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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

Electron microscopy of unlabeled LL37, Sc37, or poly(I:C) (pIC; 50 µg/ml) either alone or in combination.A) LL37, Sc37 or poly(I:C) was added to a carbon coated copper grid, stained with uranyl acetate and visualized using electron microscopy. The images were taken at a 40,000X magnification using a JEOL transmission microscope. LL37 and Sc37 were present at 10 µM and poly(I:C) was at 50 µg/ml. The bottom middle and right panels show images of LL37 and poly(I:C). B) A plot of the maximal lengths and widths of the subsets of particles present in the electron micrographs. Measurements were made using the toolbox within the EMAN package of software's [66]. Slopes calculated using linear regression with GrapPad Prism software. Slopes for pIC, LL37, Sc37, pIC+Sc37 are all significantly different from 0 (p<0.0005, 23–203 structures). Slope for poly(I:C)+LL37 is not significantly different from 0; p = 0.2; 45 structures). Number of particles measured for each treatment is indicated in parentheses.
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pone-0026632-g009: Electron microscopy of unlabeled LL37, Sc37, or poly(I:C) (pIC; 50 µg/ml) either alone or in combination.A) LL37, Sc37 or poly(I:C) was added to a carbon coated copper grid, stained with uranyl acetate and visualized using electron microscopy. The images were taken at a 40,000X magnification using a JEOL transmission microscope. LL37 and Sc37 were present at 10 µM and poly(I:C) was at 50 µg/ml. The bottom middle and right panels show images of LL37 and poly(I:C). B) A plot of the maximal lengths and widths of the subsets of particles present in the electron micrographs. Measurements were made using the toolbox within the EMAN package of software's [66]. Slopes calculated using linear regression with GrapPad Prism software. Slopes for pIC, LL37, Sc37, pIC+Sc37 are all significantly different from 0 (p<0.0005, 23–203 structures). Slope for poly(I:C)+LL37 is not significantly different from 0; p = 0.2; 45 structures). Number of particles measured for each treatment is indicated in parentheses.

Mentions: To better visualize the conformation of the LL37-dsRNA complexes, we used negative-stain transmission electron microscopy to image LL37 (10 µM), size-fractionated poly(I:C) (200–500 bp; 50 µg/ml), or a mixture of the two. Sc37 (10 µM) served as a control in this experiment. The results are shown in Figure 9A. At pH 7.4, we observed heterogeneous globular structures for LL37, Sc37, and poly(I:C). Sc37 mixed with poly(I:C) showed globular structures similar to those observed with Sc37 or poly(I:C) alone. However, the mixture of LL37 and poly(I:C) resulted in a predominantly filamentous structures. The results suggest that LL37 in complex with poly(I:C) can physically alter the conformation of poly(I:C), a feature that may influence recognition by TLR3.


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)

Electron microscopy of unlabeled LL37, Sc37, or poly(I:C) (pIC; 50 µg/ml) either alone or in combination.A) LL37, Sc37 or poly(I:C) was added to a carbon coated copper grid, stained with uranyl acetate and visualized using electron microscopy. The images were taken at a 40,000X magnification using a JEOL transmission microscope. LL37 and Sc37 were present at 10 µM and poly(I:C) was at 50 µg/ml. The bottom middle and right panels show images of LL37 and poly(I:C). B) A plot of the maximal lengths and widths of the subsets of particles present in the electron micrographs. Measurements were made using the toolbox within the EMAN package of software's [66]. Slopes calculated using linear regression with GrapPad Prism software. Slopes for pIC, LL37, Sc37, pIC+Sc37 are all significantly different from 0 (p<0.0005, 23–203 structures). Slope for poly(I:C)+LL37 is not significantly different from 0; p = 0.2; 45 structures). Number of particles measured for each treatment is indicated in parentheses.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026632-g009: Electron microscopy of unlabeled LL37, Sc37, or poly(I:C) (pIC; 50 µg/ml) either alone or in combination.A) LL37, Sc37 or poly(I:C) was added to a carbon coated copper grid, stained with uranyl acetate and visualized using electron microscopy. The images were taken at a 40,000X magnification using a JEOL transmission microscope. LL37 and Sc37 were present at 10 µM and poly(I:C) was at 50 µg/ml. The bottom middle and right panels show images of LL37 and poly(I:C). B) A plot of the maximal lengths and widths of the subsets of particles present in the electron micrographs. Measurements were made using the toolbox within the EMAN package of software's [66]. Slopes calculated using linear regression with GrapPad Prism software. Slopes for pIC, LL37, Sc37, pIC+Sc37 are all significantly different from 0 (p<0.0005, 23–203 structures). Slope for poly(I:C)+LL37 is not significantly different from 0; p = 0.2; 45 structures). Number of particles measured for each treatment is indicated in parentheses.
Mentions: To better visualize the conformation of the LL37-dsRNA complexes, we used negative-stain transmission electron microscopy to image LL37 (10 µM), size-fractionated poly(I:C) (200–500 bp; 50 µg/ml), or a mixture of the two. Sc37 (10 µM) served as a control in this experiment. The results are shown in Figure 9A. At pH 7.4, we observed heterogeneous globular structures for LL37, Sc37, and poly(I:C). Sc37 mixed with poly(I:C) showed globular structures similar to those observed with Sc37 or poly(I:C) alone. However, the mixture of LL37 and poly(I:C) resulted in a predominantly filamentous structures. The results suggest that LL37 in complex with poly(I:C) can physically alter the conformation of poly(I:C), a feature that may influence recognition by TLR3.

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