Acquired Tick Resistance: The Trail is Hot

INTRODUCTION

Ticks are obligate hematophagous ectoparasites distributed worldwide and serve as vectors of human and animal diseases ¹. The focus of this review is on Ixodid or hard ticks, one of three families within the suborder Ixodida that includes about 700 species ². Ixodid ticks are vectors of numerous human and livestock diseases ³. Since the bite of a tick is the only route of natural transmission of tick-borne pathogens, several strategies have been explored over the last few decades to prevent getting bitten by ticks. Acaricides, a first-line strategy used to control tick populations in endemic areas, may be detrimental to the environment, and ticks rapidly develop resistance to acaricides making them ineffective ^{4, 5}. Biocontrol measures using entomopathogenic fungi have also shown promise in controlling diverse Ixodid tick populations ⁶. However, the logistical difficulties in the implementation of biocontrol strategies are hampered by environmental variables including temperature and humidity. Vaccines that can target and impair tick feeding or fecundity have been shown to be effective at controlling tick populations ⁷. Tick infestations of livestock animals result not just in disease transmission, but also in weight loss and anemia and this impacts milk and meat production resulting in significant economic losses to the animal industry ⁸. From the veterinary disease perspective, controlling tick densities at specific localities is, therefore, highly valuable. This is exemplified by the success of the Bm86 protein subunit vaccine, the only commercially available anti-tick vaccine, based on a gut protein of Rhipicephalus microplus that impairs tick feeding and significantly reduces R. microplus infestations on cattle ⁹.

While reducing tick populations in endemic areas also impacts human disease prevalence by reducing the probability of tick encounters, vaccines that can efficiently prevent disease transmission are also possible ¹⁰. Traditionally, transmission-blocking vaccines have largely relied on targeting the pathogen, and not the vector. A vaccine to prevent tick-transmitted tick-borne encephalitis virus (TBEV) infection of humans is available for human use in Europe ¹¹. The vaccine is an antigen extract derived from in vitro grown TBE virus that is filtered and inactivated in formaldehyde ¹². In the USA, a vaccine to prevent Lyme disease based on OspA, a surface antigen of the Lyme disease agent, was approved for human use in 1998 ^{13, 14}, but was withdrawn from the market in 2002 by the manufacturer ¹⁵. Global warming is affecting the spread of diverse vectors of human and livestock pathogens, including ticks and tick-borne pathogens ^16-18. In the past two decades alone, several newly recognized tick-borne pathogens have been recognized in the Western hemisphere ¹⁹. Therefore, there is an urgent need to develop vaccine strategies that would simultaneously reduce tick populations and also prevent transmission of tick-borne pathogens to mammalian hosts. Towards this goal, several research efforts are focused on identifying tick antigens that may serve as effective vaccine targets to thwart tick feeding and consequently also prevent pathogen transmission. The sequencing of transcriptomes and genomes of multiple tick species in the last decade ^20-24 coupled with technological advancements have added critical molecular tools ^25-27 and collectively accelerated our efforts to gain functional insights into the tick genome.

While, we have overcome the paucity of genomic data that plagued tick research a decade ago, the task of sifting through these meta datasets to identify critical antigens that may serve as vaccine targets remains a daunting challenge. Providing a functional paradigm to address this challenge is the phenomenon of acquired tick resistance or ATR ²⁸. Seminal observations by William Trager in 1939 showed that non-permissive hosts mount a robust immune response to critical tick salivary antigens, and thwart tick feeding. Research efforts to identify salivary proteins targeted by ATR have yielded a diverse list of salivary antigens with functions relevant to tick feeding ^29-32. However, immunity elicited against these antigens individually or as subset of cocktails have only partially recapitulated ATR. It is clear that the molecular basis of this phenomenon that has remained a puzzle ^{33, 34} poses a bottleneck in our ability to fully exploit this robust paradigm towards defining tick salivary vaccine targets. This review will examine our current and expanding understanding of ATR, and highlight how this understanding might reveal key events at the tick-host interface that enable or disable tick feeding. This understanding will educate the prioritization of salivary antigens that may be vaccine targeted and guide the development of an anti-tick vaccine.

