Synthetic hookworm-derived peptides are potent modulators of primary human immune cell function that protect against experimental colitis in vivo

The prevalence of autoimmune diseases is on the rise globally. Currently, autoimmunity presents in over 100 different forms and affects around 9% world’s population. Current treatments available for are inadequate, expensive, tend to focus symptom management rather than cure. Clinical trials have shown that live helminthic therapy can decrease chronic inflammation associated with inflammatory bowel disease other gastrointestinal conditions. As an alternative better controlled approach infection, we identified characterized two peptides, Acan1 Nak1, from excretory/secretory component parasitic hookworms their therapeutic activity experimental colitis. We synthesized Nak1 peptides Ancylostoma caninum Necator americanus assessed structures protective properties human cell–based assays a mouse model acute displayed anticolitic via significantly reducing weight loss colon atrophy, edema, ulceration, necrosis 2,4,6-trinitrobenzene sulfonic acid–exposed mice. These hookworm prevented mucosal goblet cells preserved intestinal architecture. upregulated genes responsible repair restitution ulcerated epithelium, whereas downregulated epithelial cell migration apoptotic signaling within colon. were nontoxic key immunomodulatory functions peripheral blood mononuclear by suppressing CD4+ T proliferation inhibiting IL-2 TNF production. conclude warrant further development as therapeutics treatment autoimmunity, particularly There more conditions described additional 40 suspected (1Luo X. Miller S.D. Shea L.D. Immune tolerance transplantation.Annu. Rev. Biomed. Eng. 2016; 18: 181-205Crossref PubMed Scopus (46) Google Scholar). For unknown reasons, incidence rising annually both developing industrialized countries, currently affecting 3% 12% individuals, depending country (2Bach J.-F. hygiene hypothesis autoimmunity: role pathogens commensals.Nat. Immunol. 2018; 105-120Crossref (179) Treatment costing $100 billion dollars just United States (NIAID, 2020). vast majority incurable, new urgently needed. Helminth has been be promising option many like (IBD). such ulcerative colitis (UC) Crohn’s found low-dose inoculation helminths overall successfully improved patient Disease Activity Index (3Summers R.W. Elliott D.E. Urban J.F. Thompson R.A. Weinstock J.V. Trichuris suis active colitis: A randomized trial.Gastroenterology. 2005; 128: 825-832Abstract Full Text PDF (581) Scholar, 4Croese J. O'Neil Masson Cooke S. Melrose W. Pritchard D. Speare R. proof concept study establishing Crohn's patients reservoir donors.Gut. 2006; 55: 136-137Crossref (183) However, controversial idea infection will difficult standardize accept significant subset patients. Therefore, utilization proteins secreted become attractive target drug (5Navarro Pickering D.A. Ferreira I.B. Jones L. Ryan Troy Leech A. Hotez P.J. Zhan B. Laha T. Prentice Sparwasser Croese Engwerda C.R. Upham J.W. et al.Hookworm recombinant protein promotes regulatory responses suppress asthma.Sci. Transl. Med. 8362ra143Crossref (90) 6Ferreira Loukas Navarro Suppression tissue damage colitis.Clin. 2017; 6e157Crossref (20) 7Ferreira I. Smyth Gaze Aziz Giacomin P. Ruyssers N. Artis McSorley H.J. Hookworm products induce interleukin-4 (IL-4)+ IL-10+ pathology colitis.Infect. Immun. 2013; 81: 2104-2111Crossref (72) Helminths produce secrete proteins, glycoproteins, small-molecular-weight (MW) compounds oral openings or outer surfaces avoid detection expulsion host (8Mulvenna Hamilton Nagaraj S.H. Gorman J.J. Proteomics analysis blood-feeding stage hookworm, ancylostoma caninum.Mol. Cell. Proteomics. 2009; 8: 109Abstract (146) small number these molecules reported benefits treating allergic 9Smallwood T.B. P.R. Mulvenna J.P. Clark R.J. Miles immunomodulation disease.Front. 