Tick cysteine protease inhibitors suppress immune responses in mannan-induced psoriasis-like inflammation.

Protease inhibitors regulate various biological processes and prevent host tissue/organ damage. Specific inhibition/regulation of proteases is clinically valuable for treating several diseases. Psoriasis affects the skin in the limbs and scalp of the body, and the contribution of cysteine and serine proteases to the development of skin inflammation is well documented. Cysteine protease inhibitors from ticks have high specificity, selectivity, and affinity to their target proteases and are efficient immunomodulators. However, their potential therapeutic effect on psoriasis pathogenesis remains to be determined. Therefore, we tested four tick cystatins (Sialostatin L, Sialostatin L2, Iristatin, and Mialostatin) in the recently developed, innate immunity-dependent mannan-induced psoriasis model. We explored the effects of protease inhibitors on clinical symptoms and histological features. In addition, the number and percentage of immune cells (dendritic cells, neutrophils, macrophages, and γδT cells) by flow cytometry, immunofluorescence/immunohistochemistry and, the expression of pro-inflammatory cytokines (TNF-a, IL-6, IL-22, IL-23, and IL-17 family) by qPCR were analyzed using skin, spleen, and lymph node samples. Tick protease inhibitors have significantly decreased psoriasis symptoms and disease manifestations but had differential effects on inflammatory responses and immune cell populations, suggesting different modes of action of these inhibitors on psoriasis-like inflammation. Thus, our study demonstrates, for the first time, the usefulness of tick-derived protease inhibitors for treating skin inflammation in patients.

Protease-bound structure of Ricistatin provides insights into the mechanism of action of tick salivary cystatins in the vertebrate host.

Tick saliva injected into the vertebrate host contains bioactive anti-proteolytic proteins from the cystatin family; however, the molecular basis of their unusual biochemical and physiological properties, distinct from those of host homologs, is unknown. Here, we present Ricistatin, a novel secreted cystatin identified in the salivary gland transcriptome of Ixodes ricinus ticks. Recombinant Ricistatin inhibited host-derived cysteine cathepsins and preferentially targeted endopeptidases, while having only limited impact on proteolysis driven by exopeptidases. Determination of the crystal structure of Ricistatin in complex with a cysteine cathepsin together with characterization of structural determinants in the Ricistatin binding site explained its restricted specificity. Furthermore, Ricistatin was potently immunosuppressive and anti-inflammatory, reducing levels of pro-inflammatory cytokines IL-6, IL-1ß, and TNF-α and nitric oxide in macrophages; IL-2 and IL-9 levels in Th9 cells; and OVA antigen-induced CD4+ T cell proliferation and neutrophil migration. This work highlights the immunotherapeutic potential of Ricistatin and, for the first time, provides structural insights into the unique narrow selectivity of tick salivary cystatins determining their bioactivity.

Novel Amphibian Bowman-Birk-Like Inhibitor with Antioxidant and Anticoagulant Effects Ameliorates Pancreatitis Symptoms in Mice.

Acute pancreatitis (AP) is a serious inflammatory disorder and still lacks effective therapy globally. In this study, a novel Ranacyclin peptide, Ranacin, was identified from the skin of Pelophylax nigromaculatus frog. Ranacin adopted a compact ß-hairpin conformation with a disulfide bond (Cys5-Cys15). Ranacin was also demonstrated effectively to inhibit trypsin and have anticoagulant and antioxidant activities in vitro. Furthermore, the severity of pancreatitis was significantly alleviated in l-Arg-induced AP mice after treatment with Ranacin. In addition, structure-activity studies of Ranacin analogues confirmed that the sequences outside the trypsin inhibitory loop (TIL), especially at the C-terminal side, might be closely associated with the efficacy of its trypsin inhibitory activity. In conclusion, our data suggest that Ranacin can improve pancreatic injury in mice with severe AP through its multi-activity. Therefore, Ranacin is considered a potential drug candidate in AP therapy.

Recent Advances in Tick Antigen Discovery and Anti-Tick Vaccine Development.

