Amblyomma cajennense Sensu Stricto (Fabricius, 1787) and Amblyomma sculptum (Berlese, 1888) Tick Saliva Elicit Immune‐Modulatory Activity in Isolated Murine Macrophages With an Insight Into Proteomic Analysis

TL;DR

This study compares how tick saliva from two species affects mouse macrophages, revealing distinct immune-modulating effects.

Contribution

First comparative evaluation of salivas from two species in the A. cajennense complex and their immune-modulatory effects.

Findings

Saliva from both tick species increased ROS production and phagocytic activity in macrophages.

A. sculptum saliva decreased IL-1β and increased TNF-α, while A. cajennense s.s. saliva increased IL-6 and IL-10.

Proteomic analysis identified 221 and 303 secreted proteins in A. cajennense s.s. and A. sculptum, respectively.

Abstract

Tick saliva is known to cause immunosuppression and help pathogen transmission.
Amblyomma sculptum
is a public health concern as a vector of
Rickettsia rickettsii
. Another close‐related species is
Amblyomma cajennense
sensu stricto (s.s.). The impact of saliva from these species on murine macrophages remains unclear. This study evaluated saliva from
A. cajennense
s.s. and
A. sculptum
in murine peritoneal macrophages, assessing cell viability, adhesion, morphology, reactive oxygen species (ROS) production, phagocytosis and cytokine secretion. Additionally, a proteomic analysis was conducted and the proteins that were secreted in salivas were estimated. Neither A. sculptum nor A. cajennense s.s. saliva did it affect viability, adhesion or morphology, but increased ROS production and phagocytic activity.
A. sculptum
saliva decreased IL‐1β and increased TNF‐α, whereas
A. cajennense
s.s. saliva increased IL‐6 and IL‐10. Proteomic analysis revealed 221 and 303 secreted proteins in
A. cajennense
s.s. and
A. sculptum
, respectively and the more abundant were vitellogenins, microplusins, serpins, cystatins, actin, beta actin and calponins. These findings suggest that saliva from each species modulates macrophage activity in distinct ways, eliciting pro‐ and anti‐inflammatory responses. This is the first comparative evaluation of salivas from two species of the
A. cajennense
sensu lato complex, providing new insights into their interaction with innate immune cells.

Figures

Effect of the Amblyomma cajennense s.s. and Amblyomma sculptum salivas in TG‐elicited macrophages viability. TG‐elicited macrophages were obtained from mice injected with 3% thioglycolate. 2 × 105 cells/mL were incubated with RPMI (control) or tick salivas (S for A. sculptum and C for A. cajennense s.s.) for 6 h at 37°C in humidified atmosphere of 5% of CO2. After, viability was assessed by MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] metabolization method. Data were expressed in DO. Values represent the mean SEM from three animals.

Effect of the Amblyomma cajennense s.s. and Amblyomma sculptum salivas on the adhesion capacity of the TG‐elicited macrophages. For the adhesion assay, 2 × 105 TG‐elicited macrophages/mL were incubated for 1 h with RPMI (control) or tick salivas (S for A. sculptum and C for A. cajennense s.s.). Adhered cell levels were determined by optical density (550 nm) being proportional to the amount of incorporated Giemsa dye. Data were expressed in DO. Values are the mean ± SEM from three to five animals.

Representative photographs of TG‐elicited macrophages incubated with RPMI (impartial control), PMA (500 ng/mL positive control), tick salivas Amblyomma sculptum or Amblyomma cajennense s.s. salivas (25 μL/mL).

Effect of the Amblyomma cajennense s.s. and Amblyomma sculptum salivas in ROS production by TG‐elicited macrophages. 2 × 105 were incubated for 90 min with Hank's solution (negative control), PMA (positive control; 500 ng/mL). After addition of DCFDA (10 mM), DCF fluorescence was determined at excitation and emission wavelengths of 485 and 528 nm, respectively in a spectrophotometer. Data was expressed as fluorescence intensity. Values mean SEM from four animals. *p < 0.05 compared with the negative control (ANOVA).

Effect of the Amblyomma cajennense s.s. and Amblyomma sculptum salivas in zymosan macrophage phagocytic activity. 2 × 105 TG‐elicited macrophages were incubated with RPMI (negative control) or LPS (positive control; 1 μg/mL) or PMA (positive control; 500 ng/mL) or tick saliva (25 μg/mL) for 1 h at 37°C in a humid atmosphere (5% CO2) before addition of opsonized zymosan particles. Data were expressed as % of positive cell for phagocytosis and represent the mean ± SEM of three to four animals. *p < 0.05 compared to control (ANOVA).

Effect of the Amblyomma cajennense s.s. and Amblyomma sculptum salivas on cytokines production by TG‐elicited macrophages. 2 × 105 cells were incubated with RPMI (negative control), PMA (positive control; 500 ng/mL), LPS (positive control; 1 μg/mL) or both A. cajennense s.l. ticks salivas for 2 and 4 h, respectively, for IL‐1β (A, B), IL‐6 (C, D), TNF‐α (E, F) and 6 h for IL‐10 (G), at 37°C in a humid atmosphere (5% CO2). The concentrations of cytokines in the supernatant were quantified by specific EIA. Data were expressed as pg/mL and represent the mean ± SEM of three to five animals. *p < 0.05 compared to the standard control (ANOVA followed by Dunnet).

Tables

TABLE 1. List of putative proteins secreted in Amblyomma cajennense sensu stricto saliva by NanoLC/mass spectromety, with the description of orthologous proteins found in protein databases, mass weigh in kiloDaltons (MW‐kDa), exponentially modified protein abundance index (emPAI) and the accession number of the orthologous protein.

