Anti-inflammatory activities of Aedes aegypti cecropins and their protection against murine endotoxin shock

Background Mosquitoes are armed with physiologically active compounds to suppress the host immunity including host inflammatory reaction. However, the specific anti-inflammatory components in mosquitoes remain unknown. Results By searching for the immunomodulatory molecules from the mosquito Aedes aegypti (Diptera: Culicidae) at NCBI for anti-inflammatory function, five cecropins (for short in this study: AeaeCec1, 2, 3, 4 and 5) were selected. AeaeCec1-5 efficiently inhibited the expression of inducible nitric oxide synthase (iNOS), nitrite, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages and human peripheral blood mononuclear cells (PBMCs) with low toxicity to mammalian cells. Among the five analogues, AeaeCec5 had the strongest anti-inflammatory activity, and generated an additive effect with other AeaeCec peptides. In a mouse model of endotoxin shock, AeaeCec1-5 effectively reduced TNF-α, IL-1β and IL-6 expression in lungs, serum and peritoneal lavage and correspondingly reduced lung damage and edema, with AeaeCec5 showing the best protection. In mice infected with Escherichia coli or Pseudomonas aeruginosa, administration of AeaeCec5 reduced the production of TNF-α, IL-1β and IL-6 and correspondingly reduced lung tissue damage. These effects of Ae. aegypti AeaeCec1-5 were attributed to an efficient inhibition of the activation of mitogen-activated protein kinases (MAPKs) and transcriptional nuclear factor-κB (NF-κB) signaling pathways, as well as partial neutralization of LPS. Conclusions The current work characterized the specific anti-inflammatory agents in Ae. aegypti and provided AeaeCec5 as a potent anti-endotoxin peptide that could serve as the basis for the development of anti-inflammatory therapy. Electronic supplementary material The online version of this article (10.1186/s13071-018-3000-8) contains supplementary material, which is available to authorized users.


Background
Hematophagous arthropods like mosquito vectors are armed with a diverse group of active compounds with angiogenic, anticoagulant, vasodilatory and immunomodulatory properties, which facilitate adult female arthropods to finish blood meal acquisition and maintain pathogens before their transmission during blood-feeding [1][2][3][4][5][6][7][8][9][10][11]. An investigation of the crude salivary gland homogenates of Anopheles albimanus showed that the crude homogenates could oxidize noradrenalin and effectively inhibit vasoconstrictive pathways, thus promoting successful mosquito feeding [12]. Apyrases, the specific components in saliva of anopheline and culicine mosquitoes, were shown to inhibit ADP-induced platelet aggregation and limit local blood coagulation for successful blood-feeding [5,13]. Two novel neuropeptides named sialokinin-I and II, identified from the salivary gland of mosquito Ae. aegypti, shared amino acid homology with mammalian substance P and had smooth muscle contracting activity [11]. Mosquito tachykinins are highly conserved neuropeptides among anopheline mosquitoes (Anopheles gambiae) and culicine mosquitoes (Aedes triseriatus) [14].
Apart from the above anticoagulant compounds, mosquitoes are equipped with immunomodulatory molecules involved in interference with host immunity, such as antimicrobial peptides (AMPs), immunosuppressors, amongst others [15,16]. Mosquito AMPs were initially characterized for their antimicrobial activity in vitro [16][17][18]. Defensins, gambicins and cecropins comprise the three major AMP families in mosquitoes [15,16,19]. Mosquito defensins possess six conserved cysteine residues which form three intramolecular disulfide bonds linked in the Cys1-Cys4, Cys2-Cys5 and Cys3-Cys6 pattern [15]. Gambicins have eight cysteine residues which form four intramolecular disulfide bonds linked in the Cys1-Cys3, Cys2-Cys4, Cys5-Cys7 and Cys6-Cys8 pattern [16]. Cecropins are linear peptides lacking cysteine residues and form both amidated and nonamidated isoforms with a low molecular weight of about 4 kDa [20]. In vitro analyses of their antimicrobial activities indicated that they possessed different antimicrobial spectra. Mosquito defensins are primarily active to Gram-positive bacteria, while gambicins and cecropins are primarily active to both Gram-positive and Gram-negative bacteria [15]. Previous work indicated that Culex pipiens and Ae. aegypti feeding could modulate the cytokine production in C3H/HeJ mice [21]. Although the crude saliva or salivary gland extracts of mosquitoes showed serial immunomodulatory activities [8,22,23], active component composition in mosquitoes and its anti-inflammatory activity needs extensive study.
As described in our previous papers, cecropin-TY1 [25] and Sibacec [7] were characterized for the specific anti-inflammatory agents for the blood-feeding insects T. yao and S. bannaense. To identify the specific anti-inflammatory agents in the medically important blood-feeding mosquito vector Ae. aegypti, we searched the immune-related molecules from the genome sequences of Ae. aegypti at the National Center for Biotechnology Information (NCBI) [41,42], and a total of five cecropins have been identified. To understand whether Ae. aegypti cecropins possess anti-inflammatory activities like cecropin-TY1 and Sibacec derived from blood-feeding insects T. yao [25] and S. bannaense [7], respectively, these five cecropins were selected as anti-inflammatory peptide candidates. We assessed their cytotoxicity against mammalian cells. We examined the inhibitory effects of AeaeCec1-5 on iNOS, nitrite, TNF-α, IL-1β and IL-6 expression in murine peritoneal macrophages and human PBMCs. We also investigated the underlying mechanism by detecting whether Aeae-Cec1-5 suppressed MAPKs and NF-κB signaling activation, as well as neutralized LPS. We further explored the anti-inflammatory activities of AeaeCec1-5 in a murine endotoxic shock model and the protective roles of Aeae-Cec5 in Gram-negative bacteria-infected mice. Our data characterize the anti-inflammatory components in Ae. aegypti and indicate the value of the Ae. aegypti cecropins as a basis for future development of anti-inflammatory agents.

