- Open Access
A potent anti-inflammatory peptide from the salivary glands of horsefly
- Lin Wei†1,
- Chunjing Huang†1,
- Hailong Yang2,
- Min Li1,
- Juanjuan Yang3,
- Xue Qiao4,
- Lixian Mu2,
- Fei Xiong1,
- Jing Wu2Email author and
- Wei Xu1Email author
© Wei et al. 2015
- Received: 7 August 2015
- Accepted: 7 October 2015
- Published: 24 October 2015
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.
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.
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.
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.
- Salivary gland
- Hematophagous arthropod
Hematophagous arthropods have evolved effective mechanisms to suppress their host’s hemostatic system and immune response to get a blood meal successfully. Their salivary glands can produce a wide array of active compounds including antihemostatic and immunoregulatory substances [1–9]. A majority of antihemostatic compounds have been identified and can be divided into several categories including inhibitors of coagulation factors (Factors VII, V, IIa, and Xa) and platelet functions, fibrin(ogen)olytic enzymes, and vasoactive peptides [3–5]. Immunoregulatory factors from arthropods especially from ticks have been extensively investigated [10–15]. These immunoregulatory factors such as immunosuppressive peptides and antimicrobial peptides (AMPs) are crucial for blood-sucking arthropods to suppress the immune response including innate immunity, adaptive immunity and inflammation [5, 14–17]. Horseflies are economically important blood-feeding insects and vectors for pathogens such as filariasis . There have been reported a diverse group of active compounds in the salivary glands of horseflies like other hematophagous arthropods including mosquitoes , flies , and ticks [10, 12]. Although the antihemostaic substances in horsefly have been extensively exploited in previous work [5, 18, 19], comparatively few investigations on the anti-inflammatory effects of horsefly-derived AMPs were conducted.
AMPs are small gene-encoded defensive effectors and play key roles in the innate immunity in all living organisms. Insects are an important source of AMPs . Since the first observation of antimicrobial activity in the hemolymph of bacteria-challenged pupae of the giant silk moths (Samia Cynthia and Hyalophora cecropia) in 1974 and the first purification of AMP from the hemolymph of H. cecropia in 1980, over 200 AMPs have been identified or purified from insects . Generally, insect AMPs are comprised of four groups based on their structural motifs and unique sequences. They are (i) α-helical peptides (cecropin and moricin), (ii) cysteine-rich peptides with intramolecular cysteine disulfide bonds forming hairpin-like β-sheets or α-helical/β-sheet mixed structures (insect defensin and drosomycin), (iii) proline-rich peptides (apidaecin, drosocin, and lebocin), and (iv) glycine-rich peptides/proteins (attacin and gloverin) .
Among these insect-derived AMPs, cecropins constitute a large family of cationic α-helical peptides composed of 35–39 amino acids, and most of them are amidated at the C-terminus . Cecropin was the first insect AMP purified from the hemolymph of H. cecropia in 1980 . Since then, a variety of insect-derived cecropin AMPs were identified in lepidopteran, dipteran, and coleopteran . Cecropins usually have broad antimicrobial spectrum against various microorganisms including Gram-positive and Gram-negative bacteria [22, 23], fungi [24, 25], parasites [26, 27] and HIV-1 virus . In addition to antimicrobial activity, several cecropins showed strong anti-inflammatory activity in LPS-stimulated macrophages through interaction with LPS on the basis of their α-helical structures [29, 30]. Cecropins usually adopted random coil conformations in aqueous solutions. While these unordered structures converted to amphipathic α-helical structures in the membrane-like environments to exert its biological activity [20, 29, 30].
Recently, a wide array of physiologically active molecules such as antihemostatic substances (fibrin(ogen)olytic, Arg-Gly-Asp-motif containing proteins, vasodilator peptides, etc.), immunosuppressive peptides (tabimmuregulins), AMPs (attactin, defensin and cecropin) and allergens (Tab a 1, Tab a 2, Tab y 1) have been identified in the salivary glands in horsefly of T. yao [3, 5, 31–35]. To identify whether there are anti-inflammatory agents in the salivary glands of horsefly T. yao, induction expression of immune-related genes (immunosuppressive peptides and AMPs) in their salivary glands were analyzed after bacterial feeding. It was demonstrated that cecropin-TY1 expression was dramatically up-regulated among immune-related genes. Cecropin-TY1 was previously identified as an AMP with antimicrobial activity in the salivary glands of T. yao . In the current work, cecropin-TY1 showed strong anti-inflammatory effects in LPS-stimulated mouse peritoneal macrophages and low cytotoxicity. The effects of cecropin-TY1 on LPS-activated inflammatory signaling and the interaction of cecropin-TY1 with LPS were investigated. The secondary structures of cecropin-TY1 in different solutions were exploited by CD spectra and the 3D structures were homology modeled to understand the interaction between cecropin-TY1 and LPS. Anti-inflammatory effects of the derivatives of cecropin-TY1 were also investigated to understand the key residues/structures for its anti-inflammatory activity.
