Schistosomiasis (SM) is a parasitic disease caused by trematoda of the species Schistosoma mansoni. At least 800 million individuals in tropical regions worldwide are at risk of infection (1). This disease is the third most prevalent endemic parasitic disease associated with pulmonary hypertension in the world (2). Approximately 10% of infected individuals will present with the hepatosplenic clinical form, a fundamental condition for the development of pulmonary hypertension (PAH); among those presenting with the hepatosplenic form, 8–10% will develop PAH (3–5).

Parasite transmission is dependent on social factors, such as the presence of eggs of S. mansoni, the gastropod Biomphalaria in fresh water, and failure in populational educative strategies (6). Individuals infected by the parasite present with dermatitis, fever, and toxemic manifestations during the acute phase, as well as digestive and/or hepatointestinal, hepatosplenic, pulmonary arterial hypertension (PAH), glomerulonephritis, and neuroschistosomiasis during the chronic phase (7, 8). The main chronic manifestations of SM are related to the presence of S. mansoni eggs in distinct human organs, in which the immune response moves toward the egg, causing host tissue destruction and collagen/fibrosis deposition in a granulomatous process (9, 10).

PAH is a rare disease characterized by increased pressure in the pulmonary circulatory system, and consequent right atrial and ventricular overload. It is hemodynamically defined as mean pulmonary artery pressure ≥ 20 mmHg at rest with an occlusion pulmonary pressure < or = 15 mmHg (11).

Pulmonary reactions against S. mansoni eggs do not fit all the described vascular remodeling changes. Severe portal hypertension has been reported in patients with minimal liver fibrosis. However, when imaging techniques or histological evaluations are performed, they do not match the portal hypertension severity diagnosis (8, 12–14). A marked increase in hepatic and PAH resistance is a primary factor in portal hypertension and pulmonary development, respectively. Some of the liver and lung alterations are caused by vascular occlusion in the egg, embolization, and obstruction, which distort the liver architecture. This causes fibrosis, vascular remodelling, sinusoidal and vascular capillarization, and an unreversed increase in microcirculatory resistance. These dynamic mechanisms are modulated by adrenergic, renin-angiotensin and endothelin, and inflammatory systems, mediated by vasoactive neurotransmitters and peptides such as norepinephrine, angiotensin II, endothelin I, TGF-β, Th1-, Th2, Th17-like cells and cytokines. This causes vasodilatation/vasoconstriction misbalancing, leading to an increase in hepatic and pulmonary vascular tone (2, 15–18). Antibodies such as immunoglobulin (Ig)G1 and IgG4 are anti-S. mansoni antigens; thus, they have gained attention in the context of vaccine strategies (19, 20) and have been identified as key contributors to the worsening clinical prognosis of SM (21–23). However, in the presence of S. mansoni, self-reactive antibodies or fAABs can cause unexpected reactions in experimental and human tissues, resulting in adverse disturbances (24–26).

This study aimed to investigate and characterize the generation of fAABs/GPCRs in patients with SM presenting with acute and chronic (hepatic and pulmonary) forms of the disease.

In this study, we identified and characterized the fAAB patterns in individuals with acute and chronic SM. In the sera of chronic subjects, three fAABs against GPCR were identified recognizing α1-adrenoceptor, endothelin-1, and angiotensin AT1 receptors.

The agonist-like effects triggered by fAABs were blocked by specific antagonists of the α1-adrenoceptor (1 µM urapidil) and angiotensin-II AT1 receptors (1 µM losartan) ( Figure 1A ) and, of the endothelin-1 receptor (0.1 µM BQ123) ( Figure 1B ). In addition, we identified fAABs in the sera of SM individuals with PAH and hepatointestinal clinical forms. In those individuals in the acute phase of SM, only fAABs against α1-adrenergic ( Figure 2A ) and endothelin-1 ( Figure 2B ) receptors were changed, but not fAABs against angiotensin-II AT1 receptor ( Figure 2C ). There were no fAABs against GPCR in the uninfected subjects.

