We previously reported the identification of a novel family of immunomodulatory proteins, termed helminth defense molecules (HDMs), that are secreted by medically important trematode parasites. Since HDMs share biochemical, structural, and functional characteristics with mammalian cathelicidin‐like host defense peptides (HDPs), we proposed that HDMs modulate the immune response via molecular mimicry of host molecules. In the present study, we report the mechanism by which HDMs influence the function of macrophages. We show that the HDM secreted by Fasciola hepatica (FhHDM‐1) binds to macrophage plasma membrane lipid rafts via selective interaction with phospholipids and/or cholesterol before being internalized by endocytosis. Following internalization, FhHDM‐1 is rapidly processed by lysosomal cathepsin L to release a short C‐terminal peptide (containing a conserved amphipathic helix that is a key to HDM function), which then prevents the acidification of the endolysosomal compartments by inhibiting vacuolar ATPase activity. The resulting endolysosomal alkalization impedes macrophage antigen processing and prevents the transport of peptides to the cell surface in conjunction with MHC class II for presentation to CD4 ⁺ T cells. Thus, we have elucidated a novel mechanism by which helminth pathogens alter innate immune cell function to assist their survival in the host.—Robinson, M. W., Alvarado, R., To, J., Hutchinson, A. T., Dowdell, S. N., Lund, M., Turnbull, L., Whitchurch, C. B., O'Brien, B. A., Dalton, J. P., Donnelly, S. A helminth cathelicidin‐like protein suppresses antigen processing and presentation in macrophages via inhibition of lysosomal vATPase. FASEB J. 26, 4614–4627 (2012). www.fasebj.org

Infections with helminth parasites prevent/attenuate auto-inflammatory disease. Here we show that molecules secreted by a helminth parasite could prevent Type 1 Diabetes (T1D) in nonobese diabetic (NOD) mice. When delivered at 4 weeks of age (coincident with the initiation of autoimmunity), the excretory/secretory products of Fasciola hepatica (FhES) prevented the onset of T1D, with 84% of mice remaining normoglycaemic and insulitis-free at 30 weeks of age. Disease protection was associated with suppression of IFN-γ secretion from autoreactive T cells and a switch to the production of a regulatory isotype (from IgG2a to IgG1) of autoantibody. Following FhES injection, peritoneal macrophages converted to a regulatory M2 phenotype, characterised by increased expression levels of Ym1, Arg-1, TGFβ and PD-L1. Expression of these M2 genetic markers increased in the pancreatic lymph nodes and the pancreas of FhES-treated mice. In vitro, FhES-stimulated M2 macrophages induced the differentiation of Tregs from splenocytes isolated from naïve NOD mice. Collectively, our data shows that FhES contains immune-modulatory molecules that mediate protection from autoimmune diabetes via the induction and maintenance of a regulatory immune environment.

The NLRP3 inflammasome is a multimeric protein complex that controls the production of IL‐1β, a cytokine that influences the development of both innate and adaptive immune responses. Helminth parasites secrete molecules that interact with innate immune cells, modulating their activity to ultimately determine the phenotype of differentiated T cells, thus creating an immune environment that is conducive to sustaining chronic infection. We show that one of these molecules, FhHDM‐1, a cathelicidin‐like peptide secreted by the helminth parasite, Fasciola hepatica , inhibits the activation of the NLRP3 inflammasome resulting in reduced secretion of IL‐1β by macrophages. FhHDM‐1 had no effect on the synthesis of pro‐IL‐1β. Rather, the inhibitory effect was associatedwith the capacity of the peptide to prevent acidification of the endolysosome. The activation of cathepsin B protease by lysosomal destabilization was prevented in FhHDM‐1‐treated macrophages. By contrast, peptide derivatives of FhHDM‐1 that did not alter the lysosomal pH did not inhibit secretion of IL‐1β. Wepropose a novel immunemodulatory strategy usedby F. hepatica , whereby secretion of the FhHDM‐1 peptide impairs the activation of NLRP3 by lysosomal cathepsin B protease, which prevents the downstream production of IL‐1β and the development of protective T helper 1 type immune responses that are detrimental to parasite survival.—Alvarado, R., To, J., Lund, M. E., Pinar, A., Mansell, A., Robinson, M. W., O'Brien, B. A., Dalton, J. P., Donnelly, S. The immune modulatory peptide FhHDM‐1 secreted by the helminth Fasciola hepatica prevents NLRP3 inflammasome activation by inhibiting endolysosomal acidification in macrophages. FASEB J. 31, 85–95 (2017) www.fasebj.org

