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LL37 and cationic peptides enhance TLR3 signaling by viral double-stranded RNAs.

Lai Y, Adhikarakunnathu S, Bhardwaj K, Ranjith-Kumar CT, Wen Y, Jordan JL, Wu LH, Dragnea B, San Mateo L, Kao CC - PLoS ONE (2011)

Bottom Line: Using a human bronchial epithelial cell line (BEAS2B) and human embryonic kidney cells (HEK 293T) transiently transfected with TLR3, we found that LL37 enhanced poly(I:C)-induced TLR3 signaling and enabled the recognition of viral dsRNAs by TLR3.To separate the effects of LL37 on TLR3 and TLR4, other peptides that bind RNA and transport the complex into cells were tested and found to activate TLR3 signaling in response to dsRNAs, but had no effect on TLR4 signaling.LL37 and several cell-penetrating peptides can enhance signaling by TLR3 and enable TLR3 to respond to viral dsRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana, United States of America. yylai@indiana.edu

ABSTRACT

Background: Toll-like Receptor 3 (TLR3) detects viral dsRNA during viral infection. However, most natural viral dsRNAs are poor activators of TLR3 in cell-based systems, leading us to hypothesize that TLR3 needs additional factors to be activated by viral dsRNAs. The anti-microbial peptide LL37 is the only known human member of the cathelicidin family of anti-microbial peptides. LL37 complexes with bacterial lipopolysaccharide (LPS) to prevent activation of TLR4, binds to ssDNA to modulate TLR9 and ssRNA to modulate TLR7 and 8. It synergizes with TLR2/1, TLR3 and TLR5 agonists to increase IL8 and IL6 production. This work seeks to determine whether LL37 enhances viral dsRNA recognition by TLR3.

Methodology/principal findings: Using a human bronchial epithelial cell line (BEAS2B) and human embryonic kidney cells (HEK 293T) transiently transfected with TLR3, we found that LL37 enhanced poly(I:C)-induced TLR3 signaling and enabled the recognition of viral dsRNAs by TLR3. The presence of LL37 also increased the cytokine response to rhinovirus infection in BEAS2B cells and in activated human peripheral blood mononuclear cells. Confocal microscopy determined that LL37 could co-localize with TLR3. Electron microscopy showed that LL37 and poly(I:C) individually formed globular structures, but a complex of the two formed filamentous structures. To separate the effects of LL37 on TLR3 and TLR4, other peptides that bind RNA and transport the complex into cells were tested and found to activate TLR3 signaling in response to dsRNAs, but had no effect on TLR4 signaling. This is the first demonstration that LL37 and other RNA-binding peptides with cell penetrating motifs can activate TLR3 signaling and facilitate the recognition of viral ligands.

Conclusions/significance: LL37 and several cell-penetrating peptides can enhance signaling by TLR3 and enable TLR3 to respond to viral dsRNA.

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Related in: MedlinePlus

LL37 enhanced dsRNA-induced signaling in 293T cells transiently expressing TLR3.16 h after transfection with plasmids to express TLR3 and the luciferase reporters, reovirus dsRNA or JFH1 ssRNA (both at 1 µg/ml) was added to the media of the transfected cells ± 2 µM of LL37 or Sc37. The cells were analyzed for luciferase activity 20 h after induction using the normalized ratio of firefly/Renilla luciferase activities. The data are presented as fold induction over media control. Each bar shows mean (±SEM) of three independent experiments and the results are representative of more than five independent experiments. *Indicates p<0.05 compared to basal level of reporter activity while ** indicates p<0.05 compared to reporter activity in the presence of reovirus dsRNA.
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pone-0026632-g004: LL37 enhanced dsRNA-induced signaling in 293T cells transiently expressing TLR3.16 h after transfection with plasmids to express TLR3 and the luciferase reporters, reovirus dsRNA or JFH1 ssRNA (both at 1 µg/ml) was added to the media of the transfected cells ± 2 µM of LL37 or Sc37. The cells were analyzed for luciferase activity 20 h after induction using the normalized ratio of firefly/Renilla luciferase activities. The data are presented as fold induction over media control. Each bar shows mean (±SEM) of three independent experiments and the results are representative of more than five independent experiments. *Indicates p<0.05 compared to basal level of reporter activity while ** indicates p<0.05 compared to reporter activity in the presence of reovirus dsRNA.

Mentions: HEK293T (293T) cells transiently transfected to express TLRs and an interferon stimulated response element (ISRE) reporter-driven firefly luciferase have been extensively used to study TLR signaling [36]. 293T cells transiently transfected with TLR3 (293T/TLR3) did have a modest response in reporter activity in the presence of Reovirus dsRNA (5.8 ± 0.2 fold above the mock-treated control; p<0.005, n = 8) (Figure 4). LL37 (2 µM) increased reporter levels by an additional 1.7-fold above that of the Reovirus dsRNAs (Figure 4; p<0.0005, n = 8). The control reaction with JFH1 ssRNA had no effect on reporter levels whether or not LL37 was present (p>0.5). In addition, Sc37 also did not affect reporter activity from 293T/TLR3 cells treated with Reovirus dsRNA (Figure 4, p = 0.5, n = 4). Reporter activity in 293T cells transfected with vector or RIG-I was not increased by the addition of Reovirus dsRNA or JFH1 ssRNA in the culture media (data not shown). Taken together these results show that LL37 can enhance TLR3 signaling by viral dsRNAs in 293T/TLR3 cells. We note, however, that while 293T/TLR3 cells can respond to poly(I:C), exogenously-provided LL37 did not further increase poly(I:C)-induced reporter activity despite numerous attempts (data not shown). These results show that there are subtle differences in the effects of ligands in 293T/TLR3 cells when compared to the BEAS2B cells.