In another set of experiments, we evaluated the binding sites of fAABs on the second extracellular loop of the receptors and IgG subclasses. Figure 3A shows the epitope analysis for the α1-adrenoceptor, whose fAABs were neutralized by the peptide APEDE, located in the second extracellular loop peptide of the adrenoceptor. The other short overlapping peptides did not exhibit significant inhibitory effects ( Figure 3A ). The endothelin-1 receptor was neutralized by peptides EQHKTCM and MLNATSK; however, since both overlapped, we assumed that against endothelin-1 receptors, fAABs recognized the largest epitope ( Figure 3B ). In contrast, fAABs anti-angiotensin II AT1 receptors were neutralized by the N-terminal part of the second extracellular loop of the peptide VFFIEN and, by one peptide of the middle region of the second extracellular loop ITVCAF ( Figure 3C ). The other overlapping short peptide in the second extracellular domain of the AT1 receptor did not have a significant effect.

Next, the three identified fAABs were precipitated with monoclonal mouse anti-human antibodies belonging to the IgG1, IgG2, IgG3, and IgG4 subclasses. An anti-mouse polyclonal antibody was added to enlarge the mouse anti-human antibody complex. This antibody complex was centrifuged at 16,030xg and the supernatant was used in the experiments. As shown in Figure 4A , the activity of the α1-adrenoceptor fAABs was neutralized by mouse anti-human antibodies of the IgG1 subtype. In contrast, AT1 receptor fAABs were neutralized by mouse anti-human IgG1 and IgG3 antibodies ( Figure 4B ). The activity of the agonist-like AAB against the ETA1 receptor was blocked by anti-human IgG1, except in one patient who developed the IgG3 subtype fAAB ( Figure 4C ).

In the current investigation, individuals chronically infected by S. mansoni developed fAABs against three different GPCRs and, in this sense, we evaluated them in the presence of agonist-like fAABs against α1-adrenoceptor, ETA1, and AT1 receptors.

GPCRs are seven-transmembrane receptor domains that can use GTP-binding proteins for signal transduction and are involved in various physiological and pathophysiological processes. Studies have demonstrated the participation of GPCRs in tropical disease agents such as protozoan and trematode infections (29, 30). However, no investigation has been performed to identify fAABs targeting GPCRs in (acute and chronic) SM, or whether they exert disturbances in distinct clinical forms of the disease.

Individuals in acute SM were able to generate fAABs against the α1-adrenoceptor and ETA1 receptor, but not against the AT1 receptor. The activity of fAABs was significantly lower than that of chronic SM. Interestingly, the same fAAB pattern was observed in individuals diagnosed with pulmonary hypertension without SM (31). It seems that vasoactive fAABs, like α1-adrenoceptor or endothelin ETA-receptor, may play an important role in pulmonary hypertension pathogenesis, since the removal of such fAAB by immunoadsorption promotes a fast reduction in the dilated diameter of the right atrium and ventricle, reducing the resistance of the pulmonary vessels and increasing the oxygen saturation. Symptoms of PAH have also been observed in individuals chronically infected with S. mansoni. Therefore, we assume that the vasoactive fAABs against the α1-adrenoceptor and the endothelin-1 ETA receptor were important players in PAH related to SM, since fAABs activate the receptors as classical agonists. Moreover, these fAABs could be used as an early marker for patients with pulmonary arterial hypertension, in the late phase of SM. In addition, fAABs (eg. GPCR-autoantibodies) can permanently stimulate cells without desensitization or internalization of receptors (32).

Additionally, a third vasoactive fAAB that recognized the angiotensin II (AT1) receptor was associated with chronic hepatosplenic individuals. This idea is supported by the observation of AT1 receptor activation in inflamed and ischemic kidney arteries in rats (33). In healthy kidney arteries, the authors demonstrated that fAABs against the AT1 receptor did not have any effect, although angiotensin II was able to activate this receptor. AT1 fAABs may also play an important role in preeclampsia and kidney diseases (34).

