Commentary TLR signalling and phagosome maturation: an alternative viewpoint David G. Russell* and Robin M. Yates Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA. In the February issue of Cellular Microbiology Blander expounds on the role of Toll-like receptors (TLRs) in intraphagosomal sensing and the modulation of the nascent phagosome (Blander, 2007a). The review extends the hypothesis of Medzhitov and Blander that the innate sensing machinery is capable of triggering an accelerated or ‘inducible’ maturation that results in a more hostile intraphagosomal environment (Blander and Medzhitov, 2004; 2006a). Attractive though this hypoth- esis of TLR-mediated phagosomal autonomy may be it is contradicted directly by data from our study that used real-time, kinetic analysis of the rates of phagosome acidi- fication and phagosome–lysosome fusion in the presence and absence of stimulation by particle-associated TLR agonists (Yates and Russell, 2005; Yates et al., 2005). Given that these papers have been overlooked in recent reviews (Blander, 2007a,b), we feel it important to inform readers of the substantive data arguing that TLR stimula- tion does not impact on phagosome maturation. In our study we employed novel fluorometric assays that facilitate real-time measurement of pH through ratio- metric fluorescence and phagosome–lysosome fusion through fluorescence resonance energy transfer to probe the kinetics of phagosome maturation (Yates and Russell, 2005; Yates et al., 2005). Initial experiments examined the rate of maturation of IgG bead- and mannosylated bead- containing phagosomes in the presence or absence of lipopolysaccharide (LPS) or Pam3Cys. The experiments were performed on both wild-type and TLR2- and TLR4- deficient macrophages. Although we could observe TLR- mediated signalling in the presence of coupled TLR agonist and TLR, detected by p38 phosphorylation and a-IkB degradation, we could not observe differences in the rate or extent of phagosome maturation. In a recent review by Blander and Medzhitov the authors argue that the use of Fc receptors or the mannose receptor provides the ‘inducible’ signal without the need for stimulation of TLRs (Blander and Medzhitov, 2006a). Unfortunately the authors failed to acknowledge that in our study we then went on to examine the phagocytic processing of additional particles (Yates and Russell, 2005). We studied uptake of Staphylococcus aureus, one of the particles also used by Blander and Medzhitov, and employed phosphatidylserine-coated beads as a surrogate for the apoptotic cells to reproduce the ‘non- inflammatory’ uptake pathway invoked in their earlier pub- lication (Blander and Medzhitov, 2004). We looked at the kinetics of phagosome–lysosome fusion in phagosomes containing Staphylococcus aureus with and without absorbed LPS in both wild-type and TLR2-deficient macrophages. Once again although TLR-dependent sig- nalling was detected under appropriate conditions, no differences in phagosome maturation was observed. Furthermore, the rate of maturation of phosphatidylserine- coated bead-containing phagosomes, mimicking apop- totic cells, also revealed no alterations in the kinetics of phagosome acidification despite addition of LPS or Pam3Cys to the particle. So why the discrepancy? One set of experiments in our study may hold the key. When we compared the rates of phagosome maturation in macrophages from MyD88- deficient mice with those from parental strain controls we found that MyD88-deficient macrophages demonstrated diminished phagosome–lysosome fusion in the presence and absence of TLR agonist (Yates and Russell, 2005). Intriguingly however, LPS altered the kinetics and degree of phagosome–lysosome fusion in both wild-type and MyD88-deficient macrophages. This result has two impor- tant implications. First, MyD88-deficient macrophages have a phagosome maturation defect that is independent of the absence of short-term TLR-dependent signalling. Second, LPS has the capacity to modulate phagosome maturation independently of the action of TLR stimulation through MyD88. How do we interpret the data from Blander and Medzhitov in the context of our results? Analysis of the publication reporting the TLR-dependent modulation of Received 8 February, 2007; accepted 15 February, 2007. *For correspondence. E-mail dgr8@cornell.edu; Tel. (+1) 607 2533401; Fax (+1) 607 2534058. Cellular Microbiology (2007) 9(4), 849–850 doi:10.1111/j.1462-5822.2007.00920.x © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd