Abstract
The anti-endotoxin activity of the cationic peptide LL-37 and its derivative IG-19 is attributed to electrostatic interaction of the peptides’ positive charge with negatively charged bacterial lipopolysaccharides (LPS), and in part to the alteration of intracellular mechanisms independent of peptide binding to LPS. We examined the immunomodulatory responses induced by IG-19 and four IG-19-derived scrambled peptides (IG-19a–d), in the presence and absence of LPS, in macrophages and peripheral blood-derived mononuclear cells. All peptides had identical net charge (+5) and amino acid composition, but different hydrophobicity and α-helical propensity. Peptide IG-19 suppressed LPS-induced cytokine/chemokine production by >90%, IG-19a and IG-19b suppressed it by 40–50%, and IG-19c and IG-19d did not suppress cytokine/chemokine production at all. In silico prediction algorithms and the peptide retention time (RT) on a C18 RP HPLC column indicated a linear association between α-helical propensity and hydrophobicity with the ability of the peptides to inhibit LPS-induced responses. Peptide RT exhibited a significant correlation (>70%) between the suppression of LPS-induced cytokine/chemokine production and peptide-induced production of the anti-inflammatory cytokine IL-1RA. These results indicate that RT on a C18 column can be used as a predictor for the immunomodulatory functions of cationic peptides. Overall, we demonstrated that the immunomodulatory functions of LL-37-derived peptides with identical positive charge and amino acid composition are directly associated with the predicted α-helical propensity and hydrophobicity of the peptides.
Highlights
Cationic host defense peptides (CHDPs) play a critical role in antimicrobial and immunity-related functions
We have previously shown that LL-37 can suppress inflammatory responses in the absence of microbial pathogens, such as the response induced by the cytokine IL-32, while inducing the production anti-inflammatory cytokines e.g., interleukin-1-receptor antagonist (IL-1RA) and IL-10 [9,10,11,12]
Production of pro-inflammatory cytokine TNF and chemokines GRO-α and IL-8 was evaluated by Production of pro‐inflammatory cytokine TNF and chemokines GRO‐α and IL‐8 was evaluated by Enzyme-Linked Immunosorbent Assay (ELISA) in tissue culture (TC) supernatants after 24 h (A) Correlation analysis between the percentage inhibition of LPS-induced responses and predicted hydrophobicity index (HI) of the peptides. (B) Correlation
Summary
Cationic host defense peptides (CHDPs) play a critical role in antimicrobial and immunity-related functions. IG-19 suppresses inflammatory cytokine production by engaging various signaling intermediates in the presence of bacterial endotoxin or cytokine IL-32 [10,13,14] Both LL-37 and IG-19 share structural similarities; these are both α-helical amphipathic peptides with net positive charges of +6 and +5, respectively [13,14]. Previous studies have suggested that the ability of cationic peptides such as LL-37 to suppress bacterial lipopolysaccharide (LPS)-induced pro-inflammatory responses is largely due to peptide binding to negatively charged. Suppression of LPS-induced pro-inflammatory responses by LL-37 is not solely dependent on peptide binding to LPS, as it involves the ability of the peptide to intervene in TLR-to-NF-κB signaling mechanisms [8,12,17]. Based on the results of this study, we propose that peptide RT may be a useful tool with which to screen and predict immunomodulatory functions of CHDP and related synthetic derivatives
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