Modulation of Host Immunity by Helminths: The Expanding Repertoire of Parasite Effector Molecules

1Helminths are extremely successful parasites, affecting around a quarter of the world’s population. They establish chronic infections and if untreated can persist for the lifetime of the host. While the immune system is capable of parasite expulsion (often incurring collateral damage), in natural infections there is frequently a muted immune response in which the host accommodates, and tolerates, the invader. Such re-setting of host immunity results from both host reparative responses to physical damage from tissue-migrating parasites and/or through active immunomodulation by their molecular products. The ability of parasites to defy host immunity reflects their masterful manipulation of the immune system, which as detailed below, is achieved through the release of a spectrum of finely tuned and highly evolved immuno-modulatory factors (Table 1).

2Recent advances in helminth genomics and proteomics have uncovered a wealth of such immunomodulatory products. These act on every phase of the immune response, which for the purposes of this review we have divided into: (1) initiation, (2) antigen recognition and processing, (3) adaptive responses, (4) effector cell responses, and (5) coagulation, healing, and remodeling. Parasite immunomodulators active at each phase can be found that either share ancestral homology with host genes or have developed de novo, sharing no identifiable homology to the host (Figure 1). Here, we summarize the molecular actors known to interact at each step and their evolutionary and structural provenance, and we discuss future prospects for this exciting field.

3Immune responses are initiated by danger signals, through detection of damage-associated molecular patterns (DAMPs) or introduction of pathogen-associated molecular patterns (PAMPs). Parasite migration induces DAMPs and introduces PAMPs from either the parasite itself or bacteria entering tissues through compromised barriers. The imperative to mute barrier surface reactions has driven the evolution of potent mechanisms to suppress innate immune responses (Figure 2).

4Activation of epithelial cells results in the release of “alarmin” cytokines such as thymic stromal lymphopoietin (TSLP), interleukin-25 (IL-25), and IL-33. IL-33 is tightly spatiotemporally controlled: it is released on epithelial necrosis and then rapidly oxidized and inactivated. The murine intestinal nematode Heligmosomoides polygyrus suppresses IL-33 release through HpARI (H. polygyrus alarmin release inhibitor) within its excretory-secretory (ES) products. HpARI has a selective mode of action, binding DNA via its first domain, while the second and third domains bind to reduced (active) but not oxidized (inactive) IL-33. Binding obstructs interaction of the complex with the IL-33 receptor, ST2, while interaction with DNA tethers IL-33 within the nucleus of necrotic cells, preventing IL-33 release. HpARI administration ablates type 2 cell-mediated inflammation and improves lung function in an Alternaria allergen-dependent asthma model, while in Nippostrongylus brasiliensis infection, HpARI administration suppresses type 2 responses and increased worm burden. Crucially, these effects translate to the human setting, as HpARI prevents IL-33 release from human lung explants and blocks human IL-33 release in a transgenic mouse model.

5H. polygyrus suppresses the IL-33 pathway at multiple levels additionally to HpARI, with an undefined H. polygyrus product further downregulating IL-33 production through induction of IL-1β and through the release of small RNA-containing extracellular vesicles that suppress transcription of the IL-33 receptor.

6IL-33 release can be provoked by signals of cell stress or death, in particular extracellular ATP, which induces epithelial and mast cell IL-33 production. Many parasite secretions contain apyrases, which degrade ATP to non-inflammatory AMP, reducing inflammatory DAMP signals, and hence these could inhibit this important arm of the damage detection response.

7A pivotal point in host immunity is recognition of and reaction to pathogen molecules, typically by pathogen- or damage-associated molecules patterns (PAMPs and DAMPs) ligating pattern recognition receptors (PRRs) on myeloid cells, such as toll-like receptors (TLRs) and C-type lectin receptors (CLRs). These reactions are intensively targeted by helminth molecules (Figure 2), which block TLR ligand-induced dendritic cell and macrophage activation, interfering with receptors and their signaling, as well as antigen presentation and downstream effector mechanisms.

8The MyD88 adaptor protein is required for signaling via all TLRs except TLR3, and also IL-1 family cytokine receptors (including the IL-33 receptor). ES-62, a multifunctional glycoprotein secreted by the rodent filarial nematode Acanthocheilonema viteae, protects against pathology in mouse models of rheumatoid arthritis (RA), asthma, and lung fibrosis. ES-62 induces sequestration of the MyD88 signaling protein, leading to suppression of TLR and IL-33 signaling. The immunomodulatory principle of ES-62 is phosphorylcholine (PC) side groups carried on N-linked glycan moieties, and synthetic small molecule variants of PC can reproduce many anti-inflammatory effects of the parent molecule.

9A different mechanism is deployed by the immunomodulatory ES protein Fh12 (and its recombinant form, Fh15) from the liver fluke Fasciola hepatica. The Fh12 and Fh15 proteins induce alternative activation in human monocyte-derived macrophages (Mo-Macs), suppress macrophage activation by TLR2, TLR4, TLR5, and TLR8 ligands, and inhibit inflammatory cytokine production in septic shock. They bind to the TLR4 cofactor CD14 and reduce its expression, mediating suppression of TLR4 responses. The Fh12 and Fh15 proteins also suppress responses of CD14-deficient cells, suggesting further inhibitory functions. Furthermore, cysteine proteases from the flukes F. hepatica (FhCL1) and S. mansoni (SmCB1) both directly suppress myeloid cell TLR signaling by interfering with TLR-driven inflammation in vivo, resulting in the intracellular degradation of TLR3 and TLR4.

10Conversely, some helminth molecules stimulate, rather than inhibit, TLRs. The phospholipid lysophosphatidylserine (lyso-PS) is enriched in the tegument of S. mansoni adult worms. LysoPS is a TLR2 ligand that increases the capacity of human DCs to drive IL-10-producing T cells, suppressing T cell proliferation. In contrast, the 66-amino acid mucin-like polypeptide from F. hepatica (Fhmuc), while not itself a TLR4 ligand, promotes LPS-induced TLR4 activation of DCs, enhancing T helper 1 (Th1) cell responses, both in vitro and in vivo.

11CLR signaling is important in responses to helminths, and parasite-specific glycans are a rich source of ligands. The schistosome-specific GalNAc-di-GalNAc (LDN) motif is bound by soluble galectin-3, while fucosylated (LDN-F) derivatives and fucose-containing Lewis X (LeX) structures are bound by dendritic cell-specific ICAM3-grabbing non-integrin (DC-SIGN). Mannose-containing structures are recognized by mannose receptor (MR) and macrophage galactose-type lectin (MGL). Similar glycan ligands for these receptors are also present in many other parasites including F. hepatica. During F. hepatica infection, glycan-CLR signaling induces a mixed type 2 and regulatory phenotype with IL-10 and transforming growth factor-β (TGFβ) production.

12Schistosome glycans are strongly immunomodulatory with a predominant role in Th2 cell polarization through DCs: S. mansoni soluble egg antigen (SEA) induces Th2 cell responses in vivo in a glycan-dependent manner. The LNFPIII (lactose-N-fucopentose III) motif, present in both on S. mansoni and in human milk, has similar DC-modulating effects as total SEA and can act as an adjuvant for a type 2 immune response. LNFPIII also stimulates murine B cells to produce the immunoregulatory cytokine IL-10. SEA and human milk also contain LNnT (lacto-N-neotetraose), while intraperitoneally injected milk-derived LNnT induces the influx of a macrophage population that produces IL-10 and TGF-β and suppresses T cell proliferation. Furthermore, schistosome glycans bind to the C-type lectin receptors Dectin-1 and Dectin-2 and prime DCs for enhanced Th2 cell development via autocrine prostaglandin-E2 (PGE2) and OX40L, similar to core α(1-3)-fucose- and β(1-2)-xylose-linked glycans. Other prominent helminth glycans include chitin, a long-chain polysaccharide that is a potent type 2 response inducer.

13If and when host myeloid cells are triggered through one or more innate receptors, they initiate the proteolytic processing of exogenous antigens and expression of a suite of key stimulatory markers and mediators. Helminths counter with a palette of protein modulators, from protease inhibitors to receptor ligands, that target these pathways. Among the inhibitors, cystatins interfere with cysteine proteases involved in antigen processing, such as lysosomal cathepsins and asparaginyl endopeptidase (AEP). The filarial cystatins, including CPI-2 from B. malayi, also specifically inhibit AEP, allowing it to block antigen processing in human cells, while LsCystatin (from Litomosoides sigmodontis) reduces nitric oxide- and antigen-specific proliferative responses. In vitro, Onchocystatin (from Onchocerca volvulus) induces IL-10 from human monocytes, together with reduced MHC-II and CD86 expression. In mouse models, AvCystatin (from A. viteae) similarly drives macrophage IL-10 expression, dampening both airway allergy and colitis. Furthermore, AvCystatin induces a regulatory PD-L1+ and PD-L2+ macrophage populations, which on adoptive transfer protect against both airway allergy and intestinal inflammation and reduce pollen-specific responses in PBMCs from allergic patients.

