambridge, MA, (2017).

  47. 47

    Zhao, H., Hakala, M. & Lappalainen, P. ADF/cofilin binds phosphoinositides in a multivalent manner to act as a PIP(2)-density sensor. Biophys. J. 98, 2327–2336 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. 48

    Knight, Z.A., Feldman, M.E., Balla, A., Balla, T. & Shokat, K.M. A membrane capture assay for lipid kinase activity. Nat. Protoc. 2, 2459–2466 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank W. Pear (University of Pennsylvania) for NGFR vector; M. Lemmon (University of Pennsylvania) for the peGFP-GRP1-PH vector and for discussions; G. Luo, N. Li, D. Johnson, A. Stout, J. Zhao, G. Ruthel, the CDB Microscopy Core, and the PennVet Imaging Core for discussions and/or technical assistance. Supported by the US National Institutes of Health (AI121166, AI099216, and AI50059 to Y.H.C.; and T32CA009140 to A.E.B.) and the National Multiple Sclerosis Society (1501-02782 to Y.H.C.).

Author information

Affiliations

Authors

Contributions

S.A.F., Z.W. and Y.H.C. conceived of the study; S.A.F. and Y.H.C. wrote the article; S.A.F. designed and performed the experiments and analyzed the data; Z.W. designed and performed the in vivo experiments; A.Z., A.E.B., T.P.P., W.X., M.L., T.C., J.R.G and A.V. were involved in the design or execution of several experiments; H.S. bred mice and performed μ-slide migration assay; and Y.H.C. supervised the study.

Corresponding author

Correspondence to Youhai H Chen.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 The effect of TIPE2 on chemotaxis.

a, The expression of TIPE2 in dHL-60C, dHL-60T, TIPE2-expressing and 15/16Q-expressing dHL-60T neutrophils was detected by Western blot. b and c, Migration tracks of individual wild type (b) and Tipe2−/− (c) blood neutrophils treated with CXCL1 (200 ng/ml) on μ-slides. A total of 40 cells from each genotype were selected as described in Methods. a and b, The experiments were performed three times. The results of a representative experiment are shown. c, Live cell imaging of PtdIns(3,4,5)P3 distribution in dHL-60C and dHL-60T neutrophils subjected to point-source stimulation with CXCL8 for 300 seconds. The PtdIns(3,4,5)P3 distribution was visualized using the eGFP-GRP1-PH domain. Scale bar is 5 μm. The experiments were performed two times. The results of a representative experiment are shown.

Supplementary Figure 2 The effect of TIPE2 on the polarization of bone marrow neutrophils and dHL-60 neutrophils.

a-e, The subcellular distributions of the indicated molecules in rested wild type (WT) or Tipe2−/− bone marrow neutrophils (BMNs) (a-c), and WT or Tipe2−/− BMNs subjected to point-source stimulation with CXCL2 (d and e) were determined by confocal microscopy. Data shown are representative images (a-d, scale bars are 5 μm) and the percentages of cells with polarized (pol) or unpolarized (unpol) distributions of F-actin (e). f-h, The subcellular distribution of F-actin, human (h) and murine (m) TIPE2 in dHL-60C, dHL-60T and mTIPE2-expressing dHL-60T cells rested or subjected to point-source stimulation with CXCL8 was determined by confocal microscopy. Data shown are the percentages of cells with polarized (pol) or unpolarized (unpol) distributions of F-actin (f) and representative images (g and h, scale bars are 5 μm). a-h, The experiments were repeated three times; e and f, n ≥ 46.

Supplementary Figure 3 Quantitative measurements of the responses of wild type and Tipe2−/− myeloid cells to chemoattractant stimulation.

a-f, Flow cytometric analyses of actin polymerization (F-actin) (a, c and f) and AKT phosphorylation [pAKT(308)] (b, d and e) in wild type (WT) and Tipe2−/− bone marrow neutrophils stimulated with fMLP (a and b) or CXCL2 (c and d), and dHL-60T and TIPE2-expressing dHL-60T cells stimulated with fMLP (e and f), at the indicated times. Values represent means ± SD; *, P < 0.05. The experiments were performed in triplicates and repeated three times (a-d, n=9) or two times (e and f, n=6).

Supplementary Figure 4 Selective regulation of signaling pathways by TIPE2.

