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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 co-localizes with TLR3 and poly(I:C).A) The location of LL37 in BEAS2B cells. BEAS2B cells were cultured on coverslips and FAM-LL37 was added at a final concentration of 2 µM. Five hours after the addition of FAM-LL37, the cells were fixed and examined using confocal microscopy. B) LL37 can co-localize with Cy5-poly(I:C). FAM-LL37 (2 µM) and Cy5-poly(I:C) (1 µg/ml) were added to BEAS2B cells. The cells were fixed and imaged 5 h after the addition of the fluorescently-tagged molecules. C) TLR3 can co-localize with poly(I:C) independent of exogenously-provided LL37. BEAS2B cells were treated with Cy5-poly(I:C), then permeabilized and stained with a goat anti-TLR3 antibody in complex with Texas Red-labeled secondary antibody. D) Co-localization of FAM-LL37, endogenous TLR3 and Cy5-poly(I:C). The scale bar is 10 microns.
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pone-0026632-g010: LL37 co-localizes with TLR3 and poly(I:C).A) The location of LL37 in BEAS2B cells. BEAS2B cells were cultured on coverslips and FAM-LL37 was added at a final concentration of 2 µM. Five hours after the addition of FAM-LL37, the cells were fixed and examined using confocal microscopy. B) LL37 can co-localize with Cy5-poly(I:C). FAM-LL37 (2 µM) and Cy5-poly(I:C) (1 µg/ml) were added to BEAS2B cells. The cells were fixed and imaged 5 h after the addition of the fluorescently-tagged molecules. C) TLR3 can co-localize with poly(I:C) independent of exogenously-provided LL37. BEAS2B cells were treated with Cy5-poly(I:C), then permeabilized and stained with a goat anti-TLR3 antibody in complex with Texas Red-labeled secondary antibody. D) Co-localization of FAM-LL37, endogenous TLR3 and Cy5-poly(I:C). The scale bar is 10 microns.

Mentions: We next used confocal microscopy to determine whether LL37, poly(I:C) and TLR3 co-localized in BEAS2B cells. FAM-LL37 and Cy5 poly(I:C) retained bioactivity at levels comparable to unmodified counterparts (data not shown). Using confocal microscopy, FAM-LL37 (2 µM) was found inside cells 3 to 5 h after its addition to the cell culture media in the absence of poly(I:C) (Figure 10A) [42]. Cy5-poly(I:C) (0.15 µg/ml) also entered cells in the absence of exogenous LL37 (data not shown), consistent with our previous observations [43]. When both FAM-37 and Cy5-poly(I:C) were added to BEAS2B cells there was a significant overlap in the distribution of the two molecules (Figure 10B). These results indicate that both LL37 and poly(I:C) are internalized independently into BEAS2B cells and may also be internalized as a complex.


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 co-localizes with TLR3 and poly(I:C).A) The location of LL37 in BEAS2B cells. BEAS2B cells were cultured on coverslips and FAM-LL37 was added at a final concentration of 2 µM. Five hours after the addition of FAM-LL37, the cells were fixed and examined using confocal microscopy. B) LL37 can co-localize with Cy5-poly(I:C). FAM-LL37 (2 µM) and Cy5-poly(I:C) (1 µg/ml) were added to BEAS2B cells. The cells were fixed and imaged 5 h after the addition of the fluorescently-tagged molecules. C) TLR3 can co-localize with poly(I:C) independent of exogenously-provided LL37. BEAS2B cells were treated with Cy5-poly(I:C), then permeabilized and stained with a goat anti-TLR3 antibody in complex with Texas Red-labeled secondary antibody. D) Co-localization of FAM-LL37, endogenous TLR3 and Cy5-poly(I:C). The scale bar is 10 microns.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026632-g010: LL37 co-localizes with TLR3 and poly(I:C).A) The location of LL37 in BEAS2B cells. BEAS2B cells were cultured on coverslips and FAM-LL37 was added at a final concentration of 2 µM. Five hours after the addition of FAM-LL37, the cells were fixed and examined using confocal microscopy. B) LL37 can co-localize with Cy5-poly(I:C). FAM-LL37 (2 µM) and Cy5-poly(I:C) (1 µg/ml) were added to BEAS2B cells. The cells were fixed and imaged 5 h after the addition of the fluorescently-tagged molecules. C) TLR3 can co-localize with poly(I:C) independent of exogenously-provided LL37. BEAS2B cells were treated with Cy5-poly(I:C), then permeabilized and stained with a goat anti-TLR3 antibody in complex with Texas Red-labeled secondary antibody. D) Co-localization of FAM-LL37, endogenous TLR3 and Cy5-poly(I:C). The scale bar is 10 microns.
Mentions: We next used confocal microscopy to determine whether LL37, poly(I:C) and TLR3 co-localized in BEAS2B cells. FAM-LL37 and Cy5 poly(I:C) retained bioactivity at levels comparable to unmodified counterparts (data not shown). Using confocal microscopy, FAM-LL37 (2 µM) was found inside cells 3 to 5 h after its addition to the cell culture media in the absence of poly(I:C) (Figure 10A) [42]. Cy5-poly(I:C) (0.15 µg/ml) also entered cells in the absence of exogenous LL37 (data not shown), consistent with our previous observations [43]. When both FAM-37 and Cy5-poly(I:C) were added to BEAS2B cells there was a significant overlap in the distribution of the two molecules (Figure 10B). These results indicate that both LL37 and poly(I:C) are internalized independently into BEAS2B cells and may also be internalized as a complex.

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