Tick Immune System: What Is Known, the Interconnections, the Gaps, and the Challenges.

Ticks are ectoparasitic arthropods that necessarily feed on the blood of their vertebrate hosts. The success of blood acquisition depends on the pharmacological properties of tick saliva, which is injected into the host during tick feeding. Saliva is also used as a vehicle by several types of pathogens to be transmitted to the host, making ticks versatile vectors of several diseases for humans and other animals. When a tick feeds on an infected host, the pathogen reaches the gut of the tick and must migrate to its salivary glands via hemolymph to be successfully transmitted to a subsequent host during the next stage of feeding. In addition, some pathogens can colonize the ovaries of the tick and be transovarially transmitted to progeny. The tick immune system, as well as the immune system of other invertebrates, is more rudimentary than the immune system of vertebrates, presenting only innate immune responses. Although simpler, the large number of tick species evidences the efficiency of their immune system. The factors of their immune system act in each tick organ that interacts with pathogens; therefore, these factors are potential targets for the development of new strategies for the control of ticks and tick-borne diseases. The objective of this review is to present the prevailing knowledge on the tick immune system and to discuss the challenges of studying tick immunity, especially regarding the gaps and interconnections. To this end, we use a comparative approach of the tick immune system with the immune system of other invertebrates, focusing on various components of humoral and cellular immunity, such as signaling pathways, antimicrobial peptides, redox metabolism, complement-like molecules and regulated cell death. In addition, the role of tick microbiota in vector competence is also discussed.

Cytokine Profiling of Subclinical Tick-Borne Infections in Humans.

Over many years, tick-borne infections remain one of the most serious threats to human health worldwide. The immune response to these infections in a human after confirmed bite by an infected carrier at the early stages of infection in the absence of clinical symptoms can be the first indicator of the presence of the infectious agent in the body. During viral infection, the concentration of IL-1α, IL-8, IL-10, IL-17A, and IFNγ increases; superoxide dismutase also increases, in contrast to bacterial infections. A slight decrease in the concentration is observed only for receptor antagonist IL-1Ra. During the infection caused by bacterial pathogens, very similar profiles of the innate human immune response are observed: activation of IL-1α, IL-8, and IFNα and suppression of superoxide dismutase, IL-1Ra, and IL-17A production. It has been demonstrated, that the immune response is triggered immediately after infection, and changes in the concentration of the main cytokines in the blood plasma can be detected as early as on days 2-5 after tick bite. These results can be useful in developing new methods of emergency diagnosis and prevention of tick-borne infections.

Tick-Pathogen Interactions: The Metabolic Perspective.

The first tick genome published in 2016 provided an invaluable tool for studying the molecular basis of tick-pathogen interactions. Metabolism is a key element in host-pathogen interactions. However, our knowledge of tick-pathogen metabolic interactions is very limited. Recently, a systems biology approach, using omics datasets, has revealed that tick-borne pathogen infection induces transcriptional reprograming affecting several metabolic pathways in ticks, facilitating infection, multiplication, and transmission. Results suggest that the response of tick cells to tick-borne pathogens is associated with tolerance to infection. Here we review our current understanding of the modulation of tick metabolism by tick-borne pathogens, with a focus on the model intracellular bacterium Anaplasma phagocytophilum.

Crystal Structure of Borrelia turicatae protein, BTA121, a differentially regulated gene in the tick-mammalian transmission cycle of relapsing fever spirochetes.

Tick-borne relapsing fever (RF) borreliosis is a neglected disease that is often misdiagnosed. RF species circulating in the United States include Borrelia turicatae, which is transmitted by argasid ticks. Environmental adaptation by RF Borrelia is poorly understood, however our previous studies indicated differential regulation of B. turicatae genes localized on the 150 kb linear megaplasmid during the tick-mammalian transmission cycle, including bta121. This gene is up-regulated by B. turicatae in the tick versus the mammal, and the encoded protein (BTA121) is predicted to be surface localized. The structure of BTA121 was solved by single-wavelength anomalous dispersion (SAD) using selenomethionine-derivative protein. The topology of BTA121 is unique with four helical domains organized into two helical bundles. Due to the sequence similarity of several genes on the megaplasmid, BTA121 can serve as a model for their tertiary  structures. BTA121 has large interconnected tunnels and cavities that can accommodate ligands, notably long parallel helices, which have a large hydrophobic central pocket. Preliminary in-vitro studies suggest that BTA121 binds lipids, notably palmitate with a similar order of binding affinity as tablysin-15, a known palmitate-binding protein. The reported data will guide mechanistic studies to determine the role of BTA121 in the tick-mammalian transmission cycle of B. turicatae.

Retrospective survey of severe fever with thrombocytopenia syndrome in patients with suspected rickettsiosis in Japan.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne infectious disease caused by the SFTS virus (SFTSV). The aim of this study was to clarify whether SFTS is potentially mis-diagnosed as rickettsioses, including spotted fever, typhus fever, and scrub typhus, which are also tick-borne disease. A total of 464 serum samples collected from 222 patients with clinically suspected rickettsiosis between 1999 and 2012 were tested for antibodies against the SFTSV. Of the 464 serum samples, one was positive for antibodies against the virus in an enzyme-linked immunosorbent assay and indirect immunofluorescence assay. The patient of SFTSV antibody-positive sample (15 days after disease onset) was positive for SFTSV genome in the acute phase sample (3 days after disease onset) as determined via reverse transcription-quantitative polymerase chain reaction. This patient, who was a resident of the Yamaguchi prefecture in Western Japan, was in his 40s when he showed symptoms in 2011. As the result, 1 of 222 patients, who was clinically suspected of rickettsiosis, was retrospectively diagnosed with SFTS. In this case, both the C-reactive protein and white blood cell count levels were lower than the ranges of these parameters for patients diagnosed with rickettsiosis. Therefore, SFTS should be considered in the differential diagnosis for rickettsiosis in Japan.