LL37 and cationic peptides enhance TLR3 signaling by viral double-stranded RNAs.

Lai Y, Adhikarakunnathu S, Bhardwaj K, Ranjith-Kumar CT, Wen Y, Jordan JL, Wu LH, Dragnea B, San Mateo L, Kao CC - PLoS ONE (2011)

LL37 enhanced dsRNA-induced signaling in 293T cells transiently expressing TLR3.16 h after transfection with plasmids to express TLR3 and the luciferase reporters, reovirus dsRNA or JFH1 ssRNA (both at 1 µg/ml) was added to the media of the transfected cells ± 2 µM of LL37 or Sc37. The cells were analyzed for luciferase activity 20 h after induction using the normalized ratio of firefly/Renilla luciferase activities. The data are presented as fold induction over media control. Each bar shows mean (±SEM) of three independent experiments and the results are representative of more than five independent experiments. *Indicates p<0.05 compared to basal level of reporter activity while ** indicates p<0.05 compared to reporter activity in the presence of reovirus dsRNA.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198786&req=5

pone-0026632-g004: LL37 enhanced dsRNA-induced signaling in 293T cells transiently expressing TLR3.16 h after transfection with plasmids to express TLR3 and the luciferase reporters, reovirus dsRNA or JFH1 ssRNA (both at 1 µg/ml) was added to the media of the transfected cells ± 2 µM of LL37 or Sc37. The cells were analyzed for luciferase activity 20 h after induction using the normalized ratio of firefly/Renilla luciferase activities. The data are presented as fold induction over media control. Each bar shows mean (±SEM) of three independent experiments and the results are representative of more than five independent experiments. *Indicates p<0.05 compared to basal level of reporter activity while ** indicates p<0.05 compared to reporter activity in the presence of reovirus dsRNA.
Mentions: HEK293T (293T) cells transiently transfected to express TLRs and an interferon stimulated response element (ISRE) reporter-driven firefly luciferase have been extensively used to study TLR signaling [36]. 293T cells transiently transfected with TLR3 (293T/TLR3) did have a modest response in reporter activity in the presence of Reovirus dsRNA (5.8 ± 0.2 fold above the mock-treated control; p<0.005, n = 8) (Figure 4). LL37 (2 µM) increased reporter levels by an additional 1.7-fold above that of the Reovirus dsRNAs (Figure 4; p<0.0005, n = 8). The control reaction with JFH1 ssRNA had no effect on reporter levels whether or not LL37 was present (p>0.5). In addition, Sc37 also did not affect reporter activity from 293T/TLR3 cells treated with Reovirus dsRNA (Figure 4, p = 0.5, n = 4). Reporter activity in 293T cells transfected with vector or RIG-I was not increased by the addition of Reovirus dsRNA or JFH1 ssRNA in the culture media (data not shown). Taken together these results show that LL37 can enhance TLR3 signaling by viral dsRNAs in 293T/TLR3 cells. We note, however, that while 293T/TLR3 cells can respond to poly(I:C), exogenously-provided LL37 did not further increase poly(I:C)-induced reporter activity despite numerous attempts (data not shown). These results show that there are subtle differences in the effects of ligands in 293T/TLR3 cells when compared to the BEAS2B cells.

Bottom Line: Using a human bronchial epithelial cell line (BEAS2B) and human embryonic kidney cells (HEK 293T) transiently transfected with TLR3, we found that LL37 enhanced poly(I:C)-induced TLR3 signaling and enabled the recognition of viral dsRNAs by TLR3.To separate the effects of LL37 on TLR3 and TLR4, other peptides that bind RNA and transport the complex into cells were tested and found to activate TLR3 signaling in response to dsRNAs, but had no effect on TLR4 signaling.LL37 and several cell-penetrating peptides can enhance signaling by TLR3 and enable TLR3 to respond to viral dsRNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana, United States of America. yylai@indiana.edu

ABSTRACT

Background: Toll-like Receptor 3 (TLR3) detects viral dsRNA during viral infection. However, most natural viral dsRNAs are poor activators of TLR3 in cell-based systems, leading us to hypothesize that TLR3 needs additional factors to be activated by viral dsRNAs. The anti-microbial peptide LL37 is the only known human member of the cathelicidin family of anti-microbial peptides. LL37 complexes with bacterial lipopolysaccharide (LPS) to prevent activation of TLR4, binds to ssDNA to modulate TLR9 and ssRNA to modulate TLR7 and 8. It synergizes with TLR2/1, TLR3 and TLR5 agonists to increase IL8 and IL6 production. This work seeks to determine whether LL37 enhances viral dsRNA recognition by TLR3.

Methodology/principal findings: Using a human bronchial epithelial cell line (BEAS2B) and human embryonic kidney cells (HEK 293T) transiently transfected with TLR3, we found that LL37 enhanced poly(I:C)-induced TLR3 signaling and enabled the recognition of viral dsRNAs by TLR3. The presence of LL37 also increased the cytokine response to rhinovirus infection in BEAS2B cells and in activated human peripheral blood mononuclear cells. Confocal microscopy determined that LL37 could co-localize with TLR3. Electron microscopy showed that LL37 and poly(I:C) individually formed globular structures, but a complex of the two formed filamentous structures. To separate the effects of LL37 on TLR3 and TLR4, other peptides that bind RNA and transport the complex into cells were tested and found to activate TLR3 signaling in response to dsRNAs, but had no effect on TLR4 signaling. This is the first demonstration that LL37 and other RNA-binding peptides with cell penetrating motifs can activate TLR3 signaling and facilitate the recognition of viral ligands.

Conclusions/significance: LL37 and several cell-penetrating peptides can enhance signaling by TLR3 and enable TLR3 to respond to viral dsRNA.

Show MeSH
Related in: MedlinePlus