IL-23-mediated Th-17 cell activation and stimulation of IL-17-driven pro-inflammatory axis has been associated with autoimmunity disorders such as Inflammatory Bowel Disease (IBD) or Crohn’s Disease (CD). Recently we developed a unique class of IL-23-specific protein blockers, called ILP binding proteins that inhibit binding of IL-23 to its cognate cell-surface receptor (IL-23R) and exhibit immunosuppressive effect on human primary blood leukocytes ex vivo. In this study, we aimed to generate a recombinant Lactococcus lactis strain which could serve as in vivo producer/secretor of IL-23 protein blockers into the gut. To achieve this goal, we introduced ILP030, ILP317 and ILP323 cDNA sequences into expression plasmid vector containing USP45 secretion signal, FLAG sequence consensus and LysM-containing cA surface anchor (AcmA) ensuring cell-surface peptidoglycan anchoring. We demonstrate that all ILP variants are expressed in L. lactis cells, efficiently transported and secreted from the cell and displayed on the bacterial surface. The binding function of AcmA-immobilized ILP proteins is documented by interaction with a recombinant p19 protein, alpha subunit of human IL-23, which was assembled in the form of a fusion with Thioredoxin A. ILP317 variant exhibits the best binding to the human IL-23 cytokine, as demonstrated for particular L.lactis-ILP recombinant variants by Enzyme-Linked ImmunoSorbent Assay (ELISA). We conclude that novel recombinant ILP-secreting L. lactis strains were developed that might be useful for further in vivo studies of IL-23-mediated inflammation on animal model of experimentally-induced colitis.

Department of Biotechnology Jožef Stefan Institute Jamova 39 SI 1000 Ljubljana Slovenia

Faculty of Pharmacy University of Ljubljana Aškerčeva 7 SI 1000 Ljubljana Slovenia

Graduate School of Biomedicine Faculty of Medicine University of Ljubljana SI 1000 Ljubljana Slovenia

Laboratory of Ligand Engineering Institute of Biotechnology of the Czech Academy of Sciences v v i BIOCEV Research Center Průmyslová 595 252 50 Vestec Czech Republic

Laboratory of Structural Bioinformatics of Proteins Institute of Biotechnology of the Czech Academy of Sciences v v i BIOCEV Research Center Průmyslová 595 252 50 Vestec Czech Republic

Zobrazit více v PubMed

Girolomoni G., Strohal R., Puig L., Bachelez H., Barker J., Boehncke W.H., Prinz J.C. The role of IL-23 and the IL-23/TH 17 immune axis in the pathogenesis and treatment of psoriasis. J. Eur. Acad Dermatol. Venereol. 2017;31:1616–1626. doi: 10.1111/jdv.14433. PubMed DOI PMC

Razawy W., van Driel M., Lubberts E. The role of IL-23 receptor signaling in inflammation-mediated erosive autoimmune arthritis and bone remodeling. Eur. J. Immunol. 2018;48:220–229. doi: 10.1002/eji.201646787. PubMed DOI PMC

Luo C., Zhang H. The Role of Proinflammatory Pathways in the Pathogenesis of Colitis-Associated Colorectal Cancer. Mediat. Inflamm. 2017;2017:5126048. doi: 10.1155/2017/5126048. PubMed DOI PMC

Sarra M., Pallone F., Macdonald T.T., Monteleone G. IL-23/IL-17 axis in IBD. Inflamm. Bowel. Dis. 2010;16:1808–1813. doi: 10.1002/ibd.21248. PubMed DOI

Siakavellas S.I., Bamias G. Role of the IL-23/IL-17 axis in Crohn’s disease. Discov. Med. 2012;14:253–262. PubMed

Floss D.M., Schroder J., Franke M., Scheller J. Insights into IL-23 biology: From structure to function. Cytokine Growth Factor Rev. 2015;26:569–578. doi: 10.1016/j.cytogfr.2015.07.005. PubMed DOI

Gaffen S.L., Jain R., Garg A.V., Cua D.J. The IL-23-IL-17 immune axis: From mechanisms to therapeutic testing. Nat. Rev. Immunol. 2014;14:585–600. doi: 10.1038/nri3707. PubMed DOI PMC

Bilal J., Berlinberg A., Bhattacharjee S., Trost J., Riaz I.B., Kurtzman D.J.B. A Systematic Review and Meta-Analysis of the Efficacy and Safety of the Interleukin (IL)-12/23 and IL-17 Inhibitors Ustekinumab, Secukinumab, Ixekizumab, Brodalumab, Guselkumab, and Tildrakizumab for the Treatment of Moderate to Severe Plaque Psoriasis. J. Dermatol. Treat. 2018:1–37. doi: 10.1080/09546634.2017.1422591. PubMed DOI

