M. tuberculosis and SR-B1 Figure 3. The role of SR-B1 in mycobacterial recognition in primary cells. (A) Western Blot assessing expression of SR-B1 in different macrophage populations derived from wild type C57/BL6 and SR-B12/2 mice, as indicated. (B) Alveolar macrophages and BMDmØ isolated from wild type (black bars) and SR-B12/2 mice (white bars) were tested for BCG-lux binding in the absence of serum. Shown is the relative luciferase activity (R.L.U.), normalised to cell number by CFSE staining. (C) TNF production at 4 hr or 24 hr after binding of BCG-lux to alveolar macrophages or BMDmØ isolated from wild type (black bars) and SR-B12/2 mice (white bars). Shown is x-fold increase in TNF production relative to control (no BCG binding). Experiments were normalised to cell number by CFSE staining. (D) Survival of BCG-lux in infected BMDmØ isolated from wild type (solid lines) and SR- B12/2 mice (dashed lines). After infection of macrophages, unbound mycobacteria were removed and samples taken after the indicated time points to assess luciferase activity. Experiments were performed in triplicate, normalised to cell number by CFSE staining and shown as R.L.U. relative to time point 1. doi:10.1371/journal.pone.0008448.g003 function in cholesterol metabolism, we show here that SR-B1 is also involved in mycobacterial recognition. To our knowledge, this activity so far has only been attributed to the Drosophila homologue Pes [19]. As SR-B1 is involved in cholesterol transport across the membrane [12–14] and as mycobacteria have been shown to display high binding capacity for free cholesterol [26], we wondered if the expression of SR-B1 would lead to an accumulation of cholesterol in the membrane and indirectly lead to increased mycobacterial binding. However, depletion of the membrane’s cholesterol by cyclodextrins did not significantly reduce mycobacterial binding to SR-B1 expressing cells (Fig. 2F). This is supported by an earlier study on SR-B1 expression of transfected COS-7 cells, where the presence of SR-B1 had no effect on the level of free cholesterol [29]. We also demonstrated that mycobacteria bound directly to SR-B1, and that this binding could be competitively inhibited by specific antibodies, lipids, serum and surfactant (Fig. 2A–E). While we clearly show that SR-B1 is involved in recognition of mycobacteria on transfected cells we could not define a role for PLoS ONE | www.plosone.org 6 December 2009 | Volume 4 | Issue 12 | e8448