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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

TLR3 is responsible for the enhancement of IL6 production observed in BEAS2B cells.A) A demonstration that a set of three siRNAs to TLR3 (siTLR3) can reduce TLR3 mRNA in BEAS2B cells. siRNAs to RIG-I (siRIG-I) and a nonspecific siRNA (nsRNA) were used as controls. All siRNAs were used at 30 nM. 48 h after transfection with siRNAs, cells were either not stimulated () or stimulated with either poly(I:C) (0.13 µg/ml; pIC) or poly(I:C)+LL37 (3 µM) (pIC+LL37). After 20 h, total RNA was extracted and RT-PCR was performed using primers specific for TLR3 and GAPDH. Data is presented as %Control using corresponding agonist treatment. * Indicates p<0.05 compared to control. B) siRNAs to TLR3 reduced IL6 production induced by poly(I:C) or poly(I:C)+ LL37. The cells were transfected with siRNAs to TLR3 (siTLR3), siRNA to RIG-I (siRIG-I) or a control siRNA as described in A. The culture media were harvested 20 h after the addition of the ligands and the level of secreted IL6 protein determined. Each sample was performed in triplicates and the mean ± SEM shown. * Indicates p<0.05 compared nsRNA in corresponding treatment with agonist. C& D) The abundances of IL6 (right y-axis) and TLR3 messages (left y-axis) in response to the addition of poly(I:C) (C) or poly(I:C)+LL37 (D). The samples used were harvested after poly(I:C) addition to the BEAS2B cell culture media at the times specified in the horizontal axis. The RNAs were then subjected to RT-PCR as described in the materials and methods using either primers specific for IL6 or for TLR3. * Indicates p<0.05 compared to no treatment and ** indicates p<0.05 compared to poly(I:C) treatment. There is no difference (p>0.5) for TLR3 mRNA among any of the treatments or between any of the time points (0.5–3 h).
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pone-0026632-g002: TLR3 is responsible for the enhancement of IL6 production observed in BEAS2B cells.A) A demonstration that a set of three siRNAs to TLR3 (siTLR3) can reduce TLR3 mRNA in BEAS2B cells. siRNAs to RIG-I (siRIG-I) and a nonspecific siRNA (nsRNA) were used as controls. All siRNAs were used at 30 nM. 48 h after transfection with siRNAs, cells were either not stimulated () or stimulated with either poly(I:C) (0.13 µg/ml; pIC) or poly(I:C)+LL37 (3 µM) (pIC+LL37). After 20 h, total RNA was extracted and RT-PCR was performed using primers specific for TLR3 and GAPDH. Data is presented as %Control using corresponding agonist treatment. * Indicates p<0.05 compared to control. B) siRNAs to TLR3 reduced IL6 production induced by poly(I:C) or poly(I:C)+ LL37. The cells were transfected with siRNAs to TLR3 (siTLR3), siRNA to RIG-I (siRIG-I) or a control siRNA as described in A. The culture media were harvested 20 h after the addition of the ligands and the level of secreted IL6 protein determined. Each sample was performed in triplicates and the mean ± SEM shown. * Indicates p<0.05 compared nsRNA in corresponding treatment with agonist. C& D) The abundances of IL6 (right y-axis) and TLR3 messages (left y-axis) in response to the addition of poly(I:C) (C) or poly(I:C)+LL37 (D). The samples used were harvested after poly(I:C) addition to the BEAS2B cell culture media at the times specified in the horizontal axis. The RNAs were then subjected to RT-PCR as described in the materials and methods using either primers specific for IL6 or for TLR3. * Indicates p<0.05 compared to no treatment and ** indicates p<0.05 compared to poly(I:C) treatment. There is no difference (p>0.5) for TLR3 mRNA among any of the treatments or between any of the time points (0.5–3 h).

Mentions: Both TLR3 and RIG-I are activated by poly(I:C) [33]. Transfection of RNA agonists into the cell's cytoplasm activates cytoplasmic RIG-I while addition of dsRNA agonists to cell media and subsequent uptake of agonists by endocytosis activates TLR3 [32]. To confirm that TLR3 was required for LL37-mediated enhancement of IL6 production, we knocked down TLR3 or RIG-I expression with siRNAs (Figure 2A). RT-PCR was used to confirm that the siRNAs targeting TLR3 selectively reduced TLR3 message (Figure 2A). TLR3 mediates the LL37 effect as treatment of BEAS2B cells with TLR3 siRNAs reduced IL6 levels by 51 ± 3% and 51 ± 9%, in cells that had been treated with poly(I:C) alone or poly(I:C) plus LL37 as compared to a control siRNA (Figure 2B; reduction p<0.02 for basal level, treatment with poly(I:C) or poly(I:C)+LL37). In contrast, siRNA to RIG-I minimally affected IL6 levels (16 ± 13% and 0 ± 12%, for treatment with poly(I:C) alone or with LL37 respectively; p>0.2) (Figure 2B; n = 3). In control experiments we demonstrated that siRNA to RIG-I decreased RIG-I message by more than 80% (Figure S3), but did not affect IL6 levels when the poly(I:C) was added to the medium of the BEAS2B cells (Figures 2B and S3, p>0.5). Together these results demonstrate that TLR3, but not RIG-I, is required for the LL37-dependent enhancement of IL6 production in BEAS2B cells.


