Praziquantel Reverses Pulmonary Hypertension and ... - ATS Journals

Praziquantel Reverses Pulmonary Hypertension and Vascular Remodeling in Murine Schistosomiasis Alexi Crosby1, Frances M. Jones2, Ewa Kolosionek3, Mark Southwood4, Ian Purvis1, Elaine Soon1, Ghazwan Butrous3, David W. Dunne2, and Nicholas W. Morrell1 1

Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke’s Hospital, Cambridge, United Kingdom; Department of Pathology, University of Cambridge, Cambridge, United Kingdom; 3University of Kent, Canterbury, United Kingdom; and 4 Department of Pathology, Papworth Hospital, Cambridge, United Kingdom 2

Rationale: Schistosomiasis is the most common worldwide cause of pulmonary arterial hypertension. The anti-schistosome drug praziquantel has been shown to reverse the liver fibrosis associated with Schistosoma mansoni in mice. Objectives: We sought to determine whether praziquantel reverses established pulmonary vascular remodeling and pulmonary hypertension in a mouse model of S. mansoni. Methods: Mice were infected percutaneously with S. mansoni. At 17 weeks after infection mice were either killed or received two doses of praziquantel or vehicle by oral gavage. Treated mice were studied at 25 weeks after infection. Measurements and Main Results: Vehicle-treated mice demonstrated significant increases in right ventricular systolic pressures (RVSP) and right ventricular hypertrophy (RVH) at 25 weeks, accompanied by pulmonary vascular remodeling. The degree of vascular remodeling correlated with proximity to granulomas. The elevation of RVSP and RVH at 25 weeks was dependent on the presence of eggs in the lung. Praziquantel eliminated the production of eggs in feces and led to clearance of eggs from the lung and to a lesser extent from liver. Praziquantel prevented the rise in RVSP and RVH seen in vehicletreated mice and reversed established pulmonary vascular remodeling. Praziquantel significantly reduced lung mRNA expression of IL-13, IL-8, and IL-4, but did not reduce serum cytokine levels. Conclusions: The development of pulmonary hypertension associated with S. mansoni infection can be prevented by praziquantel, and established vascular remodeling can be reversed. The mechanism involves clearance of lung eggs and reduced local expression of lung cytokines. Keywords: hypertension, pulmonary; infection; lung

Schistosomiasis is the world’s third leading endemic parasitic disease, after malaria and amoebiasis, and is reputedly the world’s most common cause of pulmonary arterial hypertension (PAH) (1). After penetration of the skin by the larval form (cercariae), the schistosomes mature and migrate through the lung to the liver, gut, or bladder, depending on the species, where they elicit a marked immune response. The adult Schistosoma mansoni worms mate in the liver and lay eggs in the mesenteric venules of the intestine. Egg production commences (Received in original form January 25, 2011; accepted in final form May 31, 2011) Supported by a Pfizer “Pulmonary Vascular Disease European Young Researcher Award” grant and a project grant from the British Heart Foundation. Author contributions: A.C., F.M.J., E.S., M.S., I.P., and E.K. were involved with acquisition, analysis, or interpretation of the data. A.C., F.M.J., G.B., D.W.D., and N.W.M. contributed to the design and the conception of the study. All authors were involved with drafting/revising and approving the manuscript. Correspondence and requests for reprints should be addressed to Nicholas W. Morrell, M.D., Division of Respiratory Medicine, Department of Medicine, University of Cambridge School of Clinical Medicine, Box 157, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 0QQ, UK. E-mail: [email protected] This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Crit Care Med Vol 184. pp 467–473, 2011 Originally Published in Press as DOI: 10.1164/rccm.201101-0146OC on June 9, 2011 Internet address: www.atsjournals.org

AT A GLANCE COMMENTARY Scientific Knowledge on the Subject

Schistosomiasis is generally held to be the most common worldwide cause of pulmonary hypertension. However, little is known regarding the pathobiology of pulmonary hypertension in this condition. What This Study Adds to the Field

The presence of pulmonary hypertension was associated with the presence of lung eggs and the local expression of cytokine mRNA in the lung. Treatment with praziquantel at 17 weeks prevented pulmonary hypertension and reversed pulmonary vascular remodeling, suggesting that eradication of the parasite might allow resolution of disease in patients.

