A potent anti-inflammatory peptide from the salivary glands of horsefly.
BACKGROUND
A diverse group of physiologically active peptides/proteins are present in the salivary glands of horsefly Tabanus yao (Diptera, Tabanidae) that facilitate acquisition of blood meal. However, their roles in the regulation of local inflammation remains poorly understood.


METHODS
Induction expression profiles of immune-related molecules in the salivary glands of T. yao was analyzed by quantitative PCR (qPCR) after bacterial feeding. A significantly up-regulated molecule (cecropin-TY1) was selected for anti-inflammatory assay in lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages. The transcription levels of inducible NO synthase (iNOS) and pro-inflammatory cytokines were quantified by qPCR. Nitric oxide (NO) production was determined by Griess reagent. Pro-inflammatory cytokine production was determined by an enzyme-linked immunosorbent assay (ELISA). The inflammatory signals were assayed by Western blotting analysis. The secondary structure of cecropin-TY1 was measured by Circular dichroism (CD) spectroscopy. Interaction of cecropin-TY1 with LPS was evaluated by the dissociation of fluorescein isothiocyanate (FITC)-conjugated LPS aggregates and neutralization of LPS determined by a quantitative Chromogenic End-point Tachypleus amebocyte lysate (TAL) assay kit. Homology modeled structure analysis and mutation of key residues/structures were performed to understand its structure-activity relationship.


RESULTS
Cecropin-TY1 was demonstrated to possess high anti-inflammatory activity and low cytotoxicity toward mouse macrophages. In LPS-stimulated mouse peritoneal macrophage, addition of cecropin-TY1 significantly inhibited the production of nitric oxide (NO) and pro-inflammatory cytokines. Further study revealed that cecropin-TY1 inhibited inflammatory cytokine production by blocking activation of mitogen-activated protein kinases (MAPKs) and transcriptional nuclear factor-κB (NF-κB) signals. Cecropin-TY1 even interacted with LPS and neutralized LPS. The secondary structure analysis revealed that cecropin-TY1 adopted unordered structures in hydrophobic environment but converted to α-helical confirmation in membrane mimetic environments. Homology modeled structure analysis demonstrated that cecropin-TY1 adopted two α-helices (Leu3-Thr24, Ile27-Leu38) linked by a hinge (Leu25-Pro26) and the structure surface was partly positively charged. Structure-activity relationship analysis indicated that several key residues/structures are crucial for its anti-inflammatory activity including α-helices, aromatic residue Trp2, positively charged residues Lys and Arg, hinge residue Pro26 and N-terminal amidation.


CONCLUSIONS
We found a novel anti-inflammatory function of horsefly-derived cecropin-TY1 peptide, laying groundwork for better understanding the ectoparasite-host interaction of horsefly with host and highlighting its potency in anti-inflammatory therapy for sepsis and endotoxin shock caused by Gram-negative bacterial infections.
Jiangsu Key Laboratory of Infection and Immunity, Institutes of Biology and Medical Sciences, Soochow University, 199 Ren-Ai Road, Suzhou Industrial Park, Suzhou, 215123, Jiangsu Province, China. weilin1005@126.com.
Lin
L
Wei
Jiangsu Key Laboratory of Infection and Immunity, Institutes of Biology and Medical Sciences, Soochow University, 199 Ren-Ai Road, Suzhou Industrial Park, Suzhou, 215123, Jiangsu Province, China. huangcjibms@126.com.
Chunjing
C
Huang
School of Basic Medical Sciences, Kunming Medical University, 1168 West Chunrong Road, Yuhua Avenue, Chenggong District, Kunming, 650500, Yunnan Province, China. jxauyhl@aliyun.com.
Hailong
H
Yang
Jiangsu Key Laboratory of Infection and Immunity, Institutes of Biology and Medical Sciences, Soochow University, 199 Ren-Ai Road, Suzhou Industrial Park, Suzhou, 215123, Jiangsu Province, China. minl_zju@126.com.
Min
M
Li
College of Biological Science and Technology, Fuzhou University, Fuzhou, 350108, Fujian, China. juanyang@fzu.edu.cn.
Juanjuan
J
Yang
Institute of Marine Biological Technology, School of Life Science and Biotechnology, Dalian University of Technology, Dalian, 116024, Liaoning, China. 1184787297@qq.com.
Xue
X
Qiao
School of Basic Medical Sciences, Kunming Medical University, 1168 West Chunrong Road, Yuhua Avenue, Chenggong District, Kunming, 650500, Yunnan Province, China. mulixian77@163.com.
Lixian
L
Mu
Jiangsu Key Laboratory of Infection and Immunity, Institutes of Biology and Medical Sciences, Soochow University, 199 Ren-Ai Road, Suzhou Industrial Park, Suzhou, 215123, Jiangsu Province, China. xf123456.cool@163.com.
Fei
F
Xiong
School of Basic Medical Sciences, Kunming Medical University, 1168 West Chunrong Road, Yuhua Avenue, Chenggong District, Kunming, 650500, Yunnan Province, China. wujing_205@163.com.
Jing
J
Wu
Jiangsu Key Laboratory of Infection and Immunity, Institutes of Biology and Medical Sciences, Soochow University, 199 Ren-Ai Road, Suzhou Industrial Park, Suzhou, 215123, Jiangsu Province, China. xuweifd828@126.com.
Wei
W
Xu
D000818:Animals; D000893:Anti-Inflammatory Agents / Q000378:metabolism*; D015153:Blotting, Western; D054807:Cecropins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D002942:Circular Dichroism; D016207:Cytokines / Q000096:biosynthesis; D004175:Diptera / Q000502:physiology*; D004797:Enzyme-Linked Immunosorbent Assay; D020869:Gene Expression Profiling; D006790:Host-Parasite Interactions; D008070:Lipopolysaccharides / Q000276:immunology / Q000378:metabolism; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000276:immunology; D051379:Mice; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis; D011485:Protein Binding; D011487:Protein Conformation; D060888:Real-Time Polymerase Chain Reaction; D012469:Salivary Glands / Q000737:chemistry; D012471:Salivary Proteins and Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D000893:Anti-Inflammatory Agents; D054807:Cecropins; D016207:Cytokines; D008070:Lipopolysaccharides; D012471:Salivary Proteins and Peptides; D052247:Nitric Oxide Synthase Type II