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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

Effects of LL37 on cytokine production by BEAS2B cells.A) LL37 can enhance IL6 production in response to dsRNA and repress IL6 levels in response to LPS. Cells were stimulated with agonists ± LL37 or scrambled LL37 (Sc37). After 20 h, the culture media were collected. An aliquot of the media was assessed for secreted IL6 using a human IL6 ELISA assay and the amount of IL6 normalized to total volume. The final concentrations of LL37, Sc37, poly(I:C) and LPS were 2.2 µM, 2.2 µM, 0.13 µg/ml and 1 µg/ml, respectively. LL37 enhanced poly(I:C) induced IL6 production (p<0.0001;) while Sc37 had no effect (p = 0.2). B) Dose-dependent effects of LL37 on poly(I:C)-induced and LPS-induced IL6 levels. Culture media were harvested 20 h after the addition of ligands and proteins. Each sample was performed in duplicate or triplicate and data plotted as mean ± SEM.
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pone-0026632-g001: Effects of LL37 on cytokine production by BEAS2B cells.A) LL37 can enhance IL6 production in response to dsRNA and repress IL6 levels in response to LPS. Cells were stimulated with agonists ± LL37 or scrambled LL37 (Sc37). After 20 h, the culture media were collected. An aliquot of the media was assessed for secreted IL6 using a human IL6 ELISA assay and the amount of IL6 normalized to total volume. The final concentrations of LL37, Sc37, poly(I:C) and LPS were 2.2 µM, 2.2 µM, 0.13 µg/ml and 1 µg/ml, respectively. LL37 enhanced poly(I:C) induced IL6 production (p<0.0001;) while Sc37 had no effect (p = 0.2). B) Dose-dependent effects of LL37 on poly(I:C)-induced and LPS-induced IL6 levels. Culture media were harvested 20 h after the addition of ligands and proteins. Each sample was performed in duplicate or triplicate and data plotted as mean ± SEM.

Mentions: Bronchial epithelial cells are the first line defense against foreign microbes in our respiratory system and initiate immune response by producing cytokines and chemokines, resulting in recruitment of inflammatory cells [30]. The human bronchial epithelial cell line, BEAS2B, which endogenously expresses TLR3, 4, 9, and RIG-I has been extensively used to study TLR3 function [31] and was used in this study to examine whether LL37 modulates TLR3 signaling. Since LL37 and TLR3 ligands may likely encounter TLR3 after endocytosis of materials outside of cells, adding them to the media of cultured cells should be a suitable model to study the roles of LL37 and dsRNAs. We note that this mode of uptake does not activate cytoplasmic innate immune receptors, such as RIG-I like receptors, which requires transection of the ligands [32]. In the absence of TLR3 agonist poly(I:C), the basal level of IL6 was at 0.06 ± 0.10 µg/ml (n = 32). Addition of poly(I:C) to the medium increased IL6 levels 15 ± 2.2 fold (n = 12) above basal levels (Fig. 1A) [31]. LL37 (2.2 µM) further enhanced poly(I:C)-induced IL6 levels by an average of 4.5 ± 0.67 fold (n = 23; p<0.0001) when compared to poly(I:C) alone. A peptide with the scrambled LL37 sequence (Sc37) did not enhance IL6 production (Fig. 1A; n = 7; p = 0.2). Similar results were observed for LL37 and Sc37 with IL8 (data not shown). LL37 also enhanced the poly(I:C)-induced increase in IL6 and interferon beta (IFNβ) mRNAs as determined by RT-PCR (Figure S1; p<0.05 for both mRNAs).


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)

Effects of LL37 on cytokine production by BEAS2B cells.A) LL37 can enhance IL6 production in response to dsRNA and repress IL6 levels in response to LPS. Cells were stimulated with agonists ± LL37 or scrambled LL37 (Sc37). After 20 h, the culture media were collected. An aliquot of the media was assessed for secreted IL6 using a human IL6 ELISA assay and the amount of IL6 normalized to total volume. The final concentrations of LL37, Sc37, poly(I:C) and LPS were 2.2 µM, 2.2 µM, 0.13 µg/ml and 1 µg/ml, respectively. LL37 enhanced poly(I:C) induced IL6 production (p<0.0001;) while Sc37 had no effect (p = 0.2). B) Dose-dependent effects of LL37 on poly(I:C)-induced and LPS-induced IL6 levels. Culture media were harvested 20 h after the addition of ligands and proteins. Each sample was performed in duplicate or triplicate and data plotted as mean ± SEM.
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pone-0026632-g001: Effects of LL37 on cytokine production by BEAS2B cells.A) LL37 can enhance IL6 production in response to dsRNA and repress IL6 levels in response to LPS. Cells were stimulated with agonists ± LL37 or scrambled LL37 (Sc37). After 20 h, the culture media were collected. An aliquot of the media was assessed for secreted IL6 using a human IL6 ELISA assay and the amount of IL6 normalized to total volume. The final concentrations of LL37, Sc37, poly(I:C) and LPS were 2.2 µM, 2.2 µM, 0.13 µg/ml and 1 µg/ml, respectively. LL37 enhanced poly(I:C) induced IL6 production (p<0.0001;) while Sc37 had no effect (p = 0.2). B) Dose-dependent effects of LL37 on poly(I:C)-induced and LPS-induced IL6 levels. Culture media were harvested 20 h after the addition of ligands and proteins. Each sample was performed in duplicate or triplicate and data plotted as mean ± SEM.
Mentions: Bronchial epithelial cells are the first line defense against foreign microbes in our respiratory system and initiate immune response by producing cytokines and chemokines, resulting in recruitment of inflammatory cells [30]. The human bronchial epithelial cell line, BEAS2B, which endogenously expresses TLR3, 4, 9, and RIG-I has been extensively used to study TLR3 function [31] and was used in this study to examine whether LL37 modulates TLR3 signaling. Since LL37 and TLR3 ligands may likely encounter TLR3 after endocytosis of materials outside of cells, adding them to the media of cultured cells should be a suitable model to study the roles of LL37 and dsRNAs. We note that this mode of uptake does not activate cytoplasmic innate immune receptors, such as RIG-I like receptors, which requires transection of the ligands [32]. In the absence of TLR3 agonist poly(I:C), the basal level of IL6 was at 0.06 ± 0.10 µg/ml (n = 32). Addition of poly(I:C) to the medium increased IL6 levels 15 ± 2.2 fold (n = 12) above basal levels (Fig. 1A) [31]. LL37 (2.2 µM) further enhanced poly(I:C)-induced IL6 levels by an average of 4.5 ± 0.67 fold (n = 23; p<0.0001) when compared to poly(I:C) alone. A peptide with the scrambled LL37 sequence (Sc37) did not enhance IL6 production (Fig. 1A; n = 7; p = 0.2). Similar results were observed for LL37 and Sc37 with IL8 (data not shown). LL37 also enhanced the poly(I:C)-induced increase in IL6 and interferon beta (IFNβ) mRNAs as determined by RT-PCR (Figure S1; p<0.05 for both mRNAs).

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