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.

• Klíčová slova
• Cathepsin, Crystal structure, Immune responses, Ixodes ricinus, Saliva,
• MeSH
• cystatiny klasifikace   genetika   farmakologie   MeSH
• cytokiny metabolismus   MeSH
• epoxidové sloučeniny metabolismus   MeSH
• fylogeneze MeSH
• imunosupresiva chemie   metabolismus   farmakologie   MeSH
• klíště chemie   genetika   metabolismus   MeSH
• krystalografie rentgenová MeSH
• makrofágy účinky léků   metabolismus   MeSH
• oxid dusnatý metabolismus   MeSH
• proteiny členovců chemie   genetika   farmakologie   MeSH
• proteolýza účinky léků   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• slinné cystatiny chemie   genetika   farmakologie   MeSH
• T-lymfocyty účinky léků   metabolismus   MeSH
• tyrosin analogy a deriváty   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cathestatin C MeSH Prohlížeč
• cystatiny MeSH
• cytokiny MeSH
• epoxidové sloučeniny MeSH
• imunosupresiva MeSH
• oxid dusnatý MeSH
• proteiny členovců MeSH
• slinné cystatiny MeSH
• tyrosin MeSH

To ensure successful feeding tick saliva contains a number of inhibitory proteins that interfere with the host immune response and help to create a permissive environment for pathogen transmission. Among the potential targets of the salivary cystatins are two host cysteine proteases, cathepsin S, which is essential for antigen- and invariant chain-processing, and cathepsin C (dipeptidyl peptidase 1, DPP1), which plays a critical role in processing and activation of the granule serine proteases. Here, the effect of salivary cystatin OmC2 from Ornithodoros moubata was studied using differentiated MUTZ-3 cells as a model of immature dendritic cells of the host skin. Following internalization, cystatin OmC2 was initially found to inhibit the activity of several cysteine cathepsins, as indicated by the decreased rates of degradation of fluorogenic peptide substrates. To identify targets, affinity chromatography was used to isolate His-tagged cystatin OmC2 together with the bound proteins from MUTZ-3 cells. Cathepsins S and C were identified in these complexes by mass spectrometry and confirmed by immunoblotting. Furthermore, reduced increase in the surface expression of MHC II and CD86, which are associated with the maturation of dendritic cells, was observed. In contrast, human inhibitor cystatin C, which is normally expressed and secreted by dendritic cells, did not affect the expression of CD86. It is proposed that internalization of salivary cystatin OmC2 by the host dendritic cells targets cathepsins S and C, thereby affecting their maturation.

• Klíčová slova
• DPP1, cathepsin C, cathepsin S, cystatin OmC2, dendritic cells, dipeptidyl peptidase 1, lysosomal proteases, tick saliva,
• MeSH
• antigeny CD86 MeSH
• antigeny diferenciační B-lymfocytární MeSH
• buněčné linie MeSH
• cystatiny metabolismus   MeSH
• dendritické buňky imunologie   metabolismus   MeSH
• epoxidové sloučeniny imunologie   metabolismus   MeSH
• geny MHC třídy II imunologie   MeSH
• histokompatibilita - antigeny třídy II MeSH
• kathepsin C metabolismus   MeSH
• kathepsiny chemie   imunologie   metabolismus   MeSH
• klíšťata enzymologie   MeSH
• lidé MeSH
• lyzozomy enzymologie   MeSH
• Ornithodoros enzymologie   MeSH
• rekombinantní proteiny MeSH
• sliny enzymologie   MeSH
• tyrosin analogy a deriváty   imunologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny CD86 MeSH
• antigeny diferenciační B-lymfocytární MeSH
• cathepsin S MeSH Prohlížeč
• cathestatin C MeSH Prohlížeč
• CTSC protein, human MeSH Prohlížeč
• cystatiny MeSH
• epoxidové sloučeniny MeSH
• histokompatibilita - antigeny třídy II MeSH
• invariant chain MeSH Prohlížeč
• kathepsin C MeSH
• kathepsiny MeSH
• rekombinantní proteiny MeSH
• tyrosin MeSH