Description	MW (kDa)	emPAI	Accession
Putative microplusin 4— Amblyomma cajennense	12.9	99.0	A0A023FPX2
Putative vitellogenin‐2— Amblyomma cajennense	177.1	39.8	A0A023FUV2
Putative vitellogenin‐2— Amblyomma cajennense	79.2	18.7	A0A023FLT6
Putative microplusin 4— Amblyomma cajennense	12.8	12.9	A0A023FPW4
Putative vitellogenin‐2 (Fragment) OS =  Amblyomma cajennense	94.5	12.0	A0A023FMI5
Putative vitellogenin‐2 (Fragment)— Amblyomma cajennense	177.1	11.8	A0A023FS61
Putative microplusin 4— Amblyomma cajennense	12.9	9.0	A0A023FPW2
Putative ixodegrins large 9 (Fragment)— Amblyomma cajennense	14.2	6.2	A0A023FQ30
Putative secreted salivary gland peptide ixodes scapularis secreted salivary gland peptide— Amblyomma cajennense	13.0	5.8	A0A023FPX7
Putative salivary protein— Amblyomma cajennense	25.2	5.3	A0A023FUE9
Uncharacterized protein— Amblyomma maculatum	19.5	4.6	G3MMQ5
Putative vitellogenin‐2 (Fragment)— Amblyomma americanum	30.1	3.6	A0A0C9SDR9
Putative secreted protein— Amblyomma cajennense	11.1	3.6	A0A023FPR8
Putative tick cistatins 1— Amblyomma cajennense	15.0	3.3	A0A023FQ34
Actin— Haemaphysalis flava	41.8	3.1	A0A097CK67
Putative tick serpins 7 (Fragment)— Amblyomma cajennense	35.7	2.8	A0A023FPT3
Putative secreted protein (Fragment)— Amblyomma triste	21.6	2.7	A0A023G356
Putative tick til 17— Amblyomma cajennense	17.7	2.6	A0A023FL04
Histone H4— Amblyomma maculatum	15.4	2.6	G3MMX0
Putative tick serpins 1— Amblyomma cajennense	21.4	2.5	A0A023FMR2
Putative biotinidase and vanin (Fragment)— Amblyomma cajennense	54.8	2.3	A0A023FFX5
Uncharacterized protein (Fragment)— Amblyomma sculptum	43.3	2.2	A0A1E1XM23
Putative conserved membrane protein (Fragment)— Amblyomma cajennense	23.2	2.2	A0A023FLY3
Putative tick til 20— Amblyomma cajennense	17.9	2.2	A0A023FTL7
Calponin— Amblyomma cajennense	20.6	1.9	A0A023FHM0
Putative beta actin variant (Fragment)— Ixodes scapularis	44.4	1.9	A0A4D5RZP8
Putative regulatory protein mlp— Amblyomma triste	14.1	1.7	A0A023GA35
SVWC domain‐containing protein— Amblyomma cajennense	11.9	1.7	A0A023FPR5
Putative vitellogenin‐2— Amblyomma parvum	177.4	1.6	A0A023G0E1
Putative tick til 20— Amblyomma cajennense	17.9	1.5	A0A023FRL4
Putative secreted protein— Amblyomma cajennense	15.2	1.5	A0A023FCU0
Putative secreted salivary gland peptide— Amblyomma triste	14.1	1.5	A0A023GMJ1
Uncharacterized protein (Fragment)— Amblyomma cajennense	82.8	1.5	A0A023FNW2
Putative ml domain‐containing protein— Amblyomma aureolatum	16.7	1.4	A0A1E1WZY1
Putative glycine‐rich cell wall structural protein 1— Amblyomma cajennense	37.2	1.3	A0A023FUP5
Putative inducible metalloproteinase (Fragment)— Amblyomma tuberculatum	15.7	1.3	A0A6M2E4Z6
Putative serine proteinase inhibitor (Fragment)— Amblyomma cajennense	24.7	1.3	A0A023FI79
Putative thyropin— Amblyomma cajennense	29.2	1.2	A0A023FUD9
Putative secreted glycine‐rich protein (Fragment)— Amblyomma cajennense	19.3	1.2	A0A023FQI4
Putative secreted protease inhibitor— Amblyomma cajennense	14.6	1.2	A0A023FSZ3
Putative alpha‐macroglobulin ixodes scapularis alpha‐macroglobulin (Fragment)— Amblyomma cajennense	13.4	1.2	A0A023FN13
Putative secreted protein— Amblyomma cajennense	11.2	1.2	A0A023FDH4

TABLE 2. List of putative proteins secreted in Amblyomma sculptum saliva by NanoLC/mass spectromety, with the description of orthologous proteins found in protein databases, mass weigh in kiloDaltons (MW‐kDa), exponentially modified protein abundance index (emPAI) and the accession number of the orthologous protein.

