Diversification of TAM receptor tyrosine kinase function

Abstract

The clearance of apoptotic cells is critical for both tissue homeostasis and the resolution of inflammation. We found that the TAM receptor tyrosine kinases Axl and Mer had distinct roles as phagocytic receptors in these two settings, in which they exhibited divergent expression, regulation and activity. Mer acted as a tolerogenic receptor in resting macrophages and during immunosuppression. In contrast, Axl was an inflammatory response receptor whose expression was induced by proinflammatory stimuli. Axl and Mer differed in their ligand specificities, ligand-receptor complex formation in tissues, and receptor shedding upon activation. These differences notwithstanding, phagocytosis by either protein was strictly dependent on receptor activation triggered by bridging of TAM receptor–ligand complexes to the 'eat-me' signal phosphatidylserine on the surface of apoptotic cells.

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Figure 1: Differences in the expression and activation of Axl and Mer.
Figure 2: The expression of Axl and Mer in inflammatory macrophages.
Figure 3: Axl is a phagocytic receptor in activated macrophages.
Figure 4: Axl and Mer kinase activity is necessary for the phagocytosis of apoptotic cells.
Figure 5: GAS-6 is bound to Axl in vivo and in vitro.
Figure 6: Axl- and Mer-activating antibodies.

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Acknowledgements

We thank M. Joens and J. Fitzpatrick for processing samples and acquiring scanning electron microscopic images; R. Evans (Salk Institute) for nuclear receptor agonists T0901317, GW501516 and BRL49653; J. Hash, P. Burrola and C. Mayer for technical support; and members of the Lemke laboratory and the Nomis Center for discussions. Supported by the US National Institutes of Health (R01 AI077058, R01 AI101400 and R01 NS085296 to G.L.), the Leona M. and Harry B. Helmsley Charitable Trust (2012-PG-MED002 to G.L.) the Nomis Foundation, the H.N. and Frances C. Berger Foundation, the Fritz B. Burns Foundation, the HKT Foundation, Françoise Gilot-Salk, the Human Frontiers Science Program (A.Z.), the Marie Curie Seventh Framework Programme (P.G.T.), the Leukemia and Lymphoma Society and the Nomis Foundation (E.D.L.).

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Contributions

A.Z. designed and performed the experiments; P.G.T. aided in the design and execution of in vivo experiments; E.D.L. prepared purified recombinant GAS-6; I.D. aided in the design of the flow-cytometry-based phagocytosis assay; G.L. contributed to the design of the experiments and wrote the manuscript.

Corresponding author

Correspondence to Greg Lemke.

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Competing interests

G.L. is a shareholder in Kolltan Pharmaceuticals.

Integrated supplementary information

Supplementary Figure 1 Expression of Axl and Mer on populations of cells of the immune system in vivo.

Immunohistochemistry of Axl and Mer in spleen (a), liver (b), and lung (c). Closed arrowheads: cells co-expressing Axl and Mer; open arrowheads: cells expressing mostly Axl. In the spleen, the principal Axl-Mer co-expressing cells are F4/80+ red pulp (RP) macrophages. In the splenic white pulp (WP) (d), Tingible body macrophages (F4/80-CD68+) express Mer and low levels of Axl (closed arrowheads), whereas a subpopulation of splenic CD11c+ DCs express only Axl (open arrowheads). In the lung (c and e), alveolar macrophages (CD11c+CD11b-MHCII-F4/80lo) are only Axl-positive (open arrowheads). In the liver, F4/80+ Kupffer cells are both Axl and Mer positive (b, closed arrowheads). Bars, 50 μm (a-c); 10 μm (d); and 20 μm (e). Representative images from n=3 mice.

Supplementary Figure 2 Discrete Axl+ and Mer+ cell populations in vitro.