Fotiadou C., Lazaridou E., Sotiriou E., Ioannides D. Targeting IL-23 in psoriasis: Current perspectives. Psoriasis (Auckl) 2018;8:1–5. doi: 10.2147/PTT.S98893. PubMed DOI PMC

Desmyter A., Spinelli S., Boutton C., Saunders M., Blachetot C., de Haard H., Denecker G., Van Roy M., Cambillau C., Rommelaere H. Neutralization of Human Interleukin 23 by Multivalent Nanobodies Explained by the Structure of Cytokine-Nanobody Complex. Front. Immunol. 2017;8:884. doi: 10.3389/fimmu.2017.00884. PubMed DOI PMC

Ramamurthy V., Krystek S.R., Jr., Bush A., Wei A., Emanuel S.L., Das Gupta R., Janjua A., Cheng L., Murdock M., Abramczyk B., et al. Structures of adnectin/protein complexes reveal an expanded binding footprint. Structure. 2012;20:259–269. doi: 10.1016/j.str.2011.11.016. PubMed DOI

Desmet J., Verstraete K., Bloch Y., Lorent E., Wen Y., Devreese B., Vandenbroucke K., Loverix S., Hettmann T., Deroo S., et al. Structural basis of IL-23 antagonism by an Alphabody protein scaffold. Nat. Commun. 2014;5:5237. doi: 10.1038/ncomms6237. PubMed DOI PMC

Kuchar M., Vankova L., Petrokova H., Cerny J., Osicka R., Pelak O., Sipova H., Schneider B., Homola J., Sebo P., et al. Human interleukin-23 receptor antagonists derived from an albumin-binding domain scaffold inhibit IL-23-dependent ex vivo expansion of IL-17-producing T-cells. Proteins. 2014;82:975–989. doi: 10.1002/prot.24472. PubMed DOI PMC

Krizova L., Kuchar M., Petrokova H., Osicka R., Hlavnickova M., Pelak O., Cerny J., Kalina T., Maly P. p19-targeted ABD-derived protein variants inhibit IL-23 binding and exert suppressive control over IL-23-stimulated expansion of primary human IL-17+ T-cells. Autoimmunity. 2017;50:102–113. doi: 10.1080/08916934.2016.1272598. PubMed DOI

Worledge K.L., Godiska R., Barrett T.A., Kink J.A. Oral administration of avian tumor necrosis factor antibodies effectively treats experimental colitis in rats. Dig. Dis. Sci. 2000;45:2298–2305. doi: 10.1023/A:1005554900286. PubMed DOI

Pawar V.K., Meher J.G., Singh Y., Chaurasia M., Surendar Reddy B., Chourasia M.K. Targeting of gastrointestinal tract for amended delivery of protein/peptide therapeutics: Strategies and industrial perspectives. J. Control. Release. 2014;196:168–183. doi: 10.1016/j.jconrel.2014.09.031. PubMed DOI

Braat H., Rottiers P., Hommes D.W., Huyghebaert N., Remaut E., Remon J.P., van Deventer S.J., Neirynck S., Peppelenbosch M.P., Steidler L. A phase I trial with transgenic bacteria expressing interleukin-10 in Crohn’s disease. Clin. Gastroenterol. Hepatol. 2006;4:754–759. doi: 10.1016/j.cgh.2006.03.028. PubMed DOI

Douillard F.P., de Vos W.M. Functional genomics of lactic acid bacteria: From food to health. Microb. Cell Factor. 2014:13. doi: 10.1186/1475-2859-13-S1-S8. PubMed DOI PMC

Sanders M.E. Probiotics: Definition, sources, selection, and uses. Clin. Infect. Dis. 2008;46(Suppl. 2):S58–S61. doi: 10.1086/523341. discussion S144–S51. PubMed DOI

Siezen R.J., Bayjanov J.R., Felis G.E., van der Sijde M.R., Starrenburg M., Molenaar D., Wels M., van Hijum S.A., van Hylckama Vlieg J.E. Genome-scale diversity and niche adaptation analysis of Lactococcus lactis by comparative genome hybridization using multi-strain arrays. Microb. Biotechnol. 2011;4:383–402. doi: 10.1111/j.1751-7915.2011.00247.x. PubMed DOI PMC

Ballal S.A., Veiga P., Fenn K., Michaud M., Kim J.H., Gallini C.A., Glickman J.N., Quere G., Garault P., Beal C., et al. Host lysozyme-mediated lysis of Lactococcus lactis facilitates delivery of colitis-attenuating superoxide dismutase to inflamed colons. Proc. Natl. Acad. Sci. USA. 2015;112:7803–7808. doi: 10.1073/pnas.1501897112. PubMed DOI PMC