Methods
Mice, bacteria and peptides C57BL/6 mice (female, 18-20 g) were purchased from Shanghai Slac Animal Co. Inc. and housed in a pathogen-free facility. Gram-negative bacteria including E. coli (ATCC 25922) and P. aeruginosa (ATCC 27853) were cultured in Luria-Bertani broth at 37°C.
The amino acid sequence and related information of the five Ae. aegypti cecropins are listed in Additional file 1: Table S1. All of the peptides in this study were synthesized by GL Biochem (Shanghai) Ltd. (Shanghai, China). The synthetic peptides were subjected to an automated Edman degradation protein sequencer and MALDI-TOF mass spectrometry to confirm the accuracy of amino acid sequence and the purity (> 98%), respectively.

Mammalian cell culture
Mouse peritoneal macrophages from C57BL/6 mice were collected as previously described [25,43]. Cells were cultured in RPMI-1640 supplemented with antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin, Gibco, California, USA) and 10% fetal bovine serum. Human PBMCs were isolated according to a previous method [31]. Briefly, venous blood from healthy volunteers was collected into heparin-containing Vacutainer tubes (BD Biosciences, New Jersey, USA), diluted with an equal volume of PBS (pH 7.4) and separated by density gradient centrifugation using Lympholyte-poly cell separation media (Cedarlane, Ontario, Canada). Mononuclear cell layers were collected, washed and cultured in RPMI 1640 with 10% FBS, 2 mM L-glutamine, and 1 mM sodium pyruvate (Invitrogen, California, USA). Vero E6 cells were gifted by Dr Chunsheng Dong and cultured in DMEM supplemented with antibiotics and 10% FBS. All cells were cultured in a humidified incubator under 5% CO 2 at 37°C.

Cytotoxic assay
Cell Counting Kit-8 (CCK-8) was used to detect the cytotoxicity of AeaeCec1-5 against mammalian cells. In brief, mouse peritoneal macrophages, human PBMCs and Vero E6 cells were seeded in 96-well plates (2×10 4 cells/well, 100 μl/well). After cells adhered to culture plate, the culture medium was transferred with a two-fold dilution series of AeaeCec1-5 beginning with 50 μM (100 μl) prepared in serum-free RPMI-1640 for mouse macrophages and human PBMCs, and serum-free DMEM for Vero E6 cells. After incubation at 37°C for 24 h, CCK-8 solution (10 μl/well) was added for an additional 4 h incubation. The absorbance at 450 nm was recorded on a microplate reader (Epoch Etock, BioTek, Vermont, USA).

