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Myosin light chains 9 and 12 are functional ligands for CD69 that regulate airway inflammation

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Science Immunology  16 Sep 2016:
Vol. 1, Issue 3, pp. eaaf9154
DOI: 10.1126/sciimmunol.aaf9154
  • Fig. 1 Identification of Myl9/12 as interacting proteins for CD69.

    (A) BM extracts from C57BL/6 mice mixed with recombinant GST-His-CD69EC proteins were subjected to affinity purification using anti-GST antibody, followed by SDS-PAGE and Coomassie brilliant blue staining. Black arrowhead indicates CD69EC-interacting proteins (Myl9/12), and white arrowheads indicate recombinant CD69EC protein. Input was subjected to SDS-PAGE in parallel. (B) ELISAs were used to detect interactions between GST-His-Myl9 protein and 3×Flag CD69EC protein with or without PNGase F treatment. (Left) **P = 0.00039, **P = 0.0000015, **P = 0.000003, **P = 0.0000015, and **P = 0.000018 (t test; n = 3). (Right) **P = 0.000001, **P = 0.0000001, **P = 0.0000001, **P = 0.00000006, and **P = 0.000004 (t test; n = 3). (C) PNGase F–treated 3×Flag-CD69EC proteins were mixed with GST-His-Myl9 WT or mutant proteins. The mixture was subjected to co-IP with anti-Flag antibody, followed by IB with anti-His antibody. Input was also subjected to IB in parallel. Schematic representations of the GST-His-Myl9 WT and mutants are shown (bottom). (D) ELISA was used to detect interaction between PNGase F–treated 3×Flag CD69EC protein and GST-His-Myl9 in the presence of anti-Myl9/12 antibodies. Red: *P = 0.015, **P = 0.000018, **P = 0.000000099, and **P = 0.00000003 (t test; n = 3). Green: **P = 0.0015, **P = 0.000023, **P = 0.0000045, and **P = 0.000000075 (t test; n = 3). Blue: *P = 0.024, **P = 0.0045, and **P = 0.000000026 (t test; n = 3). Data are representative of at least three (A, B, D) or two (C) independent experiments.

  • Fig. 2 Attenuation of allergic airway inflammation by administration of anti-Myl9/12 antibody.

    (A) Schematic outline of the OVA-induced allergic airway inflammation model. (B) Leukocyte infiltration and mucus hyperproduction in the lung, as assessed by H&E staining (top; scale bars, 50 μm) and PAS staining (bottom; scale bars, 50 μm). (C) Absolute numbers of eosinophils (Eos.), neutrophils (Neu.), lymphocytes (Lym.), and macrophages (Mac.) in BAL fluid shown with the SEM (**P = 0.0009, **P = 0.0084, **P = 0.0002, **P = 0.0002, and **P = 0.003, t test; n = 5). (D) Cytokines in BAL fluid shown with the SEM. (E) Methacholine-induced AHR shown with the SEM (*P = 0.032 and *P = 0.043, t test; n = 5). RL, lung resistance; ND, not detected. (F) Schematic outline of HDM-induced airway inflammation model. i.n., intranasally. (G) Leukocyte infiltration in the lung, as assessed by H&E staining (scale bars, 50 μm). (H) Absolute numbers of eosinophils (Eos.), neutrophils (Neu.), lymphocytes (Lym.), and macrophages (Mac.) in BAL fluid shown with the SEM (**P = 0.009 and **P = 0.0092, t test; n = 7). All data are representative of more than three (A to E) or two (F to H) independent experiments. At least five mice per group were analyzed.

  • Fig. 3 Increased Myl9/12 expression on the luminal surface of blood vessels in the lungs during airway inflammation.

    (A) Myl9/12 expression in the lung with or without OVA inhalation, as detected by IB. (B) Immunohistological analysis of lung sections with or without OVA inhalation, stained as indicated. White square indicates blood vessels, and asterisk indicates bronchi. (C) Frequencies of Myl9/12-positive blood vessels in the lung (**P = 0.00074, t test; n = 3). Data are representative of at least three (A to C) independent experiments. At least three mice per group were analyzed in all experiments.

  • Fig. 4 Platelet-dependent formation of Myl9/12 net-like structures during airway inflammation.