4 to 6 weeks after infection and then continues for the lifetime of the worms (2). The majority of eggs are excreted in the stool, where upon contact with water the eggs hatch, releasing miracidia, which penetrate a specific fresh water snail and continue the parasitic life cycle. However, some are retained in the tissues and find their way to the hepatic sinusoids. Chronic infection with S. mansoni can result in periportal fibrosis, portal hypertension, and splenomegaly. As a result of portal hypertension, portocaval collaterals enlarge, allowing eggs to embolize to the lung. In the lung, the eggs migrate through the vessel wall into the parenchyma (3, 4), resulting in the formation of granulomas and a subsequent fibrotic reaction (5). It is not clear whether it is the presence of eggs in the lung, the underlying liver disease, or systemic inflammation that gives rise to pulmonary vascular remodeling and pulmonary hypertension. Praziquantel is commonly used as first-line treatment in humans with schistosomiasis. It is a pyrazinoisoquinoline derivative (2) and is only active against the adult worms. It is believed to increase the permeability of the membranes of the parasite cells to calcium ions, thereby inducing contraction resulting in paralysis, and causes damage to the outer tegumental surface of the parasite. Praziquantel has been reported to cure 60 to 90% of patients with schistosomiasis and to decrease the worm burden in those who are not cured (2). In mice, hepatic fibrosis has been shown to be reversed by praziquantel therapy, and lung granuloma formation has been shown to partly resolve after treatment with other antihelminthics (6), but nothing is known about the capacity of these agents to alter the course of pulmonary vascular remodeling and pulmonary hypertension (2, 7). The aim of the present study was to determine the impact of praziquantel therapy on established pulmonary vascular remodeling and the development of PAH in a murine model of schistosomiasis. In addition, we sought to determine whether killing of adult worm pairs leads to clearance of lung eggs and alters the

468

AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE

circulating or local lung expression of cytokines. We found that two oral doses of praziquantel markedly reduced lung egg deposition, prevented the subsequent development of PAH, and reversed established vascular remodeling. Importantly, we found that the presence of eggs in the lung was associated with increased local expression of cytokines, which was reversed by praziquantel.

METHODS Experimental Mouse Model of S. mansoni Infection A Puerto Rican strain of S. mansoni was used in all experiments (8). Thirty female C57/BL6 adult mice were infected transcutaneously with a suspension containing 30 cercariae, as previously described (9). Female mice were chosen because they are known to develop heavier worm burden than males during chronic infection (10, 11). Animals were killed at 17 or 25 weeks after infection. Control noninfected animals were killed at 17 and 25 weeks. At defined time points (0, 7, 12, and 23 wk after infection) venous blood was taken from the tail vein for cytokine analysis. Fecal samples were collected at 12, 16, 20, 23, and 24 weeks after infection. After killing, mice were exsanguinated and the plasma was retained for cytokine analysis. All protocols and surgical procedures were approved by the local animal care committee.

Praziquantel Treatment At 17 weeks after infection, animals were treated with either praziquantel at a dose of 250 mg/kg by oral gavage or vehicle control. Three days later the same dose was repeated.

Measurement of Right Ventricular Systolic Pressure (RVSP) At 17 and 25 weeks after infection, groups of mice were anesthetized (hypnorm/hypnovel) for hemodynamic assessment. Body weight was recorded and right-sided heart catheterization was performed to measure RVSP, as previously described (12).

VOL 184

2011

Shelmersdale, UK). Arteries of different size were scored according to whether they were completely muscular, partially muscular, or nonmuscular (18).To determine whether vascular remodeling was more extensive in arteries that were in proximity to granulomas, we used EVG-stained lung sections. First, a granuloma containing a clearly identifiable egg was located in a lung section. Then, an image was captured of the granuloma and surrounding lung tissue at 1003 magnification. The distance was measured from the center of each egg to the center of each visible artery and the wall thickness of that artery was determined. The distance between eggs and vessels and medial thickness was measured using Image pro software as previously described (13) (MediaCybernetics, Bethesda, MD). All arteries were measured on each lung section from three animals in which the presence of eggs had been established.

Cytokine mRNA Expression and Serum Levels Serum cytokines levels were measured using a multiplexed mouse cytokine assay from R&D Systems (Abingdon, UK). In brief, beads coated with the analyte-specific antibodies were incubated with serum and the amount bound was read by a Luminex analyzer. The assay was performed according to the manufacturer’s protocol. Lung cytokine mRNA expression was determined after TRIzol extraction. Real-time polymerase chain reaction was performed using the SYBR Green Jumpstart Taq Readymix (Sigma, Dorset, UK). The following primers were obtained from QIAGEN (West Sussex, UK): IL-4, IL-13, IL-6, and Kc (mouse homolog of IL-8). Reactions were amplified on the iCycler (ABI, Paisley, UK).

