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Novel Cathelicidins from Pigeon Highlights Evolutionary Convergence in Avain Cathelicidins and Functions in Modulation of Innate Immunity.

Yu H, Lu Y, Qiao X, Wei L, Fu T, Cai S, Wang C, Liu X, Zhong S, Wang Y - Sci Rep (2015)

Bottom Line: In macrophages primed by LPS, Cl-CATH2 significantly down-regulated the gene and protein expressions of inducible nitric oxide synthase and pro-inflammatory cytokines while enhancing the anti-inflammatory cytokine, acting through MAPK and NF-κB signaling pathways.Molecular docking shows for the first time that cathelicidin binds to the opening region of LPS-binding pocket on myeloid differentiation factor 2 (MD-2) of toll-like receptor (TLR)4-MD-2 complex, which in turn inhibits the TLR4 pathway.Our results, therefore, provide new insight into the mechanism underlying the blockade of TLR4 signaling by cathelicidins.

View Article: PubMed Central - PubMed

Affiliation: School of Life Science and Biotechnology, Dalian University of Technology, Dalian, Liaoning 116024, China.

ABSTRACT
Cathelicidins are short cationic host defense peptides and play a central role in host innate immune system. Here we identified two novel cathelicidins, Cl-CATH2 and 3, from Columba livia. Evolutionary analysis of avian cathelicidins via phylogenetic tree and Ka/Ks calculations supported the positive selection that prompted evolution of CATH2 to CATH1 and 3, which originate from common ancestor and could belong to one superfamily. Cl-CATH2 and 3 both adopt amphipathic α-helical comformations identified by circular dichroism and the 3D structures built by Rosetta. Cl-CATH2 of CATH2 family with the most expression abundance in bird, exhibited relatively weak antimicrobial activity, but acted instead on the innate immune response without showing undesirable toxicities. In macrophages primed by LPS, Cl-CATH2 significantly down-regulated the gene and protein expressions of inducible nitric oxide synthase and pro-inflammatory cytokines while enhancing the anti-inflammatory cytokine, acting through MAPK and NF-κB signaling pathways. Molecular docking shows for the first time that cathelicidin binds to the opening region of LPS-binding pocket on myeloid differentiation factor 2 (MD-2) of toll-like receptor (TLR)4-MD-2 complex, which in turn inhibits the TLR4 pathway. Our results, therefore, provide new insight into the mechanism underlying the blockade of TLR4 signaling by cathelicidins.

No MeSH data available.


Related in: MedlinePlus

(a) Effects of Cl-CATH2 on iNOS and pro-inflammatory cytokine expression induced by LPS. Expression of the target genes iNOS, TNF-α, IL-1β, IL-6 and GAPDH was measured by real-time PCR and presented as fold change in the target genes expression normalized to GAPDH. Fold change of target genes expression in untreated cells was normalized to 1. Data are presented as the mean ± SD from three independent experiments (*, P < 0.05; **, P < 0.01; by unpaired t test). (b) Effects of Cl-CATH2 on NO and cytokine secretions stimulated by LPS. NO, TNF-α, IL-1β, IL-6 and IL-10 in RAW264.7 cell culture supernatant was determined by Griess reagent and ELISA, respectively. Blank: without LPS and Cl-CATH2. Control: cells only treated with LPS. Data are presented as the mean ± SD from three independent experiments (*, P < 0.05; **, P < 0.01; by unpaired t test).
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f5: (a) Effects of Cl-CATH2 on iNOS and pro-inflammatory cytokine expression induced by LPS. Expression of the target genes iNOS, TNF-α, IL-1β, IL-6 and GAPDH was measured by real-time PCR and presented as fold change in the target genes expression normalized to GAPDH. Fold change of target genes expression in untreated cells was normalized to 1. Data are presented as the mean ± SD from three independent experiments (*, P < 0.05; **, P < 0.01; by unpaired t test). (b) Effects of Cl-CATH2 on NO and cytokine secretions stimulated by LPS. NO, TNF-α, IL-1β, IL-6 and IL-10 in RAW264.7 cell culture supernatant was determined by Griess reagent and ELISA, respectively. Blank: without LPS and Cl-CATH2. Control: cells only treated with LPS. Data are presented as the mean ± SD from three independent experiments (*, P < 0.05; **, P < 0.01; by unpaired t test).

