Toll-Like Receptor 2- and MyD88-Dependent Phosphatidylinositol 3-Kinase and Rac1 Activation Facilitates the Phagocytosis of Listeria monocytogenes by Murine Macrophages

Toll-like receptors (TLRs) play a key role in the innate immune response by sensing bacterial ligands. The mechanisms involved in the TLR-mediated cytokine response are well established; however, the possible contribution of TLR-dependent recognition of bacteria to macrophage phagocytosis remains unclear. Listeria monocytogenes is an intracellular, parasitic, Gram-positive bacterium recognized mainly by TLR2. In this study, we investigated whether TLR2-dependent signaling is involved in the phagocytosis of L. monocytogenes by macrophages. We found no difference in the number of L. monocytogenes cells associating with wild-type (WT) and TLR2^−/− macrophages 1 h after infection. However, the number of L. monocytogenes cells phagocytosed in TLR2^−/− and MyD88^−/− macrophages was significantly lower than that of WT macrophages. In addition, lipopolysaccharide (LPS) treatment restored impaired phagocytic activity of TLR2^−/− macrophages but did not enhance the activity of MyD88^−/− macrophages. The efficiency of phagocytosis was suppressed by inhibitors of phosphatidylinositol 3-kinase (PI3K) and the small Rho GTPases but not by cycloheximide. Moreover, functional activation of PI3K and Rac1 was impaired in TLR2^−/− and MyD88^−/− macrophages. In an in vivo infection model, we found significantly lower numbers of L. monocytogenes cells phagocytosed in peritoneal macrophages of TLR2^−/− and MyD88^−/− mice after intraperitoneal infection. Moreover, a lower number of bacteria were detected in the spleens of TLR2^−/− mice 1 day after intravenous infection than in WT mice. These results clearly indicated that TLR2-MyD88-dependent signaling enhances the basal level of phagocytosis of L. monocytogenes by macrophages through activation of PI3K and Rac1, not by synthesis of proinflammatory cytokines or expression of phagocytic receptors.Listeria monocytogenes is a Gram-positive, facultative, intracellular bacterium that causes severe disease (listeriosis) in humans and various animal species, with a mortality rate of approximately 30% (14, 38). L. monocytogenes can invade various types of cells, including epithelial cells, hepatocytes, endothelial cells, fibroblasts, and macrophages. After entry into host cells, L. monocytogenes is trapped temporarily in an endosome. However, the bacterium quickly escapes from the endosome into the cell cytoplasm, where it undergoes rapid replication by means of expressing various virulence genes, including prfA, plcA, hlyA, actA, mpl, and plcB, all of which are located in a locus called Listeria pathogenicity island 1 (14).Accumulating evidence suggests that L. monocytogenes gains entry into nonprofessional phagocytes by utilizing bacterial invasion factors called internalins (11, 20, 24, 34, 42, 45). Indeed, internalin A mediates the entry of L. monocytogenes into human intestinal cells through binding to E-cadherin and the interaction of internalin B with c-Met (hepatocyte growth factor receptor) induces endocytosis of the bacterium into various types of cells (8, 25, 37). In comparison, professional phagocytes such as macrophages are able to phagocytose and subsequently kill various pathogens through phagolysosome fusion. Macrophages are known to express various receptors that recognize bacterial components or bind to opsonins attached to bacteria (54); a series of intracellular signaling pathways are then activated that lead to the dynamic and rapid reorganization of the actin cytoskeleton for phagocytic engulfment. Of these receptors, Fcγ receptor and complement receptor serve as opsonin receptors for IgG and C3b, respectively, that bind to the surface of bacteria (19, 33). Mannose receptor and CD14 can contribute to phagocytosis of bacteria through direct binding to mannosylated components (15, 17, 44). Scavenger receptor is also implicated in phagocytosis (43). These different cell surface receptors likely operate alone or in combination in the recognition and efficient internalization of bacteria into macrophages.Toll-like receptors (TLRs) are type I integral membrane glycoproteins that are expressed in all lymphoid tissues, with the highest level of expression in peripheral blood leukocytes. TLRs are pattern recognition receptors (PRR) characterized by the presence of a Toll/IL-1 receptor (TIR) domain homologous to interleukin-1 receptor (IL-1R) in their cytoplasmic portion and a variable number of leucine-rich repeats (LRR) in their extracellular portion (31). There are more than 10 TLRs identified to date in mammals, and each TLR exhibits a distinct function in innate immune recognition (1, 5, 52). For example, TLR2 recognizes lipoteichoic acid, peptidoglycan, and lipoproteins of Gram-positive bacteria. TLR4 recognizes lipopolysaccharide (LPS) from Gram-negative bacteria, and TLR9 recognizes bacterial hypomethylated CpG DNA motifs. TLR-dependent recognition of bacteria induces a signal through myeloid differentiation factor 88 (MyD88) and is accompanied by inflammatory responses in macrophages (51). TLR2-MyD88 signaling is known to be critical for the proinflammatory cytokine response during L. monocytogenes infection. Several studies in vivo have revealed that TLR2 is required for optimum control of L. monocytogenes infection (29, 53). The increased susceptibility of MyD88^−/− mice to L. monocytogenes infection may support the idea that TLR2 plays a role in resistance to L. monocytogenes (46). On the other hand, Edelson and Unanue showed that MyD88 was necessary for resistance to L. monocytogenes infection but TLR2 deficiency did not influence the propagation of L. monocytogenes in vivo (16). These conflicting findings appeared to indicate that though TLR2 participates in host resistance to L. monocytogenes through the induction of cytokine production in vivo, there are some signaling pathways that compensate for the lack of TLR2 to control bacterial infection.In addition to this established function of TLRs in the host cytokine response, recent studies suggested that TLR signaling modulates the phagocytosis of pathogens (27, 41). Indeed, Blander and Medzhitov (6) reported that TLR-mediated signaling regulates phagolysosomal maturation in bone-marrow-derived macrophages after infection with Escherichia coli, heat-killed Salmonella enterica serovar Typhimurium, and Staphylococcus aureus. Letiembre et al. (35) reported that TLR2 promotes the phagocytosis of Streptococcus pneumoniae and killing of bacteria by polymorphonuclear leukocytes. Moreover, Luther et al. (39) have shown that TLR2, MyD88, and dectin-1 are required for efficient phagocytosis of Aspergillus fumigatus conidia. Doyle et al. (13) also reported that TLR-mediated enhancement of phagocytosis is due to the upregulation of scavenger receptors, and another recent report on MyD88-mediated phagocytosis of Borrelia burgdorferi has emphasized the importance of downstream signaling through phosphatidylinositol 3-kinase (PI3K) (48). From these findings it is likely that TLR signaling contributes to the phagocytosis and phagolysosome formation in professional phagocytes upon infection with extracellular parasitic bacteria or killed bacteria. On the other hand, little is known about whether the phagocytosis of L. monocytogenes, a representative Gram-positive intracellular parasitic bacterium, is dependent on the TLR signaling pathway. In this study, we analyze the contribution of TLR2-MyD88 signaling to phagocytosis of L. monocytogenes both in vitro and in vivo.