Ticks are hematophagous arthropods that use a complex mixture of salivary proteins to evade host defenses while taking a blood meal. Little is known about the immunological and physiological consequences of tick feeding on humans. Here, we performed the first bulk and single-nucleus RNA sequencing (snRNA-seq) of skin and blood of four persons presenting with naturally acquired, attached Ixodes scapularis ticks. Pathways and individual genes associated with innate and adaptive immunity were identified based on bulk RNA sequencing, including interleukin-17 signaling and platelet activation pathways at the site of tick attachment or in peripheral blood. snRNA-seq further revealed that the Hippo signaling, cell adhesion, and axon guidance pathways were involved in the response to an I. scapularis bite in humans. Features of the host response in these individuals also overlapped with that of laboratory guinea pigs exposed to I. scapularis and which acquired resistance to ticks. These findings offer novel insights for the development of new biomarkers for I. scapularis exposure and anti-tick vaccines for human use.

• Keywords
• RNA-seq, acquired resistance, human, immune pathways, tick, tick-borne pathogens,
• MeSH
• Ixodes * genetics   MeSH
• Tick Bites * MeSH
• Humans MeSH
• Guinea Pigs MeSH
• RNA, Small Nuclear MeSH
• Base Sequence MeSH
• Feeding Behavior physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Guinea Pigs MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• RNA, Small Nuclear MeSH

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-1β, 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.

• Keywords
• Cystatins, Host–parasite interactions, Ixodes ricinus, Protease inhibition, Protein structure, Tick saliva,
• MeSH
• Cystatins * pharmacology   MeSH
• Cysteine metabolism   MeSH
• Endopeptidases metabolism   MeSH
• Cathepsins metabolism   MeSH
• Ixodes * chemistry   MeSH
• Vertebrates MeSH
• Peptide Hydrolases metabolism   MeSH
• Salivary Cystatins chemistry   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cystatins * MeSH
• Cysteine MeSH
• Endopeptidases MeSH
• Cathepsins MeSH
• Peptide Hydrolases MeSH
• Salivary Cystatins MeSH

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.

• Keywords
• anti-inflammatory protein, cell migration, iripin, ixodes ricinus, serpin, tick saliva, tick-host interaction, ticks,
• MeSH
• Anti-Inflammatory Agents pharmacology   MeSH
• Chemokines MeSH
• Endothelial Cells metabolism   MeSH
• Ixodes * metabolism   MeSH
• Monocytes metabolism   MeSH
• Mice MeSH
• Serpins * metabolism   MeSH
• Trypsin MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Intramural MeSH
• Names of Substances
• Anti-Inflammatory Agents MeSH
• Chemokines MeSH
• Serpins * MeSH
• Trypsin MeSH

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.

• Keywords
• midgut, repeated exposure, salivary glands, ticks, transcriptome,
• MeSH
• Ixodes * genetics   MeSH
• Tick Bites * MeSH
• Rabbits MeSH
• Vertebrates MeSH
• Arthropod Proteins genetics   metabolism   MeSH
• Mammals genetics   MeSH
• Salivary Glands metabolism   MeSH
• Transcriptome MeSH
• Animals MeSH
• Check Tag
• Rabbits MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Arthropod Proteins MeSH

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.

• Keywords
• anti-tick vaccine, immunomodulation, serpins, therapeutic effects, tick host interaction, tick saliva,
• MeSH
• Serine Proteinase Inhibitors physiology   MeSH
• Ticks * metabolism   MeSH
• Serpins * metabolism   MeSH
• Salivary Glands metabolism   MeSH
• Saliva metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Serine Proteinase Inhibitors MeSH
• Serpins * MeSH

Protease inhibitors (PIs) are ubiquitous regulatory proteins present in all kingdoms. They play crucial tasks in controlling biological processes directed by proteases which, if not tightly regulated, can damage the host organism. PIs can be classified according to their targeted proteases or their mechanism of action. The functions of many PIs have now been characterized and are showing clinical relevance for the treatment of human diseases such as arthritis, hepatitis, cancer, AIDS, and cardiovascular diseases, amongst others. Other PIs have potential use in agriculture as insecticides, anti-fungal, and antibacterial agents. PIs from tick salivary glands are special due to their pharmacological properties and their high specificity, selectivity, and affinity to their target proteases at the tick-host interface. In this review, we discuss the structure and function of PIs in general and those PI superfamilies abundant in tick salivary glands to illustrate their possible practical applications. In doing so, we describe tick salivary PIs that are showing promise as drug candidates, highlighting the most promising ones tested in vivo and which are now progressing to preclinical and clinical trials.

• Keywords
• drug discovery, protease inhibitors, proteases, tick saliva,
• MeSH
• Protease Inhibitors isolation & purification   therapeutic use   MeSH
• Host-Parasite Interactions genetics   immunology   MeSH
• Ticks metabolism   MeSH
• Humans MeSH
• Salivary Glands metabolism   MeSH
• Saliva chemistry   metabolism   MeSH
• Transcriptome genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Protease Inhibitors MeSH

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.

• Keywords
• lncRNA, ncRNA, vector–host–pathogen interactions,
• Publication type
• Journal Article MeSH
• Review MeSH