Journal: The Journal of Biological Chemistry

Article Title: The Gas6-Axl Protein Interaction Mediates Endothelial Uptake of Platelet Microparticles *

doi: 10.1074/jbc.M115.699058

Figure Lengend Snippet: HUVECs ingest PMPs in a Gas6-Axl-dependent manner. TAM expression on the surface of HUVECs was evaluated using flow cytometry. Antibody specificity was verified by pre-incubating the anti-TAMs with soluble TAMs prior to addition of cells (A). The relative expression of Axl and Mer on the plasma membrane was measured by Western blotting after immunoprecipitation (IP) of Axl and Mer from cell-surface biotinylated HUVECS (B). IB, immunoblot. HUVECs were incubated with fluorescent PMPs in the presence of increasing amounts of Gas6, and uptake was monitored by flow cytometry (C). TAM specificity in the uptake was evaluated by incubating the cells with polyclonal antibodies against the extracellular domain of the different TAMs before and during uptake (D). Successful biotinylation of PMPs was measured by binding of Alexa Fluor 488-conjugated streptavidin by flow cytometry (E) and by Western blot analysis followed by detection with HRP-conjugated biotin-avidin complexes (F). Several PMP proteins were found to be biotinylated, of which the most abundant around 100 kDa was used for quantification. Biotinylated PMPs pre-incubated with increasing amounts of Gas6 were added to HUVECs cells, and after 1 h at 37 °C cells were analyzed for biotin content. A representative biotin detection by Western blot is shown to the left (G). Fluorescent PMPs were incubated with Gas6 and a double amount of unlabeled PMPs before incubating with HUVECs for 1 h. The amount of phagocytosed labeled PMPs was measured in a flow cytometer (H). PMPs were incubated with Gas6 in the presence or absence of 2.5 mm EDTA before adding to the cells. MP ingestion was measured as above (I). Data in bar graphs are shown as mean and S.D. of three individual experiments except for E, which includes two separate experiments. Other panels show representative data from at least three separate experiments. ns, not significant; ****, p < 0.0001. MFI, mean fluorescent intensity.

Article Snippet: Anti-TAM antibodies (Axl, AF154 R&D Systems; Mer, ab52968 Abcam; Tyro3, AF859 R&D Systems) diluted 1:200 were preincubated with or without 10 μg/ml sTAMs in 50 μl of 1% BSA in TBS for 20 min.

Techniques: Expressing, Flow Cytometry, Membrane, Western Blot, Immunoprecipitation, Incubation, Binding Assay, Avidin-Biotin Assay, Labeling

Journal: The Journal of Biological Chemistry

Article Title: The Gas6-Axl Protein Interaction Mediates Endothelial Uptake of Platelet Microparticles *

doi: 10.1074/jbc.M115.699058

Figure Lengend Snippet: HAECs ingest PMPs in a Gas6-Axl-dependent manner. TAM expression was evaluated in the HAECs using flow cytometry (A). Relative expression of Axl and Mer at the cell surface was evaluated by Western blotting of immunoprecipitated TAMs after cell surface biotinylation (B). Fluorescent PMPs were incubated with Gas6 and fed to HAECs for 1 h at 37 °C. PMP phagocytosis was measured by flow cytometry (C). The TAM dependence of the uptake was evaluated by introducing TAM-targeting antibodies during the uptake (D) or by treating the HAECs with siRNA targeting the different TAMs (E). The knockdown efficiency of the TAM siRNA treatment is shown in F. Fluorescent PMPs were incubated with recombinant in-house purified Gas6 or commercially available non-γ-carboxylated Gas6 before incubation with HAECs (G). Uptake was measured as described in D. Fluorescent PMPs with or without Gas6 were incubated with HAECs for increasing times at 37 °C to measure the time dependence of the uptake (H). Uptake was measured using flow cytometry as described in E. Data in bar graphs are shown as mean and S.D. of three individual experiments. Other panels show representative data from at least three separate experiments. ns, not significant; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: Anti-TAM antibodies (Axl, AF154 R&D Systems; Mer, ab52968 Abcam; Tyro3, AF859 R&D Systems) diluted 1:200 were preincubated with or without 10 μg/ml sTAMs in 50 μl of 1% BSA in TBS for 20 min.

