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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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TLRs use a MyD88–IRAK4–p38 signaling pathway in order to induce expression of the SRs MARCO, LOX-1, and SR-A. (A) BMMs from MyD88−/−, IRAK4−/− mice, or wild-type BMMs pretreated with either sb202190 (sb, 10 μM) or uo126 (10 μM) were stimulated with media (m) or CpG (100 nM) for 4 or 12 h. Total RNA was collected, converted to cDNA, and then Q-PCR was used to assay the inducible expression of SR-A, LOX-1, and MARCO under the different conditions. To control for specificity of p38 and ERK 1/2 inhibition, induction of ICAM-1 and IL-1β were also measured. Q-PCR data are represented in relative expression units and normalized to L32. (B) Wild-type, MyD88−/−, IRAK4−/−, and sb202190 (10 μM) pretreated wild-type BMMs were stained for MARCO surface expression after 24 h of media, CpG (100 nM), lipid A (1 ng/ml), or poly I:C (1 μg/ml) treatment and FACS® data are represented as the mean fluorescent intensity (MFI) of each cell population. (C) RAW 264.7 cells were stimulated with CpG (100 nM) or PGN (20 μg/ml) for 24 h, either in the presence of absence or 10 μM sb202190, stained with a FITC-labeled αSR-A antibody, and then analyzed by FACS®. (D) BMMs from wild-type and IRAK4−/− mice were stimulated with media or CpG (100 nM) for the indicated times. Whole cell extract was then subjected to immunoblotting to detect phosphorylated (activated) p38 or total p38. (E) 293T cells were transfected with a MyD88 expression vector. Extract was collected and incubated with glutathione beads containing either TLR9 (intracellular domain)–GST, TLR3 (intracellular domain)–GST, or GST alone. Bound MyD88 was eluted by boiling and visualized by immunoblotting using an anti-MyD88 polyclonal antibody. The dashed line represents a cropping border where irrelevant lanes have been removed. Data are representative of at least two independent experiments.
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fig3: TLRs use a MyD88–IRAK4–p38 signaling pathway in order to induce expression of the SRs MARCO, LOX-1, and SR-A. (A) BMMs from MyD88−/−, IRAK4−/− mice, or wild-type BMMs pretreated with either sb202190 (sb, 10 μM) or uo126 (10 μM) were stimulated with media (m) or CpG (100 nM) for 4 or 12 h. Total RNA was collected, converted to cDNA, and then Q-PCR was used to assay the inducible expression of SR-A, LOX-1, and MARCO under the different conditions. To control for specificity of p38 and ERK 1/2 inhibition, induction of ICAM-1 and IL-1β were also measured. Q-PCR data are represented in relative expression units and normalized to L32. (B) Wild-type, MyD88−/−, IRAK4−/−, and sb202190 (10 μM) pretreated wild-type BMMs were stained for MARCO surface expression after 24 h of media, CpG (100 nM), lipid A (1 ng/ml), or poly I:C (1 μg/ml) treatment and FACS® data are represented as the mean fluorescent intensity (MFI) of each cell population. (C) RAW 264.7 cells were stimulated with CpG (100 nM) or PGN (20 μg/ml) for 24 h, either in the presence of absence or 10 μM sb202190, stained with a FITC-labeled αSR-A antibody, and then analyzed by FACS®. (D) BMMs from wild-type and IRAK4−/− mice were stimulated with media or CpG (100 nM) for the indicated times. Whole cell extract was then subjected to immunoblotting to detect phosphorylated (activated) p38 or total p38. (E) 293T cells were transfected with a MyD88 expression vector. Extract was collected and incubated with glutathione beads containing either TLR9 (intracellular domain)–GST, TLR3 (intracellular domain)–GST, or GST alone. Bound MyD88 was eluted by boiling and visualized by immunoblotting using an anti-MyD88 polyclonal antibody. The dashed line represents a cropping border where irrelevant lanes have been removed. Data are representative of at least two independent experiments.

Mentions: Although TLR3, TLR4, and TLR9 all induced complement, Fc, and SRs, we chose to focus on SRs due to their ability to directly interact with microbes under nonopsonized conditions (same conditions used in Fig. 1). Using Q-PCR analysis, we confirmed the inducible expression of SR-A and MARCO by the different TLR ligands (Fig. 2 C). We also tested the expression of other SRs and found that LOX-1 was also induced by TLR ligands at early time points (Fig. 2 C). Similar to the pattern of induction obtained in our phagocytosis assays, we found that TLR9 induced the expression of these genes to the highest degree, whereas TLR4-mediated induction was more intermediate and TLR3 was the weakest inducer of the SRs tested. In addition, whereas both TLR9 and TLR4 were able to up-regulate all three of these genes, TLR3 was only able to induce LOX-1 and SR-A, but not MARCO. To determine if induction of SR transcript correlated with increased levels of protein we performed flow cytometry using antibodies specific to SR-A and MARCO and found that TLRs can induce expression of these proteins (Fig. 2 D and Fig. 3, B and C) . These data suggest that TLRs promote phagocytosis though the up-regulation of a phagocytosis gene program that includes SRs.


