The role of Toll-like receptors (TLRs) in the recognition of extracellular and facultative intracellular bacteria by the innate immune system has been extensively studied, but their role in the recognition of obligate intracellular organisms remains unknown. Coxiella burnetii, the agent of Q fever, is an obligate intracellular bacterium that specifically inhabits monocytes/macrophages. We showed in this study that C. burnetii LPS is involved in the uptake of virulent organisms by macrophages but not in that of avirulent variants. The uptake of virulent organisms was dependent on TLR4 because it was reduced in macrophages from TLR4−/− mice. In addition, LPS was responsible for filamentous actin reorganization induced by virulent C. burnetii, which was prevented in TLR4−/− macrophages. In contrast, the intracellular fate of C. burnetii was not affected in TLR4−/− macrophages, suggesting that TLR4 does not control the maturation of C. burnetii phagosome and the microbicidal activity of macrophages. These results are consistent with in vivo experiments because the pattern of tissue infection and the clearance of C. burnetii were similar in wild-type and TLR4−/− mice. We also showed that the number of granulomas was decreased in the liver of infected TLR4−/− mice, and the formation of splenic granulomas was only transient. The impaired formation of granulomas was associated with decreased production of IFN-γ and TNF. Taken together, these results demonstrate that TLR4 controls early events of C. burnetii infection such as macrophage phagocytosis, granuloma formation, and cytokine production.

he clearance of cells that are dying by apoptosis is essential to the control of tissue homeostasis 1 . In contrast to classical models of phagocytosis, the molecular dissection of engulfment of apoptotic cells is still preliminary 1 . Genetic approaches in Caenorhabditis elegans have yielded several mutants, characterized by the persistence of apoptotic corpses 2-5 . In mammals, in vitro studies have mostly shed light on the macrophage receptors engaged in the recognition of apoptotic surfaces 1,6 . Both of these approaches highlighted the importance of an ATP-binding cassette (ABC)-type transporter, named CED-7 in C. elegans and ABC1 in mouse 4,7 . The requirement for ABC1 on the macrophage surface is specific to the engulfment 7 . Here we assess the distinctive features of engulfment in comparison with other forms of phagocytosis, and find that engulfment exhibits a unique sensitivity to the ABC1 blocker glyburide 8 . In addition, glyburide, as well as antimycin and oligomycin, affects membrane behaviour in both partners of engulfment: when applied to either phagocyte or prey, these drugs impair the Ca 2+ -induced transmembrane randomization of phospholipids 9 . Our results show that engulfment requires a symmetrical transbilayer movement of lipids across the membranes of both the phagocyte and the prey.To differentiate the engulfment from classical forms of phagocytosis, we analysed the effect of pretreatment of mouse macrophages with classical inhibitors of phagocytosis 10 . The phagocytosis of opsonized sheep red blood cells and of apoptotic thymocytes exhibited similar sensitivity to cytochalasin D, lysosomotropic agents, cytosolic acidification, inhibitors of protein kinase C, and nocodazole ( Fig. 1A).However, sensitivity to glyburide 8 was correlated to the particle to be ingested (Fig. 1B). The uptake of yeast or opsonized sheep red blood cells was unaffected by the drug (yeast, 97 ± 8% of control; opsonized cells, 98.8 ± 3% of control) whereas the engulfment of apoptotic thymocytes was reduced to 56 ± 8% of control. As the larger size of the apoptotic thymocytes could influence the mechanics of engulfment, we checked that the ingestion of opsonized apoptotic thymocytes was insensitive to glyburide (116 ± 11% of control). Finally, we showed that the engulfment of fixed and unfixed apoptotic cells was equally efficient and sensitive to glyburide (fixed, 66 ± 3% of control; unfixed, 56 ± 8% of control), thus ruling out the possibility that glyburide acts by blocking the ABC1-mediated transport of a substrate released from the cell.Glyburide has been reported to decrease secretion of interleukin-1β (IL-1β) 11 , and IL-1β can potentiate engulfment 12 ; however, engulfment by macrophages from IL-1β-deficient mice was similarly inhibited by the drug (46 ± 4% of control).As the phosphatidylserine exposure by the apoptotic prey is the key to recognition by macrophages 6 , we extended our analysis to other phosphatidylserine-exposing particles, that is, cells rendered necrotic by heat 13 and human red blood cel...

Phagocytosis is a highly localized event requiring the formation of spatially and temporally restricted signals. Numerous microorganisms have taken advantage of this property to invade host cells. Coxiella burnetii, the agent of Q fever, is an obligate intracellular bacterium that has developed a survival strategy in macrophages based on subversion of receptor-mediated phagocytosis. The uptake of C. burnetii is mediated by αvβ3 integrin and is restricted by impaired cross-talk of αvβ3 integrin and complement receptor 3 (CR3) (CD11b/CD18). In this study, we showed that CR3 molecules remained outside the pseudopodal extensions induced by C. burnetii in THP-1 monocytes, although αvβ3 integrin was present in the pseudopods. Chemoattractants such as RANTES restored CR3 localization to the front of pseudopodal extensions and increased C. burnetii phagocytosis, demonstrating that the localization of CR3 is critical for bacterial uptake. In addition, monocyte activation due to the expression of HIV-1 Nef protein also restored CR3-mediated phagocytosis of C. burnetii by allowing CR3 redistribution toward bacterial-induced pseudopods. The redistribution of CR3 and increased C. burnetii phagocytosis in THP-1 cells stimulated by RANTES or expressing Nef were associated with the inhibition of intracellular replication of C. burnetii. Hence, the localization of CR3 is critical for bacterial phagocytosis and also for the control of bacterial replication. This study describes a nonpreviously reported strategy of phagocytosis subversion by intracellular pathogens based on altered localization of monocyte receptors.

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