Interleukin-6-signal transducer and activator of transcription-3 signaling mediates aortic dissections induced by angiotensin II via the T-helper lymphocyte 17-interleukin 17 axis in C57BL/6 mice

Abstract

Objective: Dysregulated angiotensin II (Ang II) signaling induces local vascular interleukin-6 (IL-6) secretion, producing leukocyte infiltration and life-threatening aortic dissections. Precise mechanisms by which IL-6 signaling induces leukocyte recruitment remain unknown. T-helper 17 lymphocytes (Th17) have been implicated in vascular pathology, but their role in the development of aortic dissections is poorly understood. Here, we tested the relationship of IL-6-signal transducer and activator of transcription-3 signaling with Th17-induced inflammation in the formation of Ang II-induced dissections in C57BL/6 mice.

Approach and results: Ang II infusion induced aortic dissections and CD4(+)-interleukin 17A (IL-17A)-expressing Th17 cell accumulation in C57BL/6 mice. A blunted local Th17 activation, macrophage recruitment, and reduced incidence of aortic dissections were seen in IL-6(-/-) mice. To determine the pathological roles of Th17 lymphocytes, we treated Ang II-infused mice with IL-17A-neutralizing antibody or infused Ang II in genetically deficient IL-17A mice and found decreased aortic chemokine monocytic chemotactic protein-1 production and macrophage recruitment, leading to a reduction in aortic dissections. This effect was independent of blood pressure in IL-17A-neutralizing antibody experiment. Application of a cell-permeable signal transducer and activator of transcription-3 inhibitor to downregulate the IL-6 pathway decreased aortic dilation and Th17 cell recruitment. We also observed increased aortic Th17 infiltration and IL-17 mRNA expression in patients with thoracic aortic dissections. Finally, we found that Ang II-mediated aortic dissections occurred independent of blood pressure changes.

Conclusions: Our results indicate that the IL-6-signal transducer and activator of transcription-3 signaling pathway converges on Th17 recruitment and IL-17A signaling upstream of macrophage recruitment, mediating aortic dissections.

Keywords: T-lymphocytes, helper; angiotensin II; aortic dissection, familial; inflammation; interleukin-6; vascular inflammation.

Figure 1

Ang II promoted Th17 cell accumulation in aortic tissues. Age matched WT mice were treated with sham or Ang II for 14 d. (A) Sham and Ang II-treated WT mice were examined for aortic Th17 recruitment. Left panel: IL-17A expression was analyzed using Q-RT- PCR. Circles: sham- treated mice. Squares: Ang II-treated mice. Right panel: Aortic sections were stained for IL-17A-expressing cells. Cell numbers were quantified microscopically and expressed as cells/visual field under 200X magnification. **, p<0.01. Bottom panel, IHC for IL-17A expression in sham and Ang II infused aortas. IL-17A immunostaining is increased in the adventitial-medial border (adventitial border is indicated by arrows). (B) Flow cytometric analysis of aortic CD4 and IL-17A-positive Th17 cells was performed and amount of double-positive cells was measured. *, p<0.05. (C) Flow cytometric analysis of aortic ROR-γT-expressing cells with CD4+ gating was performed. CD4+ ROR-γT+ cells were quantified. White bars: sham-treated animals. Black bars: animals treated with Ang II for 14 d. n=4 in each group. **, p<0.01.

Figure 2. IL-6 deficiency reduced Ang II-induced macrophage and Th17 recruitment

Age matched WT and IL-6^−/− mice were treated with Ang II or saline (sham) for 14 d. (A) Flow cytometric analysis of CD11b-positive macrophages was performed using disassociated aortic cells and the number of CD11b-positive cells was measured. Black curve: Sham-treated WT. Blue curve: Ang II-treated WT. Red curve: Sham-treated IL-6^−/−. Green curve: Ang II-treated IL-6^−/−. n=4 in each group. (B) IL-17A expression was analyzed using Q-RT-PCR. White bar: Sham-treated WT. Black bar: Ang II-treated WT. Cross bars: Sham-treated IL-6^−/−. Grey bar: Ang II-treated IL-6^−/−. n= 3–5 in each group. *, p<0.05.**, p<0.01.(C) Flow cytometric analysis of CD4-positive and IL-17A-positive cells was performed and number of double-positive cells was measured. Representative panels corresponding to each group are shown (n=6). IL-6^−/− showed abated Th17 recruitment to the aorta.

