Mining Helminths for Novel Therapeutics

Abstract

Helminths are an emerging source of therapeutics for dysregulated inflammatory diseases. Excretory/secretory (ES) molecules, released during infection, are responsible for many of these immunomodulatory effects and are likely to have evolved as a means for parasite survival in the host. While the mechanisms of action of these molecules have not been fully defined, evidence demonstrates that they target various pathways in the immune response, ranging from initiation to effector cell modulation. These molecules are applied in controlling specific effector mechanisms of type 1 and type 2 immune responses. Recently, studies have further focused on their therapeutic potential in specific disease models. Here we review recent findings on ES molecule modulation of immune functions, specifically highlighting their clinical implications for future use in inflammatory disease therapeutics.

Helminths and Their ES Molecules Can Modulate Inflammation

The mammalian immune system demonstrates remarkable flexibility in response to invading pathogens. Many rapidly propagating microbial pathogens, such as viruses and bacteria, trigger an acute type 1 immune response characterized by elevations in interferon gamma (IFNγ). This response is important for host protection that can contribute to the eradication of these pathogens before they disseminate systemically. While the type 1 response is generally short lived, if chronic or excessive it can result in hyperinflammation and severe tissue pathology associated with inflammatory disease states including diabetes and Crohn’s disease [1,2]. By contrast, large multicellular helminths (Box 1) elicit a host protective type 2 immune response, associated with elevations in IL-4, IL-5, and IL-13, that may control parasite burden and mitigate tissue damage [3]. However, a persistent type 2 response can also cause tissue damage resulting from allergic inflammation and fibrosis [3,4].

Box 1. Helminth Lifecycle: An Overview.

Helminth infections are one of the most common causes of chronic infections in the world, affecting as many as 2 billion people [105]. These infections are generally transmitted to the host through contaminated food ingestion or skin penetration, so they typically correlate with deficient sanitation and limited public health resources. The geographic distribution is predominately in warmer regions throughout sub-Saharan Africa, South America, India, and East Asia. These subtropical and tropical regions contribute to transmission by providing the optimal temperature for survival outside the host along with poorly developed hygiene and sanitation, which tends to promote host exposure. Soil-transmitted helminths (STHs) are the predominant human infective nematode species [105].

Helminth hookworms typically infect the host through skin penetration, eventually migrating through the pulmonary capillaries to enter the lung parenchyma. The larvae then access the larynx, through which they enter the digestive tract, and reach the small intestine [106]. In this organ, they develop into adults and produce eggs that are eventually released back to the soil through stool. Other species of worms, such as whipworms, are ingested as eggs on soil-contaminated food, allowing direct development in the small intestine. The clinical symptoms of helminth infections can vary in presentation from abdominal pain to anorexia, weight loss, and anemia. However, these infections are rarely fatal and many individuals even remain asymptomatic [106]. The host–helminth coevolutionary dynamic may have contributed to the development of helminth ES molecules that can regulate vertebrate immune function including associated harmful inflammation that can contribute to disease.

Recent studies have provided insights into how helminths trigger type 2 responses. As these large multicellular parasites migrate through host tissues, they can trigger cellular stress and damage. These stressed/damaged cells can release danger-associated molecular patterns (DAMPs) (see Glossary) that can activate the host immune response through binding specific receptors on immune cells and nonimmune cells, including pattern recognition receptors (PRRs). Several of these DAMPs, including ATP and trefoil factor 2 (TFF2), can trigger the expression of cytokine alarmins, including IL-33, IL-25, and thymic stromal lymphopoietin (TSLP), which then drive the downstream type 2 immune response that includes the activation of innate immune cells such as ILC2s, basophils, eosinophils, mast cells, macrophages, neutrophils, and dendritic cells (DCs), providing the stimuli required for the development of the adaptive immune response, including T and B cells [5].

Epidemiological studies observed an inverse geographic distribution of allergic inflammatory responses and parasitic infections, suggesting the ‘hygiene hypothesis’, where increased hygiene in more industrialized societies may lead to immune dysregulation [6,7]. Subsequent studies have shown that helminth-induced responses include regulatory components that can apparently control dysregulated immune responses contributing to harmful inflammation [8–11].

Based on these findings, helminth therapy has rapidly developed as a treatment option for severe chronic inflammatory diseases [12]. Recent studies suggest at least some beneficial effects of helminth therapy are mediated by their release of immunomodulatory ES products. These ES molecules comprise a heterogeneous mix of proteins, lipids, and enzymes, including peptidases, proteases, and protease inhibitors, as well as host protein mimics, that arise during all stages of the helminth lifecycle [13–17] (Table 1 and Box 1). Likely to have evolved as a consequence of host/parasite coevolution, ES molecules can modulate the immune system, influencing the helminth-induced type 2 immune response by promoting and downregulating specific components, and modulating type 1 and type 17 responses. In this review, we focus on the immunoregulatory mechanisms and the therapeutic potential of recently identified ES molecules.

Table 1.

The Heterogeneous Mix of ES Molecules Comprises Proteins, Lipids, and Enzymes That Exert Various Immunoregulatory Functions in Different Inflammatory Disease Models

Molecule name	Characteristic	Source helminth	Model tested	Function	Mechanism	Refs
HES	ES molecules	Heligmosomoides polygyrus	In vitro analysis in murine intestinal DCs	Regulatory DC development	Promotes a regulatory DC phenotype through downregulation of the Syk signaling pathway	[68,95]
				Treg activation	Increased Treg differentiation and IL-10 production through Smad7 reduction
			In vivo murine OVA-induced allergy	Innate immune response modulation	Suppresses ILC2 recruitment
HpARI	Alarmin release inhibitor	H. polygyrus	In vivo Alternaria OVA-induced allergic murine model	Alarmin suppression	Prevents IL-33 release	[22]
HpBARI	Binds alarmin receptor and inhibits	H. polygyrus	In vivo Alternaria OVA-induced allergic murine model	Alarmin suppression	IL-33R/ST2 antagonist	[21]
EV (HES)	EV	H. polygyrus	In vitro co-culture with RAW264.7 and BMDMs EV immunization in an in vivo murine H. polygyrus infection	Alarmin suppression	Reduces the number of accessible IL-33 receptors	[24]
				Macrophage suppression	Suppressed M2 activation and cytokine production
TGM	TGF-β mimic	H. polygyrus	In vitro TGF-β bioassay in MFB-F11 fibroblast cell line In vitro murine CD4+ T cells and Treg cell culture In vivo murine fully allogenic skin transplant model	Treg activation	Increases FoxP3 expression and Treg differentiation	[15,49]
			In vitro human fibroblast cell culture	Tissue repair and fibrotic regulation	Reduces αSMA expression in fibroblasts
Apyrase enzymes	Enzymes	H. polygyrus	in vivo murine H. polygyrus challenge infection immunization model	DAMP activity suppression	Degrades exogenous proinflammatory ATP and ADP	[20]
Whole worm	Whole-worm infection	H. polygyrus	In vivo murine CD4+ T cell adoptive transfer-induced colitis model in Rag mice	Adaptive immune response	Increases Treg development and TGF-β production through Smad7 suppression	[95]
Hpb GDH	GDH	H. polygyrus	In vivo murine HDM-induced allergic airway model	Innate immune response modulation	Suppressed type 2 inflammation through activation of regulatory DCs through PGE2 production	[78]
HpCRT	Calreticulin	H. polygyrus	In vitro competition assay in bone marrow-derived DCs In vitro stimulation of CD4+ T cells isolated from H. polygyrus-infected mice	Innate immune response modulation	Induces a Th2 response through binding SR-A on DCs	[59]
Nb-DNase II	Deoxyribosnuclease	Nippostrongylus brasiliensis	In vitro analysis in human neutrophils In vivo murine intraperitoneal treatment	Innate immune response modulation	Neutralized NET formation	[52]
SmATPDase1	ATP diphosphohydrolase enzyme	Schistosoma mansoni tegument	In vitro schistosome model	DAMP activity suppression	Degrades exogenous proinflammatory ATP and ADP	[19]
ω-1	Glycosylated T2 RNase	S. mansoni	In vivo murine high-fat-fed chronic type 1 inflammatory obesity model	DAMP and alarmin activation	Increases IL-33 release and ILC2 recruitment	[25–27,71]
			In vitro analysis in human moDCs and naïve CD4+ T cells In vivo murine Th2 restimulation model	Cytokine production	Suppresses IL-12 production and establishes a Th2 response
				Th2 response activation	Promotes a regulatory DC phenotype through MR
Sm29	Tegument-bound antigen	S. mansoni	In vitro analysis in hPBMCs obtained from asthma patients	Humoral response modulation	Reduces IgE production	[92,93]
				Innate immune response modulation	Reduces IL-5 production limiting eosinophilic accumulation
				Treg activation	Increases CD4+CD25+ Tregs and lymphocyte co-stimulatory markers (CTLA-4 and PD-1)
					Enhances IL-10 production
				Fibrotic regulation	Suppression of TGF-β production
IPSE/alpha-1	Glycoprotein	S. mansoni	In vivo murine intraperitoneal treatment In vitro CD19+ splenic B cell antigen-binding assay	Humoral response modulation	Induces B regulatory cell differentiation and IL-10 production	[71,94]
SmSEA	Soluble egg antigens	S. mansoni	In vitro-derived human moDC cell culture	Innate immune response modulation	Induces a Th2 response by activation of regulatory DCs through PGE2 production	[71]
			In vivo murine CD4+ T cell adoptive transfer-induced colitis model in SCID mice	Adaptive immune response modulation	Enhances Th2 response in the colon
SmKl-1	Kunitz-type serine protease inhibitor	S. mansoni	In vitro murine neutrophil elastase release and inhibition assay In vivo murine MSU-induced gout arthritis model	Innate response modulation	Inhibits neutrophil migration and elastase activity	[51]
			In vivo murine acetaminophen treatment model	Enhances tissue repair response	Reversed liver tissue damage
LPC	Lipid	S. mansoni	In vitro murine peritoneal and bone marrow macrophages	Innate response modulation	Mimics oxidized low-density lipoprotein (oxLDL) to enhance M2 differentiation through the PPARγ pathway	[14]
Schistosomal EV	EV	S. mansoni	In vitro analysis in CD4+ T cells	Adaptive response modulation	Inhibits the development of Th2 class CD4+ T cells	[79]
SmP40	Protein	S. mansoni	In vivo murine OVA-induced chronic asthma model	Innate and adaptive response modulation	Contains T cell epitopes that enhance the Th1 response	[80]
T2 RNase glycoprotein	T2 RNase glycoprotein	Schistosoma japonicum	In vitro human hepatic stellate LX-2 cell line	Tissue repair and fibrotic regulation	Downregulates TGF-β	[44]
					Reduces αSMA expression
rSjP40	Recombinant purified protein from egg products	S. japonicum	In vivo murine OVA-induced chronic asthma model	Innate and adaptive response modulation	Contain T cell epitopes that enhance the Th1 response	[45,80]
			In vitro analysis in human hepatic stellate LX-2 cells	Tissue repair and fibrotic regulation	Downregulates TGF-β1 production through the ERK signaling pathway
SjSEA	Soluble egg antigens	S. japonicum	In vivo murine Leprdb/db type 2 diabetes model	Innate response modulation	Induces Th2 response through APC modulation	[46]
				Treg activation	Increases Treg differentiation
				Tissue repair and fibrotic regulation	Enhances TGF-β production
SjCP1412	RNase T2 family protein	S. japonicum	In vitro murine BMDC and RAW264.7 macrophages cultures In vivo murine immunization treatment with cercariae	Innate response modulation	Promotes M2 and tolerogenic DC phenotype	[64]
				Treg and regulatory response activation	Enhances Treg differentiation and IL-10 and TGF-β production
rSj16	Recombinant protein	S. japonicum	In vivo murine DDS-induced colitis model	Innate response modulation	Inhibits the proinflammatory PPARα signaling pathway	[62]
				Treg activation	Enhances Treg differentiation
SjEVs	EV	S. japonicum	In vivo S. japonicum-infected mouse model In vitro murine RAW264.7 macrophage cell culture	Innate response modulation	Targets the PI3K/AKT pathway to induce a classical proinflammatory macrophage	[66,67]
SJMHE1	HSP60-derived peptide	S. japonicum	In vivo murine OVA-induced DTH	Adaptive response modulation	Contains T cell epitopes to induce Treg and Th1 differentiation	[83,84]
			In vivo murine OVA-induced chronic asthma model	Innate response modulation	Modifies macrophage and DC cytokine production
SjHSP60	Heat shock protein	S. japonicum	In vivo S. japonicum-infected mice	Treg activation	Activates de novo Treg differentiation from naïve T cells and the expansion of pre-existing Tregs	[96]
Sj-Cys	Cystatin	S. japonicum	In vivo murine CLP-induced sepsis model	Treg and regulatory response activation	Enhanced IL-10 and TGF-β production	[101]
TsSPs	Soluble products	Trichuris suis	In vitro analysis in human monocyte-derived DCs	TLR signaling modulation	Downregulates TLR4 surface expression and MyD88 signaling through Rab7b expression	[29,60,61,65,77]
				Innate and adaptive response modulation	Contains active PGE2 to suppress proinflammatory activities
			In vitro human isolated monocyte cell culture	Regulatory MO development	Induces M2 differentiation
p43	Monomeric protein	T. suis	In vivo murine intranasal treatment	Innate response modulation	Binds to IL-13 suppressing the development of the IL-13-dependent immune response	[23]
ES-62	PC-containing glycoprotein	Acanthocheilonema viteae	In vitro murine bmDC cell culture	Innate response modulation	Suppresses MyD88 activity and downstream MAPK activation by autosomal degradation of TLR	[29,32–36,97]
			In vivo murine OVA-induced chronic asthma model	Innate immune and humoral response modulation	Suppresses MyD88 signaling in mast cells
					Suppresses IL-1β production through inflammasome regulation
					Enhances regulatory B cell production
					Suppresses neutrophil migration
SMAs (11a and 12b)	SMAs containing the PC moiety of ES-62	A. viteae	In vivo murine OVA-induced chronic asthma model	Innate immune and humoral response modulation	Suppresses MyD88 signaling in mast cells	[35,36]
					Suppresses IL-1β production through inflammasome regulation
					Enhances regulatory B cell production
					Suppresses neutrophil migration
FhTLM	TGF-β homolog	Fasciola hepatica	In vitro culture with F. hepatica unembryonated eggs and newly excysted juveniles	Control of parasite development	Led to enhanced F. hepatica survival and motility	[47]
FhTeg	Tegumental coat protein	F. hepatica	In vitro murine BMDC cell culture	Innate immune response modulation	Upregulates the anti-inflammatory SOCS3	[38]
					Suppresses MyD88 and downstream MAPK activation in DCs
Fhmuc	A mucin-derived synthetic peptide	F. hepatica	In vitro murine PBMCs and splenic DCs In vivo murine LPS-induced septic shock model	Innate and adaptive response modulation	Enhances proinflammatory cytokine production through DC modulation	[75]
FhESP	ES products: all	F. hepatica	In vitro murine peritoneal macrophage culture	Innate response modulation	Upregulates M2 differentiation	[55]
Fh15	Fatty-acid-binding protein	F. hepatica	In vivo murine LPS-induced septic shock model	Innate response modulation	TLR4 antagonist	[54]
FhHDM-1	Cathelicidin-like peptide	F. hepatica	In vitro analysis in murine BMDMs In vivo murine HDM-induced allergic asthma model	Adaptive response modulation	Inhibits NLRP3 inflammasome activation	[85]
FHTE	F. hepatica total extract	F. hepatica	In vivo murine MOG-immunized autoimmune encephalomyelitis model	Adaptive response modulation	Inhibits the ability of γδ T cells and Th17 to respond to IL-1β and IL-23	[86]
ES L1	Larval stage 1 ES products	Trichinella spiralis	In vitro hPBMC-derived DC cell culture	Regulatory DC development	Promotes tolerogenic DC phenotype	[70]
EV (TS)	EV	T. spiralis	In vitro PBMC stimulation assay	Innate response modulation	Increased IL-10 and IL-6 production
EV (TS): 7C2C5Ag and rTsp53	EV	T. spiralis	In vitro rat bone marrow-derived DCs and rat-derived naïve T cells	Regulatory DC development	Promotes tolerogenic DC phenotype	[73,74]
				Adaptive response modulation	Promoted Th2 differentiation from naïve T cells
				Regulatory immune response activation	Enhances IL-4 and IL-10 production from T cells
T. spiralis ES molecules	ES molecules	T. spiralis	In vitro analysis in murine BMDMs	Regulatory MO development	Upregulates M2 differentiation	[63]
			In vivo murine DSS-induced colitis and murine OVA-induced chronic asthma model
Ts-CRT	Calreticulin	T. spiralis	In vitro human THP-1 cells induced into M2 macrophages	Innate response modulation	Binds to C1q preventing receptor binding	[57]
TsCystatin	Recombinant cystatins	T. spiralis	In vivo murine TNBS-induced colitis	Treg activation	Activates Treg differentiation	[100]
P28GST	Glutathione S-transferase	Saccharomyces cerevisiae	In vivo murine TNBS-induced colitis model	Innate response modulation	Increases eosinophil population	[53]
					Decreases the Th1/Th17 response
rBmALT-2	Filaria-abundant larval transcript 2	Brugia malayi	In vivo murine streptozotocin-induced type 1 diabetes	Innate and humoral response modulation	Suppressed type 1 cytokines (IFNγ, TNFα) and increased type 2 cytokines (IL-4, IL-5, IL-10)	[82]
					Enhanced IgG1 class-switching and increased IgE antigen
rBmCys	Recombinant cystatins	B. malayi	In vivo murine DSS-induced colitis model	Cytokine modulation	Downregulates IFNγ, TNFα, and IL-5, −6, and −17 expression	[98,99]
				Treg activation	Activates Treg differentiation and IL-10 production
				Humoral response regulation	Increases IgM+ B cells
rWbL2	Stage two larvae	Wuchereria bancrofti	In vivo murine streptozotocin-induced type 1 diabetes	Cytokine modulation	Suppressed type 1 cytokines (IFNγ, TNFα) and increased type 2 cytokines (IL-4, IL-5, IL-10)	[82]
				Humoral response regulation	Enhanced IgG1 class switching and increased IgE Ag
Ac-AIP-2	Anti-inflammatory protein-2	Ancylostoma caninum	In vivo murine OVA-induced airway inflammation model	Regulatory DC development	Promotes tolerogenic DC phenotype	[72]
				Treg activation	Enhanced differentiation of Tregs

ES Molecules Target Alarmin Production and Function

Functioning as both a DAMP and a cytokine alarmin, IL-33 can be essential in driving type 2 immune responses. Various ES molecules target IL-33 activity. ATP can activate IL-33 release by epithelial cells [18]. Apyrases catalyze the hydrolysis of ATP to its noninflammatory forms [19]. Helminth ES products have now been identified with apyrase activity. These include the SmATPDase1 from Schistosoma mansoni [19] and five apyrases from Heligmosomoides polygyrus ES products [20]. Other H. polygyrus ES proteins can directly affect IL-33. These include alarmin release inhibitor (HpARI), which sequesters IL-33 in the necrotic cells, and HpBARI (binds alarmin receptor and inhibits), which binds to the IL-33/ST2 receptor rather than to the cytokine itself [21,22] (Figure 1). Similar to the activity of HpARI and, it is likely, other recently identified homologs, the p43 protein of Trichuris suis binds to the downstream cytokine IL-13, inhibiting further initiation of type 2 immunity [23]. Additionally, recently identified H. polygyrus extracellular vesicles (EVs) contain biologically active ES molecules that inhibit IL-33R surface expression by suppressing the mRNA levels of il1rl1, the IL-33 receptor subunit ST2 gene. This receptor downregulation reduces the number of accessible binding sites for IL-33 [24] (Figure 2).

Figure 1. Excretory/Secretory (ES) Molecules Suppress Exaggerated Inflammation in an Overactive Type 2 Immune Disease State.

Allergies, as well as asthma, result from a dysregulated type 2 immune response when macromolecules, such as allergens, are introduced into the host’s system. This aberrant immune response occurs when there is an excessive response or it transitions to uncontrolled inflammation. IgE production, along with IL-4 and IL-13 expression, recruits and activates mast cells, which can lead to rapid degranulation and produce resultant hypersensitivity if not controlled. ES molecules have various mechanisms of action that demonstrate the ability to control this overactive response. Some molecules suppress the initiation of the inflammatory response by reducing alarmin activity, such as IL-33. Other molecules exert their effects on innate cells to restrict their ability to exacerbate the type 2 response. However, the predominant role for many ES molecules in this disease model is the polarization of naïve T cells to the T regulatory cell (Treg) subtype through which regulatory cytokines [IL-10, transforming growth factor beta (TGF-β)] are produced. Tregs and the associated cytokines suppress leukotriene and inflammatory cytokine production and the fibrotic response that is associated with allergic damage to the airway epithelium. Black lines indicate typical cellular pathways. Red lines highlight the regulatory mechanisms generated by ES molecules. Small green circles represent individual ES molecules, small gray circles represent alarmins, and small red circles represent TGF-β.

Figure 2. Excretory/Secretory (ES) Molecules Modulate Receptor Activation and Downstream Signaling by Myeloid Cells.

Many functions associated with helminth ES molecules target the receptors and downstream mechanisms associated with myeloid cells. Myeloid cells play a central role in directing naïve T cell differentiation and regulating the immune response to the invading pathogen. The MyD88-dependent signaling pathway is shared by these myeloid cells; therefore, this common pathway is targeted by many ES molecules. Additionally, other receptor activity, such as through Dectin-1 and mannose receptors, is altered through ES receptor binding or downstream signaling manipulation resulting in tolerogenic dendritic cell differentiation and monocyte polarization toward a M2 cell subtype. Mast cells are also involved in the exacerbation of certain disease states, which makes them another target for ES molecule regulation. In mast cells, crosstalk between multiple pattern recognition receptors (PRRs) inhibits the downstream activation of degranulation. Black lines indicate typical cellular pathways. Red lines highlight the regulatory mechanisms generated by ES molecules. Small green circles represent individual ES molecules and small red circles represent lipopolysaccharide (LPS). Abbreviations: H. polygyrus, Heligmosomoides polygyrus; IFNγ, interferon gamma; PPARα, peroxisome proliferator-activated receptor alpha; T. spiralis ES, Trichinella spiralis ES; TGF-β, transforming growth factor beta; TNFα, tumor necrosis alpha.

By contrast, other ES molecules are involved in increasing the expression of type 2-inducing alarmins. The robust type 2-inducing S. mansoni secretes a glycosylated T2 RNase, Omega 1 (ω-1), that can facilitate the resolution of type 1 inflammatory disease states. Originally identified as a potent regulator of DC differentiation following internalization via the mannose receptor (MR), recent studies have uncovered further ω-1 activity at earlier initiation stages of the type 2 immune response [25,26]. In particular, ω-1 increases IL-33 production as demonstrated in a murine obesity model, where it promoted type 2 immunity, which modulated type 1 inflammation promoting metabolic homeostasis [25–27] (Figure 2).

ES Molecules Regulate Toll-Like Receptor (TLR) and C-Type Lectin Receptor (CLR) Signaling

Conserved microbial structures recognized by PRRs are referred to as pathogen-associated molecular patterns (PAMPs), and ES molecules have recently been identified that modify the associated signaling pathways. One characteristic group of PRRs involved in microbial pathogen recognition include the TLRs which, with the exception of TLR3, share the downstream myeloid differentiation primary response gene 88 (MyD88)-dependent signaling pathways. Signaling through MyD88 activates mitogen-activated protein kinases (MAPKs). This, in turn, initiates the translocation of nuclear factor kappa B (NF-κB) to the nucleus, upregulating proinflammatory type 1 cytokines [28]. Several ES molecules target MyD88-dependent signaling. TLR4 activation is generally triggered by bacterial structures; however, they are also involved in dysregulated immune responses observed in autoimmune diseases, where they may be stimulated by DAMPs [29–31]. The immunosuppressive activity of live T. suis infections has been previously studied in murine models of inflammatory diseases and current research is exploring the specific effects of its ES products [29]. T. suis soluble products (TsSPs) activate Rab7b, which can increase the degradation of cell surface TLR4 expression, suppressing the type 1 immune response [29] (Figure 2). The phosphorylcholine (PC)-containing glycoprotein from Acanthocheilonema viteae (ES-62), by contrast, downregulates the TLR4/MyD88 signaling pathway by inhibiting MyD88 recruitment and dimerization [29,32,33] (Figure 2). Further studies have demonstrated a possible role for ES-62 in activating autophagolysosomal degradation of TLR4-associated proteins [34]. As an immunogenic molecule, however, ES-62 is considered unsuitable for therapeutic use. Therefore, small molecule analogs (SMAs) have been generated based on the PC moiety, the active component of the ES-62 molecule. Two specific SMAs (11a and 12b) have now been shown to have immunomodulatory properties similar to those of ES-62 [35]. Furthermore, the ES-62 SMA 12b has also been shown to suppress inflammasome activation, a function not observed with ES-62 itself. The effects on both the TLR4 pathway and inflammasome activity inhibited IL-1β production, which has demonstrated the therapeutic potential of ES-62 and its SMAs in murine arthritis and asthma models [36]. Recently, the impact of ES-62, as well as other ES molecules, on the host microbiome has been investigated. The regulatory mechanisms induced by ES-62 normalized the gut microbiota and ameliorated rheumatoid arthritis in a mouse model [37]. Tegumental coat antigens from Fasciola hepatica (FhTeg) also reduce the downstream activation of MAPK in DCs. FhTeg is believed to suppress TLR4 signaling indirectly through the activation of the suppressor of cytokine signaling 3 (SOCS3) in mast cells. These antigens are involved in regulating the Th1 inflammatory response to TLR activation [38] (Figure 2). However, it is important to note that many recombinant ES proteins are produced through expression in bacteria and therefore residual bacterial lipopolysaccharide (LPS) could confound the interpretation and results of these studies.

ES Molecules Modulate Regulatory Cytokine Production

Various inflammatory disease states have been associated with reduced expression of regulatory cytokines, including IL-10 and transforming growth factor beta (TGF-β) [39–41]. IL-10 is one of the key anti-inflammatory cytokines expressed by various immune cells during both type 1 and type 2 immune responses [17,39]. Interestingly, many ES molecules can modulate IL-10 production. These various mechanisms are discussed in subsequent sections. TGF-β production is also targeted by various ES molecules. Enhanced TGF-β expression can be beneficial or detrimental, including the promotion of fibrosis, depending on the context of the response [42,43]. ES molecules have been shown to regulate TGF-β production and activity, reducing fibrotic accumulation and other associated pathologies. The recombinant T2 RNase glycoprotein, expressed in Schistosoma japonicum adult worms, suppresses TGF-β signaling specifically through the downregulation of Smad4. This downstream regulation resulted in suppression of the cytokine αSMA in human hepatic stellate cells [44]. Alpha-smooth muscle actin (αSMA), expressed by activated profibrogenic myofibroblasts, is a prototypical marker for increased fibrosis. Similar αSMA reduction was also observed with another S. japonicum product, rSjP40, a purified protein from the heterogeneous schistosome egg products. This protein enhances bone morphogenetic protein 7 (BMP-7) expression, which impedes downstream TGF-β receptor (TGF-βR) signaling, thereby reducing the production of profibrotic αSMA in human hepatic stellate cells [45] (Figure 3). Another S. japonicum product, soluble egg antigen (SjSEA), induced T regulatory cells (Tregs), which increased TGF-β production. This cytokine production was involved in resolving wounds associated with diabetes in an experimental type 2 diabetes mouse model with a mutation in the leptin receptor (Lepr^db/db) [46]. Recent research has identified multiple ES molecules that mimic the highly conserved cytokine TGF-β and its activities. TGF-β homologs have been found in many helminth species, including the F. hepatica FhTLM [47,48] (Figure 3). The H. polygyrus-secreted TGF-β mimic (TGM) initially stimulates fibroblast αSMA expression to a lesser degree than endogenous TGF-β. This delay in αSMA production may delay onset of fibrosis thereby providing more time for initial wound repair and suggesting potential therapeutic benefit [15] (Figure 3). TGM is structurally distinct from endogenous TGF-β; however, like TGF-β it binds to TGF-βRII, resulting in the recruitment of TGF-βRI and the formation of a heterodimer required for TGF-β signaling. Unlike TGF-β, TGM can bind with high affinity to TGF-βRI even when not associated with TGF-βRII. This unique feature of TGM may explain its greater ability than TGF-β to enhance FoxP3 expression and Treg differentiation, thereby potentially mediating enhanced regulatory and immune suppressive activity [15,49,50].

Figure 3. Excretory/Secretory (ES) Molecules Limit Tissue Damage That Results during a Type 2 Immune Response.

Many cells and cytokines involved in the modified type 2 immune response also participate in wound healing. Tissue damage during a helminth infection is induced as the worms migrate through the host, while chronic asthma pathogenesis can result in permanent fibrotic airway remodeling through the upregulation of transforming growth factor beta (TGF-β). TGF-β enhances scar formation by activating fibroblast differentiation to myofibroblasts through the induction of alpha-smooth muscle actin (αSMA). Some ES molecule activity reduces fibrotic accumulation primarily by controlling TGF-β production and the downstream pathway of the TGF-β receptor (TGF-βR), as observed with rSjp40. rSjp40 reduces liver fibrosis by binding to the bone morphogenetic protein 7 (BMP-7) receptor, which diverts the TGF-βR signaling pathway away from TGF-β production. However, the novel TGF-β mimic (TGM) enhances T regulatory cell (Treg) production while suppressing αSMA expression. Black lines indicate typical cellular pathways. Red lines highlight the regulatory mechanisms generated by ES molecules. Small green circles represent individual ES molecules and small red circles represent TGF-β. Abbreviations: IFNγ, interferon gamma; S. japonicum T2 RNase, Schistosoma japonicum T2 RNase; TNFα, tumor necrosis alpha; VEGF, vascular endothelial growth factor.

ES Molecules Regulate Myeloid Cell Functions

Granulocytes

ES molecules can act directly on innate effector cells at the site of infection. Neutrophils are one of the earliest cell types recruited, where they phagocytose invading organisms, release neutrophil extracellular traps (NETs), and secrete antimicrobials and enzymes, including serine proteases. The S. mansoni serine protease inhibitor (SmKl-1) was shown to inhibit neutrophil activity and migration by binding to neutrophil elastase. This inhibition lowered the pathological score in gout arthritis and reduced acute liver damage in an acetaminophen mouse model [51] (Figures 3 and 4). A recent study showed that Nippostrongylus brasiliensis DNase II degrades neutrophil NET formation as a means to evade parasite damage and host immune recognition, further indicating the diverse functions of helminth ES molecules [52]. Eosinophils are another early recruit during infection, which can release cytokines, like IL-4, to promote type 2 immunity. Eosinophil accumulation can lead to asthma; however, it can also release TGF-β and other immunoregulatory cytokines that control type 1 inflammation. This was observed with schistosome glutathione S-transferase (P28GST) treatment in a colitis model that promoted eosinophil recruitment and decreased inflammation [53] (Figure 4). Limiting the recruitment and activity of innate effector granulocytes is a mechanism through which ES molecules can modulate immunity at an early stage of the response.

Figure 4. Excretory/Secretory (ES) Molecules Regulate the Inflammatory Response Associated with Type 1-Associated Diseases.

Inflammatory bowel disease (IBD)/Crohn’s disease is a chronic type 1 inflammatory disease state that develops when the gastrointestinal homeostasis is disrupted and commensal bacteria of the microbiota activate the immune response. ES molecules have various mechanisms of action that demonstrate the ability to suppress this overactive immune response. Th1 and Th17 are elevated in this disease state; therefore, many of these molecules direct the polarization of naïve T cells to a Th2 and T regulatory cell (Treg) subtype. Some ES molecules generate this polarization by influencing the downstream signaling pathways of naïve T cells. For example, components of Heligmosomoides polygyrus activate the transforming growth factor beta (TGF-β) pathway and Treg development through suppression of the pathway inhibitor Smad7. Tregs result in the reduction of intestinal inflammation and disease progression. The M2 macrophage is another critical player in immunosuppression through the production of IL-10. Many ES molecules are involved in promoting M2 differentiation from naïve monocytes. Black lines indicate typical cellular pathways. Red lines highlight the regulatory mechanisms generated by ES molecules. Small green circles represent individual ES molecules, small green line represents the whole helminth, and small red circles represent TGF-β. Abbreviations: H. polygyrus EV, Heligmosomoides polygyrus EV; IFNγ, interferon gamma; LPC, lysophosphatidylcholine; NF-κB, nuclear factor kappa B; PPARγ, peroxisome proliferator-activated receptor gamma; PRR, pattern recognition receptor; TNFα, tumor necrosis alpha.

Macrophages

Recruitment and activation of macrophage subsets is crucial for the development and maintenance of various immune responses in different disease states. Macrophage subsets and alternative activation states are characterized by distinct phenotypes and cytokine production. Several ES molecules interact with and regulate macrophage surface receptors to modulate macrophage differentiation. F. hepatica ES products (FhESP) activate macrophages through the PRR CLR Dectin-1, resulting in MAPK (ERK1/2) activation and downstream IL-10 production. By contrast, the purified F. hepatica fatty-acid-binding protein (Fh15), acts as a TLR4 antagonist to suppress M1 macrophage polarization [54,55] (Figure 2). Various other protein/receptor interactions initiate M2 macrophage differentiation including complement component 1q (C1q), a member of the complement system [56]. C1q activates M2 differentiation through both the C1q complement receptor and the atypical surface receptor myosin 18A on monocyte and macrophage surfaces [56]. Trichinella spiralis calreticulin (Ts-CRT), binds to C1q, preventing receptor binding and inhibiting C1q-dependent activities including M2 macrophage differentiation. This mechanism of action is not unique to this helminth as this molecule shares similar functions with both endogenous vertebrate host CRTs and CRTs from other helminths, including Brugia malayi [57]. Calreticulin is a conserved calcium-binding protein involved in the regulation of immune functions and maintenance of cellular homeostasis [58]. As with the endogenous murine CRT, a H. polygyrus-derived CRT (HpCRT) binds to scavenger receptor A (SR-A) on murine DCs. The resulting ES uptake through this receptor binding may modulate the activity of these cells, potentially contributing to the development of a type 2 immune response [59].

Following receptor activation, other ES molecules target downstream signaling to either enhance or redirect macrophage differentiation. T. suis TsSPs can bind to the CLR mannose surface receptor CD206, while other components inhibit the downstream MAPK signaling of TLR4 and TLR9 in macrophages, suppressing M1 macrophage activation [60,61]. Downstream proinflammatory pathways are also targeted by ES molecules such as the S. japonicum protein rSj16, which inhibits the peroxisome proliferator-activated receptor alpha (PPARα) signaling pathway in macrophages. PPARα activation has been associated with colitis development, and treatment with rSj16 decreased inflammation in a dextran sulfate sodium (DSS) murine colitis model [62] (Figure 4).

Other ES molecules act on specific pathways to directly promote M2 differentiation. The S. japonicum RNase T2 family protein SjCP1412 and T. spiralis heterogeneous ES preparations increase M2 markers, the latter through upregulation of the STAT3 pathway [63,64] (Figure 2). This M2 activation was beneficial in ameliorating a type 1 murine colitis model as well as a type 2 murine allergy model [63] (Figure 4). Meanwhile, the schistosome lipid lysophosphatidylcholine (LPC) upregulated M2 markers through the alternative peroxisome proliferator-activated receptor gamma (PPARγ)-dependent signaling pathway [14].

In addition to immediate effects on macrophage differentiation, additional studies have demonstrated that ES molecules can regulate macrophage differentiation at the epigenetic level, potentially supporting long-term immune regulation. TsSP induced epigenetic changes through histone acetylation at the TNF and IL6 promoter sites in monocytes, which inhibited classical (M1) macrophage activation despite subsequent IFNγ and LPS stimulation [65]. Gene expression regulation was also observed with S. japonicum EVs (SjEVs) that release miRNA cargo into macrophages. In contrast to TsSP, however, the miRNA cargo, primarily miR-148, promotes a type 1 inflammatory state by targeting PTEN in the PI3K/AKT pathway [66,67] (Figure 2). ES molecules that target macrophage activation and associated signaling pathways offer a compelling area of investigation in the expanding search for therapeutic treatments.

ES Molecules Increase Tolerogenic DCs

DCs are a key link between the innate and adaptive immune response, playing a critical role in antigen presentation and T cell priming [68]. As with macrophages that undergo differential activation, DCs also exhibit different activation states depending on the immune response and tissue microenvironment.

The regulatory, or tolerogenic, subset of DCs, often referred to as semi-matured DCs, are activated by IL-10 and TGF-β and are characterized by low to intermediate expression of co-stimulatory markers (MHCII, CD80, and CD86) and increased production of IL-10. The reduced levels of co-stimulatory molecules dampen T cell proliferation and effector responses and instead promote a regulatory environment through Treg expansion and regulatory cytokine (IL-10 and TGF-β) production [69,70]. ES molecules influence intracellular pathways to initiate regulatory DC differentiation. The previously described enzymatic activity of the T2 RNases ω-1 and SjCP1412 also downregulates DC co-stimulatory surface molecules, impeding DC interaction with naïve T cells [64,71] (Figure 2). The reduction of DC co-stimulatory molecule expression was also observed in a murine asthma model treated with anti-inflammatory protein-2 (Ac-AIP-2), a recombinant protein secreted from the hookworm Ancylostoma caninum [72]. A similar response is observed with T. spiralis EVs, which introduce three glycoproteins, collectively termed 7C2C5Ag, into the host DCs. Recombinant protein 53 (rTsp53) is one of the components of the 7C2C5Ag complex. Both unseparated 7C2C5Ag and purified rTsp53 independently weakly activate MAPK (ERK1/2) and p38, which induces the upregulation of IL-10 production and an associated tolerogenic DC phenotype [73,74] (Figure 2). Other ES molecules target DC surface receptors to either activate or inhibit downstream activity. Heterogeneous ES preparations from T. spiralis L1 interact with TLR2 and TLR4 to induce tolerogenic properties of DCs [70] (Figure 2). However, other ES molecules utilize TLR manipulation to promote the type 1 response. Contrary to the TLR4 suppressive activities of FhTeg, another F. hepatica molecule, Fhmuc (a mucin-derived synthetic peptide), synergizes with and amplifies LPS/TLR4 signaling in DCs. This response enhances the expression of DC co-stimulatory molecules and proinflammatory cytokine production, promoting type 1 immunity [75] (Figure 2).

ES Molecules Promote DC Leukotriene Release

ES molecules target various signaling pathways modulating DC activation. The CLR activates DCs through the intracellular Syk pathway, which is targeted by heterogeneous H. polygyrus ES preparations (HES). HES downregulates Syk expression, which can promote the development of regulatory/tolerogenic DCs [68] (Figure 4). Other ES molecules, instead, utilize Syk downstream signaling to promote a type 2 response through the production of local leukotrienes. The leukotriene prostaglandin E2 (PGE2) can promote type 2 inflammation. The SmSEA glycoproteins bind the CLRs to promote PGE2 production by DCs through Syk-dependent mechanisms, while T. suis secretes active PGE2 as a main component of its soluble products (TsSPs) [76,77] (Figure 2). This PGE2 production initiates regulatory DC polarization via autocrine signaling by OX40L [76,77] (Figure 2). However, PGE2 production has also been reported to initiate a type 1 response in other diseases models. This contrasting activity may result from differing concentrations of PGE2 and the specific context of its activity [77–80]. For example, the more recently identified H. polygyrus glutamate dehydrogenase (GDH) acts as a potent PGE2 producer in macrophages resulting in reduced airway inflammation in murine allergy models [81]. By targeting signaling pathways mediating DC activation and function, these helminth-derived products can thus exert control over both the innate and the adaptive immune response.

ES Molecules Influence the Adaptive B and T Cell Response

Several ES molecules modulate the CD4⁺ T cell transcriptional programming of naïve T cells to influence the development of T cell subtypes. The adult S. mansoni-secreted EV includes components that target NF-κB activity to downregulate Gata3 transcription in Th2 cells [82]. Furthermore, the S. japonicum protein SjP40, as well as its S. mansoni homolog SmP40, contains novel epitopes that can stimulate CD4⁺ T cells and elicit IFNy production in vitro and in an allergic asthma model in vivo [83] (Figure 1). The potent sea anemone-derived peptide Stichodactyla helianthus toxin (ShK) also suppresses the proliferation and function of CD4⁺ and CD8⁺ T effector memory (Tem) cells, without impacting naïve and central memory cells. ShK administration can reduce inflammation in both type 2 asthma models and type 1 disease responses including delayed-type hypersensitivity (DTH) [84]. Interestingly, ShK-related peptides and domains (ShKTs) have recently been identified in helminths, providing another likely avenue of helminth regulation of the host immune response [84].

Transcriptional modulation of CD4⁺ T helper cell activation by ES molecules was also observed in filarial abundant larval transcript 2 from B. malayi (rBmALT-2) and stage two larvae from Wuchereria bancrofti (rWbL2) [85]. In addition to modifying TLR activity in macrophages and DC cytokine production, the S. japonicum HSP60-derived peptide (SJMHE1) is recognized by CD4⁺ T cells and influences T cell differentiation in diseases triggered by both type 1 (DTH) and type 2 (asthma and arthritis) responses [86,87] (Figure 1). The cathelicidin-like peptide from F. hepatica, helminth defense molecule 1 (FhHDM-1), modulates inflammasome activity to influence CD4⁺ Th2 cell differentiation by reducing macrophage production of cathepsin B, which is required for NOD-leucine-rich repeat and pyrin-containing protein 3 (NLRP3) inflammasome activation. Suppression of this type 2 immune response was beneficial in decreasing airway inflammation in a murine asthma model [88] (Figure 2). Similar to FhHDM-1, F. hepatica total extract (FHTE) also attenuated autoimmune encephalomyelitis in a murine model by interfering with IL-1β and IL-23 activation of γδT and Th17 cells [88,89].

During type 2 immune responses, B cells undergo class-switching resulting in IgE and IgG production. IgE can bind to FcεRI on basophils and mast cells, which leads to their activation and degranulation [90,91]. Enhanced expression of allergen-specific IgE antibodies resulting from prior exposures to allergens can contribute to allergy and asthma [90,92]. A similar antibody response is observed during helminth infections, and ES molecules can regulate antibody production [93].

In some helminth infections, antibodies can further enhance the type 2 immune response by binding to B cell receptors. Infections with the foodborne parasite Clonorchis sinensis increased the expression of CD23, a low-affinity IgE receptor, along with enhanced IgE binding, resulting in activated B cells with increased antigen presentation to T cells [91]. Additionally, murine infections with H. polygyrus resulted in increased expression of CD23, but in contrast to the typical type 2 inflammation associated with CD23, this infection saw a reduction of airway inflammation [94]. However, in this study there was increased recruitment of regulatory cells, specifically T cells, which may have influenced the resulting response [94]. Other molecules, such as Sm29, reduce IgE production, suppressing the inflammatory response and reducing the quantity of inflammatory cells and ovalbumin (OVA)-specific IgE production in an allergy model [95,96] (Figure 2). In addition to antibody production, few studies have demonstrated ES activity directly on B cells. The S. mansoni glycoprotein IPSE/alpha-1 is one of the few molecules demonstrating direct modulation through internalization by B cells resulting in B regulatory cell differentiation and IL-10 production [71,97]. ES molecules can thus directly modify CD4⁺ T helper and B cell effector responses the modulation of adaptive immunity.

ES Molecules Activate Tregs

Tregs modulate immune responsiveness through antigen-nonspecific mechanisms and their suppression can promote inflammation and associated disease. Helminth infections have been shown to modulate the immune response through the stimulation of a complex network of regulatory cytokines (IL-10 and TGF-β) produced primarily by CD4⁺CD25⁺FoxP3⁺ Tregs [95]. For example, in a murine colitis model, H. polygyrus infection increased Treg development and IL-10 production through the suppression of a TGF-βR-negative regulator, Smad7 [98] (Figure 1). Most immunoregulatory ES molecules, including many of those previously discussed, have been associated with mechanisms of regulation that involve indirect or direct activation of Tregs and their associated regulatory cytokine production. This includes molecules such as TGM, which induced Foxp3 expression and Treg differentiation in human peripheral blood mono-nuclear cells (hPBMCs) [15]. Also, AIP-2 promoted significant expansion of Tregs via the generation of CD103⁺ DCs in the mesenteric lymph node (MLN), thereby inhibiting airway damage in a murine allergy model [72]. The S. japonicum heat shock protein SjHSP60 generates TGF-β from macrophages through TLR4-dependent mechanisms. This TGF-β production stimulates de novo Treg differentiation from naïve T cells and pre-existing Treg expansion [99]. Other molecules indirectly induce Tregs through innate cell activation, including the aforementioned ES-62, LNFPIII (another SEA product), and SjCP1412, the last of which activates DCs to upregulate Treg-inducing cytokines [64,100].

ES Molecules Contain Cystatins That Promote Treg Development

Cystatins are protease inhibitors that have a prominent role in suppressing immune-mediated disorders through T cell-mediated immunosuppression and proinflammatory cytokine reduction. Multiple helminth species have been identified that secrete cystatin-like molecules [101]. B. malayi produces a cystatin, rBmCys, that when administered in a murine colitis model suppressed the disease state through downregulation of proinflammatory cytokine production. In addition, rBmCys treatment increased the expression of IL-10-producing Treg cells and M2 cell activation [101,102] (Figure 4). Similar results were observed in an inflammatory bowel disease (IBD) murine model treated with T. spiralis cystatin (TsCystatin), which reduced IL-17, tumor necrosis factor alpha (TNFα), and IFNγ production while increasing IL-4 and IL-10 secretion by CD4⁺ Tregs [103] (Figure 4). A S. japonicum cystatin, Sj-Cys, is also likely to be involved in increased IL-10 and TGF-β production from Tregs; however, it is believed to act through the suppression of MyD88 in immune cells. Treatment with Sj-Cys demonstrated reductions in disease severity and associated organ damage in a murine sepsis model [104].

While these molecules influence various other pathways of the immune response, as discussed previously, their effects on Tregs and associated regulatory cytokine production (IL-10 and TGF-β) are a primary mechanism of controlling inflammation.

Concluding Remarks

There have been recent significant advances in our understanding of the myriad immune regulatory mechanisms employed by helminth-derived ES molecules. It is likely that these products were generated as a consequence of the long and dynamic vertebrate/helminth coevolutionary process, resulting in specific molecules that can have pronounced effects in modulating host immunity. Helminths therefore provide a rich resource for the mining of novel therapeutics, with newly identified molecules showing potential as treatments for autoimmune diseases and other inflammatory conditions. Recent research provides an important basis for novel studies addressing critical questions (see Outstanding Questions). One notable observation is the redundancy of molecule activity within the heterogeneous ES products and across helminth species. This redundancy provides a robust resource for response manipulation should initial treatments be ineffective or in cases where the host response recognizes and suppresses the activity of specific products [20]. For future translational applications, research is still needed to determine whether many of the murine parasite-derived ES molecules can generate similar responses in humans, although recent studies have indicated that this may be the case, at least with several ES molecules. Some therapeutic treatments are in Phase I clinical trials. However, early therapeutic work with whole ES products in the treatment of inflammatory diseases has been inconclusive. Standardization of unseparated ES products and whole helminths is challenging, making the identification of highly purified and potent ES-derived molecules an advantage in therapeutic applications. Further studies using isolated or potentially specific combinations of highly purified molecules to treat harmful inflammation are needed. Additionally, from a pharmacological perspective, individual ES molecules can be synthesized in large standardized quantities, another advantage over live-helminth treatments or unseparated ES products. Continued isolation of highly purified ES products and investigation into their function is a burgeoning area of future research with the potential to provide new and effective therapeutic treatments targeting harmful inflammation.

Outstanding Questions.

Why does the heterogeneous ES supernatant exert different and sometimes opposing effects compared with the responses observed with the application of isolated molecules from that supernatant?

The current mechanisms attributed to specific ES molecules are based on different disease models and the assays used by individual laboratories to characterize them. Therefore, to what extent are the apparently characteristic functions of individual ES molecules artifacts of the particular assays so far used to describe them?

Do ES molecules enhance parasite survival and reproduction in the host? Although it is speculated that production of these molecules is adaptive for the parasite, few studies have directly addressed this.

Would specific combination therapy including ES molecules with endogenous molecules and cytokines lead to enhanced clinical outcomes in these disease models?

Highlights.