Techniques: Expressing, Flow Cytometry, Western Blot, Immunoprecipitation, Incubation, Recombinant, Purification

Journal: PLoS Pathogens

Article Title: Phosphatidylserine receptors enhance SARS-CoV-2 infection

doi: 10.1371/journal.ppat.1009743

Figure Lengend Snippet: A) Cells transfected with PS receptor expression plasmids, AXL or TIM-1, with or without 50 ng of ACE2 and infected 48 hours later with SARS-CoV-2 (MOI = 0.5). Resulting cellular infection was determined by viral loads 24 hours after initial infection using RT-qPCR. B-C) PS receptors, TIM-1 (B) and AXL (C) , enhance rVSV/Spike infection at low concentrations of transfected ACE2. D) Virus binding of cells transfected with PS receptor plasmids with or without 50 ng of ACE2. rVSV/Spike was bound to transfected cells at 48 h following transfection and bound virus was measured via RT-qPCR . E) PS receptors mediate internalization of rVSV/Spike. Virion internalization was measured at 24 h after transfection with 1 μg of the indicated plasmids. FITC-labeled rVSV/Spike was bound for 1-hour, unbound virions washed away, and cells shifted to 37°C for 30 minutes. Non-internalized virus was then cleaved from cell surface by trypsin. Cells were washed, and FITC retention quantified by flow cytometry. F) HEK 293T cells transfected with PS receptor plasmids, TYRO3 or TIM-4, with or without 250 ng of ACE2 and infected 48 hours later with VSV/Spike. Viral loads were determined 24 hours following infection. Data shown are pooled from at least 3 independent experiments ( A , B , C , D , E , F ). Data represented as means ± SEM. Student’s t-test (A, E) and multiple t-test (B, C) , One-Way ANOVA with multiple comparisons (D, F) ; asterisks represent p < 0.05.

Article Snippet: Specific primary antibodies used as follows: goat anti-ACE2 (R&D; AF933), goat anti-AXL (R&D; 154), goat anti-TIM-1 (R&D; 1750), goat anti- Tyro3 (R&D; AF859), goat anti-TIM-4 (R&D; 2929), goat anti-MerTK (R&D; AF891), rabbit anti-TMPRSS2 (ABCAM; ab92323).

Techniques: Transfection, Expressing, Infection, Quantitative RT-PCR, Binding Assay, Labeling, Flow Cytometry

Journal: Cells

Article Title: TGF-β1 Promotes Zika Virus Infection in Immortalized Human First-Trimester Trophoblasts via the Smad Pathway

doi: 10.3390/cells11193026

Figure Lengend Snippet: Expression of Tyro3 and AXL on the surface Swan.71 cells. ( A ) Immunofluorescence assay. The cells were cultured on glass coverslips in 6-well plates and stained for Tyro3 or AXL. Scale bar: 100 μM. ( B ) Flow cytometry analysis. Trophoblast cells were cultured in a 96-well plate and stained for Tyro3 or AXL. Numbers displayed inside each panel correspond to the percentage of positive cells for Tyro3 or AXL of the parent-gated population. ( A , B ) Data obtained from Vero cells performed in parallel were used as comparative references.

Article Snippet: The detachment medium containing appropriate primary antibodies, i.e., a rabbit polyclonal anti-Tyro3 antibody (28513-1-AP, Proteintech) or a rabbit polyclonal anti-AXL antibody (GTX129407, GeneTex), was added and incubated in an incubator for approximately 1 h. After one wash with STB, the cells were incubated with a goat anti-rabbit IgG H&L (Alexa Fluor 488) secondary antibody (ab150081, Abcam).

