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. 2020 Apr 20;10(1):6617.
doi: 10.1038/s41598-020-63629-w.

The TIPE Molecular Pilot That Directs Lymphocyte Migration in Health and Inflammation

Honghong Sun #  1 , Mei Lin #  1 , Ali Zamani  1 , Jason R Goldsmith  1 , Amanda E Boggs  1 , Mingyue Li  1 , Chin-Nien Lee  1 , Xu Chen  1 , Xinyuan Li  1 , Ting Li  1 , Brigid L Dorrity  1 , Ning Li  1 , Yunwei Lou  1 , Songlin Shi  1 , Wei Wang  1 , Youhai H Chen  2
Affiliations

The TIPE Molecular Pilot That Directs Lymphocyte Migration in Health and Inflammation

Honghong Sun et al. Sci Rep. .

Abstract

Lymphocytes are some of the most motile cells of vertebrates, constantly navigating through various organ systems. Their specific positioning in the body is delicately controlled by site-specific directional cues such as chemokines. While it has long been suspected that an intrinsic molecular pilot, akin to a ship's pilot, guides lymphocyte navigation, the nature of this pilot is unknown. Here we show that the TIPE (TNF-α-induced protein 8-like) family of proteins pilot lymphocytes by steering them toward chemokines. TIPE proteins are carriers of lipid second messengers. They mediate chemokine-induced local generation of phosphoinositide second messengers, but inhibit global activation of the small GTPase Rac. TIPE-deficient T lymphocytes are completely pilot-less: they are unable to migrate toward chemokines despite their normal ability to move randomly. As a consequence, TIPE-deficient mice have a marked defect in positioning their T lymphocytes to various tissues, both at the steady-state and during inflammation. Thus, TIPE proteins pilot lymphocytes during migration and may be targeted for the treatment of lymphocyte-related disorders.

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Conflict of interest statement

Y.H.C. is a member of the advisory boards of Amshenn Co. and Binde Co. None of the other authors have any competing interests to disclose.

