Immunomodulatory and Anti-Inflammatory Activities of Chicken Cathelicidin-2 Derived Peptides

Abstract

Host Defence Peptides and derived peptides are promising classes of antimicrobial and immunomodulatory lead compounds. For this purpose we examined whether chicken cathelicidin-2 (CATH-2)-derived peptides modulate the function and inflammatory response of avian immune cells. Using a chicken macrophage cell line (HD11) we found that full-length CATH-2 dose-dependently induced transcription of chemokines CXCLi2/IL-8, MCP-3 and CCLi4/RANTES, but not of pro-inflammatory cytokine IL-1β. In addition, CATH-2 efficiently inhibited IL-1β and nitric oxide production by HD11 cells induced by different sources of lipopolysaccharides (LPS). N-terminal truncated CATH-2 derived peptides maintained the capacity to selectively induce chemokine transcription, but despite their high LPS affinity several analogs lacked LPS-neutralizing capacity. Substitution of phenylalanine residues by tryptophan introduced endotoxin neutralization capacity in inactive truncated CATH-2 derived peptides. In contrast, amino acid substitution of phenylalanine by tyrosine abrogated endotoxin neutralization activity of CATH-2 analogs. These findings support a pivotal role for aromatic residues in peptide-mediated endotoxin neutralization by CATH-2 analogs and were shown to be independent of LPS affinity. The capacity to modulate chemokine production and dampen endotoxin-induced pro-inflammatory responses in chicken immune cells implicates that small CATH-2 based peptides could serve as leads for the design of CATH-2 based immunomodulatory anti-infectives.

Fig 1. Metabolic activity of CATH-2 derived peptide treated HD11 cells.

Metabolic activity of HD11 determined by WST-1 assays after 24 h stimulation with CATH-2 derived peptides (2.5–20 μM). Shown are means ± SEM for at least 3 independent experiments.

Fig 2. Peptide-induced LDH release from HD11 cells.

The percentage of lactate dehydrogenase (LDH) released from HD11 cells during 24 h exposure to medium containing CATH2 derived peptides (1–20 μM). Shown are means ± SEM for at least 3 independent experiments.

Fig 3. Induction of chemokine transcription in HD11 cells by truncated CATH-2 analogs.

CXCLi2, MCP-3, CCLi4 and IL-1β transcription in HD11 cells treated with CATH-2 derived peptides (1 to 20 μM). Transcription was measured by real time QPCR analysis after 4 h (open bars) or 24 h (closed bars) of stimulation. a CATH-2 dose-dependently induced transcription of chemokines chCXCLi2/IL-8, MCP-3 and chCCLi4/RANTES, but not of pro-inflammatory cytokine IL-1β. b The capacity to induce chemokine transcription was maintained for N-terminally truncated C(1–21) analogs up to 14 amino acid residues during 24 h exposure. c No chemokine induction was observed when cells were exposed (4 h) to peptide C(1–15). Data from 3 to 4 independent experiments; means ± SEM * p<0.05, ** p<0.01, *** p<0.001.

Fig 4. Neutralization of LPS-induced pro-inflammatory cytokine production by CATH-2 analogs.

IL-1β transcription in HD11 cells stimulated with S. minnesota LPS (50 ng/ml) and CATH-2 derived peptides (20 μM). Transcription was measured by real time QPCR analysis after 4 h of stimulation. Amino acid substitutions: W3, [F2W, F5W, F12W]; W2, [F5W, F12W]; W, [F12W]; Y3, [F2Y, F5Y, F12Y]; Y2, [F5Y, F12Y]; Y, [F12Y]. a LPS-induced IL-1β transcription in HD11 cells was blocked by full-length peptide and N-terminally truncated C(1–21) analogs up to 18 amino acid residues. b Phe/Trp substitution improved the capacity to neutralize LPS-induced IL-1β transcription of peptides C(1–21) and C(4–21) and introduced LPS-neutralizing capacity in formerly inactive peptides C(7–21) and C(10–21). c Phe/Tyr substitution of active peptides C(1–21) and C(4–21) abrogated neutralization of LPS-induced IL-1β expression. Data from 3 to 4 independent experiments; means ± SEM. * p<0.05, ** p<0.01, *** p<0.001.

Fig 5. CATH-2 analog neutralization of LPS-induced macrophage nitric oxide production.

Nitric oxide production by HD11 cells stimulated with LPS (50 to 100 ng/ml) pre-incubated with CATH-2 derived peptides (20 μM). Nitric oxide (NO) production was measured in supernatants after 24 h incubation using the Griess assay. Amino acid substitutions: W3, [F2W, F5W, F12W]; W2, [F5W, F12W]; W, [F12W]; Y3, [F2Y, F5Y, F12Y]; Y2, [F5Y, F12Y]; Y, [F12Y]. a Dose-dependent production of NO by HD11 cells exposed to different sources of LPS. b CATH-2 significantly inhibited NO production induced by all tested LPS (100 ng/ml) sources, except wild-type Neisseria meningitidis LPS; bars indicate LPS-induced NO production in the absence (closed) and presence (open) of CATH-2 peptide. c Inhibition of S. minnesota LPS-induced (50 ng/ml) NO production by truncated CATH-2 analogs. Production of LPS-induced NO by HD11 cells was significantly reduced in the presence of C(1–21) and N-terminally truncated C(1–21) analogs up to 17 amino acid residues. d, e Phe/Trp substitution enhanced inhibition of active peptides C(1–21) and C(4–21), whereas Phe/Tyr substitution abrogated inhibition of LPS-induced NO production by these peptides. Data from 3 to 4 independent experiments; means ± SEM. * p<0.05, ** p<0.01, *** p<0.001.

Fig 6. Structural representation of LPS-neutralizing CATH-2 and C1-21 peptide.

Representations of the known 3-dimensional structure of paradaxin and human lactoferrin-derived peptide hLF11, cationic peptides with known LPS-binding affinity and the deduced structures of CATH-2 and C(1–21). Side chains reported or deduced to be in closest proximity to lipid A are depicted in green (aromatic side chains) and blue (basic side chains). Representations were adapted from the published structures (RSCB Protein Data bank; http://www.rcsb.org/pdb/home/home.do). The structure of CATH-2 analog C(1–21) was predicted using iTasser.

Fig 7. CATH-2 analogs partially neutralize IL-1β transcription in LPS-primed cells.

HD11 cells were primed for 30 min with S. minnesota LPS (100–1000 ng/ml), washed once and incubated during 4h in the presence or absence of 20 μM peptide. IL-1β transcription was measured by real time QPCR analysis. Data from 3 to 4 independent experiments (means ± SEM).