Limits...
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.

Show MeSH

Related in: MedlinePlus

LL37 enhanced poly(I:C)-induced cytokine production in human peripheral blood mononuclear cells (PBMCs).After 24 h of poly(I:C) ±LL37 incubation, IL-1α, MCP-1 and IP-10 levels in the medium were determined using a Milliplex cytokine/chemokine kit and the amount of each cytokine normalized to the total volume of the each sample. LL37 (5.6 µM) by itself did not induce any of the cytokines measured, but significantly enhanced poly(I:C) induced production of IL-1α, MCP-1 and IP-10, while Sc37 had no effect by itself or in the presence of poly(I:C). The addition of either LL37 or Sc37 at this concentration did not affect the morphology of the cells, indicating that there was no obvious cytotoxicity (data not shown). Each bar shows the mean with one SEM (n = 3).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3198786&req=5

pone-0026632-g006: LL37 enhanced poly(I:C)-induced cytokine production in human peripheral blood mononuclear cells (PBMCs).After 24 h of poly(I:C) ±LL37 incubation, IL-1α, MCP-1 and IP-10 levels in the medium were determined using a Milliplex cytokine/chemokine kit and the amount of each cytokine normalized to the total volume of the each sample. LL37 (5.6 µM) by itself did not induce any of the cytokines measured, but significantly enhanced poly(I:C) induced production of IL-1α, MCP-1 and IP-10, while Sc37 had no effect by itself or in the presence of poly(I:C). The addition of either LL37 or Sc37 at this concentration did not affect the morphology of the cells, indicating that there was no obvious cytotoxicity (data not shown). Each bar shows the mean with one SEM (n = 3).

Mentions: To determine whether LL37 enhances the response of primary cells to dsRNA, we examined cytokine release by human PBMCs (Figure 6). The culture medium of untreated human PBMCs had low levels of IL1α, MCP-1, and IP-10 (∼0.005, 0.2, and 0 µg/ml, respectively). Treatment with either LL37 (5.6 µM) or poly(I:C) (5 µg/ml) had modest effects on IL1α, MCP1 and IP-10 levels, but the addition of LL37 and poly(I:C) increased the levels of IL1α, MCP1 and IP10 by at least ten-fold above the levels seen with either poly(I:C) or LL37 alone (Figure 6; p = 0.0001; n = 3). Sc37 did not significantly increase any cytokine/chemokine levels (Figure 6; n = 3). These results demonstrate that the combination of LL37 and poly(I:C) enhances cytokine production in primary cells as it does in immortalized cell lines.


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 enhanced poly(I:C)-induced cytokine production in human peripheral blood mononuclear cells (PBMCs).After 24 h of poly(I:C) ±LL37 incubation, IL-1α, MCP-1 and IP-10 levels in the medium were determined using a Milliplex cytokine/chemokine kit and the amount of each cytokine normalized to the total volume of the each sample. LL37 (5.6 µM) by itself did not induce any of the cytokines measured, but significantly enhanced poly(I:C) induced production of IL-1α, MCP-1 and IP-10, while Sc37 had no effect by itself or in the presence of poly(I:C). The addition of either LL37 or Sc37 at this concentration did not affect the morphology of the cells, indicating that there was no obvious cytotoxicity (data not shown). Each bar shows the mean with one SEM (n = 3).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198786&req=5

pone-0026632-g006: LL37 enhanced poly(I:C)-induced cytokine production in human peripheral blood mononuclear cells (PBMCs).After 24 h of poly(I:C) ±LL37 incubation, IL-1α, MCP-1 and IP-10 levels in the medium were determined using a Milliplex cytokine/chemokine kit and the amount of each cytokine normalized to the total volume of the each sample. LL37 (5.6 µM) by itself did not induce any of the cytokines measured, but significantly enhanced poly(I:C) induced production of IL-1α, MCP-1 and IP-10, while Sc37 had no effect by itself or in the presence of poly(I:C). The addition of either LL37 or Sc37 at this concentration did not affect the morphology of the cells, indicating that there was no obvious cytotoxicity (data not shown). Each bar shows the mean with one SEM (n = 3).
Mentions: To determine whether LL37 enhances the response of primary cells to dsRNA, we examined cytokine release by human PBMCs (Figure 6). The culture medium of untreated human PBMCs had low levels of IL1α, MCP-1, and IP-10 (∼0.005, 0.2, and 0 µg/ml, respectively). Treatment with either LL37 (5.6 µM) or poly(I:C) (5 µg/ml) had modest effects on IL1α, MCP1 and IP-10 levels, but the addition of LL37 and poly(I:C) increased the levels of IL1α, MCP1 and IP10 by at least ten-fold above the levels seen with either poly(I:C) or LL37 alone (Figure 6; p = 0.0001; n = 3). Sc37 did not significantly increase any cytokine/chemokine levels (Figure 6; n = 3). These results demonstrate that the combination of LL37 and poly(I:C) enhances cytokine production in primary cells as it does in immortalized cell lines.

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