Description	MW (kDa)	emPAI	Accession
Putative microplusin 4— Amblyomma cajennense	12.9	999.0	A0A023FPX2
Putative vitellogenin‐2— Amblyomma cajennense	79.2	55.2	A0A023FLT6
Putative microplusin 4— Amblyomma cajennense	12.9	50.8	A0A023FPW2
Putative vitellogenin‐2 (Fragment)— Amblyomma cajennense	94.5	40.9	A0A023FMI5
Putative vitellogenin‐2— Amblyomma cajennense	177.1	30.2	A0A023FUV2
Putative vitellogenin‐2 (Fragment)— Amblyomma cajennense	177.1	25.9	A0A023FS61
Putative his‐rich 1— Amblyomma cajennense	12.8	25.8	A0A023FSW5
Putative microplusin 4— Amblyomma cajennense	12.8	18.3	A0A023FPW4
Rs 05br antigen— Rhipicephalus sanguineus	19.2	9.0	A0A247ZGJ6
Putative salivary protein— Amblyomma cajennense	25.2	7.6	A0A023FUE9
Actin— Haemaphysalis flava	41.8	7.2	A0A097CK67
Putative glycine‐rich cell wall structural protein— Amblyomma cajennense	40.6	7.1	A0A023FM63
Putative glycine‐rich cell wall structural protein 1— Amblyomma cajennense	37.2	7.1	A0A023FUP5
Putative secreted protein— Amblyomma cajennense	11.1	5.8	A0A023FPR8
Putative glycine‐rich cell wall structural protein 1— Amblyomma cajennense	36.3	5.3	A0A023FRF7
Putative kunitz‐like protease inhibitor— Amblyomma cajennense	11.0	5.3	A0A023FPM8
Putative conserved membrane protein (Fragment)— Amblyomma cajennense	23.2	4.6	A0A023FLY3
Putative secreted salivary gland peptide— Amblyomma parvum	14.1	4.6	A0A023FWJ9
Heme lipoprotein— Amblyomma americanum	176.9	4.5	A0MVX0
Uncharacterized protein— Amblyomma cajennense	40.7	4.3	A0A023FUJ9
Putative beta Actin variant (Fragment)— Ixodes scapularis	44.4	3.6	A0A4D5RZP8
Putative tick til 17— Amblyomma cajennense	17.7	3.6	A0A023FL04
Putative tick serpins 1— Amblyomma cajennense	21.4	3.3	A0A023FMR2
Uncharacterized protein (Fragment)— Amblyomma cajennense	82.8	3.3	A0A023FNW2
Putative tick til 20— Amblyomma cajennense	17.9	3.0	A0A023FTL7
Putative tick kunitz 78— Amblyomma cajennense	11.0	3.0	A0A023FPN1
Putative secreted protein— Amblyomma cajennense	23.0	2.6	A0A023FDV8
Putative scp euk: scp‐like extracellular protein (Fragment)— Amblyomma cajennense	21.8	2.4	A0A023FPT8
Putative vitellogenin‐2— Amblyomma parvum	177.4	2.3	A0A023G0E1
Putative glycine‐rich cell wall structural protein— Amblyomma cajennense	31.3	2.2	A0A023FM41
Putative secreted protein— Amblyomma cajennense	24.6	2.2	A0A023FR61
Putative tick til 20— Amblyomma cajennense	17.9	2.2	A0A023FRL4
Putative cystatin— Amblyomma sculptum	14.0	2.2	A0A1E1XU98
Putative cystatin— Amblyomma cajennense	14.0	2.2	A0A023FPZ6
Calponin— Amblyomma maculatum	20.8	1.9	G3MK71
Histone H2B (Fragment)— Ixodes ricinus	16.7	1.8	A0A131Y6E4
Putative tick cistatins 1— Amblyomma cajennense	15.0	1.8	A0A023FQ34
Histone H4— Amblyomma maculatum	15.4	1.8	G3MMX0
Putative secreted protein (Fragment)— Amblyomma triste	21.6	1.7	A0A023G356
Histone H2A (Fragment)— Ixodes scapularis	16.2	1.7	A0A4D5RWU4
Putative ixodegrins large 9 (Fragment)— Amblyomma cajennense	14.2	1.7	A0A023FQ30
Uncharacterized protein— Amblyomma cajennense	13.2	1.7	A0A023FG92
Putative serine proteinase inhibitor (Fragment)— Amblyomma cajennense	24.7	1.6	A0A023FI79
Uncharacterized protein (Fragment)— Amblyomma sculptum	43.3	1.6	A0A1E1XM23
Putative secreted salivary gland peptide— Amblyomma triste	14.1	1.5	A0A023GMJ1
Putative secreted protein— Amblyomma cajennense	10.8	1.5	A0A023FBM9
Ferritin— Amblyomma triste	22.3	1.4	A0A023G561
Putative thyropin— Amblyomma cajennense	23.3	1.4	A0A023FR27
Putative ml domain‐containing protein ‐ Amblyomma aureolatum	16.7	1.4	A0A1E1WZY1
Uncharacterized protein (Fragment)— Amblyomma americanum	12.3	1.4	A0A0C9SBE2
Putative inducible metalloproteinase— Amblyomma aureolatum	17.9	1.3	A0A1E1WZT1
Putative insulin‐like growth factor binding protein‐related protein 1— Amblyomma sculptum	26.8	1.3	A0A1E1XQV2
Putative biotinidase and vanin (Fragment)— Amblyomma cajennense	54.8	1.2	A0A023FFX5
Putative tropomyosin tropomyosin— Amblyomma triste	32.9	1.2	A0A023GHG6
Putative conserved membrane protein— Amblyomma cajennense	30.3	1.2	A0A023FM03
Putative secreted protease inhibitor— Amblyomma cajennense	14.6	1.2	A0A023FSZ3
Putative alpha‐macroglobulin ixodes scapularis alpha‐macroglobulin (Fragment)— Amblyomma cajennense	13.4	1.2	A0A023FN13
Putative secreted salivary gland peptide ixodes scapularis secreted salivary gland peptide— Amblyomma cajennense	13.0	1.2	A0A023FPX7
Putative matricellular protein osteonectin/sparc/bm‐40— Amblyomma cajennense	32.6	1.1	A0A023FMZ7

Full text

Introduction

1

Ixodid ticks, commonly known as ‘hard ticks’ (Acari: Ixodidae), maintain an intimate relationship with their vertebrate hosts, remaining attached for days to weeks during the parasitic stage [1, 2, 3]. Tick saliva is essential for successful parasitism and undergoes dynamic changes throughout the feeding process, from attachment to detachment. These variations are attributed to numerous bioactive molecules that act as immunosuppressive agents, as well as vasoactive and anticoagulant factors [4].

Beyond their role in facilitating tick parasitism, salivary components also promote pathogen invasion of the host, primarily through immunosuppressive effects [5]. These effects modulate both Th1 and Th2 immune responses and reduce circulating B and T lymphocytes [5, 6, 7, 8, 9].

Most studies on the effects of tick saliva on immune cells have focused on species of public health importance in the Northern Hemisphere, particularly those from the Ixodes genus [8, 10, 11, 12, 13]. In the Neotropical region, the saliva of ticks from the Amblyomma cajennense sensu lato (s.l.) complex has been the most extensively investigated [14, 15, 16, 17, 18, 19, 20]. Sialotranscriptome analysis of the salivary glands of A. cajennense s.l. revealed 4604 novel mRNA sequences, while proteomic analysis of Amblyomma sculptum saliva identified 124 secreted proteins [17, 20].

Similar to other tick species, A. cajennense s.l. saliva promotes a Th2 immune profile, stimulating lymphocytes to produce IL‐4, IL‐10 and TGF‐β [14]. Additionally, this saliva can inhibit the differentiation of bone marrow cells into mature dendritic cells (DCs) [18], suppress the complement system [19] and interfere with blood clot formation by inhibiting factor Xa [15]. It also exhibits antiangiogenic properties [21] and antitumor activity [22, 23].

The A. cajennense sensu lato (s.l.) complex comprises six closely related tick species, two of which occur in Brazil: A. cajennense sensu stricto (s.s.), associated with the Amazon biome and A. sculptum , the primary vector of Rickettsia rickettsii , the causative agent of Brazilian Spotted Fever (BSF). A. sculptum is predominantly linked to the Cerrado and Pantanal biomes but also occurs in anthropized areas of the Atlantic Rainforest biome [24, 25].

Although the biological role of tick saliva is well characterised for species of One Health importance in the Nearctic region [26], information on Amblyomma ticks from the Neotropical region remains limited. Given the relevance of A. sculptum to human health in Brazil, this species has been the primary focus of studies on salivary composition and function [17, 18, 19, 20, 23, 27, 28]. It is important to note the synonymy between A. cajennense and A. sculptum until 2014, when the A. cajennense s.l. species complex was formally described [24]. This taxonomic clarification enabled the present study to provide, for the first time, comparative data on the modulatory effects of saliva from A. cajennense s.s. and A. sculptum on TG‐elicited macrophages. Furthermore, we discuss proteomic analyses of saliva from both species, highlighting candidate molecules responsible or not for the observed effects and others likely unrelated to macrophage modulation.