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)

TLRs use a MyD88–IRAK4–p38 signaling pathway in order to induce expression of the SRs MARCO, LOX-1, and SR-A. (A) BMMs from MyD88−/−, IRAK4−/− mice, or wild-type BMMs pretreated with either sb202190 (sb, 10 μM) or uo126 (10 μM) were stimulated with media (m) or CpG (100 nM) for 4 or 12 h. Total RNA was collected, converted to cDNA, and then Q-PCR was used to assay the inducible expression of SR-A, LOX-1, and MARCO under the different conditions. To control for specificity of p38 and ERK 1/2 inhibition, induction of ICAM-1 and IL-1β were also measured. Q-PCR data are represented in relative expression units and normalized to L32. (B) Wild-type, MyD88−/−, IRAK4−/−, and sb202190 (10 μM) pretreated wild-type BMMs were stained for MARCO surface expression after 24 h of media, CpG (100 nM), lipid A (1 ng/ml), or poly I:C (1 μg/ml) treatment and FACS® data are represented as the mean fluorescent intensity (MFI) of each cell population. (C) RAW 264.7 cells were stimulated with CpG (100 nM) or PGN (20 μg/ml) for 24 h, either in the presence of absence or 10 μM sb202190, stained with a FITC-labeled αSR-A antibody, and then analyzed by FACS®. (D) BMMs from wild-type and IRAK4−/− mice were stimulated with media or CpG (100 nM) for the indicated times. Whole cell extract was then subjected to immunoblotting to detect phosphorylated (activated) p38 or total p38. (E) 293T cells were transfected with a MyD88 expression vector. Extract was collected and incubated with glutathione beads containing either TLR9 (intracellular domain)–GST, TLR3 (intracellular domain)–GST, or GST alone. Bound MyD88 was eluted by boiling and visualized by immunoblotting using an anti-MyD88 polyclonal antibody. The dashed line represents a cropping border where irrelevant lanes have been removed. Data are representative of at least two independent experiments.
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fig3: TLRs use a MyD88–IRAK4–p38 signaling pathway in order to induce expression of the SRs MARCO, LOX-1, and SR-A. (A) BMMs from MyD88−/−, IRAK4−/− mice, or wild-type BMMs pretreated with either sb202190 (sb, 10 μM) or uo126 (10 μM) were stimulated with media (m) or CpG (100 nM) for 4 or 12 h. Total RNA was collected, converted to cDNA, and then Q-PCR was used to assay the inducible expression of SR-A, LOX-1, and MARCO under the different conditions. To control for specificity of p38 and ERK 1/2 inhibition, induction of ICAM-1 and IL-1β were also measured. Q-PCR data are represented in relative expression units and normalized to L32. (B) Wild-type, MyD88−/−, IRAK4−/−, and sb202190 (10 μM) pretreated wild-type BMMs were stained for MARCO surface expression after 24 h of media, CpG (100 nM), lipid A (1 ng/ml), or poly I:C (1 μg/ml) treatment and FACS® data are represented as the mean fluorescent intensity (MFI) of each cell population. (C) RAW 264.7 cells were stimulated with CpG (100 nM) or PGN (20 μg/ml) for 24 h, either in the presence of absence or 10 μM sb202190, stained with a FITC-labeled αSR-A antibody, and then analyzed by FACS®. (D) BMMs from wild-type and IRAK4−/− mice were stimulated with media or CpG (100 nM) for the indicated times. Whole cell extract was then subjected to immunoblotting to detect phosphorylated (activated) p38 or total p38. (E) 293T cells were transfected with a MyD88 expression vector. Extract was collected and incubated with glutathione beads containing either TLR9 (intracellular domain)–GST, TLR3 (intracellular domain)–GST, or GST alone. Bound MyD88 was eluted by boiling and visualized by immunoblotting using an anti-MyD88 polyclonal antibody. The dashed line represents a cropping border where irrelevant lanes have been removed. Data are representative of at least two independent experiments.
Mentions: Although TLR3, TLR4, and TLR9 all induced complement, Fc, and SRs, we chose to focus on SRs due to their ability to directly interact with microbes under nonopsonized conditions (same conditions used in Fig. 1). Using Q-PCR analysis, we confirmed the inducible expression of SR-A and MARCO by the different TLR ligands (Fig. 2 C). We also tested the expression of other SRs and found that LOX-1 was also induced by TLR ligands at early time points (Fig. 2 C). Similar to the pattern of induction obtained in our phagocytosis assays, we found that TLR9 induced the expression of these genes to the highest degree, whereas TLR4-mediated induction was more intermediate and TLR3 was the weakest inducer of the SRs tested. In addition, whereas both TLR9 and TLR4 were able to up-regulate all three of these genes, TLR3 was only able to induce LOX-1 and SR-A, but not MARCO. To determine if induction of SR transcript correlated with increased levels of protein we performed flow cytometry using antibodies specific to SR-A and MARCO and found that TLRs can induce expression of these proteins (Fig. 2 D and Fig. 3, B and C) . These data suggest that TLRs promote phagocytosis though the up-regulation of a phagocytosis gene program that includes SRs.

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