The cDNA of amregulin encodes a precursor protein composed of 59 amino acid (aa) residues with the sequence of MKLHMLNMLNCLLLTVCDGHLHMHGNGATQVFKPRLVLKCPNAAQLIQPGKLQRQLLLQ. The mature peptide is composed of 40 residues with the sequence of HLHMHGNGATQVFKPRLVLKCPNAAQLIQPGKLQRQLLLQ (Figure 1).

To evaluate the immunosuppression capabilities of amregulin, its effects on IL-1, IL-8, IFN-γ and TNF-α secretions induced by LPS in rat splenocytes were tested. Four different concentrations (0, 2, 4 and 8 µg/mL) of amregulin were used to stimulate rat splenocytes, either alone or in concert with LPS. Treatment with LPS alone strongly induced the secretion of IL-1, IL-8, IFN-γ and TNF-α, whereas amregulin markedly inhibited these LPS-induced cytokine secretions in a dose-dependent manner (Figure 2A–D). The inhibitory effects of amregulin on LPS-induced cytokine secretions, especially IFN-γ, became more evident with increasing concentrations. At a dose of 8 µg/mL of amregulin, the LPS-induced secretion of IFN-γ was decreased nearly five-fold compared to that of the control (699 ± 8.5 pg/mL vs. 3720 ± 25.3 pg/mL). TNF-α, IL-1 and IL-8 were also significantly inhibited by amregulin, as compared to the LPS-treated control groups. A scrambled control peptide was also used as a control. At every test concentration, the control showed no effects on the secreted amounts of the different cytokines. These results collectively indicate that amregulin markedly inhibits the LPS-induced secretions of four different inflammatory cytokines in rat splenocytes in a concentration-dependent manner.

In light of the in vitro inhibitory effects of amregulin on host secretion of diverse inflammatory factors, as well as its free radical scavenging capabilities, we used an induced inflammation model (Freund’s complete adjuvant) to evaluate the potential anti-inflammatory ability of amregulin in vivo. As indicated in the Experimental Section, the basal footpad thickness of different male Kunming mice after treatment was selected as an indicator of inflammation. Along with the increments in treatment time, the anti-inflammatory effects of amregulin were enhanced with increasing concentrations, whereas the scrambled control peptide (8 mg/kg bodyweight) showed nearly no effects on inflammation. Significant anti-inflammatory effects were present in all mice treated with different concentrations (2, 4 or 8 mg/kg body weight) of amregulin; the paw inflammation was significantly decreased compared to that of the negative controls. The mice treated with 4 and 8 mg/kg body weight recovered to normal status after 14 days of administration. These results collectively indicate that amregulin inhibits hind paw inflammation in mice in a concentration-dependent manner (Figure 3).

It has been widely recognized that investigations of the interactions between blood-feeding arthropods and their hosts can uncover transmission mechanisms for vector-borne pathogens [34]. As blood-feeding arthropods, ticks may be an excellent animal model for host/parasite relationship studies. Hard ticks ingest blood as well as lymph and lysed tissues, whereas soft ticks feed only on blood. Hard ticks take a very long time (many days) to complete their blood-feeding meals. However, soft ticks need hours or less. Because their feedings last 1–2 weeks, hard ticks were selected as an extreme example to conduct research. To successfully obtain blood meals (and ultimately survive), hard ticks secrete immunosuppressant substances to defeat host immune defenses. Previous research has shown that tick saliva or salivary gland secretions have the potential to inhibit host immune responses and to enhance the transmission of tick-borne pathogens. More than 10 proteins from tick salivary glands have been found to exert immunomodulatory functions. One of them, Salp15, is a major immunomodulatory protein in Ixodes scapularis saliva. Salp15 inhibits T-cell receptor ligation-induced T-cell signaling by binding to CD4 [35,36]. Compared to Salp15, amregulin is a small peptide, which makes it a promising candidate for a medicine template. Another two immunoregulatory peptides (hyalomin-A and -B) identified from the salivary glands of Hyalomma asiaticum also show similar host inflammatory response suppression by modulating cytokine secretion and detoxifying reactive oxygen species [37]. Amregulin shares some similar characteristics with hyalomin-A and -B, but has different effects on LPS-induced cytokine secretion. Whereas hyalomin-A and -B strongly inhibit the secretion of IFN-γ, monocyte chemotactic protein-1 (MCP-1) and TNF-α, amregulin affects IL-1 and IL-8 in addition to IFN-γ and TNF-α. The different targets indicate the different roles of these salivary peptides.