453Crossref (106) Although studies focused higher-MW (>5 kDa), there limited examples literature about lower-MW (1–5 kDa). Anti-inflammatory protein-2 ∼19-kDa generate proregulatory imprint mesenteric lymphoid tissues results long-term protection against asthma translational anti-inflammatory modulate costimulatory dendritic (DCs) obtained (PBMCs) vitro allergen-specific proliferation. AcK1 BmK1 low-MW (∼4 kDa each) filarial nematode Brugia malayi, respectively (10Chhabra Chang S.C. Nguyen H.M. Huq Tanner M.R. Londono L.M. Estrada Dhawan V. Chauhan Upadhyay S.K. Gindin M. Valenzuela J.G. Mohanty Swarbrick J.D. al.Kv1.3 channel-blocking worms: Implications diseases.FASEB 2014; 28: 3952-3964Crossref (51) Both mitogen stimulation Ack1 contain ShKT domain, which characteristically adopts fold comprising almost perpendicular stretches helices interconnected three disulfide bonds, stabilizing structure (11Dauplais Lecoq Song Cotton Jamin Gilquin Roumestand C. Vita de Medeiros C.L. Rowan E.G. Harvey A.L. Menez On convergent evolution animal toxins. Conservation diad functional residues potassium toxins unrelated structures.J. Biol. Chem. 1997; 272: 4302-4309Abstract (299) domain was originally toxin isolated Stichodactyla helianthus sea anemone (12Castañeda O. Sotolongo Amor A.M. Stöcklin Anderson A.J. Engström Å. Wernstedt Karlsson E. Characterization channel Caribbean helianthus.Toxicon. 1995; 33: 603-613Crossref (210) ShK blocks voltage-gated channels crucial activation terminally differentiated effector memory (13Norton R.S. Pennington M.W. Wulff H. Potassium blockade multiple sclerosis diseases.Curr. 2004; 11: 3041-3052Crossref (74) Many channel–blocking critical conserved “functional dyad” consisting lysine neighboring aromatic aliphatic residue (14Mouhat De Waard Sabatier J.M. Contribution dyad acting Kv1-type channels.J. Pept. Sci. 65-68Crossref possesses dyad, made up tyrosine residue, proposed contribute (15Pennington Mahnir V.M. Krafte D.S. Zaydenberg Byrnes M.E. Khaytin Crowley K. Kem W.R. Identification separate binding sites SHK toxin, potent inhibitor voltage-dependent T-lymphocytes rat brain.Biochem. Biophys. Res. Commun. 1996; 219: 696-701Crossref (66) derivative dalazatide, recently undergone phase 1b trial plaque psoriasis (16Tarcha E.J. Olsen C.M. Probst Peckham Muñoz-Elías Kruger Iadonato S.P. Safety pharmacodynamics Kv1.3 inhibitor, psoriasis: trial.PLoS One. 12e0180762Crossref (69) dalazatide well tolerated skin lesions modulating surface marker expression, mediators (NCT02435342). Transcriptomic characterization americanus, family one most abundantly expressed families adult (17Cantacessi Mitreva Jex A.R. Young N.D. Campbell B.E. Hall Doyle M.A. Ralph S.A. Rabelo E.M. Ranganathan Sternberg P.W. Gasser R.B. Massively parallel sequencing transcriptome.PLoS Negl. Trop. Dis. 2010; 4e684Crossref (65) hookworm-secreted may similar known family. Here describe possess domains. EST databases through custom blast algorithms using specific cysteine configurations. Phylogenetic used map previously another form characteristic intramolecular bonds helical segments three-dimensional those In this study, efficacy determined vivo models primary immune cells. Peptide stability additionally serum simulated fluid. Collectively, data show promise immune-modulating proteins. Using algorithm, seven sequences plurality suitably spaced transcriptomes americanus. configuration (Fig. 1). To determine sequence identity, optimal alignment amino acid and/or nucleotide conducted computerized implementations algorithms, i.e., BLAST. Nak2 Chhabra, al. Each peptide cysteines (highlighted red), putative bond connections black lines. Only green purple) activity. linear rink amide resin solid synthesis (SPPS). Name1 Name2 could not full SPPS, therefore, native chemical ligation peptide. This achieved ligating N- C-terminal together at third sequence. unable synthesized. each formed random oxidative folding aqueous buffer, optimized buffer All amenable 0.1 M ammonium bicarbonate room temperature aid reduced (2 mM) oxidized (0.5 glutathione (pH 8.2). During oxidation Name3 Name4, N-terminal glutamine cyclize pyroglutamic acid. without acid, experimentation, they separated reversed (RP)-HPLC. fractions >95% purity experimentation S1). chosen homologs suppressive initial cytometric bead array (CBA) screen proinflammatory cytokines S2). Folding synthetic initially examined one-dimensional 1H NMR spectroscopy. spectra confirmed well-defined based quality terms signal dispersion peak linewidth. Further analyses performed (3D) order compare them (18Tudor J.E. Pallaghy P.K. Norton Solution novel anemone.Nat. Struct. 3: 317-320Crossref (157) Scholar) 2). Two-dimensional TOCSY NOESY allowed sequential manual assignment S3). 3D calculated annealing structural restraints (Table S1) derived data, resemble 2A). calculations no performed, revealing ensembles S4). Structures then all 15 possible isomers connectivity generating lowest function average helminth adopt predominantly short 310 helix separating α-helices, locations lengths differ slightly between 2B). ShK, α-helices encompass 14 19 21 25 located 9 11. 