Methods

2

Animals

2.1

The procedures with animals—10 female and 5 males of 6–8 weeks Swiss mice and adult New Zealand rabbits (Oryctyolagus cuniculus)—were carried out in accordance with the ARRIVE reporting guidelines and were approved by the Ethics Committee on the Use of Animals of Fiocruz Rondônia (CEUA/Fiocruz‐RO) under protocol number 2016/09.

Tick Colonies

2.2

A. sculptum and A. cajennense s.s. are maintained at Fiocruz Rondônia facilities. The A. sculptum colony was established from specimens that were kindly provided by Dr. Matías Pablo Juan Szabó from the Laboratory of Ixodology of the Faculty of Veterinary Medicine, Federal University of Uberlandia (LABIX‐UFU). The A. cajennense s.s. colony was established from ticks collected from a riparian forest fragment located at Monte Negro municipality, Rondônia, Brazil. Both tick species were fed on New Zealand rabbits, inside chambers glued on the shaved back, according to Bechara et al. [29]. The non‐parasitic stages were maintained in biological oxygen demand incubators at 23°C–28°C with 85%–90% air humidity.

Each rabbit was infested with ticks up to five times and was subsequently discarded. For saliva collection, each animal was infested with up to 40 females and 20 males of each species, distributed into two feeding chambers affixed to the rabbit's dorsum. Each rabbit was assigned to infestation with only one tick species ( A. cajennense s.s. or A. sculptum ). After the fifth blood meal, the rabbit was euthanized and replaced with a new animal.

Because saliva was obtained from ticks feeding on rabbits during the first through the fifth infestations, saliva batches collected from each species were ultimately pooled. Thus, the saliva pools contained material from ticks that had fed on naïve hosts (first infestation) as well as on hosts undergoing the second to fifth infestations, with comparable amounts contributed from each infestation. Pooling was performed to homogenise the saliva samples obtained across infestations, thereby standardising the material used in macrophage experiments for both species and avoiding bias that could arise if saliva from one species had been collected from rabbits exhibiting greater or lesser acquired resistance than those used for the other species.

Tick Saliva Collection

2.3

The salivas were collected from adult stage females partially engorged from both tick species mentioned above. The females were removed from the rabbits manually or with Tick Lasso (Silvalure, Sweden) at the onset of the engorgement process, comprising a period of 7–14 days. After removal, the partially engorged females were washed with distilled water and ethanol 70%. Then, the ticks were glued by their dorsal face to an adhesive tape fixed on a glass slide. Approximately 10–20 μL of pilocarpine hydrochloride at 2% (Sigma‐Aldrich, USA) in phosphate‐buffered saline (PBS: 14 mM NaCl, 2 mM NaH_2_PO_4_H_2_O, 7 mM Na_2_HPO_4_12H_2_O) pH 7.4 was injected into the ventral face of the ticks into the hemolymph using a 1 mL syringe with a 29G needle, at a middle point between the third pair of spurs. The injections were repeated once in a tick after 1–2 h after the first inoculation, when a decrease in saliva secretion occurred. Saliva was collected in 10 μL tips fixed in the tick hypostomes. When the tips were filled, the saliva from all ticks was transferred to 0.2 μL microtubes kept on ice during the process, which comprised 2–3 h. Right after, the saliva was stored at −80°C until use.

The saliva from A. cajennense s.s. used in this study belonged to the same saliva pool used and published by Cerri et al. [30] and the protein concentrations of the saliva were measured by QubitTM Protein Assay Kit (Life Technologies). For this study, as it included A. sculptum tick saliva and was performed in another laboratory, the protein concentration for this tick species was determined by the bicinchoninic acid assay (BCA).

Proteomic Analysis by NanoLC/Mass Spectrometry

2.4

Shotgun proteomics analysis was performed on pooled samples of saliva from engorged females of A. sculptum and A. cajennense s.s. Protein concentrations of cleared supernatants were determined using the Qubit Protein Assay Kit (Life Technologies) to allow the enough amount of protein from the salivas used for trypsin digestion. The conditions of protein denaturation, alkylation, digestion and cleaning were the same as described previously [31, 32]. Two μg of digested peptides from both A. sculptum and A. cajennense s.s. samples were individually analysed in technical triplicate by nanoLC/MSn (Easy‐nanoLC1000 coupled to an Orbitrap QExactive Plus, Thermo Fisher) as described elsewhere [32, 33].

The triplicated saliva samples data were analysed by the Proteome Discoverer 2.1 software using Ixodidae and rabbit databases UniProt (V. Nov 2018‐https://www.uniprot.org/). The parameters used for the search space were the same as described by Pizzatti et al. [32] and Velásquez et al. [33]. The process and biological pathway validation strategies were previously used and discussed by Pizzatti et al. [31, 32] and Gjertsen and Wiig [34].

Collection of Elicited Murine Peritoneal Macrophages

2.5

To obtain the macrophages, 3% thioglycollate (TG) was inoculated intraperitoneally in Swiss mice, 10 females and 5 males from 6 to 8 weeks, according to Zuliani et al. [35]. After 96 h, the animals were euthanized by cervical dislocation and a peritoneal lavage was performed after a gentle massage of the abdominal wall using 3 mL of cold PBS (pH 7.2). After the peritoneal cells collection containing elicited macrophages, the cell counting was determined in Neubauer's chamber. The cell population consisted of more than 95% TG‐macrophages, as determined by morphological criteria.

Macrophages Viability

2.6

Activity was measured to assess cell viability. TG‐elicited macrophages (2 × 10^5^ cells/mL) were suspended in an RPMI culture medium, supplemented with gentamicin (100 μg/mL), l‐glutamine (2 mM) and 10% fetal bovine serum. Then, the cells were incubated in duplicate in 96‐well plates with RPMI (control) or filtered salivas from A. cajennense s.s. (C) and A. sculptum (S) ticks, at concentrations of 25 and 12.5 μL/mL for 6 h, at 37°C in a humid atmosphere (5% CO_2_). Next, 10 μL of MTT (5 mg/mL) was added and incubated for 2 h. After centrifugation at 400g for 5 min, the supernatant was removed and 100 μL of DMSO was added to dissolve the formed crystals. Subsequently, the plates were kept for 15 min at room temperature and evaluated in a spectrophotometer at 540 nm. The results were expressed in percentage compared to the respective controls [36, 37].

Macrophages Adhesion Capacity

2.7

Macrophage adhesion assay was conducted according to the procedure described by Setubal et al. [38]. In brief, TG‐elicited macrophages (2 × 10^5^ cells/50 μL) were plated and incubated for 1 h with 50 μL of RPMI (control group) or A. cajennense s.s. and A. sculptum tick salivas (12.5 and 25 μL/mL, at 37°C, under a humid atmosphere with 5% CO_2_). After incubation, the plates were washed three times with PBS and the adherent cells were fixed with methanol for 10 min. After staining with 0.1% Giemsa solution for 40 min, the plates were washed with deionised water and the remaining dye was solubilised with methanol. Absorbance was determined spectrophotometrically at 550 nm. The control group was considered 100%.