The progress of tick feeding not only causes direct injury to the host’s skin, but also provides opportunities for disease-causing organisms to be ingested or expelled by the parasite. Thus, the host inflammatory and immune responses must be initiated. IL-1 was first discovered because of its strong pro-inflammatory effects. It induces a complex network of pro-inflammatory cytokines that initiate and regulate inflammatory responses via the expression of various integrins on leukocytes and endothelial cells. IL-8 induces chemotaxis of target cells, primarily neutrophils, as well as other granulocytes, causing them to migrate toward the site of infection. In humans, IL-8 is believed to play an important role in the pathogenesis of bronchiolitis [38], which is caused by viral infections. This finding indicates that IL-8 may participate in the passage of tick-borne viruses. Some ixodid species can transmit several (e.g., I. ricinus, A. variegatum) or many (I. uriae) such viruses [39]. Determining the molecular adaptations that allow these viruses to infect and replicate in both vector and host cells, as well as identifying the principal ecological determinants of virus survival are unsolved key challenges. The present work demonstrated that amregulin markedly inhibits LPS-induced secretion of cytokines, such as IL-1, IL-8, IFN-γ and TNF-α, in a concentration-dependent manner. TNF-α is one of the cytokines that makes up the acute phase reaction. Its primary role is regulating diverse immune cells, and increasing its local concentration causes the typical signs of inflammation: heat, swelling, redness and pain. Inhibition of TNF-α by amregulin suppresses the initial host response to these parasites and, thus, is beneficial for ticks taking their blood meals. Furthermore, amregulin also inhibits the only member of the type II class of interferons, IFN-γ, which inhibits viral replication directly and activates macrophages, enhancing their ability to kill intracellular organisms. Mycobacteria inhibit phagolysosome maturation, which may further affect macrophage function. IFN-γ secreted from Th1 helper cells activates macrophages in this pathological process [40]. The effects of amregulin on the dendritic cells were also tested, it showed that no affect both on the maturation and function of amregulin compare to the positive control LPS (Supplementary Materials). The inhibition of different pro-inflammatory cytokines in our study showed that amregulin may help ticks successfully obtain blood from their hosts.

Beyond its strong antioxidant activity, amregulin was found to scavenge free radicals in a concentration-dependent manner. It has been demonstrated that ABTS radicals react with phenols to form purple products with a broad absorbance of approximately 550 nm [41,42]. Of the essential amino acids, only tyrosine contains a phenol side chain, and mature amregulin contains only a single tyrosine residue. However, the strong free radical-scavenging ability and antioxidant activity of amregulin would be beneficial for the protection of tick-borne pathogens from host oxidant immune stress, as there is much evidence of overlap between the production of oxidants and the inflammatory response. During their blood meals, ticks have a difficult time coping with the toxic nature of the host inflammatory response, which involves several different oxidative molecules, including iron. Although necessary for a number of physiological processes, high amounts of iron are potentially toxic because iron can also participate in the formation of free radicals, which may cause cellular damage or even death. Tick feeding causes direct damage to the skin of the host, the release of iron and the formation of free radicals, all of which contribute to the host’s inflammatory response. Our results demonstrated that amregulin shows a strong ability to overcome potential damage from Fe³⁺ iron, indicating that amregulin may be an important peptide in the blood feeding process of A. variegatum.