Statistical Analysis Data are presented as mean 6 SEM, or median, as appropriate. Data between time points were compared using one-way analysis of variance and a Tukey post hoc test for all data except the lung cytokine expression for which a Mann-Whitney post hoc test was performed. For RVSP, RVH, histology, and morphometry, we analyzed the animals that had been infected for 25 weeks as two separate groups depending on the presence or absence of eggs in the lung. Those that had eggs in the lung are referred to as “251E” and those without eggs in the lung as “25-E.”

Right Ventricular Hypertrophy To measure the extent of right ventricular hypertrophy (RVH), the heart was removed and the right ventricle (RV) free wall was dissected from the left ventricle plus septum (LV1S), and weighed separately, as previously described (13). The degree of RVH was determined from the ratio RV/LV1S.

Tissue Preparation In all animals the left lung was fixed in situ in the distended state by infusion of 0.8% agarose into the trachea, and then placed in 10% paraformaldehyde before paraffin embedding. The right caudal lobe was taken for egg counts. The remaining lung lobes were frozen in liquid nitrogen for mRNA extraction. The liver was removed and weighed. The right, caudate, and left lateral lobes of the liver were retained for egg counts.

Tissue Egg Counts To quantify the number of eggs in the lung and liver, tissues were digested in 4% KOH, and eggs were counted as described previously (14).

Fecal Egg Counts Fecal egg counts were performed as described previously (15, 16).

Pulmonary Vascular Morphometry Lung tissues were stained with hematoxylin and eosin or elastic van Gieson (EVG) stain to assess morphology (all Merck/BDH, Lutterworth, UK). For immunohistochemistry, tissue sections were treated as previously described (17). To determine the degree of muscularization of small pulmonary arteries, lung tissue sections were doubled stained with anti–smooth muscle a-actin (a-SMactin; DakoCytomation, Ely, UK) and von Willebrand factor (DakoCytomation). Antibody staining was visualized using 3–39 diaminobenzidine hydrochloride substrate (DakoCytomation) and counterstained in Carrazzi hematoxylin (Bios,

RESULTS Praziquantel Reduces Egg Burden in Experimental Schistosomiasis

Consistent with the presence of egg-laying worm pairs in infected animals, fecal egg counts (Figure 1A) were elevated 7, 12, and 16 weeks after infection. There was heterogeneity between animals at each time point, suggesting differences in the number of worm pairs in each mouse. After treatment with praziquantel, successful eradication of worm pairs was indicated by the absence of eggs in feces. There was a significant time-dependent increase in eggs deposited in the liver (Figure 1B), with maximal values (30 3 103 6 8 3 103) at 25 weeks after infection (P , 0.001). The number of eggs deposited in the lung (Figure 1C) also increased in a timedependent manner, with maximal values (0.90 3 103 6 0.7 3 103) at 25 weeks after infection. However, there was considerable heterogeneity in the extent of lung egg deposition between animals, with some mice exhibiting no lung egg deposition. Again, consistent with eradication of parasites, there was a significant reduction (P , 0.05) in liver egg deposition (Figure 1B) and lung egg deposition (Figure 1C) in animals treated with praziquantel. Liver weights (Figure 1D) were significantly elevated at 17 and 25 weeks after infection and returned back down to control levels after praziquantel treatment. There was no change in body weight between groups at any time point (Figure 1E). Praziquantel Prevents the Development of RVH and Pulmonary Hypertension in S. mansoni–Infected Mice

To determine whether S. mansoni–infected mice developed PAH, we measured RVH (Figure 2A) and pulmonary hemodynamics

Crosby, Jones, Kolosionek, et al.: Praziquantel and Pulmonary Schistosomiasis

Figure 1. (A) Scatter plot shows the number of Schistosoma mansoni eggs in 50 mg of feces of infected animals; n ¼ 6 animals per group. (B, C) Deposition of eggs in the whole liver and lung lobe, respectively, in control mice, mice infected with S. mansoni for 17 weeks, and mice infected with S. mansoni for 25 weeks, with or without praziquantel (PZQ) treatment for the final 8 weeks. n ¼ 10 at 17 weeks and 25 weeks with PZQ and n ¼ 8 at 25 weeks without PZQ treatment. ***P , 0.001, compared with control. 1P , 0.05 compared with 25 weeks without PZQ. (D, E) Liver weight and the body weight, respectively, of control mice and mice infected with S. mansoni. n ¼ 8 for control mice and mice infected with S. mansoni for 25 weeks, n ¼ 10 for mice infected for 17 weeks and for mice infected for 25 weeks 1 PZQ treatment. *P , 0.05, ***P , 0.001, compared with control. 111P , 0.001 compared with 25 weeks without PZQ treatment.