Kawahara M., Nemoto M., Nakata T., Kondo S., Takahashi H., Kimura B., Kuda T. Anti-inflammatory properties of fermented soy milk with Lactococcus lactis subsp. lactis S-SU2 in murine macrophage RAW264.7 cells and DSS-induced IBD model mice. Int. Immunopharmacol. 2015;26:295–303. doi: 10.1016/j.intimp.2015.04.004. PubMed DOI

Drouault S., Corthier G., Ehrlich S.D., Renault P. Survival, physiology, and lysis of Lactococcus lactis in the digestive tract. Appl. Environ. Microbiol. 1999;65:4881–4886. PubMed PMC

Berlec A., Ravnikar M., Strukelj B. Lactic acid bacteria as oral delivery systems for biomolecules. Pharmazie. 2012;67:891–898. PubMed

Bermudez-Humaran L.G., Aubry C., Motta J.P., Deraison C., Steidler L., Vergnolle N., Chatel J.M., Langella P. Engineering lactococci and lactobacilli for human health. Curr. Opin. Microbiol. 2013;16:278–283. doi: 10.1016/j.mib.2013.06.002. PubMed DOI

Song A.A., In L.L.A., Lim S.H.E., Rahim R.A. A review on Lactococcus lactis: From food to factory. Microb. Cell Fact. 2017;16:55. doi: 10.1186/s12934-017-0669-x. PubMed DOI PMC

Vandenbroucke K., de Haard H., Beirnaert E., Dreier T., Lauwereys M., Huyck L., Van Huysse J., Demetter P., Steidler L., Remaut E., et al. Orally administered L. lactis secreting an anti-TNF Nanobody demonstrate efficacy in chronic colitis. Mucosal. Immunol. 2010;3:49–56. doi: 10.1038/mi.2009.116. PubMed DOI

Berlec A., Perse M., Ravnikar M., Lunder M., Erman A., Cerar A., Strukelj B. Dextran sulphate sodium colitis in C57BL/6J mice is alleviated by Lactococcus lactis and worsened by the neutralization of Tumor necrosis Factor alpha. Int. Immunopharmacol. 2017;43:219–226. doi: 10.1016/j.intimp.2016.12.027. PubMed DOI

Vandenbroucke K., Hans W., Van Huysse J., Neirynck S., Demetter P., Remaut E., Rottiers P., Steidler L. Active delivery of trefoil factors by genetically modified Lactococcus lactis prevents and heals acute colitis in mice. Gastroenterology. 2004;127:502–513. doi: 10.1053/j.gastro.2004.05.020. PubMed DOI

Galipeau H.J., Wiepjes M., Motta J.P., Schulz J.D., Jury J., Natividad J.M., Pinto-Sanchez I., Sinclair D., Rousset P., Martin-Rosique R., et al. Novel role of the serine protease inhibitor elafin in gluten-related disorders. Am. J. Gastroenterol. 2014;109:748–756. doi: 10.1038/ajg.2014.48. PubMed DOI PMC

Steidler L., Hans W., Schotte L., Neirynck S., Obermeier F., Falk W., Fiers W., Remaut E. Treatment of murine colitis by Lactococcus lactis secreting interleukin-10. Science. 2000;289:1352–1355. doi: 10.1126/science.289.5483.1352. PubMed DOI

Ravnikar M., Strukelj B., Obermajer N., Lunder M., Berlec A. Engineered lactic acid bacterium Lactococcus lactis capable of binding antibodies and tumor necrosis factor alpha. Appl. Environ. Microbiol. 2010;76:6928–6932. doi: 10.1128/AEM.00190-10. PubMed DOI PMC

Skrlec K., Pucer Janez A., Rogelj B., Strukelj B., Berlec A. Evasin-displaying lactic acid bacteria bind different chemokines and neutralize CXCL8 production in Caco-2 cells. Microb. Biotechnol. 2017;10:1732–1743. doi: 10.1111/1751-7915.12781. PubMed DOI PMC

Michon C., Langella P., Eijsink V.G., Mathiesen G., Chatel J.M. Display of recombinant proteins at the surface of lactic acid bacteria: Strategies and applications. Microb. Cell Factor. 2016;15:70. doi: 10.1186/s12934-016-0468-9. PubMed DOI PMC

Dieye Y., Usai S., Clier F., Gruss A., Piard J.C. Design of a protein-targeting system for lactic acid bacteria. J. Bacteriol. 2001;183:4157–4166. doi: 10.1128/JB.183.14.4157-4166.2001. PubMed DOI PMC

