CD24 and Siglec-10 selectively repress tissue damage-induced immune responses

Abstract

Patten recognition receptors, which recognize pathogens or components of injured cells (danger), trigger activation of the innate immune system. Whether and how the host distinguishes between danger- versus pathogen-associated molecular patterns remains unresolved. We report that CD24-deficient mice exhibit increased susceptibility to danger- but not pathogen-associated molecular patterns. CD24 associates with high mobility group box 1, heat shock protein 70, and heat shock protein 90; negatively regulates their stimulatory activity; and inhibits nuclear factor kappaB (NF-kappaB) activation. This occurs at least in part through CD24 association with Siglec-10 in humans or Siglec-G in mice. Our results reveal that the CD24-Siglec G pathway protects the host against a lethal response to pathological cell death and discriminates danger- versus pathogen-associated molecular patterns.

Fig. 1

CD24 negatively regulates the immune response to AAP-induced liver injury. CD24^-/- mice or WT mice were treated with AAP (10 mg/mouse, dissolved in H₂0) or vehicle control. (A) Survival of mice 20 hours after treatment. Numbers on graph indicate the number of viable over total mice used per group. All WT mice remained healthy. (B) Serum levels of IL-6, MCP-1 and TNF-α at 6 hours after AAP injection (mean ± SD, n = 5; *P < 0.02, **P < 0.009; ***P < 0.002, student’s t-test). (C) ALT levels measured at 6 hours after treatment (mean ± SD, n = 5; ***P < 0.00004, student’s t-test). Data shown in (B) and (C) have been repeated 2 times. (D) Livers were isolated at 9 hours after treatment. Representative images (20x) of H&E staining are shown (n = 3).

Fig. 2

CD24 associates with, and negatively regulates the immune response to HMGB1. (A) Identification of CD24-associated proteins by coimmunoprecipitation. Silver-staining of the SDS-PAGE gel is shown. Arrows indicate the positions of HMGB1 and nucleolin, two abundant CD24-associated DAMP molecules. NS: proteins that coimmunoprecipitated with anti-CD24 non-specifically. (B) Confirmation of CD24-HMGB1 association by Western blot of EDTA-disassociated proteins. (C) Reciprocal immunoprecipitations of CD24 and HMGB1 were performed with splenocyte lysates isolated from WT mice. (D) Direct, cation-dependent interaction between CD24 and HMGB1. Coimmunoprecipitation of recombinant HMGB1 protein with CD24-Fc fusion protein or control IgG-Fc. The requirement for cations was confirmed by disruption of the complex with EDTA. This experiment has been repeated 3 times. (E) Mice received i.v. injections with either vehicle (PBS) or mouse HMGB1 mAb (clone 3B1, 150 μg/mouse) 30 minutes prior to i.p. injection of AAP. Composite data from two independent experiments are shown (n = 8). (F) Serum ALT at 6 hours after treatment with AAP and HMGB1 antibodies (mean ± SD, n = 5, **P < 0.005). (G) Serum cytokine levels at 6 hours after treatment with AAP and HMGB1 antibodies (mean ± SD, n = 5, *P, 0.03, **P < 0.004). Samples in (F) and (G) represent two independent experiments, the statistical significance determined by student’s t-test.

Fig. 3

The Siglec 10/G-CD24-HMGB1 axis negatively regulates immune responses to AAP-induced liver injury. (A) Interaction between CD24 and Siglec-Fc fusion proteins. Data shown are optical density and have been repeated 3 times. (B) Flow cytometric analysis of CD24 interaction with Siglec-10. Representative histograms of two independent experiments are shown. (C) COS cells were transfected with FLAG-tagged WT or mutant (*, R119A) Siglec-10 cDNA or a vector control. Coimmunoprecipitations were performed 48 hours later. (D) Lysates from WT or CD24^-/- splenocytes were used to coimmunoprecipitate Siglec-10-Fc, CD24 and HMGB1. (E) Lysates from WT and CD24^-/- spleen cells were precipitated with either Siglec-G antibodies or control mouse Ig. The precipitates were probed with Siglec-G antisera and mAbs specific for CD24 and HMGB1. (F) Percent survival 20 hours after AAP treatment. Numbers on graph represent the number of surviving mice over total mice used. (G) ALT release in serum 6 hours after AAP treatment (mean ± SD, *P < 0.005, n=5). (H) 20x images of H&E staining of livers harvested 6 hours after AAP injection. (I) Cytokine production in blood measured 6 hours after AAP treatment (mean ± SD, n=5. *P <0.05, **P < 0.009, ***P < 0.002). (J) Survival of WT and Siglecg^-/- mice 20 hours after treatment. (K) ALT release in the blood 6 hours after treatment (mean ± SD, n = 5, *P < 0.006). (L) Cytokine release in the blood 6 hours after treatment (mean ± SD, n = 5, *P < 0.03, **P < 0.0006, ***P < 0.0004). (K-L) are representative of two independent experiments. Statistical significance was determined by the student’s t-test.

Fig. 4

CD24 and Siglec-G negatively regulate immune responses to HMGB1, HSP70 and HSP90, but not to LPS and poly I:C. (A) Production of cytokines by DCs. DCs cultured from WT, CD24^-/- or Siglecg^-/- bone marrow were stimulated with LPS (100 ng/ml), polyI:C (10 μg/ml) or increasing doses (5, 10 and 20 μg/ml) of HMGB1 for 6 hours, the supernatants were analyzed for the levels of inflammatory cytokines, using cytokine beads array. Data represents the mean ± SD for three independent cultures of DCs in each genotype and have been repeated at least three times. (B) BMDCs isolated from WT, CD24^-/- or Siglecg^-/- mice were stimulated under the indicated conditions for 6 hours. The nuclear lysates were prepared and the activation of NF-κB was assessed by blotting for the p65 subunit of NF-κB. The loading of nuclear protein was determined by amounts of Sp1 protein. Fold induction over medium control are provided underneath the photograph. Data are representative of two independent experiments. (C) Age-matched male mice received i.p. injections of LPS (450μg/mouse). Kaplan Meier survival plots are shown. No statistical significance was found by log-rank tests. (D) Cytokine production in the serum 4 hours after LPS injection (mean ± SD, the statistical significance of the differences between the control and one of the treated groups were determined by student’s t-test. *P < 0.03, **P < 0.002). The numbers of mice used are the same as (C). (E) Coimmunoprecipitation of CD24 and Hsp70 and Hsp90. (F) Siglec-G associates with Hsp70 and Hsp90 through CD24. The same precipitates used in Fig. 3E were analyzed for Hsp70 and Hsp90 by immunoblot. (G) Deficiencies in CD24 and Siglec-G enhanced production of IL-6 and TNF-α at 6 hours after stimulation with HSP70 and HSP90. Data shown represent the mean ± SD of cytokines from 4 independent isolates of DCs from each genotype and have been repeated twice.