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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

Features in LL37 required to enhance dsRNA recognition by TLR3.A) The peptides used in this set of results. Amino acid substitutions to LL37 acidic residues are shown in red. The dashes indicate that the residues are missing in the peptide KR18-37. B) The effects of the various peptides on the responses to poly(I:C) (0.13 µg/ml) in BEAS2B cells. The sample identified with a ∅ was treated with poly(I:C), but not to a peptide. Other samples were all treated with poly(I:C) and indicated peptide (2 µM for Pentamide, 4 µM for KR18-37 and 5 µM for mCRAMP). IL6 was measured 24 h after the addition of the ligands. C) The effects of LL37 or RK18-37 on IL6 levels induced by Poly(I:C), Reovirus S4 dsRNA, or LPS. *Indicates p<0.05 compared to treatment with dsRNA alone. LL37 and KR18-37 inhibited LPS induced IL6 production at all concentrations tested (p<0.05).
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pone-0026632-g007: Features in LL37 required to enhance dsRNA recognition by TLR3.A) The peptides used in this set of results. Amino acid substitutions to LL37 acidic residues are shown in red. The dashes indicate that the residues are missing in the peptide KR18-37. B) The effects of the various peptides on the responses to poly(I:C) (0.13 µg/ml) in BEAS2B cells. The sample identified with a ∅ was treated with poly(I:C), but not to a peptide. Other samples were all treated with poly(I:C) and indicated peptide (2 µM for Pentamide, 4 µM for KR18-37 and 5 µM for mCRAMP). IL6 was measured 24 h after the addition of the ligands. C) The effects of LL37 or RK18-37 on IL6 levels induced by Poly(I:C), Reovirus S4 dsRNA, or LPS. *Indicates p<0.05 compared to treatment with dsRNA alone. LL37 and KR18-37 inhibited LPS induced IL6 production at all concentrations tested (p<0.05).

Mentions: To define the features of LL37 required to enhance dsRNA-dependent signaling, we compared the effects of LL37 to three related peptides: Pentamide, a peptide with substitutions in multiple acidic residues in LL37, KR18-37 that lacks the first 17 residues of LL37 and is found in sweat [41], and the mouse analog of LL37 named mCRAMP (Figure 7A). Pentamide (2 µM) retained 77 ± 2% (n = 3) of the ability of LL37 to enhance poly(I:C)-induced TLR3 signaling in BEAS2B cells, indicating that the negatively-charged residues in LL37 are not critical to enhancing dsRNA-dependent IL6 production (Figure 7B). However, KR18-37 (4 µM; Figures 7B&C; p>0.5, n = 3) and mCRAMP (5 µM; Figure 6B; p>0.5, n = 3) were unable to enhance IL6 production in response to poly(I:C). Similar to LL37 (Figure S2), no cytotoxicity was detected with KR18-37 and mCRAMP at these concentrations (data not shown). Similar results were obtained when the S4 dsRNA was used as the agonist (Figure 7C). KR18-37 did retain, however, the ability to inhibit LPS-dependent signaling through the TLR4 receptor, in agreement with the results of Durr et al. [20] (Figure 7C). These results indicate that regions within LL37 that affect TLR3 and TLR4 signaling do not completely overlap.


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)

Features in LL37 required to enhance dsRNA recognition by TLR3.A) The peptides used in this set of results. Amino acid substitutions to LL37 acidic residues are shown in red. The dashes indicate that the residues are missing in the peptide KR18-37. B) The effects of the various peptides on the responses to poly(I:C) (0.13 µg/ml) in BEAS2B cells. The sample identified with a ∅ was treated with poly(I:C), but not to a peptide. Other samples were all treated with poly(I:C) and indicated peptide (2 µM for Pentamide, 4 µM for KR18-37 and 5 µM for mCRAMP). IL6 was measured 24 h after the addition of the ligands. C) The effects of LL37 or RK18-37 on IL6 levels induced by Poly(I:C), Reovirus S4 dsRNA, or LPS. *Indicates p<0.05 compared to treatment with dsRNA alone. LL37 and KR18-37 inhibited LPS induced IL6 production at all concentrations tested (p<0.05).
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Related In: Results  -  Collection

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pone-0026632-g007: Features in LL37 required to enhance dsRNA recognition by TLR3.A) The peptides used in this set of results. Amino acid substitutions to LL37 acidic residues are shown in red. The dashes indicate that the residues are missing in the peptide KR18-37. B) The effects of the various peptides on the responses to poly(I:C) (0.13 µg/ml) in BEAS2B cells. The sample identified with a ∅ was treated with poly(I:C), but not to a peptide. Other samples were all treated with poly(I:C) and indicated peptide (2 µM for Pentamide, 4 µM for KR18-37 and 5 µM for mCRAMP). IL6 was measured 24 h after the addition of the ligands. C) The effects of LL37 or RK18-37 on IL6 levels induced by Poly(I:C), Reovirus S4 dsRNA, or LPS. *Indicates p<0.05 compared to treatment with dsRNA alone. LL37 and KR18-37 inhibited LPS induced IL6 production at all concentrations tested (p<0.05).
Mentions: To define the features of LL37 required to enhance dsRNA-dependent signaling, we compared the effects of LL37 to three related peptides: Pentamide, a peptide with substitutions in multiple acidic residues in LL37, KR18-37 that lacks the first 17 residues of LL37 and is found in sweat [41], and the mouse analog of LL37 named mCRAMP (Figure 7A). Pentamide (2 µM) retained 77 ± 2% (n = 3) of the ability of LL37 to enhance poly(I:C)-induced TLR3 signaling in BEAS2B cells, indicating that the negatively-charged residues in LL37 are not critical to enhancing dsRNA-dependent IL6 production (Figure 7B). However, KR18-37 (4 µM; Figures 7B&C; p>0.5, n = 3) and mCRAMP (5 µM; Figure 6B; p>0.5, n = 3) were unable to enhance IL6 production in response to poly(I:C). Similar to LL37 (Figure S2), no cytotoxicity was detected with KR18-37 and mCRAMP at these concentrations (data not shown). Similar results were obtained when the S4 dsRNA was used as the agonist (Figure 7C). KR18-37 did retain, however, the ability to inhibit LPS-dependent signaling through the TLR4 receptor, in agreement with the results of Durr et al. [20] (Figure 7C). These results indicate that regions within LL37 that affect TLR3 and TLR4 signaling do not completely overlap.

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