Notch2 Regulates Macrophage-Related Genes in Marginal Zone B Cells.

[Background] While Notch1 plays critical roles in early T cell development, Notch2 is indispensable for B cell development. Conditional inactivation of Notch2 in the hematopoietic compartment leads to loss of the marginal zone B (MZB) cells and defect in the particular fraction of follicular B (FOB) cells characterized as T2 B cells. Because of their position bordering the marginal sinuses and red pulp in the spleen, MZB cells are amongst the first cells that come in contact with blood-borne substances and thus thought to have critical roles in the defense against bacterial pathogens which are presumably scavenged in this anatomical site. Here we demonstrate that Notch2 regulates the expression of genes that specifically play a role in macrophages.

[Methods] Splenic T2 B cells, supposed to contain precursors of MZB cells, were sorted from Notch2 conditional knockout (N2cKO) and wild type (WT) mouse and conveyed microarray analysis. Then, different fractions of B cells in different organs from N2cKO and WT mouse were sorted and the expression of CD36, whose upregulation by Notch2 signaling was implicated by the microarray, and CD14, which has a specific function in macrophages, was examined, using real time PCR and FACS analyses. [Results] Out of 20,000 genes that were covered by the microarray analysis, only 9 had reduced expression levels with N2cKO/WT ratio < 0.5 in quadricate experiments. Surprisingly, beside Deltex1 and Hes1 that are known to be direct Notch target genes, the majority of the genes had relation with macrophages, i.e., preferential expression or particular function. Among these macrophage-related genes, we further concentrated on CD36, being known to have scavenger function in endothelial cells and macrophages. We found that among different fractions of B cells from different organs in WT mice, CD36 was specifically expressed at higher levels in MZB and T2 B cells. We further found that the level of CD36 expression was reduced in MZB and T2 B cells in the Notch2 heterozygotes (and N2cKO mice in a gene dosage-dependent manner). Since these observations suggested that Notch2 signaling regulates the expression of macrophage-related genes in MZB cells, we examined whether Notch2 signaling also regulates the expression of CD14, a representative gene preferentially expressed in macrophages coding for the LPS/LBS receptor. In the WT mouse, we found that CD14 is expressed in MZB cells at a level comparable with that in macrophages, while we did not detect its expression in FOB cells when analyzed by FACS. We again observed decreased CD14 expression in MZB cells from Notch2 heterozygotes, and in T2 B cells from the Notch2 heterozygotes and cKO mice in a gene dosage-dependent manner.

[Conclusions] MZB cells have been considered to play important roles at the border of the acquired and innate immunity. The data presented here support this paradigm, and further suggest that Notch2 signaling is crucial for the transcriptional regulation of genes being related to the innate immune function in MZB cells.