To the editor:

In a recent Perspective in Nature Immunology1, Blander and Medzhitov explored the function of Toll-like receptor (TLR) signaling in controlling the maturation of phagosomes in macrophages and dendritic cells. As the phagosome is the key organelle for the degradation of microbes and for the generation of bacterial peptides to be presented to lymphocytes, this issue is fundamental for understanding the innate-acquired immune interface.

In their Perspective, Blander and Medzhitov discuss data from their published studies that found a key function for TLR signaling in the maturation of phagosomes. However, Blander and Medzhitov also revisit our paper in which we reported no influence of TLR signaling on phagosome maturation in macrophages2. Their comments, offering an alternative interpretation of our data, are based on the idea that some phagocytic receptors are 'more equal than others'.

In our paper, we used real-time, quantitative assays to measure the rates of acidification and phagosome-lysosome fusion phagosomes formed around silica beads bearing ligands (immunoglobulin G or mannosylated BSA), both with and without TLR agonists2,3. Blander and Medzhitov speculate that the beads we used, internalized by Fc receptors and mannose receptors, 'maxed out' those receptors and thus the rapid phagosome maturation program so that possible effects of additional TLR agonists added to those particles could not be discerned. That interpretation is proposed alongside descriptions of their own studies on phagosome cargos, including Escherichia coli, Salmonella typhimurium and Staphylococcus aureus, which they state are more relevant than immunoglobulin G or mannosylated BSA for the 'nonopsonic' uptake pathways they studied4.

In their Perspective, Blander and Medzhitov did not mention that we studied real-time kinetics of maturation and that to exclude the possibility that Fc receptors and mannose receptors provided too 'dominant' a signal, we examined beads in the context of two conditions beyond use of immunoglobulin G and mannosylated BSA. We supplied wild-type and TLR2-deficient macrophages with fixed S. aureus, with and without lipopolysaccharide (LPS)2 and, as a 'surrogate' for apoptotic cells, we assessed phosphatidylserine-mediated uptake with lipid-coated beads in the presence and absence of the TLR agonists LPS and Pam3Cys (tripalmitoyl cysteinyl lipopeptide)2. In neither of those series of experiments did we find any effect on the rate of phagosome maturation, despite manipulation of both the TLR agonist and the TLR receptor and verification of appropriate TLR activation. In summary, we found no effect of TLR signaling on the phagosomes formed around particles internalized through four distinct phagocytic routes.

In contrast, we believe that the complexity of evaluating phagocytosis of E. coli, S. typhimurium and S. aureus, as used by Blander and Medzhitov, challenges any effort to establish the 'receptor hierarchy' proposed to explain the dichotomy between their results and ours. Moreover, rather than manipulating the TLR agonist makeup of their particles, Blander and Medzhitov relied soley on TLR-deficient phagocytes or phagocytes deficient in the MyD88 signaling adapter4.

Two other results emerged from our study. First, we found that MyD88-deficient macrophages have less phagosome-lysosome fusion regardless of the identity of the internalized particles (which is perhaps a more likely explanation of the discrepancy between the results of our laboratories). Second, LPS-laden beads affect phagosome-lysosome fusion similarly in both wild-type and MyD88-deficient macrophages2. That finding, we believe, should raise concerns regarding the proposal by Blander and Medzhitov that LPS modulates dendritic cell phagosomes directly through TLR4 stimulation5, a proposal, we further believe, that would have benefited from verification by experiments with MyD88- or TLR4-deficient cells. We therefore favor our conclusion that stimulation of TLRs by agonists present on the internalized particle does not affect the rate of phagosome maturation.