Ticks can seriously affect human and animal health around the globe, causing significant economic losses each year. Chemical acaricides are widely used to control ticks, which negatively impact the environment and result in the emergence of acaricide-resistant tick populations. A vaccine is considered as one of the best alternative approaches to control ticks and tick-borne diseases, as it is less expensive and more effective than chemical controls. Many antigen-based vaccines have been developed as a result of current advances in transcriptomics, genomics, and proteomic techniques. A few of these (e.g., Gavac® and TickGARD®) are commercially available and are commonly used in different countries. Furthermore, a significant number of novel antigens are being investigated with the perspective of developing new anti-tick vaccines. However, more research is required to develop new and more efficient antigen-based vaccines, including on assessing the efficiency of various epitopes against different tick species to confirm their cross-reactivity and their high immunogenicity. In this review, we discuss the recent advancements in the development of antigen-based vaccines (traditional and RNA-based) and provide a brief overview of recent discoveries of novel antigens, along with their sources, characteristics, and the methods used to test their efficiency.

Iripin-1, a new anti-inflammatory tick serpin, inhibits leukocyte recruitment in vivo while altering the levels of chemokines and adhesion molecules.

Serpins are widely distributed and functionally diverse inhibitors of serine proteases. Ticks secrete serpins with anti-coagulation, anti-inflammatory, and immunomodulatory activities via their saliva into the feeding cavity to modulate host's hemostatic and immune reaction initiated by the insertion of tick's mouthparts into skin. The suppression of the host's immune response not only allows ticks to feed on a host for several days but also creates favorable conditions for the transmission of tick-borne pathogens. Herein we present the functional and structural characterization of Iripin-1 (Ixodes ricinus serpin-1), whose expression was detected in the salivary glands of the tick Ixodes ricinus, a European vector of tick-borne encephalitis and Lyme disease. Of 16 selected serine proteases, Iripin-1 inhibited primarily trypsin and further exhibited weaker inhibitory activity against kallikrein, matriptase, and plasmin. In the mouse model of acute peritonitis, Iripin-1 enhanced the production of the anti-inflammatory cytokine IL-10 and chemokines involved in neutrophil and monocyte recruitment, including MCP-1/CCL2, a potent histamine-releasing factor. Despite increased chemokine levels, the migration of neutrophils and monocytes to inflamed peritoneal cavities was significantly attenuated following Iripin-1 administration. Based on the results of in vitro experiments, immune cell recruitment might be inhibited due to Iripin-1-mediated reduction of the expression of chemokine receptors in neutrophils and adhesion molecules in endothelial cells. Decreased activity of serine proteases in the presence of Iripin-1 could further impede cell migration to the site of inflammation. Finally, we determined the tertiary structure of native Iripin-1 at 2.10 Å resolution by employing the X-ray crystallography technique. In conclusion, our data indicate that Iripin-1 facilitates I. ricinus feeding by attenuating the host's inflammatory response at the tick attachment site.

Editorial: Special Issue on the "Molecular Biology of Disease Vectors".

Arthropod disease vectors not only transmit malaria but many other serious diseases, many of which are, to a greater or lesser degree, neglected [...].

Tick Salivary Kunitz-Type Inhibitors: Targeting Host Hemostasis and Immunity to Mediate Successful Blood Feeding.

Kunitz domain-containing proteins are ubiquitous serine protease inhibitors with promising therapeutic potential. They target key proteases involved in major cellular processes such as inflammation or hemostasis through competitive inhibition in a substrate-like manner. Protease inhibitors from the Kunitz superfamily have a low molecular weight (18-24 kDa) and are characterized by the presence of one or more Kunitz motifs consisting of α-helices and antiparallel ß-sheets stabilized by three disulfide bonds. Kunitz-type inhibitors are an important fraction of the protease inhibitors found in tick saliva. Their roles in inhibiting and/or suppressing host homeostatic responses continue to be shown to be additive or synergistic with other protease inhibitors such as cystatins or serpins, ultimately mediating successful blood feeding for the tick. In this review, we discuss the biochemical features of tick salivary Kunitz-type protease inhibitors. We focus on their various effects on host hemostasis and immunity at the molecular and cellular level and their potential therapeutic applications. In doing so, we highlight that their pharmacological properties can be exploited for the development of novel therapies and vaccines.