Mentions: To further determine whether Cl-CATH2 can confer protection against infection by triggering an immune response other than simply killing microbes, the cytokine productions were evaluated in macrophage cells after treatment with LPS and different concentrations of Cl-CATH2 using qRT-PCR. mRNA levels of the INOS (inducible nitric oxide synthase) and couple of key pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6 were all significantly upregulated after stimulation with LPS alone (Fig. 5a). However, expressions of all cytokines examined except IL-1β were markedly suppressed by Cl-CATH2 in a dose-dependent fashion, and that of IL-1β was not obviously altered until the concentration of Cl-CATH2 up to 20 μg/ml (Fig. 5a). The mRNA level of INOS was most sensitive to Cl-CATH2′s modulation. At Cl-CATH2 concentrations of 5, 10 and 20 μg/ml, the expression level of INOS was inhibited by 79%, 90% and 95%, respectively (Fig. 5a). Cl-CATH2 at 4.85 μM (20 μg/ml) significantly suppressed the INOS, TNF-α, IL-1β and IL-6 mRNA levels by 94.8%, 94.5%, 99.93% and 99.98%, respectively, which is much more potent than other cathelicidins reported so far., SMAP-29 (20 μM) failed to downregulate the expressions of IL-1β and CCL-2/MCP-120, albeit possessing immunomodulatory activity. Chicken fowlicidin1/2 (20 μM) only decreased the same gene levels by 90%30. asCATHs from salmon was found no regulating activities upon the expression of IL-1 or IL-1823.


Novel Cathelicidins from Pigeon Highlights Evolutionary Convergence in Avain Cathelicidins and Functions in Modulation of Innate Immunity.

Yu H, Lu Y, Qiao X, Wei L, Fu T, Cai S, Wang C, Liu X, Zhong S, Wang Y - Sci Rep (2015)

(a) Effects of Cl-CATH2 on iNOS and pro-inflammatory cytokine expression induced by LPS. Expression of the target genes iNOS, TNF-α, IL-1β, IL-6 and GAPDH was measured by real-time PCR and presented as fold change in the target genes expression normalized to GAPDH. Fold change of target genes expression in untreated cells was normalized to 1. Data are presented as the mean ± SD from three independent experiments (*, P < 0.05; **, P < 0.01; by unpaired t test). (b) Effects of Cl-CATH2 on NO and cytokine secretions stimulated by LPS. NO, TNF-α, IL-1β, IL-6 and IL-10 in RAW264.7 cell culture supernatant was determined by Griess reagent and ELISA, respectively. Blank: without LPS and Cl-CATH2. Control: cells only treated with LPS. Data are presented as the mean ± SD from three independent experiments (*, P < 0.05; **, P < 0.01; by unpaired t test).
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Related In: Results  -  Collection