Buist G., Kok J., Leenhouts K.J., Dabrowska M., Venema G., Haandrikman A.J. Molecular cloning and nucleotide sequence of the gene encoding the major peptidoglycan hydrolase of Lactococcus lactis, a muramidase needed for cell separation. J. Bacteriol. 1995;177:1554–1563. doi: 10.1128/jb.177.6.1554-1563.1995. PubMed DOI PMC

Steen A., Buist G., Horsburgh G.J., Venema G., Kuipers O.P., Foster S.J., Kok J. AcmA of Lactococcus lactis is an N-acetylglucosaminidase with an optimal number of LysM domains for proper functioning. FEBS J. 2005;272:2854–2868. doi: 10.1111/j.1742-4658.2005.04706.x. PubMed DOI

Kosler S., Strukelj B., Berlec A. Lactic Acid Bacteria with Concomitant IL-17, IL-23 and TNFalpha-Binding Ability for the Treatment of Inflammatory Bowel Disease. Curr. Pharm. Biotechnol. 2017;18:318–326. doi: 10.2174/1389201018666170210152218. PubMed DOI

Bloch Y., Bouchareychas L., Merceron R., Skladanowska K., Van den Bossche L., Detry S., Govindarajan S., Elewaut D., Haerynck F., Dullaers M., et al. Structural Activation of Pro-inflammatory Human Cytokine IL-23 by Cognate IL-23 Receptor Enables Recruitment of the Shared Receptor IL-12Rbeta1. Immunity. 2018;48:45–58.e6. doi: 10.1016/j.immuni.2017.12.008. PubMed DOI PMC

Holo H., Nes I.F. Transformation of Lactococcus by electroporation. Methods Mol. Biol. 1995;47:195–199. PubMed

Ahmad J.N., Li J., Biedermannova L., Kuchar M., Sipova H., Semeradtova A., Cerny J., Petrokova H., Mikulecky P., Polinek J., et al. Novel high-affinity binders of human interferon gamma derived from albumin-binding domain of protein G. Proteins. 2012;80:774–789. doi: 10.1002/prot.23234. PubMed DOI

Mierau I., Kleerebezem M. 10 years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis. Appl. Microbiol. Biotechnol. 2005;68:705–717. doi: 10.1007/s00253-005-0107-6. PubMed DOI

Webb B., Sali A. Comparative Protein Structure Modeling Using MODELLER. Curr. Protoc. Bioinform. 2014;47:561-32. doi: 10.1002/0471250953.bi0506s47. PubMed DOI

Lupardus P.J., Garcia K.C. The structure of interleukin-23 reveals the molecular basis of p40 subunit sharing with interleukin-12. J. Mol. Biol. 2008;382:931–941. doi: 10.1016/j.jmb.2008.07.051. PubMed DOI PMC

Eastman P., Pande V.S. OpenMM: A Hardware Independent Framework for Molecular Simulations. Comput. Sci. Eng. 2015;12:34–39. doi: 10.1109/MCSE.2010.27. PubMed DOI PMC

Van Der Spoel D., Lindahl E., Hess B., Groenhof G., Mark A.E., Berendsen H.J. GROMACS: Fast, flexible, and free. J. Comput. Chem. 2005;26:1701–1718. doi: 10.1002/jcc.20291. PubMed DOI

Johansson M.U., Frick I.M., Nilsson H., Kraulis P.J., Hober S., Jonasson P., Linhult M., Nygren P.A., Uhlen M., Bjorck L., et al. Structure, specificity, and mode of interaction for bacterial albumin-binding modules. J. Biol. Chem. 2002;277:8114–8120. doi: 10.1074/jbc.M109943200. PubMed DOI

Edgar R.C. MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004;32:1792–1797. doi: 10.1093/nar/gkh340. PubMed DOI PMC

Kozakov D., Beglov D., Bohnuud T., Mottarella S.E., Xia B., Hall D.R., Vajda S. How good is automated protein docking? Proteins. 2013;81:2159–2166. doi: 10.1002/prot.24403. PubMed DOI PMC

Kozakov D., Brenke R., Comeau S.R., Vajda S. PIPER: An FFT-based protein docking program with pairwise potentials. Proteins. 2006;65:392–406. doi: 10.1002/prot.21117. PubMed DOI

Engineered Lactococcus lactis Secreting IL-23 Receptor-Targeted REX Protein Blockers for Modulation of IL-23/Th17-Mediated Inflammation

ABD-Derived Protein Blockers of Human IL-17 Receptor A as Non-IgG Alternatives for Modulation of IL-17-Dependent Pro-Inflammatory Axis