A critical role of interleukin-10 in modulating hypoxia-induced preeclampsia-like disease in mice

Abstract

Hypoxia has been implicated in the pathogenesis of preeclampsia, a hypertensive disorder of pregnancy. However, in vivo evidence and mechanistic understanding remain elusive. Preeclampsia is associated with impaired placental angiogenesis. We have recently shown that interleukin (IL)-10 can support trophoblast-driven endovascular crosstalk. Accordingly, we hypothesize that pathological levels of oxygen coupled with IL-10 deficiency induce severe preeclampsia-like features coupled with elevated production of antiangiogenic factors, apoptotic pathways, and placental injury. Exposure of pregnant wild-type and IL-10(-/-) mice to 9.5% oxygen resulted in graded placental injury and systemic symptoms of renal pathology, proteinuria (wild-type 645.15 ± 115.73 versus 198.09 ± 93.45; IL-10(-/-) 819.31 ± 127.85 versus 221.45 ± 82.73 μg/mg/24 hours) and hypertension (wild-type 118.37 ± 14.45 versus 78.67 ± 14.07; IL-10(-/-) 136.03 ± 22.59 versus 83.97 ± 18.25 mm Hg). Recombinant IL-10 reversed hypoxia-induced features in pregnant IL-10(-/-) mice confirming the protective role of IL-10 in preeclampsia. Hypoxic exposure caused marked elevation of soluble fms-like tyrosine kinase 1 (110.8 ± 20.1 versus 44.7 ± 11.9 ng/mL) in IL-10(-/-) mice compared with their wild-type counterparts (81.6 ± 13.1 versus 41.2 ± 8.9 ng/mL), whereas soluble endoglin was induced to similar levels in both strains (approximately 380 ± 50 versus 180 ± 31 ng/mL). Hypoxia-induced elevation of p53 was associated with marked induction of proapoptotic protein Bax, downregulation of Bcl-2, and trophoblast-specific apoptosis in utero-placental tissue. Collectively, we conclude that severe preeclampsia pathology could be triggered under certain threshold oxygen levels coupled with intrinsic IL-10 deficiency, which lead to excessive activation of antiangiogenic and apoptotic pathways.

Figure 1. Exposure to 9.5%, not 11%, O₂ induces preeclampsia-like symptoms in pregnant mice

Pregnant mice were exposed to 21% O₂ (normoxia), 11%, or 9.5% O₂ from gestational day (gd) 7.5 to gd 17. The term hypoxia is assigned to 9.5% O₂. Comparative data on gd 17 are shown for (A) systolic blood pressure (n=6); (B) albumin/creatinine (proteinuria) levels of 24-hour urine samples (n=6); (C) representative fetal units harvested on gd 17 after exposure to 9.5% and 21% O₂ (n=6) are depicted. Panel (D) compares the average fetal weight on gd 17 (n=58) from multiple independent experiments exposed to 21%, 11% or 9.5% O₂. All values are expressed as Mean ± SEM. ** indicates p<0.05 as compared to respective wild type or IL-10^−/− mice exposed to 21% O₂, and ^aa indicate p<0.05 between wild type and IL-10^−/− mice exposed to 9.5% O₂.

Figure 2. Maternal hypoxia induces renal pathology in pregnant mice

On gd 17, kidneys were harvested and renal tissue processed for H&E staining from at least three animals per group. A representative image of renal tissue section from pregnant mice exposed to 21% (normoxia) or 9.5% (hypoxia) is shown: (A) Lower magnification (10×) shows atrophic tubules and interstitial edema in hypoxia-exposed groups. Bold arrows show the increased interstitial space due to atrophy and (B) Higher magnification (100×) shows glomerulus that is diffusely enlarged and hypertrophy of intracapillary cells upon hypoxia exposure. A representative histology of kidney section from non-pregnant mice subjected to identical conditions of hypoxia is shown at low magnification (10×, Panel C) and at higher magnification (100×, Panel D). Unlike pregnant mice, identical hypoxia treatment in non-pregnant mice did not exhibit renal pathology.