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f5: (a) Effects of Cl-CATH2 on iNOS and pro-inflammatory cytokine expression induced by LPS. Expression of the target genes iNOS, TNF-α, IL-1β, IL-6 and GAPDH was measured by real-time PCR and presented as fold change in the target genes expression normalized to GAPDH. Fold change of target genes expression in untreated cells was normalized to 1. Data are presented as the mean ± SD from three independent experiments (*, P < 0.05; **, P < 0.01; by unpaired t test). (b) Effects of Cl-CATH2 on NO and cytokine secretions stimulated by LPS. NO, TNF-α, IL-1β, IL-6 and IL-10 in RAW264.7 cell culture supernatant was determined by Griess reagent and ELISA, respectively. Blank: without LPS and Cl-CATH2. Control: cells only treated with LPS. Data are presented as the mean ± SD from three independent experiments (*, P < 0.05; **, P < 0.01; by unpaired t test).
Mentions: To further determine whether Cl-CATH2 can confer protection against infection by triggering an immune response other than simply killing microbes, the cytokine productions were evaluated in macrophage cells after treatment with LPS and different concentrations of Cl-CATH2 using qRT-PCR. mRNA levels of the INOS (inducible nitric oxide synthase) and couple of key pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6 were all significantly upregulated after stimulation with LPS alone (Fig. 5a). However, expressions of all cytokines examined except IL-1β were markedly suppressed by Cl-CATH2 in a dose-dependent fashion, and that of IL-1β was not obviously altered until the concentration of Cl-CATH2 up to 20 μg/ml (Fig. 5a). The mRNA level of INOS was most sensitive to Cl-CATH2′s modulation. At Cl-CATH2 concentrations of 5, 10 and 20 μg/ml, the expression level of INOS was inhibited by 79%, 90% and 95%, respectively (Fig. 5a). Cl-CATH2 at 4.85 μM (20 μg/ml) significantly suppressed the INOS, TNF-α, IL-1β and IL-6 mRNA levels by 94.8%, 94.5%, 99.93% and 99.98%, respectively, which is much more potent than other cathelicidins reported so far., SMAP-29 (20 μM) failed to downregulate the expressions of IL-1β and CCL-2/MCP-120, albeit possessing immunomodulatory activity. Chicken fowlicidin1/2 (20 μM) only decreased the same gene levels by 90%30. asCATHs from salmon was found no regulating activities upon the expression of IL-1 or IL-1823.

Bottom Line: In macrophages primed by LPS, Cl-CATH2 significantly down-regulated the gene and protein expressions of inducible nitric oxide synthase and pro-inflammatory cytokines while enhancing the anti-inflammatory cytokine, acting through MAPK and NF-κB signaling pathways.Molecular docking shows for the first time that cathelicidin binds to the opening region of LPS-binding pocket on myeloid differentiation factor 2 (MD-2) of toll-like receptor (TLR)4-MD-2 complex, which in turn inhibits the TLR4 pathway.Our results, therefore, provide new insight into the mechanism underlying the blockade of TLR4 signaling by cathelicidins.

View Article: PubMed Central - PubMed

Affiliation: School of Life Science and Biotechnology, Dalian University of Technology, Dalian, Liaoning 116024, China.

ABSTRACT
Cathelicidins are short cationic host defense peptides and play a central role in host innate immune system. Here we identified two novel cathelicidins, Cl-CATH2 and 3, from Columba livia. Evolutionary analysis of avian cathelicidins via phylogenetic tree and Ka/Ks calculations supported the positive selection that prompted evolution of CATH2 to CATH1 and 3, which originate from common ancestor and could belong to one superfamily. Cl-CATH2 and 3 both adopt amphipathic α-helical comformations identified by circular dichroism and the 3D structures built by Rosetta. Cl-CATH2 of CATH2 family with the most expression abundance in bird, exhibited relatively weak antimicrobial activity, but acted instead on the innate immune response without showing undesirable toxicities. In macrophages primed by LPS, Cl-CATH2 significantly down-regulated the gene and protein expressions of inducible nitric oxide synthase and pro-inflammatory cytokines while enhancing the anti-inflammatory cytokine, acting through MAPK and NF-κB signaling pathways. Molecular docking shows for the first time that cathelicidin binds to the opening region of LPS-binding pocket on myeloid differentiation factor 2 (MD-2) of toll-like receptor (TLR)4-MD-2 complex, which in turn inhibits the TLR4 pathway. Our results, therefore, provide new insight into the mechanism underlying the blockade of TLR4 signaling by cathelicidins.

No MeSH data available.


Related in: MedlinePlus