Figure 3. IL-17A neutralization ablated Ang II-induced aortic inflammation and dissection

Mice were treated with Ang II and IL-17A NAb or ICAb for 14 d.(A) IL-17A secretion was quantified in aortic explants. White bars: Sham. Black bars: Ang II and ICAb-treated. Grey bars: Ang II and IL-17A NAb-treated. n=4 in each group. *, p<0.05. (B) During Ang II treatment, in vivo imaging of aortas was performed with ultrasonography and maximum diameter of suprarenal aortas was measured. At 14 d, percentage of aortic dissection featured by presence of intramural hematomas was recorded (left panel). Grey bar: animals treated with Ang II and IL-17A NAb, n=13. Black bars: animals treated with Ang II and ICAb, n=12. Right panel, aortic diameter was quantified at d 3, 8 and 12 for each treatment group. Circles: Ang II and IL-17A NAb-treated mice. Squares: Ang II and ICAb-treated mice.*, p<0.05. (C) Flow cytometric analysis of aortic CD4 and IL-17A-positive Th17 cells was performed and number of double-positive cells was measured. n=5 in each group. (D) Aortic sections were immunostained for macrophages using MOMA-2 antibodies. Representative images of each treatment group from 3 different experiments are shown; both images magnified at 200X. (E) Quantification of aortic macrophages for each treatment condition. MOMA-2+ cells were quantified microscopically as cells/visual field at 200x magnification. *, p<0.05. (F) Systolic blood pressure measurements, recorded with tail-cuff plethysmography, were not different between Ang II and IL-17A NAb-treated mice at baseline or at 6 d of Ang II infusion. n=5 mice per group. *, p<0.05.

Figure 4

IL-17A deficiency blunted inflammatory response and aortic dissections. Age matched WT and IL-17A−/− mice were treated with Sham or Ang II for 14 d. During Ang II treatment, in vivo imaging of aortas was performed with ultrasonography and diameters of aortas were measured (A). Percentage of aortic dissection featured by presence of intramural hematomas was recorded (left panel). White bars: animals treated with Ang II for 7 d. Black bars: animals treated with Ang II for 14 d. n=12 in each group. Right panel: aortic diameters were recorded at 6 and 12 d for each treatment group. Circles: sham-treated mice, n=5 mice respectively for WT and IL-17A−/−. Squares: Ang II-treated mice, n=6 for WT mice and n=9 for IL-17A−/− mice. *, p<0.05; ns, no significance. (B) Flow cytometric analysis of aortic CD4-positive and IL-17A-positive cells was performed and number of double-positive cells was measured. n=4 in each group. (C) MCP-1 was measured in aortic explant culture medium. White bars: sham-treated WT; Black bars Ang II-treated WT; Grey bars: Ang II-treated IL-17A−/−. n=5–6 in each group. *, p<0.05. (D) Flow cytometric analysis of CD11b-positive macrophages in dissociated aortic cells was performed. A representative measurement is shown. Grey curves: Sham-treated WT at 7 d. Red curves: Ang II-treated WT at 7 d. Blue curves: Ang II-treated IL-17−/− at 7d. n=2 in each group. (E) Systolic blood pressure was measured via a non-invasive tail-cuff method in conscious WT (> 4 months old) and IL-17−/− mice (> 8 months old) at baseline and at d 7 of Ang II infusion, as indicated. *, p<0.05.

Figure 5. STAT3 signaling mediated aortic dilation and Th17 formation

WT mice were infused for 7 d with PBS (sham, n=3), Ang II (n=5) or Ang II + pSTAT3ip (n=6). Both Ang II and pSTAT3ip were delivered subcutaneously by osmotic mini-pumps. (A) Expression of aortic SOSC3 mRNA was measured by Q-RT-PCR. *, p<0.05. (B) Aortic ultrasonography was used to monitor the full diameter of the suprarenal segment of the aorta. *, p<0.05. (C) Quantification of Th17 splenic cell population was performed by flow cytometry (n=3 in each group). *, p<0.05.

Figure 6. IL-17-positive cell accumulation was observed in patients with thoracic aortic aneurysms and dissections (TAAD)

In thoracic aortic samples from patients with TGF-β receptor mutation (TGFβR2 R460C), IL-17 was detected by immunofluorescence microscopy. Positive staining is shown in green and counterstaining with DAPI in blue is shown in blue. Representative images of aortic sections from control patients (A–C) and patients with TGFβR2 mutations (D–F)are shown. (G) Quantification of IL-17-positive cells in human aortic samples. IL-17-positive cells per visual filed were counted under a microscope at 200x magnification. White bar: control patients. Black bar: patients with TGFβR2 mutations and Type A dissection. n=3 in each group. **, p<0.01. (H) Q-RT-PCR analysis for hIL-17 mRNA normalized to GAPDH. Fold change of hIL-17 mRNA in TAAD patients relative to control patients is presented.

Figure 7. Ang II induction of dissections is independent of systolic blood pressure

WT mice were infused with Ang II for 7 days in the absence or presence of hydralazine. (A) Baseline and post-Ang II infusion systolic blood pressures were measured using the tail cuff method. Black bars: Ang II-treated WT. Grey bars: Ang II and hydralazine-treated WT. n= 9–11 mice per group. **, p<0.01. (B) The percent of dissections in each group was determined at the end of the study. ns, no significance.