Murine BAFF expression is up-regulated by estrogen and interferons: implications for sex bias in the development of autoimmunity

Abstract

Systemic lupus erythematosus (SLE) in patients and certain mouse models exhibits a strong sex bias. Additionally, in most patients, increased serum levels of type I interferon (IFN-α) are associated with severity of the disease. Because increased levels of B cell activating factor (BAFF) in SLE patients and mouse models are associated with the development of SLE, we investigated whether the female sex hormone estrogen (E2) and/or IFNs (IFN-α or γ) could regulate the expression of murine BAFF. We found that steady-state levels of BAFF mRNA and protein were measurably higher in immune cells (CD11b(+), CD11c(+), and CD19(+)) isolated from C57BL/6 females than the age-matched male mice. Treatment of immune cells with IFN or E2 significantly increased levels of BAFF mRNA and protein and a deficiency of estrogen receptor-α, IRF5, or STAT1 expression in splenic cells decreased expression of BAFF. Moreover, treatment of RAW264.7 macrophage cells with IFN-α, IFN-γ, or E2 induced expression of BAFF. Interestingly, increased expression of p202, an IFN and estrogen-inducible protein, in RAW264.7 cells significantly increased the expression levels of BAFF and also stimulated the activity of the BAFF-luc-reporter. Accordingly, the increased expression of the p202 protein in lupus-prone B6.Nba2-ABC than non lupus-prone C57BL/6 and B6.Nba2-C female mice was associated with increased expression levels of BAFF. Together, our observations demonstrated that estrogen and IFN-induced increased levels of the p202 protein in immune cells contribute to sex bias in part through up-regulation of BAFF expression.

Fig. 1. Gender, sex hormone estrogen, and IFN regulate BAFF mRNA levels

A. Total RNA prepared from the indicated purified cells isolated from C57BL/6 males and age-matched females was analyzed by quantitative real-time PCR using TaqMan assays specific to the murine BAFF gene. The ratio of the BAFF mRNA levels to β2-microglobulin mRNA was calculated in units (one unit being the ratio of the BAFF mRNA to β2-microglobulin mRNA). The ratio of mRNA levels in CD3⁺ cells from male mice is indicated as 1. The error bars represent the standard deviation (*p <0.05; **p<0.01). B. Purified CD11b⁺ cells isolated from C57BL/6 males and age-matched female mice were either left untreated (lanes 1 and 4) or treated with estrogen (10 nM; lanes 2 and 5) or IFN-α (1,000 u/ml; lanes 3 and 6) for 14 h. After the treatment, total cel lysates containing equal amounts of proteins were subjected to immunoblotting using antibodies specific to the indicated proteins. Fold change (FC) in levels of BAFF protein is indicated. C. Purified B220⁺ cells isolated from C57BL/6 females mice were either left untreated or treated with IFN-α (1,000 u/ml) or estrogen (10 nM) for 14 h. Total RNA was analyzed by quantitative real-time PCR using TaqMan assays as described in panel A. The ratio of mRNA levels in untreated cells from is indicated as 1. The error bars represent the standard deviation. D. Purified B220⁺ cells isolated from C57BL/6 females mice were either left untreated or treated with IFN-α (1,000 u/ml) or estrogen (10 nM) for 14 h. Total cell lysates containing equal amounts of proteins were analyzed by immunoblotting. Fold change in BAFF protein levels is indicated.

Fig. 2. BAFF expression depends on ERα, IRF5, and STAT1

A. Total RNA preparations from splenic cells isolated from wild type or age-matched Esr1-deficient female mice were subjected to quantitative real-time PCR. The ratio of the BAFF mRNA levels to β2-microglobulin mRNA was calculated in units. The error bars represent the standard deviation. B. Total cell lysates from splenic cells isolated from wild type or age-matched Irf5-deficient female mice were subjected to immunoblotting using antibodies specific to the indicated proteins. C. Total RNA preparations from splenic cells isolated from wild type or age-matched Stat1-deficient mice were subjected to quantitative real-time PCR. The ratio of the BAFF mRNA levels to β2-microglobulin mRNA was calculated in units. The ratio of mRNA levels in splenic cells from wild type male mice is indicated as 1.The error bars represent the standard deviation. D. Total cell lysates from splenic cells isolated from wild type or age-matched Stat1-deficient mice were subjected to immunoblotting. Fold change in BAFF protein levels is indicated.