Macrophages Morphology

2.8

Adhered macrophages were incubated with PMA (positive control group, 500 ng/mL), RPMI (negative control group) and saliva A. cajennense s.s. and A. sculptum tick salivas (experimental groups) at the concentrations of 25 and 12.5 μL/mL. TG‐elicited macrophages were photo documented and the cell morphology was analysed using contrast microscopy (zoom of ×20 or ×40).

Reactive Oxygen Species (ROS) Production by Macrophages

2.9

ROS intracellular levels were measured with a peroxide‐sensitive fluorescent probe 2′,7′‐dichlorodihydrofluorescein diacetate (DCFDA). In brief, TG‐elicited macrophages (2 × 10^5^) were resuspended in Hank's solution and distributed in 96‐well black plates. Cells were incubated with Hank's solution (negative control), PMA (positive control; 500 ng/mL), the A. cajennense s.s. or A. sculptum salivas at a concentration of 25 μg/mL for 90 min [37]. Right after, 100 μL of DCFDA (10 μM) diluted in Hank's solution was added and incubated for 30 min at 37°C, under constant dark conditions. DCF fluorescence was measured with the BioTek Synergy HT Multi‐Detection (Winooski, VT) at excitation and emission wavelengths of 485 and 528 nm, respectively.

Macrophage Phagocytic Activity

2.10

Initially, this assay was performed with female mice and after it was repeated with male mice (see the results section below). Phagocytosis of zymosan particles was conducted according to Setúbal et al. [38]. In brief, 2 × 10^5^ TG‐elicited macrophages were dispensed in 13 mm glass slides, plated in 24‐well plates and fixed for 40 min at 37°C in a humid atmosphere with 5% CO_2_. After washing with PBS to remove non‐adherent cells, the monolayers containing adhered macrophages were cultivated with RPMI (negative control group), PMA (positive control; 500 ng/mL), LPS (positive control; 1 μg/mL), A. cajennense s.s. or A. sculptum , at a concentration of 25 μg/mL, diluted in RPMI for 1 h at 37°C, under a humid atmosphere with 5% CO_2_. After washing in cold PBS (4°C), the monolayers were incubated for 40 min at 37°C and 5% CO_2_ with serum‐opsonized zymosan, prepared as described below and unbound particles were removed by washing with cold PBS. Cells were fixed with 2.5% glutaraldehyde for 15 min at room temperature and the coverslips were mounted on microscope slides. The extent of phagocytosis was quantified by contrast phase microscopic observation. At least 200 elicited macrophages were counted in each determination and those containing three or more internalised particles were considered positive for phagocytosis. Results were presented as the percentage of cells positive for phagocytosis.

To prepare serum‐opsonized zymosan particles, zymosan obtained from yeast cell walls was suspended in PBS at a concentration of 5.7 mg/mL zymosan particles. For opsonization, 1 mL of zymosan particles was mixed with 1 mL normal mouse serum and incubated for 30 min at 37°C. The serum‐opsonized zymosan particles were centrifuged at 200g for 10 min and suspended in PBS for the phagocytosis assay.

Interleukin‐1β (IL‐1β), Interleukin‐6 (IL‐6), Interleukin‐10 (IL‐10) and Tumour Necrosis Factor‐α (TNF‐α) Quantification

2.11

Initially, all the cytokine assays were performed with TG‐elicited macrophages from female mice and incubated for 6 h. After the initial results, we decided to perform the assays again to quantify IL‐1β, IL‐6 and TNF‐α using male mice, with cells incubated for 2 and 4 h.

TG‐elicited macrophages (2 × 10^5^/50 μL) were incubated with RPMI medium (control group), PMA or LPS (positive controls; 0.5 and 1 μg/mL, respectively) or both tick salivas (12.5 and 25 μg/mL) for 2 and 4 h at 37°C in a humidified atmosphere (5% CO_2_). After centrifugation, the supernatant was used to determine IL‐1β, IL‐6 and TNF‐α levels by specific enzyme‐linked immunosorbent assay (EIA). In brief, 96‐well plates were coated with 100 μL of the first capture monoclonal anti‐IL‐1β (4 μg/mL), anti‐IL‐6 (2 μg/mL), anti‐IL‐10 (4 μg/mL) or anti‐TNF‐α (0.8 μg/mL) and incubated for 18 h at 37°C. Following this period, the plate was washed with washer buffer (PBS/Tween20). After that, 200 μL of blocking buffer, containing 5% bovine serum albumin (BSA) in PBS/Tween20, was added to the wells and the plates were incubated for 1 h at 37°C. Following this period_,_ the wells were washed and 50 μL of either samples or standards were dispensed into each well and incubated for 2 h at 37°C. After this period, the plate was washed and 100 μL of antibody anti‐IL‐1β (1.5 μg/mL), anti‐IL‐6 (150 ng/mL) or anti‐TNF‐α (200 ng/mL) was added for 2 h at 37°C. After incubation and washing, 100 μL of streptavidin‐peroxidase was added, followed by incubation and addition of the substrate (100 μL/mL 3,3′, 5,5′‐tetramethylenediamine). Finally, sulfuric acid (50 μL) was added to stop the reaction. Absorbances at 540 and 450 nm were recorded and cytokine concentrations were estimated from standard curves prepared with recombinant IL‐1β (1000–1.95 pg/mL), IL‐6 (1000–1.95 pg/mL) or TNF‐α (2000–3.90 pg/mL). The results were expressed as pg/mL of each cytokine. Pilocarpine hydrochloride at 2% (the same used for saliva collection) was also used as a control group (data not shown).

Statistical Analysis

2.12

All the results obtained from each macrophage parameter above were analysed using one‐way ANOVA, followed by Dunnett's multiple comparisons test, to compare the means of all test groups (including positive and negative controls) to the standard control group.

Results

3

Protein Identification by Mass Spectrometry and Bioinformatics Analysis

3.1

The protein concentration of A. sculptum saliva, measured by BCA, was 0.98 μg/μL and 1.27 μg/μL of A. cajennense s.s. saliva [30]. The secreted proteins present in the A. cajennense s.s. and A. sculptum tick salivas were determined by proteomic shotgun analysis. A total of 221 and 303 putative proteins were identified in A. cajennense s.s. and A. sculptum samples, respectively (Tables 1 and 2).

