Rhomboid domain-containing protein 3 is a negative regulator of TLR3-triggered natural killer cell activation

Abstract

Rhomboid domain-containing protein 3 (Rhbdd3), which belongs to a family of proteins with rhomboid domain, is widely expressed in immune cells; however, the roles of the Rhbdd members, including Rhbdd3, in immunity remain unknown. Natural killer (NK) cells are critical for host immune defense and also can mediate inflammatory diseases such as hepatitis. Although much is known about how NK cells are activated, the detailed mechanisms for negative regulation of NK cell activation remain to be fully understood. Using Rhbdd3-deficient mice, we reveal that Rhbdd3, selectively up-regulated in NK cells upon Toll-like receptor 3 (TLR3) stimulation, negatively regulates TLR3-mediated NK cell activation in a feedback manner. Rhbdd3 inhibits TLR3-triggered IFN-γ and granzyme B expression of NK cells in cell-cell contact dependence of accessory cells such as dendritic cells and Kupffer cells. Rhbdd3 interacts with DNAX activation protein of 12 kDa and promotes its degradation, inhibiting MAPK activation in TLR3-triggered NK cells. Furthermore, Rhbdd3 plays a critical role in attenuating TLR3-triggered acute inflammation by controlling NK cell activation and accumulation in liver and disrupting NK cell-Kupffer cell interaction. Therefore, Rhbdd3 is a feedback inhibitor of TLR3-triggered NK cell activation. Our study outlines a mechanism for the negative regulation of NK cell activation and also provides clues for the function of the rhomboid proteins in immunity.

Keywords: immune regulation; innate immunity; poly(I:C).

Fig. 1.

Rhbdd3 negatively regulates TLR3-induced NK cell activation in vitro and in vivo. (A) Splenocytes were stimulated with poly(I:C) or IL-12/15. NK cells were purified then and assessed for Rhbdd3 mRNA expression using quantitative PCR. (B) Splenic NK cells collected from mice i.p. injected with poly(I:C) and D-GalN were assessed for Rhbdd3 mRNA expression using quantitative PCR. (C and D) Rhbdd3^+/+ and Rhbdd3^−/− splenocytes were stimulated with medium (Med), poly(I:C), or IL-12/15. The levels of IFN-γ and IL-6 in supernatants (C) and intracellular expression of granzyme B, perforin, and IFN-γ in NK1.1⁺ cells (D) were, respectively, measured by cytometric bead array (CBA) and FACS. (E and F) Rhbdd3^+/+ and Rhbdd3^−/− mice (six mice per group) were i.p. injected with poly(I:C) and D-GalN. After 6 h, the liver mononuclear cells were collected. The intracellular granzyme B, perforin, and IFN-γ in liver NK1.1⁺ cells were determined by FACS (E). Liver NK cells were further isolated and assessed for their cytotoxicity against YAC-1 cells. E:T, effector versus target (F). The data shown are the means ± SD from three independent experiments (A–C and F). *P < 0.05; **P < 0.01; NS, not significant.

Fig. 2.

Accessory cells are required for the suppression of TLR3-triggered NK cell activation by Rhbdd3 in a cell–cell contact-dependent manner. (A) Rhbdd3^+/+ and Rhbdd3^−/− splenic NK cells were stimulated with poly(I:C), IL-12/15, or poly(I:C) plus IL-12/15, and IFN-γ levels in supernatants were assessed by CBA. (B) Rhbdd3^+/+ and Rhbdd3^−/− splenic NK cells were stimulated with poly(I:C) and then cultured with 5,6-carboxyfluorescein diacetate succinimidyl ester-labeled Yac-1 cells at indicated E:T ratios. The cytotoxicity of NK cells against YAC-1 cells were analyzed by FACS. (C and D) Splenic DX5⁺ NK cells and the left DX5⁻ splenocytes, which were, respectively, seeded in the upper and lower compartments of the Transwell system (transwell), or total splenocytes (total) were stimulated with poly(I:C). IFN-γ levels in supernatants (C) and intracellular granzyme B in NK1.1⁺ cells (D) were respectively assessed by CBA and FACS. The data shown are the means ± SD (A–C) from three independent experiments. *P < 0.05; **P < 0.01; NS, not significant.

Fig. 3.

