Female Bias in Systemic Lupus Erythematosus is Associated with the Differential Expression of X-Linked Toll-Like Receptor 8

Abstract

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the production of anti-nuclear antibodies. SLE is one of many autoimmune disorders that have a strong gender bias, with 70-90% of SLE patients being female. Several explanations have been postulated to account for the severity of autoimmune diseases in females, including hormonal, microbiota, and gene dosage differences. X-linked toll-like receptors (TLRs) have recently been implicated in disease progression in females. Our previous studies using the 564Igi mouse model of SLE on a Tlr7 and Tlr9 double knockout background showed that the presence of Tlr8 on both X chromosomes was required for the production of IgG autoantibodies, Ifn-I expression and granulopoiesis in females. Here, we show the results of our investigation into the role of Tlr8 expression in SLE pathogenesis in 564Igi females. Female mice have an increase in serum pathogenic anti-RNA IgG2a and IgG2b autoantibodies. 564Igi mice have also been shown to have an increase in neutrophils in vivo, which are major contributors to Ifn-α expression. Here, we show that neutrophils from C57BL/6 mice express Ifn-α in response to 564 immune complexes and TLR8 activation. Bone marrow-derived macrophages from 564Igi females have a significant increase in Tlr8 expression compared to male-derived cells, and RNA fluorescence in situ hybridization data suggest that Tlr8 may escape X-inactivation in female-derived macrophages. These results propose a model by which females may be more susceptible to SLE pathogenesis due to inefficient inactivation of Tlr8.

Keywords: IFN-I; SLE; TLR8; X-inactivation; neutrophils.

Figure 1

564Igi females have more circulating pathogenic IgG autoantibodies. (A) Anti-RNA antibodies were detected using and isotype-specific anti-RNA ELISA. Shown is the mean O.D. 405 nm ± SEM at various serum dilutions. The number of mice is shown in the key. (B) Shown is the ratio of male:female offspring born to the dam in the indicated breeding pair. Each data point represents a single litter. The average ratio is shown with a horizontal line. (C) Pregnant C57BL/6 dams were given introperitoneal injections with 1.5 μg of the indicated antibody or media control at approximately embryonic days 13–15. Shown is the ratio of male:female offspring in each litter. Each data point represents a single litter. The average ratio is shown with a horizontal line. (D,E) The mean (D) anti-RNA and (E) anti-DNA reactivities of the indicated antibodies were tested by ELISA. Shown is the mean O.D. 405 nm ± SEM at the indicated antibody concentrations. The number of replicates is shown in the key. (F) Purified 564 and 2C10 antibodies were tested for anti-nuclear reactivity by HEp-2 staining. Shown are the staining representative staining patterns of at least four independent experiments.

Figure 2

564 IgG2b IC activates neutrophils to express Ifn-α6. (A) C57BL/6 mice were given intraperitoneal injections with 40 μg of 564 antibody, an isotype control of unknown specificity or a comparable volume of saline control. Two weeks later, the number of neutrophils in the bone marrow was determined by flow cytometry. Shown is the percent of neutrophils in each mouse. The average is shown as a horizontal line. (B) Neutrophils were purified from whole bone marrow cell suspensions from the indicated mice by cell sorting (CD11b⁺ Ly6G⁺). Shown is a representative plot of the gate used to purify neutrophils. (C) Purified neutrophils were cultured for 4 h in the presence of 0.01 μg/mL purified IgG2b 564 antibody or 564 IC made by incubating purified IgG2b 564 antibody with bone marrow-derived RNA. RNA was extracted from the cell culture, converted to cDNA, and Ifn-α6 expression was measured by RT-qPCR. Expression was normalized to β-actin and compared to untreated samples from each genotype. Shown is the average ± SEM fold-increase in Ifn-α6 expression in three mice per group tested in independent experiments, each done in triplicate.

Figure 3

TLR8 agonist induces increased Ifn-α6 expression in neutrophils. Neutrophils were purified from whole bone marrow cell suspensions from the indicated mice by cell sorting as in Figure 2B. Neutrophils were cultured for 16 h in the presence of the TLR8 agonist CLO75 (1 mg/mL) or the TLR7 agonist CLO97 (1 mg/mL). RNA was extracted from the cell culture, converted to cDNA, and Ifn-α6 transcript level was measured by RT-qPCR. Expression was normalized to β-actin and compared to untreated samples from each genotype. Shown is the average ± SEM fold-increase in Ifn-α6 expression in two mice per group tested in independent experiments, each done in triplicate.

Figure 4

BMDM from 564Igi females have increased transcript levels of Ifn-α6, Tlr7, and Tlr8 compared to BMDM from male 564Igi mice. (A) Whole bone marrow cell suspensions from female and male mice were grown in cell culture media to select for the growth of macrophages (20% FCS, 25% L-292 cell culture supernatant, 10 U/mL penicillin/streptomycin, RPMI). Shown is a representative plot showing the percentage of neutrophils, monocytes, and macrophages after culturing compared to whole bone marrow and RAW 264.7 macrophage cell line. (B–D) After 7 days in culture, RNA was isolated from the cells, converted to cDNA, and (B) Ifn-α6, (C) Tlr7, and (D) Tlr8 expression was measured by RT-qPCR. Expression was normalized to β-actin and compared to Ifn-α6, Tlr7, and Tlr8 expression in the RAW 264.7 macrophage cell line. Shown is the average ± SEM fold-increase in expression two mice per group, tested in two independent experiments, each done in triplicate.

Figure 5

Tlr8 RNA co-localizes with Xist RNA. (A–F) BMDM from C57BL/6 and 564Igi mice were stained for the presence of Xist and (A) Tlr7 and Tlr8 exonic mRNA, (B) Eif2s3x intronic mRNA, (C) Tlr7 intronic and exonic mRNA or (D–F) Tlr8 intronic and exonic mRNA using fluorescent probes. (A) An example of increased Tlr8 mRNA compared to Tlr7 mRNA in female BMDM. (B) Two examples of escaped X-inactivation of Eif2s3x. (C) An example of normal X-inactivation of Tlr7. (D) An example of potential escaped X-inactivation of Tlr8 in C57BL/6 female BMDM. (E) An example of potential escaped X-inactivation of Tlr8 in 564Igi female BMDM. 1/800 cells (~0.1%) show this staining pattern. The insert shows the Xist-cloud with the Tlr8 and weak Eif2s3x signals. (F) An example of normal X-inactivation of Tlr8 in C57BL/6 female BMDM. The probes used in each set of images are shown underneath. Intronic (int) probes detect nuclear pre-mRNA before splicing. Exonic (ex) probes also detect cytoplasmic mature mRNA after splicing. White arrowheads show target RNA expression from active X chromosome. White arrows show target RNA expression from inactivated X chromosome.