Synaptogyrin-2 Promotes Replication of a Novel Tick-borne Bunyavirus through Interacting with Viral Nonstructural Protein NSs.

Synaptogyrin-2 is a non-neuronal member of the synaptogyrin family involved in synaptic vesicle biogenesis and trafficking. Little is known about the function of synaptogyrin-2. Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease characterized by high fever, thrombocytopenia, and leukocytopenia with high mortality, caused by a novel tick-borne phlebovirus in the family Bunyaviridae. Our previous studies have shown that the viral nonstructural protein NSs forms inclusion bodies (IBs) that are involved in viral immune evasion, as well as viral RNA replication. In this study, we sought to elucidate the mechanism by which NSs formed the IBs, a lipid droplet-based structure confirmed by NSs co-localization with perilipin A and adipose differentiation-related protein (ADRP). Through a high throughput screening, we identified synaptogyrin-2 to be highly up-regulated in response to SFTS bunyavirus (SFTSV) infection and to be a promoter of viral replication. We demonstrated that synaptogyrin-2 interacted with NSs and was translocated into the IBs, which were reconstructed from lipid droplets into large structures in infection. Viral RNA replication decreased, and infectious virus titers were lowered significantly when synaptogyrin-2 was silenced in specific shRNA-expressing cells, which correlated with the reduced number of the large IBs restructured from regular lipid droplets. We hypothesize that synaptogyrin-2 is essential to promoting the formation of the IBs to become virus factories for viral RNA replication through its interaction with NSs. These findings unveil the function of synaptogyrin-2 as an enhancer in viral infection.

Whole antigenic lysates of Ixodes ricinus, but not Der-p2 allergen-like protein, are potent inducers of basophil activation in previously tick-exposed human hosts.

The clinical suspicion of tick anaphylaxis is based on a history of the bite and occurs often during the warm season. Further arguments are the presence of natural hosts in the immediate environment and, eventually, the identification of the tick. The diagnosis is confirmed when immediate-type sensitization is shown by positive skin prick tests performed with specific tick extracts or the demonstration of specific IgE in vitro. In the current study, we hypothesize that hard tick-derived material contains potent inducers being able to promote basophil stimulation, which correlates with a sensitization immunological response following tick bites. To this end, biological material from two hard tick cell lines (IRE11 and IDE8 - derived from Ixodes ricinus and I. scapularis, respectively) as well as I. ricinus salivary gland and body lysates were used in a human basophil activation test (BAT) to analyse binding and cross-linking capacity of membrane-bound IgE, because basophils are one of the main effector cells of allergic reactions. Additionally, Der-p2 allergen-like gene from I. ricinus was recombinantly expressed as a 15-kDa histidine-tagged fusion protein, purified and included as a stimulus within the setup. Blood was drawn and submitted to BAT screening from a pool of 36 individuals, both bitten and who served solely as negative controls. We have found that seven subjects (19%), all of whom were at least two times tick-bitten, positively reacted to the aforementioned stimuli, whereas the reactivity level of the ones bearing single bites proved to be within the normal range. Moreover, no significant upregulation of the assessed basophil activation marker was detected in the case of Der-p2, except a faint reaction at high dosages. We conclude that at least two tick bites of the human host must occur in order to induce significant basophil activation.

Total, membrane, and immunogenic proteomes of macrophage- and tick cell-derived Ehrlichia chaffeensis evaluated by liquid chromatography-tandem mass spectrometry and MALDI-TOF methods.

Ehrlichia chaffeensis, a tick-transmitted rickettsial, is the causative agent of human monocytic ehrlichiosis. To examine protein expression patterns, we analyzed total, membrane, and immunogenic proteomes of E. chaffeensis originating from macrophage and tick cell cultures. Total proteins resolved by one-dimensional gel electrophoresis and subjected to liquid chromatography-electrospray ionization ion trap mass spectrometry allowed identification of 134 and 116 proteins from macrophage- and tick cell-derived E. chaffeensis, respectively. Because a majority of immunogenic proteins remained in the membrane fraction, individually picked total and immunogenic membrane proteins were also surveyed by liquid chromatography-tandem mass spectrometry and matrix-assisted laser desorption ionization-time of flight methods. The analysis aided the identification of 48 additional proteins. In all, 278 genes of the E. chaffeensis genome were verified as functional genes. They included genes for DNA and protein metabolism, energy metabolism and transport, membrane proteins, hypothetical proteins, and many novel proteins of unknown function. The data reported in this study suggest that the membrane of E. chaffeensis is very complex, having many expressed proteins. This study represents the first and the most comprehensive analysis of E. chaffeensis-expressed proteins. This also is the first study confirming the expression of nearly one-fourth of all predicted genes of the E. chaffeensis genome, validating that they are functionally active genes, and demonstrating that classic shotgun proteomic approaches are feasible for tick-transmitted intraphagosomal bacteria. The identity of novel expressed proteins reported in this study, including the large selection of membrane and immunogenic proteins, will be valuable in elucidating pathogenic mechanisms and developing effective prevention and control methods.