TICK SALIVARY PROTEOME: PARAMOUNT FOR TICK FEEDING

Hard ticks (Ixodidae) are obligate hematophagous arthropods and obtain their blood meal by attaching to the skin of vertebrate hosts, tearing the skin and feeding from the pool of blood formed at the bite site. Successful hematophagy is central to the completion of the life cycle of the hard tick. Since the life cycles of the pathogens transmitted by hard ticks are entwined with that of the tick, there is significant impetus to gain a molecular understanding of how ticks acquire a bloodmeal. Tick attachment to the host involves tearing the skin, which is accomplished by a pair of barbed, articulated chelicerae at the tip of the mouthparts. The chelicerae flex and retract, inserting the hypostome and pushing the tick further into the host until the mouthparts are completely embedded into the dermis of the skin ³⁵. Slicing mouthparts create a feeding lesion into which saliva is secreted at continuous intervals over the course of the 4-10 day feeding period typical for Ixodes complex ticks. In order to remain stably tethered to the host over the prolonged feeding period the tick secretes an adhesive cement within hours of attachment to the host ³⁶. Tick cement is shown to be composed of a mixture of proteins, lipids, amino acids such as glycine, serine and tyrosine, and carbohydrates ³⁷ that together form a viscous gel-like cone composed of a core cement that fits snugly around the hypostome and a cortical cement secreted outside the core cement ³⁷. Providing an impenetrable physical barrier to the tick mouth parts is thought to be one of the prime functions of tick cement ³⁸. Until a decade or so ago, only a descriptive understanding of tick cement was available ^{39, 40} . Advances in molecular techniques, and the availability of an artificial feeding system for ticks have helped circumvent qualitative and quantitative limitations in cement research ³⁷ Cement-specific proteins have been identified predominantly from Rhipicephalus species ^41-44 and from Amblyomma species ^{45, 46} with potential antimicrobial and antihemostatic activities. A cement antigen from Rhipicephalus appendiculatus named 64P ⁴¹ was shown to be represented in multiple Ixodid tick species ⁴⁷ with the potential to serve as a broad-spectrum anti-tick vaccine. Further progress in the molecular understanding of tick cement may reveal novel strategies to thwart tick feeding.

Tick saliva contains an array of immunomodulators, anticoagulants, and hemostatic compounds, which allow the tick to manipulate and maintain the feeding site and subvert host defenses ^48-50 and is pivotal for the tick to feed to repletion. An overview of the predominant functions elaborated in the saliva of hard ticks is provided in Figure 1. The first step in hemostasis is the development of a platelet plug and salivary proteins that interfere with this process are secreted by multiple hard tick species. Prostacyclin ⁵¹ and the serine protease inhibitor (serpin) IxscS-1E1 ⁵² from I. scapularis, IRS-2 from I. ricinus ⁵³, R. microplus serpins, RmS-3 and RmS-17 ⁵⁴, and Variabilin from D. variabilis ⁵⁵ disrupt platelet aggregation. In addition to platelet aggregation, multiple pro-coagulation factors are activated by the host to prevent blood loss. Several I. scapularis salivary proteins that inhibit various steps of coagulation have been identified including, Salp14, ⁵⁶, Innonexin ⁵⁷, TIX-5 ⁵⁸, Ixolaris ⁵⁹, and Penthalaris ⁶⁰. Other anticoagulants from Ixodes ticks include metalloproteases ^61-64, various serpins ^{52, 65, 66}, Ir-CPI ⁶⁷, Iris ⁶⁸ and Rhippilin-1 and −2 from R. hemaphysaloides ⁶⁹. Haemaphysalis ticks have a particularly large number of demonstrated and putative thrombin inhibitors, including: madanin-1 and −2 ^{70, 71}, chimadanin ⁷², and the serpin HLS1 ⁷³ from H. longicornis; haemathrin from H. bispinosa ⁷⁴; and serpins HDS1 and HDS2 from H. doenitzi ⁷⁵. Iris from I. ricinus ⁶⁸ has also demonstrated anti-thrombin activity, in addition to BmGTI ⁷⁶, the serpin RmS-15 ⁷⁷, BmAP ⁷⁸, and microphilin ⁷⁹ from R. microplus. Haemaphysalis proteins longistatin ⁸⁰ and enolase ⁸¹ stimulate this pathway by activating plasminogen, that negatively regulates the coagulation cascade.

Figure 1. Hard tick attached to host skin and secreting pharmacologically active salivary components into the feeding lesion.