(Figure 2B). There was no significant increase in RVH or RVSP at 17 weeks after infection, consistent with our previous observations (19). However, there were significant increases in RVH (P , 0.05) and RVSP (P , 0.01) 25 weeks after infection in animals that were found to have eggs in the lung (251E group). Animals without evidence of lung egg deposition had RV ratios and RVSP measurements comparable to the control uninfected group. In animals treated with praziquantel, both RVH (P , 0.05) and RVSP (P , 0.01) were significantly decreased compared with the 251E group. Indeed, indices in animals treated with praziquantel were no different from measurements in uninfected control animals. Praziquantel Reverses Established Pulmonary Vascular Remodeling

To determine the degree of pulmonary vascular remodeling, we performed immunohistochemistry on serial lung sections. We observed frequent severely remodeled pulmonary arteries, staining positively for a-SMA, associated with terminal or respiratory bronchioles at 17 and 25 weeks after infection (Figure 3, see Figure E1 in the online supplement). These lesions were

469

Figure 2. Scatter plots show (A) right ventricular index (right ventricle/left ventricle plus septum [RV/LV1S]) in control mice n ¼ 8; mice infected with Schistosoma mansoni for 17 weeks, n ¼ 10; mice infected with S. mansoni for 25 weeks with eggs in the lung (251E), n ¼ 4; mice infected with S. mansoni for 25 weeks without eggs in the lung (25-E), n ¼ 2; mice infected with S. mansoni for 25 weeks and treated with praziquantel (PZQ), n ¼ 9; and (B) right ventricular systolic pressure (RVSP) in control n ¼ 6; mice infected with S. mansoni for 17 weeks, n ¼ 10; mice infected with S. mansoni for 25 weeks with eggs in the lung (251E), n ¼ 4; mice infected with S. mansoni for 25 weeks without eggs in the lung (25-E), n ¼ 2; mice infected with S. mansoni for 25 weeks and treated with praziquantel (PZQ), n ¼ 8. The RVSP of one mouse in the control group and two mice in the PZQ group were excluded from the analysis as they had very low heart rates (,100 bpm). *P , 0.05, **P , 0.01, compared with control; 11P , 0.01, 111 P , 0.001 compared with 25 weeks 1 PZQ treatment; #P , 0.05, compared with 17 weeks.

heterogeneously distributed in the lung and were often observed in proximity to granulomas surrounding eggs. These affected arteries were frequently almost completely obliterated and demonstrated perivascular inflammation. At 25 weeks after infection, these lesions were absent in animals without evidence of lung egg deposition (Figures 3 and E1). In animals treated with praziquantel we found no evidence of the severely remodeled arteries seen in infected animals at 17 or 25 weeks, indicating that cessation of lung egg deposition and clearance of eggs allowed reversal of the lesions in these arteries (Figure 4). EVG staining revealed perivascular collagen deposition in small arteries, which was reversed with praziquantel treatment (Figure E2). In addition to the severe, but heterogeneous, vascular remodeling in the arteries described above, we quantified the degree of

470


VOL 184

2011

Figure 3. Representative photomicrographs of serial lung sections from control mice and mice chronically infected with Schistosoma mansoni for 25 weeks and mice infected with S. mansoni and treated with praziquantel (PZQ). Sections were stained with hematoxylin and eosin (H1E), or immunostained for a-smooth muscle actin (a2SMA). Arrows indicate arteries. TB ¼ terminal bronchiole. All at magnification 3100. Bar ¼ 10 mm.

muscularization of small pulmonary arteries in the peripheral lung. In small peripheral vessels (0–70 mm) we observed a dramatic increase in the percentage of completely muscularized arteries in 251E animals and a dramatic reduction in nonmuscularized vessels in animals infected at 17 weeks and in 251E animals, compared with uninfected control mice (Figure 5A). The greatest percentage of fully muscularized vessels was observed in the 251E group. A similar pattern was observed in medium-sized vessels (71–150 mm) (Figure 5B) and large vessels (.150 mm) (Figure 5C). Praziquantel treatment at 17 weeks after infection prevented any further increase in the degree of muscularization. Furthermore, we found a significant negative correlation (P , 0.05) between the distance of vessels from the egg and the degree of medial thickness (Figure 6), indicating that the vessels in proximity to granulomas tend to exhibit a greater degree of remodeling. Effect of Praziquantel on Circulating Inflammatory Cytokines