Bioinformatic Analysis of Ixodes ricinus Long Non-Coding RNAs Predicts Their Binding Ability of Host miRNAs.

Ixodes ricinus ticks are distributed across Europe and are a vector of tick-borne diseases. Although I. ricinus transcriptome studies have focused exclusively on protein coding genes, the last decade witnessed a strong increase in long non-coding RNA (lncRNA) research and characterization. Here, we report for the first time an exhaustive analysis of these non-coding molecules in I. ricinus based on 131 RNA-seq datasets from three different BioProjects. Using this data, we obtained a consensus set of lncRNAs and showed that lncRNA expression is stable among different studies. While the length distribution of lncRNAs from the individual data sets is biased toward short length values, implying the existence of technical artefacts, the consensus lncRNAs show a more homogeneous distribution emphasizing the importance to incorporate data from different sources to generate a solid reference set of lncRNAs. KEGG enrichment analysis of host miRNAs putatively targeting lncRNAs upregulated upon feeding showed that these miRNAs are involved in several relevant functions for the tick-host interaction. The possibility that at least some tick lncRNAs act as host miRNA sponges was further explored by identifying lncRNAs with many target regions for a given host miRNA or sets of host miRNAs that consistently target lncRNAs together. Overall, our findings suggest that lncRNAs that may act as sponges have diverse biological roles related to the tick-host interaction in different tissues.

Transcriptomic analysis of the tick midgut and salivary gland responses upon repeated blood-feeding on a vertebrate host.

Ticks are blood-feeding arthropods that use the components of their salivary glands to counter the host's hemostatic, inflammatory, and immune responses. The tick midgut also plays a crucial role in hematophagy. It is responsible for managing blood meals (storage and digestion) and protecting against host immunity and pathogen infections. Previous transcriptomic studies revealed the complexity of tick sialomes (salivary gland transcriptomes) and mialomes (midgut transcriptomes) which encode for protease inhibitors, lipocalins (histamine-binding proteins), disintegrins, enzymes, and several other tick-specific proteins. Several studies have demonstrated that mammalian hosts acquire tick resistance against repeated tick bites. Consequently, there is an urgent need to uncover how tick sialomes and mialomes respond to resistant hosts, as they may serve to develop novel tick control strategies and applications. Here, we mimicked natural repeated tick bites in a laboratory setting and analyzed gene expression dynamics in the salivary glands and midguts of adult female ticks. Rabbits were subjected to a primary (feeding on a naive host) and a secondary infestation of the same host (we re-exposed the hosts but to other ticks). We used single salivary glands and midguts dissected from individual siblings adult pathogen-free female Ixodes ricinus to reduce genetic variability between individual ticks. The comprehensive analysis of 88 obtained RNA-seq data sets allows us to provide high-quality annotated sialomes and mialomes from individual ticks. Comparisons between fed/unfed, timepoints, and exposures yielded as many as 3000 putative differentially expressed genes (DEG). Interestingly, when classifying the exposure DEGs by means of a clustering approach we observed that the majority of these genes show increased expression at early feeding time-points in the mid-gut of re-exposed ticks. The existence of clearly defined groups of genes with highly similar responses to re-exposure suggests the existence of molecular swiches. In silico functional analysis shows that these early feeding reexposure response genes form a dense interaction network at protein level being related to virtually all aspects of gene expression regulation and glycosylation. The processed data is available through an easy-to-use database-associated webpage (https://arn.ugr.es/IxoriDB/) that can serve as a valuable resource for tick research.

Serpins in Tick Physiology and Tick-Host Interaction.

Tick saliva has been extensively studied in the context of tick-host interactions because it is involved in host homeostasis modulation and microbial pathogen transmission to the host. Accumulated knowledge about the tick saliva composition at the molecular level has revealed that serine protease inhibitors play a key role in the tick-host interaction. Serpins are one highly expressed group of protease inhibitors in tick salivary glands, their expression can be induced during tick blood-feeding, and they have many biological functions at the tick-host interface. Indeed, tick serpins have an important role in inhibiting host hemostatic processes and in the modulation of the innate and adaptive immune responses of their vertebrate hosts. Tick serpins have also been studied as potential candidates for therapeutic use and vaccine development. In this review, we critically summarize the current state of knowledge about the biological role of tick serpins in shaping tick-host interactions with emphasis on the mechanisms by which they modulate host immunity. Their potential use in drug and vaccine development is also discussed.

