Experimental Schistosoma haematobium pulmonary hypertension

Abstract

Whether all Schistosoma species cause pulmonary hypertension (PH) is unclear. Experimentally exposing mice to Schistosoma haematobium eggs caused PH, which was less severe than that induced by S. mansoni exposure. These findings align with the relatively uncommon reports of pulmonary arterial hypertension associated with S. haematobium.

INTRODUCTION

Schistosomiasis is a major cause of pulmonary arterial hypertension (PAH) worldwide. Schistosoma mansoni is the most prevalent species worldwide and causes intestinal schistosomiasis, whereas Schistosoma haematobium is the second most common and causes urinary schistosomiasis. Most schistosomiasis-PAH clinical reports involve S. mansoni, with limited information on PAH due to other species. One example is a 1990 report of a 21-year-old woman from Senegal presenting with dyspnea, S. haematobium eggs in the urine, and a mean pulmonary artery pressure of 62 mmHg on right heart catheterization. Another is a 1964 report of two cases in Iraq, 25- and 40-year-old women, both presenting with dyspnea and edema, S. haematobium eggs in the urine or sputum, and right heart enlargement on chest X-ray and electrocardiography.

Mice exposed to S. mansoni can model pulmonary hypertension (PH), with the severity correlating to the number of intrapulmonary eggs, the degree of lung inflammation, and proximity between vessels and regions of inflammation. Mice exposed to Schistosoma japonicum, endemic in Asia and the third most common species, also develop PH, albeit more mild than that induced by S. mansoni. Here, we tested ifS. haematobium exposed mice can manifest PH, and evaluated the severity relative to other Schistosoma species.

METHODS

Animal models

All studies were approved by the UCSF IACUC, Protocol #AN181431. S. haematobium eggs were harvested from S. haematobium infected hamsters, whereas S. mansoni eggs were harvested from S. mansoni infected mice, provided by the Biomedical Research Institute, using standard techniques. Experimental mice were C57Bl6/J from Jackson, 6 to 8 weeks old, and female. Mice were intraperitoneally sensitized with 240 Schistosoma eggs per gram body weight, followed 14 days later by intravenous challenge with 175 Schistosoma eggs per gram body weight.

Experimental endpoints

Right heart catheterization was conducted to measure right ventricle systolic pressure (RVSP) and right ventricle (RV) hypertrophy using a standard open-chest technique with a pressure-volume catheter (Millar). The left lung was formalin-fixed and paraffin-embedded (FFPE) and the right lung was frozen. FFPE tissue was stained for α-smooth muscle actin by published protocol. Images were captured, the vascular media identified, the average outer and internal medial layer radii quantified, and the fractional medial thickness calculated as the radii difference divided by the external radius. We used the optical rotator to estimate peri-egg granuloma volumes from hematoxylin and eosin-stained slides. Whole-lung lysates were prepared from frozen tissue macerated and sonicated in radioimmunoprecipitation assay buffer containing anti-proteases, and total protein content (Bradford assay) and interleukin (IL)-4 and IL-13 concentrations assessed by enzyme-linked immunosorbent assay (ELISA) (M4000B and M1300CB; R&D Systems).

Statistics

Prism (v9.5, GraphPad) was used. Normality was determined using Shapiro–Wilk testing, pairwise comparisons using unpaired t test or Mann–Whitney U test, and multiple comparisons using analysis of variance (ANOVA) or ANOVA on ranks, with post-hoc testing. Details are in the text and figure legend. p < 0.05 was considered statistically significant. The data are available from the authors upon reasonable request.

RESULTS

We adapted our PH mouse model to use S. haematobium eggs. Mice were intraperitoneally sensitized with eggs, and 2 weeks later intravenously challenged with eggs by tail vein injection. Female mice were used, as Schistosoma-PAH occurs more commonly in females, and we have not previously found differences between males and females. One week later, right heart catheterization and tissue collection was performed (Figure ). This approach models egg embolization to the lungs as occurs in chronic disease. We found that S. haematobium-exposed mice exhibited PH as compared to unexposed mice, evidenced by mean RVSP elevation from 24.3 to 26.9 mmHg (unpaired t test p = 0.0113; Figure ). There was no change in RV hypertrophy (Figure ), which is generally mild in this model, presumably related to the relatively short time course. An increase in medial thickness was observed when quantitatively analyzing the pulmonary vasculature (Figure ).

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We previously found S. japonicum causes milder PH than S. mansoni. Now comparing S. haematobium-exposed mice, we observed a milder PH phenotype compared to S. mansoni, and similar to that previously observed with S. japonicum: within the three groups, one-way ANOVA p = 0.0003, and post-hoc testing corrected for multiple comparisons was p = 0.005 between S. mansoni and haematobium; p = 0.0029 between S. mansoni and japonicum; and p = 0.99 between S. haematobium and japonicum (Figure ).

We quantified inflammation in whole lung lysates by ELISA for the Type 2 cytokines IL-4 and IL-13. S. haematobium-exposed mice showed a notable increase in both IL-4 and IL-13 compared to controls (Figure ). Compared to S. mansoni, IL-4 and IL-13 concentrations were both lower. Analyzing peri-egg granuloma volumes revealed that S. haematobium-exposed mice had smaller granulomas than S. mansoni-exposed mice, and comparable to S. japonicum-exposed mice (Figure ).