Helminth parasites secrete molecules that potently modulate the immune responses of their hosts and, therefore, have potential for the treatment of immune-mediated human diseases. FhHDM-1, a 68-mer peptide secreted by the helminth parasite Fasciola hepatica, ameliorated disease in two different murine models of autoimmunity, type 1 diabetes and relapsing-remitting immune-mediated demyelination. Unexpectedly, FhHDM-1 treatment did not affect the proliferation of auto-antigen specific T cells or their production of cytokines. However, in both conditions, the reduction in clinical symptoms was associated with the absence of immune cell infiltrates in the target organ (islets and the brain tissue). Furthermore, after parenteral administration, the FhHDM-1 peptide interacted with macrophages and reduced their capacity to secrete pro-inflammatory cytokines, such as TNF and IL-6. We propose this inhibition of innate pro-inflammatory immune responses, which are central to the initiation of autoimmunity in both diseases, prevented the trafficking of autoreactive lymphocytes from the periphery to the site of autoimmunity (as opposed to directly modulating their function per se), and thus prevented tissue destruction. The ability of FhHDM-1 to modulate macrophage function, combined with its efficacy in disease prevention in multiple models, suggests that FhHDM-1 has considerable potential as a treatment for autoimmune diseases.

Host defence peptides (HDPs) are expressed throughout the animal and plant kingdoms. They have multifunctional roles in the defence against infectious agents of mammals, possessing both bactericidal and immune-modulatory activities. We have identified a novel family of molecules secreted by helminth parasites (helminth defence molecules; HDMs) that exhibit similar structural and biochemical characteristics to the HDPs. Here, we have analyzed the functional activities of four HDMs derived from Schistosoma mansoni and Fasciola hepatica and compared them to human, mouse, bovine and sheep HDPs. Unlike the mammalian HDPs the helminth-derived HDMs show no antimicrobial activity and are non-cytotoxic to mammalian cells (macrophages and red blood cells). However, both the mammalian- and helminth-derived peptides suppress the activation of macrophages by microbial stimuli and alter the response of B cells to cytokine stimulation. Therefore, we hypothesise that HDMs represent a novel family of HDPs that evolved to regulate the immune responses of their mammalian hosts by retaining potent immune modulatory properties without causing deleterious cytotoxic effects. In mammals, secreted host defence peptides (HDPs) protect against a wide range of infectious pathogens. They also perform a range of immune modulatory functions which regulate the immune response to pathogens, ensuring that the protective inflammatory response is not exacerbated and that post-infection repair mechanisms are initiated. We identified a novel family of molecules secreted by medically-important helminth pathogens (termed helminth defence molecules; HDMs) that exhibit striking structural and biochemical similarities to the HDPs. To further investigate the extent of this similarity, we have performed a comparative functional study between several well characterized, anti-microbial, mammalian HDPs and a series of parasite-derived peptides. The parasite HDMs displayed immune modulatory properties that were similar to their HDP homologs in mammals, but possessed no antimicrobial or cytotoxic activity. We propose that HDMs of these helminth pathogens underwent specific adaptation, losing their anti-microbial activity but retaining their ability to regulate the immune responses of their mammalian hosts. This absence of cytotoxicity and retention of immune-modulatory activity offers an opportunity to design novel immunotherapeutics derived from the HDMs which could be used to combat destructive inflammatory responses associated with microbial infection and immune-related disorders.