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.

Na-AIP-1 secreted by human hookworms suppresses collagen-induced arthritis.

Proteins from helminths have been posed as new immunomodulatory agents with exciting potential in the treatment of immune-mediated diseases including rheumatoid arthritis (RA). In this study we assess the effects of a helminthic excretory/secretory (ES) protein Na-AIP-1 as monotherapy and in combination with methotrexate (MTX) in the well-described collagen-induced arthritis (CIA) model of RA. CIA was induced in DBA/1 J mice which were treated after the onset of arthritis with Na-AIP-1 monotherapy, MTX or Na-AIP-1 + MTX. The clinical scores for weight, arthritis and paw width were recorded along with joint histology as outcome measures. For the clinical parameters of weight, paw score and paw width, none of the Na-AIP-1 monotherapy, MTX therapy or Na-AIP-1 + MTX combination therapy groups displayed any significant difference when compared to the arthritis control. However, a significant reduction in histological score was identified after both monotherapy (Na-AIP-1: 0.83 ± 0.24 vs Arthritis control: 5.58 ± 1.49, p = 0.0277) and combination therapy (Na-AIP-1 + MTX: 0.55 ± 0.28 vs Arthritis control: 5.58 ± 1.49, p = 0.0233) when compared to arthritis control. Furthermore, Na-AIP-1 as both monotherapy (Na-AIP-1: 0.83 ± 0.24 vs MTX: 5.73 ± 1.82 p = 0.0261) and combination therapy (Na-AIP-1 + MTX: 0.55 ± 0.28 vs MTX: 5.73 ± 1.82, p = 0.0221) also significantly reduced histological score when compared to MTX monotherapy. Na-AIP-1 significantly reduced joint pathology in CIA. The hookworm protein Na-AIP-1 seems to be effective in the treatment of RA as monotherapy and when dosed together with MTX, constituting a potential new candidate for drug development. Research should focus on elucidating the mechanism of Na-AIP-1 action as a means to identify novel targets for therapeutics and to further our current understanding of immunobiology in RA.

Hookworm-Derived Metabolites Suppress Pathology in a Mouse Model of Colitis and Inhibit Secretion of Key Inflammatory Cytokines in Primary Human Leukocytes.

Iatrogenic hookworm therapy shows promise for treating disorders that result from a dysregulated immune system, including inflammatory bowel disease (IBD). Using a murine model of trinitrobenzenesulfonic acid-induced colitis and human peripheral blood mononuclear cells, we demonstrated that low-molecular-weight metabolites derived from both somatic extracts (LMWM-SE) and excretory-secretory products (LMWM-ESP) of the hookworm, Ancylostoma caninum, display anti-inflammatory properties. Administration to mice of LMWM-ESP as well as sequentially extracted fractions of LMWM-SE using both methanol (SE-MeOH) and hexane-dichloromethane-acetonitrile (SE-HDA) resulted in significant protection against T cell-mediated immunopathology, clinical signs of colitis, and impaired histological colon architecture. To assess bioactivity in human cells, we stimulated primary human leukocytes with lipopolysaccharide in the presence of hookworm extracts and showed that SE-HDA suppressed ex vivo production of inflammatory cytokines. Gas chromatography-mass spectrometry (MS) and liquid chromatography-MS analyses revealed the presence of 46 polar metabolites, 22 fatty acids, and five short-chain fatty acids (SCFAs) in the LMWM-SE fraction and 29 polar metabolites, 13 fatty acids, and six SCFAs in the LMWM-ESP fraction. Several of these small metabolites, notably the SCFAs, have been previously reported to have anti-inflammatory properties in various disease settings, including IBD. This is the first report showing that hookworms secrete small molecules with both ex vivo and in vivo anti-inflammatory bioactivity, and this warrants further exploration as a novel approach to the development of anti-inflammatory drugs inspired by coevolution of gut-dwelling hookworms with their vertebrate hosts.

The parasitic worm product ES-62 protects against collagen-induced arthritis by resetting the gut-bone marrow axis in a microbiome-dependent manner.