A. Ixodid ticks attach to the host skin by inserting their hypostome into the dermis of the host skin and adhere firmly with the help of salivary cement that is deposited at the bite-site and around the hypostome. Saliva secreted into the feeding lesion thwarts defense responses of the host. SG, salivary glands; Mc, macrophage; N, neutrophils; Lc, Langerhans cells; Dc, dendritic cells; E, eosinophils; Mt, mast cells; B, B-cells; L, lymphocytes; Cg, procoagulants; C, complement factors. B. Brief overview of predominant salivary functions characterized in tick saliva. Percent calculations based on the literature surveyed in this review.

Histamine released at the bite site by platelets, basophils and mast cells can be detrimental to tick attachment by inducing pain and itch responses at the bite site that eventually result in the host grooming the tick off ^{33, 82}. Inhibiting histamine-related inflammation is therefore essential for the tick to remain attached to the host ⁸³. Multiple histamine binding proteins have been identified in the saliva of R. appendiculatus ⁸³, D. reticulatis ⁸⁴, I. persulcatus ⁸⁵ I. ricinus ⁸⁶, and I. scapularis ⁶¹. Ticks also maintain their feeding lesion by interfering with host immune responses. Pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α are down-regulated by salivary proteins such as sialostatins (L, L2, Ip-sL1, and Ip-sL2) ^87-89, Salp15 ⁹⁰, Iris ^{91, 92}, Isac ⁹³, Ir-SPI ⁹⁴, IRS-2 ⁹⁵, and Iristatin ⁹⁶. Other anti-inflammatory salivary proteins include: serpins ⁹⁷, Hl-p36 ⁹⁸, and Longistatin (⁹⁹ from H. longicornis; PGE2 from D. variabilis ¹⁰⁰, and evasins from R. sanguineas ^{101, 102}. Interestingly, Japanin from R. appendiculatus and PGE2 from D. variabilis and I. scapularis ^{100, 103, 104} upregulate the anti-inflammatory cytokine IL-10. A sphingomylenase enzyme activity in I. scapularis saliva was shown to promote IL-4 production by CD4-T cells ¹⁰⁵. These functions of the tick sialome are suggested to skew the host immune response towards a Th2 profile that may be advantageous to the survival of tick-borne pathogens during transmission and is a key element of saliva-assisted pathogen transmission ¹⁰⁶. Tick sialome also encodes functions that prevents immune cell activation and proliferation ^{91, 94, 96-98, 104, 107-112}

Salivary proteins also impair the host complement system that represents a major innate immune response triggered by microbes, cell damage, antigen-antibody complex, and glycans and lead to increased inflammation detrimental to tick feeding. I. scapularis proteins Salp20 and Isac ^113-116; I. ricinus proteins IRAC I and II ^{117, 118} and R. pulchellus protein Cirp-T ¹¹⁹ interrupt the alternative pathway. Tick Salivary Lectin Pathway Inhibitors (TSLPI) in I. scapularis and I. ricinus interfere with the lectin pathway ^{120, 121}. Recent reports suggest that Amblyomma species may encode functions that also impair the classical pathway ⁵⁰. Additionally, antioxidant salivary proteins have been described in Ixodid ticks and function to quench reactive oxygen and nitrogen species generated by immune cells such as neutrophils and macrophages that migrate to the tick bite-site ^{29, 122-124}.

It is important to note that not all salivary proteins are expressed and secreted throughout the course of feeding. The tick sialome is dynamic and the composition changes over time ^{125, 126}, potentially orchestrated by the different phases of tick feeding ¹²⁷. Tick salivary protein functions are also exploited by pathogens as they transit to and from the host skin ^{106, 128, 129}. Therefore, it is logical to envision that salivary molecules may serve as viable vaccine targets to abrogate tick feeding and prevent pathogen transmission. Temporal changes in the salivary composition in conjunction with the functional complexity, and redundancy of the tick sialome have rendered the search for salivary vaccine targets a task akin to searching for the proverbial “needle in the haystack”. An opportunity to circumvent this challenge has come from the phenomenon of acquired tick resistance (ATR), originally described by William Trager in 1939 ²⁸. Since ATR results in thwarting tick feeding and pathogen transmission, expanding a molecular and mechanistic understanding of ATR may offer a robust paradigm to define the critical subset of salivary proteins that may be vaccine targeted.

