Tir8/Sigirr prevents murine lupus by suppressing the immunostimulatory effects of lupus autoantigens

Abstract

The Sigirr gene (also known as Tir8) encodes for an orphan receptor of the Toll-like receptor (TLR)/interleukin 1 receptor family that inhibits TLR-mediated pathogen recognition in dendritic cells. Here, we show that Sigirr also inhibits the activation of dendritic cells and B cells upon exposure to RNA and DNA lupus autoantigens. To evaluate the functional role of Sigirr in the pathogenesis of systemic lupus erythematosus (SLE), we generated Sigirr-deficient C57BL/6-lpr/lpr mice. These mice developed a progressive lymphoproliferative syndrome followed by severe autoimmune lung disease and lupus nephritis within 6 mo of age as compared with the minor abnormalities observed in C57BL/6-lpr/lpr mice. Lack of Sigirr was associated with enhanced activation of dendritic cells and increased expression of multiple proinflammatory and antiapoptotic mediators. In the absence of Sigirr, CD4 T cell numbers were increased and CD4(+)CD25(+) T cell numbers were reduced. Furthermore, lack of Sigirr enhanced the activation and proliferation of B cells, including the production of autoantibodies against multiple nuclear lupus autoantigens. These data identify Sigirr as a novel SLE susceptibility gene in mice.

Figure 1.

Lack of Sigirr is associated with massive lymphoproliferation in B6^lpr/lpr mice. (A and B) At 6 mo of age, Sigirr-deficient B6^lpr mice revealed massive hyperplasia of cervical, axillar (A), and mesenteric lymph nodes (B) as well as splenomegaly (B). Data are means ± SEM from 12 mice in each group. *, P < 0.05. (C) PAS staining of spleen sections indicates lymph follicle hyperplasia in Sigirr-deficient B6^lpr/lpr mice. Images in A and C are representative of at least 12 mice in each group. Bars: (B) 1 cm; (C) 100 μm.

Figure 2.

Sigirr and dendritic cell activation. (A and B) Dendritic cells were prepared from bone marrow cells of B6^{lpr/lpr/Tir8−/−} and B6^lpr/lpr mice and stimulated in vitro with various TLR agonists, including immune complexes formed by U1snRNP and the anti-U1snRNP antibody Y12 (Y12) or a nucleosome-specific antibody and nucleosomes for 24 h. Supernatants were analyzed for IL-12p40 (A) by ELISA. MX1 mRNA levels, as a marker of type I IFN expression, were quantified by real-time PCR after 8 h of stimulation (B). Data are shown as mean ± SEM. *, P < 0.05 versus B6^{lpr/lpr/Tir8−/−} mice. (C) The total number of CD11c⁺ dendritic cells in the spleens of B6^{lpr/lpr/Sigirr−/−} and B6^lpr/lpr wild-type mice was quantified by flow cytometry as described in Materials and methods. Data represent means ± SEM from five mice in each group. *, P < 0.05 versus B6^lpr/lpr mice. (D) RNA was isolated from the spleens of CD11b⁺ cells from B6^{lpr/lpr/Tir8−/−} and B6^lpr/lpr mice for real-time PCR analysis. Data are expressed as means of the ratio of the specific mRNA versus that of 18S rRNA ± SEM. *, P < 0.05 versus B6^lpr/lpr mice. (E) Serum samples from 6-mo-old B6^{lpr/lpr/Tir8−/−} and B6^lpr/lpr mice were analyzed for IL-12p40 by ELISA. Data are means ± SEM from at least 10 mice in each group. *, P < 0.05 versus B6^lpr/lpr mice.

Figure 3.

Sigirr and T cell subsets in B6^lpr/lpr mice. (A and B) Flow cytometry was used to determine the total number of distinct T cell subsets in the spleens of 6-mo-old B6^{lpr/lpr/Tir8−/−} and B6^lpr/lpr mice. The histogram presents means ± SEM of at least five mice in each group. *, P < 0.05 versus B6^lpr/lpr mice. (C) Cellular mRNA was prepared from CD4⁺ and CD4⁺CD25⁺ spleen cell isolates from B6^{lpr/lpr/Tir8−/−} and B6^lpr/lpr mice and analyzed by real-time PCR. Data are expressed as means of the ratio of the specific mRNA versus that of 18S rRNA ± SEM. *, P < 0.05 versus B6^lpr/lpr mice.

Figure 4.

Sigirr suppresses the proliferation of B cells from B6^lpr/lprmice. (A) Immunostaining of spleen sections for B220 illustrates the expansion of B cell areas in Tir8-deficient B6^lpr/lpr mice. Images are representative of at least 12 mice in each group. (B) The total number of spleen B220⁺ cells was quantified in B6^{lpr/lpr/Tir8−/−} and B6^lpr/lpr wild-type mice by flow cytometry. Data represent means ± SEM from five mice in each group. *, P < 0.05 versus B6^lpr/lpr mice. (C) Spleen B cells from B6^{lpr/lpr/Tir8−/−} and B6^lpr/lpr mice were stimulated in vitro with anti-IgM antibodies, various TLR agonists, including immune complexes formed by U1snRNP, and the anti-U1snRNP antibody Y12 (Y12) or a nucleosome-specific antibody and nucleosomes for 72 h. B cell proliferation was assessed as described in Materials and methods. Data are shown as mean ± SEM of the OD at 490 nm. Data are shown as mean ± SEM. *, P < 0.05 versus B6^lpr/lpr mice. Bar: (A) 100 μm.

Figure 5.

Sigirr and the production of Igs and DNA autoantibodies in B6^lpr/lprmice. Mice from both groups were bled at monthly intervals to determine serum levels of IgG (A) and dsDNA autoantibody isotypes (B) by ELISA. Serum from 6-mo-old female MRL^lpr/lpr mice served as positive control. Data represent means ± SEM from at least 10 mice in each group. *, P < 0.05 versus B6^lpr/lpr mice of the same time point; #, P < 0.05 versus month 1 of mice from the same strain. (C) C. luciliae slides were incubated with 1:50 diluted serum of 6-mo-old mice from both strains, and autoantibody binding to the flagellate's kinetoplast was detected using an FITC-labeled anti-mIgG. Signal intensity was scored applying a semiquantitative score from 0 to 3. Images on the left show anti-dsDNA IgG in green (top), and staining the kinetoplast DNA itself with DAPI is in blue (not depicted). The merged pictures are shown below demonstrating that FITC positivity matches with the kinetoplast at the flagella pole of C. luciliae. Data on the right show mean scores ± SEM from at least 6–10 mice in each group. *, P < 0.05 versus B6^lpr/lpr mice. (D) Serum levels of autoantibodies against Sm antigen, U1snRNP, nucleosomes, or rheumatoid factor (RF) were determined at monthly intervals by ELISA. Serum from 6-mo-old female MRL^lpr/lpr mice served as positive control. *, P < 0.05 versus B6^lpr/lpr mice of the same time point; #, P < 0.05 versus month 1 of mice from the same strain; N.d., not done.

Figure 6.

Lupus nephritis and lung injury in B6^lpr/lprmice. Lung and renal sections were stained with PAS. On renal sections, immunostaining was also performed for IgG and complement factor C3c. Images are representative for 10 mice in each group. Bar, 200 μm.