11 17, 23 32 21, 7 15, 31 18 20. Despite similarities structures, only 2C). side chains Lys22 (blue) Tyr23 (cyan) region orientating outward. Compared orientation, appearing angle away other. whether able inhibit channel, two-electrode voltage clamp electrophysiology examine effects Xenopus laevis oocytes 3). control IC50 57 pM (data shown), replicating low picomolar values previous (19Pennington Tajhya Chhabra Beeton Development highly selective Kv1.3-blocking ShK.Mar. Drugs. 2015; 13: 529-542Crossref (37) inhibited value 829 nM, concentration 15,000 times higher positive control, ShK. little blocking likely due lack LC/MS quantification during 24-h incubations 100% serum, porcine fluid 4). (purple) (green) stable time course, (red) began degrade after 4 h point 4A). fluid, 4B). completely degraded 6 half-life 0.8 h. By contrast, did fully until 24 had 2.4 4.4 h, 4C). R3 B1-22R I15A (20Haugaard-Kedstrom Lee H.S. M.V. Rathod Hossain Bathgate R.A.D. Rosengren K.J. Binding conformation determinants single-chain antagonist relaxin-3 receptor RXFP3.J. 293: 15765-15776Crossref (7) first hour experiment. lower demonstrates alone sufficient confer high stability. It hostile protease-rich environments resulted evolutionary adaptations intestine decades. Owing origin scaffold it hypothesized would modulatory investigate hypothesis, potential toxicity PBMC quantifying phosphatidylserine following treatment. do this, populations flow cytometry Annexin V-FITC propidium iodide staining S5). Cells treated either (10 μg/ml) retained percentage (unstained) untreated heat-treated (positive control) showed increase earlyFITC+ lateFITC+Propidium+ 5A). Consequently, cytokine suppression assay. preliminary intracellular PBMC, revealed LPS-induced productions IL-6, IL-8, MIP-1β classical (CD14+) intermediate (CD14+/CD16+) monocytes S6). Additional alter viability/proliferation metabolically mixed lymphocyte reaction days LPS S7). principally inhibits ion modulation calcium-dependent (21Fung-Leung W.-P. Edwards Liu Y. Ngo Angsana Castro G. Wu Swanson R.V. Wickenden A.D. strength-dependent blockers.PLoS 12e0170102Crossref Accordingly, PBMCs activated phorbol 12-myristate 12-acetate ionomycin (PI) Ca2+ levels Activated measured CBA 5B). suppressed PI-induced release dose-dependent manner, decreased secretion. Next, given observed Acan1, antiproliferative capacity MACS-purified CD3+ CellTrace Violet (CTV)-stained TCR anti-CD3/CD28 beads 10 μg/ml ciclosporin control), unfolded (negative days. Following incubation, division evaluated CTV fluorescence S8). Folded but donors, albeit lesser extent calcineurin 5C). Of interest, effect CD8+ division. suggest helper mechanism requires investigation. inflammation, mice once prophylactically varying doses (20, 10, 5, 1, μg) (40, 20, 6A). Over course vehicle (2,4,6-trinitrobenzene [TNBS]) group experienced 15% 6B). Mice 1 μg (0.05 mg/kg−1) intraperitoneal (i.p.) route day 3 (p < 0.05), compared 6B), improvement Nak1-treated group. Colon 6C) consistent enhanced TNBS-induced Acan1-treated visibly longer normal-looking colons when = 0.0079), manifested shortened, thickened, erythematous tissue. also 0.0238) 6D). scored macroscopically parameters: adhesion, wall thickening, ulceration 6E). reduction 0.008) macroscopic score. Macroscopic histological observations 6F) naive normal included anatomy lamina propria, gastric mucosa, cells, colonic musculature. Colons vehicle-treated exhibited extensive severe lesions, including thickening propria wall, evidence edema erosion destruction healthy colons, possessing architecture comparable (healthy group, crypts good integrity, ulceration. Together, hookworm-derived vivo. next transcriptomics cohort gene NanoString panel targeted regulation, gut barrier homeostasis, destruction. multivariate principal identify largest sources variation dataset 7). No differences contributions groups along PC1 (x-axis) 7A) 7B). difference contribution alongside PC2 0.00182), 0.04369), (linear) 0.0363), 0.0382). Together components captured 42% datasets 41% Nak1. arrows. Tnfrsf14, Foxo1, Ctla4, Tgfb1, Mki67 Tnfaip3, Nfkb2, Nfkb1, Mmp9 visualized differential expression (DE) volcano plots. pairing log2 change (FC) relative p comparison. shown, comparing DE 16 identified, largely FC 7C). Unlike negative 7D). Our controls, 7E) 7F) versus insignificant very cluster 7E). validation folded version required well-characterized widely shares clinical, histological, biochemical features UC (22Shepherd Wangchuk medicinal plant compound, capnoidine, prevents onset colitis.J. Ethnopharmacol. 211: 17-28Crossref (23) An candidate dosage S9). pilot dose wer