Nitric oxide assay
Mouse peritoneal macrophages or human PBMCs in 24-well plates (2.5×10 5 cells/well) were incubated at 37°C with LPS (100 ng/ml, from E. coli 0111:B4; Sigma-Aldrich, Missouri, USA) in the presence or absence of AeaeCec1-5 (5 μM). Cells were harvested for determination of the transcription level of iNOS by qPCR after incubation for 6 h [33,34]. After incubation for 24 h, the culture supernatant was collected, mixed with an equal volume of Griess reagent (Beyotime, Jiangsu, China), and incubated for 10 min at room temperature. NO production (nitrite accumulation) was measured on a microplate reader at 540 nm [26].

ELISA assay of cytokines
The expression levels of pro-inflammatory cytokines were evaluated after the addition of 100 ng/ml of LPS (from E. coli 0111:B4; Sigma-Aldrich) to mouse peritoneal macrophages or human PBMCs in 24-well plates (2.5×10 5 cells/ well) in the presence or absence of AeaeCec1-5 (5 μM). The cells were incubated for 6 h at 37°C. TNF-α, IL-1β and IL-6 levels in the supernatant were measured using mouse or human cytokine ELISA kits (eBioscience, California, USA) according to the instructions.

qPCR
Total RNA from cells and tissues were extracted using Trizol reagent (Life Tech, California, USA). cDNA was synthesized using PrimeScript® reverse transcriptase kit (Takara, Dalian, China). SYBR green master mix (Takara) was used for a two-step qPCR assay on a Realplex Mastercycler real-time PCR system (Eppendorf, Hamburg, Germany) according to the manufacturer's instructions. Transcription levels of target genes were normalized to GAPDH and calculated by the ΔΔCt method. The accuracy of qPCR results was verified by melting curve analysis. Primers used in the qPCR assay are listed in Additional file 1: Table S2.

LPS neutralization assay
LPS neutralization properties of peptides were measured using a ToxinSensor™ chromogenic LAL endotoxin assay kit (GenScript, Nanjing, China) according to the kit instructions [34]. Briefly, a two-fold dilution series of peptides (20 μl/well) and an equal volume of E. coli LPS solution (5 endotoxin units/ml) provided by the kit were mixed in a pyrogen-free 96-well plate. LAL water (20 μl/ well) and an equal volume of LPS were mixed as a blank control. After incubation at 37°C for 30 min, LAL reagent (20 μl/well) was added and incubated at 37°C for 10 min. Then chromogenic substrate (40 μl/well) reagent was added and incubated at 37°C for another 6 min. Finally, color stabilizers 1, 2 and 3 (40 μl/well) were added and the absorbance at 545 nm was monitored on a microplate reader (Epoch Etock, BioTek). LPS-neutralizing activity was calculated as (A blank -A peptide )/A blank × 100%.
In vivo anti-inflammatory effect in LPS-challenged mice C57BL/6 mice (female, 18-20 g, n=10) were intraperitoneally injected with 10 mg/kg LPS (from E. coli 0111:B4; Sigma-Aldrich). Individual AeaeCec1-5 (10 mg/kg), co-administration of AeaeCec1-5 (2 mg/kg each) or an equal volume of vehicle (PBS) was intraperitoneally injected into the mice 30 min after LPS injection. Mice were sacrificed 4 h after treatment. Blood, peritoneal lavage and lungs were collected. For lung edema evaluation, the wet weight was taken, and the lungs were dried in an oven at 80°C for 48 h until achieving stable dry weight [44].

Histopathological assay
Tissues were collected and fixed in 10% formalin solution for 24 h. After dehydration by an increasing concentration of alcohol, tissues were embedded in paraffin and sectioned into a thickness of 5 μm section using a Histocut (Leica, Solms, Germany). Sections were stained with hematoxylin and eosin (H&E), and observed by light microscopy (Nikon Eclipse TE2000-S, Tokyo, Japan).

Statistical analysis
GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA) was used to perform statistical analysis. Data are presented as mean ± SEM, and compared using two-tailed equal variance Student's t-test. Values corresponding to P<0.05 were considered statistically significant.

AeaeCec1-5 showed low toxicity against mammalian cells
To determine the cytotoxicity of AeaeCec1-5 against mammalian cells, Vero E6 cells, mouse peritoneal macrophages and human PBMCs were exposed to different peptide concentrations. As shown in Additional file 2: Figure S1, AeaeCec1-4 incubation resulted in 100% cell viability at concentration up to 50 μM. At a concentration below 6.25 μM, AeaeCec5 exhibited no cytotoxicity toward all tested cells, and showed minimal cytotoxicity at 12.5 μM towards mouse macrophages (9.5%) and PBMCs (8.9%). At a concentration of 50 μM, AeaeCec5 caused 28.9 and 17.5% cell death toward mouse macrophages and human PBMCs, respectively, but no cytoxicity against Vero E6 cells. In addition, the co-treatment of AeaeCec1-5 (1 μM each) did not induce any cell death toward all tested cells (data not shown). Thus, AeaeCec1-5 showed efficiently anti-inflammatory activities with low toxicity against mammalian cells.