Techniques: Expressing, Immunofluorescence, Cell Culture, Staining, Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Viral Infection sensitizes Human Fetal Membranes to Bacterial LPS by MERTK Inhibition and Inflammasome Activation 1

doi: 10.4049/jimmunol.1700870

Figure Lengend Snippet: Primers used for qRT-PCR

Article Snippet: TYRO3, AXL, and MERTK levels were evaluated by Western blot as previously described ( 8 ) using the anti-human primary antibodies against TYRO3 (#MAB859, R&D Systems), AXL (#AF154, R&D Systems), and MERTK (#AF891, R&D Systems).

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Viral Infection sensitizes Human Fetal Membranes to Bacterial LPS by MERTK Inhibition and Inflammasome Activation 1

doi: 10.4049/jimmunol.1700870

Figure Lengend Snippet: (A) Untreated FM explants (n=3) were homogenized and analyzed for expression of TYRO3, AXL, MERTK, GAS6 and PROS1 mRNA by qRT-PCR. (B–C) Human FM explants were treated with no treatment (NT), LPS (100ng/ml), MHV-68 (1.5×104/ml PFU) or both MHV-68 and LPS in the presence of media or rGas6 (50ng/ml) (n=5). Tissues were homogenized for protein and Western blot performed for (B) AXL (~140kDa) and (C) MERTK (~180kDa). Blots are from one representative experiment. Bar charts show AXL and MERTK expression as determined by densitometry and normalized to b-actin. (D) FM explants were treated with NT, LPS (1ng/ml), Poly(I:C) (20µg/ml) or both Poly(I:C) and LPS (n=4). Tissues were homogenized for protein and Western blot performed for AXL and MERTK. Blots are from one representative experiment. Bar charts show AXL and MERTK expression as determined by densitometry and normalized to β-actin. (E–F) Human FM explants were treated with no treatment (NT), LPS, MHV-68 or both MHV-68 and LPS in either the presence of media or rGAS6. Tissues were homogenized for protein and ELISA performed for (E) sMERTK (n=7); (F) GAS6 (n=5), and (G) PROS1 (n=8). *p<0.05 relative to the NT control unless otherwise indicated. Data are expressed as mean±SEM.

Article Snippet: TYRO3, AXL, and MERTK levels were evaluated by Western blot as previously described ( 8 ) using the anti-human primary antibodies against TYRO3 (#MAB859, R&D Systems), AXL (#AF154, R&D Systems), and MERTK (#AF891, R&D Systems).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Viral Infection sensitizes Human Fetal Membranes to Bacterial LPS by MERTK Inhibition and Inflammasome Activation 1

doi: 10.4049/jimmunol.1700870

Figure Lengend Snippet: (A) Human FM explants were treated with no treatment (NT), LPS (100ng/ml), MHV-68 (1.5×104/ml PFU) or both MHV-68 and LPS in the presence of media or rGas6 (50ng/ml). (i) IL-1β secretion in supernatants was measured by ELISA (n=6). Lysates were evaluated by Western blot for expression of (ii) pro-IL-1β and (iii) active IL-1β. Bar charts show pro- and active-IL-1β expression as determined by densitometry and normalized to β-actin (n=4–5) *p<0.05 relative to the NT control unless otherwise indicated. (B) Human FM explants were treated with or without LPS (1ng/ml) in the presence of blocking anti-TAM Abs or isotype control Abs. IL-1β secretion was measured and levels expressed as LPS-induced fold change (FC) relative to the NT control (n=4; *p<0.05 relative to the isotype control). (C–D) Pregnant wildtype or AXL−/−MERTK−/− mice were injected on E15.5 with either PBS or LPS (20µg/kg). After 6hrs mice were sacrificed. The FMs were harvested and homogenized for RNA. (D) TYRO3, AXL, MERTK, GAS6 and PROS1 mRNA expression levels in FMs from PBS treated wildtype mice (n=3) were measured by qRT-PCR. (E) IL-1B mRNA expression levels in FMs from PBS and LPS treated wildtype or AXL−/−MERTK−/− mice were measured by qRT-PCR. *p<0.05 relative to the control groups unless otherwise indicated. Data are expressed as mean±SEM.