As dual exposure to S. haematobium and mansoni occurs, we explored the potential for cross sensitization. Mice sensitized to S. mansoni and then challenged with S. haematobium displayed a PH severity comparable to mice sensitized and challenged solely with S. haematobium, indicating S. mansoni's ability to substitute for S. haematobium for sensitization (Figure ). However, mice sensitized to S. haematobium and challenged with S. mansoni had PH comparable to the S. haematobium-only group, and milder than S. mansoni-only, indicating that S. haematobium cannot effectively replace S. mansoni during sensitization.

DISCUSSION

In our study, mice experimentally exposed to S. haematobium developed PH, with a phenotype milder than that induced by S. mansoni and comparable to that induced by S. japonicum. The severity of Schistosoma-PH correlated with the degree of inflammation, as has been observed in other contexts. A prior mouse study also reported S. haematobium causes smaller granulomas than S. mansoni. An unexpected observation wasS. haematobium is less effective than S. mansoni at sensitizing before challenge with S. mansoni, whereas S. haematobium and S. mansoni were equally effective at sensitizing for S. haematobium challenge.

The three predominant Schistosoma species globally are S. mansoni, haematobium, and japonicum. There are other less common species restricted to specific locales, or that infect animals. Only S. haematobium causes urinary disease, as it homes to the bladder venous plexus where it lays its eggs. The other Schistosoma species home to the portal venous system. Despite differing clinical manifestations, S. mansoni and haematobium share 89% genetic similarity (synteny), and trace to a common ancestor 1 million years ago. In contrast, S. japonicum's shared ancestry with S. mansoni dates back 3.8 million years, with a genetic similarity of only 67% and 52% to S. mansoni and haematobium, respectively. Differences in egg antigenicity likely modulates inflammation, and thus PH; this includes antigen ability to bind host immune receptors, and antigen quantity released by the egg.

We previously observed that S. mansoni and japonicum effectively cross-sensitize for each other. The observed relative inability of S. haematobium to cross-sensitize for S. mansoni is paradoxical given these two species have a closer genetic relationship than S. japonicum. Investigating this incongruence further, possibly through in vitro systems, might shed light on mechanisms by which the immune system responds to Schistosoma and drives PAH.

The absence of well-documented schistosomiasis-PAH reports from regions of the world where only S. haematobium is endemic suggests that this species might less frequently lead to PAH than S. mansoni. Alternatively, there could be limited testing or reporting in these regions. Publication of more reports or systematic screening would be helpful to determine disease prevalence, including distinguishing specific species and evaluating for dual infection where appropriate. For example, Farrag et al. screened 370 individuals in the Nile River delta, where both species are endemic, finding 8.6% of seropositive individuals had an estimated RVSP > 40 mmHg by echocardiography. The serologic test they used detects antibodies against both S. haematobium and mansoni, and clinical characteristics of the cohort included both hematuria (as from S. haematobium) and hepatomegaly (as from S. mansoni).

It is likely that chronic infection with any Schistosoma species can result in organ fibrosis and egg embolization through shunts to systemic veins and the lungs. In chronic S. haematobium infection, the bladder is fibrosed; with intestinal Schistosoma species, preportal fibrosis develops. Differences in fibrosis and vascular networks between the liver and bladder could result in different egg burdens in the lungs, and thus disease prevalence.

A limitation of the Schistosoma mouse model is the number of eggs administered by body weight is much greater than that present in humans. In mice, PH severity correlates with egg burden. The higher dose facilitates studying experimental PH in a feasible duration of weeks, rather than the years it takes infected humans to develop PAH. Another limitation is we administered the same number of eggs for all Schistosoma species, whereas fecundity varies, with S. japonicum particularly laying more eggs per worm pair.

In summary, our findings identify that mice exposed to S. haematobium can develop PH, albeit milder than S. mansoni-induced PH. This aligns with the sparse clinical documentation of PAH cases associated with S. haematobium.

AUTHOR CONTRIBUTIONS

Brian B. Graham conceived the study. Biruk Kassa, Rahul Kumar, and Brian B. Graham designed the study. Biruk Kassa, Dara C. Fonseca-Balladares, Rahul Kumar, Michael H. Lee, Claudia Mickael, Linda Sanders, and Kevin Nolan performed experiments. All authors interpreted the data. Brian B. Graham wrote the manuscript. All authors reviewed and approved the manuscript.

ACKNOWLEDGMENTS

The reagent was provided by the Schistosomiasis Resource Center of the Biomedical Research Institute (Rockville, MD) through NIH-NIAID Contract HHSN272201700014I. This work was supported by grant funding provided by R01HL135872 and P01HL152961 to B. B. G.; W81XWH2210457 from the US Department of Defense, Fulbright Scholarship (12398-BR), and K08HL168310 to M. H. L.; American Heart Association Grant 19CDA34730030, ATS Foundation/Pulmonary Hypertension Association Research Fellowship, the Cardiovascular Medical Research Fund and United Therapeutics Jenesis Innovative Research Award to R. K.; R01HL135872-06S1 to D. F. B.; and 1K01HL161024-01 to C. M.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT

The data are available from the authors upon reasonable request.

ETHICS STATEMENT

All studies were approved by the UCSF IACUC, Protocol #AN181431.

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