Diminution of signal transducer and activator of transcription 3 signaling inhibits vascular permeability and anaphylaxis

Abstract

Background: During IgE-mediated immediate hypersensitivity reactions, vascular endothelial cells permeabilize in response to mast cell mediators. We have demonstrated previously that patients and mice with signal transducer and activator of transcription 3 (STAT3) mutations (autosomal dominant hyper-IgE syndrome [AD-HIES]) are partially protected from anaphylaxis.

Objectives: We sought to study the mechanism by which STAT3 contributes to anaphylaxis and determine whether small-molecule inhibition of STAT3 can prevent anaphylaxis.

Methods: Using unaffected and STAT3-inhibited or genetic loss-of-function samples, we performed histamine skin prick tests, investigated the contribution of STAT3 to animal models of anaphylaxis, and measured endothelial cell permeability, gene and protein expression, and histamine receptor-mediated signaling.

Results: Although mouse mast cell degranulation was minimally affected by STAT3 blockade, mast cell mediator-induced anaphylaxis was blunted in Stat3 mutant mice with AD-HIES and in wild-type mice subjected to small-molecule STAT3 inhibition. Histamine skin prick test responses were diminished in patients with AD-HIES. Human umbilical vein endothelial cells derived from patients with AD-HIES or treated with a STAT3 inhibitor did not signal properly through Src or cause appropriate dissolution of the adherens junctions made up of the proteins vascular endothelial-cadherin and β-catenin. Furthermore, we found that diminished STAT3 target microRNA17-92 expression in human umbilical vein endothelial cells from patients with AD-HIES is associated with increased phosphatase and tensin homolog (PTEN) expression, which inhibits Src, and increased E2F transcription factor 1 expression, which regulates β-catenin cellular dynamics.

Conclusions: These data demonstrate that STAT3-dependent transcriptional activity regulates critical components for the architecture and functional dynamics of endothelial junctions, thus permitting vascular permeability.

Keywords: Allergy; autosomal dominant hyper-IgE syndrome; immunology; innate immunity; signal transducer and activator of transcription 3.

Figure 1. Reduction in STAT3 confers resistance to anaphylaxis

(A) Effect of C188–9 pretreatment (7 days) on body temperature changes in male C57Bl/6 mice during anaphylactic shock. Mice (n=5–6/group) were sensitized with 3 µg DNP-specific IgE and challenged 24 h later with 200 µg DNP-HSA. (B) Histamine and MCPT-1 released into circulation (90 sec) after challenging sensitized C188–9 or vehicle treated mice with Ag (n=6/group). Results are presented as boxplots with min/max range. Temperature changes during anaphylaxis induced by histamine (5 µmol) in C188–9 and vehicle treated C57BL/6 (C) or AD-HIES mice (D) (n=6/group). Temperature changes during anaphylaxis induced by PAF (0.3 mg) in C188–9 and vehicle treated mice (E). (F) Survival curve of mice after PAF treatment (n=6/group in B and n=5/group in C).

Figure 2. C188–9 decreases histamine or PAF-induced vascular leakage

Hematocrit values at baseline and after passive systemic anaphylaxis (PSA) induced by (A) histamine or (B) PAF in C188–9 (n=5/group) and vehicle treated mice (n=6/group). (C–D) Evan’s blue leakage in response to IgE/Ag-challenge (C; n=10/group) or compound 48/80 (D; n=5/group) in C188–9 and vehicle treated mice. (E–F) In vitro permeability assay of mouse lung endothelial cells exposed to histamine (E; 1 µM) or PAF (F; 10ug) following pretreatment with DMSO or C188–9 (1 µM; n=3/group). (UT: untreated; His: histamine; PAF: platelet activating factor). Data are representative of 3 independent experiments. Data are expressed as the mean ± S.E.M. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Figure 3. C188–9 decreases vascular permeability through strengthening VE-cadherin junctions

In vitro permeability assay of human umbilical vascular endothelial cells (HUVECs) pretreated with DMSO or C188–9 (1 µM) and subsequently exposed to (A) histamine (100 µM) or (B) PAF (10 ug/ml) (UT: untreated; His: histamine; PAF: platelet activating factor). Data are representative of 3 independent experiments. (C) Confocal microscopy of VE-cadherin (green), total β-catenin (red), and DAPI (blue) in DMSO and C188–9 (1µM) pre-treated HUVECs then subsequently exposed to histamine (100 µM). (D) Confocal microscopy of active (non-phosphorylated) β-Catenin (green) and DAPI (blue) in DMSO and C188–9 (1 µM) pre-treated HUVECs then subsequently exposed to histamine (100 µM). (E) Western analysis of Stat3, and VE-cadherin signaling in DMSO and C188–9 treated HUVECs. Data are represented as the mean ± S.E.M. *p<0.05.

