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

Abstract

The prevalence of autoimmune diseases is on the rise globally. Currently, autoimmunity presents in over 100 different forms and affects around 9% of the world’s population. Current treatments available for autoimmune diseases are inadequate, expensive, and tend to focus on symptom management rather than cure. Clinical trials have shown that live helminthic therapy can decrease chronic inflammation associated with inflammatory bowel disease and other gastrointestinal autoimmune inflammatory conditions. As an alternative and better controlled approach to live infection, we have identified and characterized two peptides, Acan1 and Nak1, from the excretory/secretory component of parasitic hookworms for their therapeutic activity on experimental colitis. We synthesized Acan1 and Nak1 peptides from the Ancylostoma caninum and Necator americanus hookworms and assessed their structures and protective properties in human cell–based assays and in a mouse model of acute colitis. Acan1 and Nak1 displayed anticolitic properties via significantly reducing weight loss and colon atrophy, edema, ulceration, and necrosis in 2,4,6-trinitrobenzene sulfonic acid–exposed mice. These hookworm peptides prevented mucosal loss of goblet cells and preserved intestinal architecture. Acan1 upregulated genes responsible for the repair and restitution of ulcerated epithelium, whereas Nak1 downregulated genes responsible for epithelial cell migration and apoptotic cell signaling within the colon. These peptides were nontoxic and displayed key immunomodulatory functions in human peripheral blood mononuclear cells by suppressing CD4+ T cell proliferation and inhibiting IL-2 and TNF production. We conclude that Acan1 and Nak1 warrant further development as therapeutics for the treatment of autoimmunity, particularly gastrointestinal inflammatory conditions. The prevalence of autoimmune diseases is on the rise globally. Currently, autoimmunity presents in over 100 different forms and affects around 9% of the world’s population. Current treatments available for autoimmune diseases are inadequate, expensive, and tend to focus on symptom management rather than cure. Clinical trials have shown that live helminthic therapy can decrease chronic inflammation associated with inflammatory bowel disease and other gastrointestinal autoimmune inflammatory conditions. As an alternative and better controlled approach to live infection, we have identified and characterized two peptides, Acan1 and Nak1, from the excretory/secretory component of parasitic hookworms for their therapeutic activity on experimental colitis. We synthesized Acan1 and Nak1 peptides from the Ancylostoma caninum and Necator americanus hookworms and assessed their structures and protective properties in human cell–based assays and in a mouse model of acute colitis. Acan1 and Nak1 displayed anticolitic properties via significantly reducing weight loss and colon atrophy, edema, ulceration, and necrosis in 2,4,6-trinitrobenzene sulfonic acid–exposed mice. These hookworm peptides prevented mucosal loss of goblet cells and preserved intestinal architecture. Acan1 upregulated genes responsible for the repair and restitution of ulcerated epithelium, whereas Nak1 downregulated genes responsible for epithelial cell migration and apoptotic cell signaling within the colon. These peptides were nontoxic and displayed key immunomodulatory functions in human peripheral blood mononuclear cells by suppressing CD4+ T cell proliferation and inhibiting IL-2 and TNF production. We conclude that Acan1 and Nak1 warrant further development as therapeutics for the treatment of autoimmunity, particularly gastrointestinal inflammatory conditions. There are more than 100 autoimmune conditions described with an additional 40 diseases suspected (1Luo X. Miller S.D. Shea L.D. Immune tolerance for autoimmune disease and cell transplantation.Annu. Rev. Biomed. Eng. 2016; 18: 181-205Crossref PubMed Scopus (46) Google Scholar). For unknown reasons, autoimmune incidence is rising annually in both developing and industrialized countries, currently affecting 3% to 12% of individuals, depending on the country (2Bach J.-F. The hygiene hypothesis in autoimmunity: The role of pathogens and commensals.Nat. Rev. Immunol. 2018; 18: 105-120Crossref PubMed Scopus (179) Google Scholar). Treatment is costing more than $100 billion dollars annually just in the United States (NIAID, 2020). The vast majority of autoimmune conditions are incurable, and new treatments are urgently needed. Helminth therapy has been shown to be a promising treatment option for many autoimmune diseases like inflammatory bowel disease (IBD). Clinical trials for diseases such as ulcerative colitis (UC) and Crohn’s disease have found that treatment with a low-dose inoculation of helminths has overall successfully improved patient Disease Activity Index (3Summers R.W. Elliott D.E. Urban J.F. Thompson R.A. Weinstock J.V. Trichuris suis therapy for active ulcerative colitis: A randomized controlled trial.Gastroenterology. 2005; 128: 825-832Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar, 4Croese J. O'Neil J. Masson J. Cooke S. Melrose W. Pritchard D. Speare R. A proof of concept study establishing Necator americanus in Crohn's patients and reservoir donors.Gut. 2006; 55: 136-137Crossref PubMed Scopus (183) Google Scholar). However, helminthic therapy is controversial and the idea of live infection will be difficult to standardize and accept for a significant subset of patients. Therefore, the utilization of the immunomodulatory proteins and peptides secreted by helminths have become a more attractive target for drug development (5Navarro S. Pickering D.A. Ferreira I.B. Jones L. Ryan S. Troy S. Leech A. Hotez P.J. Zhan B. Laha T. Prentice R. Sparwasser T. Croese J. Engwerda C.R. Upham J.W. et al.Hookworm recombinant protein promotes regulatory T cell responses that suppress experimental asthma.Sci. Transl. Med. 2016; 8362ra143Crossref PubMed Scopus (90) Google Scholar, 6Ferreira I.B. Pickering D.A. Troy S. Croese J. Loukas A. Navarro S. Suppression of inflammation and tissue damage by a hookworm recombinant protein in experimental colitis.Clin. Transl. Immunol. 2017; 6e157Crossref PubMed Scopus (20) Google Scholar, 7Ferreira I. Smyth D. Gaze S. Aziz A. Giacomin P. Ruyssers N. Artis D. Laha T. Navarro S. Loukas A. McSorley H.J. Hookworm excretory/secretory products induce interleukin-4 (IL-4)+ IL-10+ CD4+ T cell responses and suppress pathology in a mouse model of colitis.Infect. Immun. 2013; 81: 2104-2111Crossref PubMed Scopus (72) Google Scholar). Helminths produce and secrete immunomodulatory proteins, glycoproteins, and small-molecular-weight (MW) compounds from their oral openings or outer surfaces to avoid detection and expulsion from the host (8Mulvenna J. Hamilton B. Nagaraj S.H. Smyth D. Loukas A. Gorman J.J. Proteomics analysis of the excretory/secretory component of the blood-feeding stage of the hookworm, ancylostoma caninum.Mol. Cell. Proteomics. 2009; 8: 109Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). A small number of these molecules have been characterized and reported to have benefits in treating allergic or autoimmune diseases (5Navarro S. Pickering D.A. Ferreira I.B. Jones L. Ryan S. Troy S. Leech A. Hotez P.J. Zhan B. Laha T. Prentice R. Sparwasser T. Croese J. Engwerda C.R. Upham J.W. et al.Hookworm recombinant protein promotes regulatory T cell responses that suppress experimental asthma.Sci. Transl. Med. 2016; 8362ra143Crossref PubMed Scopus (90) Google Scholar, 6Ferreira I.B. Pickering D.A. Troy S. Croese J. Loukas A. Navarro S. Suppression of inflammation and tissue damage by a hookworm recombinant protein in experimental colitis.Clin. Transl. Immunol. 2017; 6e157Crossref PubMed Scopus (20) Google Scholar, 9Smallwood T.B. Giacomin P.R. Loukas A. Mulvenna J.P. Clark R.J. Miles J.J. Helminth immunomodulation in autoimmune disease.Front. Immunol. 