panels). Importantly, Mtb-lux bound to SR-B1 transfected R6F cells and this binding could be inhibited by the presence of serum, LDL and BAL (Fig. 2A, C and D, right panels), which was due to direct competition for the receptor (Fig. 2B, right panel). Further, the binding of Mtb-lux to SR-B1 expressing cells could be inhibited in the presence of an antibody against SR-B1 (Fig. 2E, right panel). Thus overall, these data suggest that SR-B1 can directly recognize mycobacteria and that this interaction can be inhibited by serum and surfactant components. The Role of SR-B1 for Mycobacterial Infection of Primary Cells Having demonstrated that SR-B1 can mediate the recognition of mycobacteria in transfected cells, we next investigated the contribution of this receptor on primary cells. Initially, different macrophage populations (alveolar macrophages, bone-marrow derived macrophages (BMDmØ), resident as well as thioglycollate- elicited peritoneal macrophages) were isolated from wild type C57BL/6 mice and tested for the expression of SR-B1 by Western Blotting (Fig. 3A). This analysis demonstrated the expression of this receptor on all macrophage populations examined, and was absent in macrophages derived from SR-B12/2 mice. Important- ly, alveolar macrophages and BMDmØ expressed highly SR-B1, therefore these cell populations were tested for binding of BCG-lux (Fig. 3B). Surprisinlgy, no statistically significant differences in binding could be observed between wild type and SR-B12/2 macrophages in the absence of serum. Notably, mycobacterial binding to resident alveolar macrophages was inefficient when compared to BMDmØ, as has been demonstrated in a previous study [27]. When analysed for TNF production 4 hr or 24 hr after mycobacterial binding, no differences between wild type and SR-B12/2 macrophages were detected (Fig. 3C). Although the level of TNF was equally low after 4 hr in both macrophage populations, it increased considerably in BMDmØ after 24 hr, presumably reflecting the differences in bacterial binding as described above. These experiments were repeated with varying parameters in bacterial binding, including different incubation times and temperatures, but no differences between wild type and SR-B12/2 macrophages were detected under any of the conditions tested (data not shown). We also found that the presence of serum inhibited binding of mycobacteria to BMDmØ equally well in both wild type and SR-B12/2 macrophages, while the addition of MbCD (to deplete cholesterol membrane) had no effect on binding (data not shown). Furthermore, when we blocked SR-B1 with a specific antibody, binding of mycobacteria to the macrophages was unaltered compared to untreated controls (data not shown). Finally we investigated whether the survival of mycobacteria in macrophages was affected by the presence or absence of SR-B1. To this end, wild type and SR-B12/2 BMDmØ were infected with BCG-lux and mycobacterial viability analysed over time by measuring luciferase activity. As shown in Fig. 3D, both wild type and SR2B12/2 macrophages could equally efficiently kill mycobacteria within the first 24 hr after infection, following which the bacteria started to replicate. Thus loss of SR-B1 has minimal impact on mycobacterial recognition by primary macrophages in vitro. SR-B1 Is Dispensable for Infection with Mycobacterium tuberculosis In Vivo To explore a potential role for SR-B1 in vivo, wild type and SR- B12/2 mice were aerosol infected with 100 CFU Mycobacterium tuberculosis H37Rv, and the development of disease monitored after M. tuberculosis and SR-B1 2 and 4 months. As shown in Fig. 4A, both wild type and SR- B12/2 mice survived and gained weight throughout the course of the infection. The lungs of both groups displayed inflammatory foci, consisting of patchy infiltrates of inflammatory cells, predominantly foamy macrophages (Fig. 4B). Typical granulomas were not observed. Some SR-B12/2 mice showed evidence of clefts of accumulated cholesterol, which were not observed in the wild type animals (Fig. 4B and data not shown). When the sizes of inflammatory foci were measured by morphometric analysis (Fig. 4C), SR-B12/2 mice did not show significant differences to the wild type mice. Furthermore, no differences in bacterial burdens (Fig. 4D) or production of TNF, IFNc, IL10, IL12p70 or IL6 (Fig. 4E) were detected between wild type and SR-B12/2 mice. We also tested the effects of infection with a high dose of Mtb, i.e 1000 CFU per lung (Fig. 5). As before, both groups of mice gained weight and did not display any adverse effects during the course of the infection (Fig. 5A). Although we detected significantly less pulmonary TNF, IL10 and IFNc in the SR-B12/2 animals (Fig. 5C), this did not correlate with differences in bacterial burdens (Fig. 5B). Although the inflammatory pathology was similar between both groups of mice, except for the presence of occasional cholesterol clefts (Fig. 5D and data not shown), the size of the inflammatory foci in the SR-B12/2 mice were found to be slightly increased (p-value of 0.032) (Fig. 5E). Elevated serum cholesterol levels are known to correlate with susceptibility to tuberculosis [28], and as SR-B1 is involved in cholesterol metabolism, we assessed the influence of cholesterol in the diet on susceptibility to mycobacterial infection in wild type and SR-B12/2 mice. Appropriate numbers of animals were fed either a low cholesterol (LC, 0.15% cholesterol) or a high cholesterol (HC, 1.25% cholesterol) diet and then infected with 100 CFU Mtb. Cholesterol levels at the time of infection were significantly higher in SR-B12/2 mice compared to wild type mice on either diet, and higher levels of cholesterol were detected in SR- B12/2 mice on the HC diet compared to animals on the LC diet (Fig. 6A). As observed in our previous experiments (Fig. 4A and 5A), all animals gained weight and did not show any adverse effects during the course of the infection (Fig. 6B). When the experiment was terminated after 4 months, there were similar relative differences between the various mouse strains in serum cholesterol levels, although the SR-B12/2 mice showed an overall general increase (Fig. 6C, compare with Fig. 6A). We also observed a slight, but significant, diet-dependent difference in bacterial burden of the lungs, with elevated bacterial levels in both wild type and SR-B12/2 mice on the HC diet compared to the corresponding animals on the LC diet (Fig. 6E). However, there was no difference in bacterial burden between wild type and SR- B12/2 mice on either diet (Fig. 6E). Despite the slightly elevated bacterial burdens in mice on HC diets, there were no significant differences in histopathology, except for the occasional presence of cholesterol clefts in SR-B12/2 lungs (Fig. 6D), or in the levels of pulmonary cytokine (Fig. 6F). Discussion The identification of host pattern recognition receptors that recognise Mtb represents a potential opportunity for anti- mycobacterial prophylaxis, as the interruption of binding may functionally disrupt Mtb entry into its host cell niche. By screening a retroviral cDNA expression library derived from RAW264.7 murine macrophages, we identified SR-B1 as a receptor for mycobacteria (Fig. 1A and B). As a member of the CD36 family of scavenger receptors, which have been well described for their PLoS ONE | www.plosone.org 5 December 2009 | Volume 4 | Issue 12 | e8448