TABLE 1: List of putative proteins secreted in Amblyomma cajennense sensu stricto saliva by NanoLC/mass spectromety, with the description of orthologous proteins found in protein databases, mass weigh in kiloDaltons (MW‐kDa), exponentially modified protein abundance index (emPAI) and the accession number of the orthologous protein.

TABLE 2: List of putative proteins secreted in Amblyomma sculptum saliva by NanoLC/mass spectromety, with the description of orthologous proteins found in protein databases, mass weigh in kiloDaltons (MW‐kDa), exponentially modified protein abundance index (emPAI) and the accession number of the orthologous protein.

Protein size ranged from 7.50 to 513.9 kDa in both samples. We considered the Exponentially Modified Protein Abundance Index (emPAI) values to estimate the protein abundances from peptide counts in the saliva samples. A. cajennense s.s. saliva presented 42 proteins with emPAI values above 1 belonging to several protein families; among the most abundant proteins there were: six vitellogenins, three microplusins, three serine proteinase inhibitors (serpins), two actins, one cystatin, one beta actin, one calponin and one biotinidase (Table 1). A. sculptum saliva supposedly secreted 59 proteins with emPAI values above 1, belonging to almost the same protein families: five vitellogenins, three microplusins, two serpins, two Kunitz protease inhibitors, three cystatins, one calponin, one actin, one beta‐actin and one biotinidase, among others (Table 2). Some host proteins (rabbit) presented emPAI values above 1 in both saliva samples: mainly albumin, alpha‐globin and haemoglobin (data not shown).

Viability, Adhesion and Morphology

3.2

The TG‐elicited macrophages viability assay was conducted with saliva from A. cajennense s.s. (C) and A. sculptum (S) at 12.5 and 25 μL/mL showed nontoxicity or decreased viability (Figure 1). Although the cell viability of the control group was lower than the viability of the macrophages incubated with both tick salivas, no statistical significance was found (p = 0.585).

Effect of the Amblyomma cajennense s.s. and Amblyomma sculptum salivas in TG‐elicited macrophages viability. TG‐elicited macrophages were obtained from mice injected with 3% thioglycolate. 2 × 105 cells/mL were incubated with RPMI (control) or tick salivas (S for A. sculptum and C for A. cajennense s.s.) for 6 h at 37°C in humidified atmosphere of 5% of CO2. After, viability was assessed by MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] metabolization method. Data were expressed in DO. Values represent the mean SEM from three animals.

In the adhesion assay, both A. cajennense s.s. and A. sculptum salivas did not significantly change TG‐elicited macrophages' adhesion capacity (p = 0.4322) after 1 h of incubation, using both concentrations (Figure 2).

Effect of the Amblyomma cajennense s.s. and Amblyomma sculptum salivas on the adhesion capacity of the TG‐elicited macrophages. For the adhesion assay, 2 × 105 TG‐elicited macrophages/mL were incubated for 1 h with RPMI (control) or tick salivas (S for A. sculptum and C for A. cajennense s.s.). Adhered cell levels were determined by optical density (550 nm) being proportional to the amount of incorporated Giemsa dye. Data were expressed in DO. Values are the mean ± SEM from three to five animals.

The macrophage morphology of RPMI‐incubated elicited macrophages presented both round and spread macrophages. PMA‐incubated cells presented a little more spread morphology, probably because PMA activates protein kinase C (PKC) signalling events, inducing cytoskeleton action‐rearrangements. The same observation as the control cells was noticed with saliva‐incubated macrophages at the concentration of 25 μg/mL, indicating that both tick salivas did not interfere with cell morphology (Figure 3).

Representative photographs of TG‐elicited macrophages incubated with RPMI (impartial control), PMA (500 ng/mL positive control), tick salivas Amblyomma sculptum or Amblyomma cajennense s.s. salivas (25 μL/mL).

ROS Production

3.3

ROS production of TG‐elicited macrophages was measured by DFDA. The negative control cells, incubated with Hank's solution, did not induce ROS production. However, when TG‐elicited macrophages were incubated with PMA, a well‐known ROS activator, there was an increase in ROS production. The same effect was observed in cells incubated with both tick salivas, which were significantly higher than the PMA, a positive control (p < 0.05), with A. sculptum being higher than A. cajennense s.s. saliva (Figure 4).

*Effect of the Amblyomma cajennense s.s. and Amblyomma sculptum salivas in ROS production by TG‐elicited macrophages. 2 × 105 were incubated for 90 min with Hank's solution (negative control), PMA (positive control; 500 ng/mL). After addition of DCFDA (10 mM), DCF fluorescence was determined at excitation and emission wavelengths of 485 and 528 nm, respectively in a spectrophotometer. Data was expressed as fluorescence intensity. Values mean SEM from four animals. p < 0.05 compared with the negative control (ANOVA).

Zymosan Phagocytosis

3.4

Initially, using female mice, the basal level of phagocytosis in the RPMI group was above 60%, which does not allow for a fair comparison (data not shown). When using TG‐elicited macrophages from male mice, with respect to phagocytosis function, in Figure 5, it is possible to observe that the basal level of isolated elicited macrophages incubated with RPMI was 30%. This percentage is enhanced when the cells were incubated with PMA or LPS, positive controls, indicating activation of macrophages for zymosan phagocytosis. This effect was also observed when the TG‐elicited macrophages were incubated with tick salivas: A. cajennense s.s. (p = 0.008) or A. sctulptum (p = 0.001).

*Effect of the Amblyomma cajennense s.s. and Amblyomma sculptum salivas in zymosan macrophage phagocytic activity. 2 × 105 TG‐elicited macrophages were incubated with RPMI (negative control) or LPS (positive control; 1 μg/mL) or PMA (positive control; 500 ng/mL) or tick saliva (25 μg/mL) for 1 h at 37°C in a humid atmosphere (5% CO2) before addition of opsonized zymosan particles. Data were expressed as % of positive cell for phagocytosis and represent the mean ± SEM of three to four animals. p < 0.05 compared to control (ANOVA).

Cytokines Production

3.5

Initially, the assays using female mice have only shown significant differences among control and test groups for IL‐10, but not for the other cytokines after 6 h of incubation (data not shown). Therefore, the assays were repeated for the other cytokines (IL‐1β, IL‐6 and TNF‐α) using TG‐elicited macrophages from male mice, applying shorter incubation times (2 and 4 h). Thus, IL‐1β, IL‐6 and TNF‐α liberation (2 and 4 h) and IL‐10 (6 h) were presented in Figure 6. TG‐elicited macrophages produced a basal level of the studied cytokines, as can be observed in the graphs (RPMI control). At the time interval studied, TG‐elicited macrophages incubated with LPS or PMA (positive controls) induced a significant decrease of IL‐1 β, a significant increase of IL‐10, but not IL‐6 and TNF‐α release. It was observed that A. sculptum saliva induced a significant decrease in IL‐1β release (4 h) and an increase in TNF‐α release (2 h) and A. cajennese s.s. saliva induced a significant increase of IL‐6 (2 h) and IL‐10 (6 h).