Induction expression analysis
Horseflies were collected from Shanxi province as previously described [3, 5]. The collected T. yao (~800 flies) were randomly grouped in two cages (100 × 80 × 60 cm) covered with grenadine and kept at temperature of 25 ± 2 °C, humidity of 80–90 % and a 12 h/12 h photoperiod. After a 12-h starvation, horseflies were fed with fresh chicken blood containing 1 % sodium citrate (w/v) supplemented with Gram-negative bacteria Escherichia coli ATCC 8739 (2 × 106/mL) . The control group was fed with the same chicken blood without bacteria. The salivary glands of horseflies were dissected under a microscope at 0, 6, 12, 24, 36, 48 and 72 h after blood meal. The salivary gland of each horsefly was excised in phosphate buffer solution (100 mM, pH 6.0) on ice immediately [3, 5]. Total RNA extraction was performed by Trizol reagent (Life Tech, USA) according to the kit instruction. cDNA was synthesized with PrimeScript® Reverse Transcriptase Kit (Takara, Japan). The induction expression of immune-related genes including tabimmuregulins and AMPs was analyzed by qPCR to screen anti-inflammatory agents in the salivary glands of horseflies as described in qPCR section.
The study was approved by the Animal Care and Use Ethics Committee of Kunming Medical University.
qPCR was performed using SYBR green master mix (Takara, Japan) on a Realplex Mastercycler real-time PCR system (Eppendorf, Germany) according to the manufacturer’s instruction. The gene transcription levels were normalized to GAPDH or β-actinas as illustrated in figure legends and calculated by ΔΔCt method. The accuracy of qPCR results were checked by melting curve analysis. qPCR primers were listed in Additional file 1: Table S1.
The amino acid sequence of the derivatives of cecropin-TY1
Amino acid sequence
N39CONH2 → N39COOH
W2 → A2
lacked the hinge residue P26
K4 → A4, K5 → A5, K8 → A8, K9 → A9, R12 → A12, R18 → A18
Scrambled cecropin-TY1, disrupted the helices
Mouse peritoneal macrophages were prepared according to previous method . Brewer thioglycollate medium (3 %, w/v, Sigma-Aldrich, USA) was intraperitoneally injected to C57BL/6 mouse. After 3 days, the mouse was euthanized and intraperitoneally injected with 20 mL RPMI-1640 medium to collect peritoneal macrophages. RAW264.7 cells were cultured in RPMI 1640 medium (10 % FBS, 100 U/mL penicillin and 100 μg/mL streptomycin, Gibco, USA) at 37 °C humidified with 5 % CO2. Mouse macrophages, peritoneal macrophages and RAW264.7 cells, were seeded in a 96-well (2 × 104 cells/well) plate, and cultured in RPMI 1640 medium (100 μL) supplemented with 2 % FBS, 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco, USA) at 37 °C in a humidified 5 % CO2 incubator. The cytotoxicity of cecropin-TY1 against mouse macrophages was determined by Cell Counting Kit-8 assay (CCK-8) following the kit instruction. In brief, serial 2-fold peptide dilutions were added to each well, and control wells received the same volume of phosphate-buffered solution (PBS, 8 g/L NaCl, 0.2 g/L KCl, 0.2 g/L KH2PO4, 2.89 g/L Na2HPO4 · 12H2O, pH 7.4). After 24-h incubation, CCK-8 solution (10 μL/well) was added and incubated for an additional 4 h. The absorbance at 450 nm was monitored on a microplate reader (Epoch Etock, BioTek, USA).
Detection of NO production in macrophages
Peritoneal macrophages were seeded in two 24-well plates (2.5 × 105 cells/well) and cultured in RPMI-1640 containing 2 % FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, USA). Cells were incubated with peptides (0, 5, 10, and 20 μg/mL) in the presence or absence of LPS (100 ng/mL, from Escherichia coli 0111:B4, Sigma-Aldrich, USA). After 6-h incubation, cells in one plate were washed twice with ice-cold PBS and lysed by Trizol reagent (Life Tech, USA) for total RNA extraction. PrimeScript® Reverse Transcriptase Kit (Takara, Japan) was used to synthesize cDNA for qPCR to examine the transcription levels of inducible nitric oxide synthase (iNOS), which is necessary for NO production . After 24-h incubation, culture medium of each wells of another plate was harvested for nitrite detection, which indirectly reflected the NO production [39, 40]. Nitrite accumulation levels were determined by NO detection kit (Beyotime, China) following the kit instruction.
Cytokine production analysis by qPCR and ELISA
Peritoneal macrophages were cultured in a 24-well (2.5 × 105/well) plate in RPMI-1640 containing 2 % FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, USA). Cells were incubated with peptides (0, 5, 10, and 20 μg/mL) with or without 100 ng/mL LPS (from E. coli 0111:B4, Sigma-Aldrich, USA) for 6 h. After treatment, culture medium was collected for TNF-α, IL-1β and IL-6 quantification using enzyme-linked immunosorbent assay (ELISA) kits (Dakewei, China). Cells were washed with ice-cold PBS and lysed by Trizol reagent (Life Tech, USA). Total RNA was extracted for cDNA synthesis to quantify TNF-α, IL-1β and IL-6 transcription levels by qPCR as described in qPCR section.
