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.

Department of Medical Biology Faculty of Science University of South Bohemia in České Budějovice České Budějovice Czechia

Laboratory of Genomics and Proteomics of Disease Vectors Institute of Parasitology Biology Centre of the Czech Academy of Sciences České Budějovice Czechia

Laboratory of Structural Chemistry Institute of Chemistry Faculty of Science University of South Bohemia in České Budějovice České Budějovice Czechia

Zobrazit více v PubMed

Rizzoli A, Silaghi C, Obiegala A, Rudolf I, Hubálek Z, Földvári G, et al. PubMed DOI PMC

Castelli E, Caputo V, Morello V, Tomasino RM. Local reactions to tick bites. Am J Dermatopathol (2008) 30:241–8.   10.1097/DAD.0b013e3181676b60 PubMed DOI

Heinze DM, Carmical JR, Aronson JF, Thangamani S. Early immunologic events at the tick-host interface. PLoS One (2012) 7:e47301.   10.1371/journal.pone.0047301 PubMed DOI PMC

Boppana DKV, Wikel SK, Raj DG, Manohar MB, Lalitha J. Cellular infiltration at skin lesions and draining lymph nodes of sheep infested with adult PubMed DOI

Mbow ML, Rutti B, Brossard M. Infiltration of CD4+ CD8+ T cells, and expression of ICAM-1, Ia antigens, IL-1 alpha and TNF-alpha in the skin lesion of BALB/c mice undergoing repeated infestations with nymphal PubMed PMC

Glatz M, Means T, Haas J, Steere AC, Müllegger RR. Characterization of the early local immune response to PubMed DOI PMC

Nithiuthai S, Allen JR. Langerhans cells present tick antigens to lymph node cells from tick-sensitized guinea-pigs. Immunology (1985) 55:157–63. PubMed PMC

Kazimírová M, Štibrániová I. Tick salivary compounds: their role in modulation of host defences and pathogen transmission. Front Cell Infect Microbiol (2013) 3:43.   10.3389/fcimb.2013.00043 PubMed DOI PMC

Šimo L, Kazimirova M, Richardson J, Bonnet SI. The essential role of tick salivary glands and saliva in tick feeding and pathogen transmission. Front Cell Infect Microbiol (2017) 7:281.   10.3389/fcimb.2017.00281 PubMed DOI PMC

Mans BJ. Chemical equilibrium at the tick–host feeding interface: a critical examination of biological relevance in hematophagous behavior. Front Physiol (2019) 10:530.   10.3389/fphys.2019.00530 PubMed DOI PMC

Štibrániová I, Bartíková P, Holíková V, Kazimírová M. Deciphering biological processes at the tick-host interface opens new strategies for treatment of human diseases. Front Physiol (2019) 10:830.   10.3389/fphys.2019.00830 PubMed DOI PMC

Hovius JWR, Levi M, Fikrig E. Salivating for knowledge: potential pharmacological agents in tick saliva. PLoS Med (2008) 5:e43.   10.1371/journal.pmed.0050043 PubMed DOI PMC

Chmelar J, Calvo E, Pedra JHF, Francischetti IMB, Kotsyfakis M. Tick salivary secretion as a source of antihemostatics. J Proteomics (2012) 75:3842–54.   10.1016/j.jprot.2012.04.026 PubMed DOI PMC

Chmelař J, Kotál J, Kovaříková A, Kotsyfakis M. The use of tick salivary proteins as novel therapeutics. Front Physiol (2019) 10:812.   10.3389/fphys.2019.00812 PubMed DOI PMC

Rego ROM, Trentelman JJA, Anguita J, Nijhof AM, Sprong H, Klempa B, et al. Counterattacking the tick bite: towards a rational design of anti-tick vaccines targeting pathogen transmission. Parasit Vectors (2019) 12:229.   10.1186/s13071-019-3468-x PubMed DOI PMC

Schwarz A, von Reumont BM, Erhart J, Chagas AC, Ribeiro JMC, Kotsyfakis M. PubMed DOI PMC

Martins LA, Kotál J, Bensaoud C, Chmelař J, Kotsyfakis M. Small protease inhibitors in tick saliva and salivary glands and their role in tick-host-pathogen interactions. Biochim Biophys Acta Proteins Proteom (2020) 1868:140336.   10.1016/j.bbapap.2019.140336 PubMed DOI

Law RH, Zhang Q, McGowan S, Buckle AM, Silverman GA, Wong W, et al. An overview of the serpin superfamily. Genome Biol (2006) 7:216.   10.1186/gb-2006-7-5-216 PubMed DOI PMC