Figure 4. AD-HIES patient HUVECs exhibit intrinsically decreased vascular permeability through increased VE-cadherin junctions

AD-HIES and healthy control (n=10/group) patients were skin prick tested with increasing amounts of histamine. The area of the (A) Wheal and (B) Flare was recorded. (C) Image of the bottom of a transwell chamber following wild type and AD-HIES HUVECs exposure to the fluorescent reporter FITC-Dex (1 mg/well) for 30 min. (D–E) In vitro permeability assay of wild type HUVECs and AD-HIES HUVECs exposed to histamine (D; 100 µM) or PAF (E; 10ug/ml) (UT: untreated; His: histamine; PAF: platelet activating factor; n=3/group). Data are representative of 3 independent experiments. (F) Immunofluorescent analysis of VE-cadherin (green), total β-catenin (red), and DAPI (blue) in wild type and AD-HIES HUVECs following exposure to histamine (100 µM). (G) Western analysis of VE-cadherin in wild type and AD-HIES HUVECs following exposure to Wnt3A (200 ng/ml). Data are representative of 2 independent experiments. (H) In vitro permeability assay of wild type (left panel; n=3/group) and AD-HIES (right panel; n=3/group) HUVECs pretreated with Wnt3A (200 ng/ml) and subsequently exposed to histamine. Data are representative of 3 independent experiments. Data are represented as the mean ± SEM. *p<0.05.

Figure 5. HUVECs permeability is regulated by STAT3-induced mir17–92 and can be restored in AD-HIES patient cells through the canonical Wnt signaling pathway

(A) Western blot analysis of phosphorylated Src, total Src, and phosphorylated PTEN, in DMSO and C188–9 HUVECs following exposure to histamine (100 µM). (B–C) Western blot analysis of phosphorylated VE-cadherin, total VE-cadherin, active (non-phosphorylated) β-catenin, total β-catenin, phosphorylated Src, total Src, phosphorylated PTEN, total PTEN, and phosphorylated STAT3, in wild type and AD-HIES HUVECs following exposure to histamine (100 µM). Data are represented as the mean ± S.E.M. *p<0.05. (D) Real time PCR analysis of mir17–95 RNA expression in IL-11 (100ng/ml) treated wild type and AD-HIES HUVECs. Data are representative of 3 independent experiments. (E) Western analysis of SIAH1 and E2F1 expression in wild type and AD-HIES. Data are representative of 2 independent experiments. (F) Analysis of cytoplasmic verses nuclear active β-catenin in wild type and AD-HIES HUVECs.

Figure 6. PTEN inhibition restores STAT3-inhibited or mutated HUVECs permeability

(A) PTEN expression in wild-type and AD-HIES HUVECs following transfection with a mir19 mimic. (B) PTEN expression in STAT3 inhibited wild-type HUVECs over 7 days. (C) Expression of phosphorylated Src in PTEN inhibitor pre-treated HUVECs (1h) that were subsequently exposed to histamine (100µM). (D–E) In vitro permeability assay of (D) DMSO or C188–9 treated HUVECs or (E) wild-type and AD-HIES HUVECs treated with a PTEN inhibitor (1h) and subsequently exposed to histamine (100µM). (F) Model of STAT3-regulation of vascular permeability in wild type and AD-HIES HUVECs. In wild type cells, histamine treatment leads to Src phosphorylation resulting in the dissociation of β-catenin and the internalization and degradation of VE-cadherin. Under conditions of normal STAT3 signaling, STAT3 induces expression of the mir17–92 microRNA cluster that suppresses PTEN, E2F1 and SIAH1 levels. This allows for the normal regulation of Src signaling and normal distribution of β-catenin cellular dynamics and thus, directly, regulates the amount of VE-cadherin and β-catenin. In STAT3-mutated cells, histamine treatment leads to no Src phosphorylation, due to decreased expression of mir17–92 resulting in increased PTEN. This prevents the internalization and degradation of VE-cadherin, ultimately increasing its expression. Mir17–92 deficiency also allows for increased E2F1 and SIAH1. This will degrade any phosphorylated β-catenin in the cytoplasm and inhibits nuclear translocation of non-phosphorylated cytoplasmic β-catenin. This provides more non-phosphorylated β-catenin to form adherin junctions, leading to decreased vascular permeability.