    (A) Immunohistological analysis of megakaryocytes in a BM section, stained as indicated. (B) IB of Myl9/12 protein expression in purified PRP and splenic T cells. (C) ELISA to detect Myl9/12 protein in the supernatant from the culture where platelets were stimulated with or without thrombin for 2 hours in vitro (**P = 0.000068, t test; n = 3). (D) mRNA expression of Myl9, Myl12a, and Myl12b in platelets and sorted lung endothelial cells. Gene expression levels were normalized to those of Hprt. (E and F) Immunohistological analysis of lung sections after OVA inhalation, stained as indicated and merged with differential interference contrast (DIC) as indicated. (G) Three-dimensional image from immunohistological analysis using 20 μm lung sections of mice with OVA inhalation, stained as indicated. (H) Schematic outline of the allergic airway inflammation model with anti-GPIbα antibody treatment. (I) Immunohistological analysis of lung sections after OVA inhalation with anti-GPIbα antibody treatment, stained as indicated. (J) Myl9/12 expression in the lung after OVA inhalation with anti-GPIbα antibody treatment. Data are representative of at least three (A to F) or two (H to J) independent experiments. At least three mice per group were analyzed in all experiments.

  • Fig. 5 Myl9/12 expression on polyps from human ECRS patients.

    (A) Immunohistological analysis of polyps from patients with ECRS (right) and noninflamed nasal mucosa as controls (left), stained as indicated. Fluorescence intensity of Myl9/12 staining is shown (right). Five normal mucosae and nine polyps from ECRS patients were analyzed (**P = 0.000039, t test; n = 5 and n = 9). (B to D) Immunohistological analysis of polyps from ECRS patients, stained as indicated. At least four polyps from ECRS patients were analyzed.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/1/3/eaaf9154/DC1

    Materials and Methods

    Fig. S1. Identification of Myl9/12 as interacting proteins for CD69.

    Fig. S2. Impacts of polyclonal anti-Myl9/12 antibodies on OVA-induced airway inflammation.

    Fig. S3. Myl9/12 expression in the lung.

    Fig. S4. Myl9/12 protein expression in platelets.

    Fig. S5. Detection of CD69+ antigen-specific CD4 T cells in the lung vasculature during airway inflammation.

    Fig. S6. Effects of anti-Myl9/12 and antihuman CD69 antibodies on human CD69-mediated allergic airway inflammation.

    Fig. S7. Distribution of CD69+ T cells and Myl9/12 expression in polyps of ECRS patients.

    Fig. S8. Unmodified images and isotype control staining in immunohistochemistry.

    Movie S1. Myl9/12 form net-like structures in blood vessels of the inflamed lung.

  • Supplementary Materials

    Supplementary Material for:

    Myosin light chains 9 and 12 are functional ligands for CD69 that regulate airway inflammation

    Koji Hayashizaki, Motoko Y. Kimura, Koji Tokoyoda, Hiroyuki Hosokawa, Kenta Shinoda, Kiyoshi Hirahara, Tomomi Ichikawa, Atsushi Onodera, Asami Hanazawa, Chiaki Iwamura, Jungo Kakuta, Kenzo Muramoto, Shinichiro Motohashi, Damon J. Tumes, Tomohisa Iinuma, Heizaburo Yamamoto, Yuzuru Ikehara, Yoshitaka Okamoto, Toshinori Nakayama*

    *Corresponding author. Email: tnakayama{at}faculty.chiba-u.jp

    Published 16 September 2016, Sci. Immunol. 1, eaaf9154 (2016)
    DOI: 10.1126/sciimmunol.aaf9154

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Identification of Myl9/12 as interacting proteins for CD69.
    • Fig. S2. Impacts of polyclonal anti-Myl9/12 antibodies on OVA-induced airway inflammation.
    • Fig. S3. Myl9/12 expression in the lung.
    • Fig. S4. Myl9/12 protein expression in platelets.
    • Fig. S5. Detection of CD69+ antigen-specific CD4 T cells in the lung vasculature during airway inflammation.
    • Fig. S6. Effects of anti-Myl9/12 and antihuman CD69 antibodies on human CD69-mediated allergic airway inflammation.
    • Fig. S7. Distribution of CD69+ T cells and Myl9/12 expression in polyps of ECRS patients.
    • Fig. S8. Unmodified images and isotype control staining in immunohistochemistry.
    • Legend for movie S1

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). Myl9/12 form net-like structures in blood vessels of the inflamed lung.

    Download Movie S1

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