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.
- Klíčová slova
- Cystatins, Host–parasite interactions, Ixodes ricinus, Protease inhibition, Protein structure, Tick saliva,
- MeSH
- cystatiny * farmakologie MeSH
- cystein metabolismus MeSH
- endopeptidasy metabolismus MeSH
- kathepsiny metabolismus MeSH
- klíště * chemie MeSH
- obratlovci MeSH
- proteasy metabolismus MeSH
- slinné cystatiny chemie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- cystatiny * MeSH
- cystein MeSH
- endopeptidasy MeSH
- kathepsiny MeSH
- proteasy MeSH
- slinné cystatiny 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.
- 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
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
- bronchiální astma genetika imunologie patologie MeSH
- cystatiny imunologie farmakologie MeSH
- degranulace buněk imunologie MeSH
- genetická transkripce MeSH
- imunosupresiva farmakologie MeSH
- interakce hostitele a parazita imunologie MeSH
- interferonové regulační faktory nedostatek genetika imunologie MeSH
- interleukin-1beta genetika imunologie MeSH
- interleukin-6 genetika imunologie MeSH
- interleukin-9 antagonisté a inhibitory genetika imunologie MeSH
- mastocyty účinky léků imunologie patologie MeSH
- myši inbrední BALB C MeSH
- myši inbrední C57BL MeSH
- myši knockoutované MeSH
- myši MeSH
- přirozená imunita účinky léků MeSH
- promotorové oblasti (genetika) MeSH
- receptory interleukinu-1 genetika imunologie MeSH
- regulace genové exprese MeSH
- signální transdukce MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Intramural MeSH
- Názvy látek
- cystatiny MeSH
- imunosupresiva MeSH
- interferon regulatory factor-4 MeSH Prohlížeč
- interferonové regulační faktory MeSH
- interleukin-1beta MeSH
- interleukin-6 MeSH
- interleukin-9 MeSH
- receptory interleukinu-1 MeSH
- sialostatin L, Ixodes scapularis MeSH Prohlížeč