AeaeCec1-5 inhibited the activation of LPS-induced MAPKs and NF-κB signaling pathways
Recognition of LPS by Toll like receptor 4 (TLR 4) initiates signaling pathways involving activation of MAPKs and NF-κB, which play critical roles in the upregulation of pro-inflammatory factors. To understand the anti-inflammatory mechanisms of AeaeCec1-5, their effects on LPS-induced activation of MAPKs and NF-κB signaling pathways were detected. In mouse peritoneal macrophages, we detected significantly enhanced expression levels of phospho-JNK, ERK, p38 and NF-κB p65 after LPS (100 ng/ml) stimulation, which was selectively decreased by the treatment of AeaeCec1-5 with different efficiency. This inhibitory effect occurred in a dose-dependent manner (Fig. 3). At a concentration of 5 μM, AeaeCec1 only moderately reduced phosphorylation of NF-κB p65 by 56.9% (t (4) = 6.29, P = 0.0033, Fig. 3a, f).
Consistent with these findings, lungs following E. coli 0111:B4 LPS-treated mice were significantly damaged with multi-inflammatory infiltration foci, but AeaeCec5 administration significantly reversed this inflammatory damage (Fig. 5d), and the AeaeCec1-5 mixture treatment further enhanced the protection. In addition to that, AeaeCec5 and the AeaeCec1-5 mixture also significantly reduced the wet/dry lung weight ratio to a level comparable to that seen in the naïve mice (AeaeCec5: t (8) = 2.538, P = 0.0348; AeaeCec1-5: t (8) = 2.846, P = 0.0216; Fig. 5e), indicating protection against LPS-induced lung edema. These data indicate that AeaeCec5 is a strong inhibitor of endotoxin activity in C57BL/6 mice.
AeaeCec5 protected mice against E. coli and P. aeruginosa infection To further test the protective activity of AeaeCec5 in Gram-negative bacteria infection models, mice were intraperitoneally infected with two major infective bacteria in clinical infectious diseases, E. coli or P. aeruginosa (2×10 7 CFUs/mouse), and then treated with AeaeCec5 (10 mg/kg). After treatment for 18 h, a significant decrease in the levels of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) in the lung, serum, and peritoneal lavage was observed as compared to the levels of PBS-treated mice (Fig. 6a-c). Consistently, the pathology and the inflammatory injury in the lung were also significantly improved (Fig. 6d). The results indicated that AeaeCec5 offered anti-inflammatoy protection against Gram-negative bacterial infection.

