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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

Effects of homopolymeric RNAs and viral dsRNA on IL6 production by BEAS2B cells.A) LL37 enhancement of IL6 production increased with increasing length of poly(I:C). Size-selected poly(I:C) was added to BEAS2B cells at 0.5 µg/ml ± 2.2 µM LL37. B) Effect of homopolymeric single or double-stranded RNAs on TLR3 signaling. BEAS2B cells were either untreated () or induced by the single or double-stranded RNAs (0.5 µg/ml) ± LL37 (2.2 µM). Culture media were harvested 24 h after ligand addition and IL6 levels in the medium were quantified by ELISA. C) LL37 significantly enhanced IL6 production induced by viral dsRNAs. The dsRNAs were the genomic RNAs from Reovirus, extracts of plants expressing high levels of the Bell pepper endornavirus (BPEV), and Reovirus-derived S4 dsRNA (S4/Reo). The ssRNA from Hepatitis C virus JFH1 served as a control. Where present, LL37 and Sc37 were both at a final concentration of 2.2 µM. *Indicates p<0.05 compared to RNA ligand alone.
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pone-0026632-g003: Effects of homopolymeric RNAs and viral dsRNA on IL6 production by BEAS2B cells.A) LL37 enhancement of IL6 production increased with increasing length of poly(I:C). Size-selected poly(I:C) was added to BEAS2B cells at 0.5 µg/ml ± 2.2 µM LL37. B) Effect of homopolymeric single or double-stranded RNAs on TLR3 signaling. BEAS2B cells were either untreated () or induced by the single or double-stranded RNAs (0.5 µg/ml) ± LL37 (2.2 µM). Culture media were harvested 24 h after ligand addition and IL6 levels in the medium were quantified by ELISA. C) LL37 significantly enhanced IL6 production induced by viral dsRNAs. The dsRNAs were the genomic RNAs from Reovirus, extracts of plants expressing high levels of the Bell pepper endornavirus (BPEV), and Reovirus-derived S4 dsRNA (S4/Reo). The ssRNA from Hepatitis C virus JFH1 served as a control. Where present, LL37 and Sc37 were both at a final concentration of 2.2 µM. *Indicates p<0.05 compared to RNA ligand alone.

Mentions: We next sought to better define the RNA features that determine its interactions with LL37 to modulate TLR3 signaling. A feature of poly(I:C)-dependent TLR3 activation is that the minimal length for stable binding is ∼50 bp [34]. In HEK 293T cells transiently transfected with TLR3 (293T/TLR3), TLR3 signaling responds to ligands of ∼85 bp [35]. To test whether LL37 affects the length of poly(I:C) recognized by TLR3, size-fractionated poly(I:C) preparations were used as TLR3 ligands in BEAS2B cells (Figure 3A). In the presence of LL37, poly(I:C) of 50 to 85-bp in length significantly increased IL6 production in a length-dependent manner (P<0.004, n = 3; Figure 3A).


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)

Effects of homopolymeric RNAs and viral dsRNA on IL6 production by BEAS2B cells.A) LL37 enhancement of IL6 production increased with increasing length of poly(I:C). Size-selected poly(I:C) was added to BEAS2B cells at 0.5 µg/ml ± 2.2 µM LL37. B) Effect of homopolymeric single or double-stranded RNAs on TLR3 signaling. BEAS2B cells were either untreated () or induced by the single or double-stranded RNAs (0.5 µg/ml) ± LL37 (2.2 µM). Culture media were harvested 24 h after ligand addition and IL6 levels in the medium were quantified by ELISA. C) LL37 significantly enhanced IL6 production induced by viral dsRNAs. The dsRNAs were the genomic RNAs from Reovirus, extracts of plants expressing high levels of the Bell pepper endornavirus (BPEV), and Reovirus-derived S4 dsRNA (S4/Reo). The ssRNA from Hepatitis C virus JFH1 served as a control. Where present, LL37 and Sc37 were both at a final concentration of 2.2 µM. *Indicates p<0.05 compared to RNA ligand alone.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198786&req=5

pone-0026632-g003: Effects of homopolymeric RNAs and viral dsRNA on IL6 production by BEAS2B cells.A) LL37 enhancement of IL6 production increased with increasing length of poly(I:C). Size-selected poly(I:C) was added to BEAS2B cells at 0.5 µg/ml ± 2.2 µM LL37. B) Effect of homopolymeric single or double-stranded RNAs on TLR3 signaling. BEAS2B cells were either untreated () or induced by the single or double-stranded RNAs (0.5 µg/ml) ± LL37 (2.2 µM). Culture media were harvested 24 h after ligand addition and IL6 levels in the medium were quantified by ELISA. C) LL37 significantly enhanced IL6 production induced by viral dsRNAs. The dsRNAs were the genomic RNAs from Reovirus, extracts of plants expressing high levels of the Bell pepper endornavirus (BPEV), and Reovirus-derived S4 dsRNA (S4/Reo). The ssRNA from Hepatitis C virus JFH1 served as a control. Where present, LL37 and Sc37 were both at a final concentration of 2.2 µM. *Indicates p<0.05 compared to RNA ligand alone.
Mentions: We next sought to better define the RNA features that determine its interactions with LL37 to modulate TLR3 signaling. A feature of poly(I:C)-dependent TLR3 activation is that the minimal length for stable binding is ∼50 bp [34]. In HEK 293T cells transiently transfected with TLR3 (293T/TLR3), TLR3 signaling responds to ligands of ∼85 bp [35]. To test whether LL37 affects the length of poly(I:C) recognized by TLR3, size-fractionated poly(I:C) preparations were used as TLR3 ligands in BEAS2B cells (Figure 3A). In the presence of LL37, poly(I:C) of 50 to 85-bp in length significantly increased IL6 production in a length-dependent manner (P<0.004, n = 3; Figure 3A).

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