*Effect of the Amblyomma cajennense s.s. and Amblyomma sculptum salivas on cytokines production by TG‐elicited macrophages. 2 × 105 cells were incubated with RPMI (negative control), PMA (positive control; 500 ng/mL), LPS (positive control; 1 μg/mL) or both A. cajennense s.l. ticks salivas for 2 and 4 h, respectively, for IL‐1β (A, B), IL‐6 (C, D), TNF‐α (E, F) and 6 h for IL‐10 (G), at 37°C in a humid atmosphere (5% CO2). The concentrations of cytokines in the supernatant were quantified by specific EIA. Data were expressed as pg/mL and represent the mean ± SEM of three to five animals. p < 0.05 compared to the standard control (ANOVA followed by Dunnet).

It was also observed that both tick salivas induced a decrease of IL‐1β (2 h) and an increase of TNF‐α release (4 h), A. sculptum saliva induced an increase of IL‐6 (2 h) and IL‐10 (6 h) and A. cajennense s.s. saliva induced an increase in TNF‐α release (2 h). Notwithstanding, all these phenomena have no statistical significance (p > 0.05).

The pilocarpine used in the cytokine assays has only significantly increased the release of IL‐1β (4 h). No other cytokine production was observed with pilocarpine (data not shown).

Discussion

4

Tick saliva is a crucial component in their interaction with hosts, playing a significant role in modulating the immune response to ensure attachment and blood feeding [6, 9, 39, 40]. Saliva from A. cajennense s.s. and A. sculptum differentially stimulated or inhibited the production of various cytokines. A. sculptum saliva stimulated TNF‐α production and inhibited IL‐1β, whereas A. cajennense s.s. saliva stimulated IL‐6 and IL‐10 production. Neither saliva affected murine macrophage viability at the tested concentrations, consistent with previous findings for DCs incubated with A. sculptum saliva (published as A. cajennense ), indicating that tick saliva lacks cytotoxic effects that could influence other results in this study [18].

The distinct regulatory effects of these two tick species' salivas on cultured murine macrophages may be related to species‐specific protein composition. Although similar in protein families, proteomic analyses revealed a greater number of secreted proteins in A. sculptum saliva compared to A. cajennense s.s. The protein composition of A. cajennense s.s. saliva was previously described by this research group [30]. The present study used the same saliva characterised earlier, stored at −80°C, with continued saliva and tick production under identical conditions and collection protocols.

It is important to note that saliva from both species was collected under identical conditions during the final (rapid) phase of parasitism. At this stage, ticks have already completed the establishment of feeding and the salivary glands are primarily engaged in osmoregulation, returning excess water to the host. Therefore, the results of the present study indicate that differences exist in the salivary composition of these two species during the late phase of parasitism and such differences may be associated with the distinct responses observed in the murine macrophages used in this study.

Although salivation induced by secretagogue injection does not represent a physiological method, it is a widely employed approach for obtaining tick saliva in functional and comparative proteomic analyses [41, 42]. The presence of intracellular proteins, such as actins, histones and calponins, as well as hemolymph‐associated proteins such as vitellogenins and heme‐binding proteins, may at least partially reflect cellular leakage associated with the salivation process. Nevertheless, this proteomic profile has been consistently reported in tick sialome studies using this methodology [20, 43], including investigations demonstrating that some of these proteins may exert immunomodulatory functions [6, 18], display multitasking properties or act as ‘moonlighting’ proteins [26, 44]. In any case, the relative proportion of putative intracellular proteins was similar in the saliva of A. sculptum and A. cajennense s.s. and the major protein components in both salivas were microplusins and vitellogenins (Tables 1 and 2); however, subtle differences in the proportion of these cellular components and bioactive secreted proteins may have influenced the magnitude of the macrophage responses observed.

A. sculptum saliva exhibited a broader protein repertoire per family and secreted proteins that were absent in A. cajennense s.s. saliva. This richer proteomic profile may underlie A. sculptum 's parasitic success across diverse vertebrate hosts and its geographic expansion into degraded Atlantic Forest areas, where it was not originally present, becoming the primary epidemiological driver of the BSF scenario in these regions as a vector of R.rickettsii to multiple animals and humans [45]. Conversely, the closely related A. cajennense s.s. remains restricted to Amazon‐Cerrado interface areas, with no evidence of geographic expansion or significant role in pathogen transmission to humans or animals, despite its low host specificity [25].

Although neither saliva affected macrophage viability, both appeared to alter cell morphology, making them more rounded and compact, similar to the effect of the steroidal anti‐inflammatory dexamethasone and distinct from PMA stimulation. Both salivas increased ROS production and enhanced zymosan phagocytic activity, particularly with A. sculptum saliva.

Unlike Rhipicephalus microplus saliva, which reduces cell adhesion [46], the Amblyomma saliva in this study did not influence macrophage adhesion. However, adhesion here was assessed on polymer plates, differing from the previous study that evaluated R. microplus saliva's effect on peripheral blood mononuclear cells (PBMCs) adhesion to bovine umbilical vein endothelial monolayers (BUVECs), reducing leukocyte‐endothelium adhesion. Cell‐to‐cell adhesion was not tested in this study and tick saliva is known to interfere with adhesion by competing with surface adhesion molecules or downregulating their gene expression [46]. Adhesion to polymer plates may involve mechanisms distinct from those targeted by tick salivary proteins.

Saliva from Ixodes ricinus at 20 μg/mL inhibited superoxide and nitric oxide (NO) production by macrophages activated by Borrelia afzelii , preventing bacterial killing and demonstrating saliva's ability to modulate ROS release [47]. Intriguingly, in this study, ROS release was significantly higher in macrophages incubated with A. sculptum saliva. Components in this Amblyomma saliva may activate ROS production independently of pro‐inflammatory pathways, possibly via anti‐inflammatory modulation [48] or non‐inflammatory contexts [49]. Serpins are unlikely candidates, as R. microplus serpins do not affect NO production by peritoneal macrophages [40]. The molecules responsible for ROS stimulation remain unidentified in this study, as does their link to inflammatory processes.