Discussion
Mosquito vectors have been proven to produce a variety of physiologically active compounds that suppress their host's hemostatic system and immune response to successfully get a blood meal [15,25]. These physiologically active factors can be distinguished into two major classes, including anti-hemostatic and immunoregulatory substances [15,24]. So far, much work has been done to study anti-hemostatic and immunoregulatory substances from mosquito vectors. This includes work on: (i) apyrases, which inhibited ADP-induced platelet aggregation and limited local blood coagulation for successful blood-feeding on their host [5,13]; (ii) Sialokinin-I and Sialokinin-II, two mosquito neuropeptides of tachykinins, which had smooth muscle contracting activity [11]; (iii) salivary gland homogenates of A. albimanus which oxidized noradrenalin and effectively inhibited vasoconstrictive pathways [12]; (iv) AMPs, defensins, gambicins and cecropins comprising the three main antimicrobial peptide families, which were initially identified for their antimicrobial activity in vitro [15][16][17][18][19]38]; (v) immunosuppressors, three pentapeptides derived from the C-terminal fragments of tachykinins with the general structure Phe-X-Gly-Leu-Met-NH2 (with Tyr, Val and Ile in X position) which showed potent immunosuppressive effects [45]; (vi) crude saliva of Culex pipiens and Ae. aegypti which modulated the cytokine production in C3H/HeJ mice [21]; (vii) cured saliva of Ae. aegypti which modulated murine lymphocyte function [46]; (viii) crude salivary gland extracts of Ae. aegypti which inhibited the production of TNF-α in tumour cell-stimulated mast cells [23]; and (ix) crude salivary gland extract of Ae. aegypti which modulated murine cellular and host immune responses [8,22]. Collectively, several specific anticoagulant compounds have been well characterized from mosquitoes, and the immunomodulatory effects of crude saliva or salivary gland extract were described in mosquitoes. However, the specific anti-inflammatory compound from mosquito vectors is currently unknown.  [25] at 37°C. The LPS-neutralization activity was evaluated using an endotoxin quantification kit. Data are presented as mean ± SEM from three independent experiments. *P < 0.05, **P < 0.01 In this study, we characterized AeaeCec1-5 as anti-inflammatory AMPs in Ae. aegypti. We confirmed the anti-inflammatory activities of AeaeCec1-5 against LPS-induced NO and pro-inflammatory cytokines production and the co-treatment of AeaeCec1-5 generated the best anti-inflammatory effects. Among the five cecropins, AeaeCec5 displayed the strongest anti-inflammatory activity induced by LPS in vitro and in vivo with low toxicity. Treatment with AeaeCec5 in E. coli or P. aeruginosa infected mice also decreased the pro-inflammatory cytokines production and lung damage. AeaeCec1-5 might modulate both cellular and host inflammatory response by reducing the production of inflammatory mediators from monocytes/macrophages at the bite site. A reduction of inflammatory mediator release may be beneficial for the feeding of Ae. aegypti by reducing immediate inflammatory response at the feeding site, suggesting The arrows showed inflammatory infiltration. e The ratio of wet weight to dry weight of the lungs was evaluated. Data are presented as mean ± SEM from three independent experiments. *P < 0.05, **P < 0.01 that cecropins of Ae. aegypti might be the specific components that suppressed the inflammatory response of the mammalian host during feeding.
Mosquitoes have many chances to imbibe various microorganisms during feeding. Cecropins in mosquitoes were initially identified as antimicrobial compounds in vitro [15,17] which may facilitate the killing of microorganisms in blood meals, and protect them from pathogenic infection during feeding. Herein, we characterized the anti-inflammatory effects of mosquito cecropins (AeaeCec1-5) in vivo. Of the five Ae. aegypti cecropins, AeaeCec5 showed the best protective effect against LPS-stimulated inflammatory response both in vitro and in vivo. LPS, also known as endotoxin, comprises the main components of the cell wall of Gram-negative bacteria. Gram-negative bacteria infection directly leads to the release of LPS that triggers the excessive production of systemic pro-inflammatory cytokines and NO, which is called cytokine storm, and ultimately results in sepsis [47,48]. AeaeCec5 was chosen to investigate the Fig. 6 AeaeCec5 offered anti-inflammatory protection against E. coli and P. aeruginosa infection. Mice were i.p. infected with E. coli or P. aeruginosa (2 × 10 7 CFUs/mouse), and treated with one dose of AeaeCec5 (10 mg/kg) or PBS 30 min post-infection. a-c 18 h after treatment, all the mice were analyzed for TNF-α, IL-1β and IL-6 production in the lung (a), blood (b) and peritoneal lavage (c) by ELISA assay. Data are presented as mean ± SEM from three independent experiments. d Representative H&E staining of the lung sections 18 h post-infection. The arrows showed typical injury sites. Scale-bars: 100 μm, *P < 0.05, **P < 0.01 anti-inflammatory effects caused by Gram-positive bacteria infection in mice. The in vivo anti-inflammatory analyses of AeaeCec5 induced by bacterial infection illustrated that AeaeCec5 significantly ameliorated lung injury and pro-inflammatory