ANIMAL MODELS OF ACQUIRED TICK RESISTANCE

Trager, in 1939 ²⁸, showed that upon repeated tick infestations of guinea pigs with Dermacentor variabilis nymphs, animals developed an immune response that was potent enough to derail subsequent tick challenges. Tick-immune animals rapidly rejected ticks within the first 24 hours of tick attachment. This phenomenon of acquired tick resistance (ATR) is characterized by visible erythema at the tick bite-site due to the rapid recruitment of immune cells, predominantly composed of basophils and eosinophils and hence termed cutaneous basophilic hypersensitivity in guinea pigs ¹³⁰, also known as Jones-Mote hypersensitivity in humans ¹³¹. The immune responses recruited to the tick bite site on tick-resistant animals are thought to be detrimental to tick feeding resulting in early tick detachment and decreased engorgement weights. Similar observations with additional tick species, as well as the generation of acquired tick resistance in rabbits, mice and cattle ^132-135 have demonstrated that the phenomenon of ATR is widespread in tick-host interactions.

Ticks deposit a diverse set of salivary proteins into the host skin in order to facilitate acquisition of a blood meal, as outlined above. It is generally believed that when ticks feed on non-natural hosts, the host mounts a robust immune response towards the deposited salivary proteins ³³. Attesting to this hypothesis, experiments wherein animals were immunized with salivary gland extract or saliva have shown that immunity against salivary components is indeed capable of eliciting several parameters of tick resistance including erythema at the bite-site, and impaired feeding ¹³⁶. It is however, important to note that that none of the effects were as robust as naturally acquired tick resistance. These immune responses potentially neutralize salivary functions essential for feeding, and salivary functions essential for keeping detrimental cells at bay. Activation and degranulation of basophils is thought to result in the release of basophil components including histamine that is noxious to the tick during the early stages of feeding ^{82, 137}. Whether basophilic components released upon degranulation impairs tick feeding due to host responses such as vasodilation, pain and itching or whether it enters the tick gut and causes damage to the organ is not clear.

Guinea pigs have been shown to acquire tick immunity following repeated tick infestations with Amblyomma americanum ^{138, 139}, Rhipicephalus species ¹⁴⁰, I. scapularis ^{125, 141} and Dermacentor andersoni ¹⁴². Resistance has been observed at the larval, nymphal and adult stages. Guinea pigs and rabbits support I. scapularis feeding, but they are not the natural host species. Peromyscus leucopus, are the natural host for I. scapularis larvae and nymphs and do not develop tick immunity following repeated tick infestations ¹⁴³. Similarly, laboratory mouse models also do not acquire tick resistance after repeated I. scapularis tick infestations by mechanisms that are not understood ^{144, 145}.

ATR has been observed in C57BL/6 mice repeatedly infested with Haemaphysalis longicornis larvae ¹³⁷. Larger mammals such as deer, horse, and cattle, and birds are the natural hosts for H. longicornis, whereas mice are not natural hosts. Cattle have been shown to develop tick resistance following repeated exposures to Rhipicephalus microplus. Importantly, ATR impaired Babesia transmission to cattle ¹⁴⁶. Unlike guinea pigs, mice and rabbits, generation of tick immunity in cattle is dependent on the breed of cattle ¹⁴⁷. Bos taurus indicus cattle develop immunity to R. microplus, whereas Bos taurus taurus do not develop immunity ¹⁴⁷. Several studies have attempted to identify the genetic markers that could account for the differences, but, no clear markers have emerged ¹⁴⁸. Histological analysis of the bite site revealed robust infiltration of basophils, mast cells and eosinophils in the skin of the resistant Bos Taurus indicus cattle. In comparison, the bite site of permissive Bos taurus taurus cattle was primarily infiltrated by neutrophils ^{39, 149, 150}.

Evidence for tick immunity in humans is primarily anecdotal. There have been reported cases of individuals developing hypersensitivity reactions at the tick bite site, similar to the observation in tick immune animals ^{151, 152}. Additionally, people with frequent exposures to tick bites have been shown to develop antibodies to tick proteins ^{153, 154}. Importantly, individuals that report itching at the tick bite site may have a decreased chance of acquiring B. burgdorferi ¹⁵⁵. Collectively, these results suggest that prior exposure to ticks and the development of immunity towards tick proteins could potentially trigger itching at the bite site and grooming to remove ticks early. Since B. burgdorferi is transmitted only after 24-36 hours of tick attachment, ^{156, 157}, provoking the immunological parameters of acquired tick resistance upon tick attachment may offer an effective strategy for preventing tick transmission of Lyme disease ^{125, 141}. Pathogens such as Babesia microti, Anaplasma phagocytophilum and Powassan virus that reside primarily in the salivary glands may be transmitted within 24 hours or earlier ^{158, 159}. Whether, ATR may impact the transmission of these tick-borne pathogens remains to be seen.