14Other helminth cystatins with broad immunomodulatory properties including those from S. japonicum, Ascaris lumbricoides, and the liver fluke Clonorchis sinensis all ameliorate colitis in vivo, while Nippocystatin from N. brasiliensis blocks responses to ovalbumin. The physiological target of these cystatins in vivo has yet to be identified. One possible target might be protease-dependent inflammasome activation, as their effects appear more generalized than interference with the antigen processing machinery alone would predict.

15In addition to interfering in myeloid PRR responses, helminths also modulate myeloid cells through mimics of the host’s innate immune messengers. Macrophage migration inhibitory factor (MIF) proteins are evolutionarily ancient, but in mammals they activate myeloid cells. MIF homologs are also found in nematodes: those from Brugia malayi and Trichinella spiralis mirror the activity of the host protein, inducing IL-8 release from monocytes, while B. malayi MIF also potentiates alternative activation of macrophages, in synergy with IL-4. MIF from Anisakis simplex is directly anti-inflammatory, ameliorating both allergy and colitis in mice. Therefore, homology to the host cytokine does not always predict biological activity.

16Further macrophage modulation is mediated by a F. hepatica-secreted protein termed helminth defense molecule, or FhHDM-1. FhHDM-1 is a homolog of the mammalian cathelicidin-like host defense peptide (HDP) designated CAP18/LL-37. HDPs are antimicrobial peptides that can disrupt bacterial membranes and suppress pro-inflammatory macrophage responses. FhHDM-1 binds LPS, preventing LPS ligation and activation of macrophages. Furthermore, FhHDM-1 is internalized by macrophages and processed by lysosomal cathepsin L, releasing a short C-terminal peptide which in turn prevents the acidification of the endolysosomal compartment, impairing antigen processing and preventing NLRP3 inflammasome activation and IL-1β secretion. In addition to its immune-modulatory effects, FhHDM-1 may have parasite-intrinsic functions, as it also binds and detoxifies heme.

17More broadly, and beyond signaling pathways and antigen processing, myeloid cell protein expression can be profoundly affected by helminth molecules. For example, ω-1 (Omega-1), a glycoprotein released by S. mansoni eggs, is a T2 ribonuclease (RNase). It is taken up by DCs through recognition of its glycan moiety, Lewis-X, by the mannose receptor, and extinguishes protein synthesis through the degradation of ribosomal RNA and mRNA within the cell. Omega-1 does not degrade specific transcripts, but rather targets the global pool of RNA. In this way omega-1, and its close homolog from Schistosoma japonicum, CP1412, suppress DC activation and maturation, preventing upregulation of CD86 and MHCII and synthesis of IL-12 in response to CD40 ligation, favoring both Th2 cell and regulatory T (Treg) cell induction. T cell priming in the absence of IL-12 and with diminished co-stimulation may favor Th2 cell polarization.

18The Th2 cell-mediated priming capacity of omega-1 has also been confirmed in vivo using lentivirus-based transduction to silence gene expression in schistosome eggs, as intravenous injections of those eggs shows reduced lung granuloma formation compared to sham-transduced eggs. Lastly, omega-1 induces inflammasome-dependent IL-1β in TLR2-stimulated peritoneal macrophages, which is abrogated when blocking Dectin-1, suggesting that omega-1 can modulate multiple PRR pathways.

19The modulatory effects of omega-1 were recently extended to changes in host metabolism. A series of recent studies have demonstrated the beneficial effects of helminth infection on host metabolism, which can be replicated by helminth products such as SEA or recombinant omega-1. Injection of omega-1 into obese mice improves insulin sensitivity via IL-33 release from white adipose tissue, which in turn induces ILC2 activation and alternatively activated M2 macrophage differentiation. Mechanistically, the release of IL-33 is mediated by the RNase activity of omega-1, while the improved insulin sensitivity was dependent on the ligation of the mannose receptor.

20Another example showing myeloid cell signaling manipulation is from B. malayi abundant larval transcript (ALT). The mosquito-borne B. malayi larvae stockpile quantities of ALT proteins prior to entry into the mammalian host. Investigation of gene function by a transfection approach within macrophages identified ALT-induced upregulation of SOCS1, the inhibitor of IFN-γ signaling in inflammation.

21Finally, some helminth proteins contain a nuclear localization signal (NLS) for rapid translocation to the nucleus, including the S. japonicum-derived molecule Sj16, which induces IL-10 and inhibits DC maturation following nuclear translocation. IL-4-inducing principle from schistosome eggs (IPSE) is also rapidly translocated to the nucleus of DCs upon uptake, while the IPSE homolog from S. haematobium (H-IPSE) translocates to the nucleus of bladder epithelial cells. Here, H-IPSE reduces bladder hemorrhage and accelerates urothelial repair in a chemotherapy-induced hemorrhagic cystitis (CHC) model. The therapeutic effects are IL-4 dependent and may relate to its IL-4-inducing capacity described in basophils (detailed below). The effects of H-IPSE are, however, superior to IL-4 alone, implying that additional nuclear functions are involved.

