Immune System and Its Relationships with Pathogens: Structure, Physiology, and Molecular Biology

2.1. Ticks’ immunobiological response

The immune response of ticks as well as arthropods includes both cellular and humoral mechanisms, where the hemolymph and other tissues, such as salivary glands, midgut, hemolymph, and fat body, provide the principal source of molecules and cells involved in the immunological attack of pathogens. In the case of tick’s hemolymph, many pathways involved in the immune response still remain unclear [4]. Currently, few reports explain the type of response that ticks have against different infectious agents that, in turn, could be used as target to pest biocontrol.

3.1. Hemolymph

All tissues in ticks, and other invertebrates, are bathed in a fluid known as the hemolymph, which is the first source of nutrients, osmoregulation, and molecules and hormones transport, and provide protection to pathogen agents to which ticks are exposed [1]. Likewise, hemolymph coagulates at the site of some injury, preventing microbes spreading into the body tissues. The hemolymph consists of protein-rich plasma and different types of cells called hemocytes that play a transcendental role in immune response [2].

3.2. Phagocytosis

Phagocytosis is a complex mechanism that involves the recognition, engulfment, and destruction of pathogens. In this process, the immune cells recognize pathogen-associated molecular patterns (PAMPs) produced by several bacteria and fungi. In all arthropods, phagocytosis is carried out by the hemocytes and represents the first primary defense response to pathogen infection [11, 14]. In ticks, the phagocytosis process has been regulated by granulocytes type I and plasmocytes and sometimes by granulocytes type II, suggesting that differences in hemocytes’ population have different roles and contributions to the tick’s immune response [15]. In initial steps, the phagocytic cell response is binding receptor-mediated to pathogen cell surface; subsequently, signal transduction pathways are activated and followed by filopodia projections that surround and engulf the bound particle [16]. The particle is internalized by endocytosis into a vesicle, subsequently, with lysosomal compartments that in turn form the phagolysosome. Inside, intracellular enzymes are activated such as acid phosphatases, type c lysozyme, cystatins, and proteases completing the cellular lysis. Little is known about the molecular regulation in tick immune response, some reports suggested that as in insects the most important signal transduction pathways are mitogen-activated protein kinase (MAPK) and FAK/Src pathways that in turn are involved in proPO activation [16]. Moreover, several external factors are capable to enhance this process. Currently, recent evidence indicates that R. microplusproduces reactive oxygen species (ROS)-mediated oxidative burst modulated by protein kinase C, similar to that found in leucocytes [17].

3.3. Nodulation

Tick hemocytes are capable of expressing lectins on membrane surface involved in pathogen recognition. These molecules can join with lipopolysaccharides (LPS) also present on the pathogen surface. Currently, several lectins involved in the immune response and other mechanisms have been identified in tick hemocytes and different cells [18, 19, 20, 21]. In soft ticks, O. moubatawas described to have a protein called Dorin-M, lectin with high hemagglutinating activity and isolated from hemocytes, and hemolymph plasma [22]; likewise, I. ricinuswas described to have the Ixoderin A, lectin found in midgut and hemocytes [23]. The protein-carbohydrate interaction confers the ability to hemocyte aggregation that results in the pathogen entrapment and, in turn, the opsonization through lectins that may also cause bacteria aggregates [24].Thus, lectin recognition leads hemocyte recruitment that builds a sticky mass around the bacterial aggregate (nodules), preventing the dissemination of pathogens and eventually digesting it. The formation of this nodule represents a predominant cellular immunity defense mechanism to bacterial challenges [25].

3.4. Encapsulation

Encapsulation is the immunological process whereby the arthropods are capable of attacking pathogens that are very large to eliminate by nodulation or phagocytosis. Other immunological processes, such as the proteolytic degradation of microbial products (LPS and peptidoglycan), can result in the prophenoloxidase activation. This activation generates phenoloxidase expression that in turn, along with tyrosine metabolism, is directly related to melanin synthesis. In all insects, pathogen encapsulation involves melanization, where hemocytes, mainly type I granulocytes and plasmatocytes, form a capsule of thick layer around the pathogen that leads to asphyxiation and toxic-free radical production, such as quinones and semiquinones [3, 26, 27], with melanin deposition as the final step [10]. In ticks, phenoloxidase is present in O. moubatahemolymph [28], in contrast to A. americanum, D. variabilis, and I. scapularis, where there are no reports to phenoloxidase activity [29]. In this regard, in hard tick D. variabilis, the simple injection of plastic bead can induce the capsule formation, but without the presence of melanization [30]. These findings suggest that this pathway is present in ticks; however, it has a distinct role in metabolism or immune response. Moreover, genomic analyses in VectorBase indicate the absence of gene homologs for the complete pathway in I. scapularisgenome sequence [31].