Article Snippet: TYRO3, AXL, and MERTK levels were evaluated by Western blot as previously described ( 8 ) using the anti-human primary antibodies against TYRO3 (#MAB859, R&D Systems), AXL (#AF154, R&D Systems), and MERTK (#AF891, R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Control, Blocking Assay, Injection, Quantitative RT-PCR

Journal: PLoS Pathogens

Article Title: GAS6 signaling tempers Th17 development in patients with multiple sclerosis and helminth infection

doi: 10.1371/journal.ppat.1009176

Figure Lengend Snippet: A ) Representative dot plots showing the three main sub-populations of mononuclear cells based on the expression of CD11b, CD4, and CD1c. TYRO3, AXL, and MERTK expression were evaluated in circulating monocytes (CD11b high CD4 mid ) and DCs (CD1c high CD11b low ) of patients with MS, patients with HIMS, and healthy controls by flow cytometry. B-G ) The percentage of TYRO3-positive as well as the mean fluorescent intensity (MFI) of the receptor on monocytes ( B, C, and D ) and DCs ( E, F, and G ) are graphed. H, I, and J ) The percentage of AXL high as well as the MFI of the receptor on DCs are shown. K, L, and M ) The expression of MERTK on DCs is shown as both percentage and MFI. N, O, and P ) The percentage of circulating CD4 + T cell expressing GAS6 as well as its MFI are shown. Q, R, and S ) The percentage of circulating CD4 + T cell expressing PROS1 and its MFI are shown. Data is presented as a pool of independent samples included in the specific staining (Control N = 21–31; MS N = 10–27; and HIMS N = 11–16). One-way ANOVA with a Fisher post hoc test was performed to determine statistical significances, *p<0.05 **p≤0.01 ****p≤0.001. MS = multiple sclerosis, HIMS = helminth-infected multiple sclerosis.

Article Snippet: Rabbit anti-human TYRO3 (Novus Biological), biotin-conjugated goat anti-human AXL, and APC anti-human MERTK mAb IgG1 (R&D Systems) were used to evaluate TAM receptor expression after fixation and permeabilization (Cytofix/Cytoperm Kit, BD Bioscience, San Diego, CA, USA).

Techniques: Expressing, Flow Cytometry, Staining, Control, Infection

Journal: PLoS Pathogens

Article Title: GAS6 signaling tempers Th17 development in patients with multiple sclerosis and helminth infection

doi: 10.1371/journal.ppat.1009176

Figure Lengend Snippet: The host type 2 immune response is elicited to clear parasitic helminth infections; however, the co-evolution of these parasites has promoted a variety of mechanisms to oppose and redirect immune responses by manipulating immune cell programming and function. Integrating our results with the current knowledge, we propose that gastrointestinal ( 1 ) helminths infections can enhance the regulatory axis of TAM receptors (TYRO3, AXL, and MERTK) in peripheral blood ( 2 ) CD11b high and CD1c high cells, and their ligand GAS6 in CD4 + T cells of patients with MS. This negative regulatory pathway could be essential for dampening co-stimulatory signals (CD40, CD80, and CD86) of antigen-presenting cells and controlling the pathological Th17 (pTh17) response at secondary lymphoid organs ( 3 ). The active chronic infection resets T helper response toward Th2/regulatory signals, limiting pathological Th17 (pTh17) effector response. This reprogramming of CD4 + T cells and GAS6 signaling under a helminth-induced type 2 environment could be essential for maintaining the balance between CD4 + IL-10 + and Th17 cells and reducing inflammatory IL-17 and IFNγ signals in the central nervous system ( 4 ). In summary, GAS6 tempers not only the innate immune response but also regulates Th17 development in an autocrine/paracrine manner.

Article Snippet: Rabbit anti-human TYRO3 (Novus Biological), biotin-conjugated goat anti-human AXL, and APC anti-human MERTK mAb IgG1 (R&D Systems) were used to evaluate TAM receptor expression after fixation and permeabilization (Cytofix/Cytoperm Kit, BD Bioscience, San Diego, CA, USA).

Techniques: Infection