cytokines production in mice after infected with Gram-negative bacteria E. coli and P. aeruginosa, which are two major infective bacteria in clinical infectious diseases (Fig.  6). Besides anti-inflammatory activity, AeaeCec5 also showed strong antimicrobial activity against the four tested E. coli strains in vitro with minimal inhibitory concentrations ranging from 1.17 to 2.34 μg/ml (Additional file 1: Table S3), and bacterial load in peritoneal lavage of E. coli-infected mice was significantly reduced (appropriately 85.6%) after treatment of AeaeCec5 (10 mg/kg) as compared to PBS treatment (Additional file 3: Figure S2). As described above, such defensive peptides from mosquito vectors were initially identified for their antimicrobial activity in vitro [15,17]. In addition to antimicrobial activity, perhaps anti-inflammatory activity of these molecules like AeaeCec5 is another strategy of Ae. aegypti to suppress the inflammatory response of mammalian host. As a result of co-evolution, cecropins are possibly the specific components that Ae. aegypti has developed to protect mammalian hosts from pathogen infection and pathogen-induced inflammatory response at the bite site during feeding. In our previous work, two cecropins from blood-feeding insects, the horsefly and black fly, showed anti-inflammatory properties similar to cecropins from the blood-feeding arthropod Ae. aegypti [7,25], implying that cecropins might be common anti-inflammatory agents for blood-feeding.
As described previously, LPS is an agonist that target TLR 4 and subsequently activate the inflammatory pathways [26,27]. The effects of AeaeCec1-5 on LPS-activated inflammatory pathways were investigated to address the anti-inflammatory mechanisms (Fig. 3). In general, although AeaeCec1-5 showed different inhibitory efficiency on the activation of inflammatory signal pathways, AeaeCec1-5 selectively inhibited the activation (phosphorylation) of MAPKs and/or NF-κB signals in LPS-stimulated mouse macrophages. The results implied that cecropins of Ae. aegypti exerted their anti-inflammatory activities by blocking the activation (phosphorylation) of MAPKs and NF-κB signaling pathways, which in turn ameliorated the inflammatory response induced by LPS both in vitro and in vivo. In addition, Aeae-Cec1-5 showed different effects on the inhibition of the phosphorylation of ERK, JNK, p38 and NF-κB p65, suggesting that this may be the molecular basis for the best anti-inflammatory effects of the co-treatment of AeaeCec1-5. To understand the potential interactions between AeaeCec peptides, the percent of inhibition of mixtures of two, three, four, and five peptides in LPS-stimulated NO production in mouse peritoneal macrophages were investigated systematically. Among these different sets of mixture of peptides, the best protective effect in mixtures of two, three, four, or five peptides was only generated in the presence of AeaeCec5 (Additional file 1: Table  S4). The interactive effects between any two peptides of AeaeCec1-5 on inhibition of NO production in LPS-stimulated mouse peritoneal macrophages were assayed using chequerboard assays. The results indicated that additive effects were observed in mixtures of AeaeCec1 and AeaeCec5, AeaeCec2 and AeaeCec5, AeaeCec3 and AeaeCec5, and AeaeCec4 and AeaeCec5 (Additional file 1: Table S5). Insect-derived cecropins adopt a random coil structure in aqueous solution but convert to an α-helical structure in the hydrophobic environments by forming an N-terminal helix region and a C-terminal helix region linked by hinge [35]. Upon the interaction with LPS micelles (hydrophobic environments), AeaeCec1-5 possibly possess an N-terminal amphipathic α-helix and a more hydrophobic C-terminal α-helix connected by a hinge region as AeaeCec4 (Aedesin) detected by nuclear magnetic resonance spectroscopy. These helical structures in such peptides are critical for their neutralization of endotixion (LPS) and anti-inflammatory activities [25,27]. In addition to the helical structures, AeaeCec5 has a tryptophan residue (Trp2) in the N-terminal helix, which is missing from Aeae Cec1, 2, 3 and 4. As described previously, aromatic residues like Trp in the N-terminus are required for the interaction of cecropin peptides with LPS [25,49], and AeaeCec5 did exhibit the strongest LPS-neutralizing activity and anti-inflammatory activities among these five AeaeCec peptides. Collectively, we suggest that these structural properties may contribute to the additive effects generated by AeaeCec5 with other AeaeCecs. To test this hypothesis, we substituted theTrp2 with Ala2, and the additive effects generated by AeaeCec5(2W→2A) with other AeaeCec peptides were absent (Additional file 1: Table S6). Additionally, another anti-inflammatory mechanism was addressed by evaluation of the LPS-neutralization activity of cecropins of Ae. aegypti. As shown in Fig. 4, AeaeCec1-5 exhibited LPS-neutralization activity, suggesting that LPS-neutralization activity of Ae. aegypti cecropins at least partly comprised their anti-inflammatory mechanism. Some anti-inflammatory peptides, including cathelididins and cecropins, were also known to have LPS-neutralization activity [7, 25-27, 33, 34, 50]. Such LPS-neutralization activity comprised a critical mechanism of their anti-inflammatory effects.