Toll-like receptor expression and responsiveness of distinct murine splenic and mucosal B-cell subsets

Abstract

Background: Toll-like receptors (TLRs) are pattern recognition receptors that recognize pathogen associated molecular patterns and trigger innate immunity leading to initiation of adaptive immunity. TLR-mediated activation of dendritic cells (DCs) is known to be a critical event in the initiation of cellular and humoral immune responses. Recent work however suggests that B cells also express TLRs, and that they can be activated via TLR ligands. However, whether such B cell activation occurs only on memory B cells, or whether it can also occur on truly naïve B cells remains controversial. Furthermore, the expression and functional relevance of TLRs on distinct subsets of B cells, which are known to play differential roles in humoral responses is not known.

Methodology/principal findings: In this study, we investigated the expression pattern of different TLRs in distinct subsets of murine B cells (naïve, memory, follicular, marginal zone, B-1 and peyer's patch). In contrast to the reported restricted expression pattern of TLRs in human peripheral blood naïve B cells, murine splenic naïve B cells express a variety of TLRs with the exception of TLR5 and 8. Consistent with this relatively broad expression pattern, murine naive B cells proliferate and secrete antibody to a variety of TLR agonists in vitro, in the absence of B-cell receptor cross-linking. In addition, we observed subtle differences in the antibody secretion pattern of follicular, marginal zone, B-1 and peyer's patch B-cell subsets.

Conclusions/significance: Thus various B cell subsets, including truly naïve B cells, express multiple TLRs, and signaling via such TLRs results in their robust proliferation and antibody secretion, even in the absence of dendritic cell activation, or T-cell help.

Figure 1. TLR expression profile of murine B-cell subsets.

Real-time PCR profile of TLR expression in FACS sorted follicular B-cells (CD19⁺ B220⁺CD23⁺CD21⁻, panel A), marginal Zone B (CD19⁺ B220⁺CD23⁻CD21⁺, panel B), peritoneal B-1 (CD19⁺B220⁺CD23⁻, panel C) and peyer's patch B cells (CD19⁺B220⁺, panel D) as indicated with β-actin as loading control. CD11c+ dendritic cells were used as a positive control (panel E). Values represent the ratio of the TLR to β-actin.

Figure 2. TLR-induced proliferation of murine B-cell subsets.

A, FACS sorted follicular B-cells (CD19⁺ B220⁺CD23⁺CD21⁻) were cultured in vitro with various TLR ligands as indicated for 3 days and proliferation measured as described in Materials and Methods. B-D, FACS sorted marginal zone, B-1, and peyer's patch B-cells (CD19⁺ B220⁺CD23⁻CD21⁺ for marginal zone B, CD19⁺B220⁺CD23⁻ for peritoneal B-1 B and CD19⁺B220⁺ for peyer's patch B cells) were cultured in vitro with various TLR ligands as indicated for 3 days and proliferation measured by thymidine incorporation as described in Materials and Methods.

Figure 3. TLR-induced B-cell differentiation and immunoglobulin secretion of murine B-cell subsets.

A, FACS sorted follicular B-cells (CD19⁺ B220⁺CD23⁺CD21⁻) were cultured in vitro with various TLR ligands as indicated for 5 days and antibody profile of culture supernatants measured by ELISA as described in Materials and Methods. B-D, FACS sorted marginal zone, B-1 and peyer's patch B-cells (CD19⁺ B220⁺CD23⁻CD21⁺ for marginal zone B, CD19⁺B220⁺CD23⁻for peritoneal B-1 B and CD19⁺B220⁺ for peyer's patch B cells) were cultured in vitro with various TLR ligands as indicated for 5 days and antibody secretion measured by ELISA as described in Materials and Methods.

Figure 4. TLR induced proliferation and immunoglobulin secretion of naïve follicular B cells.

A, Splenocytes taken from naive GCCxRosaYFP mice were stained with anti-B220-allophycocyanin and anti-CD23-phycoerythrin to detect follicular B cells. B220⁺ CD23⁺ cells were further gated for YFP and IgM expression. Numbers shown in each plot indicate frequency of gated cells within total lymphocytes. FACS profile of Naïve (B220⁺ CD23⁺ eYFP⁻ IgM⁺) B cells pre-sort and post-sort. The purity of post-sort was ∼99%. B, FACS sorted naïve B cells as described in panel A and memory B cells (B220⁺ CD23⁺ IgG⁺) from 10% v/v SRBC immunized mice (day 12) were probed for TLR expression by Real-time PCR, C, FACS sorted naïve B cells were cultured in vitro in the presence of various TLR ligands for 3 days and proliferation measured by thymidine incorporation as described in Materials and Methods. D, FACS sorted naïve B cells were cultured in vitro in the presence of TLR ligands for 5 days, culture supernatants were harvested and ELISA was performed as described in Materials and Methods. Experiments were performed three times with similar results and a representative profile is shown.

Figure 5. A heat map summary of TLR expression and responsiveness to different TLR ligands by distinct murine B cell subsets.

The values for TLR expression profile, proliferation and antibody secretion in response to various TLR ligands by different B-cell subsets shown in Figures 1– 3 were plotted on a log scale, and represented as a heat map. For TLR expression, blue represents an 1 fold increase relative to β-actin, green represents 2 fold increase, yellow represents 3 fold increase, red represents 4 fold increase and dark brown represents ≥5 fold increase. For proliferation, blue represents an 1 fold increase relative to unstimulated medium controls, green represents 2 fold increase, yellow represents 3 fold increase, red represents 4 fold increase and dark brown represents 5 fold increase. For antibody secretion, blue represents an 1 fold increase relative to unstimulated medium controls, green represents 2 fold increase, yellow represents 3 fold increase, red represents 4 fold increase and dark brown represents 5 fold increase.