Heit C, Jackson BC, McAndrews M, Wright MW, Thompson DC, Silverman GA, et al. Update of the human and mouse SERPIN gene superfamily. Hum Genomics (2013) 7:22.   10.1186/1479-7364-7-22 PubMed DOI PMC

Khan MS, Singh P, Azhar A, Naseem A, Rashid Q, Kabir MA, et al. Serpin inhibition mechanism: a delicate balance between native metastable state and polymerization. J Amino Acids (2011) 2011:606797.   10.4061/2011/606797 PubMed DOI PMC

Porter L, Radulovic Z, Kim T, Braz GRC, Da Silva Vaz I, Mulenga A. Bioinformatic analyses of male and female PubMed DOI PMC

Tirloni L, Islam MS, Kim TK, Diedrich JK, Yates JR, Pinto AFM, et al. Saliva from nymph and adult females of PubMed DOI PMC

Kotsyfakis M, Schwarz A, Erhart J, Ribeiro JMC. Tissue- and time-dependent transcription in PubMed DOI PMC

Mulenga A, Khumthong R, Chalaire KC. PubMed DOI PMC

de Castro MH, de Klerk D, Pienaar R, Latif AA, Rees DJG, Mans BJ. PubMed DOI

Tirloni L, Seixas A, Mulenga A, da Silva Vaz I, Termignoni C. A family of serine protease inhibitors (serpins) in the cattle tick PubMed DOI

Rodriguez-Valle M, Xu T, Kurscheid S, Lew-Tabor AE. PubMed DOI PMC

Prevot P-P, Adam B, Boudjeltia KZ, Brossard M, Lins L, Cauchie P, et al. Anti-hemostatic effects of a serpin from the saliva of the tick PubMed DOI

Yu Y, Cao J, Zhou Y, Zhang H, Zhou J. Isolation and characterization of two novel serpins from the tick PubMed DOI

Ibelli AMG, Kim TK, Hill CC, Lewis LA, Bakshi M, Miller S, et al. A blood meal-induced PubMed DOI PMC

Kim TK, Tirloni L, Radulovic Z, Lewis L, Bakshi M, Hill C, et al. Conserved PubMed DOI PMC

Chmelar J, Oliveira CJ, Rezacova P, Francischetti IMB, Kovarova Z, Pejler G, et al. A tick salivary protein targets cathepsin G and chymase and inhibits host inflammation and platelet aggregation. Blood (2011) 117:736–44.   10.1182/blood-2010-06-293241 PubMed DOI PMC

Tirloni L, Kim TK, Coutinho ML, Ali A, Seixas A, Termignoni C, et al. The putative role of PubMed DOI PMC

Tirloni L, Kim TK, Berger M, Termignoni C, da Silva Vaz I, Mulenga A. PubMed DOI PMC

Coutinho ML, Bizzarro B, Tirloni L, Berger M, Freire Oliveira CJ, Sá-Nunes A, et al. PubMed DOI PMC

Kim TK, Tirloni L, Berger M, Diedrich JK, Yates JR, Termignoni C, et al. PubMed DOI PMC

Wang F, Song Z, Chen J, Wu Q, Zhou X, Ni X, et al. The immunosuppressive functions of two novel tick serpins, HlSerpin-a and HlSerpin-b, from PubMed DOI PMC

Leboulle G, Crippa M, Decrem Y, Mejri N, Brossard M, Bollen A, et al. Characterization of a novel salivary immunosuppressive protein from PubMed DOI

Páleníková J, Lieskovská J, Langhansová H, Kotsyfakis M, Chmelař J, Kopecký J. PubMed DOI PMC

Toyomane K, Konnai S, Niwa A, Githaka N, Isezaki M, Yamada S, et al. Identification and the preliminary PubMed DOI

Imamura S, Da Silva Vaz I, Sugino M, Ohashi K, Onuma M. A serine protease inhibitor (serpin) from PubMed DOI

Prevot P-P, Couvreur B, Denis V, Brossard M, Vanhamme L, Godfroid E. Protective immunity against PubMed DOI

Kim TK, Radulovic Z, Mulenga A. Target validation of highly conserved PubMed DOI PMC

Xu Z, Yan Y, Cao J, Zhou Y, Zhang H, Xu Q, et al. A family of serine protease inhibitors (serpins) and its expression profiles in the ovaries of PubMed DOI

