Trichuris trichiura egg extract proteome reveals potential diagnostic targets and immunomodulators.

Embryonated eggs are the infectious developmental stage of Trichuris trichiura and are the primary stimulus for the immune system of the definitive host. The intestinal-dwelling T. trichiura affects an estimated 465 million people worldwide with an estimated global burden of disease of 640 000 DALYs (Disability Adjusted Life Years). In Latin America and the Caribbean, trichuriasis is the most prevalent soil transmitted helminthiasis in the region (12.3%; 95% CI). The adverse health consequences impair childhood school performance and reduce school attendance resulting in lower future wage-earning capacity. The accumulation of the long-term effects translates into poverty promoting sequelae and a cycle of impoverishment. Each infective T. trichiura egg carries the antigens needed to face the immune system with a wide variety of proteins present in the shell, larvae's surface, and the accompanying fluid that contains their excretions/secretions. We used a proteomic approach with tandem mass spectrometry to investigate the proteome of soluble non-embryonated egg extracts of T. trichiura obtained from naturally infected African green monkeys (Chlorocebus sabaeus). A total of 231 proteins were identified, 168 of them with known molecular functions. The proteome revealed common proteins families which are known to play roles in energy and metabolism; the cytoskeleton, muscle and motility; proteolysis; signaling; the stress response and detoxification; transcription and translation; and lipid binding and transport. In addition to the study of the T. trichiura non-embryonated egg proteome, the antigenic profile of the T. trichiura non-embryonated egg and female soluble proteins against serum antibodies from C. sabaeus naturally infected with trichuriasis was investigated. We used an immunoproteomic approach by Western blot and tandem mass spectrometry from the corresponding SDS-PAGE gels. Vitellogenin N and VWD and DUF1943 domain containing protein, poly-cysteine and histidine tailed protein isoform 2, heat shock protein 70, glyceraldehyde-3-phosphate dehydrogenase, actin, and enolase, were among the potential immunoactive proteins. To our knowledge, this is the first study on the T. trichiura non-embryonated egg proteome as a novel source of information on potential targets for immunodiagnostics and immunomodulators from a neglected tropical disease. This initial list of T. trichiura non-embryonated egg proteins (proteome and antigenic profile) can be used in future research on the immunobiology and pathogenesis of human trichuriasis and the treatment of human intestinal immune-related diseases.

Antihypertensive effects of Trichiurus lepturus myosin hydrolysate in spontaneously hypertensive rats.

In this study, the blood pressure-lowering effects of Trichiurus lepturus myosin hydrolysate (TMH) and its possible mechanism were investigated in spontaneously hypertensive rats (SHRs). After gavage administration of TMH for 4 h, systolic blood pressure (SBP) was significantly decreased in SHRs. Furthermore, the SBP of SHRs remained low at 1 month after daily TMH treatment at 400 mg kg-1. Meanwhile, plasma levels of angiotensin II, bradykinin and nitric oxide in SHRs were ameliorated by TMH. Western blotting also suggested that TMH down-regulated the expression of ICAM-1 and VCAM-1, indicating a strong anti-inflammatory effect. Additionally, nitrotyrosine and collagen I were down-regulated, revealing a significant anti-oxidant effect of TMH. No obvious side effects or toxicity were observed in normal Wistar rats given TMH. Various pathogenic factors related to hypertension were improved by TMH, which may explain the underlying mechanism by which TMH synergistically reduces blood pressure.

Treatment with Trichuris suis soluble products during monocyte-to-macrophage differentiation reduces inflammatory responses through epigenetic remodeling.

Helminths have strong immunoregulatory properties that may be exploited in treatment of chronic immune disorders, such as multiple sclerosis and inflammatory bowel disease. Essential players in the pathogenesis of these diseases are proinflammatory macrophages. We present evidence that helminths modulate the function and phenotype of these innate immune cells. We found that soluble products derived from the Trichuris suis (TsSP) significantly affect the differentiation of monocytes into macrophages and their subsequent polarization. TsSPs reduce the expression and production of inflammatory cytokines, including IL-6 and TNF, in human proinflammatory M1 macrophages. TsSPs induce a concomitant anti-inflammatory M2 signature, with increased IL-10 production. Furthermore, they suppress CHIT activity and enhance secretion of matrix metalloproteinase 9. Short-term triggering of monocytes with TsSPs early during monocyte-to-macrophage differentiation imprinted these phenotypic alterations, suggesting long-lasting epigenetic changes. The TsSP-induced effects in M1 macrophages were completely reversed by inhibiting histone deacetylases, which corresponded with decreased histone acetylation at the TNF and IL6 promoters. These results demonstrate that TsSPs have a potent and sustained immunomodulatory effect on human macrophage differentiation and polarization through epigenetic remodeling and provide new insights into the mechanisms by which helminths modulate human immune responses.-Hoeksema, M. A., Laan, L. C., Postma, J. J., Cummings, R. D., de Winther, M. P. J., Dijkstra, C. D., van Die, I., Kooij, G. Treatment with Trichuris suis soluble products during monocyte-to-macrophage differentiation reduces inflammatory responses through epigenetic remodeling.