Western blot analysis
Peritoneal macrophages were seeded in a 6-well (2 × 106/well) plate and cultured in RPMI-1640 supplemented with 2 % FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, USA). Cells were treated with different concentrations of cecropin-TY1 (0, 5, 10, and 20 μg/mL) in the presence or absence of 100 ng/mL LPS (from E. coli 0111:B4, Sigma-Aldrich, USA). After 30-min incubation, cells were washed twice with ice-cold PBS and lysed with RIPA lysis buffer (Beyotime, China) on ice for 30 min according to our previous method . Protein concentration was quantified by BCA Protein Assay Kit (Thermo, Germany). About 40 μg protein was separated on a 10 % SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane. The membrane was blocked by incubating with 5 % BSA (BD, USA) dissolved in Tris-buffered solution Tween-20 (TBST, 2.42 g/L Trisbase, 8 g/L NaCl, 0.1 % Tween 20, pH 7.6) for 2 h at room temperature. The membrane was then incubated with primary antibodies against ERK, phospho-ERK, p38, phospho-p38, JNK, phospho-JNK, phospho-IκBα, NF-κB p65 and phospho-NF-κB p65 (1:2000, Cell Signaling Technology, USA) and GAPDH/β-actin (1:5000, Santa Cruz Biotechnology, USA) overnight at 4 °C, respectively. After incubation, the immunoblot was washed three times with TBST for 5 min each time, and incubated with secondary antibody (1:5000, Cell Signaling Technology, USA) at room temperature for 1 h. Signals were measured by enhanced chemiluminescence kit in a dark room after washing three times with TBST for 10 min each time (TIANGEN, China).
Interaction between cecropin-TY1 and LPS
Fluorescein isothiocyanate (FITC)-conjugated LPS (1 μg/mL, Sigma-Aldrich, USA) was excited at 480 nm and monitored the changes in the emission of FITC-LPS at 515 nm in the incubation of different concentrations of peptides (0, 12.5, 25, 50, 100 μg/mL). Peptides were dissolved in 10 mM phosphate buffer at pH 6.0. The interactions between peptides and LPS were further assessed by a quantitative Chromogenic End-point Tachypleus amebocyte lysate (TAL) assay kit (Xiamen Houshiji, China) following the kit instruction. Briefly, different concentrations of peptides (0, 12.5, 25, 50, 100 μg/mL) were incubated with LPS (1 μg/mL, Sigma-Aldrich, USA) at 37 °C for 30 min. After incubation, 100 μL TAL solution was added to 100 μL LPS-peptide mixtures in a pyrogen-free tube and incubated at 37 °C for 10 min. Then, LPS-peptide mixtures were added with pre-warmed substrate solution for additional 6-min incubation at 37 °C. Lastly, the absorbance at 545 nm was measured on a microplate reader (Epoch Etock, BioTek, USA). The percentages of LPS-neutralizing activity were calculated.
Circular dichroism analysis
CD spectra were collected on a Jasco-810 spectropolarimeter (Jasco, Tokyo, Japan) with a 1-mm path-length cell at 25 °C and 0.2-nm intervals from 190 to 260 nm. Cecropin-TY1 was dissolved in H2O, TFE/water solution, SDS/water solution and LPS/water solution at the concentration of 0.2 mg/mL. The data from three scans were averaged and smoothed using the Jasco-810 software for each spectrum. CD data were expressed as the mean residue ellipticity (θ) in deg.cm2.dmol−1.
Structure modeling analysis
Three-dimensional (3D) structure of cecropin-TY1 was modeled by Easymodeller version 2.0. The solution NMR structures of papiliocin (59 % identity, PDB entry code 2LA2) from swallowtail butterfly of Papilio xuthus was selected as the template for homology modeling. The comparative 3D structure model of cecropin-TY1 was optimized using MODELLER and visualized using PYMOL software (http://www.pymol.org/) .
Statistical analysis was performed using GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA) and Stata 10.0 software (Stata Corporation, College Station, TX, USA). Data were presented as mean ± SEM, and compared using two-tailed equal variance Student’s t-test. *P < 0.05 and **P < 0.01 were considered as statistical significance.
Induction expression of cecropin-TY1 in the salivary glands of T. yao
Cecropin-TY1 is non-toxic to mouse macrophages
In order to evaluate the effects of cecropin-TY1 on mouse macrophages, the cell viability was determined in the presence of serial concentrations of peptides incubated with peritoneal macrophages and RAW264.7 cells. At the concentration as high as 200 μg/mL, cecropin-TY1 didn’t show any cytotoxicity against mouse peritoneal macrophages and RAW264.7 cells.
