Schistosomiasis-induced experimental pulmonary hypertension: role of interleukin-13 signaling

Abstract

The mechanisms underlying schistosomiasis-induced pulmonary hypertension (PH), one of the most common causes of PH worldwide, remain unclear. We sought to determine whether Schistosoma mansoni causes experimental PH associated with pulmonary vascular remodeling in an interleukin (IL)-13-dependent manner. IL-13Ralpha1 is the canonical IL-13 signaling receptor, whereas IL-13Ralpha2 is a competitive nonsignaling decoy receptor. Wild-type, IL-13Ralpha1(-/-), and IL-13Ralpha2(-/-) C57BL/6J mice were percutaneously infected with S. mansoni cercariae, followed by i.v. injection of eggs. We assessed PH with right ventricular catheterization, histological evaluation of pulmonary vascular remodeling, and detection of IL-13 and transforming growth factor-beta signaling. Infected mice developed pulmonary peri-egg granulomas and arterial remodeling involving predominantly the vascular media. In addition, gain-of-function IL-13Ralpha2(-/-) mice had exacerbated vascular remodeling and PH. Mice with loss of IL-13Ralpha1 function did not develop PH and had reduced pulmonary vascular remodeling. Moreover, the expression of resistin-like molecule-alpha, a target of IL-13 signaling, was increased in infected wild-type and IL-13Ralpha2(-/-) but not IL-13Ralpha1(-/-) mice. Phosphorylated Smad2/3, a target of transforming growth factor-beta signaling, was increased in both infected mice and humans with the disease. Our data indicate that experimental schistosomiasis causes PH and potentially relies on up-regulated IL-13 signaling.

Figure 1

Vascular remodeling and PH after S. mansoni infection. A: Arterial remodeling was observed by H&E staining, as was a perivascular inflammatory cell infiltrate (arrowheads; Scale bars: 50 μm). B: With S. mansoni infection, thickening of the medial (representative images of wild-type (WT), IL-13Rα1^−/−, and IL-13Rα2^−/− mouse lungs) and intimal (seen predominantly in IL-13Rα2^−/− mouse lungs) layers was observed based on immunofluorescence for α-smooth muscle actin (SMA), identifying vascular media, and thrombomodulin (TM), identifying vascular intima (Scale bars: 50 μm). C: Quantification of vascular remodeling confirmed increased media thickness in the wild-type and IL-13Rα2^−/− infected mice (three to five animals per group; analysis of variance, P < 0.001 for media comparison; for full posthoc analysis results see Supplemental Table 1 at http://ajp.amjpathol.org). D: Right ventricular catheterization demonstrated increased pressure in the infected groups (four to five animals per group; P = 0.018 by Kruskal-Wallis one-way analysis of variance on ranks, *P < 0.005 by posthoc pairwise multiple comparison by Dunn’s method; for full posthoc analysis results see Supplemental Table 3 at http://ajp.amjpathol.org) E: Quantification of intima thickness demonstrated an increase only in the IL-13Rα2^−/− infected mice (three to five animals per group; analysis of variance P = 0.003; *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001 by posthoc Tukey test for both media and intima analysis; for full posthoc analysis results see Supplemental Table 2 at http://ajp.amjpathol.org).

Figure 2

Right ventricular hypertrophy occurred in response to PH. Right ventricular mass relative to septum and left ventricular mass was increased in the infected IL-13Rα2^−/− group (three to five animals per group; analysis of variance, P = 0.004; *P < 0.05, **P < 0.01 by posthoc Tukey test; for full posthoc analysis results, see Supplemental Table 5 at http://ajp.amjpathol.org).

Figure 3

Similar egg burden and granuloma volumes after S. mansoni infection. A: Mean egg burden for each model after cercarial and egg infection as determined by KOH digest (five animals per group; P = 0.39 by analysis of variance). B: Intraparenchymal peri-egg granulomas were present in the infected mice (representative H&E stained lungs; left panel; Scale bar: 100 μm). The granuloma volumes were similar in all genetic models as determined by the rotator method (five animals per group; P = 0.85 by analysis of variance).

Figure 4

Vascular and inflammatory cell composition and evidence of cell proliferation in pulmonary peri-egg granulomas in infected mice. Granulomas in wild-type (upper panels), IL-13Rα1^−/− (middle panels), and IL-13Rα2^−/− (lower panels) infected mice are composed of smooth muscle cells or myofibroblasts (αSM-actin, α-smooth muscle actin), macrophages (MAC-3) and eosinophils (MBP, major basic protein). Considerable proliferation was also detected (PCNA, proliferating cell nuclear antigen) (Scale bars: 100 μm).

Figure 5

RELM-α and p-Smad2 are up-regulated in response to S. mansoni infection. A: RELM-α, a downstream target of IL-4 and IL-13 signaling, was expressed in pulmonary peri-egg granulomas in infected mice, but suppressed in the absence of IL-13Rα1, as demonstrated by immunostaining (arrowheads mark positive cells) and immunoblot (B; densitometry: five animals per group). There was no evidence of pulmonary intravascular RELM-α expression (representative vessel from wild-type infected animal shown). (The full Western blot is shown in Supplemental Figure 13, see http://ajp.amjpathol.org). C: p-Smad2/3 activity, a downstream target of TGF-β signaling, is increased in both granulomas and vessels with S. mansoni infection (arrowheads mark positive cells), and quantified by immunoblot normalized by total SMAD2 (D; densitometry: three to five animals per group; P = 0.44 by analysis of variance). Only bands for Smad2 (61 kDa) were seen (Scale bars are 50 μm.).

Figure 6

p-Smad2/3 signaling is present in vascular lesions of patients who died of schistosomiasis-associated PAH. Tissue from two patients was immunostained. A: Costaining for α-smooth muscle actin (αSM-actin) and p-Smad2/3 reveals nuclear activity in the media of vessels with thickened media (arrowheads mark representative positive cells). B: Costaining for CD31 and p-Smad2/3 reveals nuclear activity in many cells present within plexiform lesions (arrowheads mark representative positive cells) (Scale bars: 50 μm.)