4.1. Antimicrobial peptides (AMPs)

Antimicrobial peptides (AMPs) represent the most effective humoral immune response, for their ability to kill several pathogens, for their fast response, and for their effectiveness at micronanomolar concentrations. AMPs are small peptides (3–20 kDa), and their action mechanism is based on their capacity to cell membrane or cell wall binding, causing structural disruption that results in loss of pathogen membrane potential. AMPs are secreted mainly by the fat body and hemocytes; however, midgut is capable to produce some peptides [2]. Many authors reported and identified several AMPs in ticks, including microplusin [35], hebraein [36], ixodidin [37], antimicrobial peptide (ISAMP) [38], and some peptides from Amblyomma hebraeum[39].

4.2. Hemagglutination (lectins)

Lectins are proteins whose structure has domains with specific binding sites for carbohydrate [54]. The bacteria membrane or cell wall including the fungi and protozoan pathogens has different carbohydrate moieties that can be recognized by lectins. These proteins exhibit different molecular sizes from 30 to 85 kDa and have been identified in the membrane surface of hemocytes, cell gut, and salivary glands, or synthesized by hemocytes and released in the hemolymph plasma of soft and hard tick species. In invertebrates, lectins are important mediators of immune response. Initially, these molecules are defined by their participation in a hemagglutination process; however, these proteins also bind to pathogens that, in turn, enable hemocytes to recognize and engulf (opsonization). This process includes carbohydrate recognition by ficolins and mannose-binding gal-lectins, among others [55, 56]. Insects, ascidians, crustaceans, and ticks contain molecule type TLP-1 and TLP-2 lectins (Tachypleustridentatus), to form molecule clusters that bind and immobilize pathogens [10, 23, 55, 56, 57]. The mechanism causes pathogen be trapped and immobilized forming aggregates, which later are surrounded by hemocytes and destroyed by encapsulation or nodulation [58]. Tick lectins are involved in processes of cell adhesion, recognition, opsonization, phagocytosis, and cytolysis of infecting pathogens [59]. The first tick lectin reports were in the papillipes Ornithodoros tartakovskyand O. tholozani[60, 61]. Subsequently, in O. moubatahemolymph plasma was identified the lectin Dorin M, lectin to 640 kDa, synthesized in the hemocytes and secreted into the hemolymph plasma, which has a high hemagglutinating capacity [19, 57, 62]. In this regard, molecular structure studies showed that Dorin M lectin has a fibrinogen-like domain related to the ficolin family of proteins that recognizes carbohydrate sequences, especially sialic acid and N-acetyl-D-glucosamine, similar to the tachylectins of T. tridentatus[57]. OMFREP is a potential lectin in O. moubataand has been identified in hemocytes [23]. The use of the bioinformatics approach complemented with molecular studies results in the identification of fibrinogen-related protein that presents a 65% identity and a similar tissue distribution to Dorin M [23]. Likewise, novel galectin (OmGalec) has a different tissue and stage distribution in O. moubata[21]. This protein has galactose-binding properties and consists of tandem repeated carbohydrate recognition domains, where the carbohydrate affinity typical motif is present [21]. In hard tick I. ricinus, Ixoderin A is expressed by hemocytes and is present in midgut and salivary glands, while Ixoderin B is only expressed in salivary glands. These findings suggest that the lectin and isoforms have a selective expression in different tissue, plasma, and cells, suggesting that they have specific roles; however, many pathways are still unknown. In salivary glands and the midgut of Rhipicephalus appendiculatuswas described a lectin that was related to a significant increase of sugar, which inhibits hemagglutination during Theileria parvainfection, suggesting a decisive role in this process [63, 64]. Recently, it was demonstrated that some lectins are involved in various processes related to feeding [65], ticks’ immune cell regulation, and molecule recognition. Interestingly, the cloning and protein expression in ticks’ hemocytes and salivary glands of two fibrinogen-related proteins, which present high homology with Dorin M lectin, showed that they are essential in the pathogen transmission [20].