Xu Z, Yan Y, Zhang H, Cao J, Zhou Y, Xu Q, et al. A serpin from the tick PubMed DOI

Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, et al. Protein Identification and Analysis Tools on the ExPASy Server. In: Walker JM, editor. The Proteomics Protocols Handbook Springer Protocols Handbooks. Totowa, NJ: Humana Press; (2005). p. 571–607.   10.1385/1-59259-890-0:571 DOI

Petersen TN, Brunak S, von Heijne G, Nielsen H. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods (2011) 8:785–6.   10.1038/nmeth.1701 PubMed DOI

de Castro E, Sigrist CJA, Gattiker A, Bulliard V, Langendijk-Genevaux PS, Gasteiger E, et al. ScanProsite: detection of PROSITE signature matches and ProRule-associated functional and structural residues in proteins. Nucleic Acids Res (2006) 34:W362–5.   10.1093/nar/gkl124 PubMed DOI PMC

Mulenga A, Khumthong R, Blandon MA. Molecular and expression analysis of a family of the PubMed DOI

Steentoft C, Vakhrushev SY, Joshi HJ, Kong Y, Vester-Christensen MB, Schjoldager KT-BG, et al. Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology. EMBO J (2013) 32:1478–88.   10.1038/emboj.2013.79 PubMed DOI PMC

Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol (1990) 215:403–10.   10.1016/S0022-2836(05)80360-2 PubMed DOI

Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, et al. Clustal W and Clustal X version 2.0. Bioinformatics (Oxford Engl) (2007) 23:2947–8.   10.1093/bioinformatics/btm404 PubMed DOI

Robert X, Gouet P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res (2014) 42:W320–4.   10.1093/nar/gku316 PubMed DOI PMC

Mueller U, Förster R, Hellmig M, Huschmann FU, Kastner A, Malecki P, et al. The macromolecular crystallography beamlines at BESSY II of the Helmholtz-Zentrum Berlin: current status and perspectives. Eur Phys J Plus (2015) 130:141.   10.1140/epjp/i2015-15141-2 DOI

Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser (1999) 41:95–8. 10.14601/Phytopathol_Mediterr-14998u1.29 DOI

Jones DT, Taylor WR, Thornton JM. The rapid generation of mutation data matrices from protein sequences. Comput Appl Biosci (1992) 8:275–82.   10.1093/bioinformatics/8.3.275 PubMed DOI

Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol (1987) 4:406–25.   10.1093/oxfordjournals.molbev.a040454 PubMed DOI

Gascuel O. BIONJ: an improved version of the NJ algorithm based on a simple model of sequence data. Mol Biol Evol (1997) 14:685–95.   10.1093/oxfordjournals.molbev.a025808 PubMed DOI

Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol (2018) 35:1547–9.   10.1093/molbev/msy096 PubMed DOI PMC

Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods (2001) 25:402–8.   10.1006/meth.2001.1262 PubMed DOI

Vechtova P, Fussy Z, Cegan R, Sterba J, Erhart J, Benes V, et al. Catalogue of stage-specific transcripts in PubMed DOI PMC

Koči J, Šimo L, Park Y. Validation of internal reference genes for real-time quantitative polymerase chain reaction studies in the tick, PubMed DOI PMC

Kýcková K, Kopecký J. Effect of tick saliva on mechanisms of innate immune response against PubMed DOI

Pfaffl MW. A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Res (2001) 29:e45.   10.1093/nar/29.9.e45 PubMed DOI PMC

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol (2002) 3:research0034.1.   10.1186/gb-2002-3-7-research0034 PubMed DOI PMC

Lew M. Good statistical practice in pharmacology Problem 2. Br J Pharmacol (2007) 152:299–303.   10.1038/sj.bjp.0707372 PubMed DOI PMC

Schechter I, Berger A. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun (1967) 27:157–62.   10.1016/s0006-291x(67)80055-x PubMed DOI

Hopkins PC, Carrell RW, Stone SR. Effects of mutations in the hinge region of serpins. Biochemistry (1993) 32:7650–7.   10.1021/bi00081a008 PubMed DOI

Olsen JV, Ong S-E, Mann M. Trypsin cleaves exclusively C-terminal to arginine and lysine residues. Mol Cell Proteomics (2004) 3:608–14.   10.1074/mcp.T400003-MCP200 PubMed DOI

Kovářová Z, Chmelař J, Šanda M, Brynda J, Mareš M, Řezáčová P. Crystallization and diffraction analysis of the serpin IRS-2 from the hard tick PubMed DOI PMC