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)

TLR3 is responsible for the enhancement of IL6 production observed in BEAS2B cells.A) A demonstration that a set of three siRNAs to TLR3 (siTLR3) can reduce TLR3 mRNA in BEAS2B cells. siRNAs to RIG-I (siRIG-I) and a nonspecific siRNA (nsRNA) were used as controls. All siRNAs were used at 30 nM. 48 h after transfection with siRNAs, cells were either not stimulated () or stimulated with either poly(I:C) (0.13 µg/ml; pIC) or poly(I:C)+LL37 (3 µM) (pIC+LL37). After 20 h, total RNA was extracted and RT-PCR was performed using primers specific for TLR3 and GAPDH. Data is presented as %Control using corresponding agonist treatment. * Indicates p<0.05 compared to control. B) siRNAs to TLR3 reduced IL6 production induced by poly(I:C) or poly(I:C)+ LL37. The cells were transfected with siRNAs to TLR3 (siTLR3), siRNA to RIG-I (siRIG-I) or a control siRNA as described in A. The culture media were harvested 20 h after the addition of the ligands and the level of secreted IL6 protein determined. Each sample was performed in triplicates and the mean ± SEM shown. * Indicates p<0.05 compared nsRNA in corresponding treatment with agonist. C& D) The abundances of IL6 (right y-axis) and TLR3 messages (left y-axis) in response to the addition of poly(I:C) (C) or poly(I:C)+LL37 (D). The samples used were harvested after poly(I:C) addition to the BEAS2B cell culture media at the times specified in the horizontal axis. The RNAs were then subjected to RT-PCR as described in the materials and methods using either primers specific for IL6 or for TLR3. * Indicates p<0.05 compared to no treatment and ** indicates p<0.05 compared to poly(I:C) treatment. There is no difference (p>0.5) for TLR3 mRNA among any of the treatments or between any of the time points (0.5–3 h).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198786&req=5

pone-0026632-g002: TLR3 is responsible for the enhancement of IL6 production observed in BEAS2B cells.A) A demonstration that a set of three siRNAs to TLR3 (siTLR3) can reduce TLR3 mRNA in BEAS2B cells. siRNAs to RIG-I (siRIG-I) and a nonspecific siRNA (nsRNA) were used as controls. All siRNAs were used at 30 nM. 48 h after transfection with siRNAs, cells were either not stimulated () or stimulated with either poly(I:C) (0.13 µg/ml; pIC) or poly(I:C)+LL37 (3 µM) (pIC+LL37). After 20 h, total RNA was extracted and RT-PCR was performed using primers specific for TLR3 and GAPDH. Data is presented as %Control using corresponding agonist treatment. * Indicates p<0.05 compared to control. B) siRNAs to TLR3 reduced IL6 production induced by poly(I:C) or poly(I:C)+ LL37. The cells were transfected with siRNAs to TLR3 (siTLR3), siRNA to RIG-I (siRIG-I) or a control siRNA as described in A. The culture media were harvested 20 h after the addition of the ligands and the level of secreted IL6 protein determined. Each sample was performed in triplicates and the mean ± SEM shown. * Indicates p<0.05 compared nsRNA in corresponding treatment with agonist. C& D) The abundances of IL6 (right y-axis) and TLR3 messages (left y-axis) in response to the addition of poly(I:C) (C) or poly(I:C)+LL37 (D). The samples used were harvested after poly(I:C) addition to the BEAS2B cell culture media at the times specified in the horizontal axis. The RNAs were then subjected to RT-PCR as described in the materials and methods using either primers specific for IL6 or for TLR3. * Indicates p<0.05 compared to no treatment and ** indicates p<0.05 compared to poly(I:C) treatment. There is no difference (p>0.5) for TLR3 mRNA among any of the treatments or between any of the time points (0.5–3 h).
Mentions: Both TLR3 and RIG-I are activated by poly(I:C) [33]. Transfection of RNA agonists into the cell's cytoplasm activates cytoplasmic RIG-I while addition of dsRNA agonists to cell media and subsequent uptake of agonists by endocytosis activates TLR3 [32]. To confirm that TLR3 was required for LL37-mediated enhancement of IL6 production, we knocked down TLR3 or RIG-I expression with siRNAs (Figure 2A). RT-PCR was used to confirm that the siRNAs targeting TLR3 selectively reduced TLR3 message (Figure 2A). TLR3 mediates the LL37 effect as treatment of BEAS2B cells with TLR3 siRNAs reduced IL6 levels by 51 ± 3% and 51 ± 9%, in cells that had been treated with poly(I:C) alone or poly(I:C) plus LL37 as compared to a control siRNA (Figure 2B; reduction p<0.02 for basal level, treatment with poly(I:C) or poly(I:C)+LL37). In contrast, siRNA to RIG-I minimally affected IL6 levels (16 ± 13% and 0 ± 12%, for treatment with poly(I:C) alone or with LL37 respectively; p>0.2) (Figure 2B; n = 3). In control experiments we demonstrated that siRNA to RIG-I decreased RIG-I message by more than 80% (Figure S3), but did not affect IL6 levels when the poly(I:C) was added to the medium of the BEAS2B cells (Figures 2B and S3, p>0.5). Together these results demonstrate that TLR3, but not RIG-I, is required for the LL37-dependent enhancement of IL6 production in BEAS2B cells.

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