2017; 8: 453Crossref PubMed Scopus (106) Google Scholar). Although many studies focused on higher-MW proteins (>5 kDa), there are limited examples in the literature about lower-MW products (1–5 kDa). Anti-inflammatory protein-2 is an ∼19-kDa protein shown to generate a proregulatory imprint on mesenteric lymphoid tissues that results in long-term protection against allergic asthma (5Navarro S. Pickering D.A. Ferreira I.B. Jones L. Ryan S. Troy S. Leech A. Hotez P.J. Zhan B. Laha T. Prentice R. Sparwasser T. Croese J. Engwerda C.R. Upham J.W. et al.Hookworm recombinant protein promotes regulatory T cell responses that suppress experimental asthma.Sci. Transl. Med. 2016; 8362ra143Crossref PubMed Scopus (90) Google Scholar). The protein has promising translational properties as anti-inflammatory protein-2 can modulate costimulatory molecules on human dendritic cells (DCs) obtained from human peripheral blood mononuclear cells (PBMCs) in vitro and suppress allergen-specific T cell proliferation. AcK1 and BmK1 are two low-MW peptides (∼4 kDa each) found in the excretory/secretory component of the hookworm Ancylostoma caninum and the human filarial nematode Brugia malayi, respectively (10Chhabra S. Chang S.C. Nguyen H.M. Huq R. Tanner M.R. Londono L.M. Estrada R. Dhawan V. Chauhan S. Upadhyay S.K. Gindin Hotez P.J. B. et immunomodulatory peptides from parasitic for autoimmune J. PubMed Scopus Google Scholar). peptides were found to suppress mouse T cell proliferation in vitro and suppress in T cells (10Chhabra S. Chang S.C. Nguyen H.M. Huq R. Tanner M.R. Londono L.M. Estrada R. Dhawan V. Chauhan S. Upadhyay S.K. Gindin Hotez P.J. B. et immunomodulatory peptides from parasitic for autoimmune J. PubMed Scopus Google Scholar). and BmK1 a a two of that are by the A. J. J. N. B. A. the of of a of in with Full Text Full Text PDF PubMed Scopus Google Scholar). The identified in a from the V. R. of a from the PubMed Scopus Google Scholar). The that are for the of T cells by the for the treatment of and other autoimmune Med. PubMed Scopus Google Scholar). peptides a of a and a or S. of the of on 2005; PubMed Scopus (46) Google Scholar). the of a and a are to to activity I. I. of on a of in human and PubMed Scopus Google Scholar). A of the has a randomized for the treatment of the autoimmune disease P. D. and of a in the treatment of A randomized 2017; PubMed Scopus Google Scholar). The study reported that and improved by T cell and as as inhibiting of inflammation within the blood of the human hookworm, Necator has shown that the of proteins are of the protein in hookworms S. Loukas A. and analysis of the Necator americanus PubMed Scopus Google Scholar). Therefore, we that or more peptides have immunomodulatory activity to other immunomodulatory proteins of the we two low-MW peptides identified from the N. americanus and A. caninum hookworms that Acan1 identified from analysis to Nak1, identified in study (10Chhabra S. Chang S.C. Nguyen H.M. Huq R. Tanner M.R. Londono L.M. Estrada R. Dhawan V. Chauhan S. Upadhyay S.K. Gindin Hotez P.J. B. et immunomodulatory peptides from parasitic for autoimmune J. PubMed Scopus Google Scholar). peptides the and within their to in the the therapeutic of Acan1 and Nak1 in a colitis model and in in vitro human human and human intestinal and gastrointestinal these for the two new a peptides with a of were identified in the of A. caninum and N. These peptides the of the of by of The Nak1 and peptides were identified by et (10Chhabra S. Chang S.C. Nguyen H.M. Huq R. Tanner M.R. Londono L.M. Estrada R. Dhawan V. Chauhan S. Upadhyay S.K. Gindin Hotez P.J. B. et immunomodulatory peptides from parasitic for autoimmune J. PubMed Scopus Google Scholar). is shown to the in and the are shown by the Acan1 found to the in and that is responsible for the The hookworm peptides were synthesized on peptides The of and be synthesized in and a approach to successfully produce the by the and the of the to be The of were by in different conditions. peptides were to in with the of both