The parasitic worm-derived immunomodulator, ES-62 rescues defective levels of IL-10-producing regulatory B cells (Bregs) and suppresses chronic Th1/Th17-driven inflammation to protect against joint destruction in the mouse collagen-induced arthritis (CIA) model of rheumatoid arthritis. Such autoimmune arthritis is also associated with dysbiosis of the gut microbiota and disruption of intestinal barrier integrity. We recently further exploited the CIA model to show that ES-62's prevention of joint destruction is associated with protection of intestinal barrier integrity and normalization of the gut microbiota, thereby suppressing the gut pathology that precedes the onset of autoimmunity and joint damage in CIA-mice. As the status of the gut microbiota impacts on immune responses by influencing haematopoiesis, we have therefore investigated whether ES-62 harnesses the homeostatic mechanisms regulating this gut-bone marrow (BM) axis to resolve the chronic inflammation promoting autoimmunity and joint destruction in CIA. Reflecting this, ES-62 was found to counteract the BM myeloid/lymphoid bias typically associated with chronic inflammation and infection. This was achieved primarily by ES-62 acting to maintain the levels of lymphoid lineages (B220+ and CD3+ cells) observed in naïve, healthy mice but lost from the BM of CIA-mice. Moreover, ES-62's ability to prevent bone-destroying osteoclastogenesis was found to be associated with its suppression of CIA-induced upregulation of osteoclast progenitors (OCPs) in the BM. Critically, and supporting ES-62's targeting of the gut-BM axis, this rewiring of inflammatory haematopoiesis was lost in mice with a depleted microbiome. Underlining the importance of ES-62's actions in restoring steady-state haematopoiesis, the BM levels of B and T lymphoid cells were shown to be inversely correlated, whilst the levels of OCPs positively correlated, with the severity of joint damage in CIA-mice.

Conquering rheumatic diseases: are parasitic worms the answer?

Despite the introduction of novel treatment strategies, management of rheumatic disorders remains associated with substantial unmet clinical need. Of interest therefore, it has recently become apparent that there is a global inverse relationship between the incidence of such conditions and parasitic helminth infection, with striking examples involving rheumatoid arthritis (RA)/systemic lupus erythematosus (SLE) patients and filarial nematode worm infection in studies in India. Such findings reflect that helminths are master manipulators of the immune system, particularly in being able to modulate proinflammatory responses. The aim of this article is thus to consider findings to date on this exciting and intriguing research area to form an opinion on whether parasitic worms may be exploited to generate novel therapies for rheumatic diseases.

Protection against lung pathology during obesity-accelerated ageing in mice by the parasitic worm product ES-62.

Mice develop pathology in the lungs as they age and this may be accelerated by a high calorie diet (HCD). ES-62 is a protein secreted by the parasitic worm Acanthocheilonema viteae that is immunomodulatory by virtue of covalently attached phosphorylcholine (PC) moieties. In this study, we show that weekly treatment of C57BL/6J mice with ES-62 protected against pathology in the lungs in male but not female mice fed a HCD from 10 weeks of age as shown by reductions in cellular infiltration and airway remodelling, particularly up to 160 days of age. ES-62 also reduced gene expression of the cytokines IL-4 and IL-17 and in addition the TLR/IL-1R adaptor MyD88, in the lungs of male mice although HCD-induced increases in these inflammatory markers were not detected until between 340 and 500 days of age. A combination of two drug-like ES-62 PC-based small molecule analogues (SMAs), produced broadly similar protective effects in the lungs of male mice with respect to both lung pathology and inflammatory markers, in addition to a decrease in HCD-induced IL-5 expression. Overall, our data show that ES-62 and its SMAs offer protection against HCD-accelerated pathological changes in the lungs during ageing. Given the targeting of Th2 cytokines and IL-17, we discuss this protection in the context of ES-62's previously described amelioration of airway hyper-responsiveness in mouse models of asthma.

Solution structure of the N-terminal extension domain of a Schistosoma japonicum asparaginyl-tRNA synthetase.