Gettins PGW. Serpin structure, mechanism, and function. Chem Rev (2002) 102:4751–804.   10.1021/cr010170+ PubMed DOI

Chmelař J, Kotál J, Langhansová H, Kotsyfakis M. Protease inhibitors in tick saliva: the role of serpins and cystatins in tick-host-pathogen interaction. Front Cell Infect Microbiol (2017) 7:216.   10.3389/fcimb.2017.00216 PubMed DOI PMC

Raber MN. Coagulation Tests, in: Clinical Methods: The History, Physical, and Laboratory Examinations. Boston: Butterworths. Available at: http://www.ncbi.nlm.nih.gov/books/NBK265/ (Accessed October 12, 2020).

Bakshi M, Kim TK, Porter L, Mwangi W, Mulenga A. PubMed DOI PMC

Zheng W, Flavell RA. The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4+ T cells. Cell (1997) 89:587–96.   10.1016/S0092-8674(00)80240-8 PubMed DOI

Szabo SJ, Kim ST, Costa GL, Zhang X, Fathman CG, Glimcher LH. A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell (2000) 100:655–69.   10.1016/S0092-8674(00)80702-3 PubMed DOI

Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol (2003) 4:330–6.   10.1038/ni904 PubMed DOI

Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science (2003) 299:1057–61.   10.1126/science.1079490 PubMed DOI

Ivanov II, McKenzie BS, Zhou L, Tadokoro CE, Lepelley A, Lafaille JJ, et al. The orphan nuclear receptor RORγt directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell (2006) 126:1121–33.   10.1016/j.cell.2006.07.035 PubMed DOI

Francischetti IMB, Sá-Nunes A, Mans BJ, Santos IM, Ribeiro JMC. The role of saliva in tick feeding. Front Biosci (2009) 14:2051–88. 10.2741/3363 PubMed DOI PMC

O’Reilly MS. Antiangiogenic antithrombin. Semin Thromb Hemost (2007) 33:660–6.   10.1055/s-2007-991533 PubMed DOI

Rau JC, Beaulieu LM, Huntington JA, Church FC. Serpins in thrombosis, hemostasis and fibrinolysis. J Thromb Haemost (2007) 5:102–15.   10.1111/j.1538-7836.2007.02516.x PubMed DOI PMC

Gál P, Dobó J, Beinrohr L, Pál G, Závodszky P. Inhibition of the serine proteases of the complement system. Adv Exp Med Biol (2013) 735:23–40.   10.1007/978-1-4614-4118-2_2 PubMed DOI

Mkaouar H, Akermi N, Kriaa A, Abraham A-L, Jablaoui A, Soussou S, et al. Serine protease inhibitors and human wellbeing interplay: new insights for old friends. PeerJ (2019) 7:e7224.   10.7717/peerj.7224 PubMed DOI PMC

Harris JL, Backes BJ, Leonetti F, Mahrus S, Ellman JA, Craik CS. Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries. Proc Natl Acad Sci U S A (2000) 97:7754–9.   10.1073/pnas.140132697 PubMed DOI PMC

Björkqvist J, Jämsä A, Renné T. Plasma kallikrein: the bradykinin-producing enzyme. Thromb Haemost (2013) 110:399–407.   10.1160/TH13-03-0258 PubMed DOI

Paterson KJ, Zambreanu L, Bennett DLH, McMahon SB. Characterisation and mechanisms of bradykinin-evoked pain in man using iontophoresis. Pain (2013) 154:782–92.   10.1016/j.pain.2013.01.003 PubMed DOI PMC

List K, Bugge TH, Szabo R. Matriptase: potent proteolysis on the cell surface. Mol Med (2006) 12:1–7.   10.2119/2006-00022.List PubMed DOI PMC

Chen Y-W, Wang J-K, Chou F-P, Wu B-Y, Hsiao H-C, Chiu H, et al. Matriptase regulates proliferation and early, but not terminal, differentiation of human keratinocytes. J Invest Dermatol (2014) 134:405–14.   10.1038/jid.2013.320 PubMed DOI PMC

Bardou O, Menou A, François C, Duitman JW, von der Thüsen JH, Borie R, et al. Membrane-anchored serine protease matriptase is a trigger of pulmonary fibrogenesis. Am J Respir Crit Care Med (2016) 193:847–60.   10.1164/rccm.201502-0299OC PubMed DOI PMC

