\begin{figure}
\centering
\begin{subfigure}{0.45\textwidth}
\centering
\includegraphics{figure_2_1.pdf}
\end{subfigure}
\begin{subfigure}{0.45\textwidth}
\centering
\includegraphics{figure_2_2.pdf}
\end{subfigure}
\caption{\textbf{Fig. 2.} $\gamma3$ germ line transcript is dependent on TLR2, TLR4 and RP105/MD-1. (A) Expression of mRNA encoding $\mu$ (left), $\gamma3$ (middle) and $\gamma2b$ (right) chain in enriched splenic B cells was measured by real-time reverse transcription, RT--PCR. Enriched splenic B cells were obtained from wild-type, RP105$^{-/-}$, MD-1$^{-/-}$, MyD88$^{-/-}$ or TLR2$^{-/-}$TLR4$^{-/-}$ mice as indicated in the figure. RT--PCR was conducted as described in Methods. The results are represented as mean value with standard deviation from triplicate samples. (B) Splenic B cells from indicated mice were stimulated with LPS (1 $\mu$g ml$^{-1}$), PC3CSK4 (1 $\mu$g ml$^{-1}$) or CpG (1.0 $\mu$M) for 2 days. Quantitative RT--PCR was conducted for measuring AID mRNA expression. Each expression level was normalized by mouse HPRT expression. Results are representatives of more than three independent experiments.}
\label{figure_2}
\end{figure}

To address a role of microbial flora, we studied germ-free mice (Fig.~\ref{fig:figure_3A}). Serum IgA in germ-free mice was reported to be reduced \cite{28}. In keeping with this, serum IgA was significantly down-regulated in the germ-free mice used in this study, demonstrating the effectiveness of germ-free condition. Nonetheless, the rest of isotypes including IgG3 and IgG2b was not reduced. Compared with control mice, IgM and IgG3 were rather up-regulated. We next employed antibiotics treatment to deplete microbial flora. Mice were given antibiotics-containing water from 4--8 weeks of age. These mice were similar to germ-free mice in that serum IgA was significantly reduced, confirming the reduction of microbial flora (Fig.~\ref{fig:figure_3B}). Despite the effective treatment with antibiotics, serum IgG3 was not decreased at all. Instead, IgG2b was significantly down-regulated by antibiotics treatment (Fig.~\ref{fig:figure_3B}). The exact reason for IgG2b reduction is currently unknown. These results did not necessarily negate roles for microbial flora, but strongly suggested that microbial flora is dispensable for tonic B cell activation driven by TLR2, TLR4 and RP105/MD-1.

\subsection{Roles for RP105 in serum Ig titers of lupus-prone mice}

TLRs have been implicated in autoimmune diseases. Especially, in MRL/lpr mice, TLR7 was reported to deteriorate the autoimmune disease \cite{5}. MRL/lpr mice reared in a germ-free state were similar to those in conventional conditions with regard to lymphoproliferation and B cell autoimmunity \cite{29}, indicating that autoimmunity in MRL/lpr is independent of live, non-pathogenic microbes. This report suggests that B cells are excessively activated by non-microbial, environmental stimuli in MRL/lpr mice. It is possible that such excessive B cell activation is driven not only by TLR7 but also by TLR2, TLR4 and RP105. To address this possibility, we established RP105$^{-/-}$ MRL/lpr mice. We considered that RP105$^{-/-}$ mutation is more appropriate than TLR2$^{-/-}$TLR4$^{-/-}$ double mutation. The lack of RP105 impairs TLR2/4 responses only in B cells \cite{16}, whereas the lack of TLR2/4 blunts all the TLR2/4 responses not only in B cells but also in dendritic cells, macrophages, endothelial cells or fibroblasts. It is difficult to discriminate the effect of TLR2/4 mutation in B cells from that in non-B cells. We also introduced RP105 deficiency into another type of lupus-prone mice, BWF1 mice. We studied the levels of serum Igs at 20 weeks of age and found that RP105$^{-/-}$ MRL/lpr mice were significantly lower than RP105$^{+/+}$ MRL/lpr mice in serum IgG3 and IgG2a. RP105$^{-/-}$ BWF1 mice were lower than RP105$^{+/+}$ BWF1 mice in serum IgG3 and IgG2b (Fig.~\ref{fig:figure_4}).

\subsection{Roles for RP105 in autoimmunity in MRL/lpr}

RP105$^{-/-}$ mutation ameliorated mortality in the MRL/lpr strain (Fig.~\ref{fig:figure_5A}). In contrast, the lack of RP105 did not influence mortality in BWF1 mice despite IgG3 reduction (Fig.~\ref{fig:figure_5A}). Elevation of BUN, which correlated with renal dysfunction, was significantly improved in RP105$^{-/-}$ MRL/lpr mice, whereas no significant difference was detected between RP105$^{+/+}$ and RP105$^{-/-}$ BWF1 mice (Fig.~\ref{fig:figure_5B}). We further examined glomerulonephritis in MRL/lpr mice. Despite the improvement in BUN accumulation (Fig.~\ref{fig:figure_5B}), any significant change was not found in the intensity and extent of glomerular alterations when assessed at 18--19 weeks of age (Fig.~\ref{fig:figure_5C}). MRL/lpr mice spontaneously develop systemic necrotizing arteritis of small- and medium-sized muscular arteries, which is characterized by the infiltration of mononuclear and/or polynuclear leukocytes and by the destruction of the arterial wall \cite{30}. Notably, necrotizing arteritis in kidneys was observed less frequently in RP105$^{-/-}$ MRL/lpr than in RP105$^{+/+}$ MRL/lpr mice (Fig.~\ref{fig:figure_5D}). Impaired renal perfusion may be responsible for deterioration of renal function revealed by elevated BUN.

MRL/lpr mice develop severe splenomegaly and lymphadenopathy due to accumulation of lymphocytes expressing an activated phenotype in these organs \cite{31}. The weight of lymphoid organs including spleen, mesenteric lymph nodes and axillary lymph nodes was examined at 19 weeks of age. We found that splenomegaly and lymphadenopathy were both significantly reduced in the absence of RP105 in MRL/lpr mice (Fig.~\ref{fig:figure_6A} and \ref{fig:figure_6B}). Due to unknown reason, axillary lymph nodes were most apparent in difference between RP105$^{-/-}$ MRL/lpr and RP105$^{+/+}$ MRL/lpr mice (Fig.~\ref{fig:figure_6B}). We next examined lymphocyte subpopulation in the spleen. Decreases in the percentages of CD4$^{+}$ T cells and CD8$^{+}$ T cells were significantly improved, whereas no significant difference was observed in the percentages of CD4/CD8 double-negative T cells (DNTCs) and B cells (Fig.~\ref{fig:figure_6C}).