Inhibition of LPS-induced NO production
Inhibition of LPS-induced pro-inflammatory cytokine production
Inhibition of LPS-induced inflammatory signal pathways
Cecropin-TY1 interacts with LPS and neutralizes its activity
Secondary structures of cecropin-TY1
Secondary structural components of cecropin-TY1 in different solutions
Homology modeled structure analysis of cecropin-TY1
Horseflies have been used as anti-thrombosis materials for hundreds of years in China and some other eastern countries . Recently, a variety of physiologically active compounds have been identified in the salivary glands of horsefly, T. yao. They are (i) fibrin(ogen)olytic enzymes, (ii) Arg-Gly-Asp-motif containing proteins, (iii) a serine protease inhibitor, (iv) a serine protease, (v) a protease, (vi) a apyrase, (vii) vasodilator peptides, (viii) a peroxidase, (ix) two metallothioneins, (x) a hyaluronidase, (xi) immunosuppressive peptides and (xii) three family of AMPs [3, 5, 31–35]. All these active compounds can be generally classified into several groups such as antihemostatic factors, immunosuppressive factors, antimicrobial factors and allergens. But anti-inflammatory agents were poorly understood in the salivary glands of T. yao.
In the present work, a novel anti-inflammatory molecule (cecropin-TY1) was identified in the salivary glands of T. yao. The mRNA transcription levels of cecropin-TY1 were dramatically up-regulated upon Gram-negative bacteria challenge at different time course (Fig. 1), suggesting that cecropin-TY1 might involve in the inflammatory response induced by the release of LPS during Gram-negative bacteria infection. In LPS-stimulated mouse peritoneal macrophages, cecropin-TY1 showed strong anti-inflammatory effects by inhibiting the transcription of iNOS and pro-inflammatory cytokines including TNF-α, IL-1β and IL-6, as well as the production of NO and these pro-inflammatory cytokines (Fig. 2 & Fig. 3). Cecropin-TY1 showed no cytotoxicity toward mouse macrophages at the concentration as high as 200 μg/mL, suggesting that its anti-inflammatory effects in LPS-stimulated macrophages were not dependent on cytotoxicity. LPS, also called endotoxin, is the characteristic components of the cell wall of Gram-negative bacteria. LPS acts as a strong stimulator to activate the innate immune system of cells, which are components of the innate immunity of diverse organisms [41, 44, 45]. Release of LPS during Gram-negative bacteria infections triggers the production of higher concentration of systemic pro-inflammatory cytokines and NO, and results in sepsis. The excessive production of systemic pro-inflammatory cytokines and NO called cytokines storm, which are responsible for the pathophysiology of septic shock and other immune diseases . To address the anti-inflammatory mechanisms, the effects of cecropin-TY1 on LPS-triggered inflammatory pathways were investigated as illustrated in Fig. 4. Cecropin-TY1 significantly inhibited the activation (phosphorylation) of MAPKs and NF-κB signals induced by LPS in mouse peritoneal macrophage. These results suggested that cecropin-TY1 exerted its anti-inflammatory effects via blockade the activation (phosphorylation) of MAPKs and NF-κB signal pathways, which in turn suppressed iNOS and pro-inflammatory cytokines mRNA transcription, finally reduced NO and pro-inflammatory cytokines production.
A majority of anti-inflammatory peptides are also known to have high LPS-binding affinity and LPS-neutralizing activity such as papiliocin, cecropin A, LL-37, SMAP-29, and fowlicidin-1. These anti-inflammatory peptides can interact with LPS through binding to LPS and neutralizing LPS [29, 30, 43, 47, 48]. To clarify whether cecropin-TY1 could interact with LPS, we monitored the fluorescence change after the incubation of FITC-conjugated LPS with cecropin-TY1. As indicated in Fig. 5a, the addition of cecropin-TY1 caused a dose-dependent increase in FITC-conjugated LPS fluorescence, implying that the interaction between cecropin-TY1 and LPS resulted in the dissociation of large LPS aggregates into smaller sizes. The interaction of cecropin-TY1 with LPS was further evaluated by TAL assay, and cecropin-TY1 showed a reduced activation of LPS-induced TAL enzyme in a dose-dependent manner, suggesting that the addition of cecropin-TY1 caused the neutralization of LPS (Fig. 6b). The CD spectra indicated that cecropin-TY1 adopted a random coil structure in aqueous solution (H2O), but it converted to α-helical structures in the hydrophobic and/or negatively charged environments including TFE/water solution, LPS/water solution, and SDS/water solution (Fig. 6 and Table 2). The structural characterizations were further investigated by 3D structure homology modeling. It revealed that cecropin-TY1 adopted two α-helical regions in residues Leu3-Thr24 and Ile27-Leu38 linked by a hinge region (Leu25-Pro26) (Fig. 7). Like papiliocin (37 residues, helical regions in residues 3–21 and 25–36) from P. xuthus, cecropin A (37 residues, helical regions in residues 5–21 and 24–37) from H. cecropia and sarcotoxin-IA (39 residues, helical region in residues 3–23), α-helical confirmations are amphipathic structures which are responsible for the interaction of cecropins with LPS [30, 49, 50]. The same situation as cecropin-TY1, papiliocin also adopted a random coil structure in aqueous solution but converted to α-helical structure in the presence of LPS micelles, and the N-terminal amphiphilic α-helix (residues 3–18) of sarcotoxin-IA was formed upon interaction with micelles [30, 50]. In addition to amphiphilic interaction, electrostatic