4.3. Proteases and protease inhibitors

5.1. Nitric oxide synthase

The nitric oxide (NO) is an unstable radical, capable to act with a key factor in several physiological and pathological pathways, and it is synthesized by the nitric oxide synthase (NOS) [91]. In invertebrates, including ticks, NO is related with a cytotoxic action against pathogens from hemocytes, derivates to phagocyte process during microbial infection [92]. Now, three NOS isoforms have been described: the classic isoform inducible nitric oxide synthase (iNOS), the endothelial isoform (eNOS), and the neuronal isoform (nNOS) [91, 93]. Currently, the gene that codified for NOS has been identified and cloned from the insects: Drosophila melanogaster, Anopheles stephensi, Anopheles gambiae, and Rhodnius prolixus, suggesting the NO activity is present in these arthropods [94, 95, 96, 97, 98]. Moreover, the activity of NOS was reported in the salivary gland of hematophagous insect Rhodnius prolixus, and the enzyme activity was FAD, NADPH, tetrahydrobiopterin, calmodulin, and Ca²⁺ dependent, suggesting high functionality, similar to NOS enzyme expressed in vertebrates [99]. Likewise, Litopenaeus vannameishrimp is capable of producing nitric oxide, in response to Vibrio harveyiinoculation, derivates to NOS activity [93]. In ticks, the activity of eNOS enzyme was reported in Dermacentor variabilissalivary glands, and by in silicoanalysis, the presence of NOS gene sequence was demonstrated in Ixodes scapularisembryonated eggs [91, 100].

5.2. Oxidative stress and detoxifying protein

In hematophagous arthropods, blood ingestion is the determinant of survival. However, during feeding and digestion, several toxic molecules are produced, such as reactive nitrogen species (RNOS) and reactive oxygen species (ROS) [101]. The protection against nitrosative and oxidative stress is carried out by detoxification agents, produced largely by the midgut epithelial cells. In many insects, enzymes such as peroxiredoxins, catalases, and many members of antioxidant peroxidase family function as antioxidant agents. However, in arthropods, as in many organisms, the microbial infections are capable to induce oxidative stress. Suppression of pathogen ROS and RNOS induction in midgut facilitates the infection and microbial tissue dispersion [102]. Interestingly, many arthropods have the capacity of enhancing ROS and RNOS against pathogen infection while simultaneously protecting their tissue cells with antioxidants. In this regard, the oxidize enzyme nicotinamide adenide dinucleotide phosphate (NADPH) of D. melanogaster, known as dual oxidase (dDuox), is capable to kill and/or inhibit the pathogen proliferation, through the oxidative burst [103]. Moreover, the glutathione S-transferases (GST) family plays an important role during oxidative stress caused by pathogens, through detoxification enzyme reactions and, in turn, removing the formatted ROS and RNOS [104]. In midgut from D. variabilistick, GST isoforms DvGST1 and DvGST2 are upregulated during blood ingestion [105], and during the B. burgdorferiinfection in tick I. ricinus, several GTSs are overexpressed in response to bacterial invasion [106]. In ticks, other detoxification enzymes have been reported, such as glutaredoxins, glutathione peroxidases, phospholipid-hydroperoxidases, thioredoxins, and one superoxide dismutase [107, 108, 109]. However, in ticks, the precise role in antimicrobial control of detoxification agents is still unclear.

