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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

Dose-dependent enhancement of IL6 production by BEAS2B cells in response to poly(I:C) and S4dsRNA in the presence of two cell penetrating peptides.A) The T3 peptide enhanced IL6 production in the presence of dsRNAs, but not LPS. The final concentrations of the T3 peptides in each reaction are shown on the horizontal axis. Poly(I:C) and S4/Reo dsRNA were at 0.13 µg/ml and LPS was at 1 µg/ml. B) The T4 peptide also induced IL6 production in the presence of dsRNAs, but not when LPS was used as an agonist. *Indicates p<0.05 compared to treatment with dsRNA alone. Neither T3 nor T4 peptide had any effect on LPS induced IL6 level (p>0.05).
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pone-0026632-g011: Dose-dependent enhancement of IL6 production by BEAS2B cells in response to poly(I:C) and S4dsRNA in the presence of two cell penetrating peptides.A) The T3 peptide enhanced IL6 production in the presence of dsRNAs, but not LPS. The final concentrations of the T3 peptides in each reaction are shown on the horizontal axis. Poly(I:C) and S4/Reo dsRNA were at 0.13 µg/ml and LPS was at 1 µg/ml. B) The T4 peptide also induced IL6 production in the presence of dsRNAs, but not when LPS was used as an agonist. *Indicates p<0.05 compared to treatment with dsRNA alone. Neither T3 nor T4 peptide had any effect on LPS induced IL6 level (p>0.05).

Mentions: In BEAS2B cells, the Tat peptide, Penetrin, and Arg(9) all enhanced poly(I:C)-induced IL-6 production by at least 2.9-fold over the levels induced by poly(I:C) alone (Table 1; p<0.05). Peptides 3, T4 and T3 also enhanced poly(I:C)-induced signaling (Table 1 and Figure 11; p<0.05). None of the CPPs had an effect on IL6 production when added in the absence of dsRNA (data not shown). Furthermore, a variant of the peptide T3 named T3ser that had the arginines substituted with serines lost the ability to enhance IL6 production (Table 1). Interestingly, P22(14-30) [44] and Buforin II [46] which also contain a number of arginines did not enhance TLR3 signaling in BEAS2B cells (Table 1; p>0.5).


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)

Dose-dependent enhancement of IL6 production by BEAS2B cells in response to poly(I:C) and S4dsRNA in the presence of two cell penetrating peptides.A) The T3 peptide enhanced IL6 production in the presence of dsRNAs, but not LPS. The final concentrations of the T3 peptides in each reaction are shown on the horizontal axis. Poly(I:C) and S4/Reo dsRNA were at 0.13 µg/ml and LPS was at 1 µg/ml. B) The T4 peptide also induced IL6 production in the presence of dsRNAs, but not when LPS was used as an agonist. *Indicates p<0.05 compared to treatment with dsRNA alone. Neither T3 nor T4 peptide had any effect on LPS induced IL6 level (p>0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198786&req=5

pone-0026632-g011: Dose-dependent enhancement of IL6 production by BEAS2B cells in response to poly(I:C) and S4dsRNA in the presence of two cell penetrating peptides.A) The T3 peptide enhanced IL6 production in the presence of dsRNAs, but not LPS. The final concentrations of the T3 peptides in each reaction are shown on the horizontal axis. Poly(I:C) and S4/Reo dsRNA were at 0.13 µg/ml and LPS was at 1 µg/ml. B) The T4 peptide also induced IL6 production in the presence of dsRNAs, but not when LPS was used as an agonist. *Indicates p<0.05 compared to treatment with dsRNA alone. Neither T3 nor T4 peptide had any effect on LPS induced IL6 level (p>0.05).
Mentions: In BEAS2B cells, the Tat peptide, Penetrin, and Arg(9) all enhanced poly(I:C)-induced IL-6 production by at least 2.9-fold over the levels induced by poly(I:C) alone (Table 1; p<0.05). Peptides 3, T4 and T3 also enhanced poly(I:C)-induced signaling (Table 1 and Figure 11; p<0.05). None of the CPPs had an effect on IL6 production when added in the absence of dsRNA (data not shown). Furthermore, a variant of the peptide T3 named T3ser that had the arginines substituted with serines lost the ability to enhance IL6 production (Table 1). Interestingly, P22(14-30) [44] and Buforin II [46] which also contain a number of arginines did not enhance TLR3 signaling in BEAS2B cells (Table 1; p>0.5).

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