interaction also plays key roles in the interaction between such anti-inflammatory peptides and LPS micelles [30, 40]. There are 4 Lys residues and 2 Arg residues in the amphipathic helix in the N-terminus of cecropin-TY1, which may involve in the electrostatic interaction of cecropin-TY1 with anionic LPS (Table S2 and Fig. 7a). The electrostatic surface analyzed by 3D structure homology modeling revealed that its surface was partly positively charged (Fig. 7c). The C-terminal amidation of cecropin is important for its interaction with liposome . Cecropin-TY1 is also amidated at C-terminus, which may contribute to its interaction with LPS. Besides, aromatic residues like Trp2 in N-terminus are required for the interaction of cecropin peptides with LPS [52, 53]. The N-terminus of cecropin-TY1 localized Trp2 residue, which is possibly important for its interaction with LPS (Table S2). The presence of Gly and Pro residues in the hinge region is important for the flexibility of the hinge . Thereafter, the presence of Pro26 in the hinge between two helical regions may contribute to the flexibility and bending potential in the central part of cecropin-TY1 that allows the hydrophobic α-helix of the N-terminus and C-terminus to interact deeply with LPS. The mutation of these predicted structures/residues indicated that the derivatives were less active than cecropin-TY1 (Fig 3c, Fig 4g–i, Fig 6c), suggesting that these key structures/residues are crucial for the anti-inflammatory activity of cecropin-TY1.
Cecropin-TY1 was demonstrated to be a potent cecropin antimicrobial peptide in previous report . In vertebrates, insects, and plants, antimicrobial peptides play pivotal roles in contribution to host defense against infections by pathogenic microorganisms. In insects, the cecropin AMPs constitute a large family of cationic α-helical peptides with antimicrobial activities against Gram-positive bacteria, Gram-negative bacteria, fungi, parasites and HIV-1 virus . However, anti-inflammatory effects of cecropin family AMPs and their associated mechanisms are comparatively less investigated. As far as we know, the anti-inflammatory activities and the underlying mechanisms of insect-derived cecropin peptides have not been extensively investigated except papiliocin and cecropin A. Papiliocin and cecropin A are naturally occurred in either fat bodies or hemocytes and then release into the hemolymph [21, 30]. But no cecropins with anti-inflammatory effects were identified in salivary glands of any insects. The present work provide the first investigation of anti-inflammatory effects of cecropin antimicrobial peptide (cecropin-TY1) and its associated mechanisms of action from the salivary glands of T. yao.
Due to the special feeding pattern, horseflies have many chances to be infected with various microorganisms. Such defensive peptides in the salivary glands of horseflies can facilitate them to kill the microorganisms in blood meal, and protect them from pathogenic infection during feeding . It has been reported that female horseflies required large amounts of blood (up to 0.5 mL) for egg production. Approximately ten feeding episodes on a host are essential for horseflies to complete a blood meal, and one landing point lasts for 3–5 min [55, 56]. The multiple landings points, continued landing time and substantial amounts of blood meal of horseflies on a host suggest that they must possess diverse potent strategies to overcome the immune response of host . Not surprisingly, horseflies have evolved various countermeasures to affect the host immune response. So far, a total of 17 immunosuppressive peptides belonging to immunoregulin family have been identified and characterized from salivary glands of the horseflies T. yao, Hybomitra atriperoides and Tabanus pleskei, respectively [5, 18, 19]. These immunosuppressive peptides are highly conserved and composed of 30 or 35 amino acid residues. All the immuregulins exerted a decreased effect on IFN-γ and/or MCP-1 production but an increased effect on IL-10 production in LPS-stimulated mouse splenocytes. Among these immunosuppressive peptides, up to 12 members (tabimmuregulins 1–12) were identified from the salivary of T. yao . In addition to the immunosuppressive effects of immuregulins, perhaps the anti-inflammatory effect of cecropin-TY1 is another strategy of horsefly to affect the immune response of host. As a result of co-evolution, cecropin-TY1 is possibly a potent molecule that the horsefly of T. yao has developed to protect their host from pathogen infection and pathogen-induced inflammatory response during blood sucking.
Taken together, a potent anti-inflammatory peptide, cecropin-TY1, was identified from the horsefly salivary glands of T. yao. Cecropin-TY1 was demonstrated to interact with LPS and neutralize LPS, which in turn endowed cecropin-TY1 with strong anti-inflammatory effects in LPS-induced mouse macrophages without cytotoxicity. These properties make cecropin-TY1 a potential peptide candidate for the future treatment of sepsis and endotoxin shock caused by Gram-negative bacterial infections. The results also reveal a novel hint for understanding the ectoparasite-host interaction between horsefly and their host, and more work does need to be eventually done to show the true role of this peptide in the saliva in future.
This work was supported by Chinese National Natural Science Foundation (81402830, 81373380, 81360253, 81260258, 81560581), Jiangsu Province Natural Science Foundation (BK20140362, 14KJA310005, 14KJD350003), and Chinese Postdoctor Science Foundation (2015 M571815).