22The end result of parasite modulation of dendritic cell and macrophage modulation is often aberrant, skewed, or tolerized T cell responses. In the next section we will deal with helminth products that act directly on adaptive immune responses.

23On activation by APCs, naive T cells can differentiate into Th1, Th2, Th17, or Treg cell subsets. Modulation of adaptive immune responses has the potential for lasting effects in the host: if the parasite can appear as harmless or commensal, a memory response can be raised that treats the parasite as “self.” Parasitic helminths have developed multiple suppressive and modulatory pathways that suppress effector and/or induce regulatory T helper and B cell responses (Figure 3).

24An example of both homologous and de novo-generated immunomodulators from helminth parasites can be found in parasite agonists of the TGF-β receptor. The effects of host immune TGF-β signaling are diverse, but are largely immunosuppressive, with generation of induced regulatory T cells, suppression of dendritic cell, macrophage, and T cell activation, and skewing of antibody isotypes to IgA. Thus, stimulation of the TGF-β pathway would be advantageous to an immunity-shy parasite.

25The ancestral function of the TGF-β pathway, across the animal kingdom, is in development: the immunomodulatory role of TGF-β is a more recent adaptive branch within vertebrates. Notably, ligands and receptors show a high degree of conservation allowing, for example, S. mansoni and Echinococcus multilocularis TGF-β receptors to ligate human TGF-β family ligands. The question is whether TGF-β family members from helminths activate immunosuppressive mechanisms in mammalian cells; indeed, B. malayi TGH-2 can signal through the TGF-βR while extracts from L. sigmodontis also ligate the TGF-βR in a manner that cannot be inhibited with antibodies to TGF-β itself. Finally, a TGF-β homolog from F. hepatica, FhTLM, binds host receptors, but with lower affinity and this was inhibited by polyclonal anti-TGF-β antibodies. Immunologically, FhTLM induces IL-10 production in macrophages, indicative of a pro-regulatory outcome.

26In contrast to these instances of evolutionarily conserved members of the TGF-β family activating host receptors, a striking example of convergent evolution has been found in H. polygyrus. Secreted products from this intestinal nematode have been shown to activate the TGF-β signaling pathway to induce regulatory T cells in vitro, even in the presence of pro-inflammatory cytokines and to a similar degree as TGF-β itself. Subsequently, through biochemical fractionation, the factor responsible has been identified as an unrelated structure named TGM (TGF-β Mimic), a 5-domain protein around three times larger than TGF-β and able to bind with high affinity to both TGF-βRI and II, in distinction to mammalian TGF-β which ligates only to RII. TGM is also active in vivo, for example in prolonging survival of fully allogeneic skin grafts. Similarly to HpARI, HpTGM is also effective in cultures of human T cells, despite H. polygyrus lacking infectivity in humans.

27While the TGF-β pathway directly induces regulatory T cells, other helminth mediators exploit indirect pathways to modulate host T cells. Hookworm-derived homologs of the tissue inhibitor of metalloproteases (TIMPs) modulate DC function, resulting in tolerogenic T cell responses and more recently these proteins (now called AIP-1 and AIP-2, for anti-inflammatory proteins) have been demonstrated to be effective quenchers of pathology in mouse models of airway allergy and TNBS-induced colitis. Protection is associated with increased Treg cell and IL-10 responses, alongside abated inflammatory cytokines and modified DC function. Effects are also seen in cultured human DCs and T cells, indicating potential as therapeutics for immune disorders.

28Treg cell induction in vivo is also seen with PAS-1, a 200-kDa native protein purified from Ascaris suum, which is reported to inhibit airway allergy in ovalbumin-alum primed mice. PAS-1 induces an expands CD25+ Treg cell populations, which can suppress allergic responses on adoptive transfer. PAS-1 is similar to ABA-1, a previously described nematode tandemly repeated polyprotein with lipid-binding properties.

29Host galectins promote Treg cell functionality and stability, and a homolog of human galectin-9 from Toxascaris leonina reduces DSS-induced colonic pathology in mice with increased production of IL-10 and TGF-β. While the mechanism is assumed to parallel that of human galectin-9, this has not been verified for the helminth homolog.