Several secreted proteins from helminths (parasitic worms) have been shown to have immunomodulatory activities. Asparaginyl-tRNA synthetases are abundantly secreted in the filarial nematode Brugia malayi (BmAsnRS) and the parasitic flatworm Schistosoma japonicum (SjAsnRS), indicating a possible immune function. The suggestion is supported by BmAsnRS alleviating disease symptoms in a T-cell transfer mouse model of colitis. This immunomodulatory function is potentially related to an N-terminal extension domain present in eukaryotic AsnRS proteins but few structure/function studies have been done on this domain. Here we have determined the three-dimensional solution structure of the N-terminal extension domain of SjAsnRS. A protein containing the 114 N-terminal amino acids of SjAsnRS was recombinantly expressed with isotopic labelling to allow structure determination using 3D NMR spectroscopy, and analysis of dynamics using NMR relaxation experiments. Structural comparisons of the N-terminal extension domain of SjAsnRS with filarial and human homologues highlight a high degree of variability in the ß-hairpin region of these eukaryotic N-AsnRS proteins, but similarities in the disorder of the C-terminal regions. Limitations in PrDOS-based intrinsically disordered region (IDR) model predictions were also evident in this comparison. Empirical structural data such as that presented in our study for N-SjAsnRS will enhance the prediction of sequence-homology based structure modelling and prediction of IDRs in the future.Communicated by Ramaswamy H. Sarma.

The parasitic worm product ES-62 protects the osteoimmunology axis in a mouse model of obesity-accelerated ageing.

Despite significant increases in human lifespan over the last century, adoption of high calorie diets (HCD) has driven global increases in type-2 diabetes, obesity and cardiovascular disease, disorders precluding corresponding improvements in healthspan. Reflecting that such conditions are associated with chronic systemic inflammation, evidence is emerging that infection with parasitic helminths might protect against obesity-accelerated ageing, by virtue of their evolution of survival-promoting anti-inflammatory molecules. Indeed, ES-62, an anti-inflammatory secreted product of the filarial nematode Acanthocheilonema viteae, improves the healthspan of both male and female C57BL/6J mice undergoing obesity-accelerated ageing and also extends median lifespan in male animals, by positively impacting on inflammatory, adipose metabolic and gut microbiome parameters of ageing. We therefore explored whether ES-62 affects the osteoimmunology axis that integrates environmental signals, such as diet and the gut microbiome to homeostatically regulate haematopoiesis and training of immune responses, which become dysregulated during (obesity-accelerated) ageing. Of note, we find sexual dimorphisms in the decline in bone health, and associated dysregulation of haematopoiesis and consequent peripheral immune responses, during obesity-accelerated ageing, highlighting the importance of developing sex-specific anti-ageing strategies. Related to this, ES-62 protects trabecular bone structure, maintaining bone marrow (BM) niches that counter the ageing-associated decline in haematopoietic stem cell (HSC) functionality highlighted by a bias towards myeloid lineages, in male but not female, HCD-fed mice. This is evidenced by the ability of ES-62 to suppress the adipocyte and megakaryocyte bias and correspondingly promote increases in B lymphocytes in the BM. Furthermore, the consequent prevention of ageing-associated myeloid/lymphoid skewing is associated with reduced accumulation of inflammatory CD11c+ macrophages and IL-1ß in adipose tissue, disrupting the perpetuation of inflammation-driven dysregulation of haematopoiesis during obesity-accelerated ageing in male HCD-fed mice. Finally, we report the ability of small drug-like molecule analogues of ES-62 to mimic some of its key actions, particularly in strongly protecting trabecular bone structure, highlighting the translational potential of these studies.

A netrin domain-containing protein secreted by the human hookworm Necator americanus protects against CD4 T cell transfer colitis.

The symbiotic relationships shared between humans and their gastrointestinal parasites present opportunities to discover novel therapies for inflammatory diseases. A prime example of this phenomenon is the interaction of humans and roundworms such as the hookworm, Necator americanus. Epidemiological observations, animal studies and clinical trials using experimental human hookworm infection show that hookworms can suppress inflammation in a safe and well-tolerated way, and that the key to their immunomodulatory properties lies within their secreted proteome. Herein we describe the identification of 2 netrin domain-containing proteins from the N. americanus secretome, and explore their potential in treating intestinal inflammation in mouse models of ulcerative colitis. One of these proteins, subsequently named Na-AIP-1, was effective at suppressing disease when administered prophylactically in the acute TNBS-induced model of colitis. This protective effect was validated in the more robust CD4 T cell transfer model of chronic colitis, where prophylactic Na-AIP-1 reduced T-cell-dependent type-1 cytokine responses in the intestine and the associated intestinal pathology. Mechanistic studies revealed that depletion of CD11c+ cells abrogated the protective anticolitic effect of Na-AIP-1. Next generation sequencing of colon tissue in the T-cell transfer model of colitis revealed that Na-AIP-1 induced a transcriptomic profile associated with the downregulation of metabolic and signaling pathways involved in type-1 inflammation, notably TNF. Finally, co-culture of Na-AIP-1 with a human monocyte-derived M1 macrophage cell line resulted in significantly reduced secretion of TNF. Na-AIP-1 is now a candidate for clinical development as a novel therapeutic for the treatment of human inflammatory bowel diseases.