Beyond showing the antioxidant and anti-inflammatory effects of amregulin at molecular and cellular levels, we also performed in vivo research by using an adjuvant-induced inflamed mouse paw model to demonstrate the potential of amregulin to suppress inflammation and noted concentration-dependent inhibition of inflammation. At Day 11, the group treated with 8 mg/kg amregulin differed significantly from the negative control group. At Day 14, the same group showed no difference in paw thickness from normal mice. The 4 mg/kg amregulin-treated group showed greatly decreased paw inflammation, whereas the scrambled control peptide showed virtually no effects on inflammation. Each amregulin-treated group showed that the administration of this peptide was able to substantially control the development of inflammation. This result demonstrates the biological significance of this immunosuppressant peptide derived from hard tick salivary glands and contributes to further understanding of the mechanism underlying hard tick immunosuppressive properties against host inflammatory responses.

Although the synthetic peptide may not be the same as the endogenous form because of posttranslational modifications, such as glycosylation or interactions with other proteins, or may even form a dimer via the cys residue, our findings may still provide some implications for understanding the mechanisms of successful tick hematophagy and tick-host interactions.

A cDNA (Accession BK007793.1) encoding the precursor of amregulin was obtained from the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA) [19]. The cDNA was cloned from the salivary glands of the hard tick A. variegatum. The protein precursor encoded by the cDNA was analyzed by SignalP 4.0 (The Center for Biological Sequence Analysis, Technical University of Denmark, Copenhagen, Denmark, 2016 [43]) to predict signal and mature peptides. Amregulin (HLHMHGNGATQVFKPRLVLKCPNAAQLIQPGKLQRQLLLQ) and a scrambled control peptide (VLVVAGNGATQVAAPRLVLKLPNAAQLIQIGELQRQLALG) were synthesized by GL Biochem Ltd. (Shanghai, China). Analysis with high performance liquid chromatography (HPLC) and mass spectrometry confirmed that its purity was higher than 98%. The peptide was dissolved in phosphate-buffered saline (PBS; 0.1 M, pH 6.0) solution at a concentration of 2 mg/mL and stored at −20 °C. This stock solution was used to prepare samples for the cytokine assays.

The wells of a 96-well plate were seeded with a suspension of splenocytes from Wistar rats in RPMI 1640 medium (Gibco Life Technologies, Grand island, NY, USA) supplemented with 5% fetal bovine serum and 100 U/mL penicillin and streptomycin. A total of 6 × 10⁶ cells (100 µL per well) were incubated at 37 °C and 3.5% CO₂. Different concentrations of the peptide were prepared by taking 20-µL samples of previously prepared peptide stock solution and dissolving them in different volumes of RPMI 1640 medium with LPS (final concentration, 2 µg/mL, Sigma, Saint louis, MO, USA). These different peptide solutions were then co-cultured with splenocytes. After a 48-h cultivation, culture supernatants were harvested and stored at −70 °C. All combinations were replicated three times. Cytokine assays for IL-1, IFN-γ, IL-8 and TNF-α were conducted by using antibody-sandwich ELISAs (Adlitteram Diagnostic Laboratories, Inc., Beijing, China) according to the manufacturer’s instructions.

Twenty microliters of vehicle (saline) or Freund’s complete adjuvant (Sigma) was administered to the plantar surface of the right hind paws of male Kunming mice (12/group). The basal footpad thickness of each mouse was measured with a vernier caliper at the beginning of the experiment. Amregulin (2, 4 or 8 mg/kg of mouse body weight) was administered to the rear femoral muscle on alternating days from Day 2 after injection of Freund's complete adjuvant (or saline as a control) to Day 14. The control groups of mice received the same volume of saline (vehicle). Right hind paw thickness was used as an indicator of inflammation and measured with a vernier caliper on Days 0, 1, 4, 8, 11, and 14. The experimental protocols were approved by the Animal Care and Use Committee at Nanjing Agricultural University (identification code: M.110/305; date of approval: 22 April 2015).

All data are presented as means ± SD and were analyzed by a one-way or two-way analysis of variance (ANOVA), followed by Duncan’s test or an unpaired t-test. p-values less than 0.05 were considered to be statistically significant, whereas p-values less than 0.01 were considered extremely statistically significant.

The Animal Care and Use Committee of Nanjing Agricultural University evaluated and approved all procedures used in this study. All in vivo experiments were performed according to sound practices of laboratory animal management.