The application of machine learning to imaging in hematological oncology: A scoping review.

Background: Here, we conducted a scoping review to (i) establish which machine learning (ML) methods have been applied to hematological malignancy imaging; (ii) establish how ML is being applied to hematological cancer radiology; and (iii) identify addressable research gaps. Methods: The review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis Extension for Scoping Reviews guidelines. The inclusion criteria were (i) pediatric and adult patients with suspected or confirmed hematological malignancy undergoing imaging (population); (ii) any study using ML techniques to derive models using radiological images to apply to the clinical management of these patients (concept); and (iii) original research articles conducted in any setting globally (context). Quality Assessment of Diagnostic Accuracy Studies 2 criteria were used to assess diagnostic and segmentation studies, while the Newcastle-Ottawa scale was used to assess the quality of observational studies. Results: Of 53 eligible studies, 33 applied diverse ML techniques to diagnose hematological malignancies or to differentiate them from other diseases, especially discriminating gliomas from primary central nervous system lymphomas (n=18); 11 applied ML to segmentation tasks, while 9 applied ML to prognostication or predicting therapeutic responses, especially for diffuse large B-cell lymphoma. All studies reported discrimination statistics, but no study calculated calibration statistics. Every diagnostic/segmentation study had a high risk of bias due to their case-control design; many studies failed to provide adequate details of the reference standard; and only a few studies used independent validation. Conclusion: To deliver validated ML-based models to radiologists managing hematological malignancies, future studies should (i) adhere to standardized, high-quality reporting guidelines such as the Checklist for Artificial Intelligence in Medical Imaging; (ii) validate models in independent cohorts; (ii) standardize volume segmentation methods for segmentation tasks; (iv) establish comprehensive prospective studies that include different tumor grades, comparisons with radiologists, optimal imaging modalities, sequences, and planes; (v) include side-by-side comparisons of different methods; and (vi) include low- and middle-income countries in multicentric studies to enhance generalizability and reduce inequity.

Ixodes ricinus Salivary Serpin Iripin-8 Inhibits the Intrinsic Pathway of Coagulation and Complement.

Tick saliva is a rich source of antihemostatic, anti-inflammatory, and immunomodulatory molecules that actively help the tick to finish its blood meal. Moreover, these molecules facilitate the transmission of tick-borne pathogens. Here we present the functional and structural characterization of Iripin-8, a salivary serpin from the tick Ixodes ricinus, a European vector of tick-borne encephalitis and Lyme disease. Iripin-8 displayed blood-meal-induced mRNA expression that peaked in nymphs and the salivary glands of adult females. Iripin-8 inhibited multiple proteases involved in blood coagulation and blocked the intrinsic and common pathways of the coagulation cascade in vitro. Moreover, Iripin-8 inhibited erythrocyte lysis by complement, and Iripin-8 knockdown by RNA interference in tick nymphs delayed the feeding time. Finally, we resolved the crystal structure of Iripin-8 at 1.89 Å resolution to reveal an unusually long and rigid reactive center loop that is conserved in several tick species. The P1 Arg residue is held in place distant from the serpin body by a conserved poly-Pro element on the P' side. Several PEG molecules bind to Iripin-8, including one in a deep cavity, perhaps indicating the presence of a small-molecule binding site. This is the first crystal structure of a tick serpin in the native state, and Iripin-8 is a tick serpin with a conserved reactive center loop that possesses antihemostatic activity that may mediate interference with host innate immunity.

Structural and biochemical characterization of the novel serpin Iripin-5 from Ixodes ricinus.

Iripin-5 is the main Ixodes ricinus salivary serpin, which acts as a modulator of host defence mechanisms by impairing neutrophil migration, suppressing nitric oxide production by macrophages and altering complement functions. Iripin-5 influences host immunity and shows high expression in the salivary glands. Here, the crystal structure of Iripin-5 in the most thermodynamically stable state of serpins is described. In the reactive-centre loop, the main substrate-recognition site of Iripin-5 is likely to be represented by Arg342, which implies the targeting of trypsin-like proteases. Furthermore, a computational structural analysis of selected Iripin-5-protease complexes together with interface analysis revealed the most probable residues of Iripin-5 involved in complex formation.