and of and the found to to and with and were for further as were to be via with were for Acan1 and Nak1 were of the for further immunomodulation as were shown to be the in an of of the peptides These that has a on the of the in of and were to the structures of the hookworm peptides Acan1 and Nak1, in to with of a from a PubMed Scopus Google and for The structures for Acan1 and Nak1 were and from the and both peptides were shown to that of The with were were for for both peptides, with the the target Although the peptides a with a the two the and of the two to and to and a is and The in Acan1 are and and and with the and whereas in Nak1, are and and and and a is and the in Acan1 found to the that is for the activity of on the The of and in are on a with the of the in the in Acan1 are shown to a different to from Acan1 and Nak1 are to human a to their on in as a and found to with an of the described in the literature Chang S.C. Chauhan S. Huq R. S. of peptides on the PubMed Scopus Google Scholar). Acan1 the with an of a than the Nak1 found to have on the to the of within the The of via in human and gastrointestinal and intestinal Acan1 and Nak1 were found to be in human over the whereas to the gastrointestinal peptides were shown to be intestinal with a of Acan1 and Nak1 and a of and respectively The L.M. Jones A. V. and of a the 2018; PubMed Scopus Google found to be within the of The of that the are to is that the of the hookworm peptides in have from by helminths that can live in the small for to their and of peptides Acan1 and Nak1 to the that both peptides have on human T the of Acan1 and Nak1 to human assessed by cell apoptotic cell were identified and with a of live cell as the whereas the cells an in to apoptotic the anti-inflammatory of Acan1 and Nak1 a assays human that Nak1 of or in or treatment shown that Acan1 significantly the of active cells in a of and treatment has been reported that T cell and signaling W. J. N. X. T cell subset and of T cell by 2017; PubMed Scopus Google Scholar). human were with and to and induce T cell production. cells were with for and IL-2 and TNF by Acan1 and Nak1 IL-2 in a whereas Acan1 TNF the of TNF by the of the assessed on T T cells were via and with 100 or 100 Acan1 for T cell by and Acan1 CD4+ T cell proliferation in two different to a than the Acan1 significant on T cell these a CD4+ T cell for Acan1 further the therapeutic of hookworm peptides in a model of acute were with of Acan1 and or Nak1 and and assessed over the of the the sulfonic a weight loss with of Acan1 via the displayed significantly weight loss with the significant in weight loss in the pathology assessed on and with protection against colitis. and with the and were found to have significantly with disease The were on the edema, and found that with Acan1 displayed a significant in the overall pathology of mouse colon displayed colon tissue architecture. the of the goblet and of damage and of the and colon of and mucosal with of goblet with Acan1 or Nak1 with the with and goblet mucosal and these results that peptides, particularly Acan1 and to a Nak1, can suppress intestinal inflammation in We on the of mouse a that genes in epithelial and tissue A analysis component analysis to genes that the to the of within the significant the of the were identified for both Acan1 and Nak1 However, a significant the of Acan1 and Acan1 and and and Acan1 as as Nak1 and the two of the in the for Acan1 and for The genes with the to and are shown by the These and and and We treatment and by the and for within As the Acan1 with the a significant of genes characterized by the Nak1 with the significant were identified in genes and were characterized by the Acan1 and Nak1 the of genes and significant from a small of genes These are further that the of both peptides is for The colitis mouse model is a in model that many and with human Giacomin P. Navarro S. Miller Loukas A. P. A the of inflammation in a mouse model of 2018; PubMed Scopus Google Scholar). colitis mouse