Helminth infections can activate regulatory components of the immune response, which can mitigate harmful inflammation.

Helminth excretory/secretory (ES) molecules mediate many of the immunoregulatory effects of helminths.

ES molecules include various proteins, lipids, and host protein mimics that influence various signaling pathways regulating inflammatory responses.

Current research suggests that the immunomodulatory effects of these ES molecules have clinical implications for disease treatment.

Clinician’s Corner.

Current treatments for chronic inflammatory disease states have limited efficacy for many patients, especially related to failure to achieve full remission [107]. While the management of these disorders is in some cases effective, they can result in long-term adverse effects [108].

Experimental models of helminth infections have demonstrated immunoregulatory effects that may have potential as therapy for these inflammatory disease states.

Many of the early clinical trials in human autoimmune disease therapeutics were performed with the zoonotic whipworm Trichuris suis. However, these studies in inflammatory gastrointestinal disease and arthritis have shown inconclusive effects [109–113].

Necator americanus hookworms have shown promising therapeutic potential in human autoimmune celiac diseases. A randomized double-blinded placebo-controlled Phase Ib/IIa clinical trial with N. americanus larvae in celiac disease led to inconclusive results [114]. However, a follow-up study, currently in Phase II, has demonstrated that N. americanus infection in addition to a gluten challenge led to increased Treg production and a reduction of autoanti-bodies, implying a potential therapeutic treatment for immune regulation in celiac patients (ClinicalTrials.gov identifier: NCT01661933) [115].

Whole-worm treatments can generate undesirable side effects and are difficult to standardize. Purified ES molecules provide more-specific effects and are more readily generated as standardized products.

There are few studies focusing on the human application of purified ES molecules. One of the earlier Phase II clinical trials involved the application of a neutrophil inhibitory factor (NIF) in stroke patients [113,116]. Previous studies in in vivo rat middle cerebral artery occlusion models demonstrated that NIF administration reduced neutrophil infiltration following ischemic initiation that would contribute to inflammation. However, human trials in stroke patients did not provide significant recovery [116]. The rSh28GST antigen, from schistosomes, has already successfully completed Phase I immunogenicity clinical trials and a pilot Phase IIa study where safety was demonstrated and there was an observed reduction of both the disease activity value and calprotectin levels in patients with Crohn’s disease (ClinicalTrials.gov identifier: NCT02281916) [53,117,118].

Immunogenicity plays a critical role in the development of novel therapeutics. Foreign molecules, particularly proteins, can potentially induce immunity, which may neutralize therapeutic effectiveness. Studies have demonstrated that collections of ES molecules can activate a greater immune response, beyond that of the preferred targeted immune mechanism [72].

Isolated ES molecules have proved beneficial in disease resolution by acting on a specific mechanism without generating a broad immunogenic response [72]. Furthermore, recent efforts have been directed toward the synthesis of less immunogenic versions of various ES molecules. For example, the active PC moiety of ES-62 was used to generate SMAs that mimic the immunosuppressive activity [35]. Smaller peptide structures or smaller versions of active molecules reduce immunogenicity and are likely to be important future therapeutic applications.

Glossary

Alpha-smooth muscle actin (αSMA)

involved in fibroblast-to-myofibroblast differentiation in wound repair

Cathepsin B

a member of the lysosomal cysteine protease family. It is found in endosomes and lysosomes where it mediates intracellular proteolysis and contributes to autophagy and catabolism. It is also involved in NLRP3 inflammasome activation

Complement component 1q (C1q)

this protein is a component of the C1 complex, which on activation initiates the classical complement pathway

C-type lectin receptors (CLRs)

a class of PRRs found on many innate cells that recognize various pathogens including fungi. They are involved in the initiation of the innate immune response

Danger-associated molecular patterns (DAMPs)

a group of molecules released by stressed or damaged cells that can activate the innate immune response through recognition by PRRs

Dectin-1

a member of the CLR family that is expressed predominantly on myeloid cells and recognizes fungi. It activates downstream signaling pathways associated with the production of proinflammatory cytokines

Gata3

a member of the GATA family of transcription factors that generally promotes type 2 responses and may also be involved in Treg function and associated FoxP3 expression

Lipopolysaccharide (LPS)

the main endotoxin on the membrane of Gram-negative bacteria that functions as a PAMP and is recognized by PRRs, including TLR4

Myeloid differentiation primary response gene 88 (MyD88)

a protein associated with the downstream signaling of all TLRs except TLR3

Myosin 18A

an unconventional member of the myosin superfamily that functions as a cell surface receptor and mediates the innate and adaptive immune response

Neutrophil extracellular traps (NETs)

networks of DNA and globular proteins released by neutrophils that are used for defense against extracellular pathogens

NOD-leucine-rich repeat and pyrin-containing protein 3 (NLRP3) inflammasome

a multimeric protein complex that on activation triggers the release of the proinflammatory cytokines IL-1β and IL-18

Pathogen-associated molecular patterns (PAMPS)

conserved microbial structures that are specifically recognized by PRRs with the resultant signaling contributing to the initiation of innate immunity

Pattern recognition receptors (PRRs)

a group of receptors, including TLRs, that recognize DAMPS and initiate the appropriate immune response

Peroxisome proliferator-activated receptor alpha (PPARα)

one of the three isoforms of a nuclear receptor family. It is expressed in cells of both the innate and the adaptive immune response, predominantly macrophages. While typically involved in fatty acid metabolism, it is also involved in the regulation of intestinal inflammation through cytokine production

Peroxisome proliferator-activated receptor gamma (PPARγ)

one of the three isoforms of a nuclear receptor family. It is expressed in cells of both the innate and the adaptive immune response, predominantly the activation and recruitment of macrophages, DCs, T cells, and intestinal epithelial cells. It modulates the immune response by regulating the production of cytokines including TGF-β, IL-10, TNFα, and IL-6

Prostaglandin E2 (PGE2)

an arachidonic acid derivative that is produced by activated immune cells and other cell types. It is involved in innate immune cell activation, recruitment, and cytokine release

Rab7b

a GTPase that acts as a TLR4 negative regulator. It is involved with vesicular transportation to late endosomes and the lysosome

Scavenger receptor A (SR-A)

a surface glycoprotein found predominantly on macrophages and other antigen-presenting cells (APCs). It regulates the uptake and internalization of lipoproteins and endotoxins and is involved in the process of antigen presentation and activation of the appropriate immune response

Suppressor of cytokine signaling 3 (SOCS3)

part of an eight-member protein family. This protein negatively regulates the JAK/STAT pathway. In addition to cytokine stimulation, TLR/MyD88 signaling is also involved in the induction of SOCS3 protein

Toll-like receptors (TLRs)

a group of PRRs that activate the innate immune response. This family of 13 receptors recognizes various molecules, including specific PAMPs and DAMPs

Thymic stromal lymphopoietin (TSLP)

an epithelial-cell-released cytokine that serves as an alarmin and can contribute to the initiation of type 2 immune responses

Trefoil factor 2 (TFF2)

a small protein that is part of the trefoil factor family. It acts as a DAMP on release from damaged cells and induces the type 2 immune response through the initiation of IL-33 production