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The human host defense peptide LL-37 interacts with Neisseria meningitidis capsular polysaccharides and inhibits inflammatory mediators release.

Zughaier SM, Svoboda P, Pohl J, Stephens DS, Shafer WM - PLoS ONE (2010)

Bottom Line: LL-37 also inhibited the ability of meningococcal CPS to induce nitric oxide release, as well as TNFα and CXCL10 (IP-10) release from TLR4-sufficient and TLR4-deficient murine macrophages.Thus, LL-37 interaction with CPS was independent of specific glucan structure.We conclude that the capacity of meningococcal CPS to activate macrophages via TLR2 and TLR4-MD-2 can be inhibited by the human cationic host defense peptide LL-37 and propose that this impacts CPS-based vaccine responses.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine and Laboratories of Microbial Pathogenesis, Atlanta, Georgia, United States of America. szughai@emory.edu

ABSTRACT
Capsular polysaccharides (CPS) are a major virulence factor in meningococcal infections and form the basis for serogroup designation and protective vaccines. Our work has identified meningococcal CPS as a pro-inflammatory ligand that functions through TLR2 and TLR4-MD2-dependent activation. We hypothesized that human cationic host defense peptides interact with CPS and influence its biologic activity. Accordingly, the interaction of meningococcal CPS with the human-derived cationic peptide LL-37, which is expressed by phagocytic and epithelial cells that interface with meningococci during infection, was investigated. LL-37 neutralized the pro-inflammatory activity of endotoxin-free CPS as assessed by TLR2 and TLR4-MD-2-dependent release of TNFα, IL-6 and IL-8 from human and murine macrophages. The cationic and hydrophobic properties of LL-37 were crucial for this inhibition, which was due to binding of LL-37 to CPS. LL-37 also inhibited the ability of meningococcal CPS to induce nitric oxide release, as well as TNFα and CXCL10 (IP-10) release from TLR4-sufficient and TLR4-deficient murine macrophages. Truncated LL-37 analogs, especially those that retained the antibacterial domain, inhibited vaccine grade CPS and meningococcal CPS prepared from the major serogroups (A, B C, Y and W135). Thus, LL-37 interaction with CPS was independent of specific glucan structure. We conclude that the capacity of meningococcal CPS to activate macrophages via TLR2 and TLR4-MD-2 can be inhibited by the human cationic host defense peptide LL-37 and propose that this impacts CPS-based vaccine responses.

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Electrophoretic mobility gel shift assay of meningococcal CPS-lpxA polymers in the presence of LL-37, LBP and CD14.CPS-lpxA complexes were prepared as described in the Methods section, were run on 8% native PAGE, and detected by Western blot using biotinylated LBP antibody. Lane 1 (MWt): molecular weight marker; lane 2 CPS-lpxA alone; lane 3: CPS-lpxA-CD14-LBP; lane 4: CD14-LBP alone; lane 5: CPS-lpxA-LL-37-CD14-LBP; lane 6: LL-37-CD14-LBP; lane 7: CPS-lpxA-LL-37 alone.
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pone-0013627-g008: Electrophoretic mobility gel shift assay of meningococcal CPS-lpxA polymers in the presence of LL-37, LBP and CD14.CPS-lpxA complexes were prepared as described in the Methods section, were run on 8% native PAGE, and detected by Western blot using biotinylated LBP antibody. Lane 1 (MWt): molecular weight marker; lane 2 CPS-lpxA alone; lane 3: CPS-lpxA-CD14-LBP; lane 4: CD14-LBP alone; lane 5: CPS-lpxA-LL-37-CD14-LBP; lane 6: LL-37-CD14-LBP; lane 7: CPS-lpxA-LL-37 alone.

Mentions: LL-37 and other cationic peptides have the ability to bind LPS by virtue of their cationic and hydrophobic properties [17]. To test if LL-37 and CPS can form a complex, we employed an electrophoretic mobility shift assay (EMSA) to resolve potential CPS:LL-37 complexes. Since CPS polymers are hydrophilic and highly water soluble, CD14 and LBP were used to stabilize the carbohydrate polymers for the EMSA assay [18]. Complexes were separated by 8% native PAGE and Western blotting using biotinylated anti-LBP and anti-CD14 antibodies to detect the complexes. Both CD14 and LBP were required to stabilize CPS complexes and visualize the shift. The additions of LL-37 to CPS-lpxA-CD14-LBP complexes caused a gel-shift (Figure 8, lane 5) compared to CPS-lpxA-CD14-LBP complexes (Figure 8 lane 3), to CD14-LBP alone (Figure 8, lane 4) and to LL-37-CD14-LBP complexes (Figure 8, lane 6). The bioactivity of the CPS-lpxA complexes used in gel-shift was further confirmed using RAW 264 macrophages. In contrast to CPS-lpxA complexes alone or with CD14 and LBP that were very active and induced large nitric oxide release, CPS-lpxA complexes that contained LL-37 gel-shifted in lane 5, showed significant reduction in nitric oxide release (data not shown). The results demonstrate that LL-37 binds to meningococcal CPS-lpxA polymers in the absence of endotoxin and that this specific interaction results in decreased CPS immuno-stimulatory activity. Thus, the interaction of this host-derived peptide with meningococcal CPS modulates innate immune responses.