Tick extracellular vesicles enable arthropod feeding and promote distinct outcomes of bacterial infection.

Extracellular vesicles are thought to facilitate pathogen transmission from arthropods to humans and other animals. Here, we reveal that pathogen spreading from arthropods to the mammalian host is multifaceted. Extracellular vesicles from Ixodes scapularis enable tick feeding and promote infection of the mildly virulent rickettsial agent Anaplasma phagocytophilum through the SNARE proteins Vamp33 and Synaptobrevin 2 and dendritic epidermal T cells. However, extracellular vesicles from the tick Dermacentor andersoni mitigate microbial spreading caused by the lethal pathogen Francisella tularensis. Collectively, we establish that tick extracellular vesicles foster distinct outcomes of bacterial infection and assist in vector feeding by acting on skin immunity. Thus, the biology of arthropods should be taken into consideration when developing strategies to control vector-borne diseases.

Mialostatin, a Novel Midgut Cystatin from Ixodes ricinus Ticks: Crystal Structure and Regulation of Host Blood Digestion.

The hard tick Ixodes ricinus is a vector of Lyme disease and tick-borne encephalitis. Host blood protein digestion, essential for tick development and reproduction, occurs in tick midgut digestive cells driven by cathepsin proteases. Little is known about the regulation of the digestive proteolytic machinery of I. ricinus. Here we characterize a novel cystatin-type protease inhibitor, mialostatin, from the I. ricinus midgut. Blood feeding rapidly induced mialostatin expression in the gut, which continued after tick detachment. Recombinant mialostatin inhibited a number of I. ricinus digestive cysteine cathepsins, with the greatest potency observed against cathepsin L isoforms, with which it co-localized in midgut digestive cells. The crystal structure of mialostatin was determined at 1.55 Å to explain its unique inhibitory specificity. Finally, mialostatin effectively blocked in vitro proteolysis of blood proteins by midgut cysteine cathepsins. Mialostatin is likely to be involved in the regulation of gut-associated proteolytic pathways, making midgut cystatins promising targets for tick control strategies.

Characterization and functional analysis of cathelicidin-MH, a novel frog-derived peptide with anti-septicemic properties.

Antimicrobial peptides form part of the innate immune response and play a vital role in host defense against pathogens. Here we report a new antimicrobial peptide belonging to the cathelicidin family, cathelicidin-MH (cath-MH), from the skin of Microhyla heymonsivogt frog. Cath-MH has a single α-helical structure in membrane-mimetic environments and is antimicrobial against fungi and bacteria, especially Gram-negative bacteria. In contrast to other cathelicidins, cath-MH suppresses coagulation by affecting the enzymatic activities of tissue plasminogen activator, plasmin, ß-tryptase, elastase, thrombin, and chymase. Cath-MH protects against lipopolysaccharide (LPS)- and cecal ligation and puncture-induced sepsis, effectively ameliorating multiorgan pathology and inflammatory cytokine through its antimicrobial, LPS-neutralizing, coagulation suppressing effects as well as suppression of MAPK signaling. Taken together, these data suggest that cath-MH is an attractive candidate therapeutic agent for the treatment of septic shock.

Iripin-3, a New Salivary Protein Isolated From Ixodes ricinus Ticks, Displays Immunomodulatory and Anti-Hemostatic Properties In Vitro.

