B cell development in IRF5^+/+ and IRF5^−/− MRL/lpr mice.

<p>Bone marrow (A) and spleen cells (B) from 2, 3, and 4-month-old IRF5^+/+ (filled circles) and IRF5^−/− (open circles) MRL/lpr mice were stained with antibodies against B220, AA4.1, IgM, CD19, CD21 and CD23. A. In bone marrow, percentages of B220⁺AA4.1⁺ (pro-B, pre-B and immature B) and B220⁺AA4.1⁻ (mature B) cells, percentages of Hardy fractions B-D (pro-B and pre-B; B220⁺IgM⁻), fraction E (immature B; B220^intermediateIgM⁺), and fraction F (mature or re-circulating B; B220^highIgM⁺) were determined. B. In spleen, percentages and cell numbers of B cells (CD19⁺), immature B cells (B220⁺AA4.1⁺) and mature B cells (B220⁺AA4.1⁻) were determined. Mature B cells were further classified as marginal zone (MZ) B cells (CD21⁺CD23^low) and follicular (FO) B cells (CD23⁺) based on CD21 and CD23 expression. Each dot represents an individual mouse. Bars represent mean ± SEM. *p<0.05; **p<0.01.</p

Increased survival in IRF5-deficient MRL/lpr mice.

<p>IRF5^+/+ (n = 12), IRF5^+/− (n = 14) and IRF5^−/− (n = 14) female MRL/lpr littermates were observed until they met the criteria for euthanasia based on predetermined humane endpoints. ** p<0.01.</p

Serum BLyS levels are elevated in MRL/lpr mice but are not affected by IRF5 deficiency.

<p>A and B. BLyS levels in the sera of IRF5^+/+ MRL/lpr mice and IRF5^−/− MRL/lpr mice (A), and C57BL/6 mice (B) at several ages were measured by ELISA. C. BLyS levels in the sera of sixteen week old IRF5^+/+ (n = 11), IRF5^+/− (n = 6) and IRF5^−/− (n = 11) female MRL/lpr mice were measured by ELISA. The BLyS serum levels of the C57BL/6 mice (n = 10) shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103478#pone-0103478-g007" target="_blank">figure 7B</a> (age range 13–32 weeks) are included for data comparison. Bars represent mean ± SEM. No significant differences were found between any of the MRL/lpr experimental groups.</p

IRF5-deficient MRL/lpr mice have greatly reduced numbers of spleen plasmablasts and bone marrow plasma cells.

<p>A. Spleen cells from IRF5^+/+ and IRF5^−/− MRL/lpr mice at 4 months of age were stained with antibodies against CD19, CD3, B220, CD22, CD44 and CD138. Plasmablasts (B220^lowCD22^lowCD44⁺CD138⁺) were identified within the splenic B cell (CD19⁺CD3⁻) population. Bone-marrow cells from IRF5^+/+ and IRF5^−/− MRL/lpr mice were stained with antibodies against CD4, CD8, F4/80, Gr-1, B220 and CD138. CD4⁻CD8⁻F4/80⁻Gr-1⁻ cells were gated to analyze plasma cells (B220⁻CD138⁺). B. Numbers and percentages of spleen plasmablasts and bone marrow plasma cells of IRF5^+/+ (filled circles) and IRF5^−/− (open circles) MRL/lpr mice at 2, 3 and 4 months of age are shown. Each dot represents an individual mouse. Bars represent mean ± SEM. *p<0.05; **p<0.01.</p

Decreased autoantibody production in IRF5-deficient MRL/lpr mice.

<p>Sera from IRF5^+/+, IRF5^+/− and IRF5^−/− female MRL/lpr littermates were analyzed at 16 weeks of age. Anti-nuclear antibody (ANA) titers were measured by HEp2 cell immunofluorescence (left-hand panel) and anti-double stranded DNA (dsDNA) antibodies were measured by Crithidia lucillae luminescence (right-hand panel). Bars represent mean ± SEM. *p<0.05; **p<0.01.</p

Less severe renal disease in IRF5-deficient MRL/lpr mice.

<p>A and B, Kidneys from IRF5^+/+ (n = 13), IRF5^+/− (n = 10) and IRF5^−/− (n = 10) female MRL/lpr littermates were analyzed at 16 weeks of age. Renal disease was quantified by measuring the percentage of glomeruli in each mouse showing evidence of mesangial expansion (A), and the percentage of glomeruli in each mouse with crescents or necrosis (B). C, Serum BUN levels in IRF5^+/+ (n = 12), IRF5^+/− (n = 12) and IRF5^−/− (n = 12) female MRL/lpr littermates at 16 weeks of age. Bars represent mean ± SEM. *p<0.05; **p<0.01.</p

The experimental mice used in this study did not have the DOCK2 mutation.

<p>PCR to detect the DOCK2 mutation was performed on tail DNA of all mice used in this study. Experimental mice are indicated by number. DNA from a mouse with the DOCK2 mutation (Mu) was used as a positive control and gave a PCR product for the DOCK2 mutation (305 bp). DNA from a mouse without the DOCK2 mutation (WT) was used as a negative control. CD19 PCR (477 bp) was used as an internal control to verify the adequacy of DNA preparation in each sample.</p

Lymphadenopathy, splenomegaly and splenic CD4⁺ T cell numbers are reduced in IRF5-deficient MRL/lpr mice.

<p>A. Lymph node weight and B. Spleen weight and splenocyte number in IRF5^+/+, IRF5^+/− and IRF5^−/− female MRL/lpr littermates. C. Percentages (upper panels) and total number (lower panels) of splenic B cells and T cell populations in IRF5^+/+, IRF5^+/− and IRF5^−/− female MRL/lpr littermates analyzed by flow cytometry. Mice were analyzed at 16 weeks of age. Bars represent mean ± SEM. *p<0.05; **p<0.01.</p