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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

LL37 can enhance the cytokine response to Rhinovirus (RV) infection in cultured BEAS2B cells.LL37 was present at a final concentration of 3 µM and cytokine levels determined using a Milliplex cytokine/chemokine kit. The symbol ∅ denotes mock-infected cells. LL37 enhanced cytokine production in RV infected cells and the effects were blocked by increasing concentrations of a monoclonal antibody specific to TLR3 (denoted by the grey triangle; [40]). * Indicates p<0.05 compared to cytokine levels in RV infected cells while ** indicates p<0.05 compared to cytokine levels induced by addition of LL37 to RV infected cells. Each bar shows mean (±SEM; n = 3).
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pone-0026632-g005: LL37 can enhance the cytokine response to Rhinovirus (RV) infection in cultured BEAS2B cells.LL37 was present at a final concentration of 3 µM and cytokine levels determined using a Milliplex cytokine/chemokine kit. The symbol ∅ denotes mock-infected cells. LL37 enhanced cytokine production in RV infected cells and the effects were blocked by increasing concentrations of a monoclonal antibody specific to TLR3 (denoted by the grey triangle; [40]). * Indicates p<0.05 compared to cytokine levels in RV infected cells while ** indicates p<0.05 compared to cytokine levels induced by addition of LL37 to RV infected cells. Each bar shows mean (±SEM; n = 3).

Mentions: Rhinovirus (RV) infections activate TLR3-mediated responses in the respiratory tract, exacerbating asthma and chronic obstructive pulmonary disease [37], [38]. Moreover, TLR3 acts as an initial endosomal sensor of rhinovirus infection in human epithelial cells [39]. To determine whether LL37 can affect TLR3-mediated responses to viral infections, we infected BEAS2B cells with RV in the presence or absence of LL37. LL37 (3 µM) alone minimally induced the levels of IL6 (Figures 1B and 5), IP10 and MCP-1 by 2 ± 0.12, 1.5 ± 0.04 and 1.07 ± 0.13 fold above basal respectively (n = 3; Figure 5) while RV infection alone induced the production of IL6, IP10 and MCP-1 by 3.9 ± 0.25, 12.8 ± 0.02 and 1.31 ± 0.1 fold respectively (Figure 5; n = 3; p = 0.0001 for IL6; p<0.005 for IP10; P = 0.056 for MCP-1). The addition of LL37 (3 µM) to the infection medium enhanced IL6 production above that of RV infection by 1.6 ± 0.04 fold (p<0.002; n = 3), IP10 production by 2.8 ± 0.3 fold (p<0.005, n = 3) and MCP-1 by 2.4 ± 0.1 fold (p = 0.003, N = 3) (Figure 5). To determine whether TLR3 was responsible for the elevated cytokine production, a monoclonal antibody to TLR3, previously demonstrated to inhibit TLR3 signaling [40], decreased the enhancement of RV-induced IL6 production by LL37 by 64% ± 3.4 (Figure 5; n = 3), IP10 production by 93 ± 1% (Figure 5, n = 3) and MCP-1 by 48 ± 3% when compared to RV infection alone. These results show that LL37 can facilitate the recognition of viral infection by host cells through the activation of 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)

LL37 can enhance the cytokine response to Rhinovirus (RV) infection in cultured BEAS2B cells.LL37 was present at a final concentration of 3 µM and cytokine levels determined using a Milliplex cytokine/chemokine kit. The symbol ∅ denotes mock-infected cells. LL37 enhanced cytokine production in RV infected cells and the effects were blocked by increasing concentrations of a monoclonal antibody specific to TLR3 (denoted by the grey triangle; [40]). * Indicates p<0.05 compared to cytokine levels in RV infected cells while ** indicates p<0.05 compared to cytokine levels induced by addition of LL37 to RV infected cells. Each bar shows mean (±SEM; n = 3).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198786&req=5

pone-0026632-g005: LL37 can enhance the cytokine response to Rhinovirus (RV) infection in cultured BEAS2B cells.LL37 was present at a final concentration of 3 µM and cytokine levels determined using a Milliplex cytokine/chemokine kit. The symbol ∅ denotes mock-infected cells. LL37 enhanced cytokine production in RV infected cells and the effects were blocked by increasing concentrations of a monoclonal antibody specific to TLR3 (denoted by the grey triangle; [40]). * Indicates p<0.05 compared to cytokine levels in RV infected cells while ** indicates p<0.05 compared to cytokine levels induced by addition of LL37 to RV infected cells. Each bar shows mean (±SEM; n = 3).
Mentions: Rhinovirus (RV) infections activate TLR3-mediated responses in the respiratory tract, exacerbating asthma and chronic obstructive pulmonary disease [37], [38]. Moreover, TLR3 acts as an initial endosomal sensor of rhinovirus infection in human epithelial cells [39]. To determine whether LL37 can affect TLR3-mediated responses to viral infections, we infected BEAS2B cells with RV in the presence or absence of LL37. LL37 (3 µM) alone minimally induced the levels of IL6 (Figures 1B and 5), IP10 and MCP-1 by 2 ± 0.12, 1.5 ± 0.04 and 1.07 ± 0.13 fold above basal respectively (n = 3; Figure 5) while RV infection alone induced the production of IL6, IP10 and MCP-1 by 3.9 ± 0.25, 12.8 ± 0.02 and 1.31 ± 0.1 fold respectively (Figure 5; n = 3; p = 0.0001 for IL6; p<0.005 for IP10; P = 0.056 for MCP-1). The addition of LL37 (3 µM) to the infection medium enhanced IL6 production above that of RV infection by 1.6 ± 0.04 fold (p<0.002; n = 3), IP10 production by 2.8 ± 0.3 fold (p<0.005, n = 3) and MCP-1 by 2.4 ± 0.1 fold (p = 0.003, N = 3) (Figure 5). To determine whether TLR3 was responsible for the elevated cytokine production, a monoclonal antibody to TLR3, previously demonstrated to inhibit TLR3 signaling [40], decreased the enhancement of RV-induced IL6 production by LL37 by 64% ± 3.4 (Figure 5; n = 3), IP10 production by 93 ± 1% (Figure 5, n = 3) and MCP-1 by 48 ± 3% when compared to RV infection alone. These results show that LL37 can facilitate the recognition of viral infection by host cells through the activation of 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