UNLABELLED: Next generation sequencing and proteomics have helped to comprehensively characterize gene expression in tick salivary glands at both the transcriptome and the proteome level. Functional data are, however, lacking. Given that tick salivary secretions are critical to the success of the tick transmission lifecycle and, as a consequence, for host colonization by the pathogens they spread, we thoroughly review here the literature on the known interactions between tick saliva (or tick salivary gland extracts) and the innate and adaptive vertebrate immune system. The information is intended to serve as a reference for functional characterization of the numerous genes and proteins expressed in tick salivary glands with an ultimate goal to develop novel vector and pathogen control strategies. SIGNIFICANCE: We overview all the known interactions of tick saliva with the vertebrate immune system. The provided information is important, given the recent developments in high-throughput transcriptomic and proteomic analysis of gene expression in tick salivary glands, since it may serve as a guideline for the functional characterization of the numerous newly-discovered genes expressed in tick salivary glands.

• Klíčová slova
• Adaptive immunity, Innate immunity, Saliva, Salivary glands, Tick,
• MeSH
• hmyzí proteiny imunologie   MeSH
• interakce hostitele a parazita imunologie   MeSH
• klíšťata imunologie   MeSH
• modely imunologické MeSH
• přirozená imunita imunologie   MeSH
• sliny imunologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, N.I.H., Intramural MeSH
• Názvy látek
• hmyzí proteiny MeSH

BACKGROUND: Transmission of pathogens by ticks is greatly supported by tick saliva released during feeding. Dendritic cells (DC) act as immunological sentinels and interconnect the innate and adaptive immune system. They control polarization of the immune response towards Th1 or Th2 phenotype. We investigated whether salivary cystatins from the hard tick Ixodes scapularis, sialostatin L (Sialo L) and sialostatin L2 (Sialo L2), influence mouse dendritic cells exposed to Borrelia burgdorferi and relevant Toll-like receptor ligands. METHODS: DCs derived from bone-marrow by GM-CSF or Flt-3 ligand, were activated with Borrelia spirochetes or TLR ligands in the presence of 3 μM Sialo L and 3 μM Sialo L2. Produced chemokines and IFN-β were measured by ELISA test. The activation of signalling pathways was tested by western blotting using specific antibodies. The maturation of DC was determined by measuring the surface expression of CD86 by flow cytometry. RESULTS: We determined the effect of cystatins on the production of chemokines in Borrelia-infected bone-marrow derived DC. The production of MIP-1α was severely suppressed by both cystatins, while IP-10 was selectively inhibited only by Sialo L2. As TLR-2 is a major receptor activated by Borrelia spirochetes, we tested whether cystatins influence signalling pathways activated by TLR-2 ligand, lipoteichoic acid (LTA). Sialo L2 and weakly Sialo L attenuated the extracellular matrix-regulated kinase (Erk1/2) pathway. The activation of phosphatidylinositol-3 kinase (PI3K)/Akt pathway and nuclear factor-κB (NF-κB) was decreased only by Sialo L2. In response to Borrelia burgdorferi, the activation of Erk1/2 was impaired by Sialo L2. Production of IFN-β was analysed in plasmacytoid DC exposed to Borrelia, TLR-7, and TLR-9 ligands. Sialo L, in contrast to Sialo L2, decreased the production of IFN-β in pDC and also impaired the maturation of these cells. CONCLUSIONS: This study shows that DC responses to Borrelia spirochetes are affected by tick cystatins. Sialo L influences the maturation of DC thus having impact on adaptive immune response. Sialo L2 affects the production of chemokines potentially engaged in the development of inflammatory response. The impact of cystatins on Borrelia growth in vivo is discussed.

• MeSH
• Borrelia burgdorferi imunologie   MeSH
• cystatiny farmakologie   MeSH
• dendritické buňky účinky léků   fyziologie   MeSH
• klíště fyziologie   MeSH
• kyseliny teichoové MeSH
• lipopolysacharidy MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• signální transdukce fyziologie   MeSH
• sliny chemie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cystatiny MeSH
• kyseliny teichoové MeSH
• lipopolysacharidy MeSH
• lipoteichoic acid MeSH Prohlížeč
• sialostatin L, Ixodes scapularis MeSH Prohlížeč