Figures

Figure 1
Figure 1
Complete loss of directionality, but not speed, of TIPE-deficient T cells. (a–c) Migration tracks (a) directionality (b) and speed (c) of CD4+ T cells from WT, Tnfaip8−/−, Tipe2−/−, and Tnfaip8−/−Tipe2−/− (DKO) mice (4 mice per group), in response to CCL21, as determined in the μ-slide migration assay. n = 25 cells per group (a); n = 30 cells for WT, 31 for Tnfaip8−/−, 29 for Tipe2−/− and 36 for DKO group (b,c). (d–f) Transmigration of CD4+ T cells from mice of the indicated genotypes (4 mice per group) (d, n = 18 samples per group), together with DKO cells transfected with the expression plasmids for TIPE2 or TIPE2 mutant (TIPE2mt) (e, n = 8 per group), or cells treated with the PI3K inhibitor LY290004 or Rac inhibitor NSC23766 (f, n = 12 per group), as determined in the transwell transmigration assay with CCL21. Data are representative of at least three independent experiments (a-c), or are pooled from three (e,f) or four experiments (d). Values are mean ± s.e.m. of n biologically independent samples (b–f). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (Mann-Whitney U test (b) or Student’s t-test (c–f)).
Figure 2
Figure 2
Essential roles of TIPE proteins in chemokine-induced PtdIns(3,4,5)P3 generation, and morphological and Rac1-GTP polarization. (a) PtdIns(3,4,5)P3 levels detected by AKT-PH-GFP biosensor in live WT and DKO CD4+ T cells, in response to point source stimulation of CCL21, as visualized by super-resolution fluorescence video microscopy. Upper panels show a WT and a DKO T cells 60 sec after CCL21 stimulation, with arrows pointing to the source of CCL21. The lower panel shows the changes in PtdIns(3,4,5)P3 levels relative to Time 0 when CCL21 was applied; values are mean ± s.e.m., with n = 20 cells for each group. (b) Morphological polarization of WT and DKO CD4+ T cells, in response to point source stimulation of CCL21, as visualized by phase contrast microscopy. Upper panels show a polarized WT and an unpolarized DKO T cells 10 min after CCL21 stimulation. The lower panel shows the percentages of polarized and unpolarized cells 10 min after CCL21 stimulation; n = 308 cells for WT and 304 cells for DKO group. (c,d) Rac1-GTP polarization in WT and DKO CD4+ T cells, in response to point source stimulation of CCL21, as visualized by immunofluorescence microscopy. Panel-c shows WT and DKO T cells 60 sec after incubation with CCL21 or medium alone (Control). Panel-d shows the calculated polarization index of each group treated as in Panel-c; n = 16–24 cells for WT and 17–18 for DKO group. Values are mean ± s.e.m. The experiments were repeated independently at least three times (a–d) with similar results. *P < 0.05; **P < 0.01; ****P < 0.0001 (Mann-Whitney U test (a) or Student’s t-test (b,d)).
Figure 3
Figure 3
TNFAIP8 binding to phosphoinositides, and its effect on PtdIns(3,4,5)P3 generation by PI3Ks. (a,b) SPR analysis of TNFAIP8 binding to DOPC membranes containing 10% (mole/mole) PtdIns(4,5)P2 (a) or PtdIns(3,4,5)P3 (b) on L1 sensor chip. Purified PLCδ-PH and GRP1-PH domains were used as positive controls, and trypsin inhibitor as a negative control (Control). The percent of maximal binding (Left Panels) and equilibrium KD (Right Panels) are shown. (c) Sedimentation-based phosphoinositide binding assay showing the proportion of TNFAIP8 and control protein trypsin inhibitor bound to SUVs containing 100% PC, 10% PtdIns(4,5)P2 or 10% PtdIns(3,4,5)P3. (d) Phosphorylation of PtdIns(4,5)P2 by PI3Ks as measured in the ADP-Glo kinase assay, in the presence of increasing concentrations of murine TNFAIP8 protein or 2 μM BSA (Control). The values are mean ± s.d. (a–c) or mean ± s.e.m. (d). Data represent three independent experiments (a,b) or are pooled from four (c) or two independent experiments done in duplicates (d). ***P < 0.001 (Student’s t-test (c)).
Figure 4
Figure 4
Abnormal positioning of leukocytes in tissues of TIPE-deficient mice under the steady state. (a) The total white blood cell, lymphocyte, and neutrophil counts of the blood of normal 6–8-week-old WT (n = 37), Tnfaip8−/− (n = 10), Tipe2−/− (n = 15), and DKO (n = 35) mice. (b) The numbers of the indicated cell subsets of thymus of WT (n = 6) and DKO (n = 8) mice as determined by flow cytometry. CD4+ and CD8+ denote the respective single positive cells; DP denotes double positive cells; DN denotes double negative cells. (c) The numbers of the indicated cell subsets of spleen of WT (n = 6) and DKO (n = 8) mice as determined by flow cytometry. (d) Relative numbers of the indicated intraepithelial cells per mg of small intestine of WT (n = 10 mice for CD45+ and 9 for CD3+ cells) and DKO (n = 13 for CD45+ and 8 for CD3+ cells) mice as determined by flow cytometry. Data are normalized to the mean of the respective WT group. The values are mean ± s.e.m. (a–d), and are pooled from two (b,c) or three (a,d) independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 (Student’s t-test (a–d)).
Figure 5
Figure 5
Abnormal positioning of leukocytes in the nervous tissue of TIPE-deficient mice during encephalomyelitis. (a) EAE scores of Rag2−/− mice (n = 4 mice per group) that were injected with MOG-specific WT or DKO CD4+ T cells. (b) The percentages of WT CD45.1+ and DKO CD45.2+ T cells among total CD3+ T cells in the blood (BLD) and spinal cord (Sp Cord) of EAE mice (n = 7 mice per group) that were injected with WT and DKO bone marrow cells at 1:1 ratio. Samples were collected one day after EAE onset. (c) The percentages of transferred CMTMR-labeled WT and CMFDA-labeled DKO cells among total leukocytes in the blood (BLD), spleen (SPL), and spinal cord (Sp Cord) of EAE mice (n = 5 mice per group) that were injected with the respective MOG-specific WT and DKO CD4+ T cells at 1:1 ratio. Samples were collected on the day of the EAE onset. The values are mean ± s.e.m. (a–c), and are pooled from two independent experiments (b,c) or are representative of four independent experiments (a). *P < 0.05; **P < 0.01; ****P < 0.0001 (Mann-Whitney U test (a,b) or Student’s t-test (c)).
Figure 6
Figure 6
The TIPE molecular pilot. Phospholipid second messengers are present in two distinct forms in cells: the lipid membrane-anchored form and the TIPE-anchored form, e.g., TIPE-PIP2 and TIPE-PIP3. The membrane-anchored form has its acyl chains inserted into the lipid bilayer and can function only at the membrane. On the other hand, the TIPE-anchored form of phosphoinositides has its acyl chains hidden inside the TIPE hydrophobic cavity and its charged head group exposed on the surface. The TIPE-anchored form is water-soluble and freely diffusible, and can function at the membrane-cytosol interface and in the cytosol. See text for more details.
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