We have previously reported that chronic schistosomal infection in mice leads to a time-dependent increase in serum levels of inflammatory cytokines (19). Because serum cytokine levels correlated with pulmonary vascular remodeling in that study, we sought to determine whether praziquantel reduced the levels of serum cytokines as a potential mechanism for the beneficial effects on pulmonary hypertension. In particular, we focused on IL-4 and IL-13, as these cytokines have been implicated in pulmonary arterial muscularization (20, 21). We measured circulating levels of Kc (the mouse homolog of IL-8), as this cytokine has also been found to be elevated in patients with PAH (21). IL-5 levels were determined, as this cytokine is known to be elevated as part of the response to parasitic infections. In the present study, increases in serum IL-4, IL-5, Kc, and IL-13 were observed in infected mice at 17 and 25 weeks. Serum IL-13 levels displayed a time-dependent increase, with maximal levels at 17 weeks after infection and a subsequent decrease. Praziquantel treatment did not significantly decrease serum levels of IL-13 compared with levels in infected vehicle-control animals at 25 weeks (Figure 7A). Similarly, serum levels of IL-4 and Kc were not reduced by praziquantel treatment at 25 weeks (Figures 7B and 7C). In contrast, serum levels of IL-5 did decrease after praziquantel treatment compared with infected vehicle-control animals (Figure 7D).

Praziquantel Reduces Lung Expression of Cytokines

Lung expression levels of IL-13 mRNA were significantly greater than uninfected control mice at 25 weeks after infection (P , 0.01, Figure 8A). A similar pattern was observed with Kc (Figure 8B) and IL-4 (Figure 8C). Although levels of IL-4 were elevated at 25 weeks after infection, this did not reach statistical significance. Praziquantel treatment normalized lung cytokine mRNA expression. A similar pattern can be seen for IL-6 (Figure 8D); however, the fold change did not reach statistical significance.

DISCUSSION The present study has demonstrated that chronic infection (25 weeks) with S. mansoni results in significant PAH accompanied by right ventricular hypertrophy. Previous studies had shown that pulmonary vascular remodeling occurs at earlier time points but without significant elevation of pulmonary artery pressures (19). Thus, our findings validate the chronic S. mansoni–infected mouse as a model of schistosomiasisassociated PAH. In addition, this study has shown that eradication of egg-laying worm pairs with two oral doses of praziquantel at 17 weeks after infection prevented the subsequent development of PAH and RVH. Furthermore, praziquantel reversed the severe vascular remodeling and perivascular inflammation in arteries associated with terminal and respiratory bronchioles.

Figure 4. Representative photomicrographs (3100 magnification) of lung sections. (A) Mice infected with Schistosoma mansoni for 25 weeks with eggs in the lung; numerous grossly remodeled vessels can be seen (arrows indicate remodeled vessels). (B) In mice infected with S. mansoni for 25 weeks and treated with praziquantel for the final 8 weeks, no grossly remodeled vessels can be seen. Sections were stained with hematoxylin and eosin. Bar represents 10 mm.

Crosby, Jones, Kolosionek, et al.: Praziquantel and Pulmonary Schistosomiasis

Figure 5. (A–C) Bar charts showing degree of muscularization of pulmonary vessels. (A) Vessels 20–70 mm; (B) vessels 71–150 mm; (C) vessels . 150 mm in control mice, mice infected with Schistosoma mansoni for 17 weeks, mice infected with S. mansoni for 25 weeks with eggs in the lung (251E), mice infected with S. mansoni for 25 weeks without eggs in the lung (25-E), mice infected with S. mansoni for 25 weeks and treated with praziquantel (PZQ). *P , 0.05, **P , 0.01, ***P , 0.001 compared with control.

These changes were accompanied by eradication of eggs from the lungs and a reduction in the local expression of lung IL-4, IL-13, and IL-6. We further demonstrated that remodeling was more severe in arterioles in proximity to granulomas, supporting the concept that it is the local presence of eggs and the surrounding granuloma that drives the process of vascular remodeling in pulmonary schistosomiasis.

