Research ArticleImmunology

CARD9 mediates dendritic cell–induced development of Lyn deficiency–associated autoimmune and inflammatory diseases

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Science Signaling  08 Oct 2019:
Vol. 12, Issue 602, eaao3829
DOI: 10.1126/scisignal.aao3829
  • Fig. 1 CARD9 promotes colitis in Lyn−/− mice.

    (A) The indicated mice were given drinking water treated with 3.5% DSS for days 0 to 5, followed by untreated drinking water for the remainder of the experiment. The weights of the animals were monitored daily and are plotted as a percentage of starting weight at day 0. (B) Bleeding/prolapse score of the indicated mice after DSS treatment described in (A) was analyzed and reported with the scales of 0, 0.5, 1.0, 1.5, and 2.0 for increasing severity. (C) Colon lengths of the indicated mice after the indicated DSS treatment. (D) The survival of mice after DSS treatment described in (A) was assessed daily and plotted as percent survival. (E and F) The indicated mice described in (A) were monitored for spontaneous rectal prolapse and the ages of prolapse development. ND, not detected. *P < 0.05, **P < 0.01, by one-way ANOVA with Tukey post hoc test for (A) to (C) and (F), and Z test for proportions (D and E). Each group represents n = 5 to 10 mice at 8 to 10 weeks of age and 17 to 19 g at day 0. Data are representative of at least three independent experiments.

  • Fig. 2 CARD9 plays a major role in the development of autoimmunity in Lyn−/− mice.

    (A to D) Serum was collected from the indicated mice at the indicated age. The amount of anti-ssRNA or anti-dsDNA antibodies in the serum was analyzed by ELISA. *P < 0.05, **P < 0.01, one-way ANOVA with Tukey post hoc test; each bar represents n = 5 to 10 mice per group. Results are representative of at least two independent experiments. OD, optical density. (E) H&E-stained kidney sections from the indicated mice at 3.5 months of age. Scale bars, 10 μm (top) and 1 μm (bottom).

  • Fig. 3 CARD9 is required for cellular activation in Lyn-deficient mice.

    (A and B) CD11c+ DCs were isolated from the spleen of the indicated mice by negative selection, plated, and stimulated with Pam3CSK4 (100 ng/ml) or LPS (100 ng/ml) for the indicated times. The amounts of TNF-α (A) or IL-6 (B) in the supernatants were measured by ELISA. (C to E) Cells were harvested from spleens of indicated mice at 2 months of age, stained with fluorescently labeled antibodies, and analyzed by flow cytometry using the gating strategy shown in fig. S7. *P < 0.05, **P < 0.01, one-way ANOVA with Tukey post hoc test; each bar represents n = 5 to 10 mice per group or cells pooled from 3 to 5 mice per group. Results are representative of at least three independent cohorts of mice.

  • Fig. 4 CARD9 is required for the increased TLR-triggered signaling and cytokine production in Lyn-deficient BMDCs, but not in macrophages.

    (A to C) BMDCs or BMMs from the indicated mice were stimulated with the indicated amount of Pam3CSK4 and LPS for 3 hours. Cell supernatants were analyzed for TNF-α and IL-6 by ELISA. (D to G) BMDCs or BMMs from the indicated mice were stimulated with Pam3CSK4 (100 ng/ml) for the indicated time [(G) is from the 30-min time point]. Cells were lysed and immunoblotted with the indicated antibodies. Densitometry analysis of the Western blots is shown in fig. S1. Each Western blot is representative of at least three independent experiments with cells pooled from three to five mice per group. *P < 0.05, one-way ANOVA with Tukey post hoc test.

  • Fig. 5 Regulation of TLR-triggered signaling and cytokine production by Lyn requires CD11b and SFKs Hck and Fgr.

    (A to C) BMDCs or BMMs from the indicated mice were stimulated with Pam3CSK4 (100 ng/ml) for the indicated time. Cells were lysed and immunoblotted with the indicated antibodies. (D) BMDCs from the indicated mice were either centrifuged onto the bottom of tissue culture–treated plates (Att) or left in suspension (Unatt) with or without the addition of antibody to CD11b (Mac-1) for 30 min (αCD11b). Cells were stimulated with Pam3CSK4 (100 ng/ml) for the indicated times, and supernatants were analyzed for TNF-α by ELISA. (E and F) BMDCs from the indicated mice were stimulated with the indicated amount of Pam3CSK4 and LPS for 3 hours. Cell supernatants were analyzed for TNF-α by ELISA. (G) BMDCs from the indicated mice were left untreated or stimulated with Pam3CSK4 (100 ng/ml) or LPS (100 ng/ml) for 30 min. Cells were lysed and immunoprecipitated with CARD9 antibody, followed by immunoblotting with phosphothreonine (P-TxR) antibody or CARD9 antibody. Densitometry analysis of the Western blots are shown in fig. S1. Blots are representative of at least three independent experiments with cells pooled from three to five mice per group. (H) DSS-induced weight loss was monitored and analyzed as in Fig. 1A. *P < 0.05, one-way ANOVA with Tukey post hoc test for (D) to (F) or Student’s t test for (H).

  • Fig. 6 CARD9 expression in DCs is critical for the development of autoimmunity and colitis in DC-specific Lyn-deficient mice.

    (A to D) Serum was collected from the indicated mice at 6 months of age. The amount of antibody to ssRNA or dsDNA in the serum was analyzed by ELISA. (E and F) Cells were harvested from spleens of indicated mice at 3 months of age, stained with fluorescently labeled antibodies, and analyzed by flow cytometry using the gating strategy shown in fig. S7. *P < 0.05, **P < 0.01, one-way ANOVA with Tukey post hoc test; each bar represents n = 5 to 10 mice per group. Results are representative of at least two independent experiments.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/602/eaao3829/DC1

    Fig. S1. Densitometry quantification of immunoblots.

    Fig. S2. PKCδ phosphorylation in Lyn−/− and Lyn−/−;Card9−/− BMDCs.

    Fig. S3. Dectin-1 signaling in Lyn−/− BMMs and BMDCs.

    Fig. S4. Knockdown of Syk and PKCδ in Lyn−/− BMDCs.

    Fig. S5. TLR2 and TLR4 signaling in WT versus Hck−/−;Fgr−/−;Lyn−/− BMDCs.

    Fig. S6. DSS colitis development in DC-specific Lyn- and Lyn;CARD9-deficient mice.

    Fig. S7. Flow cytometry gating strategy.

  • This PDF file includes:

    • Fig. S1. Densitometry quantification of immunoblots.
    • Fig. S2. PKCδ phosphorylation in Lyn−/− and Lyn−/−;Card9−/− BMDCs.
    • Fig. S3. Dectin-1 signaling in Lyn−/− BMMs and BMDCs.
    • Fig. S4. Knockdown of Syk and PKCδ in Lyn−/− BMDCs.
    • Fig. S5. TLR2 and TLR4 signaling in WT versus Hck−/−;Fgr−/−;Lyn−/− BMDCs.
    • Fig. S6. DSS colitis development in DC-specific Lyn- and Lyn;CARD9-deficient mice.
    • Fig. S7. Flow cytometry gating strategy.

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