(a) Unstimulated (Ctrl) or IFN-γ (250 U/ml, 18 h) treated BMDM cultures were stained live with Mer (green) and Axl (magenta) antibodies. Closed arrows: cells expressing mostly Axl; open arrows- cells expressing mostly Mer. The asterisk marks a single cell that is weakly positive for both Axl and Mer. Bar, 20 μm. Representative images of three independent experiments.(b,c) BMDM cultures were stimulated with 0.1 μM Dex or 10 μg/ml poly(I:C) for 24 h, fixed and stained with Mer (b) or Axl (c) antibodies and counterstained with Phalloidin-TRITC. Bar, 100 μm. Representative images of three independent experiments.

Supplementary Figure 3 Regulation of Axl and Mer by steroid hormones.

BMDM cultures were stimulated with 1 μM Dex, hydrocortisone, cortisone, aldosterone, 17β-estradiol, estrone, estriol or progesterone for 24 h. Axl and Mer expression was assayed by immunoblotting. Representative of two independent experiments.

Supplementary Figure 4 Anti-inflammatory effects of Dex are independent of Mer and Axl.

(a) RT-PCR showing the kinetics of Mertk, Fpr1, Mrc1 mRNA induction and Axl and Il21r mRNA inhibition in BMDMs in response to 0.1 μM Dex. (b) BMDMs from indicated mice were treated with 100 ng/ml LPS with or without 0.1 μM Dex. TNF secretion was measured 24 h later by ELISA in culture supernatants. Representative of three independent experiments. (c) RT-PCR showing changes in Tnf, Mfge8, Il21r and Mrc1 mRNAs in BMDMs after 24 h incubation with 0.1 μM Dex. Data in (a) and (c) are normalized to Hprt mRNA and presented as fold change relative to untreated cells. Average of two independent experiments, each done in technical duplicate, graphed as mean ± s.d. (d) Immunoblot showing changes in activity of Akt, ERK1/2 and p38 signaling pathways in response to 0.1 μM Dex treatment of BMDMs derived from indicated knock-out mice. Representative of two independent experiments.

Supplementary Figure 5 Induction of Axl by poly(I:C) and IFN-α in BMDMs.

Cells were incubated with either poly(I:C) (1 μg/ml) or IFN-α (250 U/ml) for the indicated times in hours (h), and then blotted for total Axl (top), Mer (middle), or GAPDH (bottom). Representative of two independent experiments.

Supplementary Figure 6 Flow cytometry–based phagocytosis assay.

Apoptotic cells are labeled with pH-sensitive dye, pHrodo. Once engulfed into the acidic environment of phagosomes, pHrodo fluorescence is enhanced and phagocytic macrophages are distinguished based on their side scatter (SSC-A) and pHrodo fluorescence intensity using flow cytometry. In this experiment, the percent cells in the phagocytic gate is quantified in an 1-hour assay, in absence or presence of 10 nM GAS-6. Representative plot of 6 independent experiments.

Supplementary Figure 7 Regulation of the expression of Axl and Mer and phagocytosis in BMDCs.

(a) BMDC cultures from the indicated mice were stimulated for 10 min with 10 nM GAS-6 (G) or 25 nM Protein S (S). Receptor activation was assayed by immunoprecipitation and immunoblotting. Representative of two independent experiments. (b) BMDCs were cultured for 18 h in the presence of 0.1 μM Dex or 10 μg/ml poly(I:C) and then stimulated for 10 min with 10 nM GAS-6. Receptor activation was assayed by immunoprecipitation and immunoblotting. Representative of two independent experiments. (c, d) BMDCs from mice of the indicated genotypes were cultured for 24 h in the presence of 0.1 μM Dex (c) or 10 μg/ml poly(I:C) (d) and then incubated for 1 h with pHrodo stained ACs with or without 10 nM GAS-6. Percent of phagocytosis was measured using flow cytometry. Data are presented as mean ± s.d. from two independent experiments, each done for duplicate cultures for each condition.

Supplementary Figure 8 Model for the differences in the regulation and action of Axl and Mer in inflammatory and tolerogenic environments.

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Zagórska, A., Través, P., Lew, E. et al. Diversification of TAM receptor tyrosine kinase function. Nat Immunol 15, 920–928 (2014). https://doi.org/10.1038/ni.2986

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