Effect of different intensities of physical activity on cardiometabolic markers and vascular and cardiac function in adult rats fed with a high-fat high-carbohydrate diet.

BACKGROUND: Physical activity (PA) and diet are 2 lifestyle factors that affect cardiometabolic risk. However, data on how a high-fat high-carbohydrate (HFHC) diet influences the effect of different intensities of PA on cardiometabolic health and cardiovascular function in a controlled setting are yet to be fully established. This study investigated the effect of sedentary behavior, light-intensity training (LIT), and high-intensity interval training (HIIT) on cardiometabolic markers and vascular and cardiac function in HFHC-fed adult rats. METHODS: Twelve-week-old Wistar rats were randomly allocated to 4 groups (12 rats/group): control (CTL), sedentary (SED), LIT, and HIIT. Biometric indices, glucose and lipid control, inflammatory and oxidative stress markers, vascular reactivity, and cardiac electrophysiology of the experimental groups were examined after 12 weeks of HFHC-diet feeding and PA interventions. RESULTS: The SED group had slower cardiac conduction (p = 0.0426) and greater thoracic aortic contractile responses (p < 0.05) compared with the CTL group. The LIT group showed improved cardiac conduction compared with the SED group (p = 0.0003), and the HIIT group showed decreased mesenteric artery contractile responses compared with all other groups and improved endothelium-dependent mesenteric artery relaxation compared with the LIT group (both p < 0.05). The LIT and HIIT groups had lower visceral (p = 0.0057 for LIT, p = 0.0120 for HIIT) and epididymal fat (p < 0.0001 for LIT, p = 0.0002 for HIIT) compared with the CTL group. CONCLUSION: LIT induced positive adaptations on fat accumulation and cardiac conduction, and HIIT induced a positive effect on fat accumulation, mesenteric artery contraction, and endothelium-dependent relaxation. No other differences were observed between groups. These findings suggest that few positive health effects can be achieved through LIT and HIIT when consuming a chronic and sustained HFHC diet.

Hookworm Secreted Extracellular Vesicles Interact With Host Cells and Prevent Inducible Colitis in Mice.

Gastrointestinal (GI) parasites, hookworms in particular, have evolved to cause minimal harm to their hosts, allowing them to establish chronic infections. This is mediated by creating an immunoregulatory environment. Indeed, hookworms are such potent suppressors of inflammation that they have been used in clinical trials to treat inflammatory bowel diseases (IBD) and celiac disease. Since the recent description of helminths (worms) secreting extracellular vesicles (EVs), exosome-like EVs from different helminths have been characterized and their salient roles in parasite-host interactions have been highlighted. Here, we analyze EVs from the rodent parasite Nippostrongylus brasiliensis, which has been used as a model for human hookworm infection. N. brasiliensis EVs (Nb-EVs) are actively internalized by mouse gut organoids, indicating a role in driving parasitism. We used proteomics and RNA-Seq to profile the molecular composition of Nb-EVs. We identified 81 proteins, including proteins frequently present in exosomes (like tetraspanin, enolase, 14-3-3 protein, and heat shock proteins), and 27 sperm-coating protein-like extracellular proteins. RNA-Seq analysis revealed 52 miRNA species, many of which putatively map to mouse genes involved in regulation of inflammation. To determine whether GI nematode EVs had immunomodulatory properties, we assessed their potential to suppress GI inflammation in a mouse model of inducible chemical colitis. EVs from N. brasiliensis but not those from the whipworm Trichuris muris or control vesicles from grapes protected against colitic inflammation in the gut of mice that received a single intraperitoneal injection of EVs. Key cytokines associated with colitic pathology (IL-6, IL-1ß, IFNγ, and IL-17a) were significantly suppressed in colon tissues from EV-treated mice. By contrast, high levels of the anti-inflammatory cytokine IL-10 were detected in Nb-EV-treated mice. Proteins and miRNAs contained within helminth EVs hold great potential application in development of drugs to treat helminth infections as well as chronic non-infectious diseases resulting from a dysregulated immune system, such as IBD.