DCs and KCs are required for the suppression of NK cell activation by Rhbdd3. Rhbdd3^+/+ or Rhbdd3^−/− splenic NK cells were cocultured with Rhbdd3^+/+ or Rhbdd3^−/− bone marrow-derived (BM) DCs (A and B) or liver KCs (C and D) at a ratio of 5:1 and stimulated with poly(I:C). IFN-γ levels in supernatants (A and C) and intracellular production of IFN-γ and granzyme B in NK1.1⁺ cells (B and D) were, respectively, assessed using CBA or FACS. The data shown are the means ± SD (A and C) from three independent experiments. *P < 0.05; **P < 0.01; NS, not significant.

Fig. 4.

Inhibition of TLR3-triggered NK cell DAP12 expression and MAPK activation by Rhbdd3. (A) Splenocytes were stimulated with poly(I:C) for the indicated time, and then NK cells were isolated. The intracellular localization of Rhbdd3 (red) and DAP12 (green) were determined by confocal analysis. (Objective, 40×; numerical aperture, 1.4.) Bar lengths are as indicated. (B) Cell lysates from poly(I:C)-activated splenic NK cells were immunoprecipitated and immunoblotted by anti-Rhbdd3 antibody or anti-DAP12 antibody as indicated. (C) Rhbdd3^+/+ or Rhbdd3^−/− splenic NK cells were treated with MG132 (20 μM) or DMSO (vehicle control) for 2 h and then stimulated by poly(I:C) for the indicated time. The expression of DAP12 was analyzed by Western blotting. (D) Rhbdd3^+/+ or Rhbdd3^−/− splenic NK cells were stimulated with poly(I:C) in the presence of BMDCs. The levels of p-ERK, p-JNK, and p-p38 in NK1.1⁺ NK cells were determined using a Phosflow method (BD Biosciences). (E) Splenic NK cells were, respectively, purified from Rhbdd3^+/+ and Rhbdd3^−/− mice i.p. treated with poly(I:C) and D-GalN. The expression of p-ERK, p-JNK, p-p38, and p-p65 were evaluated by Western blotting. The data shown are the means ± SD (D) from three independent experiments. *P < 0.05; **P < 0.01; NS, not significant.

Fig. 5.

Rhbdd3 attenuates poly(I:C)-induced acute liver inflammation. (A–D) Rhbdd3^+/+ and Rhbdd3^−/− mice (six mice per group) were i.p. injected with poly(I:C) and D-GalN. (A) The levels of ALT, AST, IFN-γ, and IL-6 in serum were, respectively, assayed using ELISA (ALT and AST) and CBA (IFN-γ and IL-6). (B and C) After 6 h, liver tissues were collected. The pathology of liver was analyzed by microscopy. [Original magnification, 400×; bars, 10 μm (B).] IL-6 levels in liver homogenates were determined by CBA and then normalized against milligrams of total protein determined by BCA assay (C). (D) The survival rate of Rhbdd3^+/+ and Rhbdd3^−/− mice (10 mice per group) was monitored for 48 h. P < 0.01 by Wilcoxon test. The data shown are the means ± SD (A and C) from three independent experiments. *P < 0.05; **P < 0.01.

Fig. 6.

Inhibition of NK cell function by Rhbdd3 contributes to attenuation of poly(I:C)-mediated acute liver inflammation. (A and B) Rhbdd3^+/+ and Rhbdd3^−/− mice were pretreated i.p. with anti-NK1.1 mAb (PK136) or isotype Ig (Isotype), followed by poly(I:C) and D-GalN injection 36 h later. After 24 h, serum ALT, AST, IFN-γ, and IL-6 levels were determined (six mice per group) (A). The survival rates were monitored for 48 h (10 mice per group). P < 0.01 by Wilcoxon test (B). (C) Rhbdd3^+/+ and Rhbdd3^−/− mice (six mice per group) were pretreated i.p. with clodronate liposomes or control liposomes, followed by poly(I:C) and D-GalN injection 36 h later. After 24 h, serum ALT levels were determined by ELISA. (D and E) Splenic NK cells from Rhbdd3^+/+ or Rhbdd3^−/− mice were i.v. transferred into WT mice, which were predepleted with NK cells by anti-NK1.1 mAb (4 × 10⁶ cells per mouse) 36 h before. Mice were then i.p. injected with poly(I:C) and D-GalN 24 h later. After 24 h, serum ALT, AST, IFN-γ, and IL-6 levels were determined. The data shown are the means ± SD (A and C–E) from three independent experiments. *P < 0.05; **P < 0.01; NS, not significant.