30Effector T cells are also targeted by certain products. The schistosome glutathione-S-transferase protein (P28GST) appears to promote a sufficiently strong eosinophil-rich Th2 cell response to prevent Th1 and Th17 cell-mediated inflammation in murine colitis in an eosinophil-dependent manner. In contrast, the Echinococcus multilocularis homolog of TIP (EmTIP) induces IFN-γ release from CD4+ T cells in vitro, which may explain how this parasite, unlike most other helminths, induces an initial Th1 cell response in vivo.

31Potassium channels in lymphocytes, such as voltage-gated channel Kv1.3, are crucial for the activation and proliferation of T cells. Kv1.3 is considered an interesting therapeutic target in autoimmune conditions and inhibitors include ShK, a polypeptide toxin expressed in the sea anemone Stichodactyla helianthus. ShK homologs have been conserved and adapted in helminth T cell inhibitors, e.g., AcK1, found in the hookworms Ancylostoma caninum and Ancylostoma ceylanicum, and BmK1, identified in the filarial worm B. malayi. These peptides suppress a delayed-type hypersensitivity (DTH) reaction in rats and inhibit primarily the proliferation of memory T cells without affecting naive or central memory subsets.

32An equally important target for helminth parasites is the host B cell response, which is known to be crucial for parasite ejection. Na-ASP-2 (Ancylostoma secreted protein 2 expressed by N. americanus) binds to CD79A, a component of the B cell receptor (BCR) complex, downregulating mRNA expression of targets involved in signaling and transendothelial migration while ES-62 from A. viteae uncouples the BCR signaling machinery, rendering host cells unresponsive. Other types of regulation of B cell responses in helminth infection are associated with a switch from the IgE to the IgG4 isotype, and a regulatory B cell (Breg) phenotype characterized by production of IL-10, similar to changes in B cell phenotypes found in allergen tolerance.

33The secreted products of S. mansoni eggs can induce a regulatory B (Breg) cell phenotype in murine and human cells, through or independently of the action of IPSE. Uptake of IPSE is necessary to induce B cell IL-10 and Treg cell development in vitro. IPSE also supports type 2 cell responses, through induction of IL-4 release from basophils that is IgE dependent but antigen independent and does not require uptake of IPSE by basophils, so this single protein has multiple functions and activation routes.

34By modulating CD4+ T cell differentiation, Treg cell induction, B cell isotype switching, and Breg cell induction, parasites can produce an environment most suited to them. These adaptive responses then determine effector cell responses and whether the outcome of infection is inflammation, resolution, parasite ejection, or tolerance.

35The full spectrum of cell populations are involved in defense against helminths, from “professional” immune leukocytes to stromal and epithelial cells. In particular, granulocytes (eosinophils and neutrophils) are often the final effector cells in allergic and inflammatory responses, able to target the site of infection and kill pathogens by a variety of means including delivery of toxic compounds, sequestering vital nutrients, and destabilizing the parasite environment. Therefore, parasite survival can be maintained through secretions that block effector cell recruitment and function.

36Chemokines are crucial in organizing the immune response through migration of immune cells to sites of inflammation. A key chemokine required for immune cell recruitment and activation in humans is IL-8. B. malayi produces asparaginyl-tRNA synthetase (AsnRS) which shows structural (but not sequence) homology to IL-8 and binds the IL-8 receptors CXCR1 and CXCR2, recruiting eosinophils and neutrophils. However, despite this apparently pro-inflammatory mechanism of action, administration of AsnRS in the mouse T cell transfer model of colitis induces regulatory responses and induction of IL-10.

37Conversely, human hookworms suppress chemokine responses through production of metalloproteinases which cleave eotaxin-1 (CCL11), but not IL-8 or eotaxin-2 (CCL24), specifically inhibiting the eotaxin-mediated recruitment of eosinophils. S. mansoni secretes a chemokine binding protein (SmCKBP) which binds and blocks IL-8, CCL2, CCL3, CCL5, and CX3CL1—this action inhibits neutrophil (but not eosinophil) migration and has suppressive activity in the DNFB contact hypersensitivity model, while blocking SmCKBP in vivo resulting in increased neutrophilia in S. mansoni egg-induced granulomas.

38In concert with chemokines, cell migration also requires interaction with integrins such as CD11b/CD18 to facilitate extravasation. One hookworm protein, NIF from Ancylostoma caninum, binds this integrin together with fibrinogen and prevents binding of human neutrophils to vascular endothelium. NIF is also effective in in vitro models of LPS-driven pulmonary inflammation, and a NIF-IgE Fc fusion protein (NIF-IGHE-CH4) suppresses human neutrophil migration and blister formation in an ex vivo human model of subepidermal autoimmune blistering disease. Remarkably, NIF is a member of a multi-gene family (Pfam00188, variously named CAP, SCP, TAPS, or VAL) that is prominent across a wide range of helminths, and which includes a wide variety of immune response modulators in both animals and plants.