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- Arca B, Lombardo F, de Lara Capurro M, della Torre A, Dimopoulos G, James AA, et al. Trapping cDNAs encoding secreted proteins from the salivary glands of the malaria vector Anopheles gambiae. Proc Natl Acad Sci U S A. 1999;96:1516–21.PubMed CentralView ArticlePubMedGoogle Scholar
- Charlab R, Valenzuela JG, Rowton ED, Ribeiro JM. Toward an understanding of the biochemical and pharmacological complexity of the saliva of a hematophagous sand fly Lutzomyia longipalpis. Proc Natl Acad Sci U S A. 1999;96:15155–60.PubMed CentralView ArticlePubMedGoogle Scholar
- Ma D, Wang Y, Yang H, Wu J, An S, Gao L, et al. Anti-thrombosis repertoire of blood-feeding horsefly salivary glands. Mol Cell Proteomics. 2009;8:2071–9.PubMed CentralView ArticlePubMedGoogle Scholar
- Ribeiro JM. Characterization of a vasodilator from the salivary glands of the yellow fever mosquito Aedes aegypti. J Exp Biol. 1992;165:61–71.PubMedGoogle Scholar
- Xu X, Yang H, Ma D, Wu J, Wang Y, Song Y, et al. Toward an understanding of the molecular mechanism for successful blood feeding by coupling proteomics analysis with pharmacological testing of horsefly salivary glands. Mol Cell Proteomics. 2008;7:582–90.View ArticlePubMedGoogle Scholar
- Wang J, Bian G, Pan W, Feng T, Dai J. Molecular characterization of a defensin gene from a hard tick, Dermacentor silvarum. Parasit Vectors. 2015;8:25.PubMed CentralView ArticlePubMedGoogle Scholar
- Wei L, Mu L, Wang Y, Bian H, Li J, Lu Y, et al. Purification and characterization of a novel defensin from the salivary glands of the black fly, Simulium bannaense. Parasit Vectors. 2015;8:71.PubMed CentralView ArticlePubMedGoogle Scholar
- Nayduch D, Lee MB, Saski CA. Gene discovery and differential expression analysis of humoral immune response elements in female Culicoides sonorensis (Diptera: Ceratopogonidae). Parasit Vectors. 2014;7:388.PubMed CentralView ArticlePubMedGoogle Scholar
- Tonk M, Cabezas-Cruz A, Valdes JJ, Rego RO, Chrudimska T, Strnad M, et al. Defensins from the tick Ixodes scapularis are effective against phytopathogenic fungi and the human bacterial pathogen Listeria grayi. Parasit Vectors. 2014;7:554.PubMed CentralView ArticlePubMedGoogle Scholar
- Ferreira BR, Silva JS. Successive tick infestations selectively promote a T-helper 2 cytokine profile in mice. Immunology. 1999;96:434–9.PubMed CentralView ArticlePubMedGoogle Scholar
- Ribeiro JM, Weis JJ, Telford 3rd SR. Saliva of the tick Ixodes dammini inhibits neutrophil function. Exp Parasitol. 1990;70:382–8.View ArticlePubMedGoogle Scholar
- Urioste S, Hall LR, Telford 3rd SR, Titus RG. Saliva of the Lyme disease vector, Ixodes dammini, blocks cell activation by a nonprostaglandin E2-dependent mechanism. J Exp Med. 1994;180:1077–85.View ArticlePubMedGoogle Scholar
- Kopecky J, Kuthejlova M, Pechova J. Salivary gland extract from Ixodes ricinus ticks inhibits production of interferon-gamma by the upregulation of interleukin-10. Parasite Immunol. 1999;21:351–6.View ArticlePubMedGoogle Scholar
- Wu J, Wang Y, Liu H, Yang H, Ma D, Li J, et al. Two immunoregulatory peptides with antioxidant activity from tick salivary glands. J Biol Chem. 2010;285:16606–13.PubMed CentralView ArticlePubMedGoogle Scholar
- Carvalho-Costa TM, Mendes MT, da Silva MV, da Costa TA, Tiburcio MG, Anhe AC, et al. Immunosuppressive effects of Amblyomma cajennense tick saliva on murine bone marrow-derived dendritic cells. Parasit Vectors. 2015;8:22.PubMed CentralView ArticlePubMedGoogle Scholar
- Kern A, Collin E, Barthel C, Michel C, Jaulhac B, Boulanger N. Tick saliva represses innate immunity and cutaneous inflammation in a murine model of Lyme disease. Vector Borne Zoonotic Dis. 2011;11:1343–50.View ArticlePubMedGoogle Scholar
- Liu L, Dai J, Zhao YO, Narasimhan S, Yang Y, Zhang L, et al. Ixodes scapularis JAK-STAT pathway regulates tick antimicrobial peptides, thereby controlling the agent of human granulocytic anaplasmosis. J Infect Dis. 2012;206:1233–41.PubMed CentralView ArticlePubMedGoogle Scholar
- Yan X, Feng H, Yu H, Yang X, Liu J, Lai R. An immunoregulatory peptide from salivary glands of the horsefly, Hybomitra atriperoides. Dev Comp Immunol. 2008;32:1242–7.View ArticlePubMedGoogle Scholar