MOLECULAR BASIS OF ACQUIRED TICK RESISTANCE

The mechanisms mediating tick rejection are complex and involve multiple components of the host’s adaptive immune system. As described above, cattle and guinea pigs readily acquire tick immunity following multiple tick infestations and have been used extensively to examine the tick bite site ^{143, 160, 161}. Although the reagents required to characterize the immune response in guinea pigs and cattle are not fully available, several studies have identified a response dependent on both cellular and humoral responses. Repeated tick infestation results in robust immune cell infiltration to the bite site. The bite site of I. scapularis immune guinea pigs is heavily infiltrated by heterophils and macrophages by day 2, followed by an intense infiltration of leukocytes on days 3 and 4 ¹⁴³. Infiltrating basophils have also been observed at the tick bite site in guinea pigs repeatedly infested with Dermacentor andersoni and Amblyomma americanum ^{160, 162, 163}, and at the bite site of tick immune cattle and mice ^{82, 161}.

Basophils are a major source for histamine upon activation and have an important role during tick rejection. Additionally, degranulation of basophils plays an important role in the recruitment of eosinophils to the bite site, although the role of eosinophils requires further investigation ¹³⁹. In a study to define the role of basophils, the authors demonstrated that tick rejection was abolished when immune guinea pigs were administered anti-basophil sera prior to challenge with Amblyomma americanum ¹³⁹. However, anti-histamines administered at the time of challenge with A. americanum demonstrated that the critical role of basophils occurs through a histamine-independent mechanism ¹³⁰. This is in contrast to tick rejection with other tick species that demonstrated that anti-histamines delivered to tick immune guinea pigs, mice and cattle ablate tick immunity towards R. microplus, D. andersoni and H. longicornis ^{82, 164, 165}. Overall, these results suggest that basophils are important for immunity against multiple tick species; however, the mechanism of restriction can be species-specific.

Although degranulation of basophils has been demonstrated to be critical for tick rejection, the mechanism of degranulation is not understood. Salivary proteins deposited into the skin during feeding generate homocytotropic antibodies, i.e., antibodies that only engage with cells of the same or closely related species, bind predominantly to mast cells and basophils, resulting in degranulation ¹⁶⁶. Cutaneous basophil hypersensitivity responses have been proposed to be mediated by IgG1 antibodies in guinea pigs resistant to Amblyoma americanum ¹⁶⁶, although Wada et al ¹³⁷ show that IgE antibodies play a role in rejection of H. longicornis ticks. Clearly, the role of antibodies in mediating tick rejection is complex and varies depending on the life stage of the ticks, host species, as well as the tick species ¹⁴². Passive transfer of lymph node cells, but not serum, from guinea pigs resistant to Dermacentor andersoni larvae conferred resistance to naïve guinea pigs ¹⁶⁷. Similar results have also been observed with passive transfer of lymph node cells from guinea pigs resistant to A. americanum larvae ¹⁶². However, passive transfer with sera from guinea pigs infested with adult D. andersoni was able to confer protection ¹⁴². These results suggest that the life stage of the tick may also be important for the induction of protective antibody titers ¹⁴².

The role of complement has also been demonstrated to play an important role during tick rejection. Resistance to D. andersoni larvae in guinea pigs was ablated when the animals were administered cobra venom factor, a protein which depletes serum complement, prior to tick challenge ^{168, 169}. This was further evaluated using C4-deficient guinea pigs, which are deficient in the classical complement activation pathway ¹⁷⁰. In this study, C4-deficient guinea pigs were able to acquire resistance to D. andersoni larvae, suggesting that the alternate pathway of complement activation is important for tick rejection ¹⁷⁰. This is also consistent with the observations that tick salivary proteomes, in general, do not contain proteins to inhibit the classical pathway of complement.

