Blood samples were obtained from healthy, non-pregnant adults. This procedure was approved by the Human Research Review Board (HRRB number: HS-08-135) at the University of California-Riverside. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC number: A-20110030BE) at the University of California-Riverside. We used C57BL/6 mice at 6–10 weeks of age purchased from Jackson Laboratories. Experimentation with A. phagocytophilum (HZ strain) was approved by the Biological Use Authorization Committee (BUA number: 20120020) at the University of California-Riverside. A. phagocytophilum was grown in HL-60 cells (ATCC CCL-240). HL-60 cells were maintained in Iscove’s Modified Dulbecco’s Media (IMDM) with L-glutamine and hydroxyethyl piperazineethanesulfonic acid (HEPES) (Thermo Scientific), 20% heat-inactivated fetal bovine serum (FBS) (Sigma) in 5% CO₂ and humidified air at 37°C, as previously described [33].

Lipopolysaccharide (LPS), Pam3CSK4, Zymosan, Porphyromonas gingivalis (PG)-LPS and muramyl dipeptide (MDP) were obtained from Invivogen. DOTAP was obtained from Roche. Human recombinant TNF-α was purchased from R&D Systems.

The generation of bone marrow-derived macrophages (BMDMs) and tick saliva has been previously described [33-35]. Briefly, mouse femurs were flushed and bone marrow cells were differentiated in complete Dulbecco’s Modified Eagle Medium (DMEM) (Fisher) supplemented with 30% L929 cell-conditioned media, 10% FBS and 1% PSA (100 U/mL penicillin, 100 mg/ml streptomycin, and 0.25μg/ml amphotericin) (Fisher). Cells were cultured at 37°C in a 5% CO₂ tissue culture incubator for 5–6 days, with media changed on day 3. Human peripheral blood mononuclear cells (PBMCs) were isolated by using the Polymorphprep protocol (Axis Shield).

We collected I. scapularis saliva 4–5 days after feeding because studies suggest that transmission of A. phagocytophilum initiates slowly between 24 and 48 hours and is enhanced during rapid feeding to repletion around 72 h–96 h post tick attachment [36-38]. Therefore, saliva from I. scapularis would reflect actual conditions during A. phagocytophilum transmission at the vector-host interface. In addition, I. scapularis saliva collection at 24–48 hours is technically very challenging. The alternative would be using salivary glands. However, salivary glands bring a technical artifact to the system because this organ in ticks is rich in intracellular proteins and other immune effectors such as nucleotides, which may skew cytokine response in immune cells. To isolate vector saliva, I. scapularis ticks were allowed to feed on New Zealand white rabbits. A restraining collar was placed around the neck of each rabbit, and their ears were covered prior to tick exposure. Ticks were permitted to engorge for 4–5 days on the ear of a rabbit. Upon harvesting, ticks were rinsed in distilled water and were immediately fixed to glass slides with double-sided tape. A sterile glass micropipette was placed around the hypostome to collect saliva. Salivation was induced by the application of pilocarpine to the scutum of the tick. Saliva was pooled and stored at −80°C for use.

BMDMs from C57BL/6 mice were stimulated with the TLR agonists LPS (500 ng/ml), Pam3CSK4 (1 μg/ml), Zymosan (10 μg/ml) and PG-LPS (500 ng/ml), the Nod2 stimulant MDP (10 μg/ml) or A. phagocytophilum (multiplicity of infection (MOI) 10 and 50) at indicated dilutions of tick saliva. Human PBMCs were purified by using the Polymorphprep protocol (Axis Shield), plated for 2 hours and incubated with tick saliva for 30 minutes followed by 4 hours of stimulation with 100 ng/ml of Tumor Necrosis Factor (TNF)-α. Pro-inflammatory cytokines such as TNF-α, interleukin (IL)-12p40, IL-6 and IL-1β or the chemokine IL-8 (CXCL8) were measured by ELISA.

