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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

Conformations of FAM-LL37, Cy5-poly(I:C) and the complex of FAM-LL37 and Cy5-poly(I:C).Cy5-poly(I:C) (50 µg/ml) or FAM-LL37 (2 µM) was added either singly or as a mixture to coverslips. The images were obtained on a Leica TCS SP5 confocal microscope (100x objective lens). The images separated by a white line were taken from separate areas within the sample.
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pone-0026632-g008: Conformations of FAM-LL37, Cy5-poly(I:C) and the complex of FAM-LL37 and Cy5-poly(I:C).Cy5-poly(I:C) (50 µg/ml) or FAM-LL37 (2 µM) was added either singly or as a mixture to coverslips. The images were obtained on a Leica TCS SP5 confocal microscope (100x objective lens). The images separated by a white line were taken from separate areas within the sample.

Mentions: LL37 has been shown to bind RNAs from necrotic cells and transport self-RNAs into endosomes of dendritic cells [26]. However, it is not known whether LL37 can bind dsRNAs. To determine this, we incubated Cy5-labeled poly(I:C) (50 µg/ml) with FAM-labeled LL37 (2 µM) and visualized them on coverslips using fluorescent microscopy (Figure 8). At pH 7.4, FAM-LL37 exhibited a range of shapes including occasional filamentous structures consistent with LL37's reported ability to oligomerize [20] (Figure 8). Poly(I:C), however, appeared as primarily punctate structures with some large aggregates. When FAM-LL37 was incubated with Cy5-poly(I:C), their fluorescence extensively co-localized, indicating that LL37 can bind dsRNA (Figure 8).


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)

Conformations of FAM-LL37, Cy5-poly(I:C) and the complex of FAM-LL37 and Cy5-poly(I:C).Cy5-poly(I:C) (50 µg/ml) or FAM-LL37 (2 µM) was added either singly or as a mixture to coverslips. The images were obtained on a Leica TCS SP5 confocal microscope (100x objective lens). The images separated by a white line were taken from separate areas within the sample.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198786&req=5

pone-0026632-g008: Conformations of FAM-LL37, Cy5-poly(I:C) and the complex of FAM-LL37 and Cy5-poly(I:C).Cy5-poly(I:C) (50 µg/ml) or FAM-LL37 (2 µM) was added either singly or as a mixture to coverslips. The images were obtained on a Leica TCS SP5 confocal microscope (100x objective lens). The images separated by a white line were taken from separate areas within the sample.
Mentions: LL37 has been shown to bind RNAs from necrotic cells and transport self-RNAs into endosomes of dendritic cells [26]. However, it is not known whether LL37 can bind dsRNAs. To determine this, we incubated Cy5-labeled poly(I:C) (50 µg/ml) with FAM-labeled LL37 (2 µM) and visualized them on coverslips using fluorescent microscopy (Figure 8). At pH 7.4, FAM-LL37 exhibited a range of shapes including occasional filamentous structures consistent with LL37's reported ability to oligomerize [20] (Figure 8). Poly(I:C), however, appeared as primarily punctate structures with some large aggregates. When FAM-LL37 was incubated with Cy5-poly(I:C), their fluorescence extensively co-localized, indicating that LL37 can bind dsRNA (Figure 8).

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