- Zhao R, Yu X, Yu H, Han W, Zhai L, Han J, et al. Immunoregulatory peptides from salivary glands of the horsefly, Tabanus pleskei. Comp Biochem Physiol B: Biochem Mol Biol. 2009;154:1–5.View ArticleGoogle Scholar
- Yi HY, Chowdhury M, Huang YD, Yu XQ. Insect antimicrobial peptides and their applications. Appl Microbiol Biotechnol. 2014;98:5807–22.PubMed CentralView ArticlePubMedGoogle Scholar
- Hultmark D, Steiner H, Rasmuson T, Boman HG. Insect immunity. Purification and properties of three inducible bactericidal proteins from hemolymph of immunized pupae of Hyalophora cecropia. Eur J Biochem. 1980;106:7–16.View ArticlePubMedGoogle Scholar
- Hultmark D, Engstrom A, Bennich H, Kapur R, Boman HG. Insect immunity: isolation and structure of cecropin D and four minor antibacterial components from Cecropia pupae. Eur J Biochem. 1982;127:207–17.View ArticlePubMedGoogle Scholar
- Moore AJ, Beazley WD, Bibby MC, Devine DA. Antimicrobial activity of cecropins. J Antimicrob Chemother. 1996;37:1077–89.View ArticlePubMedGoogle Scholar
- Cavallarin L, Andreu D, San Segundo B. Cecropin A-derived peptides are potent inhibitors of fungal plant pathogens. Mol Plant Microbe Interact. 1998;11:218–27.View ArticlePubMedGoogle Scholar
- DeLucca AJ, Bland JM, Jacks TJ, Grimm C, Cleveland TE, Walsh TJ. Fungicidal activity of cecropin A. Antimicrob Agents Chemother. 1997;41:481–3.PubMed CentralPubMedGoogle Scholar
- Arrowood MJ, Jaynes JM, Healey MC. In vitro activities of lytic peptides against the sporozoites of Cryptosporidium parvum. Antimicrob Agents Chemother. 1991;35:224–7.PubMed CentralView ArticlePubMedGoogle Scholar
- Barr SC, Rose D, Jaynes JM. Activity of lytic peptides against intracellular Trypanosoma cruzi amastigotes in vitro and parasitemias in mice. J Parasitol. 1995;81:974–8.View ArticlePubMedGoogle Scholar
- Wachinger M, Kleinschmidt A, Winder D, von Pechmann N, Ludvigsen A, Neumann M, et al. Antimicrobial peptides melittin and cecropin inhibit replication of human immunodeficiency virus 1 by suppressing viral gene expression. J Gen Virol. 1998;79(Pt 4):731–40.View ArticlePubMedGoogle Scholar
- Lee E, Shin A, Kim Y. Anti-inflammatory activities of cecropin A and its mechanism of action. Arch Insect Biochem Physiol. 2015;88:31–44.View ArticlePubMedGoogle Scholar
- Kim JK, Lee E, Shin S, Jeong KW, Lee JY, Bae SY, et al. Structure and function of papiliocin with antimicrobial and anti-inflammatory activities isolated from the swallowtail butterfly. Papilio xuthus J Biol Chem. 2011;286:41296–311.View ArticlePubMedGoogle Scholar
- Ma D, Gao L, An S, Song Y, Wu J, Xu X, et al. A horsefly saliva antigen 5-like protein containing RTS motif is an angiogenesis inhibitor. Toxicon. 2010;55:45–51.View ArticlePubMedGoogle Scholar
- An S, Ma D, Wei JF, Yang X, Yang HW, Yang H, et al. A novel allergen Tab y 1 with inhibitory activity of platelet aggregation from salivary glands of horseflies. Allergy. 2011;66:1420–7.View ArticlePubMedGoogle Scholar
- Ma D, Xu X, An S, Liu H, Yang X, Andersen JF, et al. A novel family of RGD-containing disintegrins (Tablysin-15) from the salivary gland of the horsefly Tabanus yao targets alphaIIbbeta3 or alphaVbeta3 and inhibits platelet aggregation and angiogenesis. Thromb Haemost. 2011;105:1032–45.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhang Z, Gao L, Shen C, Rong M, Yan X, Lai R. A potent anti-thrombosis peptide (vasotab TY) from horsefly salivary glands. Int J Biochem Cell Biol. 2014;54:83–8.View ArticlePubMedGoogle Scholar
- Ma D, Li Y, Dong J, An S, Wang Y, Liu C, et al. Purification and characterization of two new allergens from the salivary glands of the horsefly, Tabanus yao. Allergy. 2011;66:101–9.View ArticlePubMedGoogle Scholar
- Telleria EL, Sant'Anna MR, Alkurbi MO, Pitaluga AN, Dillon RJ, Traub-Cseko YM. Bacterial feeding, Leishmania infection and distinct infection routes induce differential defensin expression in Lutzomyia longipalpis. Parasit Vectors. 2013;6:12.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhang X, Goncalves R, Mosser DM. The isolation and characterization of murine macrophages. Curr Protoc Immunol. 2008;Chapter 14:Unit 14.1. doi:10.1002/0471142735.im1401s83.