5.3. Phenol oxidase and melanization

In arthropods, mechanical injury or the presence of foreign objects including pathogens results in melanin deposition around the damaged tissue or around the foreign object that in turn forms a capsule isolating the foreign particle. Melanins are molecules produced in the hemolymph by different types of hemocytes. The key enzyme for the melanization process is the phenol oxidase (PO). The metabolic pathway is initiated by hydroxylation of phenylalanine to tyrosine, followed by a series of reactions, resulting in 5,6-indolquinones, synthesized to phenol quinones, and these quinones polymerize to form melanin. The production of melanin is noticed by a dark and/or blackened color in the arthropod [110, 111, 112]. The signaling pathway starts with a hemocyte prophenol oxidase enzyme (PPO) synthesis (PO inactive form) that results in the conversion of the PPO into the active form by serine protease cascade [113]. This molecular system is capable to recognize picomolar of bacterial lipopolysaccharide (LPS), peptide glycans, and fungi β-1,3-glucane. The intermediary components of this pathway, such as semiquinones, ROS, and melanin, are all very toxic to pathogens [114]. On the other hand, the PPO-PO pathway in tick is little known. However, at the present, some studies in Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularisticks report the presence of genes involved in the PPO-PO pathway; however, the enzymatic activity has not been reported [29]. In the tick O. moubata, the PO enzyme has been reported in hemolymph plasma and in the fourth of ecdysiast nymphs [28]. However, currently, the presence of PO in ticks is controversial.

6.1. Regulation of innate immune system in ticks

The innate immune systems represent one aspect in a generalized response to several pathogens and are composed of individual factors. This variability has a particular behavior in each tick. The principal components are the hemolymph and hemocytes; however, they are not the only factors. The response depends on the pathogen type, tissue, sex, life cycle phases, and tick species, among others. In this regard, innate immunity starts when membrane receptors recognize component characteristics of bacterial cell surfaces as peptidoglycans or lipoteichoic acid, which leads to synthesis of antimicrobial peptides (AMPs) as defensins, cecropins, attacins, and lysozyme that disrupt the cell wall structure, leading to cell death [2]. Other components in the fungi cell wall are beta-1-3-glucans and beta-1-3 mannose or 2-keto-3-deoxyoctonate LPS, characteristic of Gram-negative bacteria, leading to soluble lectin synthesis [2]. These cell wall components and foreign molecular structures are known as pathogen-associated molecular patterns (PAMPs) [2, 115]. In D. variabilistick, different analyses show 56 genes involved in the immune response; however, these genes do not appear to be regulated. In sexual term, transcriptome analysis in the male reproductive structures of this tick showed seven contigs related to a dual reproductive and immune response [116]. However, the complete role of these peptides is still unknown, but their presence in seminal fluids suggests a role in the clearing of bacteria introduced during mating [117]. In the tissues, the immune response includes several factors such as AMPs, peritrophic membrane, proteases, and protease inhibitors, lectins, detoxificant proteins, and oxidative stress [3]. Transcriptome analyses in D. variabilismidgut show 8 transcripts related to the innate immune response, of which one protein (MD-2) is involved in lipid-domain recognition, lectins, and in turn involved in inhibition of macrophage activation [118]. Moreover, transcriptome analyses of tick salivary glands found AMPs, proteases, and protease inhibitors related to innate immune response [119, 120, 121]. The synganglion transcriptome of D. variabiliscontains 0.27–1.15% peptides, depending on the gene ontology, that represent between 4 and 11 genes [122] and includes AMPs, proteases, lectins, protease inhibitors, and regulatory Toll-like proteins [116]. On the other hand, the widespread response has been initiated by hemocyte cell pathogen recognition carried out by the presence of microbe-associated microbial patterns (MAMPs), expressed in pathogen’s membrane surface. However, in ticks, the hemocyte receptors to MAMP’s recognition are still unknown, but analyses reveal similar receptors to those identified in insects [26]. In this regard, homologs to peptidoglycan receptor proteins (PGRPs), Gram-negative–binding proteins (GNBPs), and gal-lectins have been reported [123]. To identify the pathogen type, the hemocyte cells need to be activated, using specific receptors that result in a specific signaling pathway [116]. The fruit fly D. melanogasterhas been used as a genetic model to elucidate the activation of the innate immune system, which is an evolutionarily conserved mechanism in eukaryotes. Drosophila has three pathways involved in an immune response: Toll, Imd, and Hop, homologs to TLR, TNFα, and Jak/STAT in mammals [124]. Different components of these three signaling pathways were found in tick’s database, such as in Toll pathway, Toll, MyD88 and Pelle. In Drosophilamodel, the fungal pathogens and Gram-positive bacteria activate the Toll cascade, which is composed of different Toll-like receptors (TLRs) capable of recognizing diverse types of PAMPs [2, 115, 125, 126]. After pathogen recognition, intermediate effectors such as myeloid differentiation factor 88 (MyDD88), Tube, and Pelle are activated followed by activation of transcription factor Dorsal (homolog of NF-kB) and Dorsal-related immunity factor (Dif) that translocates into the nucleus and regulates the AMP synthesis [126]. From the Imd pathway, Dredd, Caspar, and Relish have been found. Gram-negative bacteria infection activates the Imd cascade through the recognition of DAP-type peptidoglycan in the membrane protein peptidoglycan (PGRP-LE) [115, 125] and activates molecules such as TAB2/TAK1, JNK, IKK, and Relish inducing the transcription of AMP [115, 126]. Finally, from the Hop pathway, JAK and STAT have been found [123]. In the absence of infection, a selective repression of this IMD/Toll-dependent AMP pathways is achieved by the home box gene Caudal [127, 128]. Recently, the RNA interference (RNAi) pathway has been described that regulates the immune system in arthropods including ticks. This process is crucial in the innate response to viruses that infect and are transmitted by ticks [129]. In Drosophila, the RNAi mechanism is related to a virus penetration and regulation of innate immune response in the midgut. The RNAi pathway in ticks is unknown; however, its components are described in some ticks [130], as RNAi has been used to silence genes involved in several mechanisms, suggesting that RNAi pathway is active in some tick species, which would explain the different capacity of ticks to transfer several viruses [129].