The helminth Trichuris suis suppresses TLR4-induced inflammatory responses in human macrophages.

Recent clinical trials in patients with inflammatory diseases like multiple sclerosis (MS) or inflammatory bowel disease (IBD) have shown the beneficial effects of probiotic helminth administration, although the underlying mechanism of action remains largely unknown. Potential cellular targets may include innate immune cells that propagate inflammation in these diseases, like pro-inflammatory macrophages. We here investigated the effects of the helminth Trichuris suis soluble products (SPs) on the phenotype and function of human inflammatory (granulocyte-macrophage colony-stimulating factor (GM-CSF)-differentiated) macrophages. Interestingly, we here show that T. suis SPs potently skew inflammatory macrophages into a more anti-inflammatory state in a Toll-like receptor 4 (TLR4)-dependent manner, and less effects are seen when stimulating macrophages with TLR2 or -3 ligands. Gene microarray analysis of GM-CSF-differentiated macrophages further revealed that many TLR4-induced inflammatory mediators, including interleukin (IL)-12B, CCL1 and CXCL9, are downregulated by T. suis SPs. In particular, we observed a strong reduction in the expression and function of P2RX7, a purinergic receptor involved in macrophage inflammation, leading to reduced IL-1ß secretion. In conclusion, we show that T. suis SPs suppress a broad range of inflammatory pathways in GM-CSF-differentiated macrophages in a TLR4-dependent manner, thereby providing enhanced mechanistic insight into the therapeutic potential of this helminth for patients with inflammatory diseases.

Trichuris suis: a secretory chymotrypsin/elastase inhibitor with potential as an immunomodulator.

A serine protease inhibitor, termed TsCEI, was purified from adult-stage Trichuris suis by acid precipitation, affinity chromatography (elastase-agarose), and reverse-phase HPLC. The molecular weight of TsCEI was estimated at 6.437 kDa by laser desorption mass spectrometry. TsCEI potently inhibited both chymotrypsin (K(i) = 33.4 pM) and pancreatic elastase (K(i) = 8.32 nM). Neutrophil elastase, chymase (mouse mast cell protease-1, mMCP-1), and cathepsin G were also inhibited by TsCEI, whereas trypsin, thrombin, and factor Xa were not. The cDNA-derived amino acid sequence of the mature TsCEI consisted of 58 residues including 9 cysteine residues with a molecular mass of 6.196 kDa. TsCEI displayed 48% sequence identity to a previously characterized trypsin/chymotrypsin inhibitor of T. suis, TsTCI. TsCEI showed 36% sequence identity to a protease inhibitor from the hemolymph of the honeybee Apis mellifera. Sequence similarity was also detected with the trypsin/thrombin inhibitor of the European frog Bombina bombina, the elastase isoinhibitors of the nematode Anisakis simplex, and the chymotrypsin/elastase and trypsin inhibitors of the nematode Ascaris suum. The inhibitors of T. suis, an intestinal parasite of swine, may function as components of a parasite defense mechanism by modulating intestinal mucosal mast cell-associated, protease-mediated, host immune responses.

Trichuris suis: A secretory serine protease inhibitor.

A trypsin inhibitor was identified in extracts of adult Trichuris suis and culture fluids from 24-h in vitro cultivation of adult parasites. The inhibitor was isolated by acid precipitation, affinity chromatography (trypsin-agarose), and reverse phase HPLC as a single polypeptide with a molecular weight estimated at 6.6 kDa by laser desorption mass spectrometry. The purified inhibitor associated strongly with trypsin (equilibrium dissociation inhibitory constant (K(j)) of 3.07 nM) and chymotrypsin (K(j) = 24.5 nM) and was termed TsTCI. Elastase, thrombin, and factor Xa were not inhibited. The cDNA-derived amino acid sequence of the mature TsTCI consisted of 61 residues including 8 cysteine residues with a molecular mass of 6.687 kDa. The N-terminal region of TsTCI (46 residues) showed limited homology (36%) to a protease inhibitor from the hemolymph of the honeybee Apis mellifera, which is considered to be a member of the Ascaris inhibitor family. However, TsTCI did not display sequence homology with other members of this family or the distinctive cysteine residue pattern which distinguishes this family. However, similarity of a region of TsTCI (11 residues) with the reactive site regions of inhibitors from the nematodes Ascaris suum, Anisakis simplex, and Ancylostoma caninum was apparent.