39After responding to an infection, the host must heal after epithelial damage and inflammation. This can be mediated by immune pathways and helped (or hindered) by parasite products.

40As parasites enter the host, they must digest connective tissue to allow their migration, followed by rapid healing to maintain health of their host. They achieve this through secretion of proteases and other “chemical scissors” essential for the invasive process. Their entry is also facilitated by inhibiting coagulation, particularly prominent in blood-dwelling helminths like schistosomes with multiple strategies to manipulate host vasodilation, fibrinolysis, and coagulation. Calpain family proteases SmCalp-1 and SmCalp-2 are expressed in the tegument of S. mansoni adult worms, where they appear to cleave and inactivate key coagulation proteins fibronectin and high-molecular-weight kininogen (HK).

41As discussed above, ATP-degrading enzymes (apyrases) are commonly secreted by helminths, suppressing DAMP-induced inflammatory responses. However, these enzymes have further roles in the control of thrombosis, as extracellular ATP and ADP are potent activators of platelets. Schistosome teguments contain ATP diphosphohydrolase (SmATPDase1) and the ectonucleotide pyrophosphatase/phosphodiesterase homolog SmNPP5, both of which degrade ATP and ADP to non-thrombotic AMP.

42Conversely, a serine protease-2 (SmSP-2) has been found to activate tissue plasminogen activator and plasminogen, both key components of the fibrinolytic system. Similar activities have been described for other parasite secretions, such as the cathepsin L peptidases FhCL-1, -2, and -3 from F. hepatica, which degrade fibrinogen and fibrin, and a plasminogen-binding alpha-enolase from O. volvulus (OvENO), which may promote proteolysis and degradation of the host extracellular matrix for migration of larvae through host tissues.

43Parasite secretions can also encourage the healing of the epithelium. An extreme example of this is the bile duct-dwelling trematode Opisthorcis viverrini. This trematode feeds on bile duct epithelial cells and causes extensive inflammation in situ. It secretes a granulin homolog (Ov-GRN-1) which causes epithelial cell proliferation: this has potential as a therapeutic agent for non-healing wounds, but during infection can also result in epithelial transformation and contributes to mortality in O. viverrini infection due to cholangiocarcinoma.

44Thus, the secretions of parasitic helminths can modulate every phase of immunity, from the most proximal events in immune initiation to terminal effector responses. In the next section we will discuss how these recent research findings could lead to the discovery of further parasite immunomodulators and how these could be developed for use in human immune-mediated diseases.

45The field of helminth immunomodulators has, until now, been dominated by protein actors, a tendency likely to continue with the torrent of sequence information extending to more than 100 helminth genomes, each with >10,000 protein-coding genes. Protein sequence analyses also provide insights into the molecular evolution of the different families of helminth immunomodulators (Figure 4). However, this should not overshadow the critical role of non-protein components (alone or in concert with protein moieties), which play many critical roles in helminth immunology.

46A relatively neglected aspect of helminth biology has been their production of small molecules with potential for immune modulation, such as prostaglandins and short-chain fatty acids (SCFAs). Some helminths, for example Ascaris, release acetate and other metabolites likely to impact responses of the epithelium. In addition, helminths can modify the commensal microbiota in a fashion that raises SCFA concentrations, indirectly promoting regulatory mechanisms in the host. With metabolomics now being applied to helminths and genomics piecing together parasite biosynthetic pathways, the suite of such small molecules is likely to now expand rapidly and uncover many more critical interactions in host recognition and immune control. In a recent demonstration of this, small molecules from A. caninum suppressed pathology in a mouse model of TNBS-induced colitis.

47Lipid mediators are among the most potent small molecules in the host immune system, both activating and suppressing the immune response. They are <1 kDa, intensely active within their milieu, and have conserved chemical structures across multiple hosts. They represent a target for parasite neutralization or molecular mimicry where helminths would benefit from their effects. PGE2 is an arachadonic acid metabolite, with roles in ILC2, T helper cell, macrophage, dendritic cell, and neutrophil responses. PGE2 is strongly linked to Th2 cell and ILC2 responses, for example inducing IL-22-mediated atopic dermatitis, although it can also directly inhibit ILC2s. T. suis, S. mansoni, and filarial nematode secretions contain PGE2, and S. mansoni SEA has the ability to induce further PGE2 from host cells, in this case mediating Th2 cell induction.

48In the context of the pivotal role of lipid mediators, more attention is likely to be focused on lipid-binding proteins that are numerous among helminth products. For example, F. hepatica Fh12 (also known as fatty acid binding protein) mentioned above induces alternative activation in human Mo-Macs and suppresses LPS-induced activation via binding and blocking of CD14.

49A paradigm-shifting discovery has been that helminths release extracellular vesicles (EVs), often termed exosomes, which are able to fuse and enter host cells, carrying with them a cargo of miRNAs. In H. polygyrus infections, these EVs target epithelial cells and macrophages, resulting in their loss of expression of the IL-33 receptor (IL-33R, also termed ST2) and downregulation of other immune genes. These effects result in suppression of both alternative and inflammatory macrophage activation. These in vitro findings have been corroborated in vivo, as N. brasiliensis EVs protect against TNBS-induced colitis, and H. polygyrus EVs suppress Alternaria allergen-induced allergic airway responses. In contrast, EVs from S. japonicum actively drive M1 macrophage differentiation in vitro, joining a range of helminth-secreted proteins that counter-intuitively induce inflammatory responses. Hence EVs are an adaptable part of the armory of helminth immunomodulators, allowing extracellular pathogens to manipulate events within host cells in a receptor-independent manner. Further research is required in this area, to investigate the level of specificity of particular EV populations for specific host cells, to determine the relative importance of EV protein and nucleic acid cargoes, and to develop these findings toward potential therapeutic agents for human disease.

50As the identification of defined immunomodulators accelerates, the scale of what the future may hold is also becoming apparent. Each large and complex helminth genome can encode 10,000–50,000 genes (compared to 20,000 in the human genome), and as a result many million previously unannotated genes are now being cataloged. Moreover, parasite secretions are extremely complex, containing hundreds to thousands of protein, carbohydrate, lipid, nucleotide, and vesicular moieties, each of which may be finely tuned to a particular host range.

51Parasitism has arisen on multiple occasions within the helminth phyla, and each lineage has evolved specific molecular strategies to defeat the host immune system. As helminths are metazoan members of the animal kingdom, they share many gene families with vertebrates that might provide a template for the molecular evolution of modulators interactive with counterparts in the host. Thus, as the host-adapted genes in the evolution of the immune system, parasites may have followed suit in parallel. Some cases, described below, appear to fulfil this prediction. In contrast, however, some of the most striking parasite immunomodulators such as HpARI, HpTGM, omega-1, and IPSE show no appreciable homology to host immune genes or previously known parasite immunomodulatory factors.

52Immunomodulators derived from an ancient shared ancestor of both parasites and their hosts include the TGF-β and MIF families. In the case of TGF-β, a gene family originally required for developmental processes expanded to encompass members with cytokine functions. Thus, as the host developed an immunological role for TGF-β, some parasites have been able to adapt pre-existing developmental homologs toward an immune-modulatory role. Therefore, the TGF-β homologs encoded in parasite genomes do not necessarily indicate immune modulatory functions, while conversely a single TGF-β homolog may fulfil both developmental and immunomodulatory roles (Figure 4A).

53It was recently discovered that, rather than adapt a pre-existing TGF-β homolog to interact with host receptors, H. polygyrus has convergently evolved an unrelated structure in the form of Hp-TGM, a protein consisting of five atypical complement control protein (CCP) domains with no homology to TGF-β, which ligates host TGF-β receptor. Furthermore, the same parasite blocks IL-33 through HpARI, another CCP domain protein. Although CCP domains are present in all helminths studied, there has been a preferential expansion of these genes in H. polygyrus (Figure 4B). CCP domains occur in a wide range of organisms and serve diverse functions in neuronal signaling, cytokine receptors, hemoglobin binding, and of course, complement control. CCP domain proteins are highly stable and soluble (each tight, globular CCP domain contains two disulfide bonds and conserved glycosylation), structurally simple but functionally sophisticated with independent activities of multiple domains within a single protein. This adaptable scaffold appears to have been exploited by H. polygyrus in particular.

54Gene family expansion in helminths hints at functional adaptation to the parasitic life style. Among the most notable is Pfam00188, which in helminths include venom allergen-like (VAL) and Ancylostoma secreted (ASP) proteins. This gene family is dramatically expanded in many parasitic nematodes (especially the hookworms and strongylides) and have been implicated in lipid binding, immune modulation, and pathogen resistance, so they may, like the CCP domain family, represent an adaptable scaffold upon which a range of functions can be developed.