471

We observed marked variability between infected animals in the number of eggs deposited in the lungs at 25 weeks. We and others (3, 21, 23) have commented that for eggs to be shunted to the lung, portocaval collateral vessels must open. Portocaval collaterals are believed to open as a consequence of portal hypertension, induced by the liver granulomas and the subsequent fibrosis. The extent of lung egg deposition depends on several factors, including the degree of liver involvement and the number of egg-laying worm pairs present. The major contribution of the local presence of eggs and the surrounding granuloma in the lung to pulmonary hypertension pathobiology is strongly supported by several lines of evidence in our model. First, only mice with eggs in the lung at 25 weeks had significant pulmonary hypertension, vascular remodeling, and RVH, whereas mice without measurable lung egg deposition exhibited minimal changes in vascular morphometry or pulmonary hemodynamics. Second, eradication of lung eggs led to a disappearance of grossly remodeled arteries with perivascular inflammation and prevented the development of pulmonary hypertension by 25 weeks. In addition, we demonstrated for the first time the relationship between the distance of a vessel from a granulomas and the extent of vascular remodeling. The earliest description of egg deposition in the lungs and the formation of lung granulomas in human schistosomal infection was reported by Azmy and Effat (23) in 1932. Subsequently, a number of investigators have reported pulmonary granulomas and pulmonary arteritis in both patients and animal models of schistosomiasis (6, 22, 24). However, the present study is the first to report a significant increase in right ventricular hypertrophy and an increase in right ventricular systolic pressure in an animal model of S. mansoni infection. The chronicity of infection and the continuous replenishment of lung egg deposition may be important factors in the development of PAH. The presence of portocaval shunts in other forms of liver fibrosis, particularly cirrhosis, are also known to be associated with susceptibility to PAH (25, 26) when it is referred to as portopulmonary hypertension. Thus, additional factors, in addition to physical shunting of antigenic structures such as eggs, are likely to play a role in PAH pathobiology associated with liver disease. Recent reports suggest that 4 to 10% of patients with chronic schistosomiasis develop hepatosplenic disease (2, 27, 28), and between 7 and 30% of these patients develop PAH (27, 29, 30). Praziquantel prevented the development of PAH between 17 and 25 weeks of infection and reversed the changes of severe pulmonary vascular remodeling associated with perivascular inflammation that were already present at 17 weeks. The efficacy of praziquantel treatment was documented by the absence of eggs in the feces of treated animals and a reduction in lung and liver

Figure 6. (A) Representative example (n ¼ 3) of semiquantification of distance of vessels to egg and medial thickness (*P , 0.05). (B) Histology sections from mouse lung showing the distance from the egg to the surrounding arterioles. Sections were stained with hematoxylin and eosin and the distance from egg to vessel was measured by the number of pixels between them. Medial thickness was measured by the width of the vessel in pixels.

472


VOL 184

2011

Figure 8. Scatter plots showing fold changes in lung cytokine mRNA expression levels relative to b-actin. (A) IL-13, (B) Kc, (C) IL-4, and (D) IL-6. Control animals (0) n ¼ 4; animals infected for 17 weeks, n ¼ 7; animals infected for 25 weeks, n ¼ 6; animals infected for 25 weeks and treated with praziquantel (PZQ), n ¼ 7.

Figure 7. Bar charts showing the time course of cytokine profiles in serum from control mice, mice infected with Schistosoma mansoni, and mice infected with S. mansoni and treated with praziquantel (PZQ). Serum from control animals (0); serum from the tail vein of animals infected for 7 weeks; serum from animals infected for 17 weeks; serum from the tail vein of animals infected for 23 weeks; serum from animals infected for 25 weeks; serum from animals infected for 25 weeks and treated with praziquantel (PZQ). (A) IL-13, (B) IL-4, (C) Kc, and (D) IL-5. *P , 0.05, **P , 0.01, ***P , 0.001 compared with control.

egg deposition at 25 weeks. Despite a reduction in the egg burden in praziquantel-treated infected animals, systemic inflammation persisted, with elevated circulating levels of IL4, Kc, and IL-13. Presumably this was a result of the continuing presence of a substantial number of eggs in the liver. In contrast, the lung egg burden was greatly reduced by praziquantel treatment. This was accompanied by a reduction in the lung mRNA expression of cytokines. The association between lung egg burden, lung cytokine expression, and indices of PAH further support the contention that it is the local presence of eggs in the lung and lung cytokine expression that is primarily responsible for the changes of PAH in this model. We have previously shown that IL-13 permits