- Rochette L, Lorin J, Zeller M, Guilland JC, Lorgis L, Cottin Y, et al. Nitric oxide synthase inhibition and oxidative stress in cardiovascular diseases: possible therapeutic targets? Pharmacol Ther. 2013;140:239–57.View ArticlePubMedGoogle Scholar
- Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem. 1982;126:131–8.View ArticlePubMedGoogle Scholar
- Wei L, Yang J, He X, Mo G, Hong J, Yan X, et al. Structure and function of a potent lipopolysaccharide-binding antimicrobial and anti-inflammatory peptide. J Med Chem. 2013;56:3546–56.View ArticlePubMedGoogle Scholar
- Alexander C, Rietschel ET. Bacterial lipopolysaccharides and innate immunity. J Endotoxin Res. 2001;7:167–202.PubMedGoogle Scholar
- Mueller M, Lindner B, Kusumoto S, Fukase K, Schromm AB, Seydel U. Aggregates are the biologically active units of endotoxin. J Biol Chem. 2004;279:26307–13.View ArticlePubMedGoogle Scholar
- Rosenfeld Y, Papo N, Shai Y. Endotoxin (lipopolysaccharide) neutralization by innate immunity host-defense peptides. Peptide properties and plausible modes of action. J Biol Chem. 2006;281:1636–43.View ArticlePubMedGoogle Scholar
- Cohen J. The immunopathogenesis of sepsis. Nature. 2002;420:885–91.View ArticlePubMedGoogle Scholar
- De Castro C, Parrilli M, Holst O, Molinaro A. Microbe-associated molecular patterns in innate immunity: Extraction and chemical analysis of gram-negative bacterial lipopolysaccharides. Methods Enzymol. 2010;480:89–115.View ArticlePubMedGoogle Scholar
- Papo N, Shai Y. A molecular mechanism for lipopolysaccharide protection of Gram-negative bacteria from antimicrobial peptides. J Biol Chem. 2005;280:10378–87.View ArticlePubMedGoogle Scholar
- Jerala R, Porro M. Endotoxin neutralizing peptides. Curr Top Med Chem. 2004;4:1173–84.View ArticlePubMedGoogle Scholar
- Rosenfeld Y, Shai Y. Lipopolysaccharide (Endotoxin)-host defense antibacterial peptides interactions: role in bacterial resistance and prevention of sepsis. Biochim Biophys Acta. 2006;1758:1513–22.View ArticlePubMedGoogle Scholar
- Holak TA, Engstrom A, Kraulis PJ, Lindeberg G, Bennich H, Jones TA, et al. The solution conformation of the antibacterial peptide cecropin A: a nuclear magnetic resonance and dynamical simulated annealing study. Biochemistry. 1988;27:7620–9.View ArticlePubMedGoogle Scholar
- Iwai H, Nakajima Y, Natori S, Arata Y, Shimada I. Solution conformation of an antibacterial peptide, sarcotoxin IA, as determined by 1H-NMR. Eur J Biochem. 1993;217:639–44.View ArticlePubMedGoogle Scholar
- Nakajima Y, Qu XM, Natori S. Interaction between liposomes and sarcotoxin IA, a potent antibacterial protein of Sarcophaga peregrina (flesh fly). J Biol Chem. 1987;262:1665–9.PubMedGoogle Scholar
- Okemoto K, Nakajima Y, Fujioka T, Natori S. Participation of two N-terminal residues in LPS-neutralizing activity of sarcotoxin IA. J Biochem. 2002;131:277–81.View ArticlePubMedGoogle Scholar
- Lee E, Kim JK, Jeon D, Jeong KW, Shin A, Kim Y. Functional roles of aromatic residues and helices of papiliocin in its antimicrobial and anti-inflammatory activies. Sci Rep. 2015;5:12048.PubMed CentralView ArticlePubMedGoogle Scholar
- Oh D, Shin SY, Lee S, Kang JH, Kim SD, Ryu PD, et al. Role of the hinge region and the tryptophan residue in the synthetic antimicrobial peptides, cecropin A(1–8)-magainin 2(1–12) and its analogues, on their antibiotic activities and structures. Biochemistry. 2000;39:11855–64.View ArticlePubMedGoogle Scholar
- Hollander AL, Wright RE. Impact of tabanids on cattle: blood meal size and preferred feeding sites. J Econ Entomol. 1980;73:431–3.View ArticlePubMedGoogle Scholar
- Kazimirova M, Sulanova M, Kozanek M, Takac P, Labuda M, Nuttall PA. Identification of anticoagulant activities in salivary gland extracts of four horsefly species (Diptera, tabanidae). Haemostasis. 2001;31:294–305.PubMedGoogle Scholar