Over one-third of the world population is infected with parasitic helminths, Strongyloides ssp. accounting for approximately 30-100 million infected people. In this study, we employ the experimental system of murine Strongyloides ratti infection to investigate the interaction of this pathogenic nematode with its mammalian host. We provide a comprehensive kinetic description of the immune response to S. ratti infection that was reflected by induction of antigen-specific IgM and IgG1, mast cell activation and a Th2-like cytokine response. T cells derived from infected mice displayed an increased IL-3, IL-4, IL-5, IL-13 and IL-10 response to CD3-engagement in comparison with T cells derived from naïve mice. The IFN-gamma response to CD3-engagement that was well detectable in T cells derived from naïve mice, however, was suppressed in T cells derived from infected mice. Both, the induction of the S. ratti-specific Th2 response and the suppression of pro-inflammatory cytokines were transient and observed in strict correlation to the course of infection and the number of infective larvae used. Finally, comparing artificial infections induced by subcutaneous injection of larvae to natural infections, we observed similar antigen-specific T cell responses although the natural infection led to a significantly lower worm burden.

To escape expulsion by their host’s immune system, pathogenic nematodes exploit regulatory pathways that are intrinsic parts of the mammalian immune system, such as regulatory T cells (Tregs). Using depletion of Treg mice, we showed that Foxp3+ Treg numbers increased rapidly during infection with the nematode Strongyloides ratti. Transient depletion of Tregs during the first days of infection led to dramatically reduced worm burden and larval output, without aggravation of immune pathology. The transient absence of Tregs during primary infection did not interfere with the generation of protective memory. Depletion of Tregs at later time points of infection (i.e., day 4) did not improve resistance, suggesting that Tregs exert their counterregulatory function during the priming of S. ratti-specific immune responses. Improved resistance upon early Treg depletion was accompanied by accelerated and prolonged mast cell activation and increased production of types 1 and 2 cytokines. In contrast, the blockade of the regulatory receptor CTLA-4 specifically increased nematode-specific type 2 cytokine production. Despite this improved immune response, resistance to the infection was only marginally improved. Taken together, we provide evidence that Treg expansion during S. ratti infection suppresses the protective immune response to this pathogenic nematode and, thus, represents a mechanism of immune evasion.

The frequent von Willebrand factor (VWF) variant p.Phe2561Tyr is located within the C4 domain, which also harbors the platelet GPIIb/IIIa-binding RGD sequence. To investigate its potential effect on hemostasis, we genotyped 865 patients with coronary artery disease (CAD), 915 with myocardial infarction (MI), and 417 control patients (Ludwigshafen Risk and Cardiovascular Health Study) and performed functional studies of this variant. A univariate analysis of male and female carriers of the Tyr2561 allele aged 55 years or younger revealed an elevated risk for repeated MI (odds ratio, 2.53; 95% confidence interval [CI], 1.07-5.98). The odds ratio was even higher in females aged 55 years or younger, at a value of 5.93 (95% CI, 1.12-31.24). Cone and plate aggregometry showed that compared with Phe2561, Tyr2561 was associated with increased platelet aggregate size both in probands’ blood and with the recombinant variants. Microfluidic assays revealed that the critical shear rate for inducing aggregate formation was decreased to 50% by Tyr2561 compared with Phe2561. Differences in C-domain circular dichroism spectra resulting from Tyr2561 suggest an increased shear sensitivity of VWF as a result of altered association of the C domains that disrupts the normal dimer interface. In summary, our data emphasize the functional effect of the VWF C4 domain for VWF-mediated platelet aggregation in a shear-dependent manner and provide the first evidence that a functional variant of VWF plays a role in arterial thromboembolism.

Graphical abstract

No abstract

The multimeric plasma glycoprotein von Willebrand factor (VWF) is best known for recruiting platelets to sites of injury during primary hemostasis. Generally, mutations in the VWF gene lead to loss of hemostatic activity and thus the bleeding disorder von Willebrand Disease. By employing cone and platelet aggregometry and microfluidic assays, we uncovered a platelet glycoprotein (GP)IIb/IIIa-dependent prothrombotic gain-of-function (GOF) for variant p.Pro2555Arg, located in the C4-domain, leading to an increase in platelet aggregate size. We performed complementary biophysical and structural investigations using circular dichroism spectra, small angle X-ray scattering, NMR spectroscopy, molecular dynamics simulations on the single C4-domain and dimeric wildtype and p.Pro2555Arg constructs. C4-p.Pro2555Arg retained the overall structural conformation with minor populations of alternative conformations exhibiting increased hinge flexibility and slow conformational exchange. The dimeric protein becomes disordered and more flexible. Our data suggest that the GOF is not affecting the binding affinity of the C4-domain for GPIIb/IIIa. Instead, the increased VWF dimer flexibility enhances temporal accessibility of platelet binding sites. Using an interdisciplinary approach, we revealed that p.Pro2555Arg is the first VWF variant, which increases platelet aggregate size and show a shear-dependent function of the VWF stem region, which can become hyperactive through mutations. Prothrombotic GOF variants of VWF are a novel concept of a VWF-associated pathomechanism of thromboembolic events, which is of general interest to vascular health but which is not yet considered in diagnostics. Thus, awareness should be raised for the risk they pose. Furthermore, our data implicate the C4-domain as a novel anti-thrombotic drug target.

The frequent von Willebrand factor (VWF) variant p.Phe2561Tyr is located within the C4 domain, which also harbors the platelet GPIIb/IIIa-binding RGD sequence. To investigate its potential effect on hemostasis, we genotyped 865 patients with coronary artery disease (CAD), 915 with myocardial infarction (MI) and 417 controls (Ludwigshafen Risk and Cardiovascular Health Study (LURIC)) and performed functional studies of this variant. A univariate analysis of male and female carriers of the Tyr2561 allele ≤55 years revealed an elevated risk for repeated MI (Odds ratio: 2.53, 95% CI:1.07-5.98). The Odds ratio was even higher in females ≤55 years at a value of 5.93 (95% CI:1.12-31.24). Cone and plate aggregometry showed, that compared to Phe2561, Tyr2561 was associated with increased platelet aggregate size, both in probands' blood and with the recombinant variants. Inhibition of the VWF-GPIIb/IIIa interaction abrogated the effect. Microfluidic assays revealed that the critical shear rate for inducing aggregate formation was decreased to 50% by Tyr2561 compared to Phe2561. Differences in C-domain circular dichroism spectra resulting from Tyr2561 suggest an increased shear sensitivity of VWF, as a result of altered association of the C-domains that disrupts the normal dimer interface. In summary, our data emphasize the functional impact of the VWF C4-domain for VWF-mediated platelet aggregation in a GPIIb/IIIa-dependent manner and provide the first evidence that a functional variant of VWF plays a role in arterial thromboembolism. Disclosures Schneppenheim: SHIRE: Consultancy; CSL Behring: Consultancy.