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.

• Keywords
• autoimmune disease, immune responses, protease inhibitors, psoriasis, tick,
• MeSH
• Dermatitis * MeSH
• Endopeptidases MeSH
• Cysteine Proteinase Inhibitors MeSH
• Protease Inhibitors MeSH
• Humans MeSH
• Mannans MeSH
• Immunity, Innate MeSH
• Peptide Hydrolases MeSH
• Psoriasis * chemically induced   drug therapy   MeSH
• Inflammation drug therapy   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Endopeptidases MeSH
• Cysteine Proteinase Inhibitors MeSH
• Protease Inhibitors MeSH
• Mannans MeSH
• Peptide Hydrolases 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

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

Immunodeficiency disorders and autoimmune diseases are common, but a lack of effective targeted drugs and the side-effects of existing drugs have stimulated interest in finding therapeutic alternatives. Naturally derived substances are a recognized source of novel drugs, and tick saliva is increasingly recognized as a rich source of bioactive molecules with specific functions. Ticks use their saliva to overcome the innate and adaptive host immune systems. Their saliva is a rich cocktail of molecules including proteins, peptides, lipid derivatives, and recently discovered non-coding RNAs that inhibit or modulate vertebrate immune reactions. A number of tick saliva and/or salivary gland molecules have been characterized and shown to be promising candidates for drug development for vertebrate immune diseases. However, further validation of these molecules at the molecular, cellular, and organism levels is now required to progress lead candidates to clinical testing. In this paper, we review the data on the immuno-pharmacological aspects of tick salivary compounds characterized in vitro and/or in vivo and present recent findings on non-coding RNAs that might be exploitable as immunomodulatory therapies.

• Keywords
• drug discovery, host immunity, immunomodulation, salivary glands, tick saliva,
• MeSH
• Autoimmune Diseases immunology   therapy   MeSH
• Immunomodulation immunology   MeSH
• Ticks immunology   MeSH
• Humans MeSH
• Immune System Diseases immunology   therapy   MeSH
• Arthropod Proteins immunology   MeSH
• Saliva immunology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Arthropod Proteins MeSH

To successfully feed, ticks inject pharmacoactive molecules into the vertebrate host including cystatin cysteine protease inhibitors. However, the molecular and cellular events modulated by tick saliva remain largely unknown. Here, we describe and characterize a novel immunomodulatory cystatin, Iristatin, which is upregulated in the salivary glands of feeding Ixodes ricinus ticks. We present the crystal structure of Iristatin at 1.76 Å resolution. Purified recombinant Iristatin inhibited the proteolytic activity of cathepsins L and C and diminished IL-2, IL-4, IL-9, and IFN-γ production by different T-cell populations, IL-6 and IL-9 production by mast cells, and nitric oxide production by macrophages. Furthermore, Iristatin inhibited OVA antigen-induced CD4+ T-cell proliferation and leukocyte recruitment in vivo and in vitro. Our results indicate that Iristatin affects wide range of anti-tick immune responses in the vertebrate host and may be exploitable as an immunotherapeutic.

• Keywords
• Cathepsin, Crystal structure, Immune responses, Ixodes ricinus, Saliva,
• MeSH
• Cystatins classification   genetics   pharmacology   MeSH
• Cytokines metabolism   MeSH
• Epoxy Compounds metabolism   MeSH
• Phylogeny MeSH
• Immunosuppressive Agents chemistry   metabolism   pharmacology   MeSH
• Ixodes chemistry   genetics   metabolism   MeSH
• Crystallography, X-Ray MeSH
• Macrophages drug effects   metabolism   MeSH
• Nitric Oxide metabolism   MeSH
• Arthropod Proteins chemistry   genetics   pharmacology   MeSH
• Proteolysis drug effects   MeSH
• Amino Acid Sequence MeSH
• Sequence Homology, Amino Acid MeSH
• Salivary Cystatins chemistry   genetics   pharmacology   MeSH
• T-Lymphocytes drug effects   metabolism   MeSH
• Tyrosine analogs & derivatives   metabolism   MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• cathestatin C MeSH Browser
• Cystatins MeSH
• Cytokines MeSH
• Epoxy Compounds MeSH
• Immunosuppressive Agents MeSH
• Nitric Oxide MeSH
• Arthropod Proteins MeSH
• Salivary Cystatins MeSH
• Tyrosine MeSH

