Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011;6(10):e26525.
doi: 10.1371/journal.pone.0026525. Epub 2011 Oct 18.

Evaluation of the ability of LL-37 to neutralise LPS in vitro and ex vivo

Aaron Scott  1 , Sinéad Weldon, Paul J Buchanan, Bettina Schock, Robert K Ernst, Danny F McAuley, Michael M Tunney, Chris R Irwin, J Stuart Elborn, Clifford C Taggart
Affiliations

Evaluation of the ability of LL-37 to neutralise LPS in vitro and ex vivo

Aaron Scott et al. PLoS One. 2011.

Abstract

Background: Human cathelicidin LL-37 is a cationic antimicrobial peptide (AMP) which possesses a variety of activities including the ability to neutralise endotoxin. In this study, we investigated the role of LPS neutralisation in mediating LL-37's ability to inhibit Pseudomonas aeruginosa LPS signalling in human monocytic cells.

Methodology/principal findings: Pre-treatment of monocytes with LL-37 significantly inhibited LPS-induced IL-8 production and the signalling pathway of associated transcription factors such as NF-κB. However, upon removal of LL-37 from the media prior to LPS stimulation, these inhibitory effects were abolished. These findings suggest that the ability of LL-37 to inhibit LPS signalling is largely dependent on extracellular LPS neutralisation. In addition, LL-37 potently inhibited cytokine production induced by LPS extracted from P. aeruginosa isolated from the lungs of cystic fibrosis (CF) patients. In the CF lung, polyanionic molecules such as glycosaminoglycans (GAGs) and DNA bind LL-37 and impact negatively on its antibacterial activity. In order to determine whether such interactions interfere with the LPS neutralising ability of LL-37, the status of LL-37 and its ability to bind LPS in CF sputum were investigated. Overall our findings suggest that in the CF lung, the ability of LL-37 to bind LPS and inhibit LPS-induced IL-8 production is attenuated as a result of binding to DNA and GAGs. However, LL-37 levels and its concomitant LPS-binding activity can be increased with a combination of DNase and GAG lyase (heparinase II) treatment.

Conclusions/significance: Overall, these findings suggest that a deficiency in available LL-37 in the CF lung may contribute to greater LPS-induced inflammation during CF lung disease.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. LL-37 inhibits LPS-induced IL-8 production and IκB degradation in THP-1 monocytic cells.
(A) THP-1 monocytes were pre-treated for 1 h with 10 µg/ml LL-37 and incubated in the absence or presence of 1 µg/ml P. aeruginosa LPS. After 24 h stimulation with LPS, IL-8 levels were quantified in cell-free culture supernatants by ELISA. *** p<0.001; Con, control. (B) Cytoplasmic extracts were prepared following 30, 60 and 120 min and levels of IκBα, IκBβ, phosphorylation of IκBα (Ser32/36) and IKKα/β (Ser180/181) were determined by Western blotting. GAPDH was used to control for protein loading.
Figure 2
Figure 2. Removal of exogenous LL-37 prior to LPS stimulation diminishes its anti-endotoxin activity.
(A) THP-1 monocytes were pre-treated for 1 h with LL-37 (10 µg/ml) and then washed with sterile PBS to remove extracellular, non-incorporated, exogenous LL-37. The cells were resuspended in fresh media and stimulated with 0.01, 0.1 and 1 µg/ml LPS for 24 h. IL-8 levels in cell-free culture supernatants were quantified by ELISA. (B) THP-1 monocytes were pre-treated for 1 h with 10 µg/ml LL-37 and washed as described previously. Following resuspension, cells were stimulated with 1 µg/ml LPS for 30, 60 and 120 min. Cytoplasmic extracts were prepared and IκBα levels were determined by Western blotting. GAPDH was used to control for protein loading.
Figure 3
Figure 3. LL-37 neutralises LPS from clinical strains of P. aeruginosa isolated from CF patients with severe airway disease.
THP-1 monocytes were pre-treated with 5 µg/ml LL-37 for 1 h before cells were stimulated for 24 h with (A) SE4 and (B) SE22 LPS isolates from CF patients with severe airway disease (1, 10 and 100 ng/ml). IL-8 levels in cell-free culture supernatants were quantified by ELISA. ** p<0.01; *** p<0.001 compared to corresponding LPS alone (- LL-37). THP-1 monocytes were pre-treated for 1 h with LL-37 (5 µg/ml) and then washed with sterile PBS to remove extracellular, non-incorporated, exogenous LL-37. The cells were resuspended in fresh media and stimulated with 50 ng/ml (C) SE4 and (D) SE22 LPS for 24 h. IL-8 levels in cell-free culture supernatants were quantified by ELISA. Results are expressed as percentage of control response (100% = LPS alone). *** p<0.001.
Figure 4
Figure 4. LL-37 levels and its ability to bind LPS in CF sputum are increased following treatment with DNase and heparinase II.
(A) CF sputum samples (n = 12) were treated with Pulmozyme® DNase and/or heparinase II for 1 h at 37°C. The LL-37 content of these samples and untreated sputum samples were quantified by ELISA. (B) The ability of available LL-37 present in untreated and treated CF sputum samples to bind LPS was determined by ELISA. Results are expressed as percentage of untreated control (100% = untreated CF sputum). ** p<0.01; *** p<0.001.
Figure 5
Figure 5. Increased LL-37 in DNase and heparinase II treated CF sputum inhibits IL-8 production from THP-1 monocytes.
CF sputum samples (n = 12) were treated with Pulmozyme® DNase and/or heparinase II for 1 h at 37°C in the absence or presence of an LL-37 antibody or heat-inactivated (HI) LL-37 antibody as a negative control. Samples were incubated in LPS-immobilised 96-well microtiter plates (100 ng LPS/well) for 1 h at RT. Unbound peptides were removed by washing and THP-1 monocytes (1×106/ml) were added to each well and incubated for 6 h at 37°C. The ability of released LL-37 to inhibit LPS-induced IL-8 production by THP-1 monocytes was determined by ELISA. Results are expressed as percentage of untreated control (100% = untreated CF sputum). *** p<0.001.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Donaldson SH, Boucher RC. Sodium channels and cystic fibrosis. Chest. 2007;132:1631–1636. - PubMed
    1. Gibson RL, Burns JL, Ramsey BW. Pathophysiology and management of pulmonary infections in cystic fibrosis. Am J Respir Crit Care Med. 2003;168:918–951. - PubMed
    1. Döring G, Gulbins E. Cystic fibrosis and innate immunity: how chloride channel mutations provoke lung disease. Cell Microbiol. 2009;11:208–216. - PubMed
    1. Bals R, Weiner DJ, Wilson JM. The innate immune system in cystic fibrosis lung disease. J Clin Invest. 1999;103:303–307. - PMC - PubMed
    1. Hiemstra PS. The role of epithelial beta-defensins and cathelicidins in host defense of the lung. Exp Lung Res. 2007;33:537–542. - PubMed

MeSH terms