Moreover, AT1- and α1-adrenoceptor fAABs can maturate and degranulate mast cells in cultured neonatal rat heart cardiomyocytes which can release several cytokine and enzymes such as TNF, chymase, tryptase, and histamine (35). Additionally, AT1 receptor fAABs increase the adhesion of human monocytes to endothelial cells and induce the formation of tissue factors in other immune cells. Both effects were blocked by losartan, an AT1 receptor antagonist, and were observed in monocytes of normotensive and hypertensive individuals (36). Previous studies showed that fAABs against the AT1 receptor elevated the expression of tissue factor (TF) in monocytes, an essential element of the coagulation cascade. Furthermore, AT1 receptor fAABs have also been identified in preeclamptic patients, causing activation and expression of TFs in vascular cells (37) and in thrombocytes, mediating signalling to platelet-leukocyte-endothelial cell interactions (38).

The fAABs may signalize the presence and the intensity of the immune response and, consequently, can act as biomarkers of prognosis in autoimmune diseases (type 1 diabetes, celiac diseases, thyroiditis) or even in cardiovascular and parasite evolutive diseases (39–41). The fAABs can be identified in biological samples years before the development of the disease, which means they should be able to predict diseases. The goal of predicting diseases is the possibility of preventive intervention using pharmacology or other biotechnologies to block their actions. The matter of fact is that understanding the role and specificity of fAABs in distinct non-autoimmune diseases, such as what we are showing in schistosomiasis, opens a new window of clinical opportunity. However, for a conclusive clinical application of fAABs, three parameters must be quantified – identification of its sensitivity and specificity of prediction and, of its positive predictive values.

Altogether, beyond Schistosoma egg embolization with portal and pulmonary circulation obstruction, there will be focal arteritis, vessel destruction, and plexiform lesions. These pathological conditions suggest the release of several cytokines and neurohormones from the adrenergic, renin-angiotensin, and endothelin systems. In addition, vascular heparan sulfate proteoglycans and endothelial adhesion molecules may be overexpressed after fAAB activity, contributing to an increase in (i) vascular resistance, (ii) vasoconstriction, (iii) coagulation processes with microclot formation, and portal and PAH (42). Therefore, this study assumed that individuals with chronic schistosomiasis produce fAABs against GPCR, whose activity might be involved in the development of hypertensive forms of the disease. Under S. mansoni infection conditions, fAAB can disturb the neurohormonal balance and represents a key player in the pathophysiological development of the liver and pulmonary clinical forms of SM.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving humans were approved by UFMG Research Ethics Committee (CAAE-25095814.0.0000.5149 COEP-UFMG) and conducted according to the guidelines in the Declaration of Helsinki (2013) from the World Medical Association. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. The animal study was approved by Ethics Committee in Research (# Y9008/12 and # Tötungsanzeige Y9004/19) at the Max Delbrück Centre for Molecular Medicine Berlin, Germany. The study was conducted in accordance with the local legislation and institutional requirements.

FB: Conceptualization, Methodology, Resources, Visualization, Writing – original draft, Writing – review & editing. JL: Conceptualization, Formal analysis, Methodology, Writing – original draft, Writing – review & editing. RS: Data curation, Formal analysis, Funding acquisition, Methodology, Writing – original draft, Writing – review & editing. JM: Conceptualization, Formal analysis, Funding acquisition, Methodology, Project administration, Writing – original draft, Writing – review & editing. AT: Formal analysis, Funding acquisition, Methodology, Visualization, Writing – original draft, Writing – review & editing. GW: Conceptualization, Formal analysis, Funding acquisition, Methodology, Resources, Writing – original draft, Writing – review & editing.