Figure 3. Serum levels of sFlt-1 and sEng in pregnant wild type and IL-10^−/− mice exposed to normoxia or hypoxia

(A) A significant increase was observed in the serum levels of sFlt-1 in response to hypoxia in pregnant mice, with significantly higher levels in IL-10^−/− mice (n=6). (B) sEng levels were also elevated in response to hypoxia, although no differences were observed between wild type and IL-10^−/− mice (n=6). All values are shown as Mean ± SEM. ** indicates p<0.05 as compared to normoxia. ^aa indicates p<0.05 between wild type and IL-10^−/− mice in response to hypoxia.

Figure 4. Recombinant IL-10 (rIL-10) rescues hypoxia-induced preeclampsia like features

Pregnant IL-10^−/− mice were injected (i.p.) recombinant IL-10 daily from gd 8 to gd 16 with concomitant exposure to 9.5% O₂ in hypoxia chamber. On gd 17 the animals were removed from the hypoxia chamber and evaluated. Panel A shows a representative image of recovery of fetal size in response to IL-10 treatment. Panel B shows the average weight of fetus on gd 17 from different treatment groups (n=6 each). Panel C shows comparative systolic blood pressure measurements in response to different treatments (n=6 in each group). IL-10 treatment prevented the onset of hypoxia-induced hypertension. (D) Comparative proteinuria data from different treatment groups show that rIL-10 reverses the hypoxia-induced proteinuria. (E) Analysis of circulating levels of sFlt-1 in response to different treatments shows that rIL-10 prevents hypoxia-induced excess production of sFlt-1. All values are shown as Mean ± SEM. ** indicates p<0.05 as compared to respective experimental groups.

Figure 5. Maternal hypoxia induces injury in the placenta

(A) EF5 staining for normoxia- and hypoxia-exposed placenta in wild type and IL-10^−/− mice. Upper panel shows H&E sections mapped for mesometrium (M), decidua basalis (DB), junctional zone (JZ) and labyrinthine (LB) of utero-placental units from animals from normoxia or hypoxia (4×). Middle panel shows EF5 staining of the utero-placental section under low magnification (4×). Hypoxic injury was found localized to the junctional zone (JZ, indicated by the box). The lower panel shows the higher magnification (20×) of the junctional zone. IL-10^−/− mice exhibits more severe hypoxic injury in JZ. (B) Immunoblot of HIF-1α protein in gd 13 utero-placental tissue. Hypoxia induced HIF-1α protein in both wild type and IL-10^−/− tissue. A representative of three independent experiments is shown.

Figure 6. Maternal hypoxia induces apoptosis in trophoblasts at the maternal-fetal interface

A representative immunofluorescence staining with pancytokeratin antibody (left panel) and TUNEL (middle panel) in IL-10^−/− placenta harvested on gd 17 is shown. Overlay (right panel) for both markers indicated trophoblasts undergoing apoptosis upon exposure to hypoxia. The apoptotic region coincides with the junctional zone.

Figure 7. Maternal hypoxia induces p53-Bax-caspase3 pathway for placental cell death

(A) p53 protein expression in tissue harvested on gd 17. Hypoxia induces significant induction of p53 compared to normoxia tissue, and the induction was more robust in IL-10^−/− tissue when compared to wild type mice. (B) A representative immunoblot of p21 protein. p21 exhibits a parallel expression profile as that seen for p53. (C) Bax and Bcl-2 expression in normoxic and hypoxic tissue. Bax is induced significantly, particularly in IL-10^−/− tissue, whereas Bcl-2 expression decreased significantly. (D) Procaspase 3 and cleaved caspase 3 expression in gd 17 utero-placental tissue. Active caspase 3 bands are uniquely prominent in hypoxic tissue. Data represent at least three independent experiments for each protein.

Figure 8. Schematic model for maternal hypoxia-induced p53 signaling and anti-angiogenic pathways and preeclampsia-like disease in pregnant mice

IL-10 is depicted as a modulator of preeclampsia through partial inhibition of p53 and sFlt-1 induction.