The immune reaction at the tick bite-site of immune animals can induce significant changes in the skin, which may play a role to limit tick feeding. In guinea pigs, repeat tick infestation with I. scapularis results in epicutaneous erythema, severe epidermal hyperplasia, edema and hyperkeratosis at the tick bite site ¹⁴³. Similar results to dermal tissue have been observed in guinea pigs exposed to multiple infestations with Rhipicephalus sanguineus and A. americanum ^{160, 171}. Unlike guinea pigs, Peromyscus leucopus fail to acquire tick immunity following repeat exposures to I. scapularis nymphs ¹⁴³. Examination of the bite site in P. leucopus revealed minimal disruption to the dermal tissue following repeat tick infestation ¹⁴³. The observed changes to dermal tissue are speculated to contribute towards tick rejection in guinea pigs, whereas the lack of dermal changes in P. leucopus could support repeat tick feeding.

Basophils were mistakenly thought to be absent in mice ¹⁷², until mouse basophils were described by Dvorak et al ¹⁷³. Over the last two decades, an understanding of the enigmatic role of basophils in Th2-immunity has started to unravel ^174-176 and these studies have also provided insights into the possible mechanisms of ATR. While the lack of immunological reagents has hampered our ability to dissect the molecular basis of ATR using the guinea pig and cattle model, this has been circumvented using the mouse model of ATR that is elicited by repeated infestations with H. longicornis ¹³⁷. Using mice expressing the diphtheria toxin receptor under the control of basophil-specific mast cell protease 8 (Mcpt8) promoter, Wada et al ¹³⁷ selectively ablated basophils by administering diphtheria toxin to the animals after the first tick infestation and showed that basophils, play a non-redundant role in eliciting ATR to H. longicornis nymphs. Ohta et al ¹⁷⁷ then demonstrated that basophil recruitment is promoted by IL-3 secreted by CD4⁺ memory T cells that arrive at the skin after a tick-challenge. Dissecting the mechanism of ATR, Tabakawa et al ⁸² showed that histamine released by skin-infiltrating basophils, but not by skin-resident mast cells, promotes H. longicornis rejection. The exact mechanism/s by which histamine promotes tick rejection remains to be deciphered. While the molecular understanding of H. longicornis-provoked ATR in mice is beginning to clarify, given the heterogeneity in the immunological responses of host species to tick species and stages, a single unifying mechanism may not emerge.

DICHOTOMOUS IMMUNE RESPONSES TO TICK BITES

Although, ticks can feed on diverse hosts, each tick species appears to have a host preference in nature that is likely determined by a combination of host, tick, and ecological factors. In general, preferred hosts serve as reservoir hosts for the specific tick and are able to host multiple infestations of the tick species without developing any immune responses detrimental to tick feeding. This characteristic is essential in order to ensure the successful completion of the tick’s life cycle. For example, I. scapularis nymphs and larvae predominantly feed on P. leucopus, the reservoir host ¹⁷⁸. Expectedly, in summer and fall when nymphs and larvae are active, P. leucopus are fed upon multiple times by these ticks without any evidence of ATR. Mus musculus has served as a laboratory model of reservoir host for I. scapularis studies despite the fact that P. leucopus and M. musculus belong to different genera and M. musculus is not the natural reservoir host ¹⁷⁸. The genetic traits that allow both P. leucopus and M. musculus species to host I. scapularis ticks without developing ATR are not known. Guinea pigs, and rabbits are non-natural hosts for I. scapularis that readily develop ATR. This dichotomy in immune response to tick feeding is thought to be multifactorial as summarized in Figure 2. Proposing a lock and key hypothesis to explain this dichotomy, Ribeiro suggested that tick salivary molecules have co-evolved with reservoir hosts like P. leucopus and geared to efficiently engage with and diffuse adaptive immune responses of P. leucopus ¹⁷⁹. Conversely, I. scapularis salivary components engage poorly with immune components of non-natural hosts, hence unable to thwart host adaptive immune responses. In essence, natural/permissive hosts do not have an immunological memory of tick bites, while non-natural/non-permissive hosts do.

Figure 2. Potential mechanisms of acquired tick resistance.

Factors that may drive the dichotomous immune responses to tick bites on permissive or resistant host species include: Optimal (A) or suboptimal (A’) engagement of salivary proteins with host defense responses; genetic predisposition to decreased (B) or increased (B’) inflammatory responses to salivary proteins; structural and immunological differences in the skin (C, C’); Host-specific salivary proteome that is sufficient (D) or deficient (D’) in modulating host defense responses; and differences in wound healing without scar (E) or with scar (E’) formation.