model to the peptides Acan1 and Nak1 for their and the responses of both hookworm peptides were study in model of experimental hookworm significantly against weight as as the of colitis. Acan1 and Nak1 of gastrointestinal with and goblet cell mucosal and of ulceration, edema, and colon Although from the Nak1 to that of the the to than that of Acan1 colon of treatment genes were found to have in Acan1 with the of these genes are found to be in mucosal repair P. The against loss of epithelial and experimental Full Text Full Text PDF PubMed Scopus Google Scholar, J. A. V. S. R.J. et a key role in the of intestinal PubMed Scopus Google repair and restitution of ulcerated S. I. V. D. The in PubMed Scopus Google cell proliferation I. cells produce that the of and against Immunol. PubMed Scopus Google and and of cells dendritic cells and the for Rev. Immunol. PubMed Scopus Google J. L. D. of the dendritic cell 2009; Full Text Full Text PDF PubMed Scopus Google Scholar, A. T. H.J. S. dendritic cell have different and 2009; Full Text Full Text PDF PubMed Scopus Google A. T. H.J. S. dendritic cell have different and 2009; Full Text Full Text PDF PubMed Scopus Google T cells in and 2009; Full Text Full Text PDF PubMed Scopus Google The by Nak1 is more difficult to with a found in significant These that the of the hookworm in treating can be to the peptides secreted the by the However, and are significantly different in and and in results that the is to and that peptides from the hookworms can against a model of colitis. of inflammation in the mouse model with treatment of the hookworm peptides be to the decrease in and CD4+ T vitro that Acan1 and Nak1 were nontoxic and induce as human Acan1 and Nak1 were found to decrease IL-2 in a an further Acan1 the proliferation of CD4+ T cells that of T A T cell is as a key of chronic inflammation in T cells in and 2009; Full Text Full Text PDF PubMed Scopus Google Scholar, J. migration in inflammatory bowel Med. PubMed Scopus Google Scholar). that CD4+ T cells are the of pathology T. S. M.R. T cell in inflammatory bowel Immunol. 2018; PubMed Scopus Google Scholar, J. B. P.J. T cell in inflammatory bowel T cell to disease activity and disease 2017; Scopus Google Scholar). The activity of Acan1 on human T cell proliferation in vitro that therapeutic the inflammatory and Acan1 found to decrease TNF an in the of of of therapy in inflammatory bowel 2016; PubMed Scopus Google Scholar). The of both Acan1 and Nak1, by are on the activity in the mouse the of the hookworm is for of the Acan1 and Nak1 the as the their is than Acan1 the within the is responsible for to the of the However, in Acan1 the is with an and can be in a different in the with be responsible for the of Acan1 the studies have that the of T cell in active and a is with both and of inflammation The role of T cell and in the inflammatory in ulcerative Med. J. Google Scholar, I. of in mucosal therapeutic for inflammatory bowel PubMed Scopus Google Scholar, S. W. X. J. Clinical of and in of active ulcerative PubMed Scopus Google Scholar). activity of that Acan1 be of therapeutic in inflammatory and autoimmune As to of Nak1 found to have activity on the to be as a of the of The excretory/secretory component secreted by parasitic a vast of proteins that can the of host T.B. Giacomin P.R. Loukas A. Mulvenna J.P. Clark R.J. Miles J.J. Helminth immunomodulation in autoimmune disease.Front. Immunol. 2017; 8: 453Crossref PubMed Scopus (106) Google Scholar). A significant number of trials have shown the of hookworm therapy for the treatment of autoimmune and T.B. Giacomin P.R. Loukas A. Mulvenna J.P. Clark R.J. Miles J.J. Helminth immunomodulation in autoimmune disease.Front. Immunol. 2017; 8: 453Crossref PubMed Scopus (106) Google Scholar). we that the of peptides, rather than live infection, is to induce protection in a mouse model of colitis and tissue repair and Acan1 and Nak1 were shown to the human of and T cell proliferation. 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