The publication of the first tick sialome (salivary gland transcriptome) heralded a new era of research of tick protease inhibitors, which represent important constituents of the proteins secreted via tick saliva into the host. Three major groups of protease inhibitors are secreted into saliva: Kunitz inhibitors, serpins, and cystatins. Kunitz inhibitors are anti-hemostatic agents and tens of proteins with one or more Kunitz domains are known to block host coagulation and/or platelet aggregation. Serpins and cystatins are also anti-hemostatic effectors, but intriguingly, from the translational perspective, also act as pluripotent modulators of the host immune system. Here we focus especially on this latter aspect of protease inhibition by ticks and describe the current knowledge and data on secreted salivary serpins and cystatins and their role in tick-host-pathogen interaction triad. We also discuss the potential therapeutic use of tick protease inhibitors.

• Keywords
• cystatins, immunomodulation, protease inhibitors, serpins, tick-host interaction,
• MeSH
• Cystatins physiology   therapeutic use   MeSH
• Immunomodulation MeSH
• Protease Inhibitors classification   metabolism   therapeutic use   MeSH
• Serine Proteinase Inhibitors physiology   therapeutic use   MeSH
• Host-Parasite Interactions MeSH
• Ticks metabolism   MeSH
• Humans MeSH
• Serpins physiology   therapeutic use   MeSH
• Saliva enzymology   metabolism   MeSH
• Transcriptome MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Cystatins MeSH
• Protease Inhibitors MeSH
• Serine Proteinase Inhibitors MeSH
• Serpins MeSH

Having emerged during the early part of the Cretaceous period, ticks are an ancient group of hematophagous ectoparasites with significant veterinary and public health importance worldwide. The success of their life strategy can be attributed, in part, to saliva. As we enter into a scientific era where the collection of massive data sets and structures for biological application is possible, we suggest that understanding the molecular mechanisms that govern the life cycle of ticks is within grasp. With this in mind, we discuss what is currently known regarding the manipulation of Toll-like (TLR) and Nod-like (NLR) receptor signaling pathways by tick salivary proteins, and how these molecules impact pathogen transmission.

• Keywords
• Innate immune signaling, Nod-like receptors (NLR), Tick saliva, Tick-borne diseases, Toll-like receptors (TLR),
• Publication type
• Journal Article MeSH

The saliva of ixodid ticks contains a mixture of bioactive molecules that target a wide spectrum of host defense mechanisms to allow ticks to feed on the vertebrate host for several days. Tick salivary proteins cluster in multigenic protein families, and individual family members display redundancy and pluripotency in their action to ameliorate or evade host immune responses. It is now clear that members of different protein families can target the same cellular or molecular pathway of the host physiological response to tick feeding. We present and discuss our hypothesis that redundancy and pluripotency evolved in tick salivary immunomodulators to evade immune recognition by the host while retaining the immunomodulatory potential of their saliva.

• Keywords
• immunomodulation, multigenic protein families, pluripotency, redundancy, silent antigens, tick salivary proteins,
• MeSH
• Arachnid Vectors immunology   parasitology   MeSH
• Immune Evasion immunology   MeSH
• Host-Parasite Interactions immunology   MeSH
• Ixodidae immunology   parasitology   MeSH
• Humans MeSH
• Parasitic Diseases immunology   transmission   MeSH
• Arthropod Proteins immunology   MeSH
• Salivary Proteins and Peptides immunology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Arthropod Proteins MeSH
• Salivary Proteins and Peptides MeSH