The human host defense peptide LL-37 interacts with Neisseria meningitidis capsular polysaccharides and inhibits inflammatory mediators release.

Zughaier SM, Svoboda P, Pohl J, Stephens DS, Shafer WM - PLoS ONE (2010)

Electrophoretic mobility gel shift assay of meningococcal CPS-lpxA polymers in the presence of LL-37, LBP and CD14.CPS-lpxA complexes were prepared as described in the Methods section, were run on 8% native PAGE, and detected by Western blot using biotinylated LBP antibody. Lane 1 (MWt): molecular weight marker; lane 2 CPS-lpxA alone; lane 3: CPS-lpxA-CD14-LBP; lane 4: CD14-LBP alone; lane 5: CPS-lpxA-LL-37-CD14-LBP; lane 6: LL-37-CD14-LBP; lane 7: CPS-lpxA-LL-37 alone.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2964311&req=5

pone-0013627-g008: Electrophoretic mobility gel shift assay of meningococcal CPS-lpxA polymers in the presence of LL-37, LBP and CD14.CPS-lpxA complexes were prepared as described in the Methods section, were run on 8% native PAGE, and detected by Western blot using biotinylated LBP antibody. Lane 1 (MWt): molecular weight marker; lane 2 CPS-lpxA alone; lane 3: CPS-lpxA-CD14-LBP; lane 4: CD14-LBP alone; lane 5: CPS-lpxA-LL-37-CD14-LBP; lane 6: LL-37-CD14-LBP; lane 7: CPS-lpxA-LL-37 alone.
Mentions: LL-37 and other cationic peptides have the ability to bind LPS by virtue of their cationic and hydrophobic properties [17]. To test if LL-37 and CPS can form a complex, we employed an electrophoretic mobility shift assay (EMSA) to resolve potential CPS:LL-37 complexes. Since CPS polymers are hydrophilic and highly water soluble, CD14 and LBP were used to stabilize the carbohydrate polymers for the EMSA assay [18]. Complexes were separated by 8% native PAGE and Western blotting using biotinylated anti-LBP and anti-CD14 antibodies to detect the complexes. Both CD14 and LBP were required to stabilize CPS complexes and visualize the shift. The additions of LL-37 to CPS-lpxA-CD14-LBP complexes caused a gel-shift (Figure 8, lane 5) compared to CPS-lpxA-CD14-LBP complexes (Figure 8 lane 3), to CD14-LBP alone (Figure 8, lane 4) and to LL-37-CD14-LBP complexes (Figure 8, lane 6). The bioactivity of the CPS-lpxA complexes used in gel-shift was further confirmed using RAW 264 macrophages. In contrast to CPS-lpxA complexes alone or with CD14 and LBP that were very active and induced large nitric oxide release, CPS-lpxA complexes that contained LL-37 gel-shifted in lane 5, showed significant reduction in nitric oxide release (data not shown). The results demonstrate that LL-37 binds to meningococcal CPS-lpxA polymers in the absence of endotoxin and that this specific interaction results in decreased CPS immuno-stimulatory activity. Thus, the interaction of this host-derived peptide with meningococcal CPS modulates innate immune responses.

Bottom Line: LL-37 also inhibited the ability of meningococcal CPS to induce nitric oxide release, as well as TNFα and CXCL10 (IP-10) release from TLR4-sufficient and TLR4-deficient murine macrophages.Thus, LL-37 interaction with CPS was independent of specific glucan structure.We conclude that the capacity of meningococcal CPS to activate macrophages via TLR2 and TLR4-MD-2 can be inhibited by the human cationic host defense peptide LL-37 and propose that this impacts CPS-based vaccine responses.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Department of Medicine, Emory University School of Medicine and Laboratories of Microbial Pathogenesis, Atlanta, Georgia, United States of America. szughai@emory.edu

ABSTRACT
Capsular polysaccharides (CPS) are a major virulence factor in meningococcal infections and form the basis for serogroup designation and protective vaccines. Our work has identified meningococcal CPS as a pro-inflammatory ligand that functions through TLR2 and TLR4-MD2-dependent activation. We hypothesized that human cationic host defense peptides interact with CPS and influence its biologic activity. Accordingly, the interaction of meningococcal CPS with the human-derived cationic peptide LL-37, which is expressed by phagocytic and epithelial cells that interface with meningococci during infection, was investigated. LL-37 neutralized the pro-inflammatory activity of endotoxin-free CPS as assessed by TLR2 and TLR4-MD-2-dependent release of TNFα, IL-6 and IL-8 from human and murine macrophages. The cationic and hydrophobic properties of LL-37 were crucial for this inhibition, which was due to binding of LL-37 to CPS. LL-37 also inhibited the ability of meningococcal CPS to induce nitric oxide release, as well as TNFα and CXCL10 (IP-10) release from TLR4-sufficient and TLR4-deficient murine macrophages. Truncated LL-37 analogs, especially those that retained the antibacterial domain, inhibited vaccine grade CPS and meningococcal CPS prepared from the major serogroups (A, B C, Y and W135). Thus, LL-37 interaction with CPS was independent of specific glucan structure. We conclude that the capacity of meningococcal CPS to activate macrophages via TLR2 and TLR4-MD-2 can be inhibited by the human cationic host defense peptide LL-37 and propose that this impacts CPS-based vaccine responses.

Show MeSH
Related in: MedlinePlus