Tick saliva is a rich source of pharmacologically and immunologically active molecules. These salivary components are indispensable for successful blood feeding on vertebrate hosts and are believed to facilitate the transmission of tick-borne pathogens. Here we present the functional and structural characterization of Iripin-3, a protein expressed in the salivary glands of the tick Ixodes ricinus, a European vector of tick-borne encephalitis and Lyme disease. Belonging to the serpin superfamily of protease inhibitors, Iripin-3 strongly inhibited the proteolytic activity of serine proteases kallikrein and matriptase. In an in vitro setup, Iripin-3 was capable of modulating the adaptive immune response as evidenced by reduced survival of mouse splenocytes, impaired proliferation of CD4+ T lymphocytes, suppression of the T helper type 1 immune response, and induction of regulatory T cell differentiation. Apart from altering acquired immunity, Iripin-3 also inhibited the extrinsic blood coagulation pathway and reduced the production of pro-inflammatory cytokine interleukin-6 by lipopolysaccharide-stimulated bone marrow-derived macrophages. In addition to its functional characterization, we present the crystal structure of cleaved Iripin-3 at 1.95 Å resolution. Iripin-3 proved to be a pluripotent salivary serpin with immunomodulatory and anti-hemostatic properties that could facilitate tick feeding via the suppression of host anti-tick defenses. Physiological relevance of Iripin-3 activities observed in vitro needs to be supported by appropriate in vivo experiments.

Comparison of the hemolysis machinery in two evolutionarily distant blood-feeding arthropod vectors of human diseases.

Host blood protein digestion plays a pivotal role in the ontogeny and reproduction of hematophagous vectors. The gut of hematophagous arthropods stores and slowly digests host blood and represents the primary gateway for transmitted pathogens. The initial step in blood degradation is induced lysis of host red blood cells (hemolysis), which releases hemoglobin for subsequent processing by digestive proteolytic enzymes. The activity cycles and characteristics of hemolysis in vectors are poorly understood. Hence, we investigated hemolysis in two evolutionarily distant blood-feeding arthropods: The mosquito Culex pipiens and the soft tick Argas persicus, both of which are important human and veterinary disease vectors. Hemolysis in both species was cyclical after blood meal ingestion. Maximum digestion occurs under slightly alkaline conditions in females. Hemolytic activity appears to be of lipoid origin in C. pipiens and enzymatic activity (proteolytic) in A. persicus. We have assessed the effect of pH, incubation time, and temperature on hemolytic activity and the hemolysin. The susceptibility of red blood cells from different hosts to the hemolysin and the effect of metabolic inhibition of hemolytic activity were assessed. We conclude that in C. pipiens and A. persicus midgut hemolysins control the amplitude of blood lysis step to guarantee an efficient blood digestion.

Long Non-Coding RNAs and Their Potential Roles in the Vector-Host-Pathogen Triad.

Long non-coding (lnc)RNAs have emerged as critical regulators of gene expression and are involved in almost every cellular process. They can bind to other molecules including DNA, proteins, or even other RNA types such messenger RNA or small RNAs. LncRNAs are typically expressed at much lower levels than mRNA, and their expression is often restricted to tissue- or time-specific developmental stages. They are also involved in several inter-species interactions, including vector-host-pathogen interactions, where they can be either vector/host-derived or encoded by pathogens. In these interactions, they function via multiple mechanisms including regulating pathogen growth and replication or via cell-autonomous antimicrobial defense mechanisms. Recent advances suggest that characterizing lncRNAs and their targets in different species may hold the key to understanding the role of this class of non-coding RNA in interspecies crosstalk. In this review, we present a general overview of recent studies related to lncRNA-related regulation of gene expression as well as their possible involvement in regulating vector-host-pathogen interactions.

Tick salivary gland transcriptomics and proteomics.

'Omics' technologies have facilitated the identification of hundreds to thousands of tick molecules that mediate tick feeding and play a role in the transmission of tick-borne diseases. Deep sequencing methodologies have played a key role in this knowledge accumulation, profoundly facilitating the study of the biology of disease vectors lacking reference genomes. For example, the nucleotide sequences of the entire set of tick salivary effectors, the so-called tick 'sialome', now contain at least one order of magnitude more transcript sequences compared to similar projects based on Sanger sequencing. Tick feeding is a complex and dynamic process, and while the dynamic 'sialome' is thought to mediate tick feeding success, exactly how transcriptome dynamics relate to tick-host-pathogen interactions is still largely unknown. The identification and, importantly, the functional analysis of the tick 'sialome' is expected to shed light on this 'black box'. This information will be crucial for developing strategies to block pathogen transmission, not only for anti-tick vaccine development but also the discovery and development of new, pharmacologically active compounds for human diseases.