a, Lysates of SW480 cells transfected with control or TIPE2 plasmids were subjected to co-immunoprecipitation (co-IP) with anti-Rac or control IgG. The precipitates were analyzed by Western blot for the indicated proteins. b, Lysates of SW480 cells transfected with control or TIPE2 plasmids (left panel), and wild type (WT) and Tipe2−/− bone marrow-derived macrophages (BMDMs) cultured with supernatant of L929 cells (middle panel) or stimulated with CCL2 for 5 min (right panel), were subjected to pull-down with PAK-GST beads. The Rac in the pull-down (Rac-GTP) and in the lysates (total Rac) was detected by Western blot using Rac-specific antibodies. c, Lysates of WT and Tipe2−/− BMDMs cultured with or without L929 cell supernatant (designated as L929 sup) or CCL2 (stimulated for 5 min) were subjected to immunoprecipitation (IP) with anti-Rac or control IgG. The precipitates were analyzed by Western blot using Rac- or mTOR-specific antibodies. d-f, Lysates of SW480 cells transfected with control or TIPE2 plasmids (d), WT and Tipe2−/− BMDMs cultured with supernatant of L929 cells (e), and WT and Tipe2−/− BMDMs treated with CCL2 for the indicated times (f) were analyzed by Western blot using antibodies to the indicated proteins. a-f, The experiments were performed at least three times. The results of a representative experiments are shown. g and h, Relative adhesion of WT and Tipe2−/− BMDMs which were rested (g) or stimulated with CCL2 (h). Cell adhesion was measured at the indicated times. i, Relative adhesion of dHL-60C, dHL-60T and TIPE2-expressing dHL-60T cells without (None) or with Rac inhibitor (Rac inh) or PI(3)K inhibitor (PI(3)K inh). For panels g-i, values represent means ± SD; *, P < 0.05; **, P < 0.01; the experiments were performed in triplicates and repeated three times (g and h, n=9) or two times (i, n=6); RU, relative units.

Supplementary Figure 5 Rac-dependent and Rac-independent functions of TIPE2 in dHL-60 neutrophils.

a-d, The subcellular distribution of F-actin (a, b and d), human (h) and murine (m) TIPE2 (b-d) in dHL-60C, dHL-60T and TIPE2-expressing dHL-60T neutrophils which were stimulated with CXCL8 (point source), with or without pretreatments with Rac inhibitor (Rac inh) (a-c) or PI(3)K inhibitor (PI(3)K inh) (a, c and d). Data shown are the percentages of cells with polarized (pol) or unpolarized (unpol) distributions of F-actin and h/mTIPE2 (a and c) and representative images (b and d, scale bars are 5 μm). ns, not significant. a-d, The experiments were repeated three times; a and c, n ≥ 46.

Supplementary Figure 6 Interactions between TIPE2 and phosphoinositides.

a, Purified recombinant TIPE2, 15/16Q, α0-eGFP, α0 15/16Q-eGFP, α0 4Q-eGFP, cofilin, and control protein trypsin inhibitor, were separated by SDS-PAGE, and stained with silver. The experiments were performed at least three times. The results of a representative experiments are shown. b, Alignment of the partial sequences of murine TNFAIP8, TIPE1, TIPE2, and TIPE3 generated by CLUSTAL W2. Identical residues are highlighted in black, and similar residues are highlighted in gray (generated by the BOXSHADE 3.21). c, The percentages of TIPE2, 15/16Q, control protein, α0-eGFP, α0 15/16Q-eGFP, and α0 4Q-eGFP bound to small unilamellar vesicles containing the indicated lipids as determined by the phosphoinositide binding assay. Values represent means ± SD; **, P < 0.01; ns, not significant; the experiments were repeated at least three times (n ≥ 3). d, A schematic model of TIPE2 action in cell polarization. The plasma membrane of the neutrophil is shown in light blue; the segmented nucleus of the neutrophil is shown in light gray. e, Degree of F-actin polarization in dHL-60C, dHL-60T, TIPE2-expressing and 15/16Q-expressing dHL-60T neutrophils stimulated with CXCL8 (point source) was determined using confocal microscopy. Values represent means ± SD; *, P < 0.05; **, P < 0.01; the experiments were repeated three times; n ≥ 46.

Supplementary Figure 7 The effect of the binding of TIPE2 to phosphoinositides on cofilin-dependent depolymerization of F-actin.

a-c, Time course of cofilin-dependent F-actin depolymerization performed in the presence or absence of control protein (a-c), TIPE2 (a-c), small unilamellar vesicles (SUV) containing 10% PtdIns(4,5)P2/10% PtdIns(3,4,5)P3 (a), SUV containing 10% PtdIns(4,5)P2 (b) or SUV containing 10% PtdIns(3,4,5)P3 (c). Results are shown as the difference in the remaining F-actin over the indicated period of time between samples containing control protein and samples containing control protein + SUV or TIPE2 + SUV. FIU, fluorescence intensity units. The experiments were repeated at least three times.

Supplementary Figure 8 Competent anti-MOG responses of TIPE2-deficient T cells.

Tipe2−/− and wild type (WT) mice were immunized with myelin oligodendrocyte glycoprotein (MOG) peptide to induce experimental autoimmune encephalomyelitis (EAE). Twenty-five days after immunization, Tipe2−/− and WT mice were sacrificed, and their splenocytes were isolated and cultured with the indicated amounts of MOG peptide for 24 h. Cytokine concentrations in the culture supernatants were determined by ELISA. Values represent means ± SD. *, P < 0.05; **, P < 0.01; the experiments were repeated three times, n = 8.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Note (PDF 2365 kb)

Life Sciences Reporting Summary (PDF 172 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Fayngerts, S., Wang, Z., Zamani, A. et al. Direction of leukocyte polarization and migration by the phosphoinositide-transfer protein TIPE2. Nat Immunol 18, 1353–1360 (2017). https://doi.org/10.1038/ni.3866

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing