Excretory/secretory antigens from Trichinella spiralis muscle larvae ameliorate HFD-induced non-alcoholic steatohepatitis via driving macrophage anti-inflammatory activity.

No approved effective therapy for non-alcoholic steatohepatitis (NASH) is currently available. Trichinella spiralis (T. spiralis) infection and their products have positive impact on several metabolic diseases. Considering, we firstly investigated the effects of the T. spiralis-derived Excretory-Secretory antigens (ESA) on high fat diet (HFD)-induced NASH mouse models. To further elucidate the mechanism of action, HepG2 cells were incubated with palmitic acid (PA) to construct NASH-like cell model, and then the culture medium supernatant collected from ESA-treated macrophages was applied to intervene the cell model in vitro. In NASH mouse models, ESA significantly alleviated hepatic steatosis and hepatic inflammation, as reflected by reducing pro-inflammatory cytokines and inactivating TLR4/MYD88/NF-κB pathway and NLRP3 inflammasome. Meanwhile, the HFD-induced oxidative stress was restored by ESA through lessening the level of MDA, increasing the activity of T-SOD and enhancing Nrf2 signaling-related proteins, including p-Nrf2, NQO1, HO-1, GPX4, and p-AMPK. Notably, ESA preferentially promoted macrophages polarization toward M2 anti-inflammatory phenotype in vivo and vitro. Moreover, in vitro, intervention of PA-treated HepG2 cells with medium supernatant of ESA-treated macrophages attenuated lipid accumulation, inflammation, as well as oxidative stress. In conclusion, T. spiralis-derived ESA may serve as a novel promising candidate for the treatment of NASH via its properties of driving macrophage anti-inflammatory activity.

Enhancing the therapeutic potential of P29 protein-targeted monoclonal antibodies in the management of alveolar echinococcosis through CDC-mediated mechanisms.

Alveolar echinococcosis (AE) is a highly lethal helminth infection. Current chemotherapeutic strategies for AE primarily involve the use of benzimidazoles (BZs) such as mebendazole (MDZ) and albendazole (ABZ), which exhibit limited efficacy. In a previous study, the vaccine of recombinant Echinococcus granulosus P29 (rEgP29) showed significant immunoprotection against E. granulosus in both mice and sheep. In the current study, we utilized hybridoma technology to generate five monoclonal antibodies (mAbs) against P29, among which 4G10F4 mAb exhibited the highest antigen-specific binding capacity. This mAb was selected for further investigation of anti-AE therapy, both in vivo and in vitro. In vitro, 4G10F4 inhibited a noteworthy inhibition of E. multilocularis protoscoleces and primary cells viability through complement-dependent cytotoxicity (CDC) mechanism. In vivo, two experiments were conducted. In the first experiment, mice were intraperitoneally injected with Em protoscoleces, and subsequently treated with 4G10F4 mAb (2.5/5/10 mg/kg) at 12 weeks postinfection once per week for 8 times via tail vein injection. Mice that were treated with 4G10F4 mAb only in dosage of 5mg/kg exhibited a significant lower mean parasite burden (0.89±0.97 g) compared to isotype mAb treated control mice (2.21±1.30 g). In the second experiment, mice were infected through hepatic portal vein and treated with 4G10F4 mAb (5mg/kg) at one week after surgery once per week for 8 times. The numbers of hepatic metacestode lesions of the 4G10F4 treatment group were significantly lower in comparison to the isotype control group. Pathological analysis revealed severe disruption of the inner structure of the metacestode in both experiments, particularly affecting the germinal and laminated layers, resulting in the transformation into infertile vesicles after treatment with 4G10F4. In addition, the safety of 4G10F4 for AE treatment was confirmed through assessment of mouse weight and evaluation of liver and kidney function. This study presents antigen-specific monoclonal antibody immunotherapy as a promising therapeutic approach against E. multilocularis induced AE.

Helminth protein enhances wound healing by inhibiting fibrosis and promoting tissue regeneration.

Skin wound healing due to full thickness wounds typically results in fibrosis and scarring, where parenchyma tissue is replaced with connective tissue. A major advance in wound healing research would be to instead promote tissue regeneration. Helminth parasites express excretory/secretory (ES) molecules, which can modulate mammalian host responses. One recently discovered ES protein, TGF-ß mimic (TGM), binds the TGF-ß receptor, though likely has other activities. Here, we demonstrate that topical administration of TGM under a Tegaderm bandage enhanced wound healing and tissue regeneration in an in vivo wound biopsy model. Increased restoration of normal tissue structure in the wound beds of TGM-treated mice was observed during mid- to late-stage wound healing. Both accelerated re-epithelialization and hair follicle regeneration were observed. Further analysis showed differential expansion of myeloid populations at different wound healing stages, suggesting recruitment and reprogramming of specific macrophage subsets. This study indicates a role for TGM as a potential therapeutic option for enhanced wound healing.

Nematode Galectin Inhibits Basophilic Leukaemia RBL-2H3 Cells Apoptosis in IgE-Mediated Activation.

Mast cells are essential immune cells involved in the host's defence against gastrointestinal nematodes. To evade the immune response, parasitic nematodes produce a variety of molecules. Galectin 1, produced by Teladorsagia circumcincta (Tci-gal-1), reduces mast cell degranulation and selectively regulates mediator production and release in an IgE-dependent manner. To uncover the activity of Tci-gal-1, we have examined the effect of the protein on gene expression, protein production, and apoptosis in activated basophilic leukaemia RBL-2H3 cells. Rat RBL-2H3 cells were activated with anti-DNP IgE and DNP-HSA, and then treated with Tci-gal-1. Microarray analysis was used to examine gene expression. The levels of several apoptosis-related molecules and cytokines were determined using antibody arrays and ELISA. Early and late apoptosis was evaluated cytometrically. Degranulation of cells was determined by a ß-hexosaminidase release assay. Treatment of activated RBL-2H3 cells with Tci-gal-1 resulted in inhibited apoptosis and decreased degranulation, although we did not detect significant changes in gene expression. The production of pro-apoptotic molecules, receptor for advanced glycation end products (RAGE) and Fas ligand (FasL), and the cytokines IL-9, IL-10, IL-13, TNF-α, and IL-2 was strongly inhibited. Tci-gal-1 modulates apoptosis, degranulation, and production of cytokines by activated RBL-2H3 cells without detectable influence on gene transcription. This parasite protein is crucial for modulation of the protective immune response and the inhibition of chronic inflammation driven by mast cell activity.

Trichinella spiralis Paramyosin Alleviates Collagen-Induced Arthritis in Mice by Modulating CD4+ T Cell Differentiation.

Rheumatoid arthritis (RA) is an autoimmune disease that significantly impacts quality of life by disrupting CD4+ T cell immune homeostasis. The identification of a low-side-effect drug for RA treatment is urgently needed. Our previous study suggests that Trichinella spiralis paramyosin (Ts-Pmy) has immunomodulatory effects, but its potential effect on CD4+ T cell response in RA remains unclear. In this study, we used a murine model to investigate the role of rTs-Pmy in regulating CD4+ T cell differentiation in collagen-induced arthritis (CIA). Additionally, we assessed the impact of rTs-Pmy on CD4+ T cell differentiation towards the Th1 and Th17 phenotypes, which are associated with inflammatory responses in arthritis, using in vitro assays. The results demonstrated that rTs-Pmy administration reduced arthritis severity by inhibiting Th1 and Th17 response while enhancing Treg response. Prophylactic administration of Ts-Pmy showed superior efficacy on CIA compared to therapeutic administration. Furthermore, in vitro assays demonstrated that rTs-Pmy could inhibit the differentiation of CD4+ T cells into Th1 and Th17 while inducing the production of Tregs, suggesting a potential mechanism underlying its therapeutic effects. This study suggests that Ts-Pmy may ameliorate CIA by restoring the immune balance of CD4+ T cells and provides new insights into the mechanism through which helminth-derived proteins exert their effects on autoimmune diseases.

Succinate coenzyme A ligase ß-like protein from Trichinella spiralis is a potential therapeutic molecule for allergic asthma.

BACKGROUND: For decades, studies have demonstrated the anti-inflammatory potential of proteins secreted by helminths in allergies and asthma. Previous studies have demonstrated the immunomodulatory capabilities of Succinate Coenzyme A ligase beta-like protein (SUCLA-ß) derived from Trichinella spiralis, a crucial excretory product of this parasite. OBJECTIVE: To explore the therapeutic potential of SUCLA-ß in alleviating and controlling ovalbumin (OVA)-induced allergic asthma, as well as its influence on host immune modulation. METHODS: In this research, we utilized the rTs-SUCLA-ß protein derived from T. spiralis to investigate its potential in mitigating airway inflammation in a murine model of asthma induced by OVA sensitization/stimulation, both pre- and post-challenge. The treatment's efficacy was assessed by quantifying the extent of inflammation in the lungs. RESULTS: Treatment with rTs-SUCLA-ß demonstrated efficacy in ameliorating OVA-induced airway inflammation, as evidenced by a reduction in eosinophil infiltration, levels of OVA-specific Immunoglobulin E, interferon-γ, interleukin (IL)-9, and IL-17A, along with an elevation in IL-10. The equilibrium between Th17 and Treg cells plays a pivotal role in modulating the abundance of inflammatory cells within the organism, thereby ameliorating inflammation and alleviating symptoms associated with allergic asthma. CONCLUSIONS AND CLINICAL RELEVANCE: Our data revealed that T. spiralis-derived Ts-SUCLA-ß protein may inhibit the allergic airway inflammation by regulating host immune responses.

The helminth-derived peptide, FhHDM-1, reverses the trained phenotype of NOD bone-marrow-derived macrophages and regulates proinflammatory responses.

We implicate a phenotype of trained immunity in bone-marrow-derived macrophages in the onset and progression of type 1 diabetes in nonobese diabetic mice. Treatment with FhHDM-1 reversed immune training, reducing histone methylation and glycolysis, and decreasing proinflammatory cytokine production to the same level as macrophages from nondiabetic immune-competent BALB/c mice.

Identification and characterization of TatD DNase in planarian Dugesia japonica and its antibiofilm effect.

TatD DNase, a key enzyme in vertebrates and invertebrates, plays a pivotal role in various physiological processes. Dugesia japonica (D. japonica), a flatworm species, has remarkable regenerative capabilities and possesses a simplified immune system. However, the existence and biological functions of TatD DNase in D. japonica require further investigation. Here, we obtained the open reading frame (ORF) of DjTatD and demonstrated its conservation. The three-dimensional structure of DjTatD revealed its active site and binding mechanism. To investigate its enzymological properties, we overexpressed, purified, and characterized recombinant DjTatD (rDjTatD). We observed that DjTatD was primarily expressed in the pharynx and its expression could be significantly challenged upon stimulation with lipopolysaccharide, peptidoglycan, gram-positive and gram-negative bacteria. RNA interference results indicated that both DjTatD and DjDN2s play a role in pharyngeal regeneration and may serve as functional complements to each other. Additionally, we found that rDjTatD and recombinant T7DjTatD effectively reduce biofilm formation regardless of their bacterial origin. Together, our results demonstrated that DjTatD may be involved in the planarian immune response and pharyngeal regeneration. Furthermore, after further optimization in the future, rDjTatD and T7DjTatD can be considered highly effective antibiofilm agents.

In Vitro Modulation of Complement Activation by Therapeutically Prospective Analogues of the Marine Polychaeta Arenicin Peptides.

The widespread resistance to antibiotics in pathogenic bacteria makes the development of a new generation of antimicrobials an urgent task. The development of new antibiotics must be accompanied by a comprehensive study of all of their biological activities in order to avoid adverse side-effects from their application. Some promising antibiotic prototypes derived from the structures of arenicins, antimicrobial peptides from the lugworm Arenicola marina, have been developed. Previously, we described the ability of natural arenicins -1 and -2 to modulate the human complement system activation in vitro. In this regard, it seems important to evaluate the effect of therapeutically promising arenicin analogues on complement activation. Here, we describe the complement-modulating activity of three such analogues, Ar-1[V8R], ALP1, and AA139. We found that the mode of action of Ar-1[V8R] and ALP1 on the complement was similar to that of natural arenicins, which can both activate and inhibit the complement, depending on the concentration. However, Ar-1[V8R] behaved predominantly as an inhibitor, showing only a moderate increase in C3a production in the alternative pathway model and no enhancement at all of the classical pathway of complement activation. In contrast, the action of ALP1 was characterized by a marked increase in the complement activation through the classical pathway in the concentration range of 2.5-20 µg/mL. At the same time, at higher concentrations (80-160 µg/mL), this peptide exhibited a complement inhibitory effect characteristic of the other arenicins. Peptide AA139, like other arenicins, exhibited an inhibitory effect on complement at a concentration of 160 µg/mL, but was much less pronounced. Overall, our results suggest that the effect on the complement system should be taken into account in the development of antibiotics based on arenicins.

Novel antiinflammatory biologics shaped by parasite-host coevolution.

Parasitic helminth infections, while a major cause of neglected tropical disease burden, negatively correlate with the incidence of immune-mediated inflammatory diseases such as inflammatory bowel diseases (IBD). To evade expulsion, helminths have developed sophisticated mechanisms to regulate their host's immune responses. Controlled experimental human helminth infections have been assessed clinically for treating inflammatory conditions; however, such a radical therapeutic modality has challenges. An alternative approach is to harness the immunomodulatory properties within the worm's excretory-secretory (ES) complement, its secretome. Here, we report a biologics discovery and validation pipeline to generate and screen in vivo a recombinant cell-free secretome library of helminth-derived immunomodulatory proteins. We successfully expressed 78 recombinant ES proteins from gastrointestinal hookworms and screened the crude in vitro translation reactions for anti-IBD properties in a mouse model of acute colitis. After statistical filtering and ranking, 20 proteins conferred significant protection against various parameters of colitis. Lead candidates from distinct protein families, including annexins, transthyretins, nematode-specific retinol-binding proteins, and SCP/TAPS were identified. Representative proteins were produced in mammalian cells and further validated, including ex vivo suppression of inflammatory cytokine secretion by T cells from IBD patient colon biopsies. Proteins identified herein offer promise as novel, safe, and mechanistically differentiated biologics for treating the globally increasing burden of inflammatory diseases.

Investigations into the membrane activity of arenicin antimicrobial peptide AA139.

Arenicin-3 is an amphipathic ß-hairpin antimicrobial peptide that is produced by the lugworm Arenicola marina. In this study, we have investigated the mechanism of action of arenicin-3 and an optimized synthetic analogue, AA139, by studying their effects on lipid bilayer model membranes and Escherichia coli bacterial cells. The results show that simple amino acid changes can lead to subtle variations in their interaction with membranes and therefore alter their pre-clinical potency, selectivity and toxicity. While the mechanism of action of arenicin-3 is primarily dependent on universal membrane permeabilization, our data suggest that the analogue AA139 relies on more specific binding and insertion properties to elicit its improved antibacterial activity and lower toxicity, as exemplified by greater selectivity between lipid composition when inserting into model membranes i.e. the N-terminus of AA139 seems to insert deeper into lipid bilayers than arenicin-3 does, with a clear distinction between zwitterionic and negatively charged lipid bilayer vesicles, and AA139 demonstrates a cytoplasmic permeabilization dose response profile that is consistent with its greater antibacterial potency against E. coli cells compared to arenicin-3.

Intestinal epithelial tuft cell induction is negated by a murine helminth and its secreted products.

Helminth parasites are adept manipulators of the immune system, using multiple strategies to evade the host type 2 response. In the intestinal niche, the epithelium is crucial for initiating type 2 immunity via tuft cells, which together with goblet cells expand dramatically in response to the type 2 cytokines IL-4 and IL-13. However, it is not known whether helminths modulate these epithelial cell populations. In vitro, using small intestinal organoids, we found that excretory/secretory products (HpES) from Heligmosomoides polygyrus blocked the effects of IL-4/13, inhibiting tuft and goblet cell gene expression and expansion, and inducing spheroid growth characteristic of fetal epithelium and homeostatic repair. Similar outcomes were seen in organoids exposed to parasite larvae. In vivo, H. polygyrus infection inhibited tuft cell responses to heterologous Nippostrongylus brasiliensis infection or succinate, and HpES also reduced succinate-stimulated tuft cell expansion. Our results demonstrate that helminth parasites reshape their intestinal environment in a novel strategy for undermining the host protective response.

Fasciola hepatica fatty acid binding protein (Fh12) induces apoptosis and tolerogenic properties in murine bone marrow derived dendritic cells.

In a previous study we demonstrated that Fasciola hepatica fatty acid binding protein (Fh12) significantly suppress macrophage function by inhibiting IL-6, IL-1ß, tumor necrosis factor (TNF)-α and IL-12 production in TLR4-stimulated murine macrophages, an effect mediated through the signaling of CD14 co-receptor without affecting the viability of these cells. Given that dendritic cells (DCs) are immune cells that play a central role in the initiation of primary immune responses and that are the only antigen-presenting cells capable of stimulating naïve T-cells, in the present study we investigated the effect of Fh12 on DCs. We found that Fh12 exerts a strong suppressive effect on activation and function of DCs. However, in contrast to the effect observed on macrophages, Fh12 induces early and late apoptosis of DCs being this phenomenon dose-dependent and CD14-coreceptor independent. At low concentration Fh12 modulates the LPS-induced DCs maturation status by suppressing the MHC-II, and co-stimulatory molecules CD40 and CD80 surface expression together with the pro-inflammatory cytokines IL-12p70 and IL-6 production whereas increase the IL-10 levels. Besides, Fh12 decreased the ability of LPS-activated DCs to induce IFN-γ production against allogeneic splenocytes, while increasing IL-4 production. We have described for the first time the ability of Fh12 to modify selectively the viability of DCs by apoptosis induction. The selective diminution in DCs survival could be a F. hepatica strategy in order to prevent a host immune response during the earliest phases of infection.

Suppression of inflammatory arthritis by the parasitic worm product ES-62 is associated with epigenetic changes in synovial fibroblasts.

ES-62 is the major secreted protein of the parasitic filarial nematode, Acanthocheilonema viteae. The molecule exists as a large tetramer (MW, ~240kD), which possesses immunomodulatory properties by virtue of multiple phosphorylcholine (PC) moieties attached to N-type glycans. By suppressing inflammatory immune responses, ES-62 can prevent disease development in certain mouse models of allergic and autoimmune conditions, including joint pathology in collagen-induced arthritis (CIA), a model of rheumatoid arthritis (RA). Such protection is associated with functional suppression of "pathogenic" hyper-responsive synovial fibroblasts (SFs), which exhibit an aggressive inflammatory and bone-damaging phenotype induced by their epigenetic rewiring in response to the inflammatory microenvironment of the arthritic joint. Critically, exposure to ES-62 in vivo induces a stably-imprinted CIA-SF phenotype that exhibits functional responses more typical of healthy, Naïve-SFs. Consistent with this, ES-62 "rewiring" of SFs away from the hyper-responsive phenotype is associated with suppression of ERK activation, STAT3 activation and miR-155 upregulation, signals widely associated with SF pathogenesis. Surprisingly however, DNA methylome analysis of Naïve-, CIA- and ES-62-CIA-SF cohorts reveals that rather than simply preventing pathogenic rewiring of SFs, ES-62 induces further changes in DNA methylation under the inflammatory conditions pertaining in the inflamed joint, including targeting genes associated with ciliogenesis, to programme a novel "resolving" CIA-SF phenotype. In addition to introducing a previously unsuspected aspect of ES-62's mechanism of action, such unique behaviour signposts the potential for developing DNA methylation signatures predictive of pathogenesis and its resolution and hence, candidate mechanisms by which novel therapeutic interventions could prevent SFs from perpetuating joint inflammation and destruction in RA. Pertinent to these translational aspects of ES-62-behavior, small molecule analogues (SMAs) based on ES-62's active PC-moieties mimic the rewiring of SFs as well as the protection against joint disease in CIA afforded by the parasitic worm product.

Excretory/secretory proteins of adult Toxocara canis induce changes in the expression of proteins involved in the NOD1-RIP2-NF-κB pathway and modulate cytokine production in mouse macrophages.

Dog roundworm (Toxocara canis) is the major causative agent of toxocarosis, a parasitic disease of both veterinary and medical importance. Knowledge gaps in fundamental and applied aspects hinder the control of this important zoonotic disease. To have a better understanding of Toxocara infection and host immune responses, mouse macrophages were exposed to excretory/secretory (ES) proteins released by adult worms of T. canis in vitro. The messenger RNA transcription and protein expression of nucleotide-binding oligomerization domain-containing protein 1 (NOD1), receptor interacting protein 2 (RIP2) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in macrophages were analysed using quantitative real-time PCR (qRT-PCR) and Western blot. The levels of tumour necrosis factor alpha (TNF-ɑ), interleukin-1 beta (IL-1ß) and IL-6 released by the stimulated macrophages were analysed using enzyme-linked immunosorbent assay. It was found that 20 µg/mL ES proteins of adult T. canis induced the expression of NOD1, RIP2 and NF-κB in mouse macrophages at both transcriptional and translational levels after 9 h of incubation in vitro. Incubation with 20 µg/mL ES proteins also modulated the production of pro-inflammatory cytokines TNF-ɑ, IL-1ß and IL-6 by the macrophages. Taken together, ES proteins of adult T. canis appeared to be able to affect the macrophage NOD1-RIP2-NF-κB signalling pathway, which might play a role in regulating the production of proinflammatory cytokines. Further investigation of these aspects should lead to a better understanding of immune recognition of and modulation by Toxocara canis in host animals.

Agglutination Activity of Fasciola gigantica DM9-1, a Mannose-Binding Lectin.

The DM9 domain is a protein unit of 60-75 amino acids that has been first detected in the fruit fly Drosophila as a repeated motif of unknown function. Recent research on proteins carrying DM9 domains in the mosquito Anopheles gambiae and the oyster Crassostrea gigas indicated an association with the uptake of microbial organisms. Likewise, in the trematode Fasciola gigantica DM9-1 showed intracellular relocalization following microbial, heat and drug stress. In the present research, we show that FgDM9-1 is a lectin with a novel mannose-binding site that has been recently described for the protein CGL1 of Crassostrea gigas. This property allowed FgDM9-1 to agglutinate gram-positive and -negative bacteria with appropriate cell surface glycosylation patterns. Furthermore, FgDM9-1 caused hemagglutination across all ABO blood group phenotypes. It is speculated that the parenchymal located FgDM9-1 has a role in cellular processes that involve the transport of mannose-carrying molecules in the parenchymal cells of the parasite.

Clonorchis sinensis-Derived Protein Attenuates Inflammation and New Bone Formation in Ankylosing Spondylitis.

Helminth infections and their components have been shown to have the potential to modulate and attenuate immune responses. The objective of this study was to evaluate the potential protective effects of Clonorchis sinensis-derived protein (CSp) on ankylosing spondylitis (AS). Cytotoxicity of CSp at different doses was assessed by MTS and flow cytometry before performing experiments. Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were obtained from AS patients. Inflammatory cytokine-producing cells were analyzed using flow cytometry. The levels of INF- γ , IL-17A, TNF-α, and IL-6 were measured by enzyme-linked immunosorbent assay (ELISA). SKG mice were treated with CSp or vehicles. Inflammation and new bone formation were evaluated using immunohistochemistry, positron emission tomography (PET), and micro-computed tomography (CT). Treatment with CSp resulted in no reduced cell viability of PBMCs or SFMCs until 24 h. In experiments culturing PBMCs and SFMCs, the frequencies of IFN- γ and IL-17A producing cells were significantly reduced after CSp treatment. In the SKG mouse model, CSp treatment significantly suppressed arthritis, enthesitis, and enteritis. Micro-CT analysis of hind paw revealed reduced new bone formation in CSp-treated mice than in vehicle-treated mice. We provide the first evidence demonstrating that CSp can ameliorate clinical signs and cytokine derangements in AS. In addition, such CSp treatment could reduce the new bone formation of AS.

Schistosoma japonicum cystatin suppresses osteoclastogenesis via manipulating the NF­κB signaling pathway.