Seitz I, Hess S, Schulz H, Eckl R, Busch G, Montens HP, et al. Membrane-type serine protease-1/matriptase induces interleukin-6 and -8 in endothelial cells by activation of protease-activated receptor-2: potential implications in atherosclerosis. Arterioscler Thromb Vasc Biol (2007) 27:769–75.   10.1161/01.ATV.0000258862.61067.14 PubMed DOI

Sugino M, Imamura S, Mulenga A, Nakajima M, Tsuda A, Ohashi K, et al. A serine proteinase inhibitor (serpin) from ixodid tick PubMed DOI

Palta S, Saroa R, Palta A. Overview of the coagulation system. Indian J Anaesth (2014) 58:515–23.   10.4103/0019-5049.144643 PubMed DOI PMC

Larsen KS, Østergaard H, Bjelke JR, Olsen OH, Rasmussen HB, Christensen L, et al. Engineering the substrate and inhibitor specificities of human coagulation Factor VIIa. Biochem J (2007) 405:429–38.   10.1042/BJ20061901 PubMed DOI PMC

Lawson JH, Butenas S, Ribarik N, Mann KG. Complex-dependent inhibition of factor VIIa by antithrombin III and heparin. J Biol Chem (1993) 268:767–70. 10.1016/S0021-9258(18)53998-3 PubMed DOI

Rao LV, Rapaport SI, Hoang AD. Binding of factor VIIa to tissue factor permits rapid antithrombin III/heparin inhibition of factor VIIa. Blood (1993) 81:2600–7. 10.1182/blood.V81.10.2600.2600 PubMed DOI

Fortenberry YM, Hlavacek AC, Church FC. Protein C inhibitor inhibits factor VIIa when bound to tissue factor. J Thromb Haemost (2011) 9:861–3.   10.1111/j.1538-7836.2011.04196.x PubMed DOI

Prevot P-P, Beschin A, Lins L, Beaufays J, Grosjean A, Bruys L, et al. Exosites mediate the anti-inflammatory effects of a multifunctional serpin from the saliva of the tick PubMed DOI

Aldonyte R, Jansson L, Janciauskiene S. Concentration-dependent effects of native and polymerised α1-antitrypsin on primary human monocytes, PubMed DOI PMC

Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, et al. Reciprocal developmental pathways for the generation of pathogenic effector Th17 and regulatory T cells. Nature (2006) 441:235–8.   10.1038/nature04753 PubMed DOI

Korn T, Mitsdoerffer M, Croxford AL, Awasthi A, Dardalhon VA, Galileos G, et al. IL-6 controls Th17 immunity PubMed DOI PMC

Chen X, Das R, Komorowski R, Beres A, Hessner MJ, Mihara M, et al. Blockade of interleukin-6 signaling augments regulatory T-cell reconstitution and attenuates the severity of graft-versus-host disease. Blood (2009) 114:891–900.   10.1182/blood-2009-01-197178 PubMed DOI PMC

Plitas G, Rudensky AY. Regulatory T cells: differentiation and function. Cancer Immunol Res (2016) 4:721–5.   10.1158/2326-6066.CIR-16-0193 PubMed DOI PMC

Ferreira BR, Silva JS. Successive tick infestations selectively promote a T-helper 2 cytokine profile in mice. Immunology (1999) 96:434–9.   10.1046/j.1365-2567.1999.00683.x PubMed DOI PMC

Heinze DM, Wikel SK, Thangamani S, Alarcon-Chaidez FJ. Transcriptional profiling of the murine cutaneous response during initial and subsequent infestations with PubMed DOI PMC

Ashton-Rickardt PG. An emerging role for Serine Protease Inhibitors in T lymphocyte immunity and beyond. Immunol Lett (2013) 152:65–76.   10.1016/j.imlet.2013.04.004 PubMed DOI

Gao G, Shao C, Zhang SX, Dudley A, Fant J, Ma J-X. Kallikrein-binding protein inhibits retinal neovascularization and decreases vascular leakage. Diabetologia (2003) 46:689–98.   10.1007/s00125-003-1085-9 PubMed DOI

Latha K, Zhang W, Cella N, Shi HY, Zhang M. Maspin mediates increased tumor cell apoptosis upon induction of the mitochondrial permeability transition. Mol Cell Biol (2005) 25:1737–48.   10.1128/MCB.25.5.1737-1748.2005 PubMed DOI PMC

