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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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LL-37 neutralized meningococcal CPS bioactivity and inhibited cytokine release from human and murine macrophages.CPS polymers were purified from the endotoxin-deficient serogroup B meningococcal NMB-lpxA mutant designated CPS-lpxA. A: TNFα release from human macrophage-like THP-1 cells induced overnight with CPS-lpxA polymers pre-incubated with or without 2 µg/ml of LL-37 for 30 min at 37°C (Materials and Methods section). B. IL-6 release from THP-1 cells induced as in panel A. C. IL-8 release from HEK-TLR2/6 stably transfected cells induced with CPS-lpxA as in panel A. TNFα,IL-6 and IL-8 release was measured by ELISA. D. Nitric oxide release from murine RAW 264.7 macrophages induced with CPS-lpxA polymers as in panel A and measured by the Griess method as nitrite accumulation after 24 h of incubation at 37°C with 5% CO2. Error bars represent ±SD from the mean of quadruplicate measurements. This experiment is representative of three independent experiments.
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pone-0013627-g001: LL-37 neutralized meningococcal CPS bioactivity and inhibited cytokine release from human and murine macrophages.CPS polymers were purified from the endotoxin-deficient serogroup B meningococcal NMB-lpxA mutant designated CPS-lpxA. A: TNFα release from human macrophage-like THP-1 cells induced overnight with CPS-lpxA polymers pre-incubated with or without 2 µg/ml of LL-37 for 30 min at 37°C (Materials and Methods section). B. IL-6 release from THP-1 cells induced as in panel A. C. IL-8 release from HEK-TLR2/6 stably transfected cells induced with CPS-lpxA as in panel A. TNFα,IL-6 and IL-8 release was measured by ELISA. D. Nitric oxide release from murine RAW 264.7 macrophages induced with CPS-lpxA polymers as in panel A and measured by the Griess method as nitrite accumulation after 24 h of incubation at 37°C with 5% CO2. Error bars represent ±SD from the mean of quadruplicate measurements. This experiment is representative of three independent experiments.

Mentions: Meningococcal CPS polymers consist of distinct repeating glucan units that vary in composition and linkage among invasive serotypes [1]. Meningococcal CPS polymers are polyanionic due to the negatively charged sialic acid residues found in many invasive meningococcal serogroups (e.g. B, C, Y, W-135) or due to the presence of phosphates in the serogroup A polymers [1], [4], [12]. The immuno-stimulatory activity (bioactivity) of meningococcal CPS polymers (vaccine grade and laboratory prepared CPS) was investigated in vitro using well established and characterized human and murine cell lines [14]. CPS was purified from an endotoxin-deficient serogroup B N. meningitidis lpxA mutant (designated CPS-lpxA) and consists of (α2→8)-N-acetylneuraminic acid rendered protein-, lipopeptide-, phospholipid- and nucleic acid-free by digestion with proteinase K, DNAse and RNAse during the extraction and purification procedures (see Methods). This CPS induced a dose-dependent release of the pro-inflammatory cytokines TNFα and IL-6 from THP-1, human macrophages-like cells (Figure 1A and B), IL-8 from HEK-TLR2/6 stably transfected cells (Figure 1C) and nitric oxide (NO) release from murine RAW 264 macrophages (Figure 1D). Hence, based on its ability to induce macrophage activation, as assessed by release of cytokines and NO, we concluded that the endotoxin-free serogroup B CPS is bioactive in an LPS-independent manner; other CPS polymers from serogroup A, C, Y and W-135 strains were found to have a similar activity (see below).


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)

LL-37 neutralized meningococcal CPS bioactivity and inhibited cytokine release from human and murine macrophages.CPS polymers were purified from the endotoxin-deficient serogroup B meningococcal NMB-lpxA mutant designated CPS-lpxA. A: TNFα release from human macrophage-like THP-1 cells induced overnight with CPS-lpxA polymers pre-incubated with or without 2 µg/ml of LL-37 for 30 min at 37°C (Materials and Methods section). B. IL-6 release from THP-1 cells induced as in panel A. C. IL-8 release from HEK-TLR2/6 stably transfected cells induced with CPS-lpxA as in panel A. TNFα,IL-6 and IL-8 release was measured by ELISA. D. Nitric oxide release from murine RAW 264.7 macrophages induced with CPS-lpxA polymers as in panel A and measured by the Griess method as nitrite accumulation after 24 h of incubation at 37°C with 5% CO2. Error bars represent ±SD from the mean of quadruplicate measurements. This experiment is representative of three independent experiments.
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Related In: Results  -  Collection

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pone-0013627-g001: LL-37 neutralized meningococcal CPS bioactivity and inhibited cytokine release from human and murine macrophages.CPS polymers were purified from the endotoxin-deficient serogroup B meningococcal NMB-lpxA mutant designated CPS-lpxA. A: TNFα release from human macrophage-like THP-1 cells induced overnight with CPS-lpxA polymers pre-incubated with or without 2 µg/ml of LL-37 for 30 min at 37°C (Materials and Methods section). B. IL-6 release from THP-1 cells induced as in panel A. C. IL-8 release from HEK-TLR2/6 stably transfected cells induced with CPS-lpxA as in panel A. TNFα,IL-6 and IL-8 release was measured by ELISA. D. Nitric oxide release from murine RAW 264.7 macrophages induced with CPS-lpxA polymers as in panel A and measured by the Griess method as nitrite accumulation after 24 h of incubation at 37°C with 5% CO2. Error bars represent ±SD from the mean of quadruplicate measurements. This experiment is representative of three independent experiments.
Mentions: Meningococcal CPS polymers consist of distinct repeating glucan units that vary in composition and linkage among invasive serotypes [1]. Meningococcal CPS polymers are polyanionic due to the negatively charged sialic acid residues found in many invasive meningococcal serogroups (e.g. B, C, Y, W-135) or due to the presence of phosphates in the serogroup A polymers [1], [4], [12]. The immuno-stimulatory activity (bioactivity) of meningococcal CPS polymers (vaccine grade and laboratory prepared CPS) was investigated in vitro using well established and characterized human and murine cell lines [14]. CPS was purified from an endotoxin-deficient serogroup B N. meningitidis lpxA mutant (designated CPS-lpxA) and consists of (α2→8)-N-acetylneuraminic acid rendered protein-, lipopeptide-, phospholipid- and nucleic acid-free by digestion with proteinase K, DNAse and RNAse during the extraction and purification procedures (see Methods). This CPS induced a dose-dependent release of the pro-inflammatory cytokines TNFα and IL-6 from THP-1, human macrophages-like cells (Figure 1A and B), IL-8 from HEK-TLR2/6 stably transfected cells (Figure 1C) and nitric oxide (NO) release from murine RAW 264 macrophages (Figure 1D). Hence, based on its ability to induce macrophage activation, as assessed by release of cytokines and NO, we concluded that the endotoxin-free serogroup B CPS is bioactive in an LPS-independent manner; other CPS polymers from serogroup A, C, Y and W-135 strains were found to have a similar activity (see below).

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