Saliva from Dermacentor variabilis reduces zymosan phagocytosis by IC‐21 macrophages at 0.27–0.54 μg/mL but not at 0.135 μg/mL [50]. Another study showed inhibition of B. afzelii phagocytosis by macrophages incubated with I. ricinus salivary gland extract [51]. In contrast, both A. sculptum and A. cajennense s.s. salivas increased macrophage phagocytosis of opsonized zymosan suggests activation pathways yet to be elucidated. Recombinant serpins from R. microplus do not alter macrophage phagocytosis [40] and serpins were among the major proteins identified here, implying other proteins drive this effect. Notably, this study used total salivary protein concentrations ~50× higher than Kramer et al. [50].

A. sculptum saliva stimulated TNF‐α production, while A. cajennense s.s. saliva induced IL‐6, indicating pro‐inflammatory proteins in both. Pro‐inflammatory activity has been reported for recombinant salivary proteins from Amblyomma americanum resembling insulin‐like growth factor‐binding proteins (rAmIGFBPs), which activate both PBMC‐derived and RAW 267.4 macrophages to express co‐stimulatory markers and cytokines [52]. Conversely, I. ricinus and A. sculptum (published as A. cajennense ) salivas inhibited TNF‐α and IL‐6 production by DCs incubated with Borrelia afzelii or tick‐borne encephalitis virus ( I. Ricinus saliva) and cells not incubated with any bacteria ( A. sculptum saliva), but cytokine quantification in these records occurred at longer incubation times (24–48 h) than in this study, suggesting delayed inhibitory effects [18, 53, 54]. Curiously, the protein concentration of the salivas in this study was nearly three times higher than that reported by Carvalho‐Costa et al. [18]; however, this effect was corrected by diluting the salivas in the cytokine production assays at a ratio of 1:40, resulting in a final protein concentration similar to that employed in the cited study, which used dilutions ranging from 1:10 to 1:1000.

Proteomic analysis indicated the presence of an insulin‐like growth factor‐binding protein (AsIGFBP) in A. sculptum saliva but not in A. cajennense s.s. saliva. Although its emPAI value was lower than that of other proteins, it may have contributed to TNF‐α induction. For A. cajennense s.s., other proteins likely mediated IL‐6 induction. Further studies with isolated proteins (e.g., recombinant) are needed to confirm their roles in inflammatory events.

In contrast to these pro‐inflammatory effects, each species' saliva exhibited distinct anti‐inflammatory modulation: A. sculptum significantly reduced IL‐1β production, while A. cajennense s.s. stimulated IL‐10. IL‐1β suppression after 4 h mirrors findings for Ixodes scapularis saliva in BMDMs stimulated with Anaplasma phagocytophilum [55]. Interestingly, D. variabilis saliva stimulated IL‐1β at much lower protein concentrations (7–20 times lower than this study) and this effect was attributed to prostaglandin E2 [56], which was not measured here. IL‐10 induction by tick saliva has been reported for A. sculptum and D. variabilis [18, 50], though other studies found no effect [56].

The highest emPAI values were associated with homologous proteins to microplusin and vitellogenin, indicating that these were the most abundant proteins in the saliva of both tick species analysed. Microplusin has previously been reported as secreted in the saliva of R. microplus and expressed in the salivary glands of Amblyomma aureolatum [43, 57], whereas vitellogenin has been described in the saliva of R. microplus and Ornithodoros moubata [43, 58]. This is the first report of these proteins being secreted in the saliva of A. cajennense s.l. ticks, although the transcription of antimicrobial peptides has already been reported in salivary glands of A. sculptum [20]. Microplusin is an antimicrobial peptide, while vitellogenin functions both in energy metabolism and as a protein source for larval development within the egg [59, 60]. Although the immunomodulatory activity of these proteins has not been tested in this study, antimicrobial peptides are known to play significant roles, whereas the purpose of vitellogenin secretion in tick saliva remains poorly understood. Their abundant presence in the saliva of both species may suggest involvement in modulating the host's innate inflammatory response.

The expression of pro‐ and anti‐inflammatory salivary proteins by ticks may occur due to the need to balance these molecules in regulating the host's immune response according to the time required for each stage of parasitism—from attachment and engorgement to detachment—which may vary depending on the host species (each host provides specific immune resistance to the tick) or the presence of microorganisms that activate pro‐inflammatory pathways, such as Borrelia spirochaetes or tick‐borne encephalitis virus [52, 53, 54].

In this study, phylogenetically related species of the A. cajennense s.l. complex occurring in Brazil and fed on rabbits differentially modulated TG‐elicited macrophage activity. Although their salivary proteomes were broadly similar, subtle differences appear to influence the pro‐ versus anti‐inflammatory balance associated with each species' saliva. Further studies using isolated proteins are essential to identify inhibitory and stimulatory components and to determine whether these differences are related to vector competence for bacterial pathogens (e.g., Rickettsia) or to the adaptation of these ticks to new hosts in landscapes altered by anthropogenic pressures. Additionally, complementary normalisation strategies such as the use of internal markers or fractions enriched in secreted proteins should be employed to refine the functional comparison between species.

Author Contributions

A.d.A.R.A. participated in the idealisation of the article and writing, provided ticks for the colonies and together with A.L.P.M.C. and K.M.T., tick rearing, saliva collection and animal handling. V.P.d.R. and S.d.S.S. participated in murine macrophage collection, all assays with macrophages in culture, data collection and analysis. J.P.Z. and J.F.M. participated in contributions on experimental design and writing. All authors participated in the interpretation and discussion of the results. G.P.C.E., J.A.M.E. and F.C.S.N. performed and interpreted the proteomic analysis of the tick salivas.

Funding

This study was supported by Fundação Rondônia de Amparo ao Desenvolvimento das Ações Científicas e Tecnológicas e à Pesquisa (FAPERO) with grant number 0012427579201852.044/2018, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Grant 311696/2021‐0). The authors thank the Network Technological Platforms from FIOCRUZ for the support and financing of the services provided by the Bioprospecting and Molecular Interaction (RPT‐10B) and Microscopy (RPT‐07I) facilities/FIOCRUZ‐Rondonia. Juliana Pavan Zuliani was a recipient of a productivity grant 311696/2021‐0 from CNPq.

Ethics Statement

The procedures with animals (New Zealand rabbits— Oryctolagus cuniculus ) were carried out in accordance with the ARRIVE reporting guidelines and were approved by the Ethics Committee on the Use of Animals of Fiocruz Rondônia (CEUA/Fiocruz‐RO) under protocol number 2016/09.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Data S1: pim70072‐sup‐0001‐DataS1.xlsx.

Data S2: pim70072‐sup‐0002‐DataS2.xlsx.