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Toll-like receptors induce a phagocytic gene program through p38.

Doyle SE, O'Connell RM, Miranda GA, Vaidya SA, Chow EK, Liu PT, Suzuki S, Suzuki N, Modlin RL, Yeh WC, Lane TF, Cheng G - J. Exp. Med. (2003)

Bottom Line: However, the relationship between these two processes is not well established.Our data indicate that TLR ligands specifically promote bacterial phagocytosis, in both murine and human cells, through induction of a phagocytic gene program.Interestingly, individual TLRs promote phagocytosis to varying degrees with TLR9 being the strongest and TLR3 being the weakest inducer of this process.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Microbiology, Immunology and Molecular Genetics, University of California-Los Angeles, 8-240 Factor Building, 10833 Le Conte Avenue, Los Angeles, CA 90095, USA.

ABSTRACT
Toll-like receptor (TLR) signaling and phagocytosis are hallmarks of macrophage-mediated innate immune responses to bacterial infection. However, the relationship between these two processes is not well established. Our data indicate that TLR ligands specifically promote bacterial phagocytosis, in both murine and human cells, through induction of a phagocytic gene program. Importantly, TLR-induced phagocytosis of bacteria was found to be reliant on myeloid differentiation factor 88-dependent signaling through interleukin-1 receptor-associated kinase-4 and p38 leading to the up-regulation of scavenger receptors. Interestingly, individual TLRs promote phagocytosis to varying degrees with TLR9 being the strongest and TLR3 being the weakest inducer of this process. We also demonstrate that TLR ligands not only amplify the percentage of phagocytes uptaking Escherichia coli, but also increase the number of bacteria phagocytosed by individual macrophages. Taken together, our data describe an evolutionarily conserved mechanism by which TLRs can specifically promote phagocytic clearance of bacteria during infection.

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TLR-induced phagocytosis of bacteria is MyD88, p38, and SR dependent. (A) BMMs derived from either wild-type or MyD88-deficient mice were stimulated with media or 100 nM CpG for 24 h followed by infection with GFP-expressing E. coli at an MOI of 10 for 45 min. Cells were washed twice with cold PBS and subjected to FACS® analysis. (B) RAW 264.7 macrophage cells were stimulated with media, CpG (100 nM), or PGN (20 μg/ml) for 24 h. Cells were cotreated with 10 μM sb202190 for the entire duration of the media, CpG or PGN pretreatment, or just for the final 1 h. Cells were then challenged with GFP–E. coli for 45 min at an MOI of 25, washed, and subjected to FACS® analysis. (C) RAW 264.7 cells were pretreated with either media, CpG (100 nM), or PGN (20 μg/ml) for 24 h. For the final 1 h, the cells were treated with poly I (100 μg/ml), anti–SR-A (3 μg/ml), or IgG2b isotype control (3 μg/ml) and phagocytosis assays were performed. (D) THP-1 monocytes were treated with media or PGN (20 μg/ml) for 36 h. The same conditions were also used along with sb202190 (10 μM) for 36 h or poly I (100 μg/ml) for the final 1 h of TLR ligand pretreatment. Cells were then challenged with GFP–E. coli at an MOI of 5, washed, and subjected to FACS® analysis. Data represent at least two independent experiments.
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fig4: TLR-induced phagocytosis of bacteria is MyD88, p38, and SR dependent. (A) BMMs derived from either wild-type or MyD88-deficient mice were stimulated with media or 100 nM CpG for 24 h followed by infection with GFP-expressing E. coli at an MOI of 10 for 45 min. Cells were washed twice with cold PBS and subjected to FACS® analysis. (B) RAW 264.7 macrophage cells were stimulated with media, CpG (100 nM), or PGN (20 μg/ml) for 24 h. Cells were cotreated with 10 μM sb202190 for the entire duration of the media, CpG or PGN pretreatment, or just for the final 1 h. Cells were then challenged with GFP–E. coli for 45 min at an MOI of 25, washed, and subjected to FACS® analysis. (C) RAW 264.7 cells were pretreated with either media, CpG (100 nM), or PGN (20 μg/ml) for 24 h. For the final 1 h, the cells were treated with poly I (100 μg/ml), anti–SR-A (3 μg/ml), or IgG2b isotype control (3 μg/ml) and phagocytosis assays were performed. (D) THP-1 monocytes were treated with media or PGN (20 μg/ml) for 36 h. The same conditions were also used along with sb202190 (10 μM) for 36 h or poly I (100 μg/ml) for the final 1 h of TLR ligand pretreatment. Cells were then challenged with GFP–E. coli at an MOI of 5, washed, and subjected to FACS® analysis. Data represent at least two independent experiments.

Mentions: To determine if TLR-induced phagocytosis of bacteria is dependent on MyD88–IRAK4–p38-mediated SR up-regulation, we first compared TLR-induced phagocytosis of bacteria by BMMs derived from wild-type and MyD88 knockout mice (Fig. 4 A). Although BMMs have a high basal level of phagocytosis relative to RAW 264.7 cells, which is likely due to up-regulation of SR-A by M-CSF in the culture media (data not shown; and references 29 and 30), CpG pretreatment still significantly increases bacterial uptake by BMMs in a MyD88-dependent manner. Interestingly, the basal level of BMM phagocytosis was significantly decreased in MyD88−/− macrophage cells compared with wild-type controls.


Toll-like receptors induce a phagocytic gene program through p38.

