Liver tissue samples were snap-frozen in Optimal Cutting Temperature compound (OCT) and cryostat sections (5 μm) stained for B cells (CD19; green), DCs (CD11c; red) and nuclei (DRAQ5; blue). Fluorescent images were captured with an Olympus Fluoview 1000 confocal microscope (software version 1·7a). Differences in levels of cytokine production and surface marker expression between the various groups were analysed by unpaired Cytoskeletal Signaling inhibitor Student’s t-test. P < 0·05 was considered significant. TLRs are the best-defined innate immune sensors that detect MAMPs. Recent evidence supports a role of TLRs in B cell activation and function [19]. We thus determined the expression of

activation markers on B6 mouse freshly isolated liver versus splenic B cells from either LPS (TLR-4 ligand)-treated

or untreated wild-type mice. As shown in Fig. 1a,b, hepatic but not splenic B cells up-regulated their cell surface expression of CD39, CD40, CD80 and CD86 within 24 h of LPS administration. By day 3, expression levels had returned to the normal steady-state level. This suggests that hepatic B cells respond in situ to systemic TLR-4 stimulation more strongly than splenic B cells. Because it has been reported that LPS and poly I:C (TLR-3 ligand) may have different effects on B cells [16], we next examined B lymphocytes isolated from either poly I:C-treated or untreated wild-type mice. As shown in Supplementary Fig. S1, both hepatic learn more and splenic B cells up-regulated their expression of CD39, CD40, CD80, CD86 and PD-L1. This suggests that hepatic and splenic B cells respond in situ to systemic TLR-3 stimulation in a similar manner. In response to TLR stimulation, different mouse splenic B cell subsets exhibit different cytokine secretion profiles [19]. For instance, spleen B1 and marginal zone (MZ) B cells secrete more IL-10, while follicular B cells secrete more IFN-γ [19]. We next examined the pattern of in-vitro

LPS-induced cytokine production by hepatic and splenic B cells. Compared isothipendyl with splenic B cells, hepatic B cells secreted significantly more IFN-γ, IL-6 and TNF-α (Fig. 1c). In contrast, splenic B cells comprised significantly more IL-10 producers (Fig. 1d,e) and secreted much larger amounts of IL-10 than hepatic B cells (Fig. 1c). Consistent with this finding, the spleen exhibited significantly higher percentages of B1a and MZ B cells and a lower incidence of follicular B cells than the liver (Fig. 2). As IL-10 appears to play a pivotal role in the suppressive function of Breg [20], our findings that the liver lacks B1a and MZ-like B cells, and that LPS-stimulated hepatic B cells secrete very low levels of IL-10, suggest that B10 cells are not a prominent regulatory cell subset in mouse liver. There is evidence that the tolerogenic milieu in the normal mouse liver inhibits hepatic mDC differentiation/maturation [3].