increased migration of mouse pulmonary artery smooth muscle cells (19). One potential limitation of our study is that although hemodynamic and morphometric indices in the group of animals treated with praziquantel were similar to those in uninfected mice, we have no way of knowing how many mice in the praziquanteltreated group actually had egg deposition in the lungs at the time of treatment. However, based on the findings in the vehicle control group at 17 weeks, approximately 50% of mice demonstrated lung egg deposition at this time point. Therefore, we would anticipate that a similar proportion of the praziquantel-treated mice would have had evidence of lung egg deposition before starting treatment. Despite this, we found no evidence of granulomas or the gross remodeling of intraacinar pulmonary arteries observed in untreated mice at 17 (19) or 25 weeks, and none of these mice exhibited pulmonary hypertension. Although not significantly elevated above the levels of cytokines observed at 17 weeks after infection, there was a tendency for cytokine levels to increase at 23 weeks after infection in animals treated with praziquantel. This is likely due to the exposure of worm antigens on killing of the parasites. Interestingly, the levels of IL-5, which are associated with parasitic infections, were decreased at this time point. Apart from IL-5, which returned to baseline values in mice treated with praziquantel, none of the other serum cytokines measured returned to baseline after praziquantel. This persistent systemic inflammatory response is likely due to the continuing liver egg burden and to inflammation in the mesenteric lymph nodes as a consequence of infection. The fact that the pulmonary vascular changes largely resolved despite continuing systemic inflammation also supports the notion that it is the local cytokine expression in the lung secondary to the presence of eggs that drives the process of pulmonary vascular remodeling and pulmonary hypertension in this model. Taken together, our findings provide further evidence that the presence of eggs and granulomas in the lung result in local expression of inflammatory cytokines that contribute significantly to the process of pulmonary vascular remodeling. This process is reversible when the delivery of eggs to the lung ceases. These findings have implications for the treatment of patients with schistosomiasis-associated PAH and for our understanding of the role of cytokines in PAH.

Crosby, Jones, Kolosionek, et al.: Praziquantel and Pulmonary Schistosomiasis Author Disclosure: A.C. received institutional grant support from the British Heart Foundation and Pfizer. F.M.J. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. E.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. I.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. E.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. G.B. was a consultant for Bayer, Novartis, and Pfizer and is employed by Private Works. He received payment for the development of educational presentations and received travel accommodations from Bayer, Ltd. He owns stocks/options in Pfizer. D.W.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. N.W.M. received institutional grant support from the British Heart Foundation.

References 1. Alpert JS, Irwin RS, Dalen JE. Pulmonary hypertension. Curr Probl Cardiol 1981;5:1–39. 2. Ross AG, Bartley PB, Sleigh AC, Olds GR, Li Y, Williams GM, McManus DP. Schistosomiasis. N Engl J Med 2002;346:1212–1220. 3. Andrade ZA, Andrade SG. Pathogenesis of schistosomal pulmonary arteritis. Am J Trop Med Hyg 1970;19:305–310. 4. Bedford DE, Aidaros SM, Girgis B. Bilharzial heart disease in Egypt. Br Heart J 1946;8:87. 5. Chirakalwasan N, Coyle CM, Meisner MSJ, Chandra A. Treating a case of severe pulmonary hypertension due to schistosomiasis. Chest 2006; 130:293S–294S. 6. Cardoso de Almeida MA, Andrade ZA. Effect of chemotherapy on experimental pulmonary schistosomiasis. Am J Trop MedHyg 1983; 32:1049–1054. 7. Schwartz E. Pulmonary schistosomiasis. Clin Chest Med 2002;23:433–443. 8. Mentink-Kane MM, Cheever AW, Thompson RW, Hari DM, Kabatereine NB, Vennervald BJ, Ouma JH, Mwatha JK, Jones FM, Donaldson DD, et al. IL-13 receptor alpha 2 down-modulates granulomatous inflammation and prolongs host survival in schistosomiasis. Proc Natl Acad Sci USA 2004;101:586–590. 9. Smithers SR, Terry RJ. The infection of laboratory hosts with cercariae of Schistosoma mansoni and the recovery of adult worms. Parasitology 1965;55:695–700. 10. Berg E. Effects of castration and testosterone in male mice on Schistosoma mansoni. Trans R Soc Trop Med Hyg 1957;51:353–358. 11. Nakazawa M, Fantappie MR, Freeman GL Jr, Eloi-Santos S, Olsen NJ, Kovacs WJ, Secor WE, Colley DG. Schistosoma mansoni: susceptibility differences between male and female mice can be mediated by testosterone during early infection. Exp Parasitol 1997;85:233–240. 12. Long L, MacLean MR, Jeffery TK, Morecroft I, Yang X, Rudarakanchana N, Southwood M, James V, Trembath RC, Morrell NW. Serotonin increases susceptibility to pulmonary hypertension in BMPR2-deficient mice. Circ Res 2006;98:818–827. 13. Morrell NW, Atochina EN, Morris KG, Danilov SM, Stenmark KR. Angiotensin converting enzyme expression is increased in small pulmonary arteries of rats with hypoxia-induced pulmonary hypertension. J Clin Invest 1995;96:1823–1833. 14. Cheever AW. Conditions affecting the accuracy of potassium hydroxide digestion techniques for counting Schistosoma mansoni eggs in tissues. Bull World Health Organ 1968;39:328–331.

