this receptor in primary cells, although SR-B1 was clearly expressed in wild type macrophages (Fig. 3A) as has been shown earlier for the macrophage cell line RAW264.7 [30]. While disappointing, this was not entirely unexpected, as another study investigating the human homologue of SR-B1, was shown to recognise Gram positive and Gram negative bacteria in transfect- ed, but not in primary cells [20]. Studies on primary macrophages revealed no difference in binding of mycobacteria and subsequent TNF production in the presence or absence of SR-B1 (Fig. 3B and C). However, binding of mycobacteria to both wild type and SR-B12/2 BMDmØ was much stronger than to alveolar macrophages (Fig. 3B), and this is likely to be due to the expression of other recognition receptors on these cells, as has been shown previously [11,31]. We also found that serum blocked binding of mycobacteria to both wild type and SR-B12/2 alveolar macrophages and BMDmØ (data not shown) as opposed to an earlier observation where serum components increased binding to BMDmØ [31]. Furthermore, depletion of the membrane’s cholesterol with MbCD had no effect on binding (data not shown), which is in contrast to a previous study [26]. This can be due to different macrophage populations, preparation techniques and different sera used. Also, other macrophage receptors might be more important for mycobacterial binding which render the role of the cholesterol content in the membrane redundant. Our in vivo studies on the role of SR-B1 during mycobacterial infection also revealed no substantial difference between wild type and SR-B12/2 mice when infected with either a low or a high dose Mtb or when investigated in the context of dietary cholesterol (Fig. 4D, Fig. 5B, Fig. 6E). However, we did observe some clefts of accumulated cholesterol, limited largely to the peripheral paren- chyma, in the absence of SR-B1 after Mtb infection (Fig. 4B). The accumulation of cholesterol in the inflammatory lesions of the SR- B12/2 mouse lungs might account for the fact that cholesterol transport across the macrophage membrane is impaired in these animals. Although we did detect decreased levels of TNF, IFNc and IL10 in the SR-B12/2 animals infected with a high dose Mtb (Fig. 5C), the bacterial burdens in both groups of animals was comparable (Fig. 5B). In fact, there were still substantial amounts of TNF and IFNc present in the SR-B12/2 lungs: 76% TNF and 63% IFNc levels compared to those found in wild type animals, while the amount of IL10 was decreased to 40% compared to wild type. While the changes in cytokine levels are likely to have contributed to the slight difference in size of the inflammatory lesions that were observed following infection with a high dose Mtb (Fig. 5E), we do not feel these data represent a significant biological difference between wild type and knockout animals. The lack of a substantial effect of SR-B1 deficiency was surprising, however, considering the elevated serum cholesterol levels in SR-B12/2 mice (Fig. 6A and C). Indeed, a recent report demonstrated that high serum cholesterol levels in apolipoprotein E (ApoE) deficient mice fed on a HC diet, resulted in high susceptibility of to Mtb [28], although ApoE appeared to have a more pronounced influence on the regulation of plasma cholesterol than SR-B1. However, there were significantly increased bacterial numbers in the lungs of infected wild type and SR-B12/2 animals fed on the HC diet compared to mice on the LC diet (Fig. 6E), which was independent of SR-B1, and, interestingly, did not appear to correlate with the serum cholesterol levels (Fig. 6C and 6E). Although not detectable in the serum, animals fed the HC diet might still have elevated intracellular cholesterol stores which can be taken up and used by Mtb as a source of energy to their growth advantage [32]. This might explain why those animals on the HC diet displayed higher M. tuberculosis and SR-B1 bacterial burdens in the lungs compared to those fed a LC diet. However, the role of SR-B1 for mycobacterial growth seems to be minor and independent of the diet. While playing a role for recognition of mycobacteria in an isolated context using transfected cells, our data supports the notion that macrophage receptors show redundancy when investigated in primary cells [1,33]. This is true, for example, with the mannose receptor (MR), which has long been considered to play a central role in mycobacterial phagocytosis [34], and for the TLRs, which have recently emerged to contribute to the innate recognition of these pathogens [35,36]. The situation is even more complex in vivo. Receptors such as CR3, for example, which is able to mediate both complement-opsonized as well as non-opsonized phagocytosis of mycobacteria in vitro on transfected cells [37] was not found to play a role in vivo [4]. Similarly contradicting findings have been described for the TLRs: although important in vitro [38], TLR2, TLR4, or mice triply deficient in TLR2, TLR4 and TLR9 [5,6,39] were infected with mycobacteria, no susceptibility to Mtb was observed. However, these data were contradicted by other groups showing that TLR2 and TLR4 do play roles in controlling the infection with Mtb [40,41]. This clearly shows that when studying the interaction of Mtb with its host in vivo the particular experimental setting greatly affects the outcome. In this context, the preparation of the mycobacteria has much influence on the experimental observations: we and others found that mycobacteria bind to and activate macrophages to a much greater extent when sonicated rather than syringed ([42] and data not shown). Also, when performing the binding assay under shear conditions rather than static binding one would simulate the dynamic and physiological conditions in the lung much better and select for higher-affinity receptors [43]. With the static binding conditions as presented here, one would also identify low-affinity receptors. It is possible that SR-B1, being a true scavenger with broad ligand specificity, is a low-affinity receptor for mycobacteria and simply plays only a minor role in vivo. Experimental differences will also occur when different strains of mycobacteria (virulent vs. avirulent) and/or genetic background of the receptor-deficient mice (relatively resistant such as C57BL/6 vs. relatively susceptible such as BALB/c) are being used. To add to the complexity, different macrophage populations (i.e. resident vs. elicited vs. immune-activated as well as human vs. murine) express a different array of receptors [11,27] and therefore respond differently to infection with Mtb. This is also important in our experimental approach: we identified SR-B1 when screening a cDNA library generated from the murine macrophage cell line RAW264.7. These cells might express a completely different array of receptors compared to primary cells, leading to the identification of Mtb- binding surface molecules that might not be relevant in vivo. As discussed in the literature before, it seems that Mtb enters the host cell via a number of possible receptor/ligand interactions [1,33]. The ability of Mtb to engage multiple receptors (or receptor clusters) simultaneously might be an evolutionary advantage for its pathogenesis: in the absence of a single receptor, phagocyte infection by mycobacteria can be efficiently supported by alternative receptors. Taken together, we demonstrate that SR-B1 is another example of the diverse spectrum of receptors that recognize mycobacteria in vitro but is redundant in the extremely complex macrophage- mycobacterium interaction in vivo. On the one hand, this redundancy of SR-B1 in mycobacterial infection might be a result, at least in part, of surfactant blocking the bacterial-receptor interactions, as we demonstrated in vitro (Fig. 2D). Surfactant may directly compete with Mtb for the receptor binding site, or that aggregated mycobacteria alter their interaction with the host cells, PLoS ONE | www.plosone.org 10 December 2009 | Volume 4 | Issue 12 | e8448