Fig. 3. Treatment of RAW264.7 cells with IFNs or estrogen increases BAFF expression

A. Sub-confluent cultures of RAW264.7 cells were either left untreated or treated with IFN-α or IFN-γ for 14 h. After the treatments, total RNA was prepared and subjected to quantitative real-time PCR to analyze BAFF mRNA levels. The ratio of the BAFF mRNA levels to β2-microglobulin mRNA was calculated in units. The ratio of mRNA levels in splenic cells from control cells is indicated as 1.The error bars represent the standard deviation (***p<0.001). B. Sub-confluent cultures of RAW264.7 cells were transfected with BAFF-luc-reporter (1.8 µg) plus pRL-TK plasmid (0.2 µg). 24 h after transfections, the transfected cells were either left untreated or treated with the indicated IFN for 16 h. After the treatments cells were lysed and cell lysates were analyzed for dual luciferase activity. The ratio between firefly luciferase and Renilla luciferase in control cells is indicated as 1. The error bars represent the standard deviation (**p<0.01; ***p<0.001). C. Sub-confluent cultures of RAW264.7 cells were either left untreated or treated with IFN-α or IFN-γ for 14 h. After the treatments, total cell lysates were analyzed by immunoblotting. Fold change in BAFF protein levels is indicated. D. Sub-confluent cultures of RAW264.7 cells were either left untreated or treated with E2 (10 nM) for 24 h. After the treatment, total RNA was prepared and subjected to semi-quantitative PCR to analyze BAFF and actin mRNA levels.

Fig. 4. p202 protein regulates BAFF expression

A. Total RNA prepared from RAW264.7 cells stably transfected with vector (V; pCMV) or pCMV-202 (p202) were analyzed by semi-quantitative PCR. Fold change in BAFF mRNA levels is indicated. B. Total cell extracts prepared from RAW264.7 cells stably transfected with vector (V; pCMV) or pCMV-202 (p202) were analyzed by immunoblotting. Fold change in BAFF protein levels is indicated. C. Sub-confluent cultures of RAW264.7 cells were transfected with BAFF-luc-reporter (1 µg) and pRL-TK plasmid (0.2 µg) along with pCMV plasmid (V; 0.4 µg) or pCMV-202 plasmid (p202; 0.2 or 0.4 µg). 45 h after transfections, cells were lysed and cell lysates were analyzed for dual luciferase activity. The ratio between firefly luciferase and Renilla luciferase in vector transfected cells is indicated as 1. The error bars represent the standard deviation. D. Total cell extracts prepared from J774.A1 cells stably infected with control lentivirus (Cont) or shIfi202 lentivirus were analyzed by immunoblotting. Fold change in BAFF protein levels is indicated.

Fig. 5. Increased levels of p202 protein in lupus-prone mice are associated with increased expression levels of BAFF

A. Total cell extracts prepared from purified CD11c⁺ cells isolated from C57BL/6 (B6) or B6.Nba2 (Nba2) male and age-matched female mice were analyzed by immunoblotting using antibodies specific to the indicated proteins. B. Total cell extracts prepared from purified CD19⁺ cells isolated from B6 or B6.Nba2 (Nba2) female mice were analyzed by immunoblotting using antibodies specific to the indicated proteins. C. Total RNA prepared from splenic cells isolated from age-matched C57BL/6 (B6), B6.Nba2-C (C), or B6.Nb2 (Nba2) females was analyzed by quantitative real-time PCR using TaqMan assays specific to the murine BAFF gene. The ratio of the BAFF mRNA levels to β2-microglobulin mRNA was calculated in units (one unit being the ratio of the BAFF mRNA to β2-microglobulin mRNA). The ratio of mRNA levels in the B6 splenic cells is indicated as 1. The error bars represent the standard deviation (*p<0.05; ***p<0.001). D. Purified CD19⁺ cells from age-matched B6, B6.Nba2-C (C), or B6.Nb2 (Nba2) female mice were lysed and the cell lysates were analyzed by immunoblotting. Fold change in BAFF protein levels is indicated. E. Total splenic cell extracts from wild type MRL or MRL-lpr/lpr male and age-matched female mice were analyzed by immunoblotting.

Fig. 6. Treatment of cells with BAFF increases expression levels of p202

Purified splenic B220⁺ cells isolated from B6.Nba2-ABC male or female mice were either left untreated or treated with recombinant murine BAFF (10 ng/ml) for 16 h. After the treatment, cell extracts were analyzed by immunoblotting for the indicated proteins.

Fig. 7. Proposed model for the regulation of murine BAFF expression by estrogen and IFN-signaling