Another explanation that does not exclude the lock and key hypothesis, but adds another facet to the dichotomous immune response on natural and non-natural host is that the tick sialome is different when it feeds on different hosts. Narasimhan et al ¹⁴⁵ compared the salivary composition of I. scapularis nymphs fed on M. musculus, the laboratory model of natural host, and on guinea pigs, the model non-natural host and observed that the sialome composition is host-specific. Tirloni et al ¹⁸⁰ also observed that the protein composition of adult I. scapularis and A. americanum stimulated by exposing them to specific host skin semiochemicals of rabbit, dog or human was indeed different. This iterated that the tick salivary composition is not just temporally modulated, but also modulated by host skin components. Narasimhan et al’s study ¹⁴⁵ showed that mouse splenocytes incubated with mouse-fed salivary gland extracts elicited significantly less IL-4, a Th2-defining cytokine, when compared to the amounts elicited by guinea pig-fed salivary gland extracts. Consistent with this, Franzin et al ¹⁸¹ showed that R. microplus fed on tick susceptible Bos taurus taurus expressed significantly increased amounts of salivary transcripts for genes encoding immune modulators such evasins, and metalloproteases, when compared to that in ticks fed on tick-resistant Bos taurus indicus. This differential expression of secreted salivary immunomodulators could account for differential evasion of host defense responses. Therefore, host-modulated differences in the tick sialome composition could inadvertently account, in part, for functional differences critical to dampen host immune responses. A detailed characterization of the qualitative differences between tick-susceptible host-fed and tick-resistant host-fed tick sialomes would offer molecular insights into ATR.

The dichotomous immune response to tick feeding could also be driven by the inherent structural and immunological differences between the guinea pig and mouse skin. While the epidermis, dermis and hypodermis are well defined and thick in the guinea pig, these layers are thin in mice ^{182, 183}. Since the mouse skin is covered by fur, the fat-laden hypodermis that normally provides thermal homeostasis in guinea pig and human skin ¹⁸⁴, is significantly reduced in the murine skin ¹⁸³. Mice have an additional subcutaneous layer called panniculus carnosus, an extra layer of muscle that is thought to allow wound healing via contraction resulting in little or no scarring ¹⁸⁵. While guinea pigs also have the panniculus carnosus layer, wound healing may occur via re-epithelization leading to scar formation ¹⁸⁶ and could account for the epidermal hyperplasia and hyperkeratosis observed at the I. scapularis bite-site on guinea pigs, but not on mice ¹⁴³ . While, the immunology of the guinea pig skin is not fully understood, presumably it behaves more like the human skin than the murine skin. The Langerhans cells, the dendritic cell subset of the epidermis, in mice are not critical for priming CD8+ T cells, unlike in humans ^{187, 188}. Lymphocytes in the murine epidermis are predominantly populated by γδT cells, while in humans it is predominantly αβT cells ¹⁸⁹.

Although, paucity of immunological tools to examine traditional animal models of ATR such as the guinea pig and cattle has stymied progress in this research, Franzin et al ¹⁸¹, More et al ¹⁹⁰, and Kurokawa et al ¹⁹¹ have harnessed the power of rapidly evolving next generation RNA sequencing tools to examine host components at the tick bite-site that may serve as molecular drivers of ATR. Franzin et al ¹⁸¹ underscore the genetic predisposition to resistance or susceptibility and show that R. microplus resistant B. taurus indicus have a higher baseline expression of genes encoding proinflammatory cytokines. Consistent with histological examination of tick bite sites of resistant animals, the tick-resistant breed of Bos taurus demonstrated increased expression of basophil and T lymphocyte recruiting cytokine CCL2 when compared to tick-sensitive animals. More interestingly, their study showed that the skin of the susceptible breed expresses higher levels of enzymes involved in detoxification and this also generates volatile metabolites that serve as semiochemicals that are attractive to ticks. More et al ¹⁹⁰ showed that upon repeated infestations of tick-resistant breed of B. taurus with R. microplus expression of genes involved in skin remodeling and in basophil activation were increased at the bite-site when compared to that in tick-susceptible breed. Further, transcripts encoding for CCL13, a cytokine invoked in eosinophil recruitment was upregulated at the tick bite-site. This was consistent with the findings of Kurokawa et al ¹⁹¹ that eosinophils are increased at the tick bite-sites on tick-resistant guinea pigs. Further, Robbertse et al ¹⁹² addressed lymphocyte populations the skin and draining lymph nodes of resistant and susceptible B. taurus after challenge with R. microplus and showed that the numbers of B lymphocytes and T-helper lymphocytes were decreased in the lymph nodes of tick-susceptible animals. Increased variability in WC1+γδ T lymphocytes populations was also associated with increased susceptibility to R. microplus ticks. Kurokawa et al ¹⁹¹ demonstrated that FCεRI-signaling, complement pathways and procoagulation pathways are activated in the guinea pig skin, but not in mice skin upon repeated infestations with I. scapularis nymphs.