Chen L, Zhang SS-M, Barnstable CJ, Tombran-Tink J. PEDF induces apoptosis in human endothelial cells by activating p38 MAP kinase dependent cleavage of multiple caspases. Biochem Biophys Res Commun (2006) 348:1288–95.   10.1016/j.bbrc.2006.07.188 PubMed DOI

Becerra SP, Notario V. The effects of PEDF on cancer biology: mechanisms of action and therapeutic potential. Nat Rev Cancer (2013) 13:258–71.   10.1038/nrc3484 PubMed DOI PMC

Yao Y, Li L, Huang X, Gu X, Xu Z, Zhang Y, et al. SERPINA3K induces apoptosis in human colorectal cancer cells via activating the Fas/FasL/caspase-8 signaling pathway. FEBS J (2013) 280:3244–55.   10.1111/febs.12303 PubMed DOI

Bröker LE, Kruyt FAE, Giaccone G. Cell death independent of caspases: a review. Clin Cancer Res (2005) 11:3155–62.   10.1158/1078-0432.CCR-04-2223 PubMed DOI

Dhuriya YK, Sharma D. Necroptosis: a regulated inflammatory mode of cell death. J Neuroinflamm (2018) 15:199.   10.1186/s12974-018-1235-0 PubMed DOI PMC

Kovár L, Kopecký J, Ríhová B. Salivary gland extract from PubMed DOI

Kovár L, Kopecký J, Ríhová B. Salivary gland extract from PubMed DOI

Mejri N, Rutti B, Brossard M. Immunosuppressive effects of PubMed DOI

Skallová A, Iezzi G, Ampenberger F, Kopf M, Kopecky J. Tick saliva inhibits dendritic cell migration, maturation, and function while promoting development of Th2 responses. J Immunol (2008) 180:6186–92.   10.4049/jimmunol.180.9.6186 PubMed DOI

Arce-Sillas A, Álvarez-Luquín DD, Tamaya-Domínguez B, Gomez-Fuentes S, Trejo-García A, Melo-Salas M, et al. Regulatory T cells: molecular actions on effector cells in immune regulation. J Immunol Res (2016) 2016:1720827.   10.1155/2016/1720827 PubMed DOI PMC

Chmelař J, Kotál J, Kopecky J, Pedra JH, Kotsyfakis M. All for one and one for all on the tick-host battlefield. Trends Parasitol (2016) 32:368–77.   10.1016/j.pt.2016.01.004 PubMed DOI PMC

Kotál J, Stergiou N, Buša M, Chlastáková A, Beránková Z, Řezáčová P, et al. The structure and function of Iristatin, a novel immunosuppressive tick salivary cystatin. Cell Mol Life Sci (2019) 76:2003–13.   10.1007/s00018-019-03034-3 PubMed DOI PMC

Blisnick AA, Šimo L, Grillon C, Fasani F, Brûlé S, Le Bonniec B, et al. The immunomodulatory effect of IrSPI, a tick salivary gland serine protease inhibitor involved in PubMed DOI PMC

Visentin C, Broggini L, Sala BM, Russo R, Barbiroli A, Santambrogio C, et al. Glycosylation tunes neuroserpin physiological and pathological properties. Int J Mol Sci (2020) 21:3235.   10.3390/ijms21093235 PubMed DOI PMC

Sarkar A, Wintrode PL. Effects of glycosylation on the stability and flexibility of a metastable protein: the human serpin α(1)-antitrypsin. Int J Mass Spectrom (2011) 302:69–75.   10.1016/j.ijms.2010.08.003 PubMed DOI PMC

Kwon KS, Yu MH. Effect of glycosylation on the stability of alpha1-antitrypsin toward urea denaturation and thermal deactivation. Biochim Biophys Acta (1997) 1335:265–72.   10.1016/s0304-4165(96)00143-2 PubMed DOI

Bergin DA, Reeves EP, Meleady P, Henry M, McElvaney OJ, Carroll TP, et al. α-1 Antitrypsin regulates human neutrophil chemotaxis induced by soluble immune complexes and IL-8. J Clin Invest (2010) 120:4236–50.   10.1172/JCI41196 PubMed DOI PMC

IxsS7: A novel biomarker for Ixodes scapularis tick bite exposure in humans

Genome sequences of four Ixodes species expands understanding of tick evolution

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

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

Conformational transition of the Ixodes ricinus salivary serpin Iripin-4

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

Serpins in Tick Physiology and Tick-Host Interaction

rDromaserpin: A Novel Anti-Hemostatic Serpin, from the Salivary Glands of the Hard Tick Hyalomma dromedarii

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

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