Mouse TNF-α, IL-1β, IL-6 and human IL-8 were measured with the BD OptEIA Set from BD Biosciences. Mouse IL-12p40 was measured with capture and detection antibodies from eBiosciences. For the ELISA assays, wells were coated with recommended capture antibody dilutions in freshly prepared coating buffer (0.1 M sodium carbonate, pH 9.5). Plates were sealed and incubated overnight at 4°C followed by aspiration. Wells were then washed 3 times with ≥ 300 μL/well of freshly prepared wash buffer (phosphate buffered saline (PBS) with 0.05% Tween-20). After the last wash, plates were inverted and blotted on absorbent paper to remove any residual buffer. Wells were then blocked with ≥ 200 μL/well of assay diluent (PBS with 10% FBS, pH 7.0) and incubated at room temperature for 1 hour. Plates were washed 3 times with wash buffer. Then, standards and sample dilutions were prepared in assay diluent, as recommended (BD Biosciences). 100 μL of samples and standards were pipetted into the wells, incubated for 2 hours at room temperature followed by 5 washes. 100 μL of detection antibodies were diluted in assay diluent and added to each well. Plates were sealed and incubated for 1 hour at room temperature. Plates were washed 5 times. Then, 100 μL of enzyme reagent (BD OptEIA Set from BD Biosciences) were diluted in assay diluent, pipetted into each well and incubated for 30 minutes at room temperature. Wells were aspirated and washed 7 times with wash buffer. 100 μL of substrate solution (BD OptEIA Set from BD Biosciences) were added to each well and incubated for 30 minutes (without plate sealer) at room temperature in the dark. 50 μL of 2 N H₂SO₄ was added to each well. Absorbance was read in the ELISA plate reader (Bio-Rad) at 450 nm within 30 minutes. Background was corrected by reading the subtract absorbance at 570 nm.

Cell death was assayed by measuring lactate dehydrogenase (LDH), as recommended by the manufacturer (Takara). Briefly, 100 μL of each sample was placed into a well. Then, a catalyst solution (Takara) was added to the samples and controls. Samples were incubated for 10–30 minutes, at room temperature, protected from light. Reactions were stopped at the end of the incubation period by adding 50 μL of 1 N HCl. Absorbance was measured at 490 nm. The iMark microplate absorbance reader (Bio-Rad) was used according to the manufacturer’s instruction.

Data were expressed as means ± standard errors of the means (SEM). The following parametric analyses were used: unpaired Student’s t test (two-group comparisons); one-way analysis of variance (ANOVA) (comparisons of three or more groups); Bonferroni post hoc multiple-comparison tests were used following ANOVA. All statistical calculations were performed using GraphPad Prism version 5.04. Graphs were made using GraphPad Prism version 5.04. P < 0.05 was considered statistically significant.

Tick saliva has immunomodulatory properties [1,2]. To determine whether tick saliva inhibits macrophage function, we first stimulated mouse BMDMs with LPS in the presence or absence of different dilutions of I. scapularis saliva. Cytokine levels were not altered during BMDM stimulation with tick saliva alone, suggesting that the saliva does not carry any contaminants, pathogen-associated molecular (PAMPs) or danger-associated molecular (DAMPs) patterns. As expected, LPS induced high levels of cytokine secretion in murine BMDMs (Figures 1 and 2). However, I. scapularis saliva inhibited secretion of both TNF-α and IL-12p40 by BMDMs after stimulation with LPS (Figure 1). This effect was more pronounced for TNF-α, as a tick saliva dilution of 1:10000 (v/v) still affected cytokine secretion by BMDMs during LPS stimulation (Figure 1A). A reduction in IL-12p40 secretion by macrophages after LPS stimulation was only observed for a tick saliva dilution of 1:1000 (v/v) and below (Figure 1B).

We then stimulated mouse BMDMs with a wide range of TLR ligands and measured IL-6 and IL-12p40 secretion in the presence of I. scapularis saliva. Tick saliva 1:500 (v/v) inhibited IL-6 and IL-12p40 secretion by BMDMs when stimulated with TLR agonists, such as LPS, Pam3CSK4, Zymosan and PG-LPS (Figure 2). Next, we stimulated BMDMs with Nod1 and Nod2 agonists to determine whether the effect of I. scapularis saliva on cytokine secretion was restricted to TLRs. Nod1 and Nod2 are considered cytosolic receptors and are part of the Nod-like receptor (NLR) protein family [39]. The Nod1 and Nod2 agonists iE-DAP and MDP did not induce cytokine production in BMDMs during extracellular stimulation (Figure 3; data not shown). However, transfection of MDP to the cytosol using the cationic lipid DOTAP led to secretion of both IL-6 and IL-12p40 by BMDMs (Figure 3). Further, I. scapularis saliva inhibited IL-6 and IL-12p40 secretion mediated by the transfected Nod2 agonist MDP at the saliva dilution of 1:500 (v/v). Taken together, we report that I. scapularis saliva mitigates cytokine secretion by BMDMs during extracellular and cytosolic stimulation of TLR and NLR pathways.