These genetic, transcriptomic and immunologic studies of host responses to tick bites have begun to reveal new insights into the differential responses to tick bites on susceptible and resistant hosts. However, an understanding of what components of the tick saliva drive these differential responses on different host species will be essential to develop strategies to prevent tick feeding and tick-transmission of pathogens. An interesting facet of ATR that may additionally help to shed light on the molecular mechanisms of ATR is the observation that ATR against one tick genus may be cross-protective against another related tick genus.

ATR- MEDIATED CROSS PROTECTION

Cross protective immunity is the phenomenon wherein ATR against a tick species of primary exposure confers host resistance against a secondary tick species for which the host has no prior exposure ^{28, 193, 194}. Numerous tick antigens are conserved among hard tick species including many salivary proteins ¹⁹⁵ and could, in part, explain the acquisition of cross-protective immunity. This is also likely the reason why immunity against truncated protein constructs (64TRP) of the 64P cement antigen from Rhipicephalus appendiculatus was shown to induce immunity in rabbits and hamsters against Ixodes ricinus, and Rhipicephalus sanguineus that impacted tick feeding and mortality ⁴⁷. Cross species protection is important for several reasons related to vaccine development against ticks. Closely related tick species are frequently competent vectors of similar pathogens. For example, the western blacklegged tick, Ixodes pacificus transmits Lyme disease spirochetes (Borrelia burgdorferi) in the West Coast region of the United States, while Lyme spirochetes are transmitted by I. scapularis in other geographic parts of the US. In Europe and Eurasia, the castor bean tick, I. ricinus is the primary vector for Lyme spirochetes. Cross protective immunity could also potentially be useful in areas where multiple medically important tick species occur in close proximity and parasitize the same hosts. This could include the eastern United States, where I. scapularis, Dermacentor variabilis, and A. americanum frequently co-exist and utilize some of the same host species, including white-tail deer (Odocoileus virginianus). Therefore, a molecular understanding of salivary proteins that are involved in cross-protective immunity would help define and prioritize vaccine targets that may serve to simultaneously impair the feeding and fecundity of multiple tick species in endemic regions. It is conceivable that a broad-spectrum anti-tick vaccine applied to wild deer could limit the density of tick populations, potentially reducing enzootic transmission of Borrelia, Anaplasma, Ehrlichia, Rickettsia, and Babesia species transmitted by these ticks. Such an approach would also be operationally cost-effective.

CONCLUSIONS

The phenomenon of acquired tick resistance to ticks remains a puzzling facet of tick-host interactions, since its first description almost 80 years ago ²⁸. Technological advancements in molecular tools to address tick and host functional genomes, transcriptomes and immunomes have provided the much-needed momentum to unravel a new understanding of this phenomenon. A molecular understanding of this phenomenon will enhance approaches to define the subset of antigens that may serve as potential vaccine targets. Translating insights from ATR to tick vaccine development will also be accelerated by recent advancements in vaccine delivery platforms. Although, protein-based vaccines have been the mainstay of vaccinology for the last century ¹⁹⁶, the rapidly evolving science of vaccinomics has highlighted the exciting possibility of using DNA ^197-199 and mRNA ²⁰⁰-based vaccines to deliver nucleotide sequences encoding target antigens into the host. This enables the in-vivo generation of recombinant antigens by the host translational machinery and immunological presentation of these antigens elicit B and T cell-mediated responses critical for effective and long-lasting immunity ^200-202, These approaches may circumvent the cumbersome process of optimization of protein production and formulation and spur progress in tick vaccine development. Importantly, deciphering a mechanistic understanding of ATR will reveal interesting facets of mammalian immunology, and of tick biology that will transcend the field of tick research.

DATA AVAILABILITY

Data cited in this review are published and available on-line or upon request from the authors of the respective publications. No unpublished data is included in this manuscript.