473

15. Bell DR. A new method for counting Schistosoma mansoni eggs in faeces: with special reference to therapeutic trials. Bull World Health Organ 1963;29:525–530. 16. Doenhoff M, Bickle Q, Long E, Bain J, McGregor A. Factors affecting the acquisition of resistance against Schistosoma mansoni in the mouse. I. Demonstration of resistance to reinfection using a model system that involves perfusion of mice within three weeks of challenge. J Helminthol 1978;52:173–186. 17. Yang X, Long L, Southwood M, Rudarakanchana N, Upton PD, Jeffery TK, Atkinson C, Chen H, Trembath RC, Morrell NW. Dysfunctional Smad signaling contributes to abnormal smooth muscle cell proliferation in familial pulmonary arterial hypertension. Circ Res 2005; 96:1053–1063. 18. Schermuly RT, Dony E, Ghofrani HA, Pullamsetti S, Savai R, Roth M, Sydykov A, Lai YJ, Weissmann N, Seeger W, et al. Reversal of experimental pulmonary hypertension by PDGF inhibition. J Clin Invest 2005;115:2811–2821. 19. Crosby A, Jones FM, Southwood M, Stewart S, Schermuly R, Butrous G, Dunne DW, Morrell NW. Pulmonary vascular remodeling correlates with lung eggs and cytokines in murine schistosomiasis. Am J Respir Crit Care Med 2009;181:279–288. 20. Daley E, Emson C, Guignabert C, de Waal MR, Louten J, Kurup VP, Hogaboam C, Taraseviciene-Stewart L, Voelkel NF, Rabinovitch M, et al. Pulmonary arterial remodeling induced by a Th2 immune response. J Exp Med 2008;205:361–372. 21. Soon E, Holmes AM, Treacy CM, Doughty NJ, Southgate L, Machado RD, Trembath RC, Jennings S, Barker L, Nicklin P, et al. Elevated levels of inflammatory cytokines predict survival in idiopathic and familial pulmonary arterial hypertension. Circulation 2010;122:920– 927. 22. Warren KS. Experimental schistosomiasis. Trans R Soc Trop Med Hyg 1964;58:228–233. 23. Azmy S, Effat S. Pulmonary arteriosclerosis of a Bilharzial nature. J Egypt Med Assoc 1932;15:87. 24. Sami AA. Pulmonary manifestations of schistosomiasis. Dis Chest 1951; 19:698–705. 25. Grander W, Eller P, Fuschelberger R, Tilg H. Bosentan treatment of portopulmonary hypertension related to liver cirrhosis owing to hepatitis C. Eur J Clin Invest 2006;36:67–70. 26. Trabelsi K, Essid M, Azzouz MM. (Pulmonary arterial hypertension in cirrhosis). Tunis Med 2002;80:139–141. 27. Lapa M, Dias B, Jardim C, Fernandes CJ, Dourado PM, Figueiredo M, Farias A, Tsutsui J, Terra-Filho M, Humbert M, et al. Cardiopulmonary manifestations of hepatosplenic schistosomiasis. Circulation 2009;119:1518–1523. 28. Shaw AFB, Ghareeb AA. The pathogenesis of pulmonary schistzosomais in Egypt with special reference to Ayerza’s disease. J Pathol 1938;46:401–424. 29. Barbosa MM, Lamounier JA, Oliveira EC, Souza MV, Marques DS, Silva AA, Lambertucci J. Pulmonary hypertension in Schistosomiasis mansoni. Trans R Soc Trop Med Hyg 1996;90:663–665. 30. Ferreira RC, Domingues AL, Bandeira AP, Markman Filho B, Albuqerque Filho ES, Correiade de Araujo AC, Batista LJ, Markman M, Campelo AR. Prevalence of pulmonary hypertension in patients with schistosomal liver fibrosis. Ann Trop Med Parasitol 2009;103:129–143.