Type I interferon receptor controls B-cell expression of nucleic acid-sensing Toll-like receptors and autoantibody production in a murine model of lupus

Abstract

Introduction: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the production of high-titer IgG autoantibodies directed against nuclear autoantigens. Type I interferon (IFN-I) has been shown to play a pathogenic role in this disease. In the current study, we characterized the role of the IFNAR2 chain of the type I IFN (IFN-I) receptor in the targeting of nucleic acid-associated autoantigens and in B-cell expression of the nucleic acid-sensing Toll-like receptors (TLRs), TLR7 and TLR9, in the pristane model of lupus.

Methods: Wild-type (WT) and IFNAR2-/- mice were treated with pristane and monitored for proteinuria on a monthly basis. Autoantibody production was determined by autoantigen microarrays and confirmed using enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation. Serum immunoglobulin isotype levels, as well as B-cell cytokine production in vitro, were quantified by ELISA. B-cell proliferation was measured by thymidine incorporation assay.

Results: Autoantigen microarray profiling revealed that pristane-treated IFNAR2-/- mice lacked autoantibodies directed against components of the RNA-associated autoantigen complexes Smith antigen/ribonucleoprotein (Sm/RNP) and ribosomal phosphoprotein P0 (RiboP). The level of IgG anti-single-stranded DNA and anti-histone autoantibodies in pristane-treated IFNAR2-/- mice was decreased compared to pristane-treated WT mice. TLR7 expression and activation by a TLR7 agonist were dramatically reduced in B cells from IFNAR2-/- mice. IFNAR2-/- B cells failed to upregulate TLR7 as well as TLR9 expression in response to IFN-I, and effector responses to TLR7 and TLR9 agonists were significantly decreased as compared to B cells from WT mice following treatment with IFN-alpha.

Conclusions: Our studies provide a critical link between the IFN-I pathway and the regulation of TLR-specific B-cell responses in a murine model of SLE.

Figure 1

Serum immunoglobulin levels in pristane-treated mice. Total immunoglobulin levels were measured by enzyme-linked immunosorbent assay in serum obtained 6 months after treatment with phosphate-buffered saline (PBS) or pristane. Mean values with standard deviation are shown for each group. P values were obtained using the Student t test and are displayed above each plot. IFNAR2: interferon-I receptor 2; n.s.: not significant; WT: wild-type.

Figure 2

Autoantibody profiling of pristane-treated mice using autoantigen microarrays. Individual arrays composed of over 50 recombinant or purified antigens were incubated with diluted sera obtained 6 months after pristane treatment. Pairwise significance analysis of microarrays was used to determine antigen features with statistically significant differences in array reactivity between pristane-treated wild-type (WT) and pristane-treated IFNAR2^-/- mice (false discovery rate < 0.05, fold change > 3). IFNAR2: interferon-I receptor 2; RiboP: ribosomal phosphoprotein P0; Sm: Smith antigen; SmRNP: Smith antigen ribonucleoprotein.

Figure 3

Autoantibody production in pristane-treated IFNAR2^-/- mice. Sera obtained 6 months after treatment with pristane or phosphate-buffered saline (PBS) were analyzed for levels of IgM or IgG anti-Sm/RNP (a), anti-RiboP (b), anti-ssDNA (c), or anti-Histone (d) antibodies by enzyme-linked immunosorbent assay. Data are plotted as absorbance values for individual animals minus background. P values were determined using the Mann-Whitney t test for pristane-treated wild-type (WT) versus pristane-treated IFNAR2^-/- mice and are displayed above each graph. Closed circles represent serum from PBS-treated mice, and open circles represent serum from pristane-treated mice. IFNAR2: interferon-I receptor 2; n.s.: not significant; OD: optical density; RiboP: ribosomal phosphoprotein P0; Sm/RNP: Smith antigen/ribonucleoprotein; ssDNA: single-stranded DNA.

Figure 4

Expression of Toll-like receptors TLR7 and TLR9 in IFNAR2^-/- B cells. (a) B cells were purified from wild-type (WT) or IFNAR2^-/- mice using magnetic beads. RNA was extracted and the relative mRNA expression of TLR7 and TLR9 was measured. (b) Purified B cells were cultured in the presence or absence of interferon-alpha (IFN-α). RNA was extracted and the relative expression TLR7 and TLR9 was measured. P values were determined using the Student t test. IFNAR2: interferon-I receptor 2; n.s.: not significant.

Figure 5

Activation of Toll-like receptors TLR7 and TLR9 in IFNAR2^-/- mice. (a) B cells were purified from pristane-treated wild-type (WT) or IFNAR2^-/- mice, and proliferation in response to Loxoribine or ODN1826 was measured. Data are represented as the difference in mean counts per minute (cpm) of stimulated and unstimulated triplicate wells (Δ cpm) + standard error of the mean. (b) B cells were purified as above and the concentration of interleukin-6 (IL-6) in the supernatant was measured following stimulation with Loxoribine or ODN1826. (c) B cells were purified as above and were cultured in the presence or absence of interferon-alpha (IFN-α) for 24 hours before treatment with Loxoribine or ODN1826. The concentration of IL-6 in the supernatant was then measured. P values were determined using the Student t test. IFNAR2: interferon-I receptor 2; n.s.: not significant; ODN: oligodeoxynucleotide.