DNA microarray gene expression profile of marginal zone versus follicular B cells and idiotype positive marginal zone B cells before and after immunization with Streptococcus pneumoniae

Abstract

Marginal zone (MZ) B cells play an important role in the clearance of blood-borne bacterial infections via rapid T-independent IgM responses. We have previously demonstrated that MZ B cells respond rapidly and robustly to bacterial particulates. To determine the MZ-specific genes that are expressed to allow for this response, MZ and follicular (FO) B cells were sort purified and analyzed via DNA microarray analysis. We identified 181 genes that were significantly different between the two B cell populations. Ninety-nine genes were more highly expressed in MZ B cells while 82 genes were more highly expressed in FO B cells. To further understand the molecular mechanisms by which MZ B cells respond so rapidly to bacterial challenge, Id-positive and -negative MZ B cells were sort purified before (0 h) or after (1 h) i.v. immunization with heat-killed Streptococcus pneumoniae, R36A, and analyzed via DNA microarray analysis. We identified genes specifically up-regulated or down-regulated at 1 h following immunization in the Id-positive MZ B cells. These results give insight into the gene expression pattern in resting MZ vs FO B cells and the specific regulation of gene expression in Ag-specific MZ B cells following interaction with Ag.

Figure 1. Expression profile of differentially expressed genes between FO and MZ B cells

DNA microarray analysis identified 181 genes that were significantly different in sort-purified follicular (FO) vs. marginal zone (MZ) B cells from MD4 transgenic mice (B6 background). The identified transcripts have a fold change > 2 and a p value < 0.02 by T-test. The differentially expressed genes were grouped into various functional categories (A) Motility/Adhesion, (B) Immune Response, (C) Apoptosis, and (D) Proliferation. Shown are normalized expression values greater than (yellow), near (black), or less than (blue) the mean of that gene. Each column represents one independent sample of sort-purified FO or MZ B cells. Genes or transcripts are represented in rows. Clustering of the genes is unsupervised.

Figure 2. Expression profile of differentially expressed genes between FO and MZ B cells

DNA microarray analysis identified 181 genes that were significantly different in sort-purified follicular (FO) vs. marginal zone (MZ) B cells from MD4 transgenic mice (B6 background). The identified transcripts have a fold change > 2 and a p value < 0.02 by T-test. The differentially expressed genes were grouped into various functional categories (A) Transcription Factors and (B) Signal Transduction. Shown are normalized expression values greater than (yellow), near (black), or less than (blue) the mean of that gene. Each column represents one independent sample of sorted FO or MZ B cells. Genes or transcripts are represented in rows. Clustering of the genes is unsupervised.

Figure 3. Identification of strain-specific differences in gene expression profiles between FO and MZ B cells

Gene expression profile of splenic FO and MZ B cells from B6, SWR, and C3H mice. The profile includes 181 gene transcripts with a fold change > 2 and a p value < 0.02 by T-test. (A) Hierarchical analysis of 152 genes with consistent regulation across the three mouse strains. (B) Hierarchical analysis of 29 genes with strain-specific differences in MZ vs. FO gene expression profiles. Shown are normalized expression values greater than (yellow), near (black), or less than (blue) the mean of that strain. Each column represents one sample of sorted FO or MZ B cells. Genes or transcripts are represented in rows. Clustering of genes is unsupervised.

Figure 4. MZ B cells express higher levels of D6 and RGS10 relative to FO B cells

Resting splenic MZ and FO B cells were sort-purified and total RNA and protein isolated. Resting MZ B cells express higher (A) mRNA and (B) protein levels of D6 and RGS10, as determined by RT-PCR and Western blot, respectively. Total splenocytes were isolated and analyzed via FLOW cytometry. The expression level of (C) D6 Isotype 0.3%, MZ B cell 88.8%, and FO B cell 11.8% and (D) RGS10 Isotype 0.2%, MZ B cell 82.1%, and FO B cell 1.1% are displayed as a histogram plot.

Figure 5. Regulated genes in Idiotype Positive MZ B cells after Activation

MZ Id+ (Ag+) and Id− (Ag−) B cells were isolated from M167 Tg mice at 0 and 1 hour after i.v. immunization with heat killed S. pneumoniae, R36A. DNA microarray analysis identified genes that were significantly up regulated and down regulated in the Id+ MZ B cells 1 hr after activation. The genes specifically regulated in the Id+ MZ B cells were grouped into various functional categories (A) Chemokines, (B) Chemokine Receptors, (C) Cytokines, and (D) Cytokine Receptors. Shown are normalized expression values greater than (yellow), near (black), or less than (blue) the mean of that gene. Each column represents one independent sample. Genes or transcripts are represented in rows. Clustering of the genes is unsupervised.

Figure 6. Regulated genes in Idiotype Positive MZ B cells after Activation

MZ Id+ (Ag+) and Id− (Ag−) B cells were isolated from M167 Tg mice at 0 and 1 hour after i.v. immunization with heat killed S. pneumoniae, R36A. DNA microarray analysis identified genes that were significantly up regulated and down regulated in the Id+ MZ B cells 1 hr after activation. The genes specifically regulated in the Id+ MZ B cells were grouped into various functional categories (A) Apoptosis and (B) Immune Cell Markers. Shown are normalized expression values greater than (yellow), near (black), or less than (blue) the mean of that gene. Each column represents one independent sample. Genes or transcripts are represented in rows. Clustering of the genes is unsupervised.

Figure 7. IL-10 and Stra13 are increased following R36A immunization

M167 tg mice were crossed with an IL-10/Thy1.1 reporter mouse and immunized i.v. with R36A. MZ Id+ B cells were sort-purified at 0, 1, and 4 hours after immunization and total RNA was isolated. (A) RT-PCR was performed using gene-specific primers for IL-10, Thy1.1, Stra13, and actin. The expression level of (B) Thy1.1 was determined via FACS analysis on gated MZ Id+ B cells at 24 hours following R36A immunization. MZ Id+ B cells were approximately 5% (PBS) and 20% (R36A) positive for Thy1.1 respectively.