55Conversely, expansion of the T2 ribonuclease family (containing omega-1) appears specific to schistosomes (Figure 4C), implying that this functionality may have evolved within the trematode family relatively recently. T2 ribonucleases are broadly expressed across many parasites, plants, and animals and are implicated in recycling of RNA and in RNA degradation during the stress response. T2 ribonucleases typically enter the secretory pathway and are often found in vacuoles and autophagosomes within cells. However, some T2 ribonucleases (for instance Erns from pestiviruses) can also be secreted, for degradation of RNA in extracellular spaces or in target cells, and it is this latter function which appears to have been utilized in the schistosomes, degrading RNA in host DCs and suppressing their maturation. Thus, schistosomes have “weaponized” a normal metabolic pathway to suppress DC maturation.

56Similarly the apyrase family, which is expanded in many parasitic nematodes, may have originally been a metabolic enzyme but have subsequently used to suppress of host damage detection and inhibit blood coagulation.

57These examples of helminth modulation exemplify the techniques used by helminths to develop immunomodulatory products: convergent evolution of shared protein families, de novo production of cytokine mimics or antagonists from adaptable modular domains, and hi-jacking of pre-existing protein functions. The agenda for the future is to test whether administration of defined helminth-derived molecular entities (with known functions and in a specific context) can lead us to clinically effective therapeutic molecules.

58In 2005, the first helminth therapy trials (using live T. suis infection) showed impressive effects in IBD and generated a great deal of interest in the use of live helminths as therapeutic modalities. However, subsequent trials have shown smaller or absent effect sizes, and a lack of understanding of the mechanistic underpinning of helminth immunomodulation has hampered progression. With the recent advances in understanding of helminth immunomodulation at the molecular level, enormous potential exists for helminth-derived products for clinical use.

59ES-62 was one of the original immunomodulatory molecules identified from parasites and with its activity through the small molecule phosphorylcholine (PC) moiety, it has great potential for therapeutic drug-like molecules. Two small molecule analogs (SMAs) of PC, termed 11a and 12b, can replicate many of the suppressive effects of ES-62, and due to their small size, stability, and lack of immunogenicity and efficacy are now being developed as therapeutic agents. Similarly, many of the recently identified immunomodulatory proteins are likely to be pursued as potential anti-inflammatory therapeutics for clinical use. These proteins have evolved to function in the mammalian milieu and represent natural biologicals that may offer unique mechanisms of action not available with existing drugs; in this context, the coming years of translational research promise to be very engaging.

60We summarize here a remarkable panoply of helminth molecules which, when tested in isolation in vitro or in vivo, target distinct host populations and pathways. Little is yet known, however, of how products from parasites in specific niches (such as the intestinal lumen) are able engage the broader host immune system, whether they exploit uptake mechanisms for systemic dissemination or rely on localized effects in their preferred site. Certainly, in species such as H. polygyrus, secretion products differ markedly between immature tissue-dwelling larvae and the intestinal lumen-dwelling adults, arguing for developmental adaptability for interaction with the immune system. In other cases, blood-dwelling parasites such as schistosomes or lymphatic-dwelling filarial worms have more direct access to immune cells and may more readily exert a systemic effect.

61Although parasite immunomodulators may act potently in laboratory tests, one obstacle to clinical development has been potential immunogenicity as foreign proteins in vivo. However, immunogenicity of helminth immunomodulators would represent an adverse outcome for the parasite, and thus mechanisms may be in place (including the innately immunosuppressive nature of these molecules) for avoidance of this: the fully foreign hookworm product AIP-2 is minimally immunogenic in vivo.

62There are also good precedents for the development of pathogen-derived proteins that have been successfully developed for human use, despite concerns of immunogenicity. Streptokinase (a Streptococcus-derived thrombolytic) has been used for decades in thrombotic disease such as myocardial infarction. Although highly immunogenic, as long as the drug is not repeatedly administered, it is effective and its immunogenicity does not present a barrier to use. Likewise, the myxoma viral protein Serp1 is an anti-inflammatory inhibitor of serine proteases; administration of Serp1 causes a non-neutralizing antibody response and subsequently the functional activity of the protein has been extended (and potential immunogenicity decreased) through development of small peptide mimetics that replicate the function of the full protein. Therefore, formulations of helminth immunomodulators are being developed in which immunogenicity is not present, or is not a barrier to use, for future therapeutic applications.

63Helminth immunomodulation is coming of age. Through advances in -omics analysis of parasitic helminths, ever more information about helminth products is becoming available. However, only by careful analysis of defined recombinant products can the potential of helminth therapeutic agents be realized. In recent years this has led to the characterization of a handful of helminth immunomodulators that could be developed for use in the clinic. Considering the size and complexity of helminth secretions, the complex interaction with their host, and the intricacy of the immune response directed against them, it appears almost certain that we have only scratched the surface of the full gamut of helminth immunomodulators, and that many more will be identified in the next decade.