Previous in vitro and in vivo studies
Previous in vitro and in vivo studies have shown the potent ability of toll-like receptor (TLR) ligands and type I interferon (IFN) signaling to induce CH25H in macrophages and DCs following exposure to innate immune stimuli (Bauman et al., 2009; Diczfalusy et al., 2009; Park and Scott, 2010; Zou et al., 2011). Although we have established the essential role of stromal cells in producing and degrading EBI2 ligand in lymphoid organs to support B cell migration responses, we do not exclude the possibility that macrophages (such as marginal metallophilic or subcapsular sinus macrophages) become involved in ligand production under some inflammatory conditions. Our findings suggest that DC production of 7α,25-OHC is normally restricted by intrinsic HSD3B7-mediated degradation. It is notable that CD8+ DCs expressed more Hsd3b7 transcripts than did CD8– DCs. Within the spleen, CD8+ DCs (that coexpress DEC205) are abundant in the inner T zone, whereas CD8– DCs are abundant in the major interfollicular regions (Steinman et al., 1997). Our studies are consistent with the possibility that this difference in HSD3B7 expression contributes to the central T zone’s status as a low-EBI2-ligand zone compared to interfollicular regions. Indeed, it can be speculated that EBI2 favors encounters between 6015 and CD8– DCs by promoting movement to interfollicular regions. We anticipate that under activation conditions leading to CH25H induction (Bauman et al., 2009; Diczfalusy et al., 2009; Park and Scott, 2010; Zou et al., 2011), DC production of 7α,25-OHC exceeds HSD3B7-mediated degradation and acts to promote new interactions between DCs and EBI2-expressing cells. Although HSD3B7 transcripts were ∼10-fold more abundant in DCs than in lymphocytes, our studies do not exclude roles for hematopoietic cells other than DCs in mediating 7α,25-OHC metabolism in the T zone. CYP7B1 deficiency causes a loss of detectable EBI2 ligand production in lymphoid organs, consistent with this being the only enzyme known to catalyze 7α-hydroxylation of 25-OHC (Russell, 2003) and with the finding of reduced 7α,25-OHC in spleens of clotramizole-treated mice (Liu et al., 2011). CYP7B1 will 7α-hydroxylate 27-hydroxycholesterol to produce 7α,27-OHC, another EBI2 ligand that is about one-tenth as potent as 7α,25-OHC in migration assays (Hannedouche et al., 2011; Liu et al., 2011). The loss of EBI2 ligand function in CH25H-deficient mice, animals that fully retain the ability to synthesize 27-OHC and 7α,27-OHC, argues against a major role for 7α,27-OHC in guiding the movement of B cells in lymphoid tissues. It remains possible that 7α,27-OHC functions to control a cellular behavior not studied here. Moreover, because both 7α,25-OHC and 7α,27-OHC are substrates for HSD3B7 (Russell, 2003), it is possible that some of the increase in EBI2 ligand detectable in the absence of this enzyme is attributable to 7α,27-OHC. Additional studies will be needed to quantify 7α,25-OHC and 7α,27-OHC concentrations in mouse lymphoid and peripheral tissues. Although compromised in EBI2 ligand production, CYP7B1-deficient mice have elevated circulating 25-OHC and 27-OHC (Bauman et al., 2009; Li-Hawkins et al., 2000). These oxysterols have the capacity to modulate some immune cell functions through actions on nuclear hormone receptors (Bensinger et al., 2008; Kalaany and Mangelsdorf, 2006; Villablanca et al., 2010) and to regulate IgA production (Bauman et al., 2009). Increases in these oxysterols are unlikely to account for the defective IgM and IgG plasma cell responses we describe here, because EBI2-deficient B cells are equally compromised in the generation of plasma cells in both wild-type and CYP7B1-deficient hosts. That is, the positive influence of CYP7B1 in promoting IgM and IgG plasma cell responses appears to depend upon the generation of EBI2 ligand and signaling via EBI2. Moreover, in contrast to the altered mucosal IgA responses seen in CH25H- and CYP7B1-deficient mice (Bauman et al., 2009), we have not detected altered IgA production in EBI2-deficient mice (A. Reboldi, L.M.K., and J.G.C., unpublished data). Further supporting the conclusion that it is the amount of EBI2 ligand that is important is the discovery of similar defects in the plasma cell response in Ch25h−/− and Hsd3b7−/− mice. An important challenge for future studies will be elucidating whether EBI2, CH25H, CYP7B1, and HSD3B7 support B cell responses by promoting more efficient interactions between B and T cells at the B-T boundary and in interfollicular regions, or by promoting interactions between early plasmablasts and factors in interfollicular and outer follicular regions that support their growth and differentiation.