Coevolution of ticks and the vertebrate immune system has led to the development of immunosuppressive molecules that prevent immediate response of skin-resident immune cells to quickly fend off the parasite. In this article, we demonstrate that the tick-derived immunosuppressor sialostatin L restrains IL-9 production by mast cells, whereas degranulation and IL-6 expression are both unaffected. In addition, the expression of IL-1β and IRF4 is strongly reduced in the presence of sialostatin L. Correspondingly, IRF4- or IL-1R-deficient mast cells exhibit a strong impairment in IL-9 production, demonstrating the importance of IRF4 and IL-1 in the regulation of the Il9 locus in mast cells. Furthermore, IRF4 binds to the promoters of Il1b and Il9, suggesting that sialostatin L suppresses mast cell-derived IL-9 preferentially by inhibiting IRF4. In an experimental asthma model, mast cell-specific deficiency in IRF4 or administration of sialostatin L results in a strong reduction in asthma symptoms, demonstrating the immunosuppressive potency of tick-derived molecules.

• MeSH
• Asthma genetics   immunology   pathology   MeSH
• Cystatins immunology   pharmacology   MeSH
• Cell Degranulation immunology   MeSH
• Transcription, Genetic MeSH
• Immunosuppressive Agents pharmacology   MeSH
• Host-Parasite Interactions immunology   MeSH
• Interferon Regulatory Factors deficiency   genetics   immunology   MeSH
• Interleukin-1beta genetics   immunology   MeSH
• Interleukin-6 genetics   immunology   MeSH
• Interleukin-9 antagonists & inhibitors   genetics   immunology   MeSH
• Mast Cells drug effects   immunology   pathology   MeSH
• Mice, Inbred BALB C MeSH
• Mice, Inbred C57BL MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Immunity, Innate drug effects   MeSH
• Promoter Regions, Genetic MeSH
• Receptors, Interleukin-1 genetics   immunology   MeSH
• Gene Expression Regulation MeSH
• Signal Transduction MeSH
• Protein Binding MeSH
• Binding Sites 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
• Cystatins MeSH
• Immunosuppressive Agents MeSH
• interferon regulatory factor-4 MeSH Browser
• Interferon Regulatory Factors MeSH
• Interleukin-1beta MeSH
• Interleukin-6 MeSH
• Interleukin-9 MeSH
• Receptors, Interleukin-1 MeSH
• sialostatin L, Ixodes scapularis MeSH Browser

Th17 cells constitute a subset of CD4(+) T lymphocytes that play a crucial role in protection against extracellular bacteria and fungi. They are also associated with tissue injury in autoimmune and inflammatory diseases. Here, we report that serpin from the tick Ixodes ricinus, IRS-2, inhibits Th17 differentiation by impairment of the interleukin-6 (IL-6)/STAT-3 signaling pathway. Following activation, mature dendritic cells produce an array of cytokines, including the pleiotropic cytokine IL-6, which triggers the IL-6 signaling pathway. The major transcription factor activated by IL-6 is STAT-3. We show that IRS-2 selectively inhibits production of IL-6 in dendritic cells stimulated with Borrelia spirochetes, which leads to attenuated STAT-3 phosphorylation and finally to impaired Th17 differentiation. The results presented extend the knowledge about the effect of tick salivary serpins on innate immunity cells and their function in driving adaptive immune responses.

• MeSH
• Borrelia immunology   MeSH
• Cell Differentiation drug effects   MeSH
• Th17 Cells drug effects   physiology   MeSH
• Dendritic Cells drug effects   physiology   MeSH
• Interleukin-6 antagonists & inhibitors   metabolism   MeSH
• Ixodes MeSH
• Mice, Inbred C57BL MeSH
• Serpins metabolism   MeSH
• Signal Transduction drug effects   MeSH
• STAT3 Transcription Factor antagonists & inhibitors   metabolism   MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• interleukin-6, mouse MeSH Browser
• Interleukin-6 MeSH
• Serpins MeSH
• Stat3 protein, mouse MeSH Browser
• STAT3 Transcription Factor MeSH