Doyle SE, O'Connell RM, Miranda GA, Vaidya SA, Chow EK, Liu PT, Suzuki S, Suzuki N, Modlin RL, Yeh WC, Lane TF, Cheng G - J. Exp. Med. (2003)

TLR-induced phagocytosis of bacteria is MyD88, p38, and SR dependent. (A) BMMs derived from either wild-type or MyD88-deficient mice were stimulated with media or 100 nM CpG for 24 h followed by infection with GFP-expressing E. coli at an MOI of 10 for 45 min. Cells were washed twice with cold PBS and subjected to FACS® analysis. (B) RAW 264.7 macrophage cells were stimulated with media, CpG (100 nM), or PGN (20 μg/ml) for 24 h. Cells were cotreated with 10 μM sb202190 for the entire duration of the media, CpG or PGN pretreatment, or just for the final 1 h. Cells were then challenged with GFP–E. coli for 45 min at an MOI of 25, washed, and subjected to FACS® analysis. (C) RAW 264.7 cells were pretreated with either media, CpG (100 nM), or PGN (20 μg/ml) for 24 h. For the final 1 h, the cells were treated with poly I (100 μg/ml), anti–SR-A (3 μg/ml), or IgG2b isotype control (3 μg/ml) and phagocytosis assays were performed. (D) THP-1 monocytes were treated with media or PGN (20 μg/ml) for 36 h. The same conditions were also used along with sb202190 (10 μM) for 36 h or poly I (100 μg/ml) for the final 1 h of TLR ligand pretreatment. Cells were then challenged with GFP–E. coli at an MOI of 5, washed, and subjected to FACS® analysis. Data represent at least two independent experiments.
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Related In: Results  -  Collection

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fig4: TLR-induced phagocytosis of bacteria is MyD88, p38, and SR dependent. (A) BMMs derived from either wild-type or MyD88-deficient mice were stimulated with media or 100 nM CpG for 24 h followed by infection with GFP-expressing E. coli at an MOI of 10 for 45 min. Cells were washed twice with cold PBS and subjected to FACS® analysis. (B) RAW 264.7 macrophage cells were stimulated with media, CpG (100 nM), or PGN (20 μg/ml) for 24 h. Cells were cotreated with 10 μM sb202190 for the entire duration of the media, CpG or PGN pretreatment, or just for the final 1 h. Cells were then challenged with GFP–E. coli for 45 min at an MOI of 25, washed, and subjected to FACS® analysis. (C) RAW 264.7 cells were pretreated with either media, CpG (100 nM), or PGN (20 μg/ml) for 24 h. For the final 1 h, the cells were treated with poly I (100 μg/ml), anti–SR-A (3 μg/ml), or IgG2b isotype control (3 μg/ml) and phagocytosis assays were performed. (D) THP-1 monocytes were treated with media or PGN (20 μg/ml) for 36 h. The same conditions were also used along with sb202190 (10 μM) for 36 h or poly I (100 μg/ml) for the final 1 h of TLR ligand pretreatment. Cells were then challenged with GFP–E. coli at an MOI of 5, washed, and subjected to FACS® analysis. Data represent at least two independent experiments.
Mentions: To determine if TLR-induced phagocytosis of bacteria is dependent on MyD88–IRAK4–p38-mediated SR up-regulation, we first compared TLR-induced phagocytosis of bacteria by BMMs derived from wild-type and MyD88 knockout mice (Fig. 4 A). Although BMMs have a high basal level of phagocytosis relative to RAW 264.7 cells, which is likely due to up-regulation of SR-A by M-CSF in the culture media (data not shown; and references 29 and 30), CpG pretreatment still significantly increases bacterial uptake by BMMs in a MyD88-dependent manner. Interestingly, the basal level of BMM phagocytosis was significantly decreased in MyD88−/− macrophage cells compared with wild-type controls.

Bottom Line: However, the relationship between these two processes is not well established.Our data indicate that TLR ligands specifically promote bacterial phagocytosis, in both murine and human cells, through induction of a phagocytic gene program.Interestingly, individual TLRs promote phagocytosis to varying degrees with TLR9 being the strongest and TLR3 being the weakest inducer of this process.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Microbiology, Immunology and Molecular Genetics, University of California-Los Angeles, 8-240 Factor Building, 10833 Le Conte Avenue, Los Angeles, CA 90095, USA.

ABSTRACT
Toll-like receptor (TLR) signaling and phagocytosis are hallmarks of macrophage-mediated innate immune responses to bacterial infection. However, the relationship between these two processes is not well established. Our data indicate that TLR ligands specifically promote bacterial phagocytosis, in both murine and human cells, through induction of a phagocytic gene program. Importantly, TLR-induced phagocytosis of bacteria was found to be reliant on myeloid differentiation factor 88-dependent signaling through interleukin-1 receptor-associated kinase-4 and p38 leading to the up-regulation of scavenger receptors. Interestingly, individual TLRs promote phagocytosis to varying degrees with TLR9 being the strongest and TLR3 being the weakest inducer of this process. We also demonstrate that TLR ligands not only amplify the percentage of phagocytes uptaking Escherichia coli, but also increase the number of bacteria phagocytosed by individual macrophages. Taken together, our data describe an evolutionarily conserved mechanism by which TLRs can specifically promote phagocytic clearance of bacteria during infection.

Show MeSH
Related in: MedlinePlus