6.2. Advance in molecular, functional genomics and proteomics in tick-host-pathogen interaction

Advances in gene identification and expression in tick tissues are being achieved by the use of expressed sequence tag (EST). The EST analyses correspond to partial sequence of acid nucleic from different random clones included in a cDNA library, obtained from the interest tissue mRNA [131]. The analyses include the translation of EST sequence to amino acid sequence and compared with a public genome database. Interestingly, salivary gland genes of ticks show differential expression during blood ingestion, suggesting that processes are involved in homeostasis, tissue remodeling, immune defenses, angiogenesis, and the facilitation of the transmissible pathogen establishment [132]. The EST library from unfed hard tick larvae of R. micropluswas the first study reported [133]. However, 234 unique ESTs were identified, and 39% of them were not found in genome database. In A. americanum, cDNA libraries showed that 1462 and 480 ESTs (adult and larvae respectively) presented 56% to no-similarity identified in encoded proteins [134]. On the other hand, R. microplusgene expression analyses from cDNA library obtained from RNA tissue larvae exposed to different stimuli and infected with Babesiashowed that 8270 unique sequences were identified to 11,520 total sequenced clones and presented a 44% of shared similarity to database sequences [135]. A meta-analysis was done, which describes the transcripts from salivary glands of several species of ticks, including the salivary gland transcripts from unfed male of I. ricinusand A. americanus; fed female of I. pacificusand A. variegatum; unfed female and unfed-fed nymphs of I. scapularis; and finally unfed males and fed female of Dermacentor andersoni. All tick groups analyzed were from different ages and different feeding times, or unfed. The results showed that the secreted proteins comprised 49% from which 15% were no match with any gene reported in silicoanalyses. Interestingly, transposable elements were found in 0.5% of the transcripts, which suggest gene rearrangements. In the exclusive case of females, differential gene expressions of transcripts were showed. The unfed female showed no change in expression, while fed female showed the highest number of overexpressed variants. All biologically relevant genes are likely redundant and encode antigenic variants, in turn identifying gene families involved in hemostatic deregulation. Other identified genes include cystatins, lectins, cysteine and glycine-rich peptides, and protease inhibitors [119, 132, 136, 137, 138]. A very important finding in R. appendiculatustick showed that tick-borne pathogen presence did not modify the gene expression. In this regard, no significant differences were found in the expressed transcripts of 9162 ESTs from salivary glands of R. appendiculatusuninfected, compared with the 9844 ESTs obtained from salivary gland of R. appendiculatusinfected with Theileria parva[139]. Currently, the genome sequence of several arthropod vectors including ticks is under development, and partial results of I. scapularissequencing efforts reveal that deer tick genome is approximately 2.1 Gbp; likewise, the hard tick R. microplusgenome contains 7.1 Gbp [140]. It is remarkable that cattle tick genome is more than twice the size of the human genome that contains 3–2 Gbp [141]; furthermore, the cattle tick genomes are larger than most insect species genomes.