Abnormal osteoclastic activation and secretion of cysteine proteinases result in excessive bone resorption, which is one of the primary factors in the development of bone metabolic disorders, such as rheumatoid arthritis and osteoporosis. Mammalian cystatins have been demonstrated to restrain osteoclastic bone resorption and to alleviate severe osteolytic destruction via blocking the activity of cysteine proteinases. However, the specific effects of parasite cystatins on the formation and function of osteoclasts remain unclear. The purpose of the current study was to explore the effects of cystatins from Schistosoma japonicum (Sj­Cys) on macrophage colony­stimulating factor (M­CSF) and receptor activator of NF­κB ligand (RANKL)­induced osteoclast differentiation, as well as the underlying molecular mechanisms. Recombinant Sj­Cys (rSj­Cys) dose­dependently restrained osteoclast formation, with a half­maximal inhibitory concentration (IC50) value of 0.3 µM, and suppressed osteoclastic bone resorptive capability in vitro. The findings were based on tartrate resistant acid phosphatase (TRAP) staining and bone resorption assays, respectively. However, the cell viability assay showed that the repression of rSj­Cys on osteoclast formation did not depend on effects on cell viability or apoptosis. Based on the results of reverse transcription­quantitative PCR and western blot analysis, it was found that rSj­Cys downregulated the expression levels of osteoclastogenesis­related genes and proteins, by interfering with M­CSF and RANKL­induced NF­κB signaling and downstream transcription factors during early­phase osteoclastogenesis. Overall, the results of the present study revealed that rSj­Cys exerted an inhibitory role in osteoclast differentiation and could be a prospective biotherapeutic candidate for the treatment and prevention of bone metabolic disorders.

Inhibition of inflammatory cytokine production and proliferation in macrophages by Kunitz-type inhibitors from Echinococcus granulosus.

The genus Echinococcus of cestode parasites includes important pathogens of humans and livestock animals. Transcriptomic and genomic studies on E. granulosus and E. multilocularis uncovered striking expansion of monodomain Kunitz proteins. This expansion is accompanied by the specialization of some family members away from the ancestral protease inhibition function to fulfill cation channel blockade functions. Since cation channels are involved in immune processes, we tested the effects on macrophage physiology of two E. granulosus Kunitz-type inhibitors of voltage-activated cation channels (Kv) that are close paralogs. Both inhibitors, EgKU-1 and EgKU-4, inhibited production of the Th1/Th17 cytokine subunit IL-12/23p40 by macrophages stimulated with the TLR4 agonist LPS. In addition, EgKU-4 but not EgKU-1 inhibited production of the inflammatory cytokine IL-6. These activities were not displayed by EgKU-3, a family member that is a protease inhibitor without known activity on cation channels. EgKU-4 potently inhibited macrophage proliferation in response to M-CSF, whereas EgKU-1 displayed similar activity but with much lower potency, similar to EgKU-3. We discuss structural differences, including a heavily cationic C-terminal extension present in EgKU-4 but not in EgKU-1, that may explain the differential activities of the two close paralogs.

The helminth glycoprotein omega-1 improves metabolic homeostasis in obese mice through type 2 immunity-independent inhibition of food intake.

Type 2 immunity plays an essential role in the maintenance of metabolic homeostasis and its disruption during obesity promotes meta-inflammation and insulin resistance. Infection with the helminth parasite Schistosoma mansoni and treatment with its soluble egg antigens (SEA) induce a type 2 immune response in metabolic organs and improve insulin sensitivity and glucose tolerance in obese mice, yet, a causal relationship remains unproven. Here, we investigated the effects and underlying mechanisms of the T2 ribonuclease omega-1 (ω1), one of the major S mansoni immunomodulatory glycoproteins, on metabolic homeostasis. We show that treatment of obese mice with plant-produced recombinant ω1, harboring similar glycan motifs as present on the native molecule, decreased body fat mass, and improved systemic insulin sensitivity and glucose tolerance in a time- and dose-dependent manner. This effect was associated with an increase in white adipose tissue (WAT) type 2 T helper cells, eosinophils, and alternatively activated macrophages, without affecting type 2 innate lymphoid cells. In contrast to SEA, the metabolic effects of ω1 were still observed in obese STAT6-deficient mice with impaired type 2 immunity, indicating that its metabolic effects are independent of the type 2 immune response. Instead, we found that ω1 inhibited food intake, without affecting locomotor activity, WAT thermogenic capacity or whole-body energy expenditure, an effect also occurring in leptin receptor-deficient obese and hyperphagic db/db mice. Altogether, we demonstrate that while the helminth glycoprotein ω1 can induce type 2 immunity, it improves whole-body metabolic homeostasis in obese mice by inhibiting food intake via a STAT6-independent mechanism.