We then tested whether the effect observed for I. scapularis during cytokine secretion was restricted to murine BMDMs. We isolated human PBMCs from the blood of healthy and immunocompetent adults and stimulated these immune cells with the pro-inflammatory cytokine TNF-α. TNF-α has been extensively used to stimulate cytokine secretion in immune cells [40]. We included human PBMCs in our studies for two reasons. First, we would like to show that the inhibitory aspect of tick saliva is not only restricted to murine immune cells. To the contrary, it can also occur during stimulation of human immune cells. Second, PBMCs are a heterogenous group of immune cells. Therefore, we reasoned that PBMCs would be ideal to demonstrate the inhibitory cytokine capacity of tick saliva in a mixed group of immune cells. The chemokine IL-8 (CXCL-8) was used as a read-out in our analysis. PBMCs produced large amounts of IL-8 when stimulated with TNF-α (Figure 4). Similar to our findings observed in murine BMDMs, IL-8 (CXCL8) secretion by PBMCs stimulated with recombinant human TNF-α was diminished in the presence of tick saliva (Figure 4). Overall, our results show that I. scapularis saliva is able to mitigate cytokine and chemokine secretion by both murine and human immune cells.

To determine whether the effect of tick saliva was restricted to TLR and NLR ligands, we stimulated BMDMs with the I. scapularis rickettsial pathogen A. phagocytophilum. A. phagocytophilum induced the secretion of large amounts of cytokines by murine macrophages (Figures 5 and 6A-B). Nonetheless, tick saliva was also efficient in reducing cytokines by BMDMs, such as IL-6, IL-12p40 and TNF-α during A. phagocytophilum stimulation (Figure 5). Similar to BMDM stimulation with LPS, the effect of tick saliva was more pronounced on TNF-α secretion by BMDMs during pathogen stimulation. TNF-α secretion was completely abolished when BMDMs were stimulated with A. phagocytophilum in the presence of tick saliva (Figure 5C).

Overall, the inhibitory effect of tick saliva on cytokine secretion by BMDMs during A. phagocytophilum stimulation was best observed at a 1:150 dilution (v/v). Experiments performed with tick saliva at a 1:300 dilution (v/v) also showed reduction of cytokine secretion by BMDMs during A. phagocytophilum stimulation (Figure 5). However, the effect was milder (although statistically significant) for IL-6 and IL-12p40 (Figure 5A and B). We did not detect a dilution effect on TNF-α secretion by BMDMs at a 1:300 dilution (v/v) (Figure 5C). These results suggest that the inhibition threshold for tick saliva on TNF-α secretion by BMDMs during A. phagocytophilum stimulation is greater than 1:300 (v/v) compared to 1:150 dilution (v/v). It is unclear why more concentrated tick saliva is required to inhibit cytokine secretion during A. phagocytophilum stimulation when compared to individual TLR or NLR agonists. We reasoned that the presence of multiple PAMPs in a pathogen, such as A. phagocytophilum, may require a stronger dose of tick saliva to mitigate cytokine secretion by BMDMs.

A dogma has emerged in the last few years in which the production and release of IL-1β are the result of a two-tier system: one signal is induced by pattern recognition receptors or pro-inflammatory cytokines, which activates the transcription and translation of IL-1β via the transcription factor nuclear factor (NF)-κB. This signal is also referred to as priming and it is typically done by LPS stimulation of immune cells. The second signal is mediated by the inflammasome, a protein scaffold that cleaves IL-1β into its mature form through caspase-1 activation [41-43]. Our studies show that IL-1β secretion triggered by A. phagocytophilum during BMDM stimulation does not require LPS priming. To the contrary, LPS priming in BMDMs before A. phagocytophilum stimulation inhibited IL-1β secretion in a dose-dependent manner (Figure 6A). These results are consistent with the lack of genes for LPS and peptidoglycan synthesis in the A. phagocytophilum genome [44] and suggest that molecules other than LPS may prime BMDMs for the production of pro-IL-1β. Further, tick saliva inhibited A. phagocytophilum-induced IL-1β secretion by BMDMs (Figure 6B). IL-1β secretion may be coupled to a cell death phenomenon named pyroptosis [45]. We did not detect high levels of cell death during A. phagocytophilum stimulation of BMDMs in the absence of tick saliva (~10-15%) (Figure 6C). However, this is not entirely surprising, as A. phagocytophilum does not cause much cell death during infection [32,46]. Based on these findings, we conclude that in the